Bidirectional actions of hydrogen peroxide on endothelial nitric-oxide synthase phosphorylation and function: co-commitment and interplay of Akt and AMPK

Endothelial NO synthase (eNOS) is critically modulated by kinases via the phosphorylation of its Ser(1179) (bovine) or Ser(1177) (human) residue. Reactive oxygen species such as H(2)O(2) was reported to activate Akt, leading to increased eNOS Ser(1179) phosphorylation and activity. But reactive oxygen species are also known to attenuate eNOS function in cardiovascular diseases. Prior studies showing H(2)O(2)-stimulated eNOS phosphorylation were performed on serum-starved cells, and only the short term effect of H(2)O(2) was examined. Here we found that the effects of H(2)O(2) on eNOS Ser(1179) phosphorylation and function were bidirectional. With endothelial cells cultured with serum, H(2)O(2) initially raised eNOS Ser(1179) phosphorylation and activity. However, after the peak increase at 30 min, eNOS Ser(1179) phosphorylation dramatically declined. Parallel to the alterations of eNOS Ser(1179) phosphorylation, Akt was transiently activated by H(2)O(2) and subsequently became dormant. In contrast, AMP-activated protein kinase (AMPK) was progressively activated in H(2)O(2)-treated cells. Blocking Akt activation abolished the initial rise of eNOS Ser(1179) phosphorylation after H(2)O(2) treatment. In long term H(2)O(2)-treated cells where Akt was deactivated, significant amounts of Ser(1179)-phosphorylated eNOS remained. AMPK inhibition eradicated the remaining eNOS Ser(1179) phosphorylation. Taken together, these studies revealed that Akt and AMPK orchestrated a bidirectional action on eNOS Ser(1179) phosphorylation in H(2)O(2)-treated cells. Long term H(2)O(2) exposure decreased eNOS Ser(1179) phosphorylation, and this might account for the loss of eNOS function in cardiovascular diseases where chronic oxidative injury occurs.     

Nitric oxide production and regulation of endothelial nitric-oxide synthase phosphorylation by prolonged treatment with troglitazone: evidence for involvement of peroxisome proliferator-activated receptor (PPAR) gamma-dependent and PPARgamma-independent signaling pathways

Recently, peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been reported to increase endothelial NO, but the signaling mechanisms involved are unknown. Using troglitazone, a PPARgamma ligand known as an antidiabetic compound, we investigated the molecular mechanism of its effect on NO production in bovine aortic endothelial cells. Troglitazone increased endothelial NO production in a dose- and time-dependent manner with no alteration in endothelial nitric-oxide synthase (eNOS) expression. The maximal increase ( approximately 3.1-fold) was achieved with 20 microm troglitazone treatment for 12 h, and this increase was accompanied by increases in the expression of vascular endothelial growth factor (VEGF) and its receptor, KDR/Flk-1, and in Akt phosphorylation. Analysis with antibodies specific for each phosphorylated site demonstrated that troglitazone (20 microm treatment for 12 h) significantly increased both the phosphorylation of Ser(1179) of eNOS (eNOS-Ser(1179)) and the dephosphorylation of eNOS-Ser(116) but did not alter eNOS-Thr(497) phosphorylation. Treatment with anti-VEGF antibody to scavenge the increased VEGF induced by troglitazone partially inhibited troglitazone-stimulated NO production. This was accompanied by the attenuation of troglitazone-stimulated increases in the phosphorylation of Akt and eNOS-Ser(1179) with no alteration in eNOS-Ser(116) dephosphorylation. We also found that bisphenol A diglycidyl ether, a PPARgamma antagonist, partially inhibited troglitazone-stimulated NO production with a concomitant reduction in VEGF-KDR/Flk-1-Akt-mediated eNOS-Ser(1179) phosphorylation but with no alteration in eNOS-Ser(116) dephosphorylation induced by troglitazone. Taken together, our results demonstrate that prolonged treatment with troglitazone increases endothelial NO production by at least two independent signaling pathways: PPARgamma-dependent, VEGF-KDR/Flk-1-Akt-mediated eNOS-Ser(1179) phosphorylation and PPARgamma-independent, eNOS-Ser(116) dephosphorylation.     

Retinoic acid decreases nitric oxide production in endothelial cells: a role of phosphorylation of endothelial nitric oxide synthase at Ser(1179)

The effects of retinoic acid (RA) on nitric oxide (NO) production are controversial. Furthermore, it has never been studied whether these effects are mediated by direct modulation of phosphorylation of endothelial nitric oxide synthase (eNOS). Using bovine aortic endothelial cells, we found that all-trans RA (atRA) dose- and time-dependently decreased NO production without alteration in eNOS expression. This decrease was accompanied by reduction in eNOS-Ser(1179) phosphorylation. However, atRA did not alter the phosphorylation of eNOS-Ser(116) or eNOS-Thr(497). Concurrently, atRA also decreased the expressions of vascular endothelial growth factor (VEGF) and its receptor KDR/Flk-1, and Akt phosphorylation. Co-treatment with troglitazone, an activator of VEGF expression, reversed the atRA-induced reductions in eNOS-Ser(1179) phosphorylation and NO production, with concomitant restoration in VEGF expression. Direct treatment with VEGF also reversed these inhibitory effects, suggesting an important role for VEGF. Nonetheless, the RARalpha antagonist Ro 41-5253 did not block all the inhibitory effects of atRA, indicating that these inhibitory effects are not mediated by the RA response element (RARE). Thus, atRA decreases eNOS-Ser(1179) phosphorylation through a mechanism that depends on VEGF-KDR/Flk-1-mediated Akt phosphorylation but is independent of RARE, leading to reduction in NO production.     

Effects of epoxyeicosatrienoic acids on levels of eNOS phosphorylation and relevant signaling transduction path-ways involved

Vascular endothelial cells play crucial roles in regulating cardiovascular function, maintaining car-diovascular homeostasis and preventing the occur-rence of cardiac and cerebral vascular diseases. All these protective effects are fulfilled through various vasoactive products secreted by endothelium including nitric oxide (NO), prostacyclin (PGI2) and endothe-lium-derived hyperpolarizing factor (EDHF). NO, pro-duced from L-arginine by endothelial nitric-oxide synthase (eNOS), is an impor…     

Adiponectin stimulates production of nitric oxide in vascular endothelial cells

Adiponectin is secreted by adipose cells and mimics many metabolic actions of insulin. However, mechanisms by which adiponectin acts are poorly understood. The vascular action of insulin to stimulate endothelial production of nitric oxide (NO), leading to vasodilation and increased blood flow is an important component of insulin-stimulated whole body glucose utilization. Therefore, we hypothesized that adiponectin may also stimulate production of NO in endothelium. Bovine aortic endothelial cells in primary culture loaded with the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2 DA) were treated with lysophosphatidic acid (LPA) (a calcium-releasing agonist) or adiponectin (10 microg/ml bacterially produced full-length adiponectin). LPA treatment increased production of NO by approximately 4-fold. Interestingly, adiponectin treatment significantly increased production of NO by approximately 3-fold. Preincubation of cells with wortmannin (phosphatidylinositol 3-kinase inhibitor) blocked only adiponectin- but not LPA-mediated production of NO. Using phospho-specific antibodies, we observed that either adiponectin or insulin treatment (but not LPA treatment) caused phosphorylation of both Akt at Ser473 and endothelial nitric-oxide synthase (eNOS) at Ser1179 that was inhibitable by wortmannin. We next transfected bovine aortic endothelial cells with dominant-inhibitory mutants of Akt (Akt-AAA) or AMP-activated protein kinase (AMPK) (AMPKK45R). Neither mutant affected production of NO in response to LPA treatment. Importantly, only AMPKK45R, but not Akt-AAA, caused a significant partial inhibition of NO production in response to adiponectin. Moreover, AMPK-K45R inhibited phosphorylation of eNOS at Ser1179 in response to adiponectin but not in response to insulin. We conclude that adiponectin has novel vascular actions to directly stimulate production of NO in endothelial cells using phosphatidylinositol 3-kinase-dependent pathways involving phosphorylation of eNOS at Ser1179 by AMPK. Thus, the effects of adiponectin to augment metabolic actions of insulin in vivo may be due, in part, to vasodilator actions of adiponectin.     

A constituent of green tea, epigallocatechin-3-gallate, activates endothelial nitric oxide synthase by a phosphatidylinositol-3-OH-kinase-, cAMP-dependent protein kinase-, and Akt-dependent pathway and leads to endothelial-dependent vasorelaxation

Epidemiological studies suggest that tea catechins may reduce the risk of cardiovascular disease, but the mechanisms of benefit have not been determined. The objective of the present study was to investigate the effects of epigallocatechin-3-gallate (EGCG), the major constituent of green tea, on vasorelaxation and on eNOS expression and activity in endothelial cells. EGCG (1-50 microm) induced dose-dependent vasodilation in rat aortic rings. Vasodilation was abolished by pretreatment with Ng-nitro L-arginine methyl ester. In bovine aortic endothelial cells, EGCG increased endothelial nitric oxide (eNOS) activity dose-dependently after 15 min. Treatment with EGCG induced a sustained activation of Akt, ERK1/2, and eNOS Ser1179 phosphorylation. Inhibition of extracellular signal-regulated kinase (ERK)1/2 had no influence on eNOS activity or Ser1179 phosphorylation. Simultaneous treatment of cells with selective inhibitors for cAMP-dependent protein kinase (PKA) and Akt completely prevented the increase in eNOS activity by EGCG after 15 min, indicating that both kinases act in concert. Specific phosphatidylinositol-3-OH-kinase inhibitors yielded identical results. Akt inhibition prevented eNOS Ser1179 phosphorylation, whereas inhibition of PKA did not influence Akt and eNOS Ser1179 phosphorylation. Pretreatment of endothelial cells with EGCG for 4 h markedly enhanced the increase in eNOS activity stimulated by Ca-ionomycin, suggesting that Akt accounts for prolonged eNOS activation. Treatment of cells for 72 h with EGCG did not change eNOS protein levels. Our results indicate that EGCG-induced endothelium-dependent vasodilation is primarily based on rapid activation of eNOS by a phosphatidylinositol 3-kinase-, PKA-, and Akt-dependent increase in eNOS activity, independently of an altered eNOS protein content.     

Rapid increase in endothelial nitric oxide production by bradykinin is mediated by protein kinase A signaling pathway

Bradykinin (BK) acutely increases endothelial nitric oxide (NO) production by activating endothelial NO synthase (eNOS), and this increase is in part correlated with enhanced phosphorylation/dephosphorylation of eNOS by several protein kinases and phosphatases. However, the signaling mechanisms producing this increase are still controversial. In an attempt to delineate the acute effect of BK on endothelial NO production, confluent bovine aortic endothelial cells were incubated with BK, and NO production was measured by NO-specific chemiluminescence. Significant increase in NO levels was detected as early as 1 min after BK treatment, with concomitant increase in the phosphorylation of Ser(1179) (bovine sequence) site of eNOS (eNOS-Ser(1179)). This acute effect of BK on both increases was blocked only by treatment of protein kinase A inhibitor H-89, but not by the inhibitors of calmodulin-dependent kinase II and protein kinase B, suggesting that the rapid increase in NO production by BK is mediated by the PKA-dependent phosphorylation of eNOS-Ser(1179).     

Regulation of endothelial nitric-oxide synthase activity through phosphorylation in response to epoxyeicosatrienoic acids

Endothelial nitric oxide synthase (eNOS) is a key enzyme in NO-mediated cardiovascular homeostasis and its activity is modulated by a variety of hormonal and mechanical stimuli via phosphorylation modification. Our previous study has demonstrated that epoxyeicosatrienoic acids (EETs), the cytochrome P450 (CYP)-dependent metabolites of arachidonic acid, could robustly up-regulate eNOS expression. However, the molecular mechanism underlying the effects of EETs on eNOS remains elusive. Particularly, whether and how EETs affect eNOS phosphorylation is unknown. In the present study, we investigated the effects of EETs on eNOS phosphorylation with cultured bovine aortic endothelial cells (BAECs). BAECs were either treated with exogenous EETs or infected with recombinant adeno-associated virus (rAAV) carrying CYP2C11-CYPOR, CYP102 F87V mutant and CYP2J2, respectively, to increase endogenous EETs. Both addition of EETs and CYP epoxygenase transfection markedly increased eNOS phosphorylation at its Ser1179 and Thr497 residues. Inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 prevented EETs-induced increases of eNOS-Ser(P)1179 but had no effect on the phosphorylation status of Thr497. However, inhibitors of protein kinase B (Akt), mitogen-activated protein kinase (MAPK) and MAPK kinase could block phosphorylation of eNOS at both sites. Inhibition of these kinases also attenuated the up-regulation of eNOS expression by EETs. Finally, administration of viral CYP epoxygenases expression vectors into rats enhanced eNOS phosphorylation and function in vivo. Thus, in addition to up-regulating eNOS expression, EETs also augment eNOS function by enhancing eNOS phosphorylation. EETs-induced up-regulation of eNOS phosphorylation and expression appears to involve in both PI3K/Akt and MAPK pathways.     

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.     

c-Jun N-terminal kinase 2 phosphorylates endothelial nitric oxide synthase at serine 116 and regulates nitric oxide production

The c-Jun N-terminal kinases (JNKs) belonging to the mitogen-activated protein kinase (MAPK) superfamily play important roles in foam-cell formation, hypercholesterolemia-mediated endothelial dysfunction, and the development of obesity. Although decreased nitric oxide (NO) production via decreased phosphorylation of endothelial NO synthase at serine 1179 (eNOS-Ser(1179)) was reported to be partly involved in JNK2-derived endothelial dysfunction, JNK2 seems likely to be indirectly involved in this signaling pathway. Here, using bovine aortic endothelial cells, we examined whether JNK2 directly phosphorylated eNOS-Ser(116), a putative substrate site for the MAPK superfamily, and this phosphorylation resulted in decreased NO release. JNK inhibitor SP60012 increased NO release in a time- and dose-dependent manner, which was accompanied by increased eNOS-Ser(116) phosphorylation. Purified JNK2 directly phosphorylated eNOS-Ser(116)in vitro. Ectopic expression of dominant negative JNK2 repressed eNOS-Ser(116) phosphorylation and increased NO production. Coimmunoprecipitation and confocal microscopy studies revealed a colocalization of eNOS and JNK2. However, all these observed effects were not manifested when JNK1 probes were used. Overall, this study indicates that JNK2 is a physiological kinase responsible for eNOS-Ser(116) phosphorylation and regulates NO production.     

Adiponectin stimulates angiogenesis by promoting cross-talk between AMP-activated protein kinase and Akt signaling in endothelial cells

Adiponectin is an adipocyte-specific adipocytokine with anti-atherogenic and anti-diabetic properties. Here, we investigated whether adiponectin regulates angiogenic processes in vitro and in vivo. Adiponectin stimulated the differentiation of human umbilical vein endothelium cells (HUVECs) into capillary-like structures in vitro and functioned as a chemoattractant in migration assays. Adiponectin promoted the phosphorylation of AMP-activated protein kinase (AMPK), protein kinase Akt/protein kinase B, and endothelial nitric oxide synthesis (eNOS) in HUVECs. Transduction with either dominant-negative AMPK or dominant-negative Akt abolished adiponectin-induced eNOS phosphorylation as well as adiponectin-stimulated HUVEC migration and differentiation. Dominant-negative AMPK also inhibited adiponectin-induced Akt phosphorylation, suggesting that AMPK is upstream of Akt. Dominant-negative Akt or the phosphatidylinositol 3-kinase inhibitor LY294002 blocked adiponectin-stimulated Akt and eNOS phosphorylation, migration, and differentiation without altering AMPK phosphorylation. Finally, adiponectin stimulated blood vessel growth in vivo in mouse Matrigel plug implantation and rabbit corneal models of angiogenesis. These data indicate that adiponectin can function to stimulate the new blood vessel growth by promoting cross-talk between AMP-activated protein kinase and Akt signaling within endothelial cells.     

3-Morpholinosyndnonimine inhibits 5-hydroxytryptamine-induced phosphorylation of nitric oxide synthase in endothelial cells.

5-Hydroxytryptamine (5-HT) is a vasoactive substance that is taken up by endothelial cells to activate endothelial nitrite oxide synthase (eNOS). The activation of eNOS results in the production of nitric oxide (NO), which is responsible for vasodilation of blood vessels. NO also interacts with superoxide anion (O2*-) to form peroxynitrite (ONOO-), a potent oxidant that has been shown to induce vascular endothelial dysfunction. We examined the ability of 3-morpholinosyndnonimine (SIN-1), an ONOO- generator, to inhibit 5-HT-induced phosphorylation of eNOS in cultured bovine aortic endothelial cells (BAECs). We observed that 5-HT phosphorylates Ser1179 eNOS in a time- and concentration-dependent manner. Maximum phosphorylation occurred at 30 sec using a concentration of 1.0 microM 5-HT. BAECs treated with SIN-1 (1-1000 microM) for 30 min showed no significant increase in eNOS phosphorylation. However, 5-HT-induced eNOS phosphorylation was inhibited in cells treated with various concentrations of SIN-1 for 30 min and stimulated with 5-HT. These data suggest that an increase in ONOO- as a result of an increase in the production of O2*-, may feedback to inhibit 5-HT-induced eNOS phosphorylation at Ser1179 and therefore, contribute to endothelial dysfunction associated with cardiovascular diseases.     

Sphingosine 1-phosphate and activation of endothelial nitric-oxide synthase. differential regulation of Akt and MAP kinase pathways by EDG and bradykinin receptors in vascular endothelial cells

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that elicits numerous biological responses in endothelial cells mediated by a family of G protein-coupled EDG receptors. Stimulation of EDG receptors by S1P has been shown to activate the endothelial isoform of nitric-oxide synthase (eNOS) in heterologous expression systems (Igarashi, J., and Michel, T. (2000) J. Biol. Chem. 275, 32363-32370). However, the signaling pathways that modulate eNOS regulation by S1P/EDG in vascular endothelial cells remain less well understood. We now report that S1P treatment of bovine aortic endothelial cells (BAEC) acutely increases eNOS enzyme activity; the EC(50) for S1P activation of eNOS is approximately 10 nm. The magnitude of eNOS activation by S1P in BAEC is equivalent to that elicited by the agonist bradykinin. S1P treatment activates Akt, a protein kinase implicated in phosphorylation of eNOS. S1P treatment of BAEC leads to eNOS phosphorylation at Ser(1179), a residue phosphorylated by Akt; an eNOS mutant in which this Akt phosphorylation site is inactivated shows attenuated S1P-induced eNOS activation. S1P-induced activation both of Akt and of eNOS is inhibited by pertussis toxin, by the phosphoinositide 3-kinase inhibitor wortmannin, and by the intracellular calcium chelator BAPTA (1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). By contrast to S1P, activation of G protein-coupled bradykinin B2 receptors neither activates kinase Akt nor promotes Ser(1179) eNOS phosphorylation despite robustly activating eNOS enzyme activity. Understanding the differential regulation of protein kinase pathways by S1P and bradykinin may lead to the identification of new points for eNOS regulation in vascular endothelial cells.     

Follistatin-like 1, a secreted muscle protein, promotes endothelial cell function and revascularization in ischemic tissue through a nitric-oxide synthase-dependent mechanism

Myogenic Akt signaling coordinates blood vessel recruitment with normal tissue growth. Here, we investigated the role of Follistatin-like 1 (Fstl1) in the regulation of endothelial cell function and blood vessel growth in muscle. Transgenic Akt1 overexpression in skeletal muscle led to myofiber growth that was coupled to an increase in muscle capillary density. Myogenic Akt signaling or ischemic hind limb surgery led to the induction of Fstl1 in muscle and increased circulating levels of Fstl1. Intramuscular administration of an adenoviral vector expressing Fstl1 (Ad-Fstl1) accelerated flow recovery and increased capillary density in the ischemic hind limbs of wild-type mice, and this was associated with an increase in endothelial nitric oxide synthase (eNOS) phosphorylation at residue Ser-1179. In cultured endothelial cells, Ad-Fstl1 stimulated migration and differentiation into network structures and inhibited apoptosis under conditions of serum deprivation. These cell responses were associated with the activating phosphorylation of Akt and eNOS. Conversely, transduction with dominant-negative Akt or LY294002 blocked Fstl1-stimulated eNOS phosphorylation and inhibited Fstl1-stimulated cellular responses. Treatment with the eNOS inhibitor N(G)-nitro-L-arginine methyl ester also reduced endothelial cell migration and differentiation induced by Ad-Fstl1. The stimulatory effect of Ad-Fstl1 on ischemic limb reperfusion was abolished in mice lacking eNOS. These data indicate that Fstl1 is a secreted muscle protein or myokine that can function to promote endothelial cell function and stimulates revascularization in response to ischemic insult through its ability to activate Akt-eNOS signaling.     

AMP-activated protein kinase mediates erythropoietin-induced activation of endothelial nitric oxide synthase

We investigated whether AMP-activated protein kinase (AMPK), a multi-functional regulator of energy homeostasis, participates in the regulation of erythropoietin (EPO)-mediated activation of endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs) and mice. In ECs, treatment with EPO increased the phosphorylation of AMPK, acetyl-CoA carboxylase (ACC), and eNOS, as revealed by Western blot analysis. Inhibition of AMPK activation by compound C or dominant-negative AMPK mutant abrogated the EPO-induced increase in the phosphorylation of AMPK, ACC, and eNOS, as well as nitric oxide (NO) production. Additionally, suppression of AMPK activation abolished EPO-induced EC proliferation, migration and tube formation. Immunoprecipitation analysis demonstrated that AMPK mediated the EPO-induced increase in the phosphorylation of β common receptor (βCR) and the formation of a βCR-AMPK-eNOS complex. In mice, inhibition of AMPK activation by compound C markedly decreased EPO-elicited angiogenesis in Matrigel plugs. Furthermore, the phosphorylation of AMPK and eNOS was significantly higher in aortas from EPO transgenic mice than wild-type mice. Moreover, treatment with EPO neutralizing antibody greatly reduced the exercise training-induced increase in phosphorylation of AMPK and eNOS in aortas of wild-type mice. Taken together, EPO may trigger AMPK-dependent signaling, which leads to enhanced phosphorylation of βCR and eNOS, increased βCR-AMPK-eNOS complex formation, NO production, and, ultimately, angiogenesis.     

Asymmetric Dimethylarginine Induces Endothelial Nitric-oxide Synthase Mitochondrial Redistribution through the Nitration-mediated Activation of Akt1

We have recently demonstrated that asymmetric dimethylarginine (ADMA) induces the translocation of endothelial nitric-oxide synthase (eNOS) to the mitochondrion via a mechanism that requires protein nitration. Thus, the goal of this study was elucidate how eNOS redistributes to mitochondria and to identify the nitrated protein responsible for this event. Our data indicate that exposure of pulmonary arterial endothelial cells to ADMA enhanced eNOS phosphorylation at the Akt1-dependent phosphorylation sites Ser⁶¹⁷ and Ser¹¹⁷⁹. Mutation of these serine residues to alanine (S617A and S1179A) inhibited nitration-mediated eNOS translocation to the mitochondria, whereas the phosphormimic mutations (S617D and S1179D) exhibited increased mitochondrial redistribution in the absence of ADMA. The overexpression of a dominant-negative Akt1 also attenuated ADMA-mediated eNOS mitochondrial translocation. Furthermore, ADMA enhanced Akt1 nitration and increased its activity. Mass spectrometry identified a single nitration site in Akt1 located at the tyrosine residue (Tyr³⁵⁰) located within the client-binding domain. Replacement of Tyr³⁵⁰ with phenylalanine abolished peroxynitrite-mediated eNOS translocation to mitochondria. We also found that in the absence of ADMA, eNOS translocation decreased mitochondrial oxygen consumption and superoxide production without altering cellular ATP level. This suggests that under physiologic conditions, eNOS translocation enhances mitochondria coupling. In conclusion, we have identified a new mechanism by which eNOS translocation to mitochondria is regulated by the phosphorylation of eNOS at Ser⁶¹⁷ and Ser¹¹⁷⁹ by Akt1 and that this is enhanced when Akt1 becomes nitrated at Tyr³⁵⁰.     

Disabling a C-terminal autoinhibitory control element in endothelial nitric-oxide synthase by phosphorylation provides a molecular explanation for activation of vascular NO synthesis by diverse physiological stimuli

Calmodulin-dependent activation of endothelial nitric-oxide synthase is generally considered to follow a transient increase in intracellular calcium levels. However, a number of physiological stimuli (e.g. endothelial shear-stress, insulin) are known to activate endothelial nitric oxide (eNOS) via a non-classical, "calcium-independent" pathway. Recent findings demonstrate that such stimuli elicit the phosphorylation of a C-terminal residue in eNOS (Ser(1179) in the bovine isoform), rendering eNOS active at resting levels of intracellular calcium. However, the mechanistic basis for this mode of eNOS activation remains unknown. Protein modeling led us to consider that the C terminus of eNOS may fulfill an autoinhibitory function that can be disrupted by phosphorylation of serine 1179. To test this possibility we contrasted the phenotype of wild type bovine eNOS with that of a mutant lacking C-terminal residues 1179-1205 (CDelta27 eNOS). Despite no observed difference in calmodulin affinity, CDelta27 eNOS exhibited a 5-fold reduction in EC(50) for calcium and a 2-4-fold increase in maximal catalytic activities. In these phenotypic properties, CDelta27 accurately mimics phospho-Ser(1179) wild type eNOS. We conclude that the C terminus imposes a significant barrier to the activation of eNOS by calmodulin binding and that this barrier can be functionally disabled by Ser(1179) phosphorylation-elicited enzyme activation.     

Activation of protein kinase C zeta by peroxynitrite regulates LKB1-dependent AMP-activated protein kinase in cultured endothelial cells

We previously reported the phosphoinositide 3-kinase-dependent activation of the 5'-AMP-activated kinase (AMPK) by peroxynitrite (ONOO-) and hypoxia-reoxygenation in cultured endothelial cells. Here we show the molecular mechanism of activation of this pathway. Exposure of bovine aortic endothelial cells to ONOO- significantly increased the phosphorylation of both Thr172 of AMPK and Ser1179 of endothelial nitric-oxide synthase, a known downstream enzyme of AMPK. In addition, activation of AMPK by ONOO- was accompanied by increased phosphorylation of protein kinase Czeta (PKCzeta) (Thr410/403) and translocation of cytosolic PKCzeta into the membrane. Further, inhibition of PKCzeta abrogated ONOO- -induced AMPK-Thr172 phosphorylation as that of endothelial nitric-oxide synthase. Furthermore, overexpression of a constitutively active PKCzeta mutant enhanced the phosphorylation of AMPK-Thr172, suggesting that PKCzeta is upstream of AMPK activation. In contrast, ONOO- activated PKCzeta in LKB1-deficient HeLa-S3 but affected neither AMPK-Thr172 nor AMPK activity. These data suggest that LKB1 is required for PKCzeta-enhanced AMPK activation. In vitro, recombinant PKCzeta phosphorylated LKB1 at Ser428, resulting in phosphorylation of AMPK at Thr172. Further, direct mutation of Ser428 of LKB1 into alanine, like the kinase-inactive LKB1 mutant, abolished ONOO- -induced AMPK activation. In several cell types originating from human, rat, and mouse, inhibition of PKCzeta significantly attenuated the phosphorylation of both LKB1-Ser428 and AMPK-Thr172 that were enhanced by ONOO-. Taken together, we conclude that PKCzeta can regulate AMPK activity by increasing the Ser428 phosphorylation of LKB1, resulting in association of LKB1 with AMPK and consequent AMPK Thr172 phosphorylation by LKB1.     

Serine Phosphorylation Sites on IRS2 Activated by Angiotensin II and Protein Kinase C To Induce Selective Insulin Resistance in Endothelial Cells

Protein kinase C (PKC) activation, induced by hyperglycemia and angiotensin II (AngII), inhibited insulin-induced phosphorylation of Akt/endothelial nitric oxide (eNOS) by decreasing tyrosine phosphorylation of IRS2 (p-Tyr-IRS2) in endothelial cells. PKC activation by phorbol ester (phorbol myristate acetate [PMA]) reduced insulin-induced p-Tyr-IRS2 by 46% ± 13% and, similarly, phosphorylation of Akt/eNOS. Site-specific mutational analysis showed that PMA increased serine phosphorylation at three sites on IRS2 (positions 303, 343, and 675), which affected insulin-induced tyrosine phosphorylation of IRS2 at positions 653, 671, and 911 (p-Tyr-IRS2) and p-Akt/eNOS. Specific PKCβ2 activation decreased p-Tyr-IRS2 and increased the phosphorylation of two serines (Ser303 and Ser675) on IRS2 that were confirmed in cells overexpressing single point mutants of IRS2 (S303A or S675A) containing a PKCβ2-dominant negative or selective PKCβ inhibitor. AngII induced phosphorylation only on Ser303 of IRS2 and inhibited insulin-induced p-Tyr911 of IRS2 and p-Akt/eNOS, which were blocked by an antagonist of AngII receptor I, losartan, or overexpression of single mutant S303A of IRS2. Increases in p-Ser303 and p-Ser675 and decreases in p-Tyr911 of IRS2 were observed in vessels of insulin-resistant Zucker fatty rats versus lean rats. Thus, AngII or PKCβ activation can phosphorylate Ser303 and Ser675 in IRS2 to inhibit insulin-induced p-Tyr911 and its anti-atherogenic actions (p-Akt/eNOS) in endothelial cells.     

Identification of regulatory sites of phosphorylation of the bovine endothelial nitric-oxide synthase at serine 617 and serine 635

Endothelial nitric-oxide synthase (eNOS) is regulated by signaling pathways involving multiple sites of phosphorylation. The coordinated phosphorylation of eNOS at Ser(1179) and dephosphorylation at Thr(497) activates the enzyme, whereas inhibition results when Thr(497) is phosphorylated and Ser(1179) is dephosphorylated. We have identified two further phosphorylation sites, at Ser(617) and Ser(635), by phosphopeptide mapping and matrix-assisted laser desorption ionization time of flight mass spectrometry. Purified protein kinase A (PKA) phosphorylates both sites in purified eNOS, whereas purified Akt phosphorylates only Ser(617). In bovine aortic endothelial cells, bradykinin (BK), ATP, and vascular endothelial growth factor stimulate phosphorylation of both sites. BK-stimulated phosphorylation of Ser(617) is Ca(2+)-dependent and is partially inhibited by LY294002 and wortmannin, phosphatidylinositol 3-kinase inhibitors, suggesting signaling via Akt. BK-stimulated phosphorylation of Ser(635) is Ca(2+)-independent and is completely abolished by the PKA inhibitor, KT5720, suggesting signaling via PKA. Activation of PKA with isobutylmethylxanthine also causes Ser(635), but not Ser(617), phosphorylation. Mimicking phosphorylation at Ser(635) by Ser to Asp mutation results in a greater than 2-fold increase in activity of the purified protein, whereas mimicking phosphorylation at Ser(617) does not alter maximal activity but significantly increases Ca(2+)-calmodulin sensitivity. These data show that phosphorylation of both Ser(617) and Ser(635) regulates eNOS activity and contributes to the agonist-stimulated eNOS activation process.