**6. High D-glucose and expression and activity of L-arginine/NO pathway**

Hyperglycemia and diabetes mellitus are pathological conditions associated with fetal endothelial dysfunction [55] and type 2 diabetes mellitus (T2DM) [57] or cardiovascular disease (CVD) [58]. CVD in patients with diabetes mellitus is associated with the generation of ROS.

High concentration of D-glucose (25 mM) increases L-arginine transport and cGMP accumulation in endothelium in a similar manner to that observed in HUVEC from pregnancies with gestational diabetes [33, 59]. Increased L-arginine transport in response to incubation with high D-glucose has been related to increased mRNA levels for the hCAT-1 and eNOS activity in HUVEC [60]. In human aortic endothelial cells, prolonged incubation (7 days) with 25 mM D-glucose induces a decrease in eNOS activity (determined by nitrite content), protein abundance, and mRNA level. This effect is associated with a decrease in eNOS promoter activity [61]. In bovine aortic endothelial cells (BAECs), there is a lower production of insulin-induced NO when the cells were incubated with high extracellular concentration of D-glucose, an effect that seems to depend on a signaling pathway that involves to the type 1 insulin receptor (IR-1), phosphatidyl inositol 3 kinase, and the inhibitor of nuclear factor kappa-B kinase [62]. On the other hand, the increase of cGMP production induced by high D-glucose in HUVEC is blocked by incubating the cells with 1 nM insulin [63]. Incubation with 1 nM insulin (8 h) has been shown in this same cell type to be sufficient to block the effect that D-glucose has on the decreased transport of adenosine [64], an important vasoactive nucleoside [65].

In HUVEC, high extracellular D-glucose increases L-arginine transport, NO synthesis, and O2 .− generation through eNOS and NADPH oxidase activation. Additionally, high D-glucose increased the contractile response in the human umbilical vein. Insulin reversed these effects of high D-glucose, leading to normal hCAT-1 expression, NO synthesis, ROS generation, and vascular tone. Insulin acts like antioxidant molecules (like tempol, ascorbic acid) to restore high D-glucoseincreased oxidative stress in the fetoplacental vascular bed [66]. High D-glucose increases L-arginine transport, likely resulting from higher hCAT-1 expression and protein abundance in the plasma membrane. This mechanism could be an adaptive response of HUVEC to higher ROS generation from high D-glucose-activated

**53**

*L-Arginine/Nitric Oxide Pathway and KCa Channels in Endothelial Cells: A Mini-Review*

NADPH oxidase. In parallel, high D-glucose increased NO synthesis. Insulin reversed the high D-glucose-mediated alterations in L-arginine transport involving the modulation of *SLC7A1* gene expression, leading to altered umbilical vein reactivity. Modulation of hCAT-1 expression and activity by insulin is the key to maintaining umbilical vein tone and endothelial function in physiologic and pathophysiological

*Endothelial dysfunction induced by high D-glucose and protection by insulin in HUVEC. Exposure of HUVEC to high D-glucose leads to an increase (↑) in the plasma membrane abundance of the human cationic amino acid transporter 1 (hCAT-1) and higher L-arginine uptake. High D-glucose activates NADPH oxidase, leading* 

*.−. Insulin restores ROS and O2*

*to 5 mM D-glucose (normal), resulting in the restoration of hCAT-1-mediated L-arginine transport and nitric oxide (NO) synthesis. High D-glucose and insulin also activate the* SLC7A1 *promoter region (coding for* 

*restores hCAT-1 protein abundance and its distribution in the cells via an NADPH oxidase-independent* 

*.− generation to values in cells exposed* 

*.− generation. In addition, insulin* 

Another important mechanism that regulates the endothelial function is the activity of ion channels that modulate the cell membrane potential. The calciumactivated potassium channels (KCa) have been shown to be relevant to induce the necessary hyperpolarization to stimulate the relaxation of vascular smooth muscle cells (related with EDHF). In systemic circulation, large conductance KCa (BKCa) channels have been shown preferentially expressed in VSMC, meanwhile small (SKCa) and intermediate (IKCa) conductance KCa are preferentially expressed in endothelium [67, 68]. However, potassium currents inhibited by iberiotoxin (BKCa inhibitor) have been described in HUVEC stimulated by sildenafil or insulin [69]. In fact, insulin (10 nM) can directly activate native and recombinant BKCa currents in cell-attached patch-clamping experiments with a rapid effect that is MAPK-dependent when the hormone was added in the pipette [70]. There is evidence that insulin may induce endothelial cell hyperpolarization by modulating K channels activity [38, 71]. The insulin-induced relaxation in human placental veins (~368 μm diameter), pre-constricted with U46619, is a mechanism dependent on the BKCa channel activity. The co-incubation of vessels with genistein (tyrosine kinases inhibitor) and wortmannin (PI3K inhibitor) did not block the insulin's relaxation, and by contrast potentiated the insulin-induced

**7. Role of potassium channels in endothelial function**

*hCAT-1) up to −650 bp from the ATG via a mechanism involving ROS and O2*

*DOI: http://dx.doi.org/10.5772/intechopen.93400*

conditions (**Figure 3**) [66].

*to higher generation of ROS, including O2*

*mechanism (data from González et al. [66]).*

**Figure 3.**

*L-Arginine/Nitric Oxide Pathway and KCa Channels in Endothelial Cells: A Mini-Review DOI: http://dx.doi.org/10.5772/intechopen.93400*

#### **Figure 3.**

*Vascular Biology - Selection of Mechanisms and Clinical Applications*

cardiovascular disease (CVD).

binding in C6 rat glioma cells [56].

and cardiovascular diseases.

associated with the generation of ROS.

important vasoactive nucleoside [65].

synthesis, and O2

**pathway**

and the reduction of the promoter activity of this gene could be associated with

On the other hand, the first intron of *SLC7A1* may play a bifunctional role in regulating the *SLC7A1* transcriptional activity by the binding of the purine-rich element binding protein A (Pur alpha) in physiological conditions and by activating the transcription factor 4 (ATF4) in endoplasmic reticulum stress or by decreasing the *SLC7A1* transcriptional activity by the C/EBP homologous protein 10 (CHOP)

For the physiological regulation of hCAT-1 activity, both transcriptional regula-

tion of *SLC7A1* and/or posttranscriptional regulation of *SLC7A1* transcript are relevant for the protein expression and L-arginine transport [55]. Insulin increases the expression of *SLC7A1* gene due to an increased transcriptional activity, most likely due to higher Sp1 activity. So, hCAT-1 expression and activity are regulated by insulin in endothelium, suggesting that in insulin resistance there is a reduction of L-arginine transport and NO synthesis that contributes to endothelial dysfunction

**6. High D-glucose and expression and activity of L-arginine/NO** 

Hyperglycemia and diabetes mellitus are pathological conditions associated with fetal endothelial dysfunction [55] and type 2 diabetes mellitus (T2DM) [57] or cardiovascular disease (CVD) [58]. CVD in patients with diabetes mellitus is

High concentration of D-glucose (25 mM) increases L-arginine transport and cGMP accumulation in endothelium in a similar manner to that observed in HUVEC from pregnancies with gestational diabetes [33, 59]. Increased L-arginine transport in response to incubation with high D-glucose has been related to increased mRNA levels for the hCAT-1 and eNOS activity in HUVEC [60]. In human aortic endothelial cells, prolonged incubation (7 days) with 25 mM D-glucose induces a decrease in eNOS activity (determined by nitrite content), protein abundance, and mRNA level. This effect is associated with a decrease in eNOS promoter activity [61]. In bovine aortic endothelial cells (BAECs), there is a lower production of insulin-induced NO when the cells were incubated with high extracellular concentration of D-glucose, an effect that seems to depend on a signaling pathway that involves to the type 1 insulin receptor (IR-1), phosphatidyl inositol 3 kinase, and the inhibitor of nuclear factor kappa-B kinase [62]. On the other hand, the increase of cGMP production induced by high D-glucose in HUVEC is blocked by incubating the cells with 1 nM insulin [63]. Incubation with 1 nM insulin (8 h) has been shown in this same cell type to be sufficient to block the effect that D-glucose has on the decreased transport of adenosine [64], an

In HUVEC, high extracellular D-glucose increases L-arginine transport, NO

Additionally, high D-glucose increased the contractile response in the human umbilical vein. Insulin reversed these effects of high D-glucose, leading to normal hCAT-1 expression, NO synthesis, ROS generation, and vascular tone. Insulin acts like antioxidant molecules (like tempol, ascorbic acid) to restore high D-glucoseincreased oxidative stress in the fetoplacental vascular bed [66]. High D-glucose increases L-arginine transport, likely resulting from higher hCAT-1 expression and protein abundance in the plasma membrane. This mechanism could be an adaptive response of HUVEC to higher ROS generation from high D-glucose-activated

.− generation through eNOS and NADPH oxidase activation.

**52**

NADPH oxidase. In parallel, high D-glucose increased NO synthesis. Insulin reversed the high D-glucose-mediated alterations in L-arginine transport involving the modulation of *SLC7A1* gene expression, leading to altered umbilical vein reactivity. Modulation of hCAT-1 expression and activity by insulin is the key to maintaining umbilical vein tone and endothelial function in physiologic and pathophysiological conditions (**Figure 3**) [66].

### **7. Role of potassium channels in endothelial function**

Another important mechanism that regulates the endothelial function is the activity of ion channels that modulate the cell membrane potential. The calciumactivated potassium channels (KCa) have been shown to be relevant to induce the necessary hyperpolarization to stimulate the relaxation of vascular smooth muscle cells (related with EDHF). In systemic circulation, large conductance KCa (BKCa) channels have been shown preferentially expressed in VSMC, meanwhile small (SKCa) and intermediate (IKCa) conductance KCa are preferentially expressed in endothelium [67, 68]. However, potassium currents inhibited by iberiotoxin (BKCa inhibitor) have been described in HUVEC stimulated by sildenafil or insulin [69]. In fact, insulin (10 nM) can directly activate native and recombinant BKCa currents in cell-attached patch-clamping experiments with a rapid effect that is MAPK-dependent when the hormone was added in the pipette [70]. There is evidence that insulin may induce endothelial cell hyperpolarization by modulating K channels activity [38, 71]. The insulin-induced relaxation in human placental veins (~368 μm diameter), pre-constricted with U46619, is a mechanism dependent on the BKCa channel activity. The co-incubation of vessels with genistein (tyrosine kinases inhibitor) and wortmannin (PI3K inhibitor) did not block the insulin's relaxation, and by contrast potentiated the insulin-induced

*Endothelial dysfunction induced by high D-glucose and protection by insulin in HUVEC. Exposure of HUVEC to high D-glucose leads to an increase (↑) in the plasma membrane abundance of the human cationic amino acid transporter 1 (hCAT-1) and higher L-arginine uptake. High D-glucose activates NADPH oxidase, leading to higher generation of ROS, including O2 .−. Insulin restores ROS and O2 .− generation to values in cells exposed to 5 mM D-glucose (normal), resulting in the restoration of hCAT-1-mediated L-arginine transport and nitric oxide (NO) synthesis. High D-glucose and insulin also activate the* SLC7A1 *promoter region (coding for hCAT-1) up to −650 bp from the ATG via a mechanism involving ROS and O2 .− generation. In addition, insulin restores hCAT-1 protein abundance and its distribution in the cells via an NADPH oxidase-independent mechanism (data from González et al. [66]).*

#### **Figure 4.**

*Proposal of mechanism for KCa activation by insulin. Evidence obtained in endothelial cells (ECs) shows that insulin activates KCa (mainly BKCa) in a mechanism still not fully understood. The activation of KCa by insulin induces hyperpolarization (↑ΔV), leading to activation of eNOS for NO synthesis from L-arginine uptake by hCAT-1 (modified from Rojas et al. [71]).*

vasodilation. Also, insulin decreased perfusion pressure (34 ± 3%) in the isolated cotyledon of normal placenta with a basal perfusion pressure of 64 ± 5 mmHg (or pre-constricted with U46619) [72]. The effects of insulin on BKCa activity are associated with evidences that show that the constriction induced by U46619 and H2O2 in placental vasculature is partially decreased with 10 nM insulin preincubation (10 min) in a mechanism totally dependent of BKCa activity [72]. Recently, it has been determined that insulin-mediated NO synthesis requires the participation of both IKCa/ BKCa channels and eNOS activity in HUVECs [71]. In the same cell type, insulin increased the open probability (NPo) of BKCa, associated with hyperpolarization in single cell analysis [69]. In human placental arteries, the relaxation induced by the NO donor, SNAP, is partially blocked by charybdotoxin (BKCa inhibitor) and almost totally blocked by charybdotoxin and ODQ (sGC inhibitor) [73]. Therefore, an extracellular stimulus that increases the NO availability activates a mechanism that involves sGC and BKCa activities [71]. These findings constitute evidence for postulating a new mechanism induced by insulin in human vasculature related with the physiological regulation of KCa activity for NO synthesis (**Figure 4**).

### **8. Final remarks**

The relevance of the endothelium for cardiovascular physiology is well established, mainly by findings related to the capacity of endothelial cells to synthesize NO and regulate the plasma membrane potential of smooth muscle cells. **Figure 5** shows a graphical summary of the L-arginine/NO pathway in the human blood vessels that highlight the capacity of endothelial cells to respond to extracellular stimuli and translate the mechanical forces and endocrine signals to intracellular mechanisms leading to NO synthesis and activation of potassium channels. It is important to note that the subcellular distribution of hCAT-1 and eNOS is also relevant for endothelial cells function. In physiological state, hCAT-1 colocalizes with caveolin-1 in the plasma membrane caveolae in proximity to eNOS.

**55**

**Acknowledgements**

**Figure 5.**

Elbakyan for support the open science.

*L-Arginine/Nitric Oxide Pathway and KCa Channels in Endothelial Cells: A Mini-Review*

The authors would like to acknowledge the staff at Laboratorio de Investigación Materno-Fetal (LIMaF) and Department of Obstetrics and Gynecology from the Universidad de Concepción. The authors express special thanks to Alexandra

*Role of endothelium in the regulation of vascular tone. Endothelial cells, as a part of blood vessels walls, respond to mechanical stress induced by flow (shear stress) by activation of L-arginine/NO pathway to induce the NO release and relaxation of smooth muscle cells (SMCs). Subcellular localization of hCAT-1 in caveolae is relevant for its function, and the role of potassium channels (BKCa, mainly) has been recently described as important for endothelial cells function. The activity of the endothelium is regulated by different agonists like acetylcholine (Ach) through plasma membrane receptor (AchR) and others like insulin or serotonin, etc.*

*DOI: http://dx.doi.org/10.5772/intechopen.93400*

*L-Arginine/Nitric Oxide Pathway and KCa Channels in Endothelial Cells: A Mini-Review DOI: http://dx.doi.org/10.5772/intechopen.93400*

#### **Figure 5.**

*Vascular Biology - Selection of Mechanisms and Clinical Applications*

vasodilation. Also, insulin decreased perfusion pressure (34 ± 3%) in the isolated cotyledon of normal placenta with a basal perfusion pressure of 64 ± 5 mmHg (or pre-constricted with U46619) [72]. The effects of insulin on BKCa activity are associated with evidences that show that the constriction induced by U46619 and H2O2 in placental vasculature is partially decreased with 10 nM insulin preincubation (10 min) in a mechanism totally dependent of BKCa activity [72]. Recently, it has been determined that insulin-mediated NO synthesis requires the participation of both IKCa/ BKCa channels and eNOS activity in HUVECs [71]. In the same cell type, insulin increased the open probability (NPo) of BKCa, associated with hyperpolarization in single cell analysis [69]. In human placental arteries, the relaxation induced by the NO donor, SNAP, is partially blocked by charybdotoxin (BKCa inhibitor) and almost totally blocked by charybdotoxin and ODQ (sGC inhibitor) [73]. Therefore, an extracellular stimulus that increases the NO availability activates a mechanism that involves sGC and BKCa activities [71]. These findings constitute evidence for postulating a new mechanism induced by insulin in human vasculature related with the physiological regulation of KCa activity for

*Proposal of mechanism for KCa activation by insulin. Evidence obtained in endothelial cells (ECs) shows that insulin activates KCa (mainly BKCa) in a mechanism still not fully understood. The activation of KCa by insulin induces hyperpolarization (↑ΔV), leading to activation of eNOS for NO synthesis from L-arginine* 

The relevance of the endothelium for cardiovascular physiology is well established, mainly by findings related to the capacity of endothelial cells to synthesize NO and regulate the plasma membrane potential of smooth muscle cells. **Figure 5** shows a graphical summary of the L-arginine/NO pathway in the human blood vessels that highlight the capacity of endothelial cells to respond to extracellular stimuli and translate the mechanical forces and endocrine signals to intracellular mechanisms leading to NO synthesis and activation of potassium channels. It is important to note that the subcellular distribution of hCAT-1 and eNOS is also relevant for endothelial cells function. In physiological state, hCAT-1 colocalizes with caveolin-1 in the

**54**

NO synthesis (**Figure 4**).

*uptake by hCAT-1 (modified from Rojas et al. [71]).*

plasma membrane caveolae in proximity to eNOS.

**8. Final remarks**

**Figure 4.**

*Role of endothelium in the regulation of vascular tone. Endothelial cells, as a part of blood vessels walls, respond to mechanical stress induced by flow (shear stress) by activation of L-arginine/NO pathway to induce the NO release and relaxation of smooth muscle cells (SMCs). Subcellular localization of hCAT-1 in caveolae is relevant for its function, and the role of potassium channels (BKCa, mainly) has been recently described as important for endothelial cells function. The activity of the endothelium is regulated by different agonists like acetylcholine (Ach) through plasma membrane receptor (AchR) and others like insulin or serotonin, etc.*

### **Acknowledgements**

The authors would like to acknowledge the staff at Laboratorio de Investigación Materno-Fetal (LIMaF) and Department of Obstetrics and Gynecology from the Universidad de Concepción. The authors express special thanks to Alexandra Elbakyan for support the open science.

*Vascular Biology - Selection of Mechanisms and Clinical Applications*
