**Serotonin Effects on Expression of the LDL Receptor Family Member LR11 and 7-Ketocholesterol–Induced Apoptosis in Human Vascular Smooth Muscle Cells**

Daiji Nagayama and Ichiro Tatsuno

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67679

#### **Abstract**

We previously confirmed the effect of sarpogrelate hydrochloride (sarpogrelate), 5-hydroxytryptamine (5-HT) 2A receptor antagonist on cardio-ankle vascular index (CAVI) as a marker of systemic arterial stiffness. After 6 months of treatment with sarpogrelate for 35 type 2 diabetic patients, decreased CAVI, indicating the ameliorated arterial stiffness, was observed. Therefore, via 5-HT2A receptor blockade, sarpogrelate might effect as a vasoactive agent, as well as an inhibitor of platelet aggregation. 5-HT is a known mitogen for vascular smooth muscle cells (VSMCs). In addition, the pathogenic change of VSMCs such as dedifferentiation and proliferation/apoptosis represents one of the atherosclerotic changes. On the other hand, LR11, a mosaic LDL receptor family member, may involve in the invasion of VSMCs into neointimal thickening. We therefore investigated an *in vitro* study to clarify whether 5-HT was concerned to LR11 expression and apoptosis of human VSMCs induced by 7-ketocholesterol (7KCHO), a major oxidation product of cholesterol involved in plaque destabilization. Resultantly, 5-HT accelerated the proliferation of VSMCs, and this effect was suppressed by simultaneous addition of sarpogrelate. Sarpogrelate also attenuated the 5-HT–induced LR11 mRNA expression in VSMCs. Additionally, 5-HT attenuated the 7KCHO-induced apoptosis of VSMCs through caspase-dependent pathway. These results suggest new knowledge on the modification of human VSMCs induced by 5-HT.

**Keywords:** arterial stiffness, vascular smooth muscle cells, LR11, apoptosis, 7-ketocholesterol

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

Invasion of vascular smooth muscle cells (VSMCs) to intima takes a principle finding in the progression of atherosclerosis and the incidence of restenosis after vascular intervention [1– 3]. Lately, LR11, a mosaic low-density lipoprotein (LDL) receptor family member, is known to exist largely in VSMCs of the hyperplastic intima, but not the media and induce the invasion and migration potential of intimal VSMCs, speculated to originate from medial VSMCs [4–6]. Meanwhile, reduced extracellular matrix, reduced number of VSMCs, thin fibrous cap, and extracellular oxysterol accumulation have been observed in unstable plaques [7– 9]. We have previously shown that 7-ketocholesterol (7KCHO), a major oxidation product of cholesterol, revealed as an apoptosis inducer on VSMCs [10, 11], and attenuated the migration of VSMCs [12]. These reports propose that the presence of 7KCHO may reduce the number of VSMCs and contribute to unstable plaque. Generally, proliferation/apoptosis and dedifferentiation of VSMCs in the arterial intima identify one of the pathological findings observed in atherogenesis [2, 13, 14]. Meanwhile, the factors modulating proliferation/ apoptosis of VSMCs and the potential cellular mechanisms are not fully elucidated.

Serotonin (5-hydroxytryptamine, 5-HT), secretion from activated platelets, is recognized to be a naturally occurring vasoactive mediator involved in vascular inflammation and atherogenesis [15]. 5-HT has multiple receptor subtypes [16] and induces platelet aggregation, vasoconstriction, and VSMC proliferation [17, 18]. In addition, the plasma level of 5-hydroxyindole-3-acetic acid (5-HIAA; a derivative end product of 5-HT) is relatively high in subjects with visceral adiposity, revealing that 5-HT is one of the potential mediators for atherogenesis in lifestyle diseases [19].

Sarpogrelate hydrochloride (sarpogrelate), a selective 5-HT2A receptor antagonist, is used for diabetic patients with chronic arterial occlusive diseases [20] and is known to suppress platelet aggregation, vascular endothelial dysfunction, and smooth muscle contraction mediated via 5-HT2A receptor [21, 22]. The restorative effects of sarpogrelate on cardiovascular disturbance in experimental diabetic rats were also reported [23]. Furthermore, we investigated prospectively the effect of sarpogrelate on systemic arterial stiffness assessed by cardio-ankle vascular index (CAVI) in type 2 diabetic patients [24]. After 6 months of treatment with sarpogrelate for 35 Japanese type 2 diabetic patients, decreased CAVI, indicating the ameliorated arterial stiffness, was observed (**Table 1**). Sarpogrelate is known to inhibit 5-HT–induced vascular smooth muscle contraction and/or cell proliferation [25, 26]. Moreover, Shirai et al. have reported that CAVI might be affected by change in contractility of vascular smooth muscle [27]. Therefore, these results suggest that sarpogrelate may ameliorate arterial stiffness through inhibiting vascular smooth muscle contractility. However, the effects of 5-HT on vascular composition are not fully understood. We hypothesized that 5-HT was concerned to the invasion and migration of VSMCs through the regulation of LR11, besides the apoptosis of VSMCs.

We confirmed the effect of 5-HT on LR11 expression in human VSMCs. Additionally, whether there was an interaction of 5-HT with 7KCHO in inducing VSMC apoptosis was investigated.


Data are presented as mean ± standard deviation. Paired *t*-test was used in comparing baseline and 6-month data. BMI, body mass index; CAVI, cardio-ankle vascular index; sBP, systolic blood pressure; dBP, diastolic blood pressure; TG, triglyceride; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; FPG, fasting plasma glucose; HbA1c, glycosylated hemoglobin; ARB, angiotensin receptor blocker; ACE-I, angiotensin converting enzyme inhibitor.

**Table 1.** Participant characteristics before and after 6 months of sarpogrelate treatment.

## **2. Materials and methods**

#### **2.1. Cell cultures**

**1. Introduction**

204 Serotonin - A Chemical Messenger Between All Types of Living Cells

in lifestyle diseases [19].

of VSMCs.

Invasion of vascular smooth muscle cells (VSMCs) to intima takes a principle finding in the progression of atherosclerosis and the incidence of restenosis after vascular intervention [1– 3]. Lately, LR11, a mosaic low-density lipoprotein (LDL) receptor family member, is known to exist largely in VSMCs of the hyperplastic intima, but not the media and induce the invasion and migration potential of intimal VSMCs, speculated to originate from medial VSMCs [4–6]. Meanwhile, reduced extracellular matrix, reduced number of VSMCs, thin fibrous cap, and extracellular oxysterol accumulation have been observed in unstable plaques [7– 9]. We have previously shown that 7-ketocholesterol (7KCHO), a major oxidation product of cholesterol, revealed as an apoptosis inducer on VSMCs [10, 11], and attenuated the migration of VSMCs [12]. These reports propose that the presence of 7KCHO may reduce the number of VSMCs and contribute to unstable plaque. Generally, proliferation/apoptosis and dedifferentiation of VSMCs in the arterial intima identify one of the pathological findings observed in atherogenesis [2, 13, 14]. Meanwhile, the factors modulating proliferation/

apoptosis of VSMCs and the potential cellular mechanisms are not fully elucidated.

Serotonin (5-hydroxytryptamine, 5-HT), secretion from activated platelets, is recognized to be a naturally occurring vasoactive mediator involved in vascular inflammation and atherogenesis [15]. 5-HT has multiple receptor subtypes [16] and induces platelet aggregation, vasoconstriction, and VSMC proliferation [17, 18]. In addition, the plasma level of 5-hydroxyindole-3-acetic acid (5-HIAA; a derivative end product of 5-HT) is relatively high in subjects with visceral adiposity, revealing that 5-HT is one of the potential mediators for atherogenesis

Sarpogrelate hydrochloride (sarpogrelate), a selective 5-HT2A receptor antagonist, is used for diabetic patients with chronic arterial occlusive diseases [20] and is known to suppress platelet aggregation, vascular endothelial dysfunction, and smooth muscle contraction mediated via 5-HT2A receptor [21, 22]. The restorative effects of sarpogrelate on cardiovascular disturbance in experimental diabetic rats were also reported [23]. Furthermore, we investigated prospectively the effect of sarpogrelate on systemic arterial stiffness assessed by cardio-ankle vascular index (CAVI) in type 2 diabetic patients [24]. After 6 months of treatment with sarpogrelate for 35 Japanese type 2 diabetic patients, decreased CAVI, indicating the ameliorated arterial stiffness, was observed (**Table 1**). Sarpogrelate is known to inhibit 5-HT–induced vascular smooth muscle contraction and/or cell proliferation [25, 26]. Moreover, Shirai et al. have reported that CAVI might be affected by change in contractility of vascular smooth muscle [27]. Therefore, these results suggest that sarpogrelate may ameliorate arterial stiffness through inhibiting vascular smooth muscle contractility. However, the effects of 5-HT on vascular composition are not fully understood. We hypothesized that 5-HT was concerned to the invasion and migration of VSMCs through the regulation of LR11, besides the apoptosis

We confirmed the effect of 5-HT on LR11 expression in human VSMCs. Additionally, whether there was an interaction of 5-HT with 7KCHO in inducing VSMC apoptosis was investigated.

VSMCs prepared from human femoral artery were cultured in growth medium of Dulbecco's modified Eagle's minimal essential medium supplemented with delipidated calf serum mixture or 5–10% (v/v) heat inactivated fetal calf serum (FCS), 40-μg/mL gentamicin, and 2-mmol/L L-glutamine, maintained at 37°C in 5% carbon dioxide.

Sarpogrelate was a gifted reagent from Mitsubishi-Tanabe Pharma Co., Osaka, Japan. 5-HT, 7KCHO, and other reagents were generously provided by Sigma (St. Louis, Missouri).

## **2.2. Proliferation of VSMCs**

VSMCs were plated into 12-well microtiter plates in triplicate (10<sup>4</sup> cells per well). After 72 h of growth, the medium was changed to Dulbecco's modified Eagle medium containing 5% FCS and sarpogrelate and/or 5-HT was administered. Thereafter, proliferation of VSMCs was evaluated by a hemocytometer during 0 and 8 days after the administration of sarpogrelate and/or 5-HT.

### **2.3. Reverse transcription polymerase chain reaction**

Total RNA was isolated from VSMCs by RNeasy kit (Qiagen, Courtaboeuf, France), and complementary (c) DNA was prepared by reverse transcription (RT-) polymerase chain reaction (PCR) kit (TaKaRa, Tokyo, Japan) as already described in the manufacturer's instructions. RNA concentrations were evaluated by measuring absorbance at 260 nm. Thereafter, RT-PCR was carried out using 1 μg of reverse transcribed total RNA. Expression of β-actin internal standard was adopted as housekeeping gene for quantifying RNA levels. The specific primers were as follows:

LR11:

sense 5′-AGGAGGGCATCCTGCAGTATTGCCAAGAAG-3′

antisense 5′-TGGCGACGGTGTGCCAGTGA-3′

β-actin:

sense 5′-CTCTTCCAGCCTTCCTTCCT-3′

```
antisense 5′-AGCACTGTGTTGGCGTACAG-3′
```
PCR was run on a Gene Amp PCR System 9700 (Applied Biosystems, Foster city, CA) for 35 cycles both for β-actin and LR11. The terminal cycle for denaturation, annealing, and elongation was required at 94°C for 30 s, 56°C for 30 s, and 72°C for 60 s for each. The amplified products were electrophoresed on 1% agarose gels, stained with ethidium bromide, and visualized by UV irradiation. Furthermore, the images were photographed using an Olympus digital camera (Tokyo, Japan).

### **2.4. Caspase activity of VSMCs**

Two methods were used in order to measure caspase activity in VSMCs. First, a luminescent assay for measuring caspase-3 and -7 (caspase-3/7) activities was adopted. The other was flow cytometric analysis by the fluorescein-5–isothiocyanate (FITC) Active Caspase-3 Apoptosis Kit (BD Pharmingen, La Jolla, California). After two washes in ice-cold phosphate-buffered saline, VSMCs incubated in 96-well microplates were scraped off the tissue culture dish, and caspase activities were evaluated using the Caspase-Glo® 3/7 Assay (Promega, Wisconsin) as described in the manufacturer's protocol.

WST-8 cell counting kit (Dojindo Laboratories, Kumamoto, Japan) was adopted to evaluate bioavailability of VSMCs. When serial dilutions of VSMCs were plated into 96-well microtiter plates and analyzed, an absorbance response at 460 nm was observed linearly (data not shown). The number of VSMCs was measured using the regression equation. Caspase-3/7 activity was corrected for mean cell number measured for each.

## **2.5. Apoptosis of VSMCs**

mixture or 5–10% (v/v) heat inactivated fetal calf serum (FCS), 40-μg/mL gentamicin, and

Sarpogrelate was a gifted reagent from Mitsubishi-Tanabe Pharma Co., Osaka, Japan. 5-HT, 7KCHO, and other reagents were generously provided by Sigma (St. Louis, Missouri).

of growth, the medium was changed to Dulbecco's modified Eagle medium containing 5% FCS and sarpogrelate and/or 5-HT was administered. Thereafter, proliferation of VSMCs was evaluated by a hemocytometer during 0 and 8 days after the administration of sarpogrelate

Total RNA was isolated from VSMCs by RNeasy kit (Qiagen, Courtaboeuf, France), and complementary (c) DNA was prepared by reverse transcription (RT-) polymerase chain reaction (PCR) kit (TaKaRa, Tokyo, Japan) as already described in the manufacturer's instructions. RNA concentrations were evaluated by measuring absorbance at 260 nm. Thereafter, RT-PCR was carried out using 1 μg of reverse transcribed total RNA. Expression of β-actin internal standard was adopted as housekeeping gene for quantifying RNA levels. The specific primers

PCR was run on a Gene Amp PCR System 9700 (Applied Biosystems, Foster city, CA) for 35 cycles both for β-actin and LR11. The terminal cycle for denaturation, annealing, and elongation was required at 94°C for 30 s, 56°C for 30 s, and 72°C for 60 s for each. The amplified products were electrophoresed on 1% agarose gels, stained with ethidium bromide, and visualized by UV irradiation. Furthermore, the images were photographed using an Olympus digital

Two methods were used in order to measure caspase activity in VSMCs. First, a luminescent assay for measuring caspase-3 and -7 (caspase-3/7) activities was adopted. The other was flow

cells per well). After 72 h

2-mmol/L L-glutamine, maintained at 37°C in 5% carbon dioxide.

206 Serotonin - A Chemical Messenger Between All Types of Living Cells

VSMCs were plated into 12-well microtiter plates in triplicate (10<sup>4</sup>

**2.3. Reverse transcription polymerase chain reaction**

sense 5′-AGGAGGGCATCCTGCAGTATTGCCAAGAAG-3′

antisense 5′-TGGCGACGGTGTGCCAGTGA-3′

antisense 5′-AGCACTGTGTTGGCGTACAG-3′

sense 5′-CTCTTCCAGCCTTCCTTCCT-3′

**2.2. Proliferation of VSMCs**

and/or 5-HT.

were as follows:

camera (Tokyo, Japan).

**2.4. Caspase activity of VSMCs**

LR11:

β-actin:

VSMCs were harvested by trypsinization and kept into 5-mL fluorescence-activated cell sorting (FACS) tubes in phosphate buffered saline (PBS) (pH 7.4) containing 5% FCS. Thereafter, samples were processed in a Becton Dickinson FACScalibur (Immunocytometry Systems, San Jose, California) equipped with a 15 mW, 488 nm argon laser, and filter configuration. BD™ Biosciences Propidium Iodide Staining Solution was adopted to quantify DNA content. FITC Active Caspase-3 Apoptosis Kit (BD Pharmingen) was used to evaluate active caspase-3. Cell samples (20,000 cells) were analyzed on a FACSort cytometer using Cell Quest Pro software (BD Biosciences). The percentage of apoptotic cells in each sample was quantified by manual counting of adherent VSMCs using fluorescence microscopy.

## **2.6. Statistical analysis**

Statistical analyses were carried out using SPSS software (version 11.5, Chicago, IL, USA). The data are presented as mean ± standard deviation (SD). The effects of reagents were compared by one-way ANOVA followed by Bonferroni multiple comparison test and values of *p* < 0.05 were considered statistically significant.

## **3. Results**

## **3.1. Effects of 5-HT and/or sarpogrelate on VSMC proliferation**

**Figure 1A** proposes VSMC proliferation in the absence or presence of 5-HT for 8 days. Administration of 5-HT (100 μM) to VSMCs significantly increased proliferation at days 5 and 8. The cell counts of VSMCs on day 8 after the administration of 5-HT (1, 10, or 100 μM) with or without sarpogrelate (10 μM) were shown in **Figure 1B**. The administration of 5-HT to VSMCs induced a dose-dependent increase in cell number and simultaneous addition of sarpogrelate attenuated the effect of 5-HT.

**Figure 1.** Effects of serotonin (5-HT) and sarpogrelate on proliferation of vascular smooth muscle cells (VSMCs). (A) Changes in cell number over time. After seeding VSMCs in 12-well microplates (1 × 10<sup>4</sup> /well, in triplicate) and culturing for 72 h, 5-HT (100 μM) was added. Cell number was counted from day 0 to day 8 after the addition of 5-HT. Data are presented as mean ± SD of triplicate samples. \* Significantly higher (*p* < 0.05, unpaired *t*-test) cell count compared with control. (B) Effects of 5-HT concentration and sarpogrelate on cell proliferation. After seeding VSMCs in 12-well microplates (1 × 10<sup>4</sup> /well, in triplicate) and culturing for 72 h, 5-HT and sarpogrelate at indicated concentrations were added. Cell numbers were counted on day 8 after addition of 5-HT and/or sarpogrelate. Data are presented as mean ± SD of triplicate samples. \* *p* < 0.05, \*\**p* < 0.01; one-way ANOVA followed by Bonferroni multiple comparison test.

**Figure 2.** Effects of serotonin (5-HT) and sarpogrelate on LR11 mRNA expression in vascular smooth muscle cells (VSMCs). After seeding VSMCs in 6-well microplates (6 × 10<sup>4</sup> /well, in triplicate) and culturing for 72 h, 5-HT and sarpogrelate at indicated concentrations were added and cultured for another 72 h. Upper panel shows LR11 mRNA expression determined by reverse transcription PCR. Beta-actin expression shown in the lower panel was used as internal standard.

#### **3.2. Effects of 5-HT and/or sarpogrelate on LR11 mRNA expression in VSMCs**

RT-PCR showed that LR11 mRNA expression was enhanced dose dependently by administration of 5-HT at 1–100 μM in VSMCs. Additionally, simultaneous addition of sarpogrelate at 10 μM attenuated the effect of 5-HT (**Figure 2**).

#### **3.3. Effects of 5-HT and/or 7KCHO on caspase activity in VSMCs**

The effects of 5-HT and/or 7KCHO on caspase activity in VSMCs were examined using two methods. Flow cytometric analysis was carried out by VSMCs stained with FITC-conjugated antiactive caspase-3 monoclonal antibody. The histograms in **Figure 3A** propose the distribution of VSMCs. Administration of 5-HT to VSMCs alone caused a slight leftward shift of the peak from control, revealing an attenuation in active caspase-3 expression [28]. Contrastingly, administration of 7KCHO alone caused an enhancement in active caspase-3 expression as revealed by a rightward shift of the histogram, and this effect of 7KCHO was attenuated by simultaneous addition of 5-HT (**Figure 3B**).

Next, a luminescent assay was carried out in order to evaluate caspase-3/7 activities using the same protocol shown in the previous experiment. Administration of 5-HT alone did not change the caspase-3/7 activities in VSMCs. On the other hand, administration of 7KCHO alone enhanced caspase-3/7 activity ninefold compared to the control, and this

**Figure 3.** Effects of serotonin (5-HT) and/or 7-ketocholesterol (7KCHO) on caspase activity in vascular smooth muscle cells (VSMCs). (A and B) Caspase-3 activity assayed by antiactive caspase antibody and flow cytometry. After seeding VSMCs in 6-well microplates (8 × 10<sup>4</sup> /well, in duplicate) and culturing for 48 h, VSMCs were incubated with no addition, addition of 5-HT (100 μM) alone, or addition of 5-HT (100 μM) and 7KCHO (50 μM) for another 48 h. The cells were maintained in Dulbecco's modified Eagle's minimal essential medium containing 10% FBS and 1% non essential amino acid, and incubated at 37°C, 5% CO2 . Caspase-3 expression was analyzed using FITC-conjugated monoclonal anti active caspase-3 antibody followed by flow cytometry. Changes in active caspase-3 activity are shown in FL1 histograms. (C) Caspase-3/7 activities assayed by luminescent assay. VSMCs were seeded into 96-well microplates (1 × 10<sup>5</sup> /well, in triplicate), and incubated with or without the addition of 5-HT (100 μM) and/or 7KCHO (50 μM) for 48 h. Luciferase activity was measured according to the protocol from Promega. Data are presented as mean ± SD of triplicate samples. \* *p* < 0.01; one-way ANOVA followed by Bonferroni multiple comparison test.

**3.2. Effects of 5-HT and/or sarpogrelate on LR11 mRNA expression in VSMCs**

at 10 μM attenuated the effect of 5-HT (**Figure 2**).

(VSMCs). After seeding VSMCs in 6-well microplates (6 × 10<sup>4</sup>

**3.3. Effects of 5-HT and/or 7KCHO on caspase activity in VSMCs**

RT-PCR showed that LR11 mRNA expression was enhanced dose dependently by administration of 5-HT at 1–100 μM in VSMCs. Additionally, simultaneous addition of sarpogrelate

**Figure 2.** Effects of serotonin (5-HT) and sarpogrelate on LR11 mRNA expression in vascular smooth muscle cells

sarpogrelate at indicated concentrations were added and cultured for another 72 h. Upper panel shows LR11 mRNA expression determined by reverse transcription PCR. Beta-actin expression shown in the lower panel was used as

**Figure 1.** Effects of serotonin (5-HT) and sarpogrelate on proliferation of vascular smooth muscle cells (VSMCs).

culturing for 72 h, 5-HT (100 μM) was added. Cell number was counted from day 0 to day 8 after the addition of

count compared with control. (B) Effects of 5-HT concentration and sarpogrelate on cell proliferation. After seeding

concentrations were added. Cell numbers were counted on day 8 after addition of 5-HT and/or sarpogrelate. Data are

/well, in triplicate) and

Significantly higher (*p* < 0.05, unpaired *t*-test) cell

/well, in triplicate) and culturing for 72 h, 5-HT and

/well, in triplicate) and culturing for 72 h, 5-HT and sarpogrelate at indicated

*p* < 0.05, \*\**p* < 0.01; one-way ANOVA followed by Bonferroni multiple

(A) Changes in cell number over time. After seeding VSMCs in 12-well microplates (1 × 10<sup>4</sup>

5-HT. Data are presented as mean ± SD of triplicate samples. \*

208 Serotonin - A Chemical Messenger Between All Types of Living Cells

VSMCs in 12-well microplates (1 × 10<sup>4</sup>

comparison test.

internal standard.

presented as mean ± SD of triplicate samples. \*

The effects of 5-HT and/or 7KCHO on caspase activity in VSMCs were examined using two methods. Flow cytometric analysis was carried out by VSMCs stained with FITC-conjugated effect of 7KCHO was attenuated by simultaneous addition of 5-HT (**Figure 3C**). To sum up, almost same effects of 5-HT were shown in both methods for measuring caspase activity in VSMCs.

#### **3.4. Effects of 5-HT and/or 7KCHO on quantitation of apoptosis in VSMCs**

Apoptotic DNA fragmentation in VSMCs was evaluated by propidium iodide fluorescence to clarify the apoptosis-inducing effect of 7KCHO in the absence or presence of 5-HT (**Figure 4**). The apoptotic rate was elevated after administration of 7KCHO (50 μM) alone, but this effect of 7KCHO was suppressed by simultaneous addition of 5-HT at 100 μM.

**Figure 4.** Effects of serotonin (5-HT) and/or 7-ketocholesterol (7KCHO) on quantitative analysis of apoptosis of vascular smooth muscle cells (VSMCs). After seeding VSMCs in 6-well microplates (8 × 10<sup>4</sup> /well, in duplicate) and culturing for 48 h, VSMCs were incubated with or without the addition of 5-HT (100 μM) for another 96 h. 7KCHO (50 μM) was added to some wells 72 h after the addition of 5-HT. Cells were stained with 50 μg/ml of propidium iodide after cell lysis and analyzed by flow cytometry. Apoptotic rate is the percentage of nuclei in the sub-G1 population representing DNA fragmentation as shown in FL2 histograms. Data are presented as mean ± SD of three independent experiments. \* *p* < 0.01; one-way ANOVA followed by Bonferroni multiple comparison test.

## **4. Discussion**

In the present study, 5-HT accelerated the proliferation of human VSMCs and this effect of 5-HT was attenuated by simultaneous addition of sarpogrelate. Moreover, sarpogrelate also suppressed the 5-HT–induced enhancement of LR11 mRNA expression in VSMCs. 5-HT attenuated the 7KCHO-induced VSMC apoptosis through caspase-3/7–dependent pathway besides. There is so far no evidence providing the effect of 5-HT on LR11 expression and apoptosis in VSMCs. The present report demonstrates the effects of 5-HT on such pathogenic changes in VSMCs.

effect of 7KCHO was attenuated by simultaneous addition of 5-HT (**Figure 3C**). To sum up, almost same effects of 5-HT were shown in both methods for measuring caspase activity

Apoptotic DNA fragmentation in VSMCs was evaluated by propidium iodide fluorescence to clarify the apoptosis-inducing effect of 7KCHO in the absence or presence of 5-HT (**Figure 4**). The apoptotic rate was elevated after administration of 7KCHO (50 μM) alone, but this effect

In the present study, 5-HT accelerated the proliferation of human VSMCs and this effect of 5-HT was attenuated by simultaneous addition of sarpogrelate. Moreover, sarpogrelate also suppressed the 5-HT–induced enhancement of LR11 mRNA expression in VSMCs. 5-HT attenuated the 7KCHO-induced VSMC apoptosis through caspase-3/7–dependent pathway

**Figure 4.** Effects of serotonin (5-HT) and/or 7-ketocholesterol (7KCHO) on quantitative analysis of apoptosis of vascular

48 h, VSMCs were incubated with or without the addition of 5-HT (100 μM) for another 96 h. 7KCHO (50 μM) was added to some wells 72 h after the addition of 5-HT. Cells were stained with 50 μg/ml of propidium iodide after cell lysis and analyzed by flow cytometry. Apoptotic rate is the percentage of nuclei in the sub-G1 population representing DNA fragmentation as shown in FL2 histograms. Data are presented as mean ± SD of three independent experiments. \*

/well, in duplicate) and culturing for

*p* < 0.01;

smooth muscle cells (VSMCs). After seeding VSMCs in 6-well microplates (8 × 10<sup>4</sup>

one-way ANOVA followed by Bonferroni multiple comparison test.

**3.4. Effects of 5-HT and/or 7KCHO on quantitation of apoptosis in VSMCs**

210 Serotonin - A Chemical Messenger Between All Types of Living Cells

of 7KCHO was suppressed by simultaneous addition of 5-HT at 100 μM.

in VSMCs.

**4. Discussion**

The mechanism by which 5-HT regulates the number of intimal VSMCs has not been fully clarified, so that the modulators for migration of VSMCs from the arterial media to the intima should be examined. Previous reports have shown that LR11 expression was largely involved in the differentiation of VSMCs. Furthermore, the VSMCs with a contractile phenotype observed in the arterial media do not express LR11, whereas the VSMCs in an active synthetic phenotype located in the intima highly express LR11 [29, 30]. Additionally, circulating soluble form of LR11 concentrations in serum is known to correlate with the degree of coronary organic stenosis, carotid intima-media thickness, and pulmonary arterial hypertension [31–33]. These findings suggest that LR11 may play key role of modification in VSMCs during atherogenesis. Our data suggested that 5-HT enhanced the expression of LR11 mRNA in VSMCs, and simultaneous addition of sarpogrelate attenuated this effect of 5-HT. These results reveal that 5-HT may participate to neointimal thickening through stimulating not only proliferation, but also invasion of VSMCs accompanied by upregulation of LR11. Therefore, sarpogrelate may show the pleiotropic effect on vascular tissue, such as decreased systemic arterial stiffness, partially through down-regulation of LR11 in VSMCs. Additionally, sarpogrelate was recently reported to ameliorate the development of chronic hypoxic pulmonary hypertension through the occurrence of increased apoptosis and decreased proliferation of VSMCs [34]. However, we cannot definitely consider that the effect of 5-HT on VSMCs is absolutely malignant to human body.

Apoptosis of VSMCs located in atheroma is known to be associ ated with vulnerable plaque ruptures [35, 36]. In this study, luminescent assay and flow cytometric analysis revealed that 7KCHO enhanced the caspase-3/7–dependent apoptotic pathway. In a phase of progressive atherosclerotic plaque formation, 7KCHO is speculated to induce the absence of VSMCs, which make plaque unstable leading to rupture. Meanwhile, simultaneous addition of 5-HT suppressed 7KCHO-induced VSMC apoptosis. These findings reveal that 5-HT may reduce the occurrence of plaque rupture through the attenuation of 7KCHO-induced VSMC apoptosis.

Whether 5-HT is favorable or not for the vascular remodeling process is still controversial. In state of vascular injury, subsequent platelet activation accompanied by endothelial damage provides increasing local plasma level of 5-HT. Furthermore, 5-HT causes the proliferation, contraction, and migration of VSMCs through the 5-HT2A receptor amplified by various intracellular signaling pathways [37–39]. Thus, 5-HT plays the basic principle of vascular repair, which spreads to neointimal thickening and decrease of peripheral blood flow. Note that the administration of 5-HT to VSMCs resulted in the enhancement of potential for cell migration caused by up-regulation of LR11 in the present study. Moreover, the suppressive effect of 5-HT on VSMC apoptosis might concern to the suppression of vulnerability in atheromatous plaque caused by 7KCHO. These effects of 5-HT can be protective for vascular composition.

It is still controversial whether modification of VSMCs along with upregulation of LR11 directly concern to the attenuation in apoptosis-inducing effect of 7KCHO. Further elucidation about the causal relationship of LR11 expression with VSMC apoptosis should be examined.

Resultantly, 5-HT accelerated the proliferation of VSMCs, and this effect was suppressed by simultaneous addition of sarpogrelate. Sarpogrelate also attenuated the 5-HT–induced LR11 mRNA expression in VSMCs. Additionally, 5-HT attenuated the 7KCHO-induced apoptosis of VSMCs through caspase-dependent pathway. These results suggest new knowledge on the modification of human VSMCs induced by 5-HT.

## **Declaration of conflicting interests**

Potential conflicts of interest with any of the authors: None.

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

## **Author details**

Daiji Nagayama1,2\* and Ichiro Tatsuno2 \*

\*Address all correspondence to: deverlast96071@gmail.com and ichiro.tatsuno@med.toho-u.ac.jp

1 Center of Endocrinology and Metabolism, Shin-Oyama City Hospital, Hitotonoya, Oyama-City, Japan

2 Center of Diabetes, Endocrinology and Metabolism, Toho University, Sakura Medical Center, Shimoshizu, Sakura-City, Japan

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It is still controversial whether modification of VSMCs along with upregulation of LR11 directly concern to the attenuation in apoptosis-inducing effect of 7KCHO. Further elucidation about the causal relationship of LR11 expression with VSMC apoptosis should be

Resultantly, 5-HT accelerated the proliferation of VSMCs, and this effect was suppressed by simultaneous addition of sarpogrelate. Sarpogrelate also attenuated the 5-HT–induced LR11 mRNA expression in VSMCs. Additionally, 5-HT attenuated the 7KCHO-induced apoptosis of VSMCs through caspase-dependent pathway. These results suggest new knowledge on the

This research received no specific grant from any funding agency in the public, commercial,

\*Address all correspondence to: deverlast96071@gmail.com and ichiro.tatsuno@med.toho-u.ac.jp 1 Center of Endocrinology and Metabolism, Shin-Oyama City Hospital, Hitotonoya, Oyama-

2 Center of Diabetes, Endocrinology and Metabolism, Toho University, Sakura Medical

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[4] Clowes AW, Schwartz SM. Significance of quiescent smooth muscle migration in the

migration of smooth muscle cells in vitro. Circulation. 2002;105(15):1830-1836.

\*

occlusion and restenosis. Thromb Haemost. 1995;74(1):541-551.

injured rat carotid artery. Circ Res. 1985;56(1):139-145.

examined.

modification of human VSMCs induced by 5-HT.

212 Serotonin - A Chemical Messenger Between All Types of Living Cells

Potential conflicts of interest with any of the authors: None.

**Declaration of conflicting interests**

Daiji Nagayama1,2\* and Ichiro Tatsuno2

Center, Shimoshizu, Sakura-City, Japan

Opin Lipidol. 1994;5(5):323-330.

or not-for-profit sectors.

**Author details**

City, Japan

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[21] Nonogaki K, Nozue K, Oka Y. Increased hypothalamic 5-HT2A receptor gene expression and effects of pharmacologic 5-HT2A receptor inactivation in obese Ay mice. Biochem

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[25] Sharma SK, Del Rizzo DF, Zahradka P, Bhangu SK, Werner JP, Kumamoto H, Takeda N, Dhalla NS. Sarpogrelate inhibits serotonin-induced proliferation of porcine coronary artery smooth muscle cells: Implications for long-term graft patency. Ann Thorac Surg.

[26] Nishihira K, Yamashita A, Tanaka N, Moriguchi-Goto S, Imamura T, Ishida T, Kawashima S, Yamamoto R, Kitamura K, Asada Y. Serotonin induces vasoconstriction of smooth muscle cell-rich neointima through 5-hydroxytryptamine2A receptor in rabbit femoral arteries. J Thromb Haemost. 2008;6(7):1207-1214. DOI: 10.1111/j.1538-7836.2008.02996.

[27] Shirai K, Song M, Suzuki J, Kurosu T, Oyama T, Nagayama D, Miyashita Y, Yamamura S, Takahashi M. Contradictory effects of β1- and α1- aderenergic receptor blockers on cardio-ankle vascular stiffness index (CAVI)-CAVI independent of blood pressure. J

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[29] Jiang M, Bujo H, Zhu Y, Yamazaki H, Hirayama S, Kanaki T, Shibasaki M, Takahashi K, Schneider WJ, Saito Y. Pitavastatin attenuates the PDGF-induced LR11/uPA receptormediated migration of smooth muscle cells. Biochem Biophys Res Commun. 2006; 348

[30] Ohwaki K, Bujo H, Jiang M, Yamazaki H, Schneider WJ, Saito Y. A secreted soluble form of LR11, specifically expressed in intimal smooth muscle cells, accelerates formation of

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2001;71(6):1856-1864.


**Section 4**

## **Systems**

## **Chapter 10**

## **Serotonin in Neurological Diseases**

Jolanta Dorszewska, Jolanta Florczak-Wyspianska,

Marta Kowalska, Marcin Stanski,

Alicja Kowalewska and Wojciech Kozubski

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69035

#### **Abstract**

Serotonin (5-HT) is responsible for anxiety, aggression, and stress. Alterations in a serotonergic system play a significant role in pathogenesis of neurological diseases and neuropsychiatric disorders. A wide range of disturbances associated with serotonergic neurotransmission results from different functions of 5-HT in a nervous system. It is believed that 5-HT may be involved in the pathogenesis of migraine, epilepsy, Parkinson's disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), attention-deficit hyperactivity disorder (ADHD), and autism spectrum disorder (ASD). In these diseases, disturbances of 5-HT and its metabolites, such as 5-hydroxyindoleacetic acid (5-HIAA), were observed in the plasma, blood platelets, and cerebrospinal fluid (CSF). Changes in the level of this biogenic amine (5-HT) may be associated with malfunction of 5-HT receptors, reuptake transporter for 5-HT (5-HTT, SERT), the enzymes responsible for the synthesis and metabolism of 5-HT, and genetic variants for serotonergic system. It seems that 5-HT and its metabolites may be used as a diagnostic and prognostic marker for neurological diseases or a target for more efficient therapy in neurology in the future.

**Keywords:** serotonin, molecular factors, neurological diseases

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

Serotonin (5-HT) is a neurotransmitter responsible for anxiety, aggressive behavior, stress, blood pressure regulation, peristaltic movements, heart rate, and the coagulation system. 5-hydroxytryptamine (5-HT) is produced in neurons and gut cells, as well as in the walls of blood vessels and the heart. On the periphery, 5-HT is located in platelets, which enters via 5-HT reuptake transporter (5-HTT, SERT) [1].

The level of 5-HT in whole blood is in the range of 65–250 ng/ml and in the plasma has a lower value, 5.6–23.9 ng/ml [2]. 5-HT enhances the response of the adrenal medulla, and other sympathetic ganglia using 5-HT2A/3. It has been shown that the impairment of serotonin transporter (SERT) function in addition to the increased 5-HT in the extracellular fluid and increased turnover of 5-HT and its decreased level in nerve cells causes an abnormal stress response in the form of anxiety, as well as an excessive response of the adrenal medulla, including triggered by the hypothalamic-pituitary axis (with no effects on the expression of tyrosine hydroxylase and AT2 receptors) [3]. Furthermore, 5-HT released from the terminals of afferent vagal neurons enhances the activity of the catecholaminergic neurons of the solitary tract nucleus (pulsed through potentiation of glutamatergic) and the effect on food intake and cardiovascular reflexes [4]. 5-HT acting on 5-HT4 receptors in the human heart causes stimulation of the atrium, pro-arrhythmic effect, produces a positive inotropic effect. At the same time, stimulation of 5-HT1B/1D endings of the sympathetic cardiac causes decreased release of norepinephrine (NE) [2]. 5-HT binds competitively to the binding site of catechol-O-methyltransferase (COMT) in binding site S-adenosyl-S-methionine, inhibiting methylation substrates for this enzyme [5]. It also acts antiapoptotic by stimulating the expression of cystathionine-beta-synthase (CBS) and increases the level of hydrogen sulfide (H2 S) and antioxidant activity [6].

Currently, it is believed that disturbances in the level of 5-HT may be associated with the pathogenesis of few neurological diseases such as migraine [7], epilepsy [8], Parkinson's disease (PD) [1], multiple sclerosis (MS) [9], and amyotrophic lateral sclerosis (ALS) [10] and other disorders (attention-deficit hyperactivity disorder (ADHD) [11], autism spectrum disorder (ASD) [12]).

## **2. Biosynthesis and metabolism of serotonin**

Biosynthesis of 5-HT is a process consisted of coupled reactions, with amino acid tryptophan (Trp) as a primary substrate. The first reaction is hydroxylation of Trp yielding 5-hydroxytryptophane (5-HTP). The next step is decarboxylation of 5-HTP to 5-hydroxytryptamine (5-HT). 5-HT is further metabolized in the body. The main metabolic pathways of 5-HT are shown in **Figure 1** [1, 13].

**Figure 1.** Biosynthesis and metabolic pathways of serotonin.

**1. Introduction**

5-HT reuptake transporter (5-HTT, SERT) [1].

220 Serotonin - A Chemical Messenger Between All Types of Living Cells

tyrosine hydroxylase and AT2

antioxidant activity [6].

order (ASD) [12]).

shown in **Figure 1** [1, 13].

**2. Biosynthesis and metabolism of serotonin**

Serotonin (5-HT) is a neurotransmitter responsible for anxiety, aggressive behavior, stress, blood pressure regulation, peristaltic movements, heart rate, and the coagulation system. 5-hydroxytryptamine (5-HT) is produced in neurons and gut cells, as well as in the walls of blood vessels and the heart. On the periphery, 5-HT is located in platelets, which enters via

The level of 5-HT in whole blood is in the range of 65–250 ng/ml and in the plasma has a lower value, 5.6–23.9 ng/ml [2]. 5-HT enhances the response of the adrenal medulla, and other sympathetic ganglia using 5-HT2A/3. It has been shown that the impairment of serotonin transporter (SERT) function in addition to the increased 5-HT in the extracellular fluid and increased turnover of 5-HT and its decreased level in nerve cells causes an abnormal stress response in the form of anxiety, as well as an excessive response of the adrenal medulla, including triggered by the hypothalamic-pituitary axis (with no effects on the expression of

of afferent vagal neurons enhances the activity of the catecholaminergic neurons of the solitary tract nucleus (pulsed through potentiation of glutamatergic) and the effect on food intake and cardiovascular reflexes [4]. 5-HT acting on 5-HT4 receptors in the human heart causes stimulation of the atrium, pro-arrhythmic effect, produces a positive inotropic effect. At the same time, stimulation of 5-HT1B/1D endings of the sympathetic cardiac causes decreased release of norepinephrine (NE) [2]. 5-HT binds competitively to the binding site of catechol-O-methyltransferase (COMT) in binding site S-adenosyl-S-methionine, inhibiting methylation substrates for this enzyme [5]. It also acts antiapoptotic by stimulating the expression

of cystathionine-beta-synthase (CBS) and increases the level of hydrogen sulfide (H2

Currently, it is believed that disturbances in the level of 5-HT may be associated with the pathogenesis of few neurological diseases such as migraine [7], epilepsy [8], Parkinson's disease (PD) [1], multiple sclerosis (MS) [9], and amyotrophic lateral sclerosis (ALS) [10] and other disorders (attention-deficit hyperactivity disorder (ADHD) [11], autism spectrum dis-

Biosynthesis of 5-HT is a process consisted of coupled reactions, with amino acid tryptophan (Trp) as a primary substrate. The first reaction is hydroxylation of Trp yielding 5-hydroxytryptophane (5-HTP). The next step is decarboxylation of 5-HTP to 5-hydroxytryptamine (5-HT). 5-HT is further metabolized in the body. The main metabolic pathways of 5-HT are

receptors) [3]. Furthermore, 5-HT released from the terminals

S) and

## **3. Serotonin and its metabolites in migraine**

The disturbances in serotonergic system are a hallmark of migraine. Migraine is a common primary headache disorder that affects 11% of adult worldwide. It occurs three times more often in females (15–18%) than in males (6–8%) [14]. The disease is divided into two main clinical forms: migraine with aura (MA) and migraine without aura (MO). The exact pathomechanism of migraine is unknown, but it is postulated that disease has neurovascular origin in which cortical spreading depression (CSD) and trigeminovascular system (TGVS) play an important role [15]. The TGVS regulates vascular tone and transmission of pain signals [16]. It is believed that activation of TGVS during the head pain phase initiates a chemical cascade of vasoactive neuropeptides such as substance P, calcitonin gene-related peptide, neurokinin A, and nitric oxide. These molecules cause vasodilatation, which can contribute to headaches [17]. The TGVS transmitting migraine pain may be controlled by serotonergic neurons. 5-HT can modulate the trigeminal nerve function, as well as inhibit or promote the pain perception [18]. A decreased level of platelet 5-HT and its metabolite, N-acetylserotonin (NAS), during migraine activate TGVS by CSD [19].

It is known that migraine is a consequence of chronically low 5-HT disposition due to disturbances in its synthesis. The 5-HT metabolism has a cycling character in course of migraine. The plasma concentration of 5-HT is lower and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), is higher during attack-free period, with transient increase of 5-HT and decrease of 5-HIAA during attacks [20–22]. The changes of 5-HT and its metabolite in plasma reflect the situation in the brain as the elevated 5-HIAA level was also found in cerebrospinal fluid (CSF) of migraine suffers [23].

Although the role of 5-HT in migraine pathogenesis is known from ages, the reason of abnormalities of the central 5-HT synthesis remains unknown. The neuroimaging studies have found some answers for a serotonergic mechanism in migraine brain [24]. The electrophysiological studies of Sand et al. [25] indicated that reduced level of serotonergic neurotransmission caused the increase in visual evoked potentials (VEPs) amplitude (P100-N145) in MA patients compared with controls and individuals with MO. It may be associated with the presence of visual aura and increased sensitivity to light in patients with migraine. Authors suggested that disturbances in 5-HT metabolism may be more important in MA than in MO. Dysregulation of 5-HT in brainstem of migraine patients may be caused by a higher level of 5-HTT compared with controls. The higher the availability of 5-HTT, the lower the synaptic level of 5-HT, and in consequence, the lower the brain 5-HT level [26]. The reduction of brain 5-HT synthesis and serotonergic neurotransmission may lead to symptoms related to migraine, such as nausea, dizziness, photophobia, and pain sensitivity [27].

Numerous studies searched the polymorphisms and mutations in genes involved with 5-HT homeostasis in migraine patients. No association between migraine and polymorphisms in genes encoding tryptophan hydroxylase (TPH), aromatic l-amino acid decarboxylase (AADC), monoamine oxidase A (MAO-A), monoamine oxidase B (MAO-B), and most of 5-HT receptors (5-HT1A, 5-HT2A, 5-HT2C, 5-HT1B, and 5-HT1F) were found [13]. Genetic variants in 5-HTT gene, SLC6A4, have also been analyzed in migraine. There are two widely studied polymorphisms: the first is 5-HTTLPR insertion-deletion polymorphism located in the regulatory region of SLC6A4 and the second is STin2 VNTR (variable number of tandem repeats) with four different alleles that correspond to the number of tandem repeats (12, 10, 9, or 7). Both polymorphisms are associated with lower 5-HT reuptake. According to meta-analyses, the short allele of 5-HTTLPR is a risk factor for migraine among European women, while the non-STin2.12 alleles have the protective effect against migraine compared with STin2.12 genotype in the European population [28, 29]. According to the review of Margoob and Mushtaq [30], the S allele and S/S genotype are also associated with many neuropsychiatric diseases, such as major depressive disorder, unipolar or bipolar depression, and seasonal affective disorder. This may explain the fact that patients with migraine more often suffer from depression and anxiety disorders.

A control of the 5-HT level is a means of migraine treatment. Triptans—the 5-HT1B/1D receptor agonists—are successfully used in migraine therapy. The medications that inhibit the reuptake of 5-HT (e.g., selective 5-HT reuptake inhibitor, SSRI) are efficient in chronic pain conditions among which are chronic headaches [31].

High prevalence of migraine was noted in a population of fibromyalgia (FM) sufferers; therefore, it is suggested that both disorders share the same pathomechanism with disturbances in 5-HT metabolism [32]. FM is a chronic pain syndrome, characterized by widespread musculoskeletal pain with diffuse tenderness in specific areas. It affects 3–6% of the world population and 80% of suffers are women [33, 34]. The plasma and CSF levels of 5-HT are decreased in individuals with FM and correlate with clinical symptoms. The low level of Trp and 5-HT precursor, 5-HTTP, as well as high concentration of metabolites in the kynurenine pathway suggest that the synthesis of 5-HT is decreased in FM. Additionally, 5-HTP supplements are recommended for people with FM. A combined therapy of 5-HTP and MAO inhibitors is more effective that each substance alone [35, 36]. The disturbances in 5-HT concentrations may be associated with changes in 5-HTT, as well. The binding capacity of 5-HTT was found to be lower in FM patients compared with controls. A negative correlation was noted between the binding capacity and rate of 5-HTT and severity of symptoms. The lower expression of 5-HTT in FM patients may be caused by genetic changes [37]. The genetic studies in FM have found that short allele of 5-HTTLPR polymorphism is associated with decline in 5-HTT expression and is a risk factor for developing the disease, similarly to migraine. The T102C polymorphism in *HTR2A* gene encoding 5-HT2A is also postulated to be a risk factor for FM [38]. As 5-HT2 and 5-HT3 are involved in pain perception, the treatment with 5-HT3 antagonist or inhibition of 5-HT reuptake is effective in FM patients [39]. The SSRI administration is necessary as depression is a common disorder among FM patients and it is present in up to 80% of individuals [34]. Participation of 5-HT in the pathogenesis of migraine attacks requires further study.

## **4. Serotonin levels in epileptic patients**

Although the role of 5-HT in migraine pathogenesis is known from ages, the reason of abnormalities of the central 5-HT synthesis remains unknown. The neuroimaging studies have found some answers for a serotonergic mechanism in migraine brain [24]. The electrophysiological studies of Sand et al. [25] indicated that reduced level of serotonergic neurotransmission caused the increase in visual evoked potentials (VEPs) amplitude (P100-N145) in MA patients compared with controls and individuals with MO. It may be associated with the presence of visual aura and increased sensitivity to light in patients with migraine. Authors suggested that disturbances in 5-HT metabolism may be more important in MA than in MO. Dysregulation of 5-HT in brainstem of migraine patients may be caused by a higher level of 5-HTT compared with controls. The higher the availability of 5-HTT, the lower the synaptic level of 5-HT, and in consequence, the lower the brain 5-HT level [26]. The reduction of brain 5-HT synthesis and serotonergic neurotransmission may lead to symptoms related to migraine, such as nausea, dizziness, photophobia, and pain

222 Serotonin - A Chemical Messenger Between All Types of Living Cells

Numerous studies searched the polymorphisms and mutations in genes involved with 5-HT homeostasis in migraine patients. No association between migraine and polymorphisms in genes encoding tryptophan hydroxylase (TPH), aromatic l-amino acid decarboxylase (AADC), monoamine oxidase A (MAO-A), monoamine oxidase B (MAO-B), and most of 5-HT receptors (5-HT1A, 5-HT2A, 5-HT2C, 5-HT1B, and 5-HT1F) were found [13]. Genetic variants in 5-HTT gene, SLC6A4, have also been analyzed in migraine. There are two widely studied polymorphisms: the first is 5-HTTLPR insertion-deletion polymorphism located in the regulatory region of SLC6A4 and the second is STin2 VNTR (variable number of tandem repeats) with four different alleles that correspond to the number of tandem repeats (12, 10, 9, or 7). Both polymorphisms are associated with lower 5-HT reuptake. According to meta-analyses, the short allele of 5-HTTLPR is a risk factor for migraine among European women, while the non-STin2.12 alleles have the protective effect against migraine compared with STin2.12 genotype in the European population [28, 29]. According to the review of Margoob and Mushtaq [30], the S allele and S/S genotype are also associated with many neuropsychiatric diseases, such as major depressive disorder, unipolar or bipolar depression, and seasonal affective disorder. This may explain the fact that patients with migraine more often suffer from depres-

A control of the 5-HT level is a means of migraine treatment. Triptans—the 5-HT1B/1D receptor agonists—are successfully used in migraine therapy. The medications that inhibit the reuptake of 5-HT (e.g., selective 5-HT reuptake inhibitor, SSRI) are efficient in chronic pain

High prevalence of migraine was noted in a population of fibromyalgia (FM) sufferers; therefore, it is suggested that both disorders share the same pathomechanism with disturbances in 5-HT metabolism [32]. FM is a chronic pain syndrome, characterized by widespread musculoskeletal pain with diffuse tenderness in specific areas. It affects 3–6% of the world population and 80% of suffers are women [33, 34]. The plasma and CSF levels of 5-HT are decreased in individuals with FM and correlate with clinical symptoms. The low level of Trp and 5-HT precursor, 5-HTTP, as well as high concentration of metabolites in the kynurenine pathway

sensitivity [27].

sion and anxiety disorders.

conditions among which are chronic headaches [31].

Inhibitors of 5-HT reuptake are also used in another common neurological disorder, epilepsy. Since 1957 it is known that 5-HT can inhibit epileptic attacks [40]. Epilepsy is defined as a set of somatic, vegetative, and mental symptoms. The disease affects 1% of the world population [41]. The disease occurs with a comparable frequency in women and men. Two peaks of incidence are noted: one in childhood and the other over the age of 65 years [42]. In the pharmacotherapy of epilepsy old (e.g., carbamazepine, CBZ; valproate, VPA) and new generation (e.g., lamotrigine, LTG) of antiepileptic drugs (AEDs) are used. Their mechanisms of action among others involve also changes in serotonergic system: CBZ and VPA release the 5-HT, while LTG inhibits 5-HT uptake [8]. The increase in extracellular 5-HT level inhibits both limbic and generalized seizures [43]. Lower values of 5-HIAA concentration were observed in CSF of individuals with epilepsy; this in turns suggests hypofunctional serotonergic neurotransmission in the course of the disease [44].

Moreover, alterations in 5-HT1A, 5-HT2C, 5-HT3, and 5-HT7 receptor subtypes have been analyzed in epilepsy [8]. The binding capacity of 5-HT1A is lower in epilepsy. Reduction in 5-HT1A binding and changes in 5-HT2C and 5-HT7 are features of depression, thus it is unsurprising that 25% of epilepsy cases are accompanied by depression [45, 46]. There is also an age-related decline in 5-HT1A receptors and as it was mentioned before the onset of epilepsy increases in older people. SSRI has anticonvulsant effects because of nonspecific receptor activation, as the volume of 5-HT1A and 5-HT2C receptors is decreased in the temporal regions of brain in epileptic patients. Studies on a mouse model of epilepsy have found that disturbances in the serotonergic system may lead to postictal depression of breathing due to inadequate response to increased CO2 blood level. Moreover, SSRI drugs are thought to be effective in prevention of hypoventilation after a seizure incident, and of sudden unexpected death in epilepsy in consequence [8, 47].

## **5. Serotonin and Parkinson's disease**

PD was first described in 1817 by an English physician James Parkinson. PD is still an incurable neurological disease, and its pathological mechanism is not fully explained. It is known that in PD there is an imbalance of motor and nonmotor functions, including the autonomic system [1]. Biogenic amines: catecholamines and 5-HT are involved in the regulation of autonomic functions such as blood pressure. In PD degeneration of serotonergic system may also result in depression, psychiatric, and sleep disorders [48]. Moreover, factors regulating levels of biogenic amines such as COMT [49], MAO-A [50], and *5-HTT* gene encoding SERT [51], and bradykinin [52] are involved in the regulation of pain sensation involving neuropeptide Y (NPY). Neuropeptides, Y<sup>1</sup> and Y<sup>2</sup> , are also involved in controlling the level of calcium ions regulating by calbindin-B and inflammatory conditions, underlying degenerative changes in the course of PD [1].

Moreover, so far the participation of MAO-B enzyme in the pathogenesis of PD is well known. While the role of MAO-A enzyme in this pathogenesis is not clear. The results of association studies between genetic variants of the *MAO-A* gene and the disclosure of PD are divergent. Hotamisligil and Breakefield [53] have shown that *Eco*RV and *Msp*I polymorphisms of the *MAO-A* gene occurred with threefold higher frequency in patients with PD compared with controls. In contrast, the study of Costa-Mallen et al. [50] did not confirm this association. It was also shown that *MAO-A* polymorphism in the intron 1 in both Japanese population [54] and Caucasians [55] was not associated with PD. On the other hand, a study of Parsian et al. [56] confirmed that *MAO-A* polymorphism was linked to the general population of patients with PD but it did not demonstrate significant differences between familiar PD (FPD) and sporadic PD (SPD).

Preliminary study of Dorszewska et al. [1] indicated that the use of selective MAO inhibitors for depression treatment (by increasing the levels of biogenic amines) in PD may be a particularly effective therapy for patients with genotype *MAO-A* TT (c.1460C>T) and lower levels of NA and 5-HT. Antidepressant MAO inhibitors lead to an inactivation of MAO-A and they promote an increase of 5-HT concentration [57].

It has been shown that SERT (or 5-HTT) is involved in regulating of 5-HT level. SERT is encoded by *5-HTT* gene (SLC6A4, SLC6 member 4) located on the long arm of chromosome 17 in the region 17q11.1-q12 [58]. The *5-HTT* gene may play an important role in revealing and development of mental illness, depression, and feeling of pain as well as SPD [51, 59–63]. In SPD, changes in the SERT level are observed within the raphe nuclei, cingulate, and hypothalamus, as well as increase of SERT activity and decrease of 5-HT level in the striatum, thus leading to depression in these patients [64, 65]. It has been shown that depressive symptoms occur in 50% of patients with PD [1].

Influence of genetic variants of the *5-HTT* gene on SERT concentrations in specific brain structures in PD is not clear. The literature data indicated that 5-HTTLPR polymorphisms and the *5-HTR2* gene lead to lower SERT expression in the dentate rim and caudate nucleus. There was no correlation between the *5-HTT* polymorphism and disclosure of SPD [66]. In contrast, the study conducted on 393 Caucasian PD patients indicated the influence of 5-HTTLPR polymorphism on a risk of SPD disclosure [67]. Mutations in the *5-HTT* gene related to pathogenesis SPD are summarized in the work of Dorszewska et al. [1].

It seems that in patients with PD, there are many mechanisms involved in controlling levels of biogenic amines, including catecholamines and 5-HT, associated with the appearance of motor and nonmotor symptoms and impaired blood pressure regulation. Furthermore, changes in levels of biogenic amines may also be a consequence of genetic variants influencing their level and the activity of enzymes responsible for the metabolism.

## **6. Disturbances of serotonin levels in multiple sclerosis**

**5. Serotonin and Parkinson's disease**

224 Serotonin - A Chemical Messenger Between All Types of Living Cells

and Y<sup>2</sup>

Y (NPY). Neuropeptides, Y<sup>1</sup>

PD (FPD) and sporadic PD (SPD).

occur in 50% of patients with PD [1].

promote an increase of 5-HT concentration [57].

the course of PD [1].

PD was first described in 1817 by an English physician James Parkinson. PD is still an incurable neurological disease, and its pathological mechanism is not fully explained. It is known that in PD there is an imbalance of motor and nonmotor functions, including the autonomic system [1]. Biogenic amines: catecholamines and 5-HT are involved in the regulation of autonomic functions such as blood pressure. In PD degeneration of serotonergic system may also result in depression, psychiatric, and sleep disorders [48]. Moreover, factors regulating levels of biogenic amines such as COMT [49], MAO-A [50], and *5-HTT* gene encoding SERT [51], and bradykinin [52] are involved in the regulation of pain sensation involving neuropeptide

regulating by calbindin-B and inflammatory conditions, underlying degenerative changes in

Moreover, so far the participation of MAO-B enzyme in the pathogenesis of PD is well known. While the role of MAO-A enzyme in this pathogenesis is not clear. The results of association studies between genetic variants of the *MAO-A* gene and the disclosure of PD are divergent. Hotamisligil and Breakefield [53] have shown that *Eco*RV and *Msp*I polymorphisms of the *MAO-A* gene occurred with threefold higher frequency in patients with PD compared with controls. In contrast, the study of Costa-Mallen et al. [50] did not confirm this association. It was also shown that *MAO-A* polymorphism in the intron 1 in both Japanese population [54] and Caucasians [55] was not associated with PD. On the other hand, a study of Parsian et al. [56] confirmed that *MAO-A* polymorphism was linked to the general population of patients with PD but it did not demonstrate significant differences between familiar

Preliminary study of Dorszewska et al. [1] indicated that the use of selective MAO inhibitors for depression treatment (by increasing the levels of biogenic amines) in PD may be a particularly effective therapy for patients with genotype *MAO-A* TT (c.1460C>T) and lower levels of NA and 5-HT. Antidepressant MAO inhibitors lead to an inactivation of MAO-A and they

It has been shown that SERT (or 5-HTT) is involved in regulating of 5-HT level. SERT is encoded by *5-HTT* gene (SLC6A4, SLC6 member 4) located on the long arm of chromosome 17 in the region 17q11.1-q12 [58]. The *5-HTT* gene may play an important role in revealing and development of mental illness, depression, and feeling of pain as well as SPD [51, 59–63]. In SPD, changes in the SERT level are observed within the raphe nuclei, cingulate, and hypothalamus, as well as increase of SERT activity and decrease of 5-HT level in the striatum, thus leading to depression in these patients [64, 65]. It has been shown that depressive symptoms

Influence of genetic variants of the *5-HTT* gene on SERT concentrations in specific brain structures in PD is not clear. The literature data indicated that 5-HTTLPR polymorphisms and the *5-HTR2* gene lead to lower SERT expression in the dentate rim and caudate nucleus. There was no correlation between the *5-HTT* polymorphism and disclosure of SPD [66]. In contrast,

, are also involved in controlling the level of calcium ions

MS is a complex and not fully recognized neurological disorder. Both the environmental and genetic factors are a probable cause of this disease. MS is mainly characterized by myelin destruction and a consequent dysfunction of the central nervous system (CNS). This disease is caused by inflammatory processes, linked with increased levels of Th17 and Th1 cells and decreased levels of regulatory T cells. All the MS patients are at risk of disease progression over time. This progression affects not only physical ability but also mental functions. The disease may have different forms, such as relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), primary progressive multiple sclerosis (PPMS), and progressive relapsing multiple sclerosis (PRMS). Serotonergic system disturbances are one of the studied areas in MS patients [68].

In MS, the level of 5-HT precursor, Trp, is reduced in both plasma and CSF [68–70]. Monaco et al. [68] found that not only the Trp level in plasma was decreased, but also the level of leucine and valine was decreased. The neutral amino acids to Trp ratio were found to be significantly higher in MS than in other analyzed neurological diseases. The low concentration of Trp in CSF and plasma of MS patients stays in line with decreased of brain 5-HT synthesis and overactivation of kynurenine pathway of Trp metabolism. The kynurenine pathway competes with the melatonin pathway for Trp. Moreover, overactivation of the kynurenine pathway leads to severe imbalance between emerging neuroprotective and neurotoxic metabolites [6, 71, 72].

It is known, that in MS, the decreased of 5-HT synthesis in the brain may lead to the local 5-HT-deficit. A significant role in this deficit may play 5-HT metabolites, N-acetylserotonin (NAS) and melatonin. The levels of these metabolites are dependent on availability of 5-HT. NAS and melatonin exhibit antioxidant and anti-inflammatory properties. It also acts as immune signaling agents [73]. NAS exerts similar as a brain-derived neurotrophic factor (BDNF), activating the brain-derived neurotrophic factor (BDNF) receptor. However, melatonin decreases the number of Th1 and Th17 cell populations and the cytokines synthetized [74]. It also exerts a positive effect on mitochondrial function and reduces oxidative stress [74, 75]. It has been shown that NAS and melatonin in experimental autoimmune encephalomyelitis (EAE) in mice reduce clinical scores and the loss of mature oligodendrocytes, demyelination, and axon injury [74].

Literature data indicate that both synthesis and metabolism of 5-HT are disrupted in patients with MS. The low level of 5-HIAA was found in CSF of MS patients [9, 76]. Moreover, Markianos et al. [77] presented a negative correlation between 5-HIAA CSF level in RRMS patients and scores of disability scales: expanded disability status scale (EDSS) and multiple sclerosis severity scale (MSSS). What is interesting, the negative correlation was stronger between 5-HIAA level and MSSS than EDSS. MSSS scores not only disability status as EDSS, but also time of disease duration. Markianos et al. [77] also suggest that 5-HT turnover is more affected by the rate of accumulation of disability rather than disability itself. Reduced serotonnergic activity may lead to axonal loss. Therefore, it seems that 5-HIAA may be considered as a biomarker of severity and duration in RRMS.

It is believed that the serotonergic system also may be a target for therapy in MS. It has been shown that fluoxetine, a represent of SSRIs, reduces the formation of new enhancing lesions in magnetic resonance imaging (MRI) of nondepressed patients with RRMS. This explains the reason of elevated astrocyte-cAMP levels. The elevated levels of intracellular cAMP levels inhibit interferon-gamma induction of MHC class II in astrocytes. Normally, the MHC class II expressed on astrocytes in MS acts as antigen-presenting cells and take part in inflammation [78]. What is more, fluoxetine also promotes disease remission in acute EAE [79]. Moreover, escitalopram belonging to SSRI lowered the risk of stress-related relapse in women with MS [80]. Those studies implicated fluoxetine, and perhaps other SSRIs, may be analyzed as candidate drugs in MS.

The altered of 5-HT activity is linked not only to MS symptoms, but also to mental changes in these patients. For instance, the low concentration of platelet 5-HT may correlate with fatigue symptoms in MS [81]. Other studies have shown that SSRIs and duloxetine, which is 5-HT and NE reuptake inhibitor, are effective in depression treatment in MS [82]. Depression in MS is explained among others, as due to decreased 5-HT and melatonin synthesis.

Many studies suggested that platelet 5-HT may be used to estimate brain 5-HT level. Platelet 5-HT was found to strongly correlate with 5-HT level in CSF [83]. There are many similarities in serotonergic mechanisms in platelets and serotonergic neurons. The 5-HT uptake from plasma to platelets is similar to neuronal 5-HT uptake [84]. It is known that SERT transports 5-HT through the membrane. This transporter is encoded by the same single copy gene in platelets and neurons [85]. The 5-HT uptake in platelets and neurons is inhibited by the same drugs, tricyclic antidepressants and neuroleptics. Furthermore, 5-HT is stored in dense granules in both platelets and synaptic vesicles in neurons. Moreover, both types of cells contain MAO-B in a greater amount than MAO-A. This fact allows them to storage 5-HT which is not metabolized by MAO-B. These similarities justify treating platelets as models of serotonergic neurons [83]. Moreover, 5-HT in the blood is concentrated mainly in platelets what underlines their significant function in the serotonergic system. The 5-HT level in platelets is 24,000 times higher than in plasma [86] and the platelet 5-HT accounts for 98% of its total circulating amount [87].

As it has been mentioned that plasma 5-HT is transported to platelets by SERT. SERT is a member of the Na+/Cl-dependent solute carrier 6 (SLC6) family. In platelets, 5-HT may be deposited in dense vesicles by vesicular monoamine transporter (VMAT) or degraded by MAO [88]. Although the mechanisms of transport are recognized, the relations between 5-HT plasma level, SERT, and platelets are still not fully understood. SERT is found to compete with dopamine transporter (DAT). Moreover, the SERT expression in relation to the 5-HT plasma level seems to be complicated and biphasic [86, 88]. These facts may play a significant role in regulation of SERT in platelets.

The similarities between neurons and platelets are mainly the complex transport regulation and lack of 5-HT synthesis in platelets. Despite that it can be used to estimate the brain 5-HT in many studies of neurological diseases, such as ALS and MS. These studies will be discussed further.

## **7. Amyotrophic lateral sclerosis and serotonin level**

Literature data indicate that both synthesis and metabolism of 5-HT are disrupted in patients with MS. The low level of 5-HIAA was found in CSF of MS patients [9, 76]. Moreover, Markianos et al. [77] presented a negative correlation between 5-HIAA CSF level in RRMS patients and scores of disability scales: expanded disability status scale (EDSS) and multiple sclerosis severity scale (MSSS). What is interesting, the negative correlation was stronger between 5-HIAA level and MSSS than EDSS. MSSS scores not only disability status as EDSS, but also time of disease duration. Markianos et al. [77] also suggest that 5-HT turnover is more affected by the rate of accumulation of disability rather than disability itself. Reduced serotonnergic activity may lead to axonal loss. Therefore, it seems that 5-HIAA may be considered as

It is believed that the serotonergic system also may be a target for therapy in MS. It has been shown that fluoxetine, a represent of SSRIs, reduces the formation of new enhancing lesions in magnetic resonance imaging (MRI) of nondepressed patients with RRMS. This explains the reason of elevated astrocyte-cAMP levels. The elevated levels of intracellular cAMP levels inhibit interferon-gamma induction of MHC class II in astrocytes. Normally, the MHC class II expressed on astrocytes in MS acts as antigen-presenting cells and take part in inflammation [78]. What is more, fluoxetine also promotes disease remission in acute EAE [79]. Moreover, escitalopram belonging to SSRI lowered the risk of stress-related relapse in women with MS [80]. Those studies implicated fluoxetine, and perhaps other SSRIs, may be analyzed as can-

The altered of 5-HT activity is linked not only to MS symptoms, but also to mental changes in these patients. For instance, the low concentration of platelet 5-HT may correlate with fatigue symptoms in MS [81]. Other studies have shown that SSRIs and duloxetine, which is 5-HT and NE reuptake inhibitor, are effective in depression treatment in MS [82]. Depression in MS

Many studies suggested that platelet 5-HT may be used to estimate brain 5-HT level. Platelet 5-HT was found to strongly correlate with 5-HT level in CSF [83]. There are many similarities in serotonergic mechanisms in platelets and serotonergic neurons. The 5-HT uptake from plasma to platelets is similar to neuronal 5-HT uptake [84]. It is known that SERT transports 5-HT through the membrane. This transporter is encoded by the same single copy gene in platelets and neurons [85]. The 5-HT uptake in platelets and neurons is inhibited by the same drugs, tricyclic antidepressants and neuroleptics. Furthermore, 5-HT is stored in dense granules in both platelets and synaptic vesicles in neurons. Moreover, both types of cells contain MAO-B in a greater amount than MAO-A. This fact allows them to storage 5-HT which is not metabolized by MAO-B. These similarities justify treating platelets as models of serotonergic neurons [83]. Moreover, 5-HT in the blood is concentrated mainly in platelets what underlines their significant function in the serotonergic system. The 5-HT level in platelets is 24,000 times higher than in plasma [86] and the platelet 5-HT accounts for 98% of its total circulating amount [87].

As it has been mentioned that plasma 5-HT is transported to platelets by SERT. SERT is a member of the Na+/Cl-dependent solute carrier 6 (SLC6) family. In platelets, 5-HT may be deposited in dense vesicles by vesicular monoamine transporter (VMAT) or degraded by MAO [88]. Although the mechanisms of transport are recognized, the relations between 5-HT

is explained among others, as due to decreased 5-HT and melatonin synthesis.

a biomarker of severity and duration in RRMS.

226 Serotonin - A Chemical Messenger Between All Types of Living Cells

didate drugs in MS.

ALS is a neurodegenerative disease that affects upper and lower motor neurons. The etiology and pathogenesis of motor neuron degeneration are still not elucidated. Many of motor neuron functions are altered in ALS, especially motor neuron excitability and synaptic glutamate release. Due to disappointing results of treatment with riluzole, a glutamate action modulator, new mechanisms are under research. 5-HT system alterations may also be involved in ALS pathogenesis. The alterations of this system affect 5-HT synthesis and release. There are reports suggesting that some changes in serotonergic system may be used in clinical laboratory tests in ALS [89].

The role of 5-HT in ALS progression may be related to many mechanisms. 5-HT facilitates motor neuron activity by strengthening weak inputs—electrical impulses or excitatory neurotransmitters, such as glutamate. As in ALS 5-HT neurons are degenerated, the amount of glutamate needed to excitation of motor neuron increases. This leads to the pathological glutamate overexpression and neurotoxicity [10]. Moreover, in the brain 5-HT inhibits the glutamatergic system as a precursor of melatonin, which inhibits glutamate neurotoxicity. El Oussini et al. [89] have also indicated that 5-HT2B receptor limits degeneration of mononuclear phagocytes in CNS, which accompanied neurodegeneration in the disease.

Disturbances of serotonergic system in ALS may be found in studies of 5-HT precursor, Trp. Monaco et al. [68] shown that CSF and plasma level of Trp are reduced in ALS patients. Moreover, plasma levels of leucine and valine, which compete with Trp for uptake into the brain [90], were increased in ALS patients as a result of a larger uptake of neutral amino acids. However, its ratio was increased not only in patients with ALS, but also in patients with some other neurological diseases, such as MS. The authors of the study suggest that its different levels may be possibly used to differentiate these diseases.

The level of 5-HT itself may also have a prognostic value. Dupuis et al. [87] have shown that platelet 5-HT level is not only significantly decreased in ALS compared with controls, but it also predicts survival in ALS. In the study, the level of 5-HT was measured at one single time point in patients with diagnosed disease. The authors calculated the difference between platelet and plasma unconjugated 5-HT concentrations. The level of platelet 5-HT was more decreased in patients with bulbar onset, what corresponds with less 5-HT1A receptor binding in imaging studies [91]. Moreover, in all ALS patients, the platelet 5-HT level corresponded with survival, from time of test to death. This can be related to some role of 5-HT alterations in the disease progression [87].

As it has been mentioned before, the serotonergic receptors can also play a significant neuroprotective role and its expression may be altered in ALS. The study of El Oussini et al. [89] has shown that the 5-HT2B receptor may limit progression in ALS by some mechanisms related to mononuclear phagocytes. On the other hand, the test of *5HT2B* gene, which encodes 5-HT2B receptors, may have some value as a survival predictor. Moreover, in the same study, patients carrying the C allele of single nucleotide polymorphism (SNP) rs10199752 in *5HT2B* gene, which encodes the 5-HT2B receptor, had a longer survival than patients carrying the more common A allele. This was also accompanied by decreased mononuclear phagocyte degeneration and increased concentrations of 5-HT2B mRNA in the spinal cord.

However, the imaging studies showed also decreased concentration of 5-HT1A receptors in the brain raphe and the cortex in ALS, even more decreased in patients with bulbar ALS onset [92]. The studies showed also alterations in concentration of 5-HIAA. This can be treated as evidence of 5-HT metabolism alterations in ALS. The *postmortem* studies of ALS patients showed decreased levels of 5-HIAA and 5-HT in the spinal cord and the brain tissue. The alterations were found particularly in the cervical and thoracic level of the spinal cord. One single study showed that concentrations of 5-HIAA were lower in the cervical spine of ALS patients with no difference in 5-HT level compared with controls [93]. However lower 5-HIAA concentration may be still linked to weak 5-HT metabolism.

## **8. Neuropsychiatric disorders and serotonin**

ADHD, one of the most common childhood conditions, is categorized as a neurodevelopmental disorder. The group of behavioral symptoms of ADHD broadly encompasses inattentiveness, hyperactivity, and impulsiveness. The exact causes of ADHD remain unknown, but 5-HT plays a potential role in its pathomechanism. Studies provide evidence that altered availability and metabolism of 5-HT may lead to impulsivity [94]. Moreover, studies indicate that 5-HT deficiency leads to a failure of 5-HT-mediated inhibitory control of aggressive behavior and can occur also in adults [11]. Some of the studies have demonstrated decreased levels of 5-HT and 5-HIAA, in the blood, urine, and CSF in individuals with ADHD compared with in healthy controls, but other studies found no differences. However, the studies indicate that 5-HT levels in the platelets are much higher in impulsive children. There was no correlation between the platelet 5-HT concentration and other common ADHD symptoms, neither any significant difference between platelet 5-HT concentrations in ADHD children compared with controls [12].

Abnormalities in 5-HT receptors were observed in patients with ADHD: the aggression and impulsiveness are linked to increased 5-HT2A and decreased 5-HT1A receptor binding. Moreover, underexpression of 5-HT1B is a predictor of increased impulsive behavior, but not of impulsive choice [95]. Changes in 5-HTT activity in various brain regions are thought to be associated with ADHD [96, 97]. Alterations in the 5-HT level may also be caused by low activity of MAO-A and lead to impulsivity and aggressive tendencies in ADHD [98].

Disturbances in serotonergic system may be a result of many polymorphisms. Animal model studies have found that inactivation of the brain-specific Trp hydroxylase-2 (TPH2) gene leads to increased aggression due to impaired synthesis of neuronal 5-HT in the raphe neurons of the brain stem [99]. The several SNPs of the TPH2 gene are found to be strongly associated with altered functions of the prefrontal cortex during a response inhibition task in adults with ADHD [100].

Hyperserotonemia is one of the biomarkers of another neuropsychiatric condition, the ASD, and is presented in approximately 30% of patient. ASD is a group of neurodevelopmental disturbances, characterized by communication difficulties, social deficits, and repetitive behaviors, and associated by mental health issues, poor motor skills, gastrointestinal symptoms, and sleep problems [101]. The range of the symptoms varies from mild to severe. The pathomechanism of ASD is unknown, as well as the contribution of the 5-HT system to its pathophysiology.

One of the consequences of hyperserotonemia is increased catabolism of 5-HT. Blood 5-HT concentrations are regulated by the activity of peripheral 5-HT-associated proteins. It is suggested that an increased velocity of kinetics of MAO-B might be an answer to high 5-HT concentrations in the platelets [102, 103]. The hyperserotonemia in platelets in autism could be due to an increased uptake of 5-HT into the platelet. Children with autism carrying the short allele of 5-HTTLPR polymorphism associated with decreased 5-HTT expression showed better connectivity than youth with autism and long allele of this polymorphism [104].

Changes in 5-HT receptors were noted in patients with Asperger's syndrome. The abnormalities in 5-HT2A receptor density and reduction in 5-HT1A receptor binding density in several brain regions were demonstrated [105, 106].

Future studies are needed to understand the role of serotonergic system in ASD.

## **9. Summary**

with survival, from time of test to death. This can be related to some role of 5-HT alterations

As it has been mentioned before, the serotonergic receptors can also play a significant neuroprotective role and its expression may be altered in ALS. The study of El Oussini et al. [89] has shown that the 5-HT2B receptor may limit progression in ALS by some mechanisms related to mononuclear phagocytes. On the other hand, the test of *5HT2B* gene, which encodes 5-HT2B receptors, may have some value as a survival predictor. Moreover, in the same study, patients carrying the C allele of single nucleotide polymorphism (SNP) rs10199752 in *5HT2B* gene, which encodes the 5-HT2B receptor, had a longer survival than patients carrying the more common A allele. This was also accompanied by decreased mononuclear phagocyte degen-

However, the imaging studies showed also decreased concentration of 5-HT1A receptors in the brain raphe and the cortex in ALS, even more decreased in patients with bulbar ALS onset [92]. The studies showed also alterations in concentration of 5-HIAA. This can be treated as evidence of 5-HT metabolism alterations in ALS. The *postmortem* studies of ALS patients showed decreased levels of 5-HIAA and 5-HT in the spinal cord and the brain tissue. The alterations were found particularly in the cervical and thoracic level of the spinal cord. One single study showed that concentrations of 5-HIAA were lower in the cervical spine of ALS patients with no difference in 5-HT level compared with controls [93]. However lower 5-HIAA

ADHD, one of the most common childhood conditions, is categorized as a neurodevelopmental disorder. The group of behavioral symptoms of ADHD broadly encompasses inattentiveness, hyperactivity, and impulsiveness. The exact causes of ADHD remain unknown, but 5-HT plays a potential role in its pathomechanism. Studies provide evidence that altered availability and metabolism of 5-HT may lead to impulsivity [94]. Moreover, studies indicate that 5-HT deficiency leads to a failure of 5-HT-mediated inhibitory control of aggressive behavior and can occur also in adults [11]. Some of the studies have demonstrated decreased levels of 5-HT and 5-HIAA, in the blood, urine, and CSF in individuals with ADHD compared with in healthy controls, but other studies found no differences. However, the studies indicate that 5-HT levels in the platelets are much higher in impulsive children. There was no correlation between the platelet 5-HT concentration and other common ADHD symptoms, neither any significant difference between platelet 5-HT concentrations in ADHD children compared with controls [12]. Abnormalities in 5-HT receptors were observed in patients with ADHD: the aggression and impulsiveness are linked to increased 5-HT2A and decreased 5-HT1A receptor binding. Moreover, underexpression of 5-HT1B is a predictor of increased impulsive behavior, but not of impulsive choice [95]. Changes in 5-HTT activity in various brain regions are thought to be associated with ADHD [96, 97]. Alterations in the 5-HT level may also be caused by low activ-

ity of MAO-A and lead to impulsivity and aggressive tendencies in ADHD [98].

eration and increased concentrations of 5-HT2B mRNA in the spinal cord.

concentration may be still linked to weak 5-HT metabolism.

**8. Neuropsychiatric disorders and serotonin**

in the disease progression [87].

228 Serotonin - A Chemical Messenger Between All Types of Living Cells

Neurological diseases, such as migraine, epilepsy, PD, MS, ALS, and neuropsychiatric disorders (ADHD, ASD) may be connected to abnormal 5-HT levels in a variety of mechanisms, as shown in **Figure 2**. Synthesis and metabolism efficiency of 5-HT is changed in neurodegeneration. Patients with ALS and MS present with reduced both plasma and CSF levels of Trp, what can be linked with a decreased 5-HT synthesis. Moreover, in MS 5-HT synthesis is decreased because of overactivation of kynurenine pathway, which drives Trp away from 5-HT synthesis. This pathway is overactivated by inflammatory molecules, such as tumor necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN). In MS, 5-HT concentration is decreased due to production of its neuroprotective metabolites, such as NAS and melatonin.

In ALS, the platelet 5-HT level is decreased compared with controls and there is a positive relation between platelet 5-HT and survival. In MS, a lower platelet 5-HT level was found in patients with a more severe fatigue syndrome.

**Figure 2.** Disturbances of serotonin levels in neurological diseases.

Simultaneously, *postmortem* studies of ALS patients showed decreased levels of 5-HIAA and 5-HT in the spinal cord and brain tissue. However, in MS patients, lower levels of 5-HIAA were found in CSF. Moreover, in RRMS there was a negative correlation between 5-HIAA CSF level and scores of disability scales. A lower 5-HIAA level was also observed in CSF of epileptic patients as well as in migraine during attacks. However, a lower level of 5-HTT in FM and PD patients is associated with genetic variants.

Understanding the mechanisms of changes in the level of 5-HT and its precursors/metabolites in neurological diseases may contribute to finding new biomarkers relevant to the diagnosis and treatment of these diseases.

## **Acknowledgements**

This study was supported by the Poznan University of Medical Sciences grant no. 502-01- 11111-45-07-467.

## **Author details**

Jolanta Dorszewska<sup>1</sup> \*, Jolanta Florczak-Wyspianska2 , Marta Kowalska<sup>1</sup> , Marcin Stanski<sup>1</sup> , Alicja Kowalewska<sup>1</sup> and Wojciech Kozubski2

\*Address all correspondence to: dorszewskaj@yahoo.com

1 Laboratory of Neurobiology, Department of Neurology, Poznan University of Medical Sciences, Poznan, Poland

2 Chair and Department of Neurology, Poznan University of Medical Sciences, Poznan, Poland

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Simultaneously, *postmortem* studies of ALS patients showed decreased levels of 5-HIAA and 5-HT in the spinal cord and brain tissue. However, in MS patients, lower levels of 5-HIAA were found in CSF. Moreover, in RRMS there was a negative correlation between 5-HIAA CSF level and scores of disability scales. A lower 5-HIAA level was also observed in CSF of epileptic patients as well as in migraine during attacks. However, a lower level of 5-HTT in

FM and PD patients is associated with genetic variants.

**Figure 2.** Disturbances of serotonin levels in neurological diseases.

230 Serotonin - A Chemical Messenger Between All Types of Living Cells


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238 Serotonin - A Chemical Messenger Between All Types of Living Cells


## **The Role of Serotonin in Aggression and Impulsiveness**

Fatih Hilmi Çetin, Yasemin Taş Torun and Esra Güney

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68918

#### **Abstract**

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> Serotonin is a neuromodulator that has a critical role on the regulation of essential events in neuronal and glial development, such as cell proliferation, differentiation, migration, apoptosis, and synaptogenesis, and acts as a developmental signal. It has been known that a serotonergic system is associated with many psychiatric disorders. The seroto‐ nergic system also predominates on the etiopathogenesis of two important endopheno‐ types: impulsivity and aggression. Impulsiveness is defined as personality trait and an implusive temperament is associated with clinical conditions such as pathological gam‐ bling, eating disorders, and borderline personality disorder as well as being a risk factor for self‐harm, suicide, and emotional liability. Aggression is not a personality trait like impulsivity, but it is the behavior of harm or injury to others. Besides being a natural human behavior toward survival, aggression can be harmful to the individual and the community when it is constant and excessive. In this chapter, we aimed to review the role of the serotonergic system on impulsivity and aggression, which are two important endophenotypes that identified in many psychiatric disorders.

**Keywords:** serotonin, aggression, impulsivity, impulsive aggression, psychiatric disorders

## **1. Introduction**

Serotonin is a neuromodulator that acts as a developmental signal [1]. The serotonin is formed by decarboxylation of the 5‐hydroxy‐tripotafan that synthesized from tryptophan via the tryptophan hydroxylase enzyme [1]. Serotonin has a critical role on the regulation of essential events in neuronal and glial development, such as cell proliferation, differentiation, migration, apoptosis, and synaptogenesis [2]. Because of this broad spectrum of serotonin functions, pathologies in serotonergic system have been held to account on many psychiatric

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

disorders such as mood disorders, anxiety disorders, attention‐deficit hyperactivity disorder (ADHD), and autism spectrum disorders (ASDs) [3]. Consider of heterogeneous clinic and different symptom clusters of psychiatric disorders, the serotonergic system predominates on the etiopathogenesis of two important endophenotypes: impulsivity and aggression [4, 5]. Impulsiveness is defined as personality trait, which is a multidimensionality [6]. An implu‐ sive temperament is associated with clinical conditions, such as pathological gambling, eating disorders, and borderline personality disorder as well as being a risk factor for self‐harm, suicide, and emotional liability [3, 7]. Brain imaging and pharmacogenetic studies have dem‐ onstrated that serotonin dysfunction is associated with impulsive behaviors [8]. Aggression is not a personality trait like impulsivity, but it is the behavior of harm or injury to others [9]. It is harmful to the individual and the community when it is constant and excessive, besides being a natural human behavior toward survival [9]. Three types of aggression have been defined as psychotic, impulsive, and proactive [4]. The serotonergic system is associated with impul‐ sive aggression which is manifested by provocation rather than proactive aggression which is goal‐oriented and planned [4]. Nowadays, researchers are directed to endophenotypes in psychiatric diseases with heterogeneous clinic in order to develop new treatment methods and to elucidate etiopathogenesis. In this chapter, we aimed to review the role of the seroto‐ nergic system on impulsivity and aggression, which are two important endophenotypes that identified in many psychiatric disorders.

## **2. Impulsivity**

Impulsivity is defined as the tendency to exhibit behavior without adequate mental assess‐ ment of possible outcomes [10, 11]. From this point of view, it can be said that impulsive dimension can be mentioned in the process of thought up to behavior [12]. In this dimen‐ sion, there have been different definitions such as impulsive choice, impulsive reflection, and impulsive action that can be measured by different assessment tools that have subjective or objective qualities [10, 13, 14]. Impulsive choice described as prefer less valuable prize in soon afterwards rather than the more valuable prize in the distant future, the inability of the individual to gather adequate data on the risks describes as impulsive reflection and a lack of motor inhibition described as impulsive action [15]. The lowa gambling test provides data about impulsive choice known as delay‐discounting [16]. Stop‐signal reaction time and go/ no go tasks are objective assessment methods that assess motor inhibition. In these tasks, individuals should wait until the appropriate signal arrives and stop the movement when no go or stop signal is received. "Waiting impulsivity" described as the failure to start the move‐ ment and "stopping impulsivity" described as the failure to stop or restrict the movement. The Barratt impulsivity scale and impulsive behavior scale are subjective self‐report scales, each with different subscales and provide data on different dimensions of the impulsivity [10, 12, 13, 15, 17–19].

The main pathophysiological mechanism is the disruption of reciprocal equilibrium in corti‐ costriatal cycles [10]. Impulsive behaviors come out as a result of impaired inhibitor function of the prefrontal cortex (PFC) to delay the award and stopping or restricting the behavior, additionally increased striatal output to achieve a small and certain but definite near future reward rather than the far‐future reward, with a high value but a low degree of uncertainty [10, 13, 15].

Recent studies showed that the basic region that rejects the award postponement when the award is quick earning despite small was nucleus accumbens; contrary the basic region that provides inhibition is the orbitofrontal cortex [20, 21]. Anterior cingulate cortex and right inferior frontal gyrus are two other important regions for inhibition [22, 23]. Nucleus accum‐ bens is also associated with impulsive cycle inflicting from the striatum, also accompanied by amygdala and hippocampus [24]. This network includes dopaminergic, noradrenergic, and serotonergic neurotransmission.

Increased impulsiveness is associated with many psychiatric disorders, although healthy indi‐ viduals have a personality trait and an advantage in situations where the organism needs to move quickly [10]. ADHD, substance abuse, eating disorders, bipolar disorder, behavioral addictions, and borderline/antisocial personality disorders are typical psychopathologies asso‐ ciated with impulsivity [25]. In these disorders, impulsive behavior patterns can be described in many expressions; but aggression is the most accentuated and evidence‐based one.

## **3. Aggression**

disorders such as mood disorders, anxiety disorders, attention‐deficit hyperactivity disorder (ADHD), and autism spectrum disorders (ASDs) [3]. Consider of heterogeneous clinic and different symptom clusters of psychiatric disorders, the serotonergic system predominates on the etiopathogenesis of two important endophenotypes: impulsivity and aggression [4, 5]. Impulsiveness is defined as personality trait, which is a multidimensionality [6]. An implu‐ sive temperament is associated with clinical conditions, such as pathological gambling, eating disorders, and borderline personality disorder as well as being a risk factor for self‐harm, suicide, and emotional liability [3, 7]. Brain imaging and pharmacogenetic studies have dem‐ onstrated that serotonin dysfunction is associated with impulsive behaviors [8]. Aggression is not a personality trait like impulsivity, but it is the behavior of harm or injury to others [9]. It is harmful to the individual and the community when it is constant and excessive, besides being a natural human behavior toward survival [9]. Three types of aggression have been defined as psychotic, impulsive, and proactive [4]. The serotonergic system is associated with impul‐ sive aggression which is manifested by provocation rather than proactive aggression which is goal‐oriented and planned [4]. Nowadays, researchers are directed to endophenotypes in psychiatric diseases with heterogeneous clinic in order to develop new treatment methods and to elucidate etiopathogenesis. In this chapter, we aimed to review the role of the seroto‐ nergic system on impulsivity and aggression, which are two important endophenotypes that

Impulsivity is defined as the tendency to exhibit behavior without adequate mental assess‐ ment of possible outcomes [10, 11]. From this point of view, it can be said that impulsive dimension can be mentioned in the process of thought up to behavior [12]. In this dimen‐ sion, there have been different definitions such as impulsive choice, impulsive reflection, and impulsive action that can be measured by different assessment tools that have subjective or objective qualities [10, 13, 14]. Impulsive choice described as prefer less valuable prize in soon afterwards rather than the more valuable prize in the distant future, the inability of the individual to gather adequate data on the risks describes as impulsive reflection and a lack of motor inhibition described as impulsive action [15]. The lowa gambling test provides data about impulsive choice known as delay‐discounting [16]. Stop‐signal reaction time and go/ no go tasks are objective assessment methods that assess motor inhibition. In these tasks, individuals should wait until the appropriate signal arrives and stop the movement when no go or stop signal is received. "Waiting impulsivity" described as the failure to start the move‐ ment and "stopping impulsivity" described as the failure to stop or restrict the movement. The Barratt impulsivity scale and impulsive behavior scale are subjective self‐report scales, each with different subscales and provide data on different dimensions of the impulsivity

The main pathophysiological mechanism is the disruption of reciprocal equilibrium in corti‐ costriatal cycles [10]. Impulsive behaviors come out as a result of impaired inhibitor function of the prefrontal cortex (PFC) to delay the award and stopping or restricting the behavior,

identified in many psychiatric disorders.

242 Serotonin - A Chemical Messenger Between All Types of Living Cells

**2. Impulsivity**

[10, 12, 13, 15, 17–19].

Aggression is the pattern of behavior that an individual exhibits in such a way as to dam‐ age himself or environment [4]. Natively, aggression is necessary to survive. For example, to protect ourselves and our beloved ones from danger, to supply the food and water for survive, and to react to possible risks of the organism on threat [26]. Investigating aggressive behaviors by subcategories is beneficial both in clarifying etiopathogenesis and in adjusting the treatment process. In previous papers, aggression had been categorized as offensive and defensive such as a dangerous or evasive response to a sense of fear, the most frequently pre‐ ferred classification in the recent literature categorized into three groups: impulsive, proactive (also known as organized, instrumental, or predatory), and psychotic. Impulsive aggression (54%) is the most common category followed by proactive aggression (29%), and psychotic aggression (17%) [27, 28]. As predicted, psychotic aggression is a process related to positive symptoms of psychosis, such as hallucinations or delusional content. In proactive aggression, the individual exhibits this behavior in a planned manner to achieve a blazing benefit such as money or revenge. Impulsive aggression is a behavioral pattern which is accompanied by physical symptoms after stimulation of the sympathetic system, often associated with feel‐ ings of fear, inhibition, or anger, which are manifested by stress, threat, or provocation [28].

The main pathophysiological mechanism of impulsive aggression is the altered balance—to the detriment of prefrontal cortex—between the inhibitor stimulants from cortex to subcortex/lim‐ bic system and excitator stimulant as strong tendency to realizing behavior from cortex [4]. PFC dysfunction results in inadequate risk assessment and top‐down inhibition is reduced [4, 27]. Bottom up outputs that have increased frequency and amplitude especially from the amygdala toward the orbitofrontal cortex contribute to impulsive aggression [27, 29]. In many human and animal studies, ventral PFC has been shown to be associated with impulse and aggression [30]. Antisocial behaviors are also observed in specific lesions of ventral PFC [30, 31].

## **4. Serotonin on impulsivity**

A significant part of serotonergic innervation in brain structures is derived from the dorsal raphe nucleus (DRN) [32]. It has been shown to increase premature response in the lesion of serotonergic areas at DRN by 5‐choice serial reaction time task (5CSRT) although increased correct response [33]. These findings point to the role of serotonergic regulation in organiz‐ ing behavior to optimize the performance of the cortex [34]. What a serotonin‐stimulated neuron will ultimately do is related to the balance between the serotonergic receptors on it [35]. In addition, there are nonserotonergic neurons in the projection fields of raphe nucleus in PFC, where 5HT1A and 5HT2A receptors are postsynaptic located [36]. In PFC, 80% of the glutamatergic neurons and 25% of the GABAergic neurons have 5HT1A and 5HT2A receptors distributed in Ref. [36]. The 5HT2A receptor activates the neuron and increase glutamate release by the contrast with 5HT1A receptor that decreases glutamate release [34]. In molecular genetic studies, 5HT2A is the most prominent receptor in the role of serotonin on impulsivity.

5HTR2A is located in the genomic chromosome 13q14‐q21 and contains three exons [37]. This gene codes for a receptor associated with the G protein, and this receptor stimulates phospho‐ lipase C, which reduces protein kinase C activity [37]. 5HT2A is most commonly expressed in the hippocampus, amygdala, and nucleus accumbens [38]. The 5HT2A receptor is associated with many common psychiatric disorders such as major depression, obsessive‐compulsive disorder, anorexia nervosa, and schizophrenia. Dopamine and 5HT have also been shown to play an important role in the regulation of attention and response control in frontal cortex by animal models [39]. In the psychopathology mentioned above, impulsivity is one of the three core symptom clusters of the disease, ADHD is especially prominent at research. Therefore, in this section, ADHD/impulsivity will be discussed in the context of serotonin. Continuation of sedative effects of methylphenidate in knockout mice inhibited dopaminergic gene function supports the role of other systems. In this model, hyperactivity was also observed to be sup‐ pressed with fluoxetine. This effect is thought to be mediated by an increase in the concentra‐ tion of extracellular serotonin through blockade of the serotonin transporter. In the direction of these findings, it has been suggested that the effect of methylphenidate on impulsivity also be demonstrated by increasing serotonin levels [40, 41]. The data obtained from pharmaco‐ logical studies, which showed that stimulated striatal 5HT2A receptors increase dopamine release and regulate hyperactivity, confirm that the serotonergic and dopaminergic require‐ ments are in interaction to mediate hyperactivity behavior [42]. Serotonin may affect ADHD and other impulsive behaviors indirectly by regulating dopaminergic functions. The nature of this regulatory effect is complex. It has been demonstrated that serotonergic neurons have inhibitory effects on dopaminergic neuron bodies in the midbrain region; both excitatory and inhibitory effects on dopamine projections in striatum, nucleus accumbens, and prefrontal cortex by animal models [43, 44]. When serotonergic agonists supplied to striatum, it has been leading to inhibited striatal neuronal firing, decreased in synaptic dopamine, which may result in reduced synthesis or release of dopamine in neuronal projections. That effect has been thought to be mediated by the serotonergic receptor 5HT2A. By way of these data, it has been thought that 5HT2A receptors may contribute to the development of ADHD [45]. Interest in the 5HT2A receptor in ADHD began with the observation that decreased hyperactivity in mice given selective 5HT2A antagonists [42]. It has been shown that the 5HT neurotransmit‐ ter system, in parallel with the typical course of ADHD, develops an age‐related develop‐ mental pattern, for example in developmental studies in monkeys, the 5HT receptor binding increased during infancy and childhood, peaked before puberty and slowly decreased during adolescence and early adulthood [46]. In humans, the 5HT2 receptor binding at 6 years was found to be higher than in neonates and 13–14 years of age [47]. The main result of activation of the 5HT2A receptor by serotonin is reduced noradrenalin and dopamine levels and increased glutamate levels [35]. In this context, 5HT2A antagonism contributes to attention functions by causing an increase in dopamine noradrenalin levels. In the light of those information, it has been aim to clarify the subtypes of impulsivity by referring to some important studies that have recently been made. The 5CSRT is a test for assessing impulsivity, as well as providing information on attention functions used in animal studies [48]. In that task, the individuals learn to get food by pressing the button after a certain goal. The pushing of the button by the animal without showing the target is regarded as a premature response and displays the wait‐ ing impulsivity [48]. In a study conducted by Fletcher et al., it was observed that 5HT2C and 5HT2A antagonists given to mice have different effects on 5CSRT [49]. While 5HT2A antago‐ nists reduced prematurity responding, 5HT2C antagonists increased. As a result of that the researchers have also indicated that the impulsivity is not only related to the level of 5‐HT, but it is also related to the balance between different serotonergic receptors [49]. In a study in which the effect of 5HT2A receptor gene polymorphism [1438G/A] on impulsivity was assessed by go/no go test, individuals with polymorphism were found to have significantly more commission errors [50]. It has been found that 8‐hydroxy‐2‐(di‐n‐propylamino) tetralin (8‐OH‐DPAT), a potent 5HT1A agonist, was ineffective on 5CSRT parameters by systemic administration; however, presynaptic 5HT1A autoreceptors at DRN had a markable effect on those parameters [34]. In a study conducted on women with bulimia nervosa in which impul‐ sive behavior patterns were observed, it was determined that there was a decrease in 5HT2A binding in ventral PFC [51].

animal studies, ventral PFC has been shown to be associated with impulse and aggression [30].

A significant part of serotonergic innervation in brain structures is derived from the dorsal raphe nucleus (DRN) [32]. It has been shown to increase premature response in the lesion of serotonergic areas at DRN by 5‐choice serial reaction time task (5CSRT) although increased correct response [33]. These findings point to the role of serotonergic regulation in organiz‐ ing behavior to optimize the performance of the cortex [34]. What a serotonin‐stimulated neuron will ultimately do is related to the balance between the serotonergic receptors on it [35]. In addition, there are nonserotonergic neurons in the projection fields of raphe nucleus in PFC, where 5HT1A and 5HT2A receptors are postsynaptic located [36]. In PFC, 80% of the glutamatergic neurons and 25% of the GABAergic neurons have 5HT1A and 5HT2A receptors distributed in Ref. [36]. The 5HT2A receptor activates the neuron and increase glutamate release by the contrast with 5HT1A receptor that decreases glutamate release [34]. In molecular genetic studies, 5HT2A is the most prominent receptor in the role of serotonin

5HTR2A is located in the genomic chromosome 13q14‐q21 and contains three exons [37]. This gene codes for a receptor associated with the G protein, and this receptor stimulates phospho‐ lipase C, which reduces protein kinase C activity [37]. 5HT2A is most commonly expressed in the hippocampus, amygdala, and nucleus accumbens [38]. The 5HT2A receptor is associated with many common psychiatric disorders such as major depression, obsessive‐compulsive disorder, anorexia nervosa, and schizophrenia. Dopamine and 5HT have also been shown to play an important role in the regulation of attention and response control in frontal cortex by animal models [39]. In the psychopathology mentioned above, impulsivity is one of the three core symptom clusters of the disease, ADHD is especially prominent at research. Therefore, in this section, ADHD/impulsivity will be discussed in the context of serotonin. Continuation of sedative effects of methylphenidate in knockout mice inhibited dopaminergic gene function supports the role of other systems. In this model, hyperactivity was also observed to be sup‐ pressed with fluoxetine. This effect is thought to be mediated by an increase in the concentra‐ tion of extracellular serotonin through blockade of the serotonin transporter. In the direction of these findings, it has been suggested that the effect of methylphenidate on impulsivity also be demonstrated by increasing serotonin levels [40, 41]. The data obtained from pharmaco‐ logical studies, which showed that stimulated striatal 5HT2A receptors increase dopamine release and regulate hyperactivity, confirm that the serotonergic and dopaminergic require‐ ments are in interaction to mediate hyperactivity behavior [42]. Serotonin may affect ADHD and other impulsive behaviors indirectly by regulating dopaminergic functions. The nature of this regulatory effect is complex. It has been demonstrated that serotonergic neurons have inhibitory effects on dopaminergic neuron bodies in the midbrain region; both excitatory and inhibitory effects on dopamine projections in striatum, nucleus accumbens, and prefrontal

Antisocial behaviors are also observed in specific lesions of ventral PFC [30, 31].

**4. Serotonin on impulsivity**

244 Serotonin - A Chemical Messenger Between All Types of Living Cells

on impulsivity.

The decrease in central serotonergic activity was associated with negative emotional state, poor impulse control, aggressive behavior, increased alcohol and nicotine use, and increased food consumption [52]. The tryptophan depletion method reduces the amount of serotonin throughout the brain. In a study conducted with this method, the relationship between impulse serotonin in humans was examined and an increase in premature response was detected. By this means, it has been concluded that central serotonin levels are related to wait‐ ing impulsivity rather than stopping impulsivity. Interestingly, it has been determine that tryptophan‐depleted individuals had an increase in motivation and accuracy in compliance with individuals who do not have this depletion [53, 54].

## **5. Serotonin on aggression**

Serotonin is the main neurotransmitter in both top‐down and bottom‐up processes of neuro‐ biological cycles associated with aggression [4]. Serotonergic hypofunction has been found to be associated with impulsive aggression in aggression subtypes [5]. It has been known that polymorphism of metabolic enzymes, carrier proteins, and receptors on the serotonergic system is associated with an increased aggressive behavior pattern [55]. The essential role of serotonin in the etiopathogenesis of impulsive aggression has been determined by brain imag‐ ing studies showing an increase in 5HT2A receptor concentration in orbital PFC in aggressive individuals, tryptophan depletion studies, molecular genetic studies that showed individuals who have monoamine oxidase a gene polymorphisms and have early stressful life events lean to aggression and violence at early adulthood period [38, 56–59].

Selective serotonin reuptake inhibitors (SSRIs) are generally recommended in the treatment of impulsive aggression. However, it should be kept in mind that special approaches are needed in special patient groups. For example, SSRIs have been found to be effective in the treatment of aggression in dementia patients and ineffective in patients with traumatic brain injury [27]. SSRIs are generally recommended in the treatment of impulsive aggression [27].

## **6. Conclusion**

In this chapter, it has been argued the relationship between serotonin, one of the basic neu‐ rotransmitters, with the two endophenotypes—impulsivity and aggression—in the face of many psychiatric disorder. There is a consensus in the literature that the problems of the sub‐ units of the serotonergic system result with impulsivity and aggression. Nowadays, research‐ ers have elaborated this information and have identified impulsivity and aggression as subtypes. In the last decade, data from both animal and human studies have been suggested that serotonin has more associated with impulsive aggression than with aggression subtypes, with more "waiting impulsivity" in impulsivity subtypes. More clinical studies are needed on this issue in which genetic and neuroimaging techniques are combined in homogeneous samples that are well defined by subtypes.

## **Author details**

Fatih Hilmi Çetin1 \*, Yasemin Taş Torun2 and Esra Güney3

\*Address all correspondence to: fatihhilmicetin@gmail.com

1 Department of Child and Adolescent Psychiatry, Faculty of Medicine, Selçuk University, Konya, Turkey

2 Department of Child and Adolescent Psychiatry, Gülhane Training and Research Hospital, Ankara, Turkey

3 Department of Child and Adolescent Psychiatry, Faculty of Medicine, Gazi University, Ankara, Turkey

## **References**

**5. Serotonin on aggression**

246 Serotonin - A Chemical Messenger Between All Types of Living Cells

**6. Conclusion**

**Author details**

Fatih Hilmi Çetin1

Konya, Turkey

Ankara, Turkey

Ankara, Turkey

samples that are well defined by subtypes.

\*, Yasemin Taş Torun2

\*Address all correspondence to: fatihhilmicetin@gmail.com

Serotonin is the main neurotransmitter in both top‐down and bottom‐up processes of neuro‐ biological cycles associated with aggression [4]. Serotonergic hypofunction has been found to be associated with impulsive aggression in aggression subtypes [5]. It has been known that polymorphism of metabolic enzymes, carrier proteins, and receptors on the serotonergic system is associated with an increased aggressive behavior pattern [55]. The essential role of serotonin in the etiopathogenesis of impulsive aggression has been determined by brain imag‐ ing studies showing an increase in 5HT2A receptor concentration in orbital PFC in aggressive individuals, tryptophan depletion studies, molecular genetic studies that showed individuals who have monoamine oxidase a gene polymorphisms and have early stressful life events lean

Selective serotonin reuptake inhibitors (SSRIs) are generally recommended in the treatment of impulsive aggression. However, it should be kept in mind that special approaches are needed in special patient groups. For example, SSRIs have been found to be effective in the treatment of aggression in dementia patients and ineffective in patients with traumatic brain injury [27].

In this chapter, it has been argued the relationship between serotonin, one of the basic neu‐ rotransmitters, with the two endophenotypes—impulsivity and aggression—in the face of many psychiatric disorder. There is a consensus in the literature that the problems of the sub‐ units of the serotonergic system result with impulsivity and aggression. Nowadays, research‐ ers have elaborated this information and have identified impulsivity and aggression as subtypes. In the last decade, data from both animal and human studies have been suggested that serotonin has more associated with impulsive aggression than with aggression subtypes, with more "waiting impulsivity" in impulsivity subtypes. More clinical studies are needed on this issue in which genetic and neuroimaging techniques are combined in homogeneous

and Esra Güney3

1 Department of Child and Adolescent Psychiatry, Faculty of Medicine, Selçuk University,

2 Department of Child and Adolescent Psychiatry, Gülhane Training and Research Hospital,

3 Department of Child and Adolescent Psychiatry, Faculty of Medicine, Gazi University,

SSRIs are generally recommended in the treatment of impulsive aggression [27].

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## **Immuno-Thrombotic Effects of Platelet Serotonin**

Elmina Mammadova-Bach, Maximilian Mauler,

Attila Braun and Daniel Duerschmied

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69349

#### **Abstract**

Platelets transport and store serotonin at a high concentration in dense granules and release it upon activation. Abnormal serotonin concentrations in the blood plasma or increased platelet serotonin release promote the development of thrombosis, sepsis, allergic asthma, myocardial infarction, and stroke. Consequently, experimental data suggest possible benefits of serotonin receptor blockade or inhibition of platelet serotonin uptake in the indicated human diseases. Here, we highlight the current state of basic biological research regarding the role of platelet serotonin in normal and pathophysiological conditions focusing on thrombotic and inflammatory diseases. We also describe the possible clinical applicability of targeting thrombo-immune-modulatory effects of platelet serotonin to treat common health problems.

**Keywords:** platelets, serotonin, inflammation, thrombosis, selective serotonin reuptake inhibitors

## **1. Introduction**

Serotonin (5-HT) is a well-known neurotransmitter, which regulates neural activity and a variety of neuropsychological processes [1]. As it has been shown to be involved in the regulation of systemic and cellular functions, alterations in serotonin concentration in the body are associated with many different diseases, such as irritable bowel syndrome, restless legs syndrome, sudden infant death syndrome, autism, headache, insomnia, anxiety, depression, anorexia, schizophrenia, Parkinson's and Alzheimer's disease, pulmonary hypertension, and

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 1.** 5-HT biosynthesis and receptor distribution in brain (A) and periphery (B). Serotonin (5-HT), serotonin transporter (SERT), monoamine oxidase (MAO), 5-hydroxyindole acetic acid (5-HIAA), 5-hydroxytryptophan (5-HTP), tryptophan (TRP), and vesicular monoamine transporter (VMAT). For the details, see the text.

myocardial infarction. 5-HT was first described in 1930 by Vittrorio Erspamer who isolated it from enterochromafin cells of the gut [1]. Only a small amount of 5-HT is synthesized in brain (5%), whereas 95% is produced by the enterochromafin cells of the gastrointestinal (GI) tract. 5-HT is synthesized from the essential amino acid l-tryptophan (TRP) to 5-hydroxytryptophan (5-HTP) by the enzyme l-tryptophan hydroxylase (TPH)-1 in the brain and TPH-2 in the periphery [2, 3]. The activity of these TPH enzymes is the rate-limiting step in the production of 5-HT in both organs. After its synthesis in the gastrointestinal tract, 5-HT is released into the bloodstream. 5-HT can bind and activate several isoforms of 5-HT receptors expressed throughout the body (**Figure 1**). 5-HT receptors were identified on different blood cells and in the vessel wall including lymphocytes, endothelial, and smooth muscle cells, respectively, which can respond to 5-HT under certain physiological conditions. These receptors constitute a family of seven different receptor sub-classes: 5-HT<sup>1</sup> (A-F, P, S), 5-HT<sup>2</sup> (A-D), 5-HT<sup>3</sup> , 5-HT4 , 5-HT5 , 5-HT6 , and 5-HT7 [3, 4]. All these receptors belong to the GPCR superfamily with the exception of 5-HT<sup>3</sup> [3], which is a member of nicotinic acetylcholine receptor superfamily and is a ligand-gated ion channel.

5-HT can also be taken up from plasma into several cells—such as platelets—via the 5-HT transporter (5-HTT, SERT). After uptake, 5-HT can be then stored in vesicles and granules through the action of vesicular monoamine transporter (VMAT)-1/2 which is expressed in neurons, neuroendocrine cells, and platelets. The largest quantity of serotonin is believed to be stored in platelets, from where it can be released upon platelet activation, for example, during thrombus formation or inflammatory reactions. Interestingly, chemical precursors of 5-HT can pass across the blood-brain barrier, but 5-HT cannot, thereby effectively isolating the brain 5-HT pool from the periphery and vice versa. In the brain, 5-HT regulates several complex networks, such as mood, perception, reward, anger, memory, appetite, attention, and sexuality. There are two major routes of 5-HT metabolism, which convert 5-HT to melatonin and 5-HIAA. 5-HT is metabolized by neurons and endothelial cells by monoamine oxidases (MAOs) and the products of this breakdown are then excreted by the kidney [3, 5–7].

Peripheral 5-HT regulates heart development and rate, valvulopathy, pain, nociception, embryonic development, vasoconstriction/vasodilatation, blood flow, hemostasis, and many other important processes. Platelets are not able to synthesize serotonin, but take it up from plasma via 5-HTT, store it in dense granules (via VMAT-1), and release it into the blood during their activation. Platelet serotonin has not only well-established autocrine functions during platelet activation and thrombus growth but also paracrine functions in the vasculature including modulation of endothelial, smooth muscle, and immune cell function.

## **2. Autocrine-regulatory mechanisms of platelet serotonin**

myocardial infarction. 5-HT was first described in 1930 by Vittrorio Erspamer who isolated it from enterochromafin cells of the gut [1]. Only a small amount of 5-HT is synthesized in brain (5%), whereas 95% is produced by the enterochromafin cells of the gastrointestinal (GI) tract. 5-HT is synthesized from the essential amino acid l-tryptophan (TRP) to 5-hydroxytryptophan (5-HTP) by the enzyme l-tryptophan hydroxylase (TPH)-1 in the brain and

**Figure 1.** 5-HT biosynthesis and receptor distribution in brain (A) and periphery (B). Serotonin (5-HT), serotonin transporter (SERT), monoamine oxidase (MAO), 5-hydroxyindole acetic acid (5-HIAA), 5-hydroxytryptophan (5-HTP),

tryptophan (TRP), and vesicular monoamine transporter (VMAT). For the details, see the text.

254 Serotonin - A Chemical Messenger Between All Types of Living Cells

Platelets store 5-HT in their dense granules at millimolar range and secrete it after activation [8]. Dense granule and 5-HT release support the recruitment of circulating platelets to preformed thrombi, thereby leading to thrombus growth. This process is mediated through the interaction between 5-HT and its receptor 5HT2A expressed on circulating platelets. Activated 5-HT2A receptor transduces the signal to G<sup>q</sup> -phospholipase C (PLC) β-signaling cascade. Enhanced PLCβ activity results in intracellular Ca2+mobilization from the store through inositol 3-phosphate (IP3) receptor and mediates 1,2-diacylglycerol (DAG)-dependent protein kinase C (PKC) activation, thereby amplifying platelet reactivity (**Figure 2**).

In addition to the mobilization of cytosolic Ca2+ [9, 10], receptor-ligand interactions are also known to regulate 5-HT uptake kinetics. In human platelets, the rise of cytoplasmic Ca2+ in

**Figure 2.** Autocrine effects of platelet 5-HT. Activated platelets release 5-HT, thereby amplifying platelet activation and the recruitment of circulating platelets. Binding of platelet 5-HT to the 5-HT2A receptor induces activation of PLCβsignaling cascade and upstream effectors which support platelet reactivity. Receptor-ligand interactions also regulate 5-HTT uptake kinetics by interconnecting several signaling pathways. For the details, see the text.

the absence of exocytosis reduces 5-HT transport into the cytoplasm, thereby decreasing the release of 5-HT [9]. Interestingly, rabbit platelets activated in the presence of the extracellular Ca2+ chelator ethylene tetraacetic acid also displayed a decrease in 5-HT transport activity [11, 12]. Consistently, human platelets treated with the membrane permeant Ca2+ chelator BAPTA-AM also had reduced 5-HT transport in the presence of extracellular Ca2+ [9]. Activation of the Orai1 Ca2+ channel induces a robust Ca2+ influx called store-operated Ca2+ entry (SOCE), which is triggered through the release of Ca2+ from intracellular stores. This process is controlled by functional coupling of activated stromal interaction molecule 1 (STIM1) to Orai1 [13]. Interestingly, strongly reduced SOCE was found in *5Htt−/−* platelets [14]. This suggests that secreted platelet 5-HT contributes to the regulation of SOCE through binding to 5-HT2A which activates Gq-PLCβ-mediated Ca2+ store release, thereby further activating STIM1/Orai1 complex. Interestingly, SOCE-induced signal can strongly inhibit 5-HT uptake in human platelets via 5-HTT [9, 11]. This could be an important step to keep 5-HT outside of platelets, thereby increasing extracellular 5-HT concentration and permanently activating 5-HT2A on the platelet surface. Therefore, 5-HT cannot enter the platelet cytosol during SOCE. Interestingly, 5-HTT contains several consensus sites for PKC. It has been shown that PKC activity is required for the internalization of the transporter suggesting a link between 5-HT uptake and intracellular Ca2+ level [15–18]. Altogether, Ca2+ signaling, Ca2+ store release, and Ca2+ influx through SOCE play an important regulatory role for 5-HT cycling in human and mouse platelets.

After Ca2+ store release and PKC activation, integrins exposed and activated on the platelet surface support aggregation and thrombus formation. In β3 integrin-deficient platelets, 5-HT uptake was strongly reduced, indicating a functional crosstalk between 5-HTT and β3 integrin [19]. Integrin activation defect in response to glycoprotein VI (GPVI) or C-type lectin-like receptor 2 (CLEC-2) stimulation was found in *5Htt−/−* mouse platelets, which was fully rescued in the presence of extracellular 5-HT [14]. The physical interaction between 5-HTT and β3 seems to be dispensable for β3 integrin activation. The observed integrin activation defect is due to the lack of the secreted platelet 5-HT which further amplifies "inside-out" activation of integrins through Ca2+-dependent and independent pathways mediated by Ca2+- and DAGregulated guanine exchange factor-1 (CalDAG-GEFI) and PKC, respectively.

Although 5-HT is mainly stored in dense granules, intracellular-free 5-HT in the cytoplasm has been proposed to activate diverse biological processes called serotonylation. It has been shown that small-guanosine triphosphate-binding protein (GTP)-ases covalently bind 5-HT, thereby changing the structure and activity of GTPase, leading to α-granule exocytosis from platelets. This process requires tissue transglutaminase and factor XIIIa, both activated by mobilized Ca2+. Transglutaminase may mediate the transamidation of small GTPases, like cytoplasmic Ras homolog gene family member A (RhoA) and a small GTP-binding protein Rab4. Serotonylation in turn blocks the inactivation of both molecules. A complex composed of Ca2+ and calmodulin (CaM) may also activate guanine exchange factors (GEFs), which induce the exchange of guanosine di- (GDP) to triphosphate (GTP) on RhoA and Rab4 and thus stimulates activation of the respective protein. These two active molecules play an important role in cytoskeleton rearrangement, exocytosis of α-, and dense granule contents. Some bioactive molecules stored in platelet granules, such as fibrinogen and factor V, are also known to be serotonylated [8]. Upon platelet activation, these proteins are exposed at the platelet surface and are used to mark a subpopulation of highly activated, pro-coagulant platelets, the so-called collagen and thrombin-activated (COAT) platelets. Coated platelets express high levels of phosphatidylserine and strongly support prothrombinase activity [8, 20].

Besides the dopamine transporter (DAT), the noradrenaline transporter (NET), and the organic transporter (OCT), 5-HTT is an important 5-HT transporter to regulate 5-HT uptake from the blood plasma and reuptake of the released platelet 5-HT in certain physiological

the absence of exocytosis reduces 5-HT transport into the cytoplasm, thereby decreasing the release of 5-HT [9]. Interestingly, rabbit platelets activated in the presence of the extracellular Ca2+ chelator ethylene tetraacetic acid also displayed a decrease in 5-HT transport activity [11, 12]. Consistently, human platelets treated with the membrane permeant Ca2+ chelator BAPTA-AM also had reduced 5-HT transport in the presence of extracellular Ca2+ [9]. Activation of the Orai1 Ca2+ channel induces a robust Ca2+ influx called store-operated Ca2+ entry (SOCE), which is triggered through the release of Ca2+ from intracellular stores.

**Figure 2.** Autocrine effects of platelet 5-HT. Activated platelets release 5-HT, thereby amplifying platelet activation and the recruitment of circulating platelets. Binding of platelet 5-HT to the 5-HT2A receptor induces activation of PLCβsignaling cascade and upstream effectors which support platelet reactivity. Receptor-ligand interactions also regulate

5-HTT uptake kinetics by interconnecting several signaling pathways. For the details, see the text.

256 Serotonin - A Chemical Messenger Between All Types of Living Cells

conditions. 5-HTT is encoded by the SLC6A4 gene containing 14 exons. The protein structure of 5-HTT contains 12 transmembrane domains. In humans, the splice variants of 5-HTT and their mutations are associated with several pathologies, such as anxiety, suicide, depression, substance abuse, autism, and neurogenic disorders [21–24]. 5-HTT is abundantly expressed not only on neurons, endothelial cells, mast cells, immune cells, in intestine, and vasculature, but also in platelets [25, 26]. It is well established that in platelets 5-HTT plays an important role in the uptake of 5-HT from the circulation. Monoamine transporters are thought to be able to compensate for one another where they are co-expressed. For example, 5-HT may be taken up in venous vessels independently of 5-HTT expression [25, 27]. Interestingly, and in sharp contrast to venous vessels, genetic ablation of *5Htt* in mice completely abolished 5-HT uptake in platelets, since no detectable secreted 5-HT was observed upon platelet activation, indicating an essential role of 5-HTT for 5-HT uptake into platelets, which cannot be compensated by other transporters [14]. Altogether, these results highlight the cell-type-specific regulation of 5-HT uptake in mammalian cells.

5-HTT can be targeted by several antidepressants, such as selective serotonin reuptake inhibitors (SSRIs) (cf., Section 5), which are widely used in the treatment of psychiatric diseases to increase 5-HTT concentrations in the synaptic space. The blockade of 5-HTT with the SSRI citalopram reduces the aggregation response to collagen in human platelets [28] due to reduced phosphorylation of a tyrosine-protein kinase Syk in the GPVI signalosome. Syk can also bind and phosphorylate 5-HTT suggesting an Syk-mediated functional crosstalk between 5-HTT and GPVI complex. Interestingly, *5Htt−/−* mouse platelets could not show any abnormalities in the tyrosine phosphorylation cascade of the GPVI signalosome, as Syk phosphorylation was normal after GPVI stimuli. Consequently, Syk and 5-HTT interaction seems to be dispensable for the initial activation of GPVI complex, but enhanced Syk activity may regulate the 5-HT uptake in platelets [29].

## **3. Paracrine-regulatory mechanisms of platelet serotonin**

During degranulation, activated platelets secrete a significant amount of 5-HT from dense granules which is clinically relevant to induce acute thrombotic events [30, 31] by promoting vasoconstriction and cellular activation of neighboring platelets and lymphocytes through their 5-HT receptors.

5-HT receptors expressed on endothelial, smooth muscle, and immune cells respond to platelet-derived 5-HT (**Figure 3**). 5-HT has growth-promoting effects on endothelial cells, which may facilitate tissue healing after vascular damages [32]. However, 5-HT may also exert dual effects either stimulating constriction or dilatation of microvasculature. In the liver, 5-HT appears to mainly promote constriction of hepatic sinusoid vessels, since mice lacking peripheral 5-HT display elevated sinusoidal perfusion under physiological and pathological conditions [33]. By contrast, platelet-derived 5-HT coordinates the formation of gaps between endothelial cells in the joint microvasculature, which in arthritic conditions may contribute to inflammation [34]. How these processes are regulated is still not clear but presumably may involve differential signaling pathways through specific 5-HT receptors expressed on vascular endothelial and smooth muscle cells.

conditions. 5-HTT is encoded by the SLC6A4 gene containing 14 exons. The protein structure of 5-HTT contains 12 transmembrane domains. In humans, the splice variants of 5-HTT and their mutations are associated with several pathologies, such as anxiety, suicide, depression, substance abuse, autism, and neurogenic disorders [21–24]. 5-HTT is abundantly expressed not only on neurons, endothelial cells, mast cells, immune cells, in intestine, and vasculature, but also in platelets [25, 26]. It is well established that in platelets 5-HTT plays an important role in the uptake of 5-HT from the circulation. Monoamine transporters are thought to be able to compensate for one another where they are co-expressed. For example, 5-HT may be taken up in venous vessels independently of 5-HTT expression [25, 27]. Interestingly, and in sharp contrast to venous vessels, genetic ablation of *5Htt* in mice completely abolished 5-HT uptake in platelets, since no detectable secreted 5-HT was observed upon platelet activation, indicating an essential role of 5-HTT for 5-HT uptake into platelets, which cannot be compensated by other transporters [14]. Altogether, these results highlight the cell-type-specific

5-HTT can be targeted by several antidepressants, such as selective serotonin reuptake inhibitors (SSRIs) (cf., Section 5), which are widely used in the treatment of psychiatric diseases to increase 5-HTT concentrations in the synaptic space. The blockade of 5-HTT with the SSRI citalopram reduces the aggregation response to collagen in human platelets [28] due to reduced phosphorylation of a tyrosine-protein kinase Syk in the GPVI signalosome. Syk can also bind and phosphorylate 5-HTT suggesting an Syk-mediated functional crosstalk between 5-HTT and GPVI complex. Interestingly, *5Htt−/−* mouse platelets could not show any abnormalities in the tyrosine phosphorylation cascade of the GPVI signalosome, as Syk phosphorylation was normal after GPVI stimuli. Consequently, Syk and 5-HTT interaction seems to be dispensable for the initial activation of

GPVI complex, but enhanced Syk activity may regulate the 5-HT uptake in platelets [29].

During degranulation, activated platelets secrete a significant amount of 5-HT from dense granules which is clinically relevant to induce acute thrombotic events [30, 31] by promoting vasoconstriction and cellular activation of neighboring platelets and lymphocytes through

5-HT receptors expressed on endothelial, smooth muscle, and immune cells respond to platelet-derived 5-HT (**Figure 3**). 5-HT has growth-promoting effects on endothelial cells, which may facilitate tissue healing after vascular damages [32]. However, 5-HT may also exert dual effects either stimulating constriction or dilatation of microvasculature. In the liver, 5-HT appears to mainly promote constriction of hepatic sinusoid vessels, since mice lacking peripheral 5-HT display elevated sinusoidal perfusion under physiological and pathological conditions [33]. By contrast, platelet-derived 5-HT coordinates the formation of gaps between endothelial cells in the joint microvasculature, which in arthritic conditions may contribute to inflammation [34]. How these processes are regulated is still not clear but presumably may involve differential signaling pathways through specific 5-HT receptors expressed on vascu-

**3. Paracrine-regulatory mechanisms of platelet serotonin**

regulation of 5-HT uptake in mammalian cells.

258 Serotonin - A Chemical Messenger Between All Types of Living Cells

their 5-HT receptors.

lar endothelial and smooth muscle cells.

**Figure 3.** Paracrine effects of platelet 5-HT. Secretion of platelet 5-HT modulates the function of endothelial and smooth muscle cells either promoting vessel constriction or dilatation. Platelet 5-HT influences several functions of immune cells, indicating their importance in the regulation of immune cell response and activities under pathophysiological conditions. For the details, see the text.

Platelet-derived 5-HT can regulate the function of T- and B-cells, natural killer cells, monocytes, and neutrophils under certain conditions [35–38]. In the spleen, 5-HT increases monocyte differentiation into dendritic cells and early naive T-cell activation via the 5-HT2A receptor [38, 39]. Furthermore, it also has been shown that lymphocytic cytokine levels in mice are reduced after treatment with SSRI [40]. In a mouse model of viral hepatitis, the release of 5-HT by platelets was responsible for tissues damage caused by CD8 (+) T-cells, microcirculatory events, and reduced clearance of infiltrated viruses [33]. Moreover, specific antagonism of 5-HT receptors in mice attenuated asthmatic attacks and sepsis [37, 41].

5-HT released from dense granules upon activation by the inflamed endothelium also contributes to the recruitment of immune cells to the vascular wall [37]. Indeed, platelet-derived 5-HT promotes leukocyte migration, possibly via activation of endothelial cells, thereby enhancing P-selectin exposure and IL-8 release [37], which trigger neutrophil rolling, adhesion, and extravasation. Moreover, locally increased levels of platelet-released 5-HT had paracrine effects on endothelial cells, thereby inducing microvasculature leakage through the activation of transglutaminase and the phosphorylation of vimentin [42]. By contrast, in solid tumors platelet-released 5-HT has been described as a major regulator of the tumor vascular homeostasis that continuously prevent bleeding. Interestingly, tumor-infiltrating leukocytes have been identified as the cause of tumor bleeding [43, 44]. Altogether, these studies suggest that under specific conditions, platelet-released 5-HT promotes clot formation and modulates immune cell functions.

In humans, 5-HT levels appear elevated in infection and autoimmune diseases, suggesting that SSRI could be applicable for vascular and immune system modulation. Since platelets are the major 5-HT store in the blood, pharmacological blockage of 5-HT uptake in platelets increases the level of 5-HT in the blood plasma transiently. Unexpectedly, *5Htt−/−* mice display reduced 5-HT levels in plasma [14]. In *5Htt−/−* mice, elevated urinary 5-HIAA levels were detected suggesting a faster 5-HT metabolism in the peripheral blood. Consequently, platelet 5-HT uptake and storage play an important regulatory role for controlling systemic 5-HT metabolic cycles. Future studies are needed to specify the exact mechanisms of platelet-derived 5-HT on vascular and immune system modulation in normal physiology and diseases.

## **4. Pathophysiological consequences of abnormal platelet serotonin release**

5-HT plasma concentration was analyzed in several pathological contexts. It became widely recognized that 5-HT is an independent risk factor for platelet aggregation and for thrombus formation in animal models (cf., Section 6) and human patients [19, 45–49]. Plasma 5-HT can support platelet aggregation and thrombus growth through 5-HT2A-dependent or independent signaling pathways. Pharmacological blockade of 5-HT2A receptor increases the 5-HT uptake rates in animal models of hypertension, as well as ex vivo platelet aggregation. Vikenes et al. detected a 10-fold increase of plasma 5-HT in patients undergoing angiography after admission for myocardial infarction [50]. In these patients, high plasma 5-HT was associated with cardiac events. In another study, more than 10-fold rise in 5-HT has been noticed in coronary vessels of patients following angioplasty. Importantly, in these patients the level of 5-HT in the systemic plasma was normal [51]. Together, these studies suggest that in vivo the interplay between circulating 5-HT and platelet function could be a predictive factor.

5-HT levels are drastically increased during myocardial ischemia, and blockade of the 5-HT2 receptor improves the outcome after myocardial infarction in different mouse models [52, 53]. 5-HT also enhances the survival of cardiomyocytes via the 5-HT2B receptor. In hepatic ischemia models, platelets promote tissue repair [54], and proliferation of hepatocytes was shown to be partly mediated by platelet 5-HT after liver ischemia [55]. 5-HT also contributes to intratumoral homeostasis by dysbalancing permeability factors [44]. 5-HT-induced growth of human hepatocellular carcinoma cells and specific blockade of the 5-HT<sup>2</sup> receptor decreased recruitment of circulating tumor cells [56, 57]. It has been suggested that the inhibition of platelet granule contents might be effective to induce intratumoral bleeding, thereby decreasing tumor viability and growth. Additionally, plasma 5-HT levels are increased in patients with colorectal, liver, and intestinal cancers [58, 59].

Allergic airway inflammation provokes a local release of 5-HT in mouse models and human patients [41]. Interestingly, after challenge with an allergen, 5-HT increased 10-fold in broncho-alveolar lavage of predisposed patients, inducing asthmatic attacks. In line with these studies, 5-HT is known as a key regulator of pulmonary vascular resistance and vessel wall integrity [60, 61].

## **5. Clinical applications: effects of selective serotonin reuptake inhibitors on platelet functions**

In humans, 5-HT levels appear elevated in infection and autoimmune diseases, suggesting that SSRI could be applicable for vascular and immune system modulation. Since platelets are the major 5-HT store in the blood, pharmacological blockage of 5-HT uptake in platelets increases the level of 5-HT in the blood plasma transiently. Unexpectedly, *5Htt−/−* mice display reduced 5-HT levels in plasma [14]. In *5Htt−/−* mice, elevated urinary 5-HIAA levels were detected suggesting a faster 5-HT metabolism in the peripheral blood. Consequently, platelet 5-HT uptake and storage play an important regulatory role for controlling systemic 5-HT metabolic cycles. Future studies are needed to specify the exact mechanisms of platelet-derived 5-HT on vascu-

lar and immune system modulation in normal physiology and diseases.

260 Serotonin - A Chemical Messenger Between All Types of Living Cells

**release**

**4. Pathophysiological consequences of abnormal platelet serotonin** 

interplay between circulating 5-HT and platelet function could be a predictive factor.

hepatocellular carcinoma cells and specific blockade of the 5-HT<sup>2</sup>

tal, liver, and intestinal cancers [58, 59].

integrity [60, 61].

5-HT levels are drastically increased during myocardial ischemia, and blockade of the 5-HT2 receptor improves the outcome after myocardial infarction in different mouse models [52, 53]. 5-HT also enhances the survival of cardiomyocytes via the 5-HT2B receptor. In hepatic ischemia models, platelets promote tissue repair [54], and proliferation of hepatocytes was shown to be partly mediated by platelet 5-HT after liver ischemia [55]. 5-HT also contributes to intratumoral homeostasis by dysbalancing permeability factors [44]. 5-HT-induced growth of human

ment of circulating tumor cells [56, 57]. It has been suggested that the inhibition of platelet granule contents might be effective to induce intratumoral bleeding, thereby decreasing tumor viability and growth. Additionally, plasma 5-HT levels are increased in patients with colorec-

Allergic airway inflammation provokes a local release of 5-HT in mouse models and human patients [41]. Interestingly, after challenge with an allergen, 5-HT increased 10-fold in broncho-alveolar lavage of predisposed patients, inducing asthmatic attacks. In line with these studies, 5-HT is known as a key regulator of pulmonary vascular resistance and vessel wall

receptor decreased recruit-

5-HT plasma concentration was analyzed in several pathological contexts. It became widely recognized that 5-HT is an independent risk factor for platelet aggregation and for thrombus formation in animal models (cf., Section 6) and human patients [19, 45–49]. Plasma 5-HT can support platelet aggregation and thrombus growth through 5-HT2A-dependent or independent signaling pathways. Pharmacological blockade of 5-HT2A receptor increases the 5-HT uptake rates in animal models of hypertension, as well as ex vivo platelet aggregation. Vikenes et al. detected a 10-fold increase of plasma 5-HT in patients undergoing angiography after admission for myocardial infarction [50]. In these patients, high plasma 5-HT was associated with cardiac events. In another study, more than 10-fold rise in 5-HT has been noticed in coronary vessels of patients following angioplasty. Importantly, in these patients the level of 5-HT in the systemic plasma was normal [51]. Together, these studies suggest that in vivo the Selective serotonin reuptake inhibitors are commonly used drugs for the treatment of patients with severe depressive and anxiety disorders [62]. SSRIs were developed to selectively inhibit the uptake of 5-HT through the 5-HTT transporter in the brain, while having minimal side effects on DAT and NET proteins which can also transport 5-HT [63]. The action of SSRIs relies on the modulation of the allosteric region of the transporter, thereby leading to a conformational change and blocking of the uptake of 5-HT [63]. The uptake of 5-HT into neurons is very important for the clearance of the synaptic cleft, preventing firing rates and overstimulation of receptors [64]. This uptake and the later release are blocked upon treatment with SSRIs, such as fluvoxamine, fluoxetine, nortryptiline, citopram, and escitalopram [65]. The different SSRIs vary in kinetics being competitive and non-competitive inhibitors. Two distinct binding sites on 5-HTT have been identified, a low-affinity allosteric site, mediating the dissociation of SSRIs from their high-affinity site, which induces the blockade of 5-HT uptake [64].

There is evidence that targeting 5-HT receptors or using serotonin-like molecules is effective in the treatment of non-neuronal diseases. The use of tricyclic antidepressants, but not SSRIs, is associated with an increased risk of myocardial infarction. SSRIs have shown no cardiac toxicity, even in patients with heart disease. Several epidemiologic studies reported lower cardiovascular morbidity and mortality in patients treated with SSRI [66–68].

Depression is a significant risk factor for ischemic heart and cerebrovascular disease as well as mortality following myocardial infarction. The potential effects of SSRIs upon the cardiovascular system may therefore play an important role. These drugs had potential benefit in hypertensive patients after myocardial infarction and hypertensive responses to depression were reduced in patients who had been prescribed SSRIs [30]. In blood samples of depressive patients taking fluoxetine, the platelet aggregation response to submaximal collagen stimulation was decreased [69]. In this study, a significant decrease in 5-HT concentration was observed in platelet-rich plasma associated with the use of fluoxetine but not with the tricyclic antidepressant amitryptiline. It is intriguing whether lowered platelet 5-HT content translates into less 5-HT release during platelet activation in patients with thrombotic diseases. Enhanced platelet reactivity was observed in patients suffering from depression and chronic heart disease due to the upregulated β-thromboglobulin (β-TG) and platelet factor 4 (PF4) levels [70]. Lowered PF4 and β-TG levels have been observed upon treatment with SSRI paroxetine [71], suggesting that reduced platelet aggregation in vivo may impact coronary artery-related mortality. SSRI treatment also decreases platelet reactivity in patients with heart failure. Other SSRIs, sertraline, and N-desmethylsertraline were also shown to dampen platelet responses [72].

SSRIs have been shown to increase the risk of bleeding in patients with liver cirrhosis and liver failure. Importantly, SSRIs may also directly increase gastric acidity with ulcerogenic effect resulting in GI bleeding. The risk of SSRI-associated GI bleeding is increased with the concurrent use of nonsteroidal anti-inflammatory drugs, anticoagulants, and antiplatelet agents, and is decreased by concurrent proton pump inhibitors [73, 74]. In conclusion, SSRIs appear to be protective against cardiovascular diseases and may enhance the risk for GI bleeding. However, to date this evidence is not yet conclusive.

## **6. Experimental studies on the role of platelet serotonin in arterial thrombosis and stroke**

Over the past decades, the functions of peripheral 5-HT have received increasing attention. It has been shown that peripheral 5-HT plays a major role in a variety of important processes, including hemostasis and immune defense. This has been addressed by using *Tph1−/−* mice, which lack peripheral 5-HT in the circulation, due to the lack of the enzyme that converts hydroxylases tryptophan to 5-HT in the gut [75]. In humans, abolished or decreased level of TPH1 is associated with impulsive behavior, aggression, irritable bowel syndrome, anxiety, and other pathologies [76–79]. Genetic ablation of TPH1 function in mice not only leads to several disorders, such as mild anemia, cardiomyopathy, and diabetes, but also to other defects in hemostasis, erythropoiesis, pulmonary hypertension, and lung regeneration. The lack of 5-HT in this mouse model is associated with decreased neutrophil recruitment to inflammatory sites, diabetics, and mild anemia [37, 80].

Recent studies using wild-type mice infused with 5-HT or *Tph1−/−* mice have demonstrated that peripheral 5-HT is required for platelet aggregation [14]. Additionally, *in vivo* 5-HT infusion generates hyperreactive platelets with reduced bleeding time and shortened occlusion time of the carotid arteries in wild-type mice. *5-Htt−/−* mice have prolonged bleeding time, reflecting the increased bleeding risk described to occur using long-term SSRI treatment in human patients. In comparison to this relatively mild hemostatic defect, *5Htt−/−* mice were not able to form occlusive thrombi in response to mechanical injury of the abdominal aorta as compared to wild-type animals [14].

Platelets contribute to the progression of infarct growth after transient brain ischemia by thrombo-inflammation with platelet-immune cell interactions. SSRI treatment of stroke patients has been described to enhance brain function recovery, indicating a therapeutic benefit of the direct blockade of 5-HTT function. Neuroblast proliferation and cell migration have been shown to be enhanced and associated with increased microvessel density during SSRI treatment, explaining the possible role of 5-HTT in tissue repair after ischemic insults [81–83]. *5Htt−/−* mice have been studied in the tMCAO (transient intraluminal filament model of middle cerebral artery occlusion) model of ischemic stroke. Unexpectedly, these mice developed similar brain infarcts to wild-type controls and the 5-HTT neurological outcome was indistinguishable [14]. In line with this study, SSRI treatment could not reduce infarct size or cerebral edema in mice [82], suggesting that this treatment cannot protect neurons or other cells in the ischemic brain. Altogether, these results indicate that SSRI treatment may have a long-term effect in the ischemic brain tissue which positively influences post-stroke recovery. Further investigation is necessary to understand the specific role of peripheral and brain 5-HT in thrombo-inflammation during stroke and infarct progression.

## **7. Conclusions**

and is decreased by concurrent proton pump inhibitors [73, 74]. In conclusion, SSRIs appear to be protective against cardiovascular diseases and may enhance the risk for GI bleeding.

Over the past decades, the functions of peripheral 5-HT have received increasing attention. It has been shown that peripheral 5-HT plays a major role in a variety of important processes, including hemostasis and immune defense. This has been addressed by using *Tph1−/−* mice, which lack peripheral 5-HT in the circulation, due to the lack of the enzyme that converts hydroxylases tryptophan to 5-HT in the gut [75]. In humans, abolished or decreased level of TPH1 is associated with impulsive behavior, aggression, irritable bowel syndrome, anxiety, and other pathologies [76–79]. Genetic ablation of TPH1 function in mice not only leads to several disorders, such as mild anemia, cardiomyopathy, and diabetes, but also to other defects in hemostasis, erythropoiesis, pulmonary hypertension, and lung regeneration. The lack of 5-HT in this mouse model is associated with decreased neutrophil recruitment to

Recent studies using wild-type mice infused with 5-HT or *Tph1−/−* mice have demonstrated that peripheral 5-HT is required for platelet aggregation [14]. Additionally, *in vivo* 5-HT infusion generates hyperreactive platelets with reduced bleeding time and shortened occlusion time of the carotid arteries in wild-type mice. *5-Htt−/−* mice have prolonged bleeding time, reflecting the increased bleeding risk described to occur using long-term SSRI treatment in human patients. In comparison to this relatively mild hemostatic defect, *5Htt−/−* mice were not able to form occlusive thrombi in response to mechanical injury of the abdominal aorta as

Platelets contribute to the progression of infarct growth after transient brain ischemia by thrombo-inflammation with platelet-immune cell interactions. SSRI treatment of stroke patients has been described to enhance brain function recovery, indicating a therapeutic benefit of the direct blockade of 5-HTT function. Neuroblast proliferation and cell migration have been shown to be enhanced and associated with increased microvessel density during SSRI treatment, explaining the possible role of 5-HTT in tissue repair after ischemic insults [81–83]. *5Htt−/−* mice have been studied in the tMCAO (transient intraluminal filament model of middle cerebral artery occlusion) model of ischemic stroke. Unexpectedly, these mice developed similar brain infarcts to wild-type controls and the 5-HTT neurological outcome was indistinguishable [14]. In line with this study, SSRI treatment could not reduce infarct size or cerebral edema in mice [82], suggesting that this treatment cannot protect neurons or other cells in the ischemic brain. Altogether, these results indicate that SSRI treatment may have a long-term effect in the ischemic brain tissue which positively influences post-stroke recovery. Further investigation is necessary to understand the specific role of peripheral and brain 5-HT in

**6. Experimental studies on the role of platelet serotonin in arterial** 

However, to date this evidence is not yet conclusive.

262 Serotonin - A Chemical Messenger Between All Types of Living Cells

inflammatory sites, diabetics, and mild anemia [37, 80].

thrombo-inflammation during stroke and infarct progression.

compared to wild-type animals [14].

**thrombosis and stroke**

5-HT is an ancient molecule that is better known for its functions in the brain than in the periphery. However, literature describing the contribution of peripheral 5-HT, including platelet 5-HT, is rapidly growing. It became evident that platelet 5-HT has a complex role involving many bidirectional interactions with tissue microenvironment to regulate platelet and immune cell functions. SSRI treatment in animal models appears to improve thrombotic and inflammatory diseases. Further fundamental and preclinical studies are needed for a better understanding of platelet 5-HT functions in humans. In conclusion, targeting thromboimmune-modulatory functions of platelet serotonin may provide new important therapeutic approaches.

## **Author details**

Elmina Mammadova-Bach<sup>1</sup> , Maximilian Mauler<sup>2</sup> , Attila Braun<sup>1</sup> and Daniel Duerschmied2 \*

\*Address all correspondence to: daniel.duerschmied@universitaets-herzzentrum.de

1 Department of Experimental Biomedicine, University Hospital and Rudolf Virchow Center, Wuerzburg, Germany

2 Department of Cardiology and Angiology I, Heart Center, Faculty of Medicine, University of Freiburg, Germany

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## **Production and Function of Serotonin in Cardiac Cells**

Joachim Neumann, Britt Hofmann and Ulrich Gergs

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69111

#### **Abstract**

Serotonin [5-hydroxy-tryptamine (5-HT)] exerts a number of effects in the mammalian heart: increase in heart rate, increase in force of contraction, fibrosis of cardiac valves, coronary constriction, arrhythmias and thrombosis. These effects are, in part, mediated by 5-HT-receptors, in part, directly by 5-HT action on intracellular proteins. In the beginning, 5-HT was thought to be only produced in the gut and then transported into the heart via platelets, because platelets can take up 5-HT in the gut and enter the capillaries and thus the mammalian heart. 5-HT is to a large extent metabolized in the liver and excreted via the urine. Here, we will also overview data that argue for additional pathways, namely production and degradation of 5-HT in the cells of the heart itself.

**Keywords:** heart, human atrium, serotonin, 5-hydroxytryptophan, MAO

## **1. Introduction**

Practically, all physiological systems of the mammalian body have been reported to be affected by 5-hydroxy-tryptamine (5-HT). Prominently affected systems are the central nerve system and the peripheral nerve system but 5-HT also plays a complex role in the gut, the liver and e.g. spleen. However, 5-HT also seems to have profound (patho)-physiological roles in the heart. Some drugs that are devised to treat non-cardiac diseases alter the level of 5-HT in the heart or act as agonists/antagonists on one or more of the 5-HT-receptors in the mammalian heart. Finally, there is evidence that in cardiovascular diseases 5-HT itself can affect the heart in a compensatory or detrimental way. Some newer aspects of the action and generation of 5-HT in the mammalian heart with special emphasis on the human heart will be addressed here. Finally, gaps in our knowledge, conflicting views, some challenging hypotheses and suggestions for further research will be put forward.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **2. Historical aspects**

The history of 5-HT goes back in time. Some write that the first hint of 5-HT's existence can be traced back to the work of Otto Weiss (1896, Göttingen, Germany) [1], who noted that serum in contrast to plasma exerted vasoconstrictory responses in dogs, a finding which one could now explained by high (serum) and low (plasma) levels of 5-HT in the samples of Weiss [2].

Enterochromaffin cells were first described in the lining of the gastrointestinal tract at the end of the nineteenth century (Charkow in ancient Imperial Russia) [3]. Using various staining reactions, these enterochromaffin cells were found in the gut of many species [4]. Early work described an increase in blood pressure in one rabbit with extracts from human carcinoid tumors (tumors derived from enterochromaffin cells [5]). Stimulated by these findings, Vittorio Erspamer in Italy (working in Pavia, Rome and later in Bari, Italy) studied acetone extracts of mucosal intestinal strips from animals (mainly rabbits) for decades. In an early paper, he presented an original recording for a positive inotropic effect (PIE) of "enteramine" (the putative active ingredient of his acetone extract) on the frog heart, conceivably the first inotropic effect reported for 5-HT [6]. Later he purified the extract further and reported the presence of a putative indolalkylamine that increased force in isolated hearts of various kinds of molluscs [7]. His efforts culminated in identifying his "enteramine" as 5-hydroxy-tryptamine [8]. 5-HT was, independently from Erspamer, studied by the group of Irvine H. Page in the USA (Cleveland, Ohio) and given its current name "serotonin" [9]. The American authors were investigating naturally occurring vasoconstrictory compounds (vasoconstriction being tested in rabbit ear arteries) in serum of humans in order to find a putative cause of peripheral arterial hypertension in humans. As a first step, they partially purified a vasoconstrictory compound from clotted serum of beef [10] and later this vasoconstrictor (their "serotonin") by chemical synthesis was identified as 5-hydroxy-tryptamine [11]. Synthetic 5-HT was shown in early work to act on blood pressure in animals (cats and rabbits [12]) and hypertensive patients [12]. These authors noted in patients an initial fall in blood pressure (measured in brachial arteries, absent in the presence of atropine) followed by a modest increase in blood pressure (up to 20 mm Hg increase). Patients injected with 5-HT complained of nausea, tightness of the breast, and dizziness, but the pulse rate was not reported [12]. A clear positive chronotropic effect (PCE) of 5-HT by venous injection in humans was published some years later [13, 14]. Initial attempts using biological tests failed [6] to detect 5-HT in the heart, but several decades later, the presence of 5-HT in the mammalian heart (hamster, human heart) has been proven (using more sensitive methods (fluorescence): e.g. [15]).

## **3. Sources of cardiac 5-HT**

Ninety percent of 5-HT in the body is present in enterochromaffin cells in the gut, 5% in platelets, and about 2% in brain [16]. 5-HT in the blood is mainly assumed to be synthesized in enterochromaffin cells of the gastrointestinal tract [17, 18]. 5-HT is released from these enterochromaffin cells and is taken up rapidly by thrombocytes (review: [19, 20]). Alternatively, 5-HT enters the portal vein and passes into liver cells where it is rapidly degraded by monoamine oxidase-A (MAO-A) (see below) and its metabolites leave the body via the urine. Thus, platelets are commonly thought to be the main source of 5-HT that reaches the mammalian heart. Many cell types like mast cells also contain 5-HT (see below) and small numbers of mast cells are present in the mammalian heart [19]. Many immune cells contain 5-HT and are also found in small numbers in the heart. Newer data (using more sensitive analytical methods: HPLC, GC, MS) cast some doubt on this classical concept (see below). Thus, 5-HT was detected not only in hamster heart and human heart [15] but also in mouse heart [21], rat neonatal cardiomyocytes [22], and adult mouse cardiomyocytes [23]. The content of 5-HT in the hamster heart was reduced by the treatment of the living hamster with pargyline, an inhibitor of MAO-A [15] suggesting degradation of 5-HT in the heart via MAO-A. Moreover, the level of 5-HT in the hamster heart was not reduced by injecting in the living hamster a dose of 6-hydroxy-dopamine, sufficiently high to reduce cardiac norepinephrine levels, suggesting that cardiac 5-HT is not derived to a measurable extent from the neuronal cells in the hamster heart [15]. Compound 48/80, a substance that is known to release 5-HT from cells in other organ systems, increased force of contraction in isolated atrial preparation of 5-HT<sup>4</sup> -receptor overexpressing mice, indicating that releasable 5-HT is present in these cardiac preparations, but leaving open the question of its cellular origin [24].

**2. Historical aspects**

272 Serotonin - A Chemical Messenger Between All Types of Living Cells

The history of 5-HT goes back in time. Some write that the first hint of 5-HT's existence can be traced back to the work of Otto Weiss (1896, Göttingen, Germany) [1], who noted that serum in contrast to plasma exerted vasoconstrictory responses in dogs, a finding which one could now explained by high (serum) and low (plasma) levels of 5-HT in the samples of Weiss [2]. Enterochromaffin cells were first described in the lining of the gastrointestinal tract at the end of the nineteenth century (Charkow in ancient Imperial Russia) [3]. Using various staining reactions, these enterochromaffin cells were found in the gut of many species [4]. Early work described an increase in blood pressure in one rabbit with extracts from human carcinoid tumors (tumors derived from enterochromaffin cells [5]). Stimulated by these findings, Vittorio Erspamer in Italy (working in Pavia, Rome and later in Bari, Italy) studied acetone extracts of mucosal intestinal strips from animals (mainly rabbits) for decades. In an early paper, he presented an original recording for a positive inotropic effect (PIE) of "enteramine" (the putative active ingredient of his acetone extract) on the frog heart, conceivably the first inotropic effect reported for 5-HT [6]. Later he purified the extract further and reported the presence of a putative indolalkylamine that increased force in isolated hearts of various kinds of molluscs [7]. His efforts culminated in identifying his "enteramine" as 5-hydroxy-tryptamine [8]. 5-HT was, independently from Erspamer, studied by the group of Irvine H. Page in the USA (Cleveland, Ohio) and given its current name "serotonin" [9]. The American authors were investigating naturally occurring vasoconstrictory compounds (vasoconstriction being tested in rabbit ear arteries) in serum of humans in order to find a putative cause of peripheral arterial hypertension in humans. As a first step, they partially purified a vasoconstrictory compound from clotted serum of beef [10] and later this vasoconstrictor (their "serotonin") by chemical synthesis was identified as 5-hydroxy-tryptamine [11]. Synthetic 5-HT was shown in early work to act on blood pressure in animals (cats and rabbits [12]) and hypertensive patients [12]. These authors noted in patients an initial fall in blood pressure (measured in brachial arteries, absent in the presence of atropine) followed by a modest increase in blood pressure (up to 20 mm Hg increase). Patients injected with 5-HT complained of nausea, tightness of the breast, and dizziness, but the pulse rate was not reported [12]. A clear positive chronotropic effect (PCE) of 5-HT by venous injection in humans was published some years later [13, 14]. Initial attempts using biological tests failed [6] to detect 5-HT in the heart, but several decades later, the presence of 5-HT in the mammalian heart (hamster, human heart)

has been proven (using more sensitive methods (fluorescence): e.g. [15]).

Ninety percent of 5-HT in the body is present in enterochromaffin cells in the gut, 5% in platelets, and about 2% in brain [16]. 5-HT in the blood is mainly assumed to be synthesized in enterochromaffin cells of the gastrointestinal tract [17, 18]. 5-HT is released from these enterochromaffin cells and is taken up rapidly by thrombocytes (review: [19, 20]). Alternatively,

**3. Sources of cardiac 5-HT**

5-HT is formed from L-tryptophan by the enzyme tryptophan hydroxylase (TPH, see next paragraph). Peroral treatment of animals or humans with tryptophan (an essential amino acid) increases body concentrations of tryptophan. Uptake of tryptophan in the gut is brought about by the protein transporter enzymes SLC6A19 and SLC16A10 [25]. Protein-rich food is known to compete with these transporters and will lead to lower levels of tryptophan in the body [25]. Effects of 5-HT in the heart were usually thought to be due to 5-HT released from intact platelets. Indeed huge amounts of 5-HT can be released from activated thrombi in the heart. These thrombi are formed in atrial fibrillation and contain, of course, thrombocytes. 5-HT released from thrombi may then reach by diffusion endothelial cells. If these endothelial cells were lacking (for instance after local injury), 5-HT may act on 5-HT-receptors on the outer surface of smooth muscle cells or may reach cardiomyocytes. Conceivably, 5-HT in the plasma may reach the heart from the lungs (lung veins, left atrium, and left ventricle) and then enter the coronaries and thence affect the whole heart. In addition, 5-HT in the plasma may come from the periphery (via the right atrium and right ventricle) and exert right heart sided effects. Indeed, in tumors producing 5-HT (carcinoids, see below), such pathways are accepted to occur. Depending on the anatomic localization of the tumor, vasoconstriction would occur and should explain cases of left or right (or global) hypertrophy or contractile failure of the heart [26]. Furthermore, the 5-HT producing enzyme TPH1 (see below) was detected in pulmonary endothelial cells and could therefore generate 5-HT in the lung and it is conceivable that this 5-HT of pulmonary origin reaches the left heart via the pulmonary veins [27]:

There is precedence in the literature that a neurotransmitter like 5-HT can be formed in the heart. Here, we mention as an example noradrenaline which can act in an autocrine and paracrine fashion: there is evidence for the presence, synthesis (and degradation), and intracellular action of noradrenaline in heart muscle cells on β- [28] and α-adrenoceptors (most of the latter being detected intracellularly in the nuclear membrane, for overview [29]). We speculate that the 5-HT-receptors (which are phylogenetically assumed to be much older than adrenoceptors) might also be detectable intracellularly in cardiomyocytes when more sensitive techniques will become available (**Figure 1**). It is notoriously difficult to detect endogenous G-protein-coupled receptors with specific, highly sensitive antibodies. Progress in this regard would be highly desired. Interestingly, 5-HT can exert intracellular effects via oxidation of 5-HT in mitochondria (e.g. in mouse heart) and formation of free radicals. In that way, 5-HT

**Figure 1.** Hypothetical fates of 5-HT in the mammalian heart. Ca2+ enters the mammalian heart cell via the L-type Ca2+ channel (LTCC). This process can be enhanced by 5-HT via a cascade starting with the 5-HT<sup>4</sup> receptor (inhibitable by GR113808) occupation of which by 5-HT elevates activity of adenylyl cyclase (AC) in the sarcolemma via stimulatory G-proteins (Gs), elevates subsequent production of cAMP, and thereby activates cAMP-dependent protein kinase (PKA). PKA increases cardiac force generation and relaxation by increasing the phosphorylation state (P) of LTCC, phospholamban (PLB), and other regulatory proteins. Trigger Ca2+ initiates release of Ca2+ from the sarcoplasmic reticulum via ryanodine receptors (RYR) into the cytosol. There, Ca2+ activates myofilaments and this activation leads to increased inotropy. In diastole, Ca2+ is taken up into the sarcoplasmic reticulum via a sarcoplasmic reticulum Ca2+ ATPase (SERCA), the activity of which is enhanced due to an increased phosphorylation state of PLB. We tentatively propose that 5-HT is stored in cardiomyocytes in a hypothetical locus from which it can be released by compound 48/80 and extruded from the cardiomyocytes, possibly via OCT2 and/or OCT3 and/or PMAT (inhibitable by cortisone, decynium 22). From the outside of the cardiomyocytes, 5-HT might be pumped back into the cardiomyocyte via SERT (inhibitable by fluoxetine). 5-HT might be formed in cardiomyocytes from tryptophan (Trp) via the enzyme tryptophan hydroxylase to generate 5-hydroxytryptophan (5-HTP) and thereafter decarboxylated by AADC (inhibitable by NSD1015) to 5-HT that is degraded via MAO-A (inhibitable by tranylcypromine) and its metabolites are substrates for aldehyde dehydrogenases (ALD; disulfiram-sensitive) or xanthine oxidases (XO; allopurinol-sensitive). In addition, one can speculate that 5-HT can pass through the outer nuclear membrane via OCT and then activate the inner nuclear membrane located 5-HT<sup>4</sup> receptor which activates than AC via Gs leading to phosphorylation of substrates in the nucleus and to altered gene transcription.

can also act receptor independently, at least in high concentrations to lead to apoptosis and necrosis, at least in the mouse heart [17, 30].

latter being detected intracellularly in the nuclear membrane, for overview [29]). We speculate that the 5-HT-receptors (which are phylogenetically assumed to be much older than adrenoceptors) might also be detectable intracellularly in cardiomyocytes when more sensitive techniques will become available (**Figure 1**). It is notoriously difficult to detect endogenous G-protein-coupled receptors with specific, highly sensitive antibodies. Progress in this regard would be highly desired. Interestingly, 5-HT can exert intracellular effects via oxidation of 5-HT in mitochondria (e.g. in mouse heart) and formation of free radicals. In that way, 5-HT

274 Serotonin - A Chemical Messenger Between All Types of Living Cells

**Figure 1.** Hypothetical fates of 5-HT in the mammalian heart. Ca2+ enters the mammalian heart cell via the L-type Ca2+

GR113808) occupation of which by 5-HT elevates activity of adenylyl cyclase (AC) in the sarcolemma via stimulatory G-proteins (Gs), elevates subsequent production of cAMP, and thereby activates cAMP-dependent protein kinase (PKA). PKA increases cardiac force generation and relaxation by increasing the phosphorylation state (P) of LTCC, phospholamban (PLB), and other regulatory proteins. Trigger Ca2+ initiates release of Ca2+ from the sarcoplasmic reticulum via ryanodine receptors (RYR) into the cytosol. There, Ca2+ activates myofilaments and this activation leads to increased inotropy. In diastole, Ca2+ is taken up into the sarcoplasmic reticulum via a sarcoplasmic reticulum Ca2+ ATPase (SERCA), the activity of which is enhanced due to an increased phosphorylation state of PLB. We tentatively propose that 5-HT is stored in cardiomyocytes in a hypothetical locus from which it can be released by compound 48/80 and extruded from the cardiomyocytes, possibly via OCT2 and/or OCT3 and/or PMAT (inhibitable by cortisone, decynium 22). From the outside of the cardiomyocytes, 5-HT might be pumped back into the cardiomyocyte via SERT (inhibitable by fluoxetine). 5-HT might be formed in cardiomyocytes from tryptophan (Trp) via the enzyme tryptophan hydroxylase to generate 5-hydroxytryptophan (5-HTP) and thereafter decarboxylated by AADC (inhibitable by NSD1015) to 5-HT that is degraded via MAO-A (inhibitable by tranylcypromine) and its metabolites are substrates for aldehyde dehydrogenases (ALD; disulfiram-sensitive) or xanthine oxidases (XO; allopurinol-sensitive). In addition, one can speculate that 5-HT can pass through the outer nuclear membrane via OCT and then activate the inner nuclear

receptor which activates than AC via Gs leading to phosphorylation of substrates in the

receptor (inhibitable by

channel (LTCC). This process can be enhanced by 5-HT via a cascade starting with the 5-HT<sup>4</sup>

membrane located 5-HT<sup>4</sup>

nucleus and to altered gene transcription.

Furthermore, 5-HT can form covalent links to intracellular proteins and thence altering their functional role: transglutaminases can initiate covalent binding of 5-HT to fibrinogen, to small G-proteins, and to several other proteins present in platelets (review: [31]).

On the surface of platelets, a 5-HT2A-receptor is known to be expressed. Its activation will activate thrombosis. 5-HT is thought to enter platelets via serotonin transporter (SERT) (for review, see Refs. [32, 33]). Within the platelet, 5-HT is either degraded via oxidation or transported via Vesicular monoamine transporter (VMAT) into vesicles in platelets which will store (VMAT1 and VMAT2 are also present in other non-neuronal cells, saliva cells [34], and renal tubular cells [35]; it is worthwhile to try to detect VMAT in cardiomyocytes, which has apparently not yet been reported) 5-HT and protect 5-HT from degradation. Upon an appropriate stimulus, 5-HT containing vesicles can reach the outer membrane of the platelets, fuse, and release 5-HT out of the platelets into the plasma. It has been shown that high blood platelet levels of 5-HT can serotonylate the protein rab4, which then inhibits the shift of SERT from the sublemmal space into the plasmalemma and hence quantitatively reduces its own uptake via SERT into platelets (which has been suggested to be of pathological relevance). There, 5-HT can act via the above-mentioned receptors in an autocrine or paracrine way [31, 36, 37]. Moreover, it stands to reason that 5-HT produced within cardiomyocytes might also exit the cardiomyocyte wherein it was formed (speculatively using uptake 1 or 2 and/or SERT, see below) to act in an autocrine or paracrine fashion at least under pathophysiological conditions.

5-HT can be compartmentalized in relevant cells: in peritoneal mast cells from rats, 5-HT was not only present in storage vesicles but also in the nucleus [38]. It is possible that mast cells produce relevant amounts of 5-HT because they contain their own TPH1 [19]. Compound 48/80 can release 5-HT from mast cells and PCPA (an inhibitor of TPH activity) can reduce the levels of 5-HT in the cytosol of mast cells but not the nucleus of mast cells [38]. Likewise, clorgyline (a MAO-A inhibitor) and fluoxetine (a SERT-inhibitor) could decrease the cytosolic but surprisingly also the nuclear amounts of 5-HT in mast cells [38]. These data argue for the existence of functionally distinct subcellular pools of 5-HT. Similar studies in cardiomyocytes are apparently lacking and are keenly awaited.

5-HT is not only produced in the mammalian body but also in the plant kingdom and is found in foodstuff such as nuts, bananas, oranges, coffee, and peaches [39]. This might be an additional source of 5-HT reaching the heart. Finally, there are data that in the lumen of the gut, bacteria form 5-HT, which may be absorbed and may also reach the heart [40]. Uptake via the intestine could be achieved by SERT which present and active in epithelial cell of the gut (in crypts of intestine, rat [41]).

## **4. Enzymes for synthesis of 5-HT in the heart**

The isoform TPH1 is mainly expressed in the gut (but also in the pineal gland [21]), whereas TPH2 is mainly found in the CNS (but also in enteric nerve cells [21, 36]). Knockout of TPH1 reduced cardiac (adult mouse) 5-HT levels to about 10% of wild-type levels, indicating a relevant production of 5-HT in the heart [21]. Some TPH1-knockout mice exhibited signs of left ventricular systolic failure without histologically detectable fibrosis [21] suggesting beneficial effects of the presence of 5-HT for cardiac function. In RNA from HL-1 cells and neonatal rat heart cells in Northern blots, TPH1 was detectable and TPH2 was missing [22] (similarly in adult hamster heart [42]). In RNA prepared from whole adult mouse hearts, TPH1 but not TPH2 was detected by PCR [43, 44]. Western blotting revealed low levels of TPH1 in mouse and rat adult heart homogenates. Fittingly, with the same antibody under similar conditions, no signal for TPH1 was noted in TPH1-knockout mouse hearts [44]. However, the localization of TPH is uncertain: in rat hearts in immunohistology, TPH1 was located only in cardiac mast cells, but in mouse heart no signal was noted in cardiac mast cells [44]. This might be explained by the antibody used, as others detected in immunohistology TPH in mouse cardiomyocytes as well as human atrial cardiomyocytes [45]. Amino acid decarboxylase (AADC, which is identical to dopamine decarboxylase [46]) on mRNA level was detected in heart (by PCR in neonatal rat cardiomyocytes but not in non-cardiomyocytes from neonatal rat hearts [22]). Subsequently, the activity of AADC will result in the generation of 5-HT. In apparent contrast to neonatal cardiomyocytes, AADC was detected via Western blotting in endothelial cells but not in cardiomyocytes of adult rat hearts and adult mouse hearts [44]. Whether this is due to age differences or lack of translation of RNA or too low protein levels of AADC or the features of the antibody used is still an open question. Others, however, noted in immunohistology the presence of AADC also in cardiomyocytes using slices from adult mouse heart and human right atrium [45]. Moreover, addition of 5-hydroxytryptophan (5-HTP, the direct precursor of 5-HT) enhanced 5-HT levels in these isolated cardiac mouse myocytes [23]. Interestingly, 5-HTP can exert functional effects in the heart. More specifically, in electrically driven left atrial preparations of transgenic mice (which overexpress the human 5-HT4 -receptor in the heart, see below), 5-HTP exerted time- and concentrationdependent positive inotropic effects (PIE) or increased the beating rate [positive chronotropic effect (PCE)] of right atrial preparations [45, 47]. Injection of 5-HTP into intact mice led to an increase of 5-HTP but allows of 5-HT in the cardiac tissue of mice [44]. Injection of benzerazide in intact mice, in contrast, reduced the cardiac levels of 5-HT [44]. Likewise, 5-HTP exerted a PIE in atrium from 5-HT4 overexpressing mice or human right atrial preparations [47] (**Figure 2**). These contractile effects were blocked by NSD-1015 (**Figure 2**), suggesting they result from the enzymatic formation of 5-HT in these mouse or more importantly human cardiac preparations. Similarly, injection of 5-HPT in living whole mice (and in isolated buffer perfused hearts) led to a measurable increase in the cardiac content of 5-HT [44], and the effect was blocked by injection of benzerazide (an AADC inhibitor, used in treatment of Parkinson's disease). The authors posited that 5-HTP derived from platelets led to 5-HT synthesis in the heart [44]. In earlier work in the kidney, infusion of 5-HTP led to vasoconstriction which was reversed (or block by pretreatment) with carbidopa (a dopa decarboxylase inhibitor [48]). Infusion of 5-HTP led to increased levels of 5-HT in the renal venous effluent and in urine and these elevations of 5-HT returned to baseline values if carbidopa was additionally applied. These data are consistent with renal formation of 5-HT from 5-HTP via dopa decarboxylase activity [48].

A drawback of these pharmacological experiments is always that their interpretation is highly dependent upon the specificity of the inhibitory drugs used. It would be useful to refute or confirm these pharmacological experiments by studying cardiomyocytes from mice with

production of 5-HT in the heart [21]. Some TPH1-knockout mice exhibited signs of left ventricular systolic failure without histologically detectable fibrosis [21] suggesting beneficial effects of the presence of 5-HT for cardiac function. In RNA from HL-1 cells and neonatal rat heart cells in Northern blots, TPH1 was detectable and TPH2 was missing [22] (similarly in adult hamster heart [42]). In RNA prepared from whole adult mouse hearts, TPH1 but not TPH2 was detected by PCR [43, 44]. Western blotting revealed low levels of TPH1 in mouse and rat adult heart homogenates. Fittingly, with the same antibody under similar conditions, no signal for TPH1 was noted in TPH1-knockout mouse hearts [44]. However, the localization of TPH is uncertain: in rat hearts in immunohistology, TPH1 was located only in cardiac mast cells, but in mouse heart no signal was noted in cardiac mast cells [44]. This might be explained by the antibody used, as others detected in immunohistology TPH in mouse cardiomyocytes as well as human atrial cardiomyocytes [45]. Amino acid decarboxylase (AADC, which is identical to dopamine decarboxylase [46]) on mRNA level was detected in heart (by PCR in neonatal rat cardiomyocytes but not in non-cardiomyocytes from neonatal rat hearts [22]). Subsequently, the activity of AADC will result in the generation of 5-HT. In apparent contrast to neonatal cardiomyocytes, AADC was detected via Western blotting in endothelial cells but not in cardiomyocytes of adult rat hearts and adult mouse hearts [44]. Whether this is due to age differences or lack of translation of RNA or too low protein levels of AADC or the features of the antibody used is still an open question. Others, however, noted in immunohistology the presence of AADC also in cardiomyocytes using slices from adult mouse heart and human right atrium [45]. Moreover, addition of 5-hydroxytryptophan (5-HTP, the direct precursor of 5-HT) enhanced 5-HT levels in these isolated cardiac mouse myocytes [23]. Interestingly, 5-HTP can exert functional effects in the heart. More specifically, in electrically driven left atrial preparations of transgenic mice (which overex-

276 Serotonin - A Chemical Messenger Between All Types of Living Cells


overexpressing mice or human right atrial preparations [47] (**Figure 2**).

dependent positive inotropic effects (PIE) or increased the beating rate [positive chronotropic effect (PCE)] of right atrial preparations [45, 47]. Injection of 5-HTP into intact mice led to an increase of 5-HTP but allows of 5-HT in the cardiac tissue of mice [44]. Injection of benzerazide in intact mice, in contrast, reduced the cardiac levels of 5-HT [44]. Likewise, 5-HTP exerted a PIE

These contractile effects were blocked by NSD-1015 (**Figure 2**), suggesting they result from the enzymatic formation of 5-HT in these mouse or more importantly human cardiac preparations. Similarly, injection of 5-HPT in living whole mice (and in isolated buffer perfused hearts) led to a measurable increase in the cardiac content of 5-HT [44], and the effect was blocked by injection of benzerazide (an AADC inhibitor, used in treatment of Parkinson's disease). The authors posited that 5-HTP derived from platelets led to 5-HT synthesis in the heart [44]. In earlier work in the kidney, infusion of 5-HTP led to vasoconstriction which was reversed (or block by pretreatment) with carbidopa (a dopa decarboxylase inhibitor [48]). Infusion of 5-HTP led to increased levels of 5-HT in the renal venous effluent and in urine and these elevations of 5-HT returned to baseline values if carbidopa was additionally applied. These data are consistent with renal formation of

A drawback of these pharmacological experiments is always that their interpretation is highly dependent upon the specificity of the inhibitory drugs used. It would be useful to refute or confirm these pharmacological experiments by studying cardiomyocytes from mice with

press the human 5-HT4

in atrium from 5-HT4

5-HT from 5-HTP via dopa decarboxylase activity [48].

**Figure 2.** Typical original recording of isolated electrically stimulated trabeculae from a human atrium. The ordinate indicates force of contraction in milli Newton (mN), and the abscissae indicate time in minutes exemplified by scale bars. Of note, 5-hydroxytryptophan increases force of contraction (lane 1) and this effect was gone in the presence of NSD 1015, suggesting that 5-HT formation is necessary. NSD always exerted a small contractile effect of unknown origin. Isoproterenol, an unselective β-adrenoceptor agonist, was used as positive control.

cardiac-specific knockout of TPH1 and/or aromatic L-amino acid decarboxylase (AADC). Data for the local generation of 5-HT in peripheral arterial tissue (rat aorta, isolated human arterial coronary smooth muscle cells) are available and argue for local production and release independently of plasma or platelet levels of 5-HT [49, 50]. When one studied cardiac tissue in adult human autopsies, in 72 or 80% of neurons within cardiac ganglia, tryptophan hydroxylase or dopa-decarboxylase immune reactivity was found, respectively, using commercial antibodies [51]. These levels were reduced in the presence of p-chlorophenylalanine (PCPA, an irreversible inhibitor of tryptophan hydroxylase activity) or 3-hydroxy-benzylhydrazine (NSD-1015), an inhibitor of aromatic L-amino acid decarboxylase (AADC [23]). Moreover, addition of 5-hydroxytryptophan (the direct precursor of 5-HT) enhanced 5-HT level in these isolated adult cardiac mouse myocytes [23].

## **5. Enzymes for degradation of 5-HT in the heart**

As mentioned above in non-cardiac tissues, 5-HT is probably degraded by MAO-A. The same probably holds true for adult cardiac myocytes: levels of 5-HT were greatly elevated in the presence of tranylcypromine (clinically used as an antidepressant, inhibiting both MAO-A and MAO-B [23]) or in the presence of clorgylin (a MAO-A inhibitor [23]) but not by deprenyl (clinically used to treat Parkinson's disease, because it inhibits MAO-B [23]). MAO is especially active in gut, liver, and serotoninergic nerve cells. However, species differences exist. MAO-B is much less active in rat heart than MAO-A, and in human heart MAO-A and MAO-B are equally active [52]. The total activity of MAO is 100 times higher in the rat than in the wildtype mouse heart [53]. Likewise, MAO-B is mainly active in mouse heart, compared to MAO-A [54]. Hence, knockout of MAO-A in mice is probably not all that physiologically relevant for the human situation. At least in rat using ligand-binding experiments, even the regional cardiac distribution of MAO was found to be regionally different: there is a fivefold difference in MAO-A levels in parts of the ventricle of rat hearts [55]. The study of 5-HT levels in human cardiac tissue (preferably in cardiomyocytes, which is technically not highly reproducible, or stem cells, which have their own pitfalls) in the absence or presence of selective MAO inhibitors or genetic reduction of MAO levels in human cardiomyocytes are awaited with eagerness. Moreover, 5-HT can also be metabolized by the acrylalkylamine-N-acetyltransferase (present in the heart [56]). 5-HT can be degraded by MAO-A or MAO-B to 5-hydroxy-indole-acetaldehyde and by action of unspecific dehydrogenases and/or alcohol dehydrogenase 2 finally to 5-hydroxy-indole-acetic acid which leaves the body via the kidneys, and its concentration has been used in patients to monitor the presence of 5-HT-producing carcinoid tumors [26, 57]. Based on knockout experiments, 5-HT in the mouse is mainly degraded by MAO-A not MAO-B [58]. Inhibition of the activity of MAO by tranylcypromine potentiated the PIE of 5-HT in atrial preparations of 5-HT<sup>4</sup> -receptor overexpressing mice [24]. 5-HT can also be metabolized by an indoleamine 2,3-dioxygenase (the rate limiting step in this pathway, with immunohistology detected in cardiomyocytes and active in mouse heart [59]) to kynurenine (present in mouse heart [60]). Indoleamine 2,3-dioxygenase (IDO) can be induced in infectious diseases like cardiac viral myocarditis [59]. Studying knockout mice for this enzyme supported an important role of IDO in acute viral myocarditis [59]. Furthermore, 5-HT can be metabolized even into melatonin (recent publication on levels of melatonin in rat heart: [61]) by hydroxyindole O-methyltransferase (enzyme present and active in mammalian heart: [56]) in the heart and this melatonin may play a role in protection against cardiac ischemia [62].

## **6. Uptake 1 of 5-HT in the heart**

tissue in adult human autopsies, in 72 or 80% of neurons within cardiac ganglia, tryptophan hydroxylase or dopa-decarboxylase immune reactivity was found, respectively, using commercial antibodies [51]. These levels were reduced in the presence of p-chlorophenylalanine (PCPA, an irreversible inhibitor of tryptophan hydroxylase activity) or 3-hydroxy-benzylhydrazine (NSD-1015), an inhibitor of aromatic L-amino acid decarboxylase (AADC [23]). Moreover, addition of 5-hydroxytryptophan (the direct precursor of 5-HT) enhanced 5-HT

As mentioned above in non-cardiac tissues, 5-HT is probably degraded by MAO-A. The same probably holds true for adult cardiac myocytes: levels of 5-HT were greatly elevated in the presence of tranylcypromine (clinically used as an antidepressant, inhibiting both MAO-A and MAO-B [23]) or in the presence of clorgylin (a MAO-A inhibitor [23]) but not by deprenyl (clinically used to treat Parkinson's disease, because it inhibits MAO-B [23]). MAO is especially active in gut, liver, and serotoninergic nerve cells. However, species differences exist. MAO-B is much less active in rat heart than MAO-A, and in human heart MAO-A and MAO-B are equally active [52]. The total activity of MAO is 100 times higher in the rat than in the wildtype mouse heart [53]. Likewise, MAO-B is mainly active in mouse heart, compared to MAO-A [54]. Hence, knockout of MAO-A in mice is probably not all that physiologically relevant for the human situation. At least in rat using ligand-binding experiments, even the regional cardiac distribution of MAO was found to be regionally different: there is a fivefold difference in MAO-A levels in parts of the ventricle of rat hearts [55]. The study of 5-HT levels in human cardiac tissue (preferably in cardiomyocytes, which is technically not highly reproducible, or stem cells, which have their own pitfalls) in the absence or presence of selective MAO inhibitors or genetic reduction of MAO levels in human cardiomyocytes are awaited with eagerness. Moreover, 5-HT can also be metabolized by the acrylalkylamine-N-acetyltransferase (present in the heart [56]). 5-HT can be degraded by MAO-A or MAO-B to 5-hydroxy-indole-acetaldehyde and by action of unspecific dehydrogenases and/or alcohol dehydrogenase 2 finally to 5-hydroxy-indole-acetic acid which leaves the body via the kidneys, and its concentration has been used in patients to monitor the presence of 5-HT-producing carcinoid tumors [26, 57]. Based on knockout experiments, 5-HT in the mouse is mainly degraded by MAO-A not MAO-B [58]. Inhibition of the activity of MAO by tranylcypromine potentiated the PIE of 5-HT

lized by an indoleamine 2,3-dioxygenase (the rate limiting step in this pathway, with immunohistology detected in cardiomyocytes and active in mouse heart [59]) to kynurenine (present in mouse heart [60]). Indoleamine 2,3-dioxygenase (IDO) can be induced in infectious diseases like cardiac viral myocarditis [59]. Studying knockout mice for this enzyme supported an important role of IDO in acute viral myocarditis [59]. Furthermore, 5-HT can be metabolized even into melatonin (recent publication on levels of melatonin in rat heart: [61]) by hydroxyindole O-methyltransferase (enzyme present and active in mammalian heart: [56]) in the heart

and this melatonin may play a role in protection against cardiac ischemia [62].


level in these isolated adult cardiac mouse myocytes [23].

278 Serotonin - A Chemical Messenger Between All Types of Living Cells

**5. Enzymes for degradation of 5-HT in the heart**

in atrial preparations of 5-HT<sup>4</sup>

Classically, re-uptake of 5-HT (but also of neurotransmitters like histamine, noradrenaline, or dopamine) into nerve cells has been called uptake 1 and is assumed to be mediated for 5-HT by SERT (and by dopamine transporter (DAT) for dopamine, as well as by noradrenaline transporter (NAT = NET) for noradrenaline, however, their specificity of transport shows some overlap, which may explain compensations in knockout mouse models [63]). SERT is blocked by some antidepressant drugs like fluoxetine. Likewise, genetic deletion of SERT (total knockout) led to a decrease of 5-HT levels from 29 to 0.4 μM in whole blood, probably as a result of lack of reuptake via SERT into platelets, clearly indicating that SERT is not only active in the central nervous system but also in the periphery. Uptake 1 is energy dependent because it acts against a neurotransmitter gradient. Uptake 1 can also be blocked by cocaine (which is, however, unspecific because it blocks at least also NAT and DAT). Interestingly, the EC50 of 5-HT in the presence of cocaine for the PIE is much smaller in human isolated atrial preparations (39 nM) than in the absence of cocaine (230 nM: [64]): this could mean that cocaine inhibits the uptake 1 into nerve cells or that it inhibits reuptake of 5-HT into cardiomyocytes by inhibiting SERT in cardiomyocytes. At low concentrations of 5-HT (50 nM), about 70% of 5-HT is taken up via uptake 1 (the remainder via uptake 2 see below). SERT has been found in the lung (endothelial cells and smooth muscle cells: [65]; rat aorta: [49]) on cardiac valves (rat: [66], dog: [67], human valvular tissue: [43]), conduction system of the mouse, mouse cardiomyocytes, and mouse cardiac endothelial cells [68–70]. At least in fetal cardiomyocytes, SERT was seen in immunohistology [71]. Some detected SERT in the endocardium and endothelium of coronary arterial cells and capillaries, while they failed to detect SERT in cardiomyocytes from adult mice [43]. Others using different experimental conditions detected SERT in cardiomyocytes from adult mouse heart and human right atrium [45]. Functional evidence for the activity and therefore presence of SERT are also available: 5-HT, applied in cell culture of adult rat ventricular myocytes induced cellular hypertrophy and this hypertrophy was attenuated by imipramine [72, 73]. This is functional proof that cardiomyocytes can take up 5-HT and might argue for an involvement of SERT in this process. Knockout of SERT in mice was accompanied in whole blood by an about 10-fold reduction of 5-HT levels [43]. Interestingly, adult mice with global knockout of SERT showed left ventricular dilatation and systolic heart failure (decreased fractional shortening in echocardiography) which was accompanied and possibly caused, in part, by cardiac ventricular interstitial fibrosis as well as cardiac valve fibrosis effects present also on 5-HT1B-receptor knockout mice and hence not 5-HT1B-receptor mediated [43]. SERT is reversible in its transporter function: during ischemia, in the presence of tyramine of amphetamines, intracellular 5-HT can leave mouse cardiomyocytes [68]. The functional role of SERT in the heart is evident from the observation that fluoxetine can shift the concentration response curve for the positive inotropic effect of 5-HT to lower concentrations of 5-HT in the left atrium of mice overexpressing the 5-HT<sup>4</sup> -receptor [24]. A prominent pathway is initiated by the enzyme indoleamine-2,3-dioxygenase (IDO, which opens and destroys the indole ring system of tryptophan), which feeds into the so-called kynurenine pathway (review: [19]).

## **7. Uptake 2 of 5-HT in the heart**

Uptake of neurotransmitters (like 5-HT) into non-neuronal cells (such as smooth muscle cells, fibroblasts, endothelial cells, or cardiomyocytes—have been called "uptake 2") and is assumed to be mediated by proteins such as OCT1, OCT2, OCT3, and PMAT. Uptake 2 is not energy dependent because it follows a neurotransmitter gradient. Another difference between uptake 1 and 2 relies on the fact that uptake 2 is much less specific for 5-HT than uptake 1. Usually, proteins that comprise uptake 2 will also transport other neurotransmitters like dopamine and noradrenaline [63]. Uptake 2 is usually inhibited by cortisone (but also by synthetic dexamethasone, by aldosterone, and by budesonide) via unknown mechanisms and divergent specificity for OCT1-3 and PMAT [63, 74]) and more specifically by decynium 22 [57, 63]. At higher concentrations of 5-HT (10 μM), it is mainly transported via uptake 2 (in synaptosomes, regarding decynium 22 as uptake 2-specific [57]). In the CNS, proteins responsible for uptake 2 have been detected not only in nerve cells but also in non-nerve cells (glial cells [63]). Uptake 2 is functionally relevant in the heart because decynium 22 affects the concentration response curve of 5-HT on force of contraction in isolated atrium of 5-HT<sup>4</sup> -receptor overexpressing mice [24]. OCT2, OCT3, and PMAT have been detected by immunohistology in mouse or human cardiomyocytes [45] and by immunofluorescence (OCT1, OCT3) in the human heart [75].

## **8. Inotropic effects of 5-HT in the heart, species differences**

A positive inotropic effect (PIE) of 5-HT was described in the heart of many mammalian species. More specifically, a PIE was described in cardiac preparations from cats, guinea pigs, dogs, pigs, and rats [76–80]. The PIE in cats is indirectly mediated via release of endogenous noradrenaline [81]. The PIE in the same species can be region dependent: for instance, in rats, a PIE in left atrium but not in papillary muscle was reported [82]. Similarly, in human atrial but not ventricular preparations, 5-HT exerted a PIE [83, 84]. Later, it was noted that in ventricular preparations from patient in end-stage heart failure a noteworthy effect of 5-HT was detectable and this effect was more pronounced in the presence of the phosphodiesterase inhibitor 3-isobutyl-1methylxanthine (IBMX) [85]. Interestingly, similar findings were reported in pigs: only in the presence of IBMX in ventricular preparations of pigs, a PIE to 5-HT could be noticed [86] suggesting that the low number of 5-HT<sup>4</sup> -receptors was unable to raise cyclic-3',5'-adenosine monophosphate (cAMP) levels to inotropically relevant levels in the presence of substantial endogenous unopposed phosphodiesterase activity in ventricular preparations of humans and pigs. In isolated paced left atrial preparations of wild-type mice, no PIE in the absence [87] or presence (Käufler, Gergs, Neumann, unpublished observations, 2017) of 100 μM IBMX to 5-HT (1 nM–1 μM) was, however, observed, underscoring species differences. The EC50 value for the PIE of 5-HT in isolated preparations from human right atrium was between 309 and 230 nM [80]. In mouse adult cardiomyocytes, the 5-HT level was estimated to amount to 2.9 pmol/mg protein [23]. Concentrations of 5-HT in isolated samples from human hearts (freshly frozen, after autopsy, from the right atrium, from papillary muscles) were reported from 0.08 to 0.4 μg/g [15], recalculated as about 0.45 to 2.3 μM. Such differences might be due to contamination with platelets (for very high values) or postmortal degradation (for low levels). Assuming a homogenous distribution of 5-HT in isolated mouse adult cardiomyocytes, intracellular concentrations of 200 nM for 5-HT have been calculated [23]. These concentrations are well in the range of EC50 values for the 5-HT-receptors like those responsible for inotropy in some mammalian species including humans [20, 88]. At high concentrations of 5-HT for prolonged times in the organ bath, a second negative inotropic effect of 5-HT was noted, which was alternatively explained as desensitization by activation of phosphodiesterases [89]. Homologous desensitization in the isolated atrium (also in left ventricle of the living animal) can be clearly shown for the PIE effect of 5-HT in 5-HT<sup>4</sup> -receptor overexpressing mice [90, 91] like in isolated human cardiac preparations (review: [92]).

## **9. Chronotropic and proarrhythmic effects of 5-HT in the heart**

**7. Uptake 2 of 5-HT in the heart**

280 Serotonin - A Chemical Messenger Between All Types of Living Cells

human heart [75].

Uptake of neurotransmitters (like 5-HT) into non-neuronal cells (such as smooth muscle cells, fibroblasts, endothelial cells, or cardiomyocytes—have been called "uptake 2") and is assumed to be mediated by proteins such as OCT1, OCT2, OCT3, and PMAT. Uptake 2 is not energy dependent because it follows a neurotransmitter gradient. Another difference between uptake 1 and 2 relies on the fact that uptake 2 is much less specific for 5-HT than uptake 1. Usually, proteins that comprise uptake 2 will also transport other neurotransmitters like dopamine and noradrenaline [63]. Uptake 2 is usually inhibited by cortisone (but also by synthetic dexamethasone, by aldosterone, and by budesonide) via unknown mechanisms and divergent specificity for OCT1-3 and PMAT [63, 74]) and more specifically by decynium 22 [57, 63]. At higher concentrations of 5-HT (10 μM), it is mainly transported via uptake 2 (in synaptosomes, regarding decynium 22 as uptake 2-specific [57]). In the CNS, proteins responsible for uptake 2 have been detected not only in nerve cells but also in non-nerve cells (glial cells [63]). Uptake 2 is functionally relevant in the heart because decynium 22 affects the con-

centration response curve of 5-HT on force of contraction in isolated atrium of 5-HT<sup>4</sup>

**8. Inotropic effects of 5-HT in the heart, species differences**

5-HT could be noticed [86] suggesting that the low number of 5-HT<sup>4</sup>

overexpressing mice [24]. OCT2, OCT3, and PMAT have been detected by immunohistology in mouse or human cardiomyocytes [45] and by immunofluorescence (OCT1, OCT3) in the

A positive inotropic effect (PIE) of 5-HT was described in the heart of many mammalian species. More specifically, a PIE was described in cardiac preparations from cats, guinea pigs, dogs, pigs, and rats [76–80]. The PIE in cats is indirectly mediated via release of endogenous noradrenaline [81]. The PIE in the same species can be region dependent: for instance, in rats, a PIE in left atrium but not in papillary muscle was reported [82]. Similarly, in human atrial but not ventricular preparations, 5-HT exerted a PIE [83, 84]. Later, it was noted that in ventricular preparations from patient in end-stage heart failure a noteworthy effect of 5-HT was detectable and this effect was more pronounced in the presence of the phosphodiesterase inhibitor 3-isobutyl-1methylxanthine (IBMX) [85]. Interestingly, similar findings were reported in pigs: only in the presence of IBMX in ventricular preparations of pigs, a PIE to

raise cyclic-3',5'-adenosine monophosphate (cAMP) levels to inotropically relevant levels in the presence of substantial endogenous unopposed phosphodiesterase activity in ventricular preparations of humans and pigs. In isolated paced left atrial preparations of wild-type mice, no PIE in the absence [87] or presence (Käufler, Gergs, Neumann, unpublished observations, 2017) of 100 μM IBMX to 5-HT (1 nM–1 μM) was, however, observed, underscoring species differences. The EC50 value for the PIE of 5-HT in isolated preparations from human right atrium was between 309 and 230 nM [80]. In mouse adult cardiomyocytes, the 5-HT level was estimated to amount to 2.9 pmol/mg protein [23]. Concentrations of 5-HT in isolated samples from human hearts (freshly frozen, after autopsy, from the right atrium, from papillary muscles) were reported from 0.08 to 0.4 μg/g [15], recalculated as about 0.45 to 2.3 μM. Such



Positive chronotropic effects of 5-HT were noted in isolated atrial preparations of rats [93], cats [94], pigs [95] as well as guinea pigs [96] and awake humans [14]. Even bradycardia can be elicited by 5-HT via the von Bezold-Jarisch reflex [97]. In whole pigs and isolated pig cardiac preparations, 5-HT increased the heart rate via 5-HT<sup>4</sup> -receptors [86, 92, 98]. It is assumed that the effect is brought about by 5-HT<sup>4</sup> -receptors initiating a cascade via Gs, AC, cAMP and then activating hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCN) in the sinus node [86]. 5-HT increased the HCN-coded current called I<sup>f</sup> in isolated human atrial cardiomyocytes and was mediated by 5-HT<sup>4</sup> -receptors (using specific receptor antagonists [99–101]). In one of the first studies on humans, 5-HT induced cardiac arrhythmias *in vivo* (tachycardia and P wave inversions in two patients: [14]). Interestingly, 5-HT induced arrhythmias even in isolated electrically driven human atrial cardiomyocytes, proving that arrhythmia does not need indirect pathways but is sufficiently explained by direct activation of receptors (5-HT<sup>4</sup> -receptors) [102]. Interestingly, the incidence of arrhythmias was enhanced in isolated atria from humans treated prior to surgery with β-adrenoceptor blockers [102, 103]. The arrhythmias could be explained on a single cell basis via late afterdepolarizations [104, 105]. In addition, arrhythmogenesis due to 5-HT might also involve stimulation L-type Ca2+-channels and potassium channels [86, 106]. 5-HT may also be relevant to sustain an existing arrhythmia: during pre-existing atrial fibrillation, more 5-HT will be released from thrombocytes [107]. This can increase local concentrations of 5-HT, which can act on 5-HT<sup>4</sup> receptors to sustain fibrillation (for further hypothetical mechanisms: [92]). Mechanistically interesting is the observation that in some children autoantibodies against 5-HT<sup>4</sup> -receptors exist which have been suggested to lead to AV blocks in neonates [108]. In 5-HT<sup>4</sup> -receptor overexpressing mice, arrhythmias under basal conditions or after 5-HT stimulation have been observed [23, 87, 109]. The 5-HT<sup>3</sup> -receptor might be antiarrhythmic: general deletion of the 5-HT3 -receptor in mice led to spontaneous ventricular tachycardia and increased sudden death in pregnant mice. It was speculated that 5-HT<sup>3</sup> -receptor blockers should therefore be avoided in pregnant women [110]. Consistent with this, ondansetron, a blocker of 5-HT<sup>3</sup> receptors, has been reported to elicit arrhythmias in patients [111]. In addition, prolongation of P-waves and highly elevated T-waves (interpreted as a sign of repolarization abnormalities) were described in mice with knockout of 5-HT2B-receptors [112].

## **10. Effects of 5-HT on cardiac vasculature, species differences**

5-HT can induce vasoconstriction also in coronary arteries [20]. During reperfusion of coronary arteries, 5-HT can have detrimental effects like apoptosis and necrosis [72]. In man, a more subtle picture emerges: without endothelium or in defective endothelium (arteriosclerosis, coronaries injected *in vivo* with 5-HT in patients having received transplanted hearts), 5-HT induces vasoconstriction, but in the presence of functional endothelium, 5-HT induces vasodilation in human coronary arterial strips [113–115]. Others noted in human coronary vessel strips with intact endothelium (obtained from transplanted hearts) a 5-HT-mediated vasoconstriction that was in part ketanserin-sensitive [116]. In isolated strips from human pulmonary veins or arteries, 5-HT led to vasoconstriction (regardless of the presence or absence of endothelium) and was interpreted to be mediated by 5-HT<sup>2</sup> - and 5-HT<sup>1</sup> -receptors while ligand-binding studies presented evidence for the expression of 5-HT<sup>4</sup> -, 5-HT2A-, and 5-HT1Dreceptors in these tissues [117]. In summary, vasoconstriction can be mediated by 5-HT2A- and 5-HT1D-like receptors and the latter are more relevant for vasoconstriction [114, 118].

## **11. Use of genetically modified mice to study functional effects of 5-HT in the heart**

Gain of function animal models like mice that overexpress in a cardiac specific way 5-HT<sup>4</sup> receptors [87], MAO-A [119], 5-HT2B-receptors [120] or SERT [121] have been described and used to better understand the role(s) of 5-HT in the mammalian heart. However, animal models with loss of function are much more abundant (**Table 1**). The cardiac phenotypes of mice overexpressing SERT or 5-HT<sup>4</sup> -receptors (in the heart) have been discussed in this text. 5-HT2B-receptor overexpressing mice, however, had no defect in systolic function (unaltered ejection fraction). Interestingly, their heart weight to body weight ratio was increased (cardiac hypertrophy). This was explained by an increase in the number and size of cardiomyocytes. Further changes were an increase in the number and activity of mitochondria in hearts from transgenic mice but no cardiac fibrosis was noted. It is possible that the hypertrophy is due to constitutive activation of the PLC pathway by the overexpressed 5-HT2B-receptor [120]. Interestingly, in 5-HT2B-receptor knockout mice (made by the same group), a dilated cardiomyopathy (with decreased systolic function, small sized cardiomyocytes) was noted [112] (**Table 1**). Surprisingly, the knockout of the 5-HT2B-receptor exhibited gender-specific differences in the phenotype. For instance, ECG alterations (prolongation of P-wave) were more pronounced in female than male 5-HT2B knockout mice [112].

To the best of our knowledge, mice with cardiac-specific knockout of the genes listed in **Table 1** have not been described in the literature and might be meaningful new study systems. In addition to the mouse models listed in **Table 1**, there is also a SERT knockout rat in the literature [147]. This rat model should be useful in some regards, because historically most work on hypertension was done in rats. In this context, mice with cardiac-specific overexpression of 5-HT2B-receptors could be generated by mating 5-HT2B knockout mice and 5-HT2Breceptor overexpressing mice, an interesting line of research for other 5-HT-receptors [148].


**Table 1.** Constitutive knockouts of genes relevant for serotonin handling.

**10. Effects of 5-HT on cardiac vasculature, species differences**

282 Serotonin - A Chemical Messenger Between All Types of Living Cells

of endothelium) and was interpreted to be mediated by 5-HT<sup>2</sup>

pronounced in female than male 5-HT2B knockout mice [112].

**in the heart**

mice overexpressing SERT or 5-HT<sup>4</sup>

ligand-binding studies presented evidence for the expression of 5-HT<sup>4</sup>

5-HT can induce vasoconstriction also in coronary arteries [20]. During reperfusion of coronary arteries, 5-HT can have detrimental effects like apoptosis and necrosis [72]. In man, a more subtle picture emerges: without endothelium or in defective endothelium (arteriosclerosis, coronaries injected *in vivo* with 5-HT in patients having received transplanted hearts), 5-HT induces vasoconstriction, but in the presence of functional endothelium, 5-HT induces vasodilation in human coronary arterial strips [113–115]. Others noted in human coronary vessel strips with intact endothelium (obtained from transplanted hearts) a 5-HT-mediated vasoconstriction that was in part ketanserin-sensitive [116]. In isolated strips from human pulmonary veins or arteries, 5-HT led to vasoconstriction (regardless of the presence or absence

receptors in these tissues [117]. In summary, vasoconstriction can be mediated by 5-HT2A- and

**11. Use of genetically modified mice to study functional effects of 5-HT** 

Gain of function animal models like mice that overexpress in a cardiac specific way 5-HT<sup>4</sup>

receptors [87], MAO-A [119], 5-HT2B-receptors [120] or SERT [121] have been described and used to better understand the role(s) of 5-HT in the mammalian heart. However, animal models with loss of function are much more abundant (**Table 1**). The cardiac phenotypes of

5-HT2B-receptor overexpressing mice, however, had no defect in systolic function (unaltered ejection fraction). Interestingly, their heart weight to body weight ratio was increased (cardiac hypertrophy). This was explained by an increase in the number and size of cardiomyocytes. Further changes were an increase in the number and activity of mitochondria in hearts from transgenic mice but no cardiac fibrosis was noted. It is possible that the hypertrophy is due to constitutive activation of the PLC pathway by the overexpressed 5-HT2B-receptor [120]. Interestingly, in 5-HT2B-receptor knockout mice (made by the same group), a dilated cardiomyopathy (with decreased systolic function, small sized cardiomyocytes) was noted [112] (**Table 1**). Surprisingly, the knockout of the 5-HT2B-receptor exhibited gender-specific differences in the phenotype. For instance, ECG alterations (prolongation of P-wave) were more

To the best of our knowledge, mice with cardiac-specific knockout of the genes listed in **Table 1** have not been described in the literature and might be meaningful new study systems. In addition to the mouse models listed in **Table 1**, there is also a SERT knockout rat in the literature [147]. This rat model should be useful in some regards, because historically most work on hypertension was done in rats. In this context, mice with cardiac-specific overexpression of 5-HT2B-receptors could be generated by mating 5-HT2B knockout mice and 5-HT2Breceptor overexpressing mice, an interesting line of research for other 5-HT-receptors [148].

5-HT1D-like receptors and the latter are more relevant for vasoconstriction [114, 118].






## **12. 5-HT-receptors present in the heart: cell and species differences**

The current thinking is that 5-HT can act via membrane bound receptors called 5-HT1-7 ([20, 149], review: [150]). The 5-HT<sup>3</sup> -receptor is a ligand-gated ion channel, whereas all other 5-HT-receptors are G-protein-coupled receptors. The 5-HT1 -receptors as well as 5-HT<sup>7</sup> receptors can inhibit the activity of adenylyl cyclase via Gi/q, whereas 5-HT<sup>4</sup> -, 5-HT<sup>5</sup> -, and 5-HT<sup>6</sup> -receptors can increase the activity of adenylyl cyclase via Gs. 5-HT<sup>2</sup> -receptors, via Gq / G11, can activate PLC and thereby increase IP3 levels as well as generate diacylglycerol and subsequently diacylglycerol can activate PKC. Moreover, 5-HT2A- and 5-HT2C-receptors can also activate phospholipase A<sup>2</sup> . In the whole mouse heart, the following receptors have been described on mRNA level: 5-HT1A-, 5-HT1B-, 5-HT1D-, 5-HT2A-, 5-HT2B-, 5-HT2C-, 5-HT<sup>3</sup> -, and 5-HT4 -receptors [43]. Surprisingly, others failed to detect the 5-HT<sup>4</sup> -receptor in mouse heart and only reported on 5-HT2A- and 5-HT2B-receptors [151]. Others failed to detect 5-HT2Creceptors in neonatal rat cardiomyocytes, which offers the possibility that the cardiac expression of 5-HT-receptors might be developmentally regulated or likewise be species dependent or in different cell types of the heart [22]. Apparently, the 5-HT6 -receptor was not found by PCR in adult mouse whole hearts [43]. Four isoforms of mouse 5-HT<sup>4</sup> -receptors exist (on RNA level) in mouse atria [152]. On RNA level, 5-HT4a- and 5-HT4b-receptors are also present in human atrium [153, 154] and to a lesser extent in human cardiac ventricle [85, 155]. As mentioned before, 5-HT2A-receptors mediate the effects of 5-HT in thrombocytes [156]. The PIE of 5-HT in rat atrium is probably mediated by 5-HT2A-receptors [82]. 5-HT2A- and 5-HT4 -receptors are, however, present on RNA in rats [82], but the 5-HT<sup>4</sup> -receptors in rat hearts only become functional (mediating a PIE) in stress (myocardial infarction: overview in [157]). The 5-HT<sup>2</sup> receptors can activate phospholipase C and can elevate IP3 levels in the rat heart [82]. 5-HT2Areceptors are found in human arterial smooth muscle cells and can lead to vasoconstriction [158, 159]. Initially, 5-HT<sup>3</sup> -receptors seemed only to be present in nerve cells in the heart and might mediate the "von Bezold-Jarisch" reflex [97]. The 5-HT<sup>3</sup> -receptors seem to be found in epicardial afferent sensory nerve ending of the vagus [160]. More recently, however, using a new knockout mouse, 5-HT<sup>3</sup> -receptors were found at least in the ventricle of wild-type mice [110]. The 5-HT<sup>4</sup> -receptor (but not, for instance, a 5-HT<sup>2</sup> -receptor) mediates the PIE and PCE in the human heart [64, 92]. The study of the 5-HT<sup>4</sup> -receptor structure is complicated because many splice variants are known which might have different physiological and/or pathophysiological roles [157]. No convincing antibodies to 5-HT<sup>4</sup> -receptors let alone for splice variants have been published in the literature (and our own unpublished observations). Hence, protein levels of these receptors are difficult to assess. At least, some radioactive ligand-binding studies shed some light on the protein expression levels in the heart and found measurable but very low densities of 5-HT<sup>4</sup> -receptors in the heart [161]. Really specific antibodies for 5-HT<sup>4</sup> receptors with high affinity are highly desirable. 5-HT1 -receptors are present in endothelial cells and smooth muscle cells in human coronary arteries and mediate vasoconstrictory effects of 5-HT [158] and can inhibit AC activity [162]. PIE of 5-HT in human atrium and ventricle are 5-HT4 -receptor mediated (trabeculae: [64]). 5-HT2B-receptors are present in cardiac valves. Their simulation by 5-HT, fenfluramine (indirectly by inhibiting SERT or by releasing 5-HT from platelets), ergotamine derivatives, methysergide, and recreational drugs ("ecstacy") can lead to deadly valve ruptures [163–165]. Typically, these drugs are present in all parts of the blood circulation; hence, the valve dysfunction can take place in the right as well as in the left heart. An excellent review on 5-HT-receptors in the vascular system especially the heart of humans is to be found in the literature and will be helpful for in depth information [157].

#### **13. Signal transduction mechanisms of 5-HT-receptors in the heart**

Moreover, 5-HT in isolated atrial preparations from human hearts increased cAMP content, PKA activity [64, 80], and the phosphorylation state of phospholamban (PLB) and the inhibitory subunit of troponin (TNI, [88]), and these effects were blocked by 5-HT<sup>4</sup> -receptor antagonists [88]. Hence, these effects were probably 5-HT<sup>4</sup> -receptor mediated [88]. In electrophysiological experiments, 5-HT elevated the L-type Ca2+-current in human atrium [83, 166, 167] but not human ventricle [83, 168]. Mechanistically important, 5-HT increased the contractility in isolated atrial paced human cardiomyocytes [103]. Stimulation of 5-HT2Areceptors led to increases of IP3 content [82]. Similarly, in transgenic mice that overexpress 5-HT4 -receptors in the mouse heart, 5-HT led to PIE and PCE in intact mice (using echocardiography), in isolated perfused hearts, in isolated left atria (electrically driven), or isolated spontaneously beating right atria. These effects were accompanied by cAMP increases, increased phosphorylation state of PLB (on amino acid serine 16 and threonine 17), increase in current through L-type Ca2+-channels, and increase in the free Ca2+ content in the cytosol in mice ventricular preparations or whole hearts [87]. In addition, increased phosphorylation of PLB was also noted in atrial preparations from 5-HT<sup>4</sup> -receptor overexpressing mice [169]. In these mice, the *in vivo* activity of agonists could be studied on contractility [90]. Here, one can recapitulate findings in cloned receptors, for instance, cisapride was less potent and effective to increase force of contraction than 5-HT. Moreover, cisapride induced concentration-dependent tachycardia (and arrhythmias) in spontaneously beating isolated right atrial preparations of 5-HT4 -receptor overexpressing mice [170], similar to tachycardias described in some patients treated with cisapride [171]. However, prucalopride was less potent but equieffective compared to 5-HT [109, 170, 172]. In addition, 5-HT is able to desensitize the 5-HT4 -receptor not only in 5-HT<sup>4</sup> -receptor overexpressing mice in the atrium [90] but also in the ventricle [172, 173]. LSD and ergotamine *in vitro* displayed biased signaling for β-arrestin at 5-HT2B- and 5-HT1B-receptors [174].

## **14. Altered expression or function of cardiac 5-HT or its receptors under pathophysiological conditions**

### **14.1. Carcinoid syndrome**

subsequently diacylglycerol can activate PKC. Moreover, 5-HT2A- and 5-HT2C-receptors can

and only reported on 5-HT2A- and 5-HT2B-receptors [151]. Others failed to detect 5-HT2Creceptors in neonatal rat cardiomyocytes, which offers the possibility that the cardiac expression of 5-HT-receptors might be developmentally regulated or likewise be species dependent

level) in mouse atria [152]. On RNA level, 5-HT4a- and 5-HT4b-receptors are also present in human atrium [153, 154] and to a lesser extent in human cardiac ventricle [85, 155]. As mentioned before, 5-HT2A-receptors mediate the effects of 5-HT in thrombocytes [156]. The PIE of

functional (mediating a PIE) in stress (myocardial infarction: overview in [157]). The 5-HT<sup>2</sup>

receptors can activate phospholipase C and can elevate IP3 levels in the rat heart [82]. 5-HT2Areceptors are found in human arterial smooth muscle cells and can lead to vasoconstriction

epicardial afferent sensory nerve ending of the vagus [160]. More recently, however, using a

many splice variants are known which might have different physiological and/or pathophysi-

have been published in the literature (and our own unpublished observations). Hence, protein levels of these receptors are difficult to assess. At least, some radioactive ligand-binding studies shed some light on the protein expression levels in the heart and found measurable but

cells and smooth muscle cells in human coronary arteries and mediate vasoconstrictory effects of 5-HT [158] and can inhibit AC activity [162]. PIE of 5-HT in human atrium and ventricle

Their simulation by 5-HT, fenfluramine (indirectly by inhibiting SERT or by releasing 5-HT from platelets), ergotamine derivatives, methysergide, and recreational drugs ("ecstacy") can lead to deadly valve ruptures [163–165]. Typically, these drugs are present in all parts of the blood circulation; hence, the valve dysfunction can take place in the right as well as in the left heart. An excellent review on 5-HT-receptors in the vascular system especially the heart of humans is to be found in the literature and will be helpful for in depth information [157].

**13. Signal transduction mechanisms of 5-HT-receptors in the heart**

Moreover, 5-HT in isolated atrial preparations from human hearts increased cAMP content, PKA activity [64, 80], and the phosphorylation state of phospholamban (PLB) and the


5-HT in rat atrium is probably mediated by 5-HT2A-receptors [82]. 5-HT2A- and 5-HT4

described on mRNA level: 5-HT1A-, 5-HT1B-, 5-HT1D-, 5-HT2A-, 5-HT2B-, 5-HT2C-, 5-HT<sup>3</sup>


or in different cell types of the heart [22]. Apparently, the 5-HT6

are, however, present on RNA in rats [82], but the 5-HT<sup>4</sup>

might mediate the "von Bezold-Jarisch" reflex [97]. The 5-HT<sup>3</sup>

in the human heart [64, 92]. The study of the 5-HT<sup>4</sup>

ological roles [157]. No convincing antibodies to 5-HT<sup>4</sup>

receptors with high affinity are highly desirable. 5-HT1


PCR in adult mouse whole hearts [43]. Four isoforms of mouse 5-HT<sup>4</sup>

. In the whole mouse heart, the following receptors have been

















also activate phospholipase A<sup>2</sup>

284 Serotonin - A Chemical Messenger Between All Types of Living Cells

[158, 159]. Initially, 5-HT<sup>3</sup>

new knockout mouse, 5-HT<sup>3</sup>

very low densities of 5-HT<sup>4</sup>

[110]. The 5-HT<sup>4</sup>

are 5-HT4

5-HT4

In the carcinoid syndrome (typically due to tumors arising from enterochromaffin cells of the gut that in 10% of cases produce high levels of 5-HT: cf. [23] for a clinical example, large patient series: [26]), high circulating levels of 5-HT, which can stimulate 5-HT-receptors, and lesions of the *right* cardiac valves have been reported. Normally, the pulmonary circulation is assumed to remove free circulating 5-HT from the plasma and very little 5-HT in the plasma would reach the left ventricle (discussed in Ref. [43]). However, when a carcinoid tumor is located to the lung or an open foramen ovale is present or exceedingly high plasma 5-HT levels occur from a carcinoid in the gut or liver, which would spill over into the pulmonary veins, *left* cardiac lesions like valve failure have also been noted [43].

#### **14.2. MAO-dependent oxidative stress**

By altering cardiac levels of 5-HT, MAO might be of clinical relevance in patients. This can be tentatively concluded from the following animal studies. Cardiac-specific overexpression of MAO-A (in mice) led to decreased cardiac levels of 5-HT (and noradrenaline). This was accompanied by increased levels of free radicals in the mouse heart, as well as oxidation of mitochondrial DNA, cardiac fibrosis, and ventricular heart failure [53]. Oppositely, a knockout of MAO-A in mice was functionally beneficial because it reduced left ventricular dilatation and left ventricular dysfunction after hypertension (via aortic banding). Accordingly, aortic banding (an experimental model to increase cardiac afterload) increased protein levels of MAO-A threefold [119]. High cardiac concentrations of 5-HT can lead to cardiac hypertrophy (and later on, possibly, to heart failure, see below) via receptor-independent mechanism(s) or 5-HT-receptor-dependent mechanisms. In more detail, high cardiac intracellular levels of 5-HT are oxidized in mitochondria via MAO activity: this leads to the generation of deleterious free radicals [119]. In aging rat hearts, MAO-A activity was increased, which may exacerbate deleterious effects of cardiac 5-HT [119]. In rat cardiac myocytes, high intracellular levels of 5-HT led to enhanced MAO-dependent oxidative stress followed by release of cytochrome c from cardiac mitochondria, upregulation of proapoptotic BAX protein, downregulation of antiapoptotic Bcl2 protein, and thus to detrimental apoptosis [72].

## **14.3. Hypertension**

In peripheral arterial hypertension, more 5-HT can be found in the plasma, and via covalent modification of the protein rab4, the function of SERT in platelets is reduced and thus a circulus vitiosus might start [175]. Moreover, it has been suggested that 5-HT might act on small G-proteins (by inducing serotonylation of these proteins) in smooth muscle cells in pulmonary arteries, rendering the arteries more susceptible to 5-HT-induced vasoconstriction and thus leading to sustained pulmonary hypertension and death [36]. Serotonylation of several proteins in rat aorta has likewise been reported [176]. Pulmonary hypertension might be causally related to 5-HT: plasma levels of 5-HT are twofold to threefold higher in patients suffering from this disease and augmented 5-HT plasma levels may lead to constriction of pulmonary arteries and thus to pulmonary hypertension in humans [177]. This is in line with animal experiments: in TPH1 knockout mice, hypoxia (placing the animals into chambers with low partial pressure of oxygen) was less prone to rise right ventricular pressure compared to WT animals [178]. In a rat model of pulmonary hypertension (induced by monocrotaline), blocking 5-HT2B-receptors was protective for right ventricular function [179].

### **14.4. Cardiac hypertrophy**

Interestingly, a blocker of 5-HT2A-receptors attenuated cardiac hypertrophy after aortic banding in mice, suggesting a role of this receptor in cardiac hypertrophy, and in this context, hypertrophy (transiently) increased the expression of the 5-HT2A-receptors in mouse cardiomyocytes after aortic banding [180]. The classical isoproterenol-induced hypertrophy in a mouse model was reduced in mice treated with a 5-HT2B blocker or in 5-HT2B-receptor knockout mice, probably by inhibiting peroxide generation in mitochondria [2, 181]. Interestingly, a isoproterenol-induced cardiac hypertrophy (a classical animal model of hypertrophy) seemed to require 5-HT2B-receptors on cardiac fibroblasts [148]. Fittingly, in patients with cardiac hypertrophy, the expression of 5-HT2B-receptors was elevated (radioligand binding [148]). 5-HT2B-receptors were detected with immunohistology in human cardiomyocytes and human non-cardiomyocytes; however, it is an open question (and a mechanistically very important question) whether overexpression of these receptors in human heart failure occurs in cardiomyocytes and/or in non-cardiomyocytes [148].

## **14.5. Heart failure**

accompanied by increased levels of free radicals in the mouse heart, as well as oxidation of mitochondrial DNA, cardiac fibrosis, and ventricular heart failure [53]. Oppositely, a knockout of MAO-A in mice was functionally beneficial because it reduced left ventricular dilatation and left ventricular dysfunction after hypertension (via aortic banding). Accordingly, aortic banding (an experimental model to increase cardiac afterload) increased protein levels of MAO-A threefold [119]. High cardiac concentrations of 5-HT can lead to cardiac hypertrophy (and later on, possibly, to heart failure, see below) via receptor-independent mechanism(s) or 5-HT-receptor-dependent mechanisms. In more detail, high cardiac intracellular levels of 5-HT are oxidized in mitochondria via MAO activity: this leads to the generation of deleterious free radicals [119]. In aging rat hearts, MAO-A activity was increased, which may exacerbate deleterious effects of cardiac 5-HT [119]. In rat cardiac myocytes, high intracellular levels of 5-HT led to enhanced MAO-dependent oxidative stress followed by release of cytochrome c from cardiac mitochondria, upregulation of proapoptotic BAX protein, downregulation of

In peripheral arterial hypertension, more 5-HT can be found in the plasma, and via covalent modification of the protein rab4, the function of SERT in platelets is reduced and thus a circulus vitiosus might start [175]. Moreover, it has been suggested that 5-HT might act on small G-proteins (by inducing serotonylation of these proteins) in smooth muscle cells in pulmonary arteries, rendering the arteries more susceptible to 5-HT-induced vasoconstriction and thus leading to sustained pulmonary hypertension and death [36]. Serotonylation of several proteins in rat aorta has likewise been reported [176]. Pulmonary hypertension might be causally related to 5-HT: plasma levels of 5-HT are twofold to threefold higher in patients suffering from this disease and augmented 5-HT plasma levels may lead to constriction of pulmonary arteries and thus to pulmonary hypertension in humans [177]. This is in line with animal experiments: in TPH1 knockout mice, hypoxia (placing the animals into chambers with low partial pressure of oxygen) was less prone to rise right ventricular pressure compared to WT animals [178]. In a rat model of pulmonary hypertension (induced by monocrotaline), block-

Interestingly, a blocker of 5-HT2A-receptors attenuated cardiac hypertrophy after aortic banding in mice, suggesting a role of this receptor in cardiac hypertrophy, and in this context, hypertrophy (transiently) increased the expression of the 5-HT2A-receptors in mouse cardiomyocytes after aortic banding [180]. The classical isoproterenol-induced hypertrophy in a mouse model was reduced in mice treated with a 5-HT2B blocker or in 5-HT2B-receptor knockout mice, probably by inhibiting peroxide generation in mitochondria [2, 181]. Interestingly, a isoproterenol-induced cardiac hypertrophy (a classical animal model of hypertrophy) seemed to require 5-HT2B-receptors on cardiac fibroblasts [148]. Fittingly, in patients with cardiac hypertrophy, the expression of 5-HT2B-receptors was elevated (radioligand binding [148]). 5-HT2B-receptors were detected with immunohistology in human cardiomyocytes and human

antiapoptotic Bcl2 protein, and thus to detrimental apoptosis [72].

286 Serotonin - A Chemical Messenger Between All Types of Living Cells

ing 5-HT2B-receptors was protective for right ventricular function [179].

**14.3. Hypertension**

**14.4. Cardiac hypertrophy**

In heart failure, 5-HT might be altered: increases in plasma 5-HT levels in patients with decompensated systolic heart failure [182] or diastolic heart failure [183] have been described. These studies concluded that 5-HT elevation may be a compensatory mechanism, trying to increase cardiac output by increasing heart rate and cardiac force [182]. In atrial samples from heart failure patients, the PIE of 5-HT was reduced. Moreover, biochemical correlates of receptor coupling like the extent to which 5-HT could increase AC activity [184] or increase L-type Ca2+-currents, was attenuated in samples from heart failure patients [168]. Some of these effects were reversed after β-adrenergic blockade of patients prior to surgery [185]. In a rat model of heart failure (infarction), the mRNA of 5-HT<sup>4</sup> -receptors increased and a robust PIE of 5-HT (which was lacking in normal rats without heart failure) became apparent [186]. Moreover, there are data that in human heart failure a PIE of 5-HT and upregulation of 5-HT<sup>4</sup> receptors become measurable [85, 155]. Interestingly, the PIE of 5-HT increased with NYHA class but the PIE of β-adrenergic stimulation decreased with NYHA class [85]. In a pilot study with heart failure patients, the EF increased after being treated with a 5-HT<sup>4</sup> -receptor antagonist (piboserod [187]). This might mean that activation of 5-HT<sup>4</sup> -receptors is deleterious in human heart failure. In apparent contrast to this conclusion, in lipopolysaccharide (LPS) induced sepsis (another accepted model of heart failure), overexpression of 5-HT<sup>4</sup> -receptors seemed to protect the heart by interference with the toll-like receptor 4 pathway [188].

#### **14.6. Atrial fibrillation**

In patients with chronic (more than 1 month persistent) atrial fibrillation, the expression of 5-HT4 -receptor mRNA levels was found to be decreased by about 36% (irrespective of β-adrenoceptor treatment) in comparison to controls in sinus rhythm and this change in receptors level was suggested to be a protective mechanism [189]. Others found the expression of 5-HT4b-receptors to be downregulated (mRNA) in acute atrial fibrillation but upregulated with atrial fibrillation lasting more than 1 year [190]. Protein data for 5-HT<sup>4</sup> -receptors in atrial fibrillation would clearly be desirable to resolve these somewhat contradictory findings. In aging, the uptake of 5-HT in platelets is augmented, concentrations of 5-HT in platelets are therefore higher, and thence 5-HT is more prone to induce aggregation of platelets and thus thrombosis (e.g. [191]).

### **14.7. Aging**

At least in pigs, the PIE of 5-HT in atrium and ventricle *in vitro* is increased from neonates to adulthood [85, 86]. The opposite occurs in rats: fetal rat ventricles express highly the mRNA for 5-HT4 -receptors and are accompanied by (and probably causes) a large PIE to 5-HT in neonatal cardiac preparation. In contrast, in adult rats, as mentioned before, 5-HT is devoid of a PIE in the rat ventricle [82, 192]. In human atria, 5-HT stimulates AC less in aging, which was explained by increased levels of Gi proteins [193]. Hence, age-dependent changes in cardiac response to 5-HT are known, but much more refined data are clearly needed from further work.

So sum up these findings, it is possible that 5-HT causes or at least contributes to cardiac hypertrophy, arterial or pulmonary hypertension, heart failure, cardiac aging, and cardiac arrhythmias.

## **15. Possible cardiac side effects of serotoninergic drugs**

Drugs elevating 5-HT: SERT *inhibitors* (specific serotonin reuptake inhibitors, SSRIs), they are suggested to increase 5-HT not only in brain synapses but also in cardiac tissue and therefore they might be implicated in arrhythmias (citalopram: [194]). Warning notices have been sent out for citalopram in this regard by regulatory authorities (FDA: 2012). A recent study noted enhanced risks of valve disease in patients who take SSRI [195], presumably because high plasma membrane concentrations of 5-HT activate 5-HT2B-receptors. *5-HTP* has been suggested to be used as add on to SSRI in order to treat depression, because it is metabolized to 5-HT. *5-HTP* is therefore predicted, indirectly, to lead to arrhythmias in patients.

5-HT<sup>1</sup> -receptors: *Sumatriptan* and congeners are well known to have the ability to contract coronary arteries and can lead to myocardial infarctions [196]. Buspirone is a drug acting among others on 5-HT1A-receptors and has been shown to lead to tachycardia.

5-HT2 -receptors: *Ergotamine* and *LSD* (but also *fenfluramine*) stimulate 5-HT1B-receptors but also detrimental 5-HT2B-receptors (leading to valve fibrosis) [174]. In addition, an important metabolite of fenfluramine called *norfenfluramine* could bind with high affinity to 5-HT2B-receptors and could stimulate in the receptor-transfected HEK cells the IP3 levels, presumably initiating fibroplasia *in vivo* in humans [197]. In 2014, pimavanserin is an antagonist at 5-HT2A-receptors entered the market in the USA as an antipsychotic drug and to treat Parkinson's disease [198]. Interestingly, the producing company lists as contraindications irregular heartbeat. In some countries, *ketanserin* (a classical 5-HT2A-antagonist) has been used for many years to treat *hypertension*. However, the antihypertensive effect is probably due to an additional α1 -adrenoceptor antagonistic effect of ketanserin [2]. Moreover, its use has rapidly declined when it was suggested to lead to deadly arrhythmias (ketanserin can make QT prolongation and thereby lead to torsade de pointes). The reason for this is that the K<sup>+</sup> channel hERG (human ether-a-go-gorelated gene) is blocked by ketanserin (reviewed in [2]).

5-HT2C-receptors: A newer serotonergic drug is the 5-HT2C-agonist *lorcaserin* that was approved by the FDA in June 2012 in order to bring about weight loss by action in the CNS [198]. Lorcaserin was developed because older drugs for weight loss were withdrawn, in the past, from the market (e.g. fenfluramine) because they led to fibrosis (reviewed in Ref. [70]). Clearly, one can speculate that lorcaserin might also have agonistic properties at 5-HT2Breceptors and thus may have effects on valves. Initial studies did not detect an increased risk of lorcaserin for valvulopathies, however the incidence of headaches was increased versus placebo which may mean that lorcaserin can act agonistic on other 5-HT receptors [199] and side effects should carefully monitored by physicians and communicated to the regulatory authorities.

5-HT4 -receptors: *Metoclopramide* acts on many receptors but in this context the activation of 5-HT4 -receptors is important [200]. One has speculated that 5-HT<sup>4</sup> -receptor agonists might be useful to treat patients with sinus bradycardia, slowing of AV conduction, and autonomic dysfunction of the heart [201]. 5-HT<sup>4</sup> -receptor agonists are used in some countries to treat irritable bowel disease (e.g. prucalopride [202]), bladder dysfunction [203], and Morbus Alzheimer (e.g. [204]). However, early on a clinical study detected arrhythmias in ECG of healthy volunteers [205], which used the 5-HT<sup>4</sup> -receptor agonist *prucalopride*, and hence caution in its used is advised. The 5-HT<sup>4</sup> -receptor agonist (RS 67333) *donecopride*, in addition, inhibits the activity of acetylcholine esterases [206] and might be useful for the treatment of Morbus Alzheimer. However, this compound is expected to lead to arrhythmias in sensitive patients via its agonist activity of cardiac 5-HT<sup>4</sup> -receptors. 5-HT<sup>4</sup> -receptor agonists have been developed to treat anxiety or chronic obstipation. *5-HT4 -receptor antagonists* have been suggested for the treatment of supraventricular arrhythmias [159, 189] but were not successful due to side effects [106].

## **Author details**

PIE in the rat ventricle [82, 192]. In human atria, 5-HT stimulates AC less in aging, which was explained by increased levels of Gi proteins [193]. Hence, age-dependent changes in cardiac response to 5-HT are known, but much more refined data are clearly needed from further

So sum up these findings, it is possible that 5-HT causes or at least contributes to cardiac hypertrophy, arterial or pulmonary hypertension, heart failure, cardiac aging, and cardiac

Drugs elevating 5-HT: SERT *inhibitors* (specific serotonin reuptake inhibitors, SSRIs), they are suggested to increase 5-HT not only in brain synapses but also in cardiac tissue and therefore they might be implicated in arrhythmias (citalopram: [194]). Warning notices have been sent out for citalopram in this regard by regulatory authorities (FDA: 2012). A recent study noted enhanced risks of valve disease in patients who take SSRI [195], presumably because high plasma membrane concentrations of 5-HT activate 5-HT2B-receptors. *5-HTP* has been suggested to be used as add on to SSRI in order to treat depression, because it is metabolized




channel hERG (human ether-a-go-go-

to 5-HT. *5-HTP* is therefore predicted, indirectly, to lead to arrhythmias in patients.

among others on 5-HT1A-receptors and has been shown to lead to tachycardia.

*tension*. However, the antihypertensive effect is probably due to an additional α1

to torsade de pointes). The reason for this is that the K<sup>+</sup>

related gene) is blocked by ketanserin (reviewed in [2]).

antagonistic effect of ketanserin [2]. Moreover, its use has rapidly declined when it was suggested to lead to deadly arrhythmias (ketanserin can make QT prolongation and thereby lead

5-HT2C-receptors: A newer serotonergic drug is the 5-HT2C-agonist *lorcaserin* that was approved by the FDA in June 2012 in order to bring about weight loss by action in the CNS [198]. Lorcaserin was developed because older drugs for weight loss were withdrawn, in the past, from the market (e.g. fenfluramine) because they led to fibrosis (reviewed in Ref. [70]). Clearly, one can speculate that lorcaserin might also have agonistic properties at 5-HT2Breceptors and thus may have effects on valves. Initial studies did not detect an increased risk of lorcaserin for valvulopathies, however the incidence of headaches was increased versus placebo which may mean that lorcaserin can act agonistic on other 5-HT receptors [199] and

**15. Possible cardiac side effects of serotoninergic drugs**

288 Serotonin - A Chemical Messenger Between All Types of Living Cells

work.

5-HT<sup>1</sup>

5-HT2

arrhythmias.

Joachim Neumann1 \*, Britt Hofmann<sup>2</sup> and Ulrich Gergs1

\*Address all correspondence to: joachim.neumann@medizin.uni-halle.de

1 Institute for Pharmacology and Toxicology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany

2 Department of Cardiothoracic Surgery, Heart Centre of the University Clinics, Halle (Saale), Germany

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## *Edited by Kaneez Fatima Shad*

Serotonin - A Chemical Messenger Between All Types of Living Cells is a very interesting book on the most ancient neurotransmitter, hormone and trophic factor serotonin or 5-hydroxytryptamine (5-HT). This unique chemical is present in all living cells including plants and animals. This book will take us through a serene journey of the evolutionary history of serotonin and its role from man to mollusk. There are many interesting chapters incorporated in this book, including novel approaches for detecting minor metabolites of serotonin in human plasma, production and function of serotonin in cardiac cells, immuno-thrombotic effects of serotonin in platelets to the identification and localization of serotonin in the nervous system and gonad of bivalve mollusks.

Serotonin - A Chemical Messenger Between All Types of Living Cells

Serotonin

A Chemical Messenger Between

All Types of Living Cells

*Edited by Kaneez Fatima Shad*

Photo by xrender / iStock