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.     

Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase

Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylation coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated with eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its specificity for this site in both eNOS and the corresponding synthetic phosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Treatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases.     

AMP-activated protein kinase phosphorylation of endothelial NO synthase

The AMP-activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl-coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK co-immunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser-1177 in the presence of Ca2+-calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca2+-calmodulin, AMPK also phosphorylates eNOS at Thr-495 in the CaM-binding sequence, resulting in inhibition of eNOS activity but Thr-495 phosphorylation is unchanged during ischaemia. Phosphorylation of eNOS by the AMPK in endothelial cells and myocytes provides a further regulatory link between metabolic stress and cardiovascular function.     

Endoplasmic reticulum Ca2+ release modulates endothelial nitric-oxide synthase via extracellular signal-regulated kinase (ERK) 1/2-mediated serine 635 phosphorylation

Endothelial nitric-oxide synthase (eNOS) plays a central role in cardiovascular regulation. eNOS function is critically modulated by Ca(2+) and protein phosphorylation, but the interrelationship between intracellular Ca(2+) mobilization and eNOS phosphorylation is poorly understood. Here we show that endoplasmic reticulum (ER) Ca(2+) release activates eNOS by selectively promoting its Ser-635/633 (bovine/human) phosphorylation. With bovine endothelial cells, thapsigargin-induced ER Ca(2+) release caused a dose-dependent increase in eNOS Ser-635 phosphorylation, leading to elevated NO production. ER Ca(2+) release also promoted eNOS Ser-633 phosphorylation in mouse vessels in vivo. This effect was independent of extracellular Ca(2+) and selective to Ser-635 because the phosphorylation status of other eNOS sites, including Ser-1179 or Thr-497, was unaffected in thapsigargin-treated cells. Blocking ERK1/2 abolished ER Ca(2+) release-induced eNOS Ser-635 phosphorylation, whereas inhibiting protein kinase A or Ca(2+)/calmodulin-dependent protein kinase II had no effect. Protein phosphorylation assay confirmed that ERK1/2 directly phosphorylated the eNOS Ser-635 residue in vitro. Further studies demonstrated that ER Ca(2+) release-induced ERK1/2 activation mediated the enhancing action of purine or bradykinin receptor stimulation on eNOS Ser-635/633 phosphorylation in bovine/human endothelial cells. Mutating the Ser-635 to nonphosphorylatable alanine prevented ATP from activating eNOS in cells. Taken together, these studies reveal that ER Ca(2+) release enhances eNOS Ser-635 phosphorylation and function via ERK1/2 activation. Because ER Ca(2+) is commonly mobilized by agonists or physicochemical stimuli, the identified ER Ca(2+)-ERK1/2-eNOS Ser-635 phosphorylation pathway may have a broad role in the regulation of endothelial function.     

Akt activates NOS3 and separately restores barrier integrity in H2O2-stressed human cardiac microvascular endothelium

The integrity of microvascular endothelium is an important regulator of myocardial contractility. Microvascular barrier integrity could be altered by increased reactive oxygen species (ROS) stress seen within minutes after cardiac arrest resuscitation. Akt and its downstream target nitric oxide (NO) synthase (NOS)3 can protect barrier integrity during ROS stress, but little work has studied these oxidant stress responses in human cardiac microvascular endothelial cells (HCMVEC). We, therefore, studied how ROS affects barrier function and NO generation via Akt and its downstream target NOS3 in HCMVEC. HCMVEC exposed to 500 microM H2O2 had increased Akt phosphorylation within 10 min at both Ser-473 and Thr-308 sites, an effect blocked by the phosphatidylinositol 3-kinase inhibitor LY-294002. H2O2 also induced NO generation that was associated with NOS3 Ser-1177 site phosphorylation and Thr-495 dephosphorylation, with Ser-1177 effects attenuated by LY-294002 and an Akt inhibitor, Akt/PKB signaling inhibitor-2 (API-2). H2O2 induced significant barrier disruption in HCMVEC within minutes, but recovery started within 30 min and normalized over hours. The NOS inhibitor Nomega-nitro-L-arginine methyl ester (200 microM) blocked NO generation but had no effect on H2O2-induced barrier permeability or the recovery of barrier integrity. By contrast, the Akt inhibitor API-2 abrogated HCMVEC barrier restoration. These results suggest that oxidant stress in HCMVEC activates NOS3 via Akt. NOS3/NO are not involved in the regulation of H2O2-affected barrier function in HCMVEC. Independent of NOS3 regulation, Akt proves to be critical for the restoration of barrier integrity in HCMVEC.     

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.     

Decreased endothelial nitric-oxide synthase (eNOS) activity resulting from abnormal interaction between eNOS and its regulatory proteins in hypoxia-induced pulmonary hypertension

In the pulmonary artery isolated from 1-week hypoxia-induced pulmonary hypertensive rats, endothelial NO production stimulated by carbachol was decreased significantly in in situ visualization using diaminofluorescein-2 diacetate and also in cGMP content. This change was followed by the decrease in carbachol-induced endothelium-dependent relaxation. Protein expression of endothelial NO synthase (eNOS) and its regulatory proteins, caveolin-1 and heat shock protein 90, did not change in the hypoxic pulmonary artery, indicating that chronic hypoxia impairs eNOS activity at posttranslational level. In the hypoxic pulmonary artery, the increase in intracellular Ca(2+) level stimulated by carbachol but not by ionomycin was reduced. We next focused on changes in Ca(2+) sensitivity of the eNOS activation system. A morphological study revealed atrophy of endothelial cells and a peripheral condensation of eNOS in hypoxic endothelial cells preserving co-localization between eNOS and Golgi or plasma membranes. However, eNOS was tightly coupled with caveolin-1, and was dissociated from heat shock protein 90 or calmodulin in the hypoxic pulmonary artery in either the presence or absence of carbachol. Furthermore, eNOS Ser(1177) phosphorylation in both conditions significantly decreased without affecting Akt phosphorylation in the hypoxic artery. In conclusion, chronic hypoxia impairs endothelial Ca(2+) metabolism and normal coupling between eNOS and caveolin-1 resulted in eNOS inactivity.     

Hsp90 ensures the transition from the early Ca2+-dependent to the late phosphorylation-dependent activation of the endothelial nitric-oxide synthase in vascular endothelial growth factor-exposed endothelial cells.

Vascular endothelial growth factor (VEGF) exerts its angiogenic effects partly through the activation of endothelial nitric-oxide synthase (eNOS). Association with heat shock protein 90 (hsp90) and phosphorylation by Akt were recently shown to separately activate eNOS upon VEGF stimulation in endothelial cells. Here, we examined the interplay between these different mechanisms in VEGF-exposed endothelial cells. We documented that hsp90 binding to eNOS is, in fact, the crucial event triggering the transition from the Ca(2+)-dependent activation of eNOS to the phosphorylation-mediated potentiation of its activity by VEGF. Accordingly, we showed that early VEGF stimulation first leads to the Ca(2+)/calmodulin disruption of the caveolin-eNOS complex and promotes the association between eNOS and hsp90. eNOS-bound hsp90 can then recruit VEGF-activated (phosphorylated) Akt to the complex, which in turn can phosphorylate eNOS. Further experiments in transfected COS cells expressing either wild-type or S1177A mutant eNOS led us to identify the serine 1177 as the critical residue for the hsp90-dependent Akt-mediated activation of eNOS. Finally, we documented that although the VEGF-induced phosphorylation of eNOS leads to a sustained production of NO independently of a maintained increase in [Ca(2+)](i), this late stage of eNOS activation is strictly conditional on the initial VEGF-induced Ca(2+)-dependent stimulation of the enzyme. These data establish the critical temporal sequence of events leading to the sustained activation of eNOS by VEGF and suggest new ways of regulating the production of NO in response to this cytokine through the ubiquitous chaperone protein, hsp90.     

Activation of endothelial nitric-oxide synthase by the p38 MAPK in response to black tea polyphenols

Black tea improves endothelial function in patients with coronary artery disease. We sought to determine the responsible components of black tea and elucidate the underlying cell signaling mechanisms. We exposed porcine aortic endothelial cells to components of black tea and found that the polyphenol fraction acutely enhanced nitric oxide bioactivity. This effect involved endothelial nitric-oxide synthase (eNOS) phosphorylation at Ser-1177 and dephosphorylation at Thr-495, consistent with increased eNOS activity. These effects were calcium-dependent, as removal of extracellular calcium prevented eNOS phosphorylation at Ser-1177, whereas inhibition of intracellular calcium mobilization with TMB-8 blunted Thr-495 dephosphorylation. Black tea polyphenol-induced eNOS activation appeared dependent upon the phosphatidylinositol 3-kinase-Akt pathway, as it was significantly inhibited by LY294002 and a dominant negative Akt, respectively. Pharmacological inhibition of p38 mitogen-activated protein kinase (p38 MAPK) with either SB202190 or SB203580 as well as overexpression of a dominant negative p38 MAPKalpha attenuated both eNOS activation and phosphorylation changes in response to black tea polyphenols. Inhibition of p38 MAPKalpha also blunted Akt activation in response to black tea polyphenols, suggesting that p38alpha MAPK is upstream of Akt in this pathway. Finally, a constitutively active mutant of MKK6bE, an upstream kinase for p38 MAPK, enhanced both the basal and stimulated activity of Akt, leading to increased eNOS activity. Taken together, these data identify the p38 MAPK as an upstream component of Akt-mediated eNOS activation.     

Functional significance of cytosolic endothelial nitric-oxide synthase (eNOS): regulation of hyperpermeability

Endothelial NOS (eNOS)-derived NO is a key factor in regulating microvascular permeability. We demonstrated previously that eNOS translocation from the plasma membrane to the cytosol is required for hyperpermeability. Herein, we tested the hypothesis that eNOS activation in the cytosol is necessary for agonist-induced hyperpermeability. To study the fundamental properties of endothelial cell monolayer permeability, we generated ECV-304 cells that stably express cDNA constructs targeting eNOS to the cytosol or plasma membrane. eNOS-transfected ECV-304 cells recapitulate the eNOS translocation and permeability properties of postcapillary venular endothelial cells (Sánchez, F. A., Rana, R., Kim, D. D., Iwahashi, T., Zheng, R., Lal, B. K., Gordon, D. M., Meininger, C. J., and Durán, W. N. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 6849-6853). We used platelet-activating factor (PAF) as a proinflammatory agonist. PAF activated eNOS by increasing phosphorylation of Ser-1177 and inducing dephosphorylation of Thr-495, increasing NO production, and elevating permeability to FITC-dextran 70 in monolayers of cells expressing wild-type and cytosolic eNOS. PAF failed to increase permeability to FITC-dextran 70 in monolayers of cells transfected with eNOS targeted to the plasma membrane. Interestingly, this occurred despite eNOS Ser-1177 phosphorylation and production of comparable amounts of NO. Our results demonstrate that the presence of eNOS in the cytosol is necessary for PAF-induced hyperpermeability. Our data provide new insights into the dynamics of eNOS and eNOS-derived NO in the process of inflammation.     

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.     

Hydrogen peroxide restrains endothelium-derived nitric oxide bioactivity -- role for iron-dependent oxidative stress

Vascular diseases are characterized by impairment of endothelial-derived nitric oxide (NO) bioactivity and increased vascular levels of hydrogen peroxide (H(2)O(2)). Here we examined the implications of H(2)O(2) for agonist-stimulated endothelial NO bioactivity in rabbit aortic rings and cultured porcine aortic endothelial cells (PAEC). Vessels pre-treated with H(2)O(2) exhibited impaired endothelial-dependent relaxation induced by acetylcholine or calcium ionophore. In contrast, H(2)O(2) had no effect on endothelium-independent relaxation induced by a NO donor, indicating a defect in endothelium-derived NO. This defect was not related to eNOS catalytic activity; treatment of PAEC with H(2)O(2) enhanced agonist-stimulated eNOS activity indicated by increased eNOS phosphorylation at Ser-1177 and de-phosphorylation at Thr-495 and enhanced conversion of [(3)H]-L-arginine to [(3)H]-L-citrulline that was prevented by inhibitors of Src and phosphatidylinositol-3 kinases. Despite activating eNOS, H(2)O(2) impaired endothelial NO bioactivity indicated by attenuation of the increase in intracellular cGMP in PAEC stimulated with calcium ionophore or NO. The decrease in cGMP was not due to impaired guanylyl cyclase as H(2)O(2) treatment increased cGMP accumulation in response to BAY 41-2272, a NO-independent activator of soluble guanylyl cyclase. At concentrations that impaired endothelial NO bioactivity H(2)O(2) increased intracellular oxidative stress and size of the labile iron pool in PAEC. The increase in oxidative stress was prevented by the free radical scavenger's tempol or tiron and the iron chelator desferrioxamine and these antioxidants reversed the H(2)O(2)-induced impairment of NO bioactivity in PAEC. This study shows that despite promoting eNOS activity, H(2)O(2) impairs endothelial NO bioactivity by promoting oxidative inactivation of synthesized NO. The study highlights another way in which oxidative stress may impair NO bioactivity during vascular disease.     

(-)-Epicatechin induces calcium and translocation independent eNOS activation in arterial endothelial cells

The consumption of cacao-derived (i.e., cocoa) products provides beneficial cardiovascular effects in healthy subjects as well as individuals with endothelial dysfunction such as smokers, diabetics, and postmenopausal women. The vascular actions of cocoa are related to enhanced nitric oxide (NO) production. These actions can be reproduced by the administration of the cacao flavanol (-)-epicatechin (EPI). To further understand the mechanisms behind the vascular action of EPI, we investigated the effects of Ca(2+) depletion on endothelial nitric oxide (NO) synthase (eNOS) activation/phosphorylation and translocation. Human coronary artery endothelial cells were treated with EPI or with bradykinin (BK), a well-known Ca(2+)-dependent eNOS activator. Results demonstrate that both EPI and BK induce increases in intracellular calcium and NO levels. However, under Ca(2+)-free conditions, EPI (but not BK) is still capable of inducing NO production through eNOS phosphorylation at serine 615, 633, and 1177. Interestingly, EPI-induced translocation of eNOS from the plasmalemma was abolished upon Ca(2+) depletion. Thus, under Ca(2+)-free conditions, EPI can stimulate NO synthesis independent of calmodulin binding to eNOS and of its translocation into the cytoplasm. We also examined the effect of EPI on the NO/cGMP/vasodilator-stimulated phosphoprotein (VASP) pathway activation in isolated Ca(2+)-deprived canine mesenteric arteries. Results demonstrate that under these conditions, EPI induces the activation of this vasorelaxation-related pathway and that this effect is inhibited by pretreatment with nitro-L-arginine methyl ester, suggesting a functional relevance for this phenomenon.     

Direct activation of AMP-activated protein kinase stimulates nitric-oxide synthesis in human aortic endothelial cells

Recent studies have indicated that endothelial nitric-oxide synthase (eNOS) is regulated by reversible phosphorylation in intact endothelial cells. AMP-activated protein kinase (AMPK) has previously been demonstrated to phosphorylate and activate eNOS at Ser-1177 in vitro, yet the function of AMPK in endothelium is poorly characterized. We therefore determined whether activation of AMPK with 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) stimulated NO production in human aortic endothelial cells. AICAR caused the time- and dose-dependent stimulation of AMPK activity, with a concomitant increase in eNOS Ser-1177 phosphorylation and NO production. AMPK was associated with immunoprecipitates of eNOS, yet this was unaffected by increasing concentrations of AICAR. AICAR also caused the time- and dose-dependent stimulation of protein kinase B phosphorylation. To confirm that the effects of AICAR were indeed mediated by AMPK, we utilized adenovirus-mediated expression of a dominant negative AMPK mutant. Expression of dominant negative AMPK attenuated AICAR-stimulated AMPK activity, eNOS Ser-1177 phosphorylation and NO production and was without effect on AICAR-stimulated protein kinase B Ser-473 phosphorylation or NO production stimulated by insulin or A23187. These data suggest that AICAR-stimulated NO production is mediated by AMPK as a consequence of increased Ser-1177 phosphorylation of eNOS. We propose that stimuli that result in the acute activation of AMPK activity in endothelial cells stimulate NO production, at least in part due to phosphorylation and activation of eNOS. Regulation of endothelial AMPK therefore provides an additional mechanism by which local vascular tone may be controlled.     

Serine 1179 Phosphorylation of Endothelial Nitric Oxide Synthase Increases Superoxide Generation and Alters Cofactor Regulation

Endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis. In addition to producing NO, eNOS can also generate superoxide (O₂ ^-.) in the absence of the cofactor tetrahydrobiopterin (BH4). Previous studies have shown that bovine eNOS serine 1179 (Serine 1177/human) phosphorylation critically modulates NO synthesis. However, the effect of serine 1179 phosphorylation on eNOS superoxide generation is unknown. Here, we used the phosphomimetic form of eNOS (S1179D) to determine the effect of S1179 phosphorylation on superoxide generating activity, and its sensitivity to regulation by BH4, Ca²⁺, and calmodulin (CAM). S1179D eNOS exhibited significantly increased superoxide generating activity and NADPH consumption compared to wild-type eNOS (WT eNOS). The superoxide generating activities of S1179D eNOS and WT eNOS did not differ significantly in their sensitivity to regulation by either Ca²⁺ or CaM. The sensitivity of the superoxide generating activity of S1179D eNOS to inhibition by BH4 was significantly reduced compared to WT eNOS. In eNOS-overexpressing 293 cells, BH4 depletion with 10mM DAHP for 48 hours followed by 50ng/ml VEGF for 30 min to phosphorylate eNOS S1179 increased ROS accumulation compared to DAHP-only treated cells. Meanwhile, MTT assay indicated that overexpression of eNOS in HEK293 cells decreased cellular viability compared to control cells at BH4 depletion condition (P<0.01). VEGF-mediated Serine 1179 phosphorylation further decreased the cellular viability in eNOS-overexpressing 293 cells (P<0.01). Our data demonstrate that eNOS serine 1179 phosphorylation, in addition to enhancing NO production, also profoundly affects superoxide generation: S1179 phosphorylation increases superoxide production while decreasing sensitivity to the inhibitory effect of BH4 on this activity.     

Phosphorylation of the endothelial nitric oxide synthase at ser-1177 is required for VEGF-induced endothelial cell migration

Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration. The protein kinase Akt activates the endothelial NO synthase (eNOS) by phosphorylation of Ser-1177. Therefore, we investigated the contribution of Akt-mediated eNOS phosphorylation to VEGF-induced EC migration. Inhibition of NO synthase or overexpression of a dominant negative Akt abrogated VEGF-induced cell migration. In contrast, overexpression of constitutively active Akt was sufficient to induce cell migration. Moreover, transfection of an Akt site phospho-mimetic eNOS (S1177D) potently stimulated EC migration, whereas a non-phosphorylatable mutant (S1177A) inhibited VEGF-induced EC migration. Our data indicate that eNOS activation via phosphorylation of Ser-1177 by Akt is necessary and sufficient for VEGF-mediated EC migration.     

Antihypertensive therapy increases tetrahydrobiopterin levels and NO/cGMP signaling in small arteries of angiotensin II-infused hypertensive rats

We previously reported that small mesenteric arteries from hypertensive rats have increased NOS-derived H(2)O(2) and reduced NO/cGMP signaling. We hypothesized that antihypertensive therapy lowers blood pressure through a tetrahydrobiopterin (BH(4))-dependent mechanism restoring NO/cGMP signaling and endothelial NOS (NOS3; eNOS) phosphorylation in small arteries. To test this hypothesis, small mesenteric arteries from normotensive rats (NORM), angiotensin II-infused rats (ANG), ANG rats with triple therapy (reserperine, hydrochlorothiazide, and hydralazine), or ANG rats with oral BH(4) therapy were studied. Both triple therapy and oral BH(4) therapy attenuated the rise in systolic blood pressure in ANG rats and restored NO/cGMP signaling in small arteries similarly. Triple therapy significantly increased vascular BH(4) levels and BH(4)-to-BH(2) ratio similar to ANG rats with BH(4) supplementation. Furthermore, triple therapy (but not oral BH(4) therapy) significantly increased GTP cyclohydrolase I (GTPCH I) activity in small arteries without a change in expression. NOS3 phosphorylation at Ser1177 was reduced in small arteries from ANG compared with NORM, while NOS3 phosphorylation at Ser633 and Thr495 were similar in ANG and NORM. NOS3 phosphorylation at Ser1177 was restored with triple therapy or oral BH(4) in ANG rats. In conclusion, antihypertensive therapy regulates NO/cGMP signaling in small arteries through increasing BH(4) levels and NOS3 phosphorylation at Ser1177.     

Structural basis for endothelial nitric oxide synthase binding to calmodulin

The enzyme nitric oxide synthase (NOS) is exquisitely regulated in vivo by the Ca(2+) sensor protein calmodulin (CaM) to control production of NO, a key signaling molecule and cytotoxin. The differential activation of NOS isozymes by CaM has remained enigmatic, despite extensive research. Here, the crystallographic structure of Ca(2+)-loaded CaM bound to a 20 residue peptide comprising the endothelial NOS (eNOS) CaM-binding region establishes their individual conformations and intermolecular interactions, and suggests the basis for isozyme-specific differences. The alpha-helical eNOS peptide binds in an antiparallel orientation to CaM through extensive hydrophobic interactions. Unique NOS interactions occur with: (i). the CaM flexible central linker, explaining its importance in NOS activation; and (ii). the CaM C-terminus, explaining the NOS-specific requirement for a bulky, hydrophobic residue at position 144. This binding mode expands mechanisms for CaM-mediated activation, explains eNOS deactivation by Thr495 phosphorylation, and implicates specific hydrophobic residues in the Ca(2+) independence of inducible NOS.     

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.