3. Endothelium and sympathetic neurotransmission

The sympathetic nervous system (SNS) is known to play a fundamental role in the short- and long-term regulation of different vascular functions. Vessels contain sympathetic nerves distributed between smooth muscle and adventitia layers [41]. Sympathetic nerve fibers are enveloped in Schwann cells: most nerve fibers travel through individual channels in the Schwann cell, but small fibers are sometimes bundled together within a single channel [42]. The SNS signals to dilate or constrict the vessel, changing the lumen size, i.e., regulating vascular tone and, therefore, affecting blood pressure.

and aggregating platelets induce contractions, because they release vasoconstrictors (thromboxane A2 and serotonin). When platelet aggregation occurs in a healthy artery (i.e, with an intact and physiologically active endothelium), serotonin (and ADP) release by the platelets as well as production of thrombin will increase NO release from endothelial cells. Thus, NO will be increased in the vicinity of smooth muscle cells inducing dilation, and consequently, increasing

Another important factor influencing NO production relies on the presence of reactive oxygen/ nitrogen species (ROS/RNS). Indeed, several enzymes from endothelium can produce superoxide anions such as nicotinamide adenine dinucleotide phosphate oxidase (NOX), xanthine oxidase (XO), cyclooxygenases (COX) and also eNOS but only when there is a deficient supply of substrate or of the cofactor BH4. Under pathophysiological conditions, superoxide anions scavenge NO resulting in the formation of peroxynitrite, reducing considerably the bioavailability of NO. Moreover, ROS can inactivate eNOS through S-glutathionylation. Taken together, these may

Moreover, intake of a number of natural products, such as flavonoids and other polyphenols, favors endothelium-dependent dilations and protects endothelium from dysfunction through increased production of NO. The protective effects of polyphenols against endothelial dysfunction involve increased production of NO in response to endothelium-derived vasodilators resulting from: facilitation of the effects of NO on the vascular smooth muscle cells, increased levels of BH4, calcium-independent phosphorylation of eNOS, antioxidant properties preventing the uncoupling of eNOS, activation of estrogen receptors and upregulation of AMP-activated protein kinase (AMPK) and of NAD(+)-dependent deacetylase (SIRT1) [28–31].

Besides its direct role as a vasodilator, NO also modulates the release of other endotheliumderived mediators. Thus, in a number of larger arteries, endothelium-derived hyperpolarization (EDH)-mediated dilations become prominent only when the synthesis of NO is inhibited [32, 33]. Hence, EDH is able to take over, at least temporarily, in the case of 'classical' endothelial dysfunction associated with a loss of NO synthesis, demonstrating strong compensatory efficiency of EDH-mediated responses. Intriguingly, exogenous NO attenuates EDH-mediated responses in coronary arteries in vitro [34] and in coronary circulation in vivo [35, 36]. Moreover, NO has been shown to exert a negative feedback effect on endothelium-dependent dilation through cGMP-mediated desensitization in isolated coronary arteries [32]. Indeed, clinical studies show that chronic therapy with nitrate, used as a NO donor, in patients with ischemic heart disease does not yield a benefit on mortality [37, 38], confirming the importance of the physiological balance between NO and EDH. Moreover, the amount of NO formed in

The sympathetic nervous system (SNS) is known to play a fundamental role in the short- and long-term regulation of different vascular functions. Vessels contain sympathetic nerves distributed between smooth muscle and adventitia layers [41]. Sympathetic nerve fibers are

the endothelial cells controls the release of vasoconstrictor prostanoids [39, 40].

3. Endothelium and sympathetic neurotransmission

explain why oxidative stress is often associated with endothelial dysfunction.

2.2. Influence of NO on another EDRF

330 Endothelial Dysfunction - Old Concepts and New Challenges

blood flow.

Nowadays, it is well established that SNS contributes to the modulation of vascular function and that this relationship is a key factor in the development of cardiovascular diseases. Several factors, such as the renin-angiotensin system, NO, ROS and endothelin, influence this modulation at central and peripheral level [43–45]. Moreover, endothelial function also seems to be regulated by SNS, mainly in the control of vascular tone. Additionally, endothelial dysfunction as well as increase in sympathetic activity has been associated to cardiovascular risk factors and disease. For example, in studies carried out in healthy subjects, an increase in sympathetic activity was associated with a decrease in endothelial function [46] Moreover, in humans, stiffness of large artery was also associated with an increased activity of SNS [47]. On the other hand, large artery stiffness can interfere with autonomic regulation by impairing carotid baroreflex sensitivity [48].

The influence of endothelium in noradrenaline release has also been previously demonstrated [49, 50]. This conclusion was obtained not only in arteries without endothelium but also in a model of endothelial dysfunction (i.e, essential hypertensive arteries), which is shown in Table 1. This type of information can be obtained from experiments where synapse events are mimicked allowing the evaluation of putative players able to alter neurotransmitter release from the nerves. Indeed, in such experiments, the use of selective pharmacological tools, such as agonists/antagonists of receptors or of activators/inhibitors of proteins or enzymes, can reveal their respective role in the neurotransmission dynamic. For instance, in experiments where rat vascular tissues, preincubated with [3 H]-noradrenaline, are electrically stimulated (5 Hz, 100 pulses, 1 ms, 50 mA), the release of <sup>3</sup> H is induced (which mimics a physiological depolarization) and can be measured by liquid scintillation spectrometry. In addition, by altering the receptors or proteins activated (with pharmacological tools), it is possible to evaluate the activity/role of a specific player in neurotransmitter release (please see previous articles from our group where the methodology is described in detail [49, 51, 52]). For example, in Table 1, data refer to tissues that were stimulated twice at 30-min interval: outflow (bn) refers to the 5-min period immediately before each stimulation period. The electrically evoked tritium overflow (Sn) was calculated by subtracting the estimated basal outflow from total outflow observed during and in the 25-min period subsequent to S1 and expressed as a percentage of the tissue <sup>3</sup> H content at the onset of stimulation. Two animal models have been used: spontaneously hypertensive rats (SHR), a well-established model of essential hypertension [53, 54], and the respective controls, the Wistar Kyoto (WKY) rats. Moreover, in WKY animals, some arteries were endothelium denuded. The influence of these conditions on the release of S1 was evaluated, and the results are presented in Table 1.

The results in this table show that the outflow observed in the endothelium-denuded vascular tissue is lower than that obtained in intact tissue. Also, the S2 values obtained in the endothelium-denuded arteries are altered, with values higher than those observed in intact tissues. These data reveal the importance of a healthy endothelium to the sympathetic neurotransmission homeostasis, once it seems to present a transsynaptic influence mediated by endothelium. In pathological conditions, this influence can be impaired augmenting the amount of noradrenaline release and causing vasoconstriction.


Tissue preparations of mesenteric and tail arteries from WKY and SHR animals were pre-incubated with [<sup>3</sup> H] noradrenaline for 40 min. After pre-incubation with [<sup>3</sup> H]-noradrenaline, tissues were superfused with [<sup>3</sup> H]-noradrenaline free medium containing desipramine (400 nM). Values presented are means SEM, and n denotes the number of tissue preparations. Significant differences from WKY intact arteries: \* P < 0.05.

eNOS oxygenase domain with residual activity of the eNOS reductase domain [50], while in mesenteric arteries, nNOS, with both reductase and oxygenase domains being equally active,

P < 0.05 and from mesenteric artery: #

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333

P < 0.05 (ANOVA followed by

Figure 1. Influence of NOS inhibitors on vascular sympathetic transmission in mesenteric and tail rat arteries. Effect elicited by the NO donor, DEA-NONOate (10 μM), and the interaction exerted by the Nω-propyl-L-arginine hydrochloride, a specific neuronal NOS (nNOS) inhibitor (100 nM), and L-NIO dihydrochloride, a specific endothelial NOS (eNOS) inhibitor (500 nM), on the electrically evoked tritium overflow. Values are mean SEM from 5 to 12 artery segments.

These differences in vascular neurotransmission elicited by NO can be explained by the activation of different pathways, leading to opposite outcomes. In resistant arteries, such as tail artery, the well-established NO-cGMP/cGMP-dependent kinases activating voltagedependent-Ca2+ seem to be the predominant pathway [64], leading to vasoconstriction. However, in other vascular territories, such as the mesenteric artery, NO actions, in addition to the classically accepted activation of intracellular cGMP-dependent pathway [65], can also activate cGMP-independent pathways, namely by eliciting an energy decrease in mitochondria (i.e., ATP), particularly with an increase in ATP catabolism, with subsequent adenosine accumulation. Adenosine will then act on presynaptic A1 receptors causing a reduction in cAMP formation and, consequently, of PKA. Therefore, a reduction of Ca2+ channels phosphorylation (by PKA) will occur reducing the intracellular amount of Ca2+. Presynaptically, the amount of intracellular Ca2+ is critical for neurotransmission; therefore, lower amounts of calcium will cause a reduction of noradrenaline release and of the postsynaptic signal events triggered by

The location of enzyme isoforms is also relevant: nNOS in mesenteric arteries are located mostly in Schwann cells contrasting to tail arteries where their presence is very scarce (Figure 2).

It is well established that adenosine can act as a physiological neuromodulator through activation of four types of adenosine receptors, A1, A2A, A2B and A3 in the vasculature [67]. These receptors present differential affinities for adenosine, with adenosine A1 receptor requiring lower concentrations to get activated (KdA1, 0.3–3 nM), followed by A2A receptors with a Kd

seems to be the most relevant isoform producing NO.

Significant differences from the appropriate control: \*

post-hoc Holm-Sidak's multicomparisons t-test).

noradrenaline, leading to vasodilation [66].

3.2. Adenosine, endothelium and vascular neurotransmission

Table 1. Basal tritium outflow (b1), electrically evoked tritium overflow (S1) and S2/S1 ratios from normotensive (WKY) and hypertensive (SHR) vessels of the rat.

Several substances produced in endothelial cells, such as NO, adenosine, ROS and/or RNS (e.g. peroxides, superoxide, hydroxyl radical, and singlet oxygen) and prostaglandins can influence sympathetic transmission [55, 56]. Also, the activity of some enzymes, such as adenosine kinase, adenosine deaminase, NOX, XO and COX, can be altered leading to changes in the bioavailability of their respective products, influencing, indirectly, sympathetic neurotransmission.

#### 3.1. NO and vascular neurotransmission

There is evidence demonstrating that NO can modulate sympathetic neurotransmission modifying vascular smooth muscle tone, in various vascular beds, such as in coronary [57, 58], mesenteric [50, 59, 60] and pulmonary arteries [61–63]. Indeed, and as illustrated in Figure 1, a NO donor, DEA-NONOATE (10 μM) altered noradrenaline release (measured as explained above, i.e., by determining the amount of <sup>3</sup> H overflow using liquid scintillation spectrometry) in differential mode depending on the vascular territory: an increase of noradrenaline release occurs in tail artery contrasting to mesenteric territory where noradrenaline release is reduced.

Another relevant data are related with NO source, i.e., the type of NOS that generates NO (Figure 1): in tail arteries, NO production is ascribed mainly to eNOS isoform, particularly to

Figure 1. Influence of NOS inhibitors on vascular sympathetic transmission in mesenteric and tail rat arteries. Effect elicited by the NO donor, DEA-NONOate (10 μM), and the interaction exerted by the Nω-propyl-L-arginine hydrochloride, a specific neuronal NOS (nNOS) inhibitor (100 nM), and L-NIO dihydrochloride, a specific endothelial NOS (eNOS) inhibitor (500 nM), on the electrically evoked tritium overflow. Values are mean SEM from 5 to 12 artery segments. Significant differences from the appropriate control: \* P < 0.05 and from mesenteric artery: # P < 0.05 (ANOVA followed by post-hoc Holm-Sidak's multicomparisons t-test).

eNOS oxygenase domain with residual activity of the eNOS reductase domain [50], while in mesenteric arteries, nNOS, with both reductase and oxygenase domains being equally active, seems to be the most relevant isoform producing NO.

These differences in vascular neurotransmission elicited by NO can be explained by the activation of different pathways, leading to opposite outcomes. In resistant arteries, such as tail artery, the well-established NO-cGMP/cGMP-dependent kinases activating voltagedependent-Ca2+ seem to be the predominant pathway [64], leading to vasoconstriction. However, in other vascular territories, such as the mesenteric artery, NO actions, in addition to the classically accepted activation of intracellular cGMP-dependent pathway [65], can also activate cGMP-independent pathways, namely by eliciting an energy decrease in mitochondria (i.e., ATP), particularly with an increase in ATP catabolism, with subsequent adenosine accumulation. Adenosine will then act on presynaptic A1 receptors causing a reduction in cAMP formation and, consequently, of PKA. Therefore, a reduction of Ca2+ channels phosphorylation (by PKA) will occur reducing the intracellular amount of Ca2+. Presynaptically, the amount of intracellular Ca2+ is critical for neurotransmission; therefore, lower amounts of calcium will cause a reduction of noradrenaline release and of the postsynaptic signal events triggered by noradrenaline, leading to vasodilation [66].

The location of enzyme isoforms is also relevant: nNOS in mesenteric arteries are located mostly in Schwann cells contrasting to tail arteries where their presence is very scarce (Figure 2).

#### 3.2. Adenosine, endothelium and vascular neurotransmission

Several substances produced in endothelial cells, such as NO, adenosine, ROS and/or RNS (e.g. peroxides, superoxide, hydroxyl radical, and singlet oxygen) and prostaglandins can influence sympathetic transmission [55, 56]. Also, the activity of some enzymes, such as adenosine kinase, adenosine deaminase, NOX, XO and COX, can be altered leading to changes in the bioavailabil-

Tissue preparations of mesenteric and tail arteries from WKY and SHR animals were pre-incubated with [<sup>3</sup>

free medium containing desipramine (400 nM). Values presented are means SEM, and n denotes the number of tissue

Table 1. Basal tritium outflow (b1), electrically evoked tritium overflow (S1) and S2/S1 ratios from normotensive (WKY)

There is evidence demonstrating that NO can modulate sympathetic neurotransmission modifying vascular smooth muscle tone, in various vascular beds, such as in coronary [57, 58], mesenteric [50, 59, 60] and pulmonary arteries [61–63]. Indeed, and as illustrated in Figure 1, a NO donor, DEA-NONOATE (10 μM) altered noradrenaline release (measured as explained

in differential mode depending on the vascular territory: an increase of noradrenaline release occurs in tail artery contrasting to mesenteric territory where noradrenaline release is reduced. Another relevant data are related with NO source, i.e., the type of NOS that generates NO (Figure 1): in tail arteries, NO production is ascribed mainly to eNOS isoform, particularly to

H overflow using liquid scintillation spectrometry)

Evoked Overflow (S1) (% of tissue

S2/S1 n

H]-

H]-noradrenaline

tritium content)

0.065 0.004 0.202 0.016 1.054 0.038 12

0.081 0.002\* 0.329 0.036\* 1.002 0.026 12

0.073 0.003\* 0.310 0.041\* 1.013 0.031 10

0.084 0.004 0.217 0.012 0.932 0.037 18

0.069 0.002\* 0.317 0.049\* 0.929 0.039 14

0.063 0.002\* 0.259 0.016\* 1.034 0.096 14

P < 0.05.

H]-noradrenaline, tissues were superfused with [<sup>3</sup>

ity of their respective products, influencing, indirectly, sympathetic neurotransmission.

3.1. NO and vascular neurotransmission

noradrenaline for 40 min. After pre-incubation with [<sup>3</sup>

and hypertensive (SHR) vessels of the rat.

preparations. Significant differences from WKY intact arteries: \*

Basal outflow (b1) (fractional rate of

)

outflow; min<sup>1</sup>

332 Endothelial Dysfunction - Old Concepts and New Challenges

Mesenteric artery

Endothelium denuded

Endothelium intact

Tail artery WKY Endothelium intact

Endothelium denuded

Endothelium intact

SHR

WKY Endothelium intact

SHR

above, i.e., by determining the amount of <sup>3</sup>

It is well established that adenosine can act as a physiological neuromodulator through activation of four types of adenosine receptors, A1, A2A, A2B and A3 in the vasculature [67]. These receptors present differential affinities for adenosine, with adenosine A1 receptor requiring lower concentrations to get activated (KdA1, 0.3–3 nM), followed by A2A receptors with a Kd

1–20 nM. Pharmacological studies have also revealed that A2B and A3 receptors are low affinity receptors for adenosine requiring concentrations higher than 1 μM, although these adenosine receptor subtypes present different Kd (A3 subtype requires higher concentrations than A2B

Adenosine, a well-known nucleoside, results from the sequential catabolism of ATP, forming

can then be further converted in inosine by adenosine deaminase or, instead, can be reconverted to AMP by adenosine kinase. In addition to adenosine receptors and adenosine, adenosinergic system is also composed by nucleoside transporters (NTs), which are responsible for nucleoside transport into the cells and vice versa. Some of the NTs in particular some equilibrative nucleoside transporters (ENT)s have already been identified as capable of promoting adenosine transport in vasculature, namely the subtype ENT1 and ENT 4 [69, 70].

In vascular tissues as well as in some diseased states, such as hypertension, the bioavailability

The amount of adenosine present in the vicinity of adenosine receptors depends on the adenosinergic system dynamics which, in turn, can be influenced by innumerous factors, such as NO, ROS, lipid peroxidation, endothelium dysfunction, etc., that can be altered in several

Another factor related with the relevance of vascular adenosine-mediated effects relies on adenosine receptor subtype distribution in the vasculature. All adenosine receptor subtypes have been identified not only in arteries, such as pulmonary [72], mesenteric [73–77], ear [73], aorta [78] and tail [51, 52, 79–81], but also in veins [75, 82]. In renal vessels, a role of adenosine receptors in sympathetic regulation was also demonstrated [83], conditioning the blood efflux

Basal outflow (b1) (pmol/mg of tissue) Evoked overflow (S1) (pmol/mg of tissue) n

P < 0.05.

WKY 25.74 � 2.57 26.47 � 2.76 5 SHR 75.44 � 4.22\* 77.31 � 5.47\* <sup>5</sup>

WKY 45.64 � 3.81 49.78 � 5.29 5 SHR 64.81 � 5.01\* 67.82 � 4.03\* <sup>5</sup> Tissue preparations of mesenteric and tail arteries from WKY and SHR animals were superfused with Krebs-Henseleit. Tissues were stimulated twice at 30-min interval (S1–S2; 100 pulses, 5 Hz, 1 ms, 50 mA): b1 refers to the 5-min period immediately before S1. The superfusate was collected in 5-min period before and after stimulation, and each sample was heated at 80�C and derivatized using chloroacetaldehyde for 50 min at 70�C in a dry bath incubator. Identification of the ε-adenosine formed in this collected samples was confirmed by a gradient HPLC using a fluorescent detector at 230 nm excitation and 420 nm emission wavelengths. Values presented are means � SEM of adenosine per mg of tissue, and n

Table 2. Basal (b1) and electrically evoked (S1) adenosine release from sympathetic nerve terminals from normotensive

denotes the number of tissue preparations. Significant differences from WKY vessels: \*

(WKY) and hypertensive (SHR) vessels of the rat.

pathological conditions, namely in hypertension, diabetes, aging and inflammation.


335

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ADP, AMP and adenosine with this later step being mediated by 5<sup>0</sup>

of adenosine varies [71], as presented in Table 2.

receptors) [68].

Mesenteric artery

Tail artery

Figure 2. Adventitia mesenteric and tail rat arteries images. Images are representative reconstructions captured with a confocal microscope (Leica SP5 LSCM system fitted with an inverted microscope (x63 oil immersion lens). Stacks of 1-μmthick serial optical images. Arteries were stained for nNOS (a primary mouse monoclonal anti-NOS1 and a species-specific secondary Alexa 488 antibody), GFAP (a primary rabbit anti-GFAP polyclonal antibody and a species-specific secondary Alexa 647 antibody) and DAPI (nuclear stain).

1–20 nM. Pharmacological studies have also revealed that A2B and A3 receptors are low affinity receptors for adenosine requiring concentrations higher than 1 μM, although these adenosine receptor subtypes present different Kd (A3 subtype requires higher concentrations than A2B receptors) [68].

Adenosine, a well-known nucleoside, results from the sequential catabolism of ATP, forming ADP, AMP and adenosine with this later step being mediated by 5<sup>0</sup> -nucleotidase. Adenosine can then be further converted in inosine by adenosine deaminase or, instead, can be reconverted to AMP by adenosine kinase. In addition to adenosine receptors and adenosine, adenosinergic system is also composed by nucleoside transporters (NTs), which are responsible for nucleoside transport into the cells and vice versa. Some of the NTs in particular some equilibrative nucleoside transporters (ENT)s have already been identified as capable of promoting adenosine transport in vasculature, namely the subtype ENT1 and ENT 4 [69, 70].

In vascular tissues as well as in some diseased states, such as hypertension, the bioavailability of adenosine varies [71], as presented in Table 2.

The amount of adenosine present in the vicinity of adenosine receptors depends on the adenosinergic system dynamics which, in turn, can be influenced by innumerous factors, such as NO, ROS, lipid peroxidation, endothelium dysfunction, etc., that can be altered in several pathological conditions, namely in hypertension, diabetes, aging and inflammation.

Another factor related with the relevance of vascular adenosine-mediated effects relies on adenosine receptor subtype distribution in the vasculature. All adenosine receptor subtypes have been identified not only in arteries, such as pulmonary [72], mesenteric [73–77], ear [73], aorta [78] and tail [51, 52, 79–81], but also in veins [75, 82]. In renal vessels, a role of adenosine receptors in sympathetic regulation was also demonstrated [83], conditioning the blood efflux


Tissue preparations of mesenteric and tail arteries from WKY and SHR animals were superfused with Krebs-Henseleit. Tissues were stimulated twice at 30-min interval (S1–S2; 100 pulses, 5 Hz, 1 ms, 50 mA): b1 refers to the 5-min period immediately before S1. The superfusate was collected in 5-min period before and after stimulation, and each sample was heated at 80�C and derivatized using chloroacetaldehyde for 50 min at 70�C in a dry bath incubator. Identification of the ε-adenosine formed in this collected samples was confirmed by a gradient HPLC using a fluorescent detector at 230 nm excitation and 420 nm emission wavelengths. Values presented are means � SEM of adenosine per mg of tissue, and n denotes the number of tissue preparations. Significant differences from WKY vessels: \* P < 0.05.

Figure 2. Adventitia mesenteric and tail rat arteries images. Images are representative reconstructions captured with a confocal microscope (Leica SP5 LSCM system fitted with an inverted microscope (x63 oil immersion lens). Stacks of 1-μmthick serial optical images. Arteries were stained for nNOS (a primary mouse monoclonal anti-NOS1 and a species-specific secondary Alexa 488 antibody), GFAP (a primary rabbit anti-GFAP polyclonal antibody and a species-specific secondary

Alexa 647 antibody) and DAPI (nuclear stain).

334 Endothelial Dysfunction - Old Concepts and New Challenges

Table 2. Basal (b1) and electrically evoked (S1) adenosine release from sympathetic nerve terminals from normotensive (WKY) and hypertensive (SHR) vessels of the rat.

in the afferent arteriole and, consequently, of renal filtration. In hypertensive arteries and veins, an impairment of the neuromodulation exerted by adenosine A1 receptors [75–77, 82] was described, contrasting with a preserved adenosine A2A receptor-mediated facilitation of noradrenaline release [75–77]. Note that a redistribution of adenosine A1 receptors from sympathetic nerves to Schwann cells was reported in hypertensive state while adenosine A2A receptors, in sympathetic nerves, were preserved [77]. Particular relevant information relies on the location of adenosine receptors on the vascular wall layers contributing to the understanding of the functional role ascribed to adenosine receptors.

In endothelium, the four adenosine receptor subtypes have been identified by functional and immunohistochemical assays, for instance in tail artery [80, 84] and aorta [84, 85]. The influence of endothelium in adenosine-mediated responses has been demonstrated, with endogenous adenosine inducing an inhibition on noradrenaline release, through activation of adenosine A1 receptors, (Figure 3, effect of DPCPX, a selective A1 receptor antagonist). Adenosine availability is a crucial factor (effect demonstrated by pentostatin and α,β-methylene ADP, which inhibit adenosine deaminase and ecto-5'nucleotidase, respectively), conditioning the type of adenosine receptor that is activated. In resistant arteries, this effect is impaired when endothelium is compromised (arteries denuded of endothelium or in essential hypertensive arteries) and, instead, a facilitatory effect mediated by adenosine A2A receptors, revealed by a selective A2A receptor antagonist, the SCH 58261, and by inhibition of adenosine kinase, revealed by an adenosine kinase inhibitor, 5<sup>0</sup> -iodotubercidin (ITU), and by ecto-5'nucleotidase inhibitor, α,β-methylene ADP, demonstrating the relevance of adenosinergic dynamics both in physiological and pathophysiological contexts, such as in hypertension [49]. The adenosinergic system dynamic is adjusted to the unfavorable conditions created by endothelium injury, with enzymes involved in adenosine formation, such as adenosine deaminase and 5<sup>0</sup> -nucleotidase operating to promote an increase in the adenosine amount available and favoring the activation of A2A receptors. This occurs despite the efforts of nucleoside transporters to equilibrate the concentration of adenosine between the inner and outer space of cells. In mesenteric arteries, A2A receptor effect is enough to counteract the existing inhibitory tonus mediated by adenosine A1, but in resistant arteries, the facilitatory effect mediated by A2A receptors (upon noradrenaline release) predominates.

#### 3.3. Interplay between nitroxidergic and other pathways in neurotransmission

NO signaling events, in mesenteric arteries, cause an accumulation of adenosine (as previously described in Section 3.1). This condition may favor adenosine neuromodulation, namely by activation of adenosine A1 receptors (which is revealed when blockade of A1 receptors occurs in the presence of the NO donor, DEA-NONOATE; Figure 4) in the mesenteric artery. Activation of A1 receptors leads to a reduction of noradrenaline release, and subsequently, the activation of α1 adrenoceptors in vascular smooth muscle cells is reduced, leading to vasodilation. In the tail artery, such interplay does not occur, at least in the experimental conditions tested.

Figure 3. Influence of endogenous adenosine on vascular sympathetic transmission in mesenteric and tail arteries. Interaction with selective adenosine receptor antagonists, DPCPX (100 nM; A1 subtype antagonist) and SCH 58261 (20 nM; A2A subtype antagonist); adenosine kinase inhibitor, ITU (100 nM); adenosine deaminase inhibitor, Pentostatin

on the electrically evoked tritium overflow. Values are mean � SEM from 4 to 12 artery segments. Significant differences

P < 0.05 and from intact arteries: #


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337

P < 0.05 (ANOVA followed by post-hoc Holm-Sidak's

(10 μM); a nucleoside transporter inhibitor, NBTI (5 μM) and an 5<sup>0</sup>

from the appropriate control: \*

multicomparisons t-test).

The interplay between nitroxidergic and adenosinergic pathways can occur in neurotransmission, with NO promoting the formation of enough amounts of adenosine capable of activating inhibitory A1 receptors. However, this type of interplay is dependent on the type of vascular bed.

in the afferent arteriole and, consequently, of renal filtration. In hypertensive arteries and veins, an impairment of the neuromodulation exerted by adenosine A1 receptors [75–77, 82] was described, contrasting with a preserved adenosine A2A receptor-mediated facilitation of noradrenaline release [75–77]. Note that a redistribution of adenosine A1 receptors from sympathetic nerves to Schwann cells was reported in hypertensive state while adenosine A2A receptors, in sympathetic nerves, were preserved [77]. Particular relevant information relies on the location of adenosine receptors on the vascular wall layers contributing to the under-

In endothelium, the four adenosine receptor subtypes have been identified by functional and immunohistochemical assays, for instance in tail artery [80, 84] and aorta [84, 85]. The influence of endothelium in adenosine-mediated responses has been demonstrated, with endogenous adenosine inducing an inhibition on noradrenaline release, through activation of adenosine A1 receptors, (Figure 3, effect of DPCPX, a selective A1 receptor antagonist). Adenosine availability is a crucial factor (effect demonstrated by pentostatin and α,β-methylene ADP, which inhibit adenosine deaminase and ecto-5'nucleotidase, respectively), conditioning the type of adenosine receptor that is activated. In resistant arteries, this effect is impaired when endothelium is compromised (arteries denuded of endothelium or in essential hypertensive arteries) and, instead, a facilitatory effect mediated by adenosine A2A receptors, revealed by a selective A2A receptor antagonist, the SCH 58261, and by inhibition of adenosine kinase,

inhibitor, α,β-methylene ADP, demonstrating the relevance of adenosinergic dynamics both in physiological and pathophysiological contexts, such as in hypertension [49]. The adenosinergic system dynamic is adjusted to the unfavorable conditions created by endothelium injury, with

operating to promote an increase in the adenosine amount available and favoring the activation of A2A receptors. This occurs despite the efforts of nucleoside transporters to equilibrate the concentration of adenosine between the inner and outer space of cells. In mesenteric arteries, A2A receptor effect is enough to counteract the existing inhibitory tonus mediated by adenosine A1, but in resistant arteries, the facilitatory effect mediated by A2A receptors (upon

NO signaling events, in mesenteric arteries, cause an accumulation of adenosine (as previously described in Section 3.1). This condition may favor adenosine neuromodulation, namely by activation of adenosine A1 receptors (which is revealed when blockade of A1 receptors occurs in the presence of the NO donor, DEA-NONOATE; Figure 4) in the mesenteric artery. Activation of A1 receptors leads to a reduction of noradrenaline release, and subsequently, the activation of α1 adrenoceptors in vascular smooth muscle cells is reduced, leading to vasodilation. In the tail

The interplay between nitroxidergic and adenosinergic pathways can occur in neurotransmission, with NO promoting the formation of enough amounts of adenosine capable of activating inhibitory A1 receptors. However, this type of interplay is dependent on the type of vascular bed.

enzymes involved in adenosine formation, such as adenosine deaminase and 5<sup>0</sup>

3.3. Interplay between nitroxidergic and other pathways in neurotransmission

artery, such interplay does not occur, at least in the experimental conditions tested.



standing of the functional role ascribed to adenosine receptors.

revealed by an adenosine kinase inhibitor, 5<sup>0</sup>

336 Endothelial Dysfunction - Old Concepts and New Challenges

noradrenaline release) predominates.

Figure 3. Influence of endogenous adenosine on vascular sympathetic transmission in mesenteric and tail arteries. Interaction with selective adenosine receptor antagonists, DPCPX (100 nM; A1 subtype antagonist) and SCH 58261 (20 nM; A2A subtype antagonist); adenosine kinase inhibitor, ITU (100 nM); adenosine deaminase inhibitor, Pentostatin (10 μM); a nucleoside transporter inhibitor, NBTI (5 μM) and an 5<sup>0</sup> -nucleotidase inhibitor, α,β-methylene-ADP (10 μM), on the electrically evoked tritium overflow. Values are mean � SEM from 4 to 12 artery segments. Significant differences from the appropriate control: \* P < 0.05 and from intact arteries: # P < 0.05 (ANOVA followed by post-hoc Holm-Sidak's multicomparisons t-test).

NO has been viewed as a vasodilator substance since its direct effect on vascular smooth muscle cells causes dilation. However, NO can influence neurotransmission, and the interplay with adenosinergic and adrenergic pathways altering neurotransmission can, in some cases, cause an increase in noradrenaline release, which consequently will promote vasoconstriction. Therefore, the importance of NO is renewed as well as its ability to interplay with other signaling pathways involving sympathetic regulation such as the adrenergic and adenosinergic ones. Additional information and research on this field are, therefore, required to extend the knowledge on the insights of transsynaptic modulation of vascular neurotransmission. This is particularly important and can be useful to develop new therapeutic strategies, particularly in pathologies or clinical conditions, where the sympathetic

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This work received financial support from FCT, Fundação para a Ciência e Tecnologia) through project UID/QUI/50006/2013–POCI/01/0145/FEDER/007265 with financial support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020. PhD grant [SFRH/BD//2009] supported by FCT. Authors would like to thank Professors Silvia M. Arribas and Maria Carmen González from Department of Physiology, School of Medicine, University Autónoma de Madrid, Spain, for their assistance in the use

2 Laboratory of Pharmacology, Department of Drug Science, Faculty of Pharmacy, University

[1] Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of

[2] Moncada S, Palmer RM, Higgs EA. Nitric oxide: Physiology, pathophysiology, and phar-

[3] Vanhoutte PM, Mombouli JV. Vascular endothelium: Vasoactive mediators. Progress in

system is hyperactivated.

Acknowledgements

of the Confocal Microscopy.

Joana Sousa1,2 and Carmen Diniz1,2\*

\*Address all correspondence to: cdiniz@ff.up.pt

1 LAQV/REQUIMTE, Faculty of Pharmacy, University of Porto, Portugal

arterial smooth muscle by acetylcholine. Nature. 1980;288:373-376

macology. Pharmacological Reviews. 1991;43:109-142

Cardiovascular Diseases. 1996;39:229-238

Author details

of Porto, Portugal

References

Figure 4. Influence of adenosine A1 receptor antagonist (DPCPX, 100 nM) and adenosine A2A receptor antagonist (SCH 58261, 20 nM), adenosine kinase inhibitor (ITU, 100 nM) and nucleoside transporter inhibitor (NBTI, 5 μM) in the effect elicited by a nitric oxide donor, DEA-NONOate (10 μM) on the electrically evoked tritium overflow, in mesenteric and tail arteries. Values are mean SEM from 4 to 12 artery segments. Significant differences from DEA-NONOate effect alone: \* P < 0.05 and from mesenteric artery: # P < 0.05 (ANOVA followed by post-hoc Holm-Sidak's multicomparisons t-test).

Furthermore, in the cardiovascular system, NO can also interplay with the adrenergic pathway. NO source is, most probably, endothelial since noradrenaline release in the presence of a β-adrenoceptor agonist, isoprenaline (300 nM), caused an increase of noradrenaline release (175.10 13.8%, n = 11), but the increase observed was lower in endothelium-denuded arteries (129.92 13.1%, n = 7). Therefore, these data support the possibility, previously raised by Balligand et al. [86] and by Conti et al. [87], that NO production can lead to an increase in noradrenaline release, as a consequence of adrenergic receptors activation, namely of facilitatory β-adrenoceptors.

#### 4. Current and future developments

In addition to the direct effects exerted by several substances on smooth muscle cells, which can cause vasodilation or vasoconstriction, the evidence that endothelium-derived factors can also influence sympathetic neurotransmission that reinforces the importance of endothelium and of its putative role in pathologies. Indeed, vascular sympathetic neurotransmission and the interplay exerted by endothelium-derived substances are, therefore, relevant in the homeostasis of vascular tone. In pathophysiological conditions, especially when endothelium is injured, their impact on neurotransmission account, at least in part, for the occurring vasoconstriction.

NO has been viewed as a vasodilator substance since its direct effect on vascular smooth muscle cells causes dilation. However, NO can influence neurotransmission, and the interplay with adenosinergic and adrenergic pathways altering neurotransmission can, in some cases, cause an increase in noradrenaline release, which consequently will promote vasoconstriction. Therefore, the importance of NO is renewed as well as its ability to interplay with other signaling pathways involving sympathetic regulation such as the adrenergic and adenosinergic ones. Additional information and research on this field are, therefore, required to extend the knowledge on the insights of transsynaptic modulation of vascular neurotransmission. This is particularly important and can be useful to develop new therapeutic strategies, particularly in pathologies or clinical conditions, where the sympathetic system is hyperactivated.
