Cellular mechanisms and intracellular signaling pathways for the modulation of eNOS in pulmonary arteries by 15-HETE

The 15-hydroxyeicosatetraenoic acid (15-HETE), a lipid metabolite and vasoconstrictor, plays an important role in hypoxic contraction of pulmonary arteries (PAs) through working on smooth muscle cells (SMCs). Previous studies have shown that vascular endothelium is also involved in PAs tone regulation. However, little is known as to how the pulmonary artery endothelial cells (PAECs) are related to the 15-HETE-induced vasoconstriction and that which intracellular signaling systems are critical. To test this hypothesis, we examined PAs constriction in isolated rat PAs rings, the expression and activity of endothelial nitric oxide synthase (eNOS) with western blot, and nitric oxide (NO) production using the DAF-FM DA fluorescent indicator. The results showed that the 15-HETE-induced PAs constriction was diminished in endothelium-intact rings. In the presence of the eNOS inhibitor L-NAME, vasoconstrictor responses to KCl were greater than the control. The activation of eNOS was activated by Ca2? released from intracellular stores and the PI3K/Akt pathway. Phosphorylations of the eNOS at Ser-1177 and Akt at Ser-473 were necessary for their activity. A prolonged 15-HETE treatment (30 min) led to a decrease in NO production by phosphorylation of eNOS at Thr-495, leading to augmentation of PAs constriction. Therefore, 15-HETE initially inhibited the PAs constriction through the endothelial NO system, and both Ca2? and the PI3K/Akt signaling systems are required for the effects of 15-HETE on PAs tone regulation.     

15-羟二十碳四烯酸下调肺动脉内皮型一氧化氮合酶的活性

15-羟二十碳四烯酸（15-hydroxyeicosatetraenoic acid,15-HETE）在低氧性肺血管收缩中起着重要作用,低氧肺动脉高压下调内皮型。氧化氮合酶（endothelial nitric oxide synthase,eNOS）,使一氧化氮（nitric oxide,NO）的产量下降,但目前尚无关于15-HETE与eNOS／NO相互作用研究的报道。我们通过Wistar大鼠肺动脉环张力、牛肺动脉内皮细胞NO产量、总eNOS表达及eNOS磷酸化测定等方法对15-HETE与eNOS／NO的相互作用进行研究。首先分离人鼠肺动脉,分为eNOS抑制剂L-NAME组（0．1mmol／L）、去缸管内皮组与内皮完整组,用15-HETE作用夫鼠离体肺动脉环,测定肺动脉张力。结果表明,L-NAME组、去除内皮组与内皮完整组分别比较,15-HETE对血管的收缩作用增强,且都有统计学意义（P〈0．05）。培养牛肺动脉内皮细胞,分别用15-HETE、15-脂氧酶（15-lipoxygenase,15-LO）抑制剂[（cinnamyl 3,4-dihydroxy-[alpha]-cyanocinnamate,CDC）和（nordihydroguiairetic acid,YDGA）]处理细胞,通过Greiss方法检测亚硝酸盐含量,间接测定NO产量,与对照组比较,1μmol／L 15-HETE明显降低肺动脉内皮细胞NO水平（P〈0．05）,10μmol／L CDC和0．1mmol／L NDGA显著增加NO水平（分别是P〈0．05,P〈0．01）;通过Western blot检测不同时间（5,10,15,20,30,60min）eNOS的表达情况,结果显示,15-HETE的不同作用时间,没有引起eNOS表达的明显不同;用苏氨酸495位点磷酸化eNOS（Thr495）抗体进行免疫沉淀,再用总eNOS抗体和15-LO抗体通过Western blot检测磷酸化型含量,问接测定eNOS活性,结果表明15-HETE增强Thr495磷酸化型eNOS含量。由于Thr495为eNOS抑制性磷酸化位点,因此15-HETE降低eNOS活性。这些数据表明：15-HETE的缩血管作用有eNOS／NO参与,15-HETE可以通过磷酸化Thr495位点降低eNOS活性,并且首次发现磷酸化eNOS（Thr495）和15-LO之间存在蛋白质相互作用。     

20-Hydroxyeicosatetraenoic Acid Impairs Endothelial Insulin Signaling by Inducing Phosphorylation of the Insulin Receptor Substrate-1 at Ser616

20-hydroxyeicosatetraenoic acid (20-HETE) induces endothelial dysfunction and is correlated with diabetes. This study was designed to investigate the effects of 20-HETE on endothelial insulin signaling.Human umbilical vein endothelial cells (HUVECs) or C57BL/6J mice were treated with 20-HETE in the presence or absence of insulin, and p-ERK1/2, p-JNK, IRS-1/PI3K/AKT/eNOS pathway, were examined in endothelial cells and aortas by immunoblotting. eNOS activity and nitric oxide production were measured. 20-HETE increased ERK1/2 phosphorylation and IRS-1 phosphorylation at Ser⁶¹⁶; these effects were reversed by ERK1/2 inhibition. We further observed that 20-HETE treatment resulted in impaired insulin-stimulated IRS-1 phosphorylation at Tyr⁶³² and subsequent PI3-kinase/Akt activation. Furthermore, 20-HETE treatment blocked insulin-stimulated phosphorylation of eNOS at the stimulatory Ser¹¹⁷⁷ site, eNOS activation and NO production; these effects were reversed by inhibiting ERK1/2. Treatment of C57BL/6J mice with 20-HETE resulted in ERK1/2 activation and impaired insulin-dependent activation of the IRS-1/PI3K/Akt/eNOS pathway in the aorta. Our data suggest that the 20-HETE activation of IRS-1 phosphorylation at Ser⁶¹⁶ is dependent on ERK1/2 and leads to impaired insulin-stimulated vasodilator effects that are mediated by the IRS-1/PI3K/AKT/eNOS pathway.     

The EGCg-induced redox-sensitive activation of endothelial nitric oxide synthase and relaxation are critically dependent on hydroxyl moieties

Several rich sources of polyphenols stimulate the endothelial formation of nitric oxide (NO), a potent vasoprotecting factor, via the redox-sensitive activation of the PI3-kinase/Akt pathway leading to the phosphorylation of endothelial NO synthase (eNOS). The present study examined the molecular mechanism underlying the stimulatory effect of epicatechins on eNOS. NO-mediated relaxation was assessed using porcine coronary artery rings in the presence of indomethacin, and charybdotoxin plus apamin, inhibitors of cyclooxygenases and EDHF-mediated responses, respectively. The phosphorylation level of Akt and eNOS was assessed in cultured coronary artery endothelial cells by Western blot, and ROS formation using dihydroethidine. (−)-Epigallocatechin-3-O-gallate (EGCg) caused endothelium-dependent relaxations in coronary artery rings and the phosphorylation of Akt and eNOS in endothelial cells. These responses were inhibited by membrane-permeant analogues of superoxide dismutase and catalase, whereas native superoxide dismutase, catalase and inhibitors of major enzymatic sources of reactive oxygen species including NADPH oxidase, xanthine oxidase, cytochrome P450 and the mitochondrial respiration chain were without effect. The EGCg derivative with all hydroxyl functions methylated induced neither relaxations nor the intracellular formation of ROS, whereas both responses were observed when the hydroxyl functions on the gallate moiety were present. In conclusion, EGCg causes endothelium-dependent NO-mediated relaxations of coronary artery rings through the Akt-dependent activation of eNOS in endothelial cells. This response is initiated by the intracellular formation of superoxide anions and hydrogen peroxide, and is critically dependent on the gallate moiety and on the presence of hydroxyl functions possibly through intracellular auto-oxidation.     

Nitric oxide contributes to 20-HETE-induced relaxation of pulmonary arteries.

In contrast to its constrictor effects on peripheral arteries, 20-hydroxyeicosatetraenoic acid (20-HETE) is an endothelial-dependent dilator of pulmonary arteries (PAs). The present study examined the hypothesis that the vasodilator effects of 20-HETE in PAs are due to an elevation of intracellular calcium concentration ([Ca(2+)](i)) and the release of nitric oxide (NO) from bovine PA endothelial cells (BPAECs). BPAECs express cytochrome P-450 4A (CYP4A) protein and produce 20-HETE. 20-HETE dilated PAs preconstricted with U-46619 or norepinephrine and treated with the cytochrome P-450 inhibitor 17-octadecynoic acid and the cyclooxygenase inhibitor indomethacin. The dilator effect of 20-HETE was blocked by the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) or by removal of endothelium. 20-HETE significantly increased [Ca(2+)](i) and NO production in BPAECs. 20-HETE-induced NO release was blunted by removal of extracellular calcium, as well as NO synthase inhibitors (L-NAME). These results suggest that 20-HETE dilates PAs at least in part by increasing [Ca(2+)](i) and NO release in BPAECs.     

Acute activation and phosphorylation of endothelial nitric oxide synthase by HMG-CoA reductase inhibitors

3-Hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, statins, provide beneficial effects independent of their lipid-lowering effects. One beneficial effect appears to involve acute activation of endothelial nitric oxide (NO) synthase (eNOS) and increased NO release. However, the mechanism of acute statin-stimulated eNOS activation is unknown. Therefore, we hypothesized that eNOS activation may be coupled to altered eNOS phosphorylation. Bovine aortic endothelial cells (BAECs), passages 2-6, were treated with either lovastatin or pravastatin from 0 to 30 min. eNOS phosphorylation was examined by Western blot by use of phosphospecific antibodies for Ser-1179, Ser-635, Ser-617, Thr-497, and Ser-116. Statin stimulation of BAECs increased eNOS phosphorylation at Ser-1179 and Ser-617, which was blocked by the phosphatidylinositol 3-kinase (PI3-kinase)/Akt inhibitor wortmannin, and at Ser-635, which was blocked by the protein kinase A (PKA) inhibitor KT-5720. Statin treatment of BAECs transiently increased NO release by fourfold, measured by cGMP accumulation, and was attenuated by N-nitro-l-arginine methyl ester, wortmannin, and KT-5720 but not by mevalonate. In conclusion, these data demonstrate that eNOS is acutely activated by statins independent of HMG-CoA reductase inhibition and that in addition to Ser-1179, eNOS phosphorylation at Ser-635 and Ser-617 through PKA and Akt, respectively, may explain, in part, a mechanism by which eNOS is activated in response to acute statin treatment.     

Signaling pathways involving the sodium pump stimulate NO production in endothelial cells

The cardiac steroid ouabain, a known inhibitor of the sodium pump (Na⁺,K⁺-ATPase), has been shown to release endothelin from endothelial cells when used at concentrations below those that inhibit the pump. The present study addresses the question of which signaling pathways are activated by ouabain in endothelial cells. Our findings indicate that ouabain, applied at low concentrations to human umbilical cord endothelial cells (HUAECs), induces a reaction cascade that leads to translocation of endothelial nitric oxide synthase (eNOS) and to activation of phosphatidylinositol 3-kinase (PI3K). These events are followed by phosphorylation of Akt (also known as protein kinase B, or PKB) and activation of eNOS by phosphorylation. This signaling pathway, which results in increased nitric oxide (NO) production in HUAECs, is inhibited by the PI3K-specific inhibitor LY294002. Activation of the reaction cascade is not due to endothelin-1 (ET-1) binding to the ET-1 receptor B (ET_B), since application of the ET_B-specific antagonist BQ-788 did not have any effect on Akt or eNOS phosphorylation. The results shown here indicate that ouabain binding to the sodium pump results in the activation of the proliferation and survival pathways involving PI3K, Akt activation, stimulation of eNOS, and production of NO in HUAECs. Together with results from previous publications, the current investigation implies that the sodium pump is involved in vascular tone regulation.     

Fo Shou San,an Ancient Chinese Herbal Decoction,Protects Endothelial Function through Increasing Endothelial Nitric Oxide Synthase Activity

Fo Shou San (FSS) is an ancient herbal decoction comprised of Chuanxiong Rhizoma (CR; Chuanxiong) and Angelicae Sinensis Radix (ASR; Danggui) in a ratio of 2∶3. Previous studies indicate that FSS promotes blood circulation and dissipates blood stasis, thus which is being used widely to treat vascular diseases. Here, we aim to determine the cellular mechanism for the vascular benefit of FSS. The treatment of FSS reversed homocysteine-induced impairment of acetylcholine (ACh)-evoked endothelium-dependent relaxation in aortic rings, isolated from rats. Like radical oxygen species (ROS) scavenger tempol, FSS attenuated homocysteine-stimulated ROS generation in cultured human umbilical vein endothelial cells (HUVECs), and it also stimulated the production of nitric oxide (NO) as measured by fluorescence dye and biochemical assay. In addition, the phosphorylation levels of both Akt kinase and endothelial NO synthases (eNOS) were markedly increased by FSS treatment, which was abolished by an Akt inhibitor triciribine. Likewise, triciribine reversed FSS-induced NO production in HUVECs. Finally, FSS elevated intracellular Ca²⁺ levels in HUVECs, and the Ca²⁺ chelator BAPTA-AM inhibited the FSS-stimulated eNOS phosphorylation. The present results show that this ancient herbal decoction benefits endothelial function through increased activity of Akt kinase and eNOS; this effect is causally via a rise of intracellular Ca²⁺ and a reduction of ROS.     

20-hydroxyeicosatetraenoic acid is a vasoconstrictor in the newborn piglet pulmonary microcirculation

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome p-450 metabolite of arachidonic acid, is a vasoconstrictor in the systemic circulation and a vasodilator in the adult pulmonary circulation. Little is known about the vasoactive properties of 20-HETE in the newborn pulmonary circulation. The objectives of this study were to determine the vascular effects of 20-HETE and to explore the signaling mechanism(s) that mediate these effects in newborn pulmonary resistance-level arteries (PRA). Our findings demonstrate that, in contrast to the adult pulmonary circulation where 20-HETE mediates vasodilation, it causes constriction in newborn PRA at resting tone. Furthermore, inhibition of cyclooxygenase (COX) with indomethacin augments 20-HETE-induced constriction. The enhanced constrictor response to 20-HETE under conditions of COX inhibition is abolished in endothelium-disrupted PRA, suggesting that 20-HETE either stimulates endothelium-derived COX to release a counteracting vasodilator or is rapidly metabolized by COX to a less potent vasoconstrictor. 20-HETE-induced constriction is significantly inhibited by blocking calcium-dependent K(+) (K(Ca)) channels and the thromboxane-PGH(2) receptor. Altogether, our data indicate that the vascular actions of 20-HETE are partially mediated via the activation of K(Ca) channels and are significantly modulated by interactions with the COX-prostaglandin pathway.     

Acute Mechanical Stretch Promotes eNOS Activation in Venous Endothelial Cells Mainly via PKA and Akt Pathways

In the vasculature, physiological levels of nitric oxide (NO) protect against various stressors, including mechanical stretch. While endothelial NO production in response to various stimuli has been studied extensively, the precise mechanism underlying stretch-induced NO production in venous endothelial cells remains incompletely understood. Using a model of continuous cellular stretch, we found that stretch promoted phosphorylation of endothelial NO synthase (eNOS) at Ser¹¹⁷⁷, Ser⁶³³ and Ser⁶¹⁵ and NO production in human umbilical vein endothelial cells. Although stretch activated the kinases AMPKα, PKA, Akt, and ERK1/2, stretch-induced eNOS activation was only inhibited by kinase-specific inhibitors of PKA and PI3K/Akt, but not of AMPKα and Erk1/2. Similar results were obtained with knockdown by shRNAs targeting the PKA and Akt genes. Furthermore, inhibition of PKA preferentially attenuated eNOS activation in the early phase, while inhibition of the PI3K/Akt pathway reduced eNOS activation in the late phase, suggesting that the PKA and PI3K/Akt pathways play distinct roles in a time-dependent manner. Finally, we investigated the role of these pathways in stretch-induced endothelial exocytosis and leukocyte adhesion. Interestingly, we found that inhibition of the PI3K/Akt pathway increased stretch-induced Weibel-Palade body exocytosis and leukocyte adhesion, while inhibition of the PKA pathway had the opposite effects, suggesting that the exocytosis-promoting effect of PKA overwhelms the inhibitory effect of PKA-mediated NO production. Taken together, the results suggest that PKA and Akt are important regulators of eNOS activation in venous endothelial cells under mechanical stretch, while playing different roles in the regulation of stretch-induced endothelial exocytosis and leukocyte adhesion.     

Effects of epoxyeicosatrienoic acids on levels of eNOS phosphorylation and relevant signaling transduction pathways involved

Endothelial nitric oxide synthase (eNOS) is a key enzyme responsible for the regulation of vascular homeostasis. Many humor factors and mechanical forces can affect eNOS activity via phosphorylation modification but the mechanisms involved vary with stimuli applied. We have demonstrated that cytochrome P450 (CYP) epoxygenase-dependent metabolites of arachidonic acid, epoxyeicosatrienoic acids (EETs), can robustly up-regulate eNOS expression and its activity, however the relevant signaling pathways responsible for activity regulation are not well known. In this study, we explored the role of PI3 kinase (PI3K)/protein kinase B (Akt) signaling pathway in eNOS expression and its phosphorylation in response to EETs via direct addition of EETs into cultured bovine aorta endothelial cells (BAECs) and recombinant adeno-associated virus-mediated transfection of CYP epoxygenase genes CYPF87V and CYP2C11 to produce endogenous EETs followed by co-treatment with PI3K or Akt inhibitor. Results show that both exogenous and endogenous EETs could remarkably enhance eNOS expression and its phosphorylation at Ser1179 and Thr497 residues; PI3K inhibitor LY294002 could inhibit EETs-induced increase in eNOS-Ser(P)¹¹⁷⁹ but had no effect on the change of eNOS-Thr(P)⁴⁹⁷, while Akt inhibitor could attenuate the increase in phosphor-eNOS at both residues; both of the two inhibitors could block EETs-enhanced eNOS expression. These results lead to conclusions: (i) EETs-mediated regulation of eNOS activity may be related with the changes of phosphorylation level at eNOS-Ser¹¹⁷⁹ via PI3K/Akt and eNOS-Thr⁴⁹⁷ via Akt; (ii) PI3K/Akt signaling pathway is involved in the up-regulation of eNOS expression by EETs.     

Stimulated HSP90 binding to eNOS and activation of the PI3-Akt pathway contribute to globular adiponectin-induced NO production: vasorelaxation in response to globular adiponectin

The present study examined potential interactions between endothelial NO synthase (eNOS), heat shock protein (HSP)90, and Akt in vascular endothelial cells stimulated with globular adiponectin to produce nitric oxide (NO). Globular adiponectin-induced eNOS phosphorylation was accompanied by eNOS-HSP90-Akt complex formation, resulting in a dose-dependent increase in NO release. Globular adiponectin stimulated binding of HSP90 to eNOS, and inhibition of HSP90 significantly suppressed globular adiponectin-stimulated NO release. Globular adiponectin also caused Akt phosphorylation, and inhibition of PI3 kinase significantly suppressed globular adiponectin-stimulated NO release. This study also examined whether globular adiponectin really induces endothelial-dependent vasodilation using rings from rat thoracic aorta. It was observed that globular adiponectin caused dose-dependent vasorelaxation in the aorta. These results indicate that stimulated HSP90 binding to eNOS and activation of the PI3-Akt pathway contribute to globular adiponectin-induced eNOS phosphorylation and NO production, and to endothelium-dependent vasorelaxation.     

Insulin induces Ca<Superscript>2+</Superscript> oscillations in white fat adipocytes via PI3K and PLC

Adipocytes of white adipose tissue are the cells maintaining glucose homeostasis in an organism, which is controlled by insulin. Insulin stimulates the translocation of glucose transporter GLUT4 from the cytosol into the cell membrane, as well as glucose transport and utilization in these cells. Here we show that insulin-induced [Ca²⁺]_i oscillations are supported by the two signaling pathways involving: (1) phosphoinositide 3-kinase (PI3K), protein kinase B (Akt/PKB), endothelial NO synthase (eNOS), nitric oxide (NO), and ryanodine receptor (RyR) and (2) phospholipase C (PLC) and inositol 3-phosphate receptor (IP₃R). Thus, the PI3K Akt/PKB signaling pathway initiates not only metabolic but also Ca²⁺-signaling pathways in response to insulin.     

Hydrogen peroxide activates endothelial nitric-oxide synthase through coordinated phosphorylation and dephosphorylation via a phosphoinositide 3-kinase-dependent signaling pathway.

Endothelial nitric-oxide synthase (eNOS) is an important component of vascular homeostasis. During vascular disease, endothelial cells are exposed to excess reactive oxygen species that can alter cellular phenotype by inducing various signaling pathways. In the current study, we examined the implications of H(2)O(2)-induced signaling for eNOS phosphorylation status and activity in porcine aortic endothelial cells. We found that H(2)O(2) treatment enhanced eNOS activity and NO bioactivity as determined by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline and cellular cGMP content. Concomitant with eNOS activation, H(2)O(2) also activated Akt, increased eNOS phosphorylation at Ser-1177, and decreased eNOS phosphorylation at Thr-495. H(2)O(2)-induced promotion of eNOS activity and modulation of the eNOS phosphorylation status at Ser-1177 and Thr-495 were significantly attenuated by selective inhibitors of Src kinase, the ErbB receptor family, and phosphoinositide 3-kinase (PI 3-K). We found that Akt activation, eNOS Ser-1177 phosphorylation, and eNOS activation by H(2)O(2) were calcium-dependent, whereas eNOS dephosphorylation at Thr-495 was not, suggesting a branch point in the signaling cascade downstream from PI 3-K. Consistent with this, overexpression of a dominant negative isoform of Akt inhibited H(2)O(2)-induced phosphorylation of eNOS at Ser-1177 but not dephosphorylation of eNOS at Thr-495. Together, these data indicate that H(2)O(2) promotes calcium-dependent eNOS activity through a coordinated change in the phosphorylation status of the enzyme mediated by Src- and ErbB receptor-dependent PI 3-K activation. In turn, PI 3-K mediates eNOS Ser-1177 phosphorylation via a calcium- and Akt-dependent pathway, whereas eNOS Thr-495 dephosphorylation does not involve calcium or Akt. This response may represent an attempt by endothelial cells to maintain NO bioactivity under conditions of enhanced oxidative stress.     

Cyclosporin-A Inhibits Constitutive Nitric Oxide Synthase Activity and Neuronal and Endothelial Nitric Oxide Synthase Expressions after Spinal Cord Injury in Rats

Nitric oxide (NO) plays a role in the pathophysiology of spinal cord injury (SCI). NO is produced by three types of nitric oxide synthase (NOS) enzymes: The constitutive Ca²⁺/calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms, and the inducible calcium-independent isoform (iNOS). During the early stages of SCI, nNOS and eNOS produce significant amounts of NO, therefore, the regulation of their activity and expression may participate in the damage after SCI. In the present study, we used Cyclosporin-A (CsA) to further substantiate the role of Ca-dependent NOS in neural responses associated to SCI. Female Wistar rats were subjected to SCI by contusion, and killed 4 h after lesion. Results showed an increase in the activity of constitutive NOS (cNOS) after lesion, inhibited by CsA (2.5 mg/kg i.p.). Western blot assays showed an increased expression of both nNOS and eNOS after trauma, also antagonized by CsA administration.     

Reciprocal phosphorylation and regulation of endothelial nitric-oxide synthase in response to bradykinin stimulation

Endothelial nitric-oxide synthase (eNOS) is phosphorylated at Ser-1179 (bovine sequence) by Akt after growth factor or shear stress stimulation of endothelial cells, resulting in increased eNOS activity. Purified eNOS is also phosphorylated at Thr-497 by purified AMP-activated protein kinase, resulting in decreased eNOS activity. We investigated whether bradykinin (BK) stimulation of bovine aortic endothelial cells (BAECs) regulates eNOS through Akt activation and Ser-1179 or Thr-497 phosphorylation. Akt is transiently activated in BK-stimulated BAECs. Activation is blocked completely by wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase, suggesting that Akt activation occurs downstream from phosphatidylinositol 3-kinase. BK stimulates a transient phosphorylation of eNOS at Ser-1179 that is correlated temporally with a transient dephosphorylation of eNOS at Thr-497. Phosphorylation at Ser-1179, but not dephosphorylation at Thr-497, is blocked by wortmannin and LY294002. BK also stimulates a transient nitric oxide (NO) release from BAECs with a time-course similar to Ser-1179 phosphorylation and Thr-497 dephosphorylation. NO release is not altered by wortmannin. BK-stimulated dephosphorylation of Thr-497 and NO release are blocked by the calcineurin inhibitor, cyclosporin A. These data suggest that BK activation of eNOS in BAECs primarily involves deinhibition of the enzyme through calcineurin-mediated dephosphorylation at Thr-497.     

Signaling pathway of nitric oxide production induced by ginsenoside Rb1 in human aortic endothelial cells: a possible involvement of androgen receptor

Ginsenosides have been shown to stimulate nitric oxide (NO) production in aortic endothelial cells. However, the signaling pathways involved have not been well studied in human aortic endothelial cells. The present study was designed to examine whether purified ginsenoside Rb1, a major active component of ginseng could actually induce NO production and to clarify the signaling pathway in human aortic endothelial cells. NO production was rapidly increased by Rb1. The rapid increase in NO production was abrogated by treatment with nitric oxide synthetase inhibitor, L-NAME. Rb1 stimulated rapid phosphorylation of Akt (Ser473), ERK1/2 (Thr202/Thr204) and eNOS (Ser1177). Rapid phosphorylation of eNOS (Ser1177) was prevented by SH-5, an Akt inhibitor or wortmannin, PI3-kinase inhibitor and partially attenuated by PD98059, an upstream inhibitor for ERK1/2. Interestingly, NO production and eNOS phosphorylation at Ser1177 by Rb1 were abolished by androgen receptor antagonist, nilutamide. The results suggest that PI3kinase/Akt and MEK/ERK pathways and androgen receptor are involved in the regulation of acute eNOS activation by Rb1 in human aortic endothelial cells.     

l-Theanine promotes nitric oxide production in endothelial cells through eNOS phosphorylation

Consumption of tea (Camellia sinensis) improves vascular function and is linked to lowering the risk of cardiovascular disease. Endothelial nitric oxide is the key regulator of vascular functions in endothelium. In this study, we establish that l-theanine, a non-protein amino-acid found in tea, promotes nitric oxide (NO) production in endothelial cells. l-theanine potentiated NO production in endothelial cells was evaluated using Griess reaction, NO sensitive electrode and a NO specific fluorescent probe (4-amino-5-methylamino-2',7'-difluororescein diacetate). l-Theanine induced NO production was partially attenuated in presence of l-NAME or l-NIO and completely abolished using eNOS siRNA. eNOS activation was Ca^2 + and Akt independent, as assessed by fluo-4AM and immunoblotting experiments, respectively and was associated with phosphorylation of eNOS Ser 1177. eNOS phosphorylation was inhibited in the presence of ERK1/2 inhibitor, PD-98059 and partially inhibited by PI3K inhibitor, LY-294002 and Wortmanin suggesting PI3K-ERK1/2 dependent pathway. Increased NO production was associated with vasodilation in ex ovo (chorioallantoic membrane) model. These results demonstrated that l-theanine administration in vitro activated ERK/eNOS resulting in enhanced NO production and thereby vasodilation in the artery. The results of our experiments are suggestive of l-theanine mediated vascular health benefits of tea.