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.     

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.     

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.     

Endothelial NO synthase phosphorylated at SER635 produces NO without requiring intracellular calcium increase

Shear stress stimulates NO production involving the Ca2+-independent mechanisms in endothelial cells. We have shown that exposure of bovine aortic endothelial cells (BAEC) to shear stress stimulates phosphorylation of eNOS at S635 and S1179 by the protein kinase A- (PKA-) dependent mechanisms. We examined whether phosphorylation of S635 of eNOS induced by PKA stimulates NO production in a calcium-independent manner. Expression of a constitutively active catalytic subunit of PKA (Cqr) in BAEC induced phosphorylation of S635 and S1179 residues and dephosphorylation of T497. Additionally, Cqr expression stimulated NO production, which could not be prevented by treating cells with the intracellular calcium chelator BAPTA-AM. To determine the role of each eNOS phosphorylation site in NO production, HEK-293 cells transfected with eNOS point mutants whereby S116, T497, S635, and S1179 were mutated to either A or D. Maximum NO production from S635D-expressing cells was significantly higher than that of either wild type or S635A in both basal and elevated [Ca2+]i conditions. More interestingly, S635D cells produced NO even when [Ca2+]i was nearly depleted by BAPTA-AM. We confirmed these results obtained in HEK-293 cells in BAEC transfected with S635D, S635A, or wild-type eNOS vector. These findings suggest that, once phosphorylated at S635 residue, eNOS produces NO without requiring any changes in [Ca2+]i. PKA-dependent phosphorylation of eNOS S635 and subsequent basal NO production in a Ca2+-independent manner may play an important role in regulating vascular biology and pathophysiology.     

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.     

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.     

Coordinated regulation of endothelial nitric oxide synthase activity by phosphorylation and subcellular localization

Endothelial nitric oxide synthase (eNOS) is regulated by multiple mechanisms including Ca(2+)/calmodulin binding, protein-protein interactions, phosphorylation, and subcellular locations. Emerging evidence suggests that these seemingly independent mechanisms may be closely correlated. In the present study, the interplay between membrane targeting and phosphorylation of eNOS was investigated by using various mutants designed to target specific subcellular locations or to mimic different phospho states. Phospho-mimicking mutations of wild-type eNOS at S635 and S1179 synergistically activated the enzyme. The targeted eNOS mutants to plasma membrane and Golgi complex exhibited higher NO production activities than that of a myristoylation-deficient cytosolic mutant. Phospho-mimicking mutations at S635 and S1179 rescued the activity of the cytosolic mutant and increased those of the plasma membrane- and Golgi-targeted mutants. In contrast, phospho-deficient mutations at these sites led to inactivation of eNOS. Unlike the other targeted mutants, the cytosolic eNOS mutant was unresponsive to cAMP, indicating that membrane association and phosphorylation are required for eNOS activation. These findings suggest that the coordinated interplay between phosphorylation and subcellular localization of eNOS plays an important role in regulating NO production in endothelial cells.     

Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production

There is evidence that endothelial nitric-oxide synthase (eNOS) is regulated by reciprocal dephosphorylation of Thr497 and phosphorylation of Ser1179. To examine the interrelationship between these sites, cells were transfected with wild-type (WT), T497A, T497D, S1179D, and T497A/S1179D eNOS and activity, NO release and eNOS localization were assessed. Although eNOS T497A, S1179D and T497A/S1179D eNOS had greater enzymatic activity than did WT eNOS in lysates, basal production of NO from cells was markedly reduced in cells transfected with T497A and T497A/S1179D eNOS but augmented in cells transfected with S1179D eNOS. Stimulating cells with ATP or ionophore normalized the loss of function seen with T497A and T497A/S1179D eNOS to levels observed with WT and S1179D eNOS, respectively. Despite these functional differences, the localization of eNOS mutants were similar to WT. Because both T497A and T497A/S1179D eNOS exhibited higher enzyme activity but reduced production of NO, we examined whether these mutations were "uncoupling" NO synthesis. T497A and T497A/S1179D eNOS generated 2-3 times more superoxide anion than WT eNOS, and both basal and stimulated interactions of T497A/S1179D eNOS with hsp90 were reduced in co-immunoprecipitation experiments. Thus, the phosphorylation/dephosphorylation of Thr497 may be an intrinsic switch mechanism that determines whether eNOS generates NO versus superoxide in cells.     

Subcellular targeting and agonist-induced site-specific phosphorylation of endothelial nitric-oxide synthase

The endothelial isoform of nitric-oxide synthase (eNOS) undergoes a complex pattern of covalent modifications, including acylation with the fatty acids myristate and palmitate as well as phosphorylation on multiple sites. eNOS acylation is a key determinant for the reversible subcellular targeting of the enzyme to plasmalemmal caveolae. We transfected a series of hemagglutinin epitope-tagged eNOS mutant cDNAs deficient in palmitoylation (palm(-)) and/or myristoylation (myr(-)) into bovine aortic endothelial cells; after treatment with the eNOS agonists sphingosine 1-phosphate or vascular endothelial growth factor, the recombinant eNOS was immunoprecipitated using an antibody directed against the epitope tag, and patterns of eNOS phosphorylation were analyzed in immunoblots probed with phosphorylation state-specific eNOS antibodies. The wild-type eNOS underwent agonist-induced phosphorylation at serine 1179 (a putative site for phosphorylation by kinase Akt), but phosphorylation of the myr(-) eNOS at this residue was nearly abrogated; the palm(-) eNOS exhibited an intermediate phenotype. The addition of the CD8 transmembrane domain to the amino terminus of eNOS acylation-deficient mutants rescued the wild-type phenotype of robust agonist-induced serine 1179 phosphorylation. Thus, membrane targeting, but not necessarily acylation, is the critical determinant for agonist-promoted eNOS phosphorylation at serine 1179. In striking contrast to serine 1179, phosphorylation of eNOS at serine 116 was enhanced in the myr(-) eNOS mutant and was markedly attenuated in the CD8-eNOS membrane-targeted fusion protein. We conclude that eNOS targeting differentially affects eNOS phosphorylation at distinct sites in the protein and suggest that the inter-relationships of eNOS acylation and phosphorylation may modulate eNOS localization and activity and thereby influence NO signaling pathways in the vessel wall.     

Enhanced electron flux and reduced calmodulin dissociation may explain "calcium-independent" eNOS activation by phosphorylation

Bovine endothelial nitric oxide synthase (eNOS) is phosphorylated directly by the protein kinase Akt at serine 1179. Mutation of this residue to the negatively charged aspartate (S1179D eNOS) increases nitric oxide (NO) production constitutively, in the absence of agonist challenge. Here, we examine the potential mechanism of how aspartate at 1179 increases eNOS activity using purified proteins. Examination of NO production and cytochrome c reduction resulted in no substantial changes in the K(m)/EC(50) for L-arginine, calmodulin, and calcium, whereas there was a 2-fold increase in the rate of NO production for S1179D and a 2-4-fold increase in reductase activity (based on cytochrome c reduction). The observed increase in activity for both assays of NOS function indicates that a faster rate of electron flux through the reductase domain is likely the rate-limiting step in NO formation from eNOS. In addition, S1179D eNOS did show an increased resistance to inactivation by EGTA compared with wild type eNOS. These results suggest that a negative charge imposed at serine 1179, either by phosphorylation or by replacement with aspartate, increases eNOS catalytic activity by increasing electron flux at the reductase domain and by reducing calmodulin dissociation from activated eNOS when calcium levels are low.     

Site-specific dephosphorylation of endothelial nitric oxide synthase by protein phosphatase 2A: evidence for crosstalk between phosphorylation sites

The endothelial isoform of nitric oxide synthase (eNOS) is a calcium/calmodulin-dependent enzyme that catalyzes the synthesis of nitric oxide, a key mediator of vascular homeostasis. eNOS undergoes a variety of posttranslational modifications, including phosphorylation on at least three residues: serines 116 and 1179 and threonine 497. Although the agonist-modulated protein kinase pathways that lead to eNOS phosphorylation have been studied in detail, the signaling pathways governing eNOS dephosphorylation remain less well characterized. The present study identifies protein phosphatase 2A (PP2A) as a key determinant of eNOS dephosphorylation and enzyme activity. We transfected bovine aortic endothelial cells (BAEC) with epitope-tagged cDNAs encoding wild-type eNOS or a series of phosphorylation-deficient eNOS mutants, immunoprecipitated [(32)P(i)] biosynthetically labeled recombinant proteins using antibodies directed against the epitope tag and treated the [(32)P(i)]-phosphorylated eNOS with protein phosphatases. We found that PP2A dephosphorylates eNOS residues threonine 497 and serine 1179 but not serine 116 and that an eNOS mutant lacking these three established phosphorylation sites is robustly labeled when expressed in BAEC and is dephosphorylated by PP2A. An inhibitor of PP2A increases eNOS enzymatic activity and augments overall levels of eNOS phosphorylation, specifically increasing phosphorylation of serines 116 and 1179. When transfected into BAEC or COS-7 cells, a "phospho-mimetic" eNOS mutant in which threonine 497 is changed to aspartate shows attenuated phosphorylation at serine 1179 as well as reduced enzyme activity in COS-7 cells. Our results indicate that regulation of eNOS dephosphorylation may be a key point for control of nitric oxide-dependent signaling pathways in vascular endothelial cells.     

Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism

Shear stress stimulates nitric oxide (NO) production by phosphorylating endothelial NO synthase (eNOS) at Ser(1179) in a phosphoinositide-3-kinase (PI3K)- and protein kinase A (PKA)-dependent manner. The eNOS has additional potential phosphorylation sites, including Ser(116), Thr(497), and Ser(635). Here, we studied these potential phosphorylation sites in response to shear, vascular endothelial growth factor (VEGF), and 8-bromocAMP (8-BRcAMP) in bovine aortic endothelial cells (BAEC). All three stimuli induced phosphorylation of eNOS at Ser(635), which was consistently slower than that at Ser(1179). Thr(497) was rapidly dephosphorylated by 8-BRcAMP but not by shear and VEGF. None of the stimuli phosphorylated Ser(116). Whereas shear-stimulated Ser(635) phosphorylation was not affected by phosphoinositide-3-kinase inhibitors wortmannin and LY-294002, it was blocked by either treating the cells with a PKA inhibitor H89 or infecting them with a recombinant adenovirus-expressing PKA inhibitor. These results suggest that shear stress stimulates eNOS by two different mechanisms: 1) PKA- and PI3K-dependent and 2) PKA-dependent but PI3K-independent pathways. Phosphorylation of Ser(635) may play an important role in chronic regulation of eNOS in response to mechanical and humoral stimuli.     

Proteasome inhibition down-regulates endothelial nitric-oxide synthase phosphorylation and function

Endothelial nitric-oxide synthase (eNOS) function is fundamentally modulated by protein phosphorylation. In particular, phosphorylation of serine 1179 (bovine)/1177 (human) by Akt has been shown to be the central mechanism of eNOS regulation. Here we revealed a novel role of proteasome in controlling eNOS serine 1179 phosphorylation and function. Rather than affecting eNOS turnover, proteasomal inhibition specifically dephosphorylated eNOS serine 1179, leading to decreased enzymatic activity. Blocking protein phosphatase 2A (PP2A) by okadaic acid or PP2A knockdown restored eNOS serine 1179 phosphorylation and activity in proteasome-inhibited cells. Although total PP2A expression and activity in cells were not affected by proteasome inhibitors, proteasomal inhibition induced PP2A ubiquitination and ubiquitinated PP2A translocated from cytosol to membrane. Further biochemical analyses demonstrated that eNOS associated with PP2A on cell membranes. Proteasomal inhibition markedly enhanced PP2A association to eNOS, and this increase of PP2A dephosphorylated eNOS and its upstream kinase Akt. Taken together, these studies identified a novel pathway in which proteasome modulates eNOS phosphorylation by inducing intracellular PP2A translocation.     

Roles of 3-phosphoinositide-dependent kinase 1 in the regulation of endothelial nitric-oxide synthase phosphorylation and function by heat shock protein 90

The 90-kDa heat shock protein (Hsp90) plays an important role in endothelial nitric-oxide synthase (eNOS) regulation. Besides acting as an allosteric enhancer, Hsp90 was shown to serve as a module recruiting Akt to phosphorylate the serine 1179/1177 (bovine/human) residue of eNOS. Akt is activated by the phosphorylation of 3-phosphoinositide-dependent kinase 1 (PDK1). Whether PDK1 is involved in the actions of Hsp90 on eNOS phosphorylation and function remains unknown. To address this issue, we treated bovine eNOS stably transfected human embryonic kidney 293 cells with Hsp90 inhibitors and determined the alterations of phospho-eNOS, Akt, and PDK1. Both geldanamycin and radicicol, two structurally different Hsp90 inhibitors, selectively reduced serine 1179-phosphorylated eNOS, leading to decreased enzyme activity. In Hsp90-inhibited cells, eNOS-associated phospho-Akt was decreased, but the total amount of Akt associated with eNOS remained the same. Further studies showed that Hsp90 inhibition dramatically depleted intracellular PDK1. Proteasome but not caspase blockade prevented the loss of PDK1 caused by Hsp90 inhibition. Silencing the PDK1 gene by small interfering RNA was sufficient to induce reduction of phospho-Akt and consequent loss of serine 1179-phosphorylated eNOS. Moreover, overexpression of PDK1, but not Akt, reversed Hsp90 inhibition-induced loss of eNOS serine 1179 phosphorylation and salvaged enzymatic activity. Thus, in addition to functioning as a module to recruit Akt to eNOS, Hsp90 also critically stabilized PDK1 by preventing it from proteasomal degradation. Inhibition of Hsp90 function resulted in PDK1 depletion and thus triggered a cascade of Akt deactivation, loss of eNOS serine 1179 phosphorylation, and decrease of enzyme function.     

Chk1 and Hsp90 cooperatively regulate phosphorylation of endothelial nitric oxide synthase at serine 1179

The effects of DNA damage on NO production have not been completely elucidated. Using ultraviolet (UV) irradiation as a DNA-damaging agent, we studied its effect on NO production in bovine aortic endothelial cells (BAEC). UV irradiation acutely increased NO production, the phosphorylation of endothelial NO synthase (eNOS) at serine 1179, and eNOS activity. No alterations in eNOS expression nor phosphorylation at eNOS Thr⁴⁹⁷ or eNOS Ser¹¹⁶ were found. SB218078, a checkpoint kinase 1 (Chk1) inhibitor, inhibited UV-irradiation-stimulated eNOS-Ser¹¹⁷⁹ phosphorylation and NO production. Similarly, ectopic expression of small interference RNA for Chk1 or a dominant-negative Chk1 repressed the UV-irradiation stimulatory effect, whereas wild-type Chk1 increased basal eNOS-Ser¹¹⁷⁹ phosphorylation. Purified Chk1 directly phosphorylated eNOS Ser¹¹⁷⁹ in vitro. Confocal microscopy and coimmunoprecipitation studies revealed a colocalization of eNOS and Chk1. In basal BAEC, heat shock protein 90 (Hsp90) predominantly interacted with Chk1. This interaction, which decreased significantly in response to UV irradiation, was accompanied by increased interaction of Hsp90 with eNOS. The Hsp90 inhibitor geldanamycin attenuated UV-irradiation-stimulated eNOS-Ser¹¹⁷⁹ phosphorylation by dissociating Hsp90 from eNOS. UV irradiation and geldanamycin did not alter the interaction between eNOS and Chk1. Overall, this is the first study demonstrating that Chk1 directly phosphorylates eNOS Ser¹¹⁷⁹ in response to UV irradiation, which is dependent on Hsp90 interaction.     

Dissociation of endothelial nitric oxide synthase phosphorylation and activity in uterine artery endothelial cells

Pregnancy enhanced nitric oxide production by uterine artery endothelial cells (UAEC) is the result of reprogramming of both Ca(2+) and kinase signaling pathways. Using UAEC derived from pregnant ewes (P-UAEC), as well as COS-7 cells transiently expressing ovine endothelial nitric oxide synthase (eNOS), we investigated the role of phosphorylation of five known amino acids following treatment with physiological calcium-mobilizing agent ATP and compared with the effects of PMA (also known as TPA) alone or in combination with ATP. In P-UAEC, ATP stimulated eNOS activity and phosphorylation of eNOS S617, S635, and S1179. PMA promoted eNOS phosphorylation but without activation. PMA and ATP cotreatment attenuated ATP-stimulated activity despite no increase in phospho (p)-T497 and potentiation of p-S1179. In COS-7 cells, PMA inhibition of ATP-stimulated eNOS activity was associated with p-T497 phosphorylation. Although T497D eNOS activity was reduced to 19% of wild-type eNOS with ATP and 44% with A23187, we nonetheless observed more p-S1179 with ATP than with A23187 (3.4-fold and 1.8-fold of control, respectively). Furthermore, the S1179A eNOS mutation partly attenuated ATP- but not A23187-stimulated activity, but when combined with T497D, no further reduction of eNOS activity was observed. In conclusion, although phosphorylation of eNOS is associated with activation in P-UAEC, no single or combination of phosphorylation events predict activity changes. In COS-7 cells, phosphorylation of T497 can attenuate activity but also influences S1179 phosphorylation. We conclude that in both cell types, observed changes in phosphorylation of key residues may influence eNOS activation but are not sufficient alone to describe eNOS activation.     

Endostatin induces acute endothelial nitric oxide and prostacyclin release

Chronic exposure to endostatin (ES) blocks endothelial cell (EC) proliferation, and migration and induces EC apoptosis thereby inhibiting angiogenesis. Nitric oxide (NO) and prostacyclin (PGI(2)), in contrast, play important roles in promoting angiogenesis. In this study, we examined the acute effects of ES on endothelial NO and PGI(2) production. Unexpectedly, a cGMP reporter cell assay showed that ES-induced acute endothelial NO release in cultured bovine aortic endothelial cells (BAECs). Enzyme immunoassay showed that ES also induced an acute increase in PGI(2) production in BAECs. These results were confirmed by ex vivo vascular ring studies that showed vascular relaxation in response to ES. Immunoblot analysis showed that ES stimulated acute phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser116, Ser617, Ser635, and Ser1179, and dephosphorylation at Thr497 in BAECs, events associated with eNOS activation. Short-term exposure of EC to ES, therefore, unlike long-term exposure which is anti-angiogenic, may be pro-angiogenic.     

Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A.

Recently, we have shown that shear stress stimulates NO(*) production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91, 1574-1581). Akt has been believed to regulate shear-dependent production of NO(*) by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser(1179) residue (eNOS-S(1179)), but a critical evaluation using specific inhibitors or dominant negative mutants (Akt(AA) or Akt(AAA)) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S(1179). Here, we show that shear-dependent phosphorylation of eNOS-S(1179) is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt(AA) or Akt(AAA) in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S(1179) if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt(AA) inhibited shear-dependent NO(*) production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S(1179) and NO(*) production. In contrast, H89 did not inhibit phosphorylation of eNOS-S(1179) induced by expressing a constitutively active Akt mutant (Akt(Myr)) in BAEC, showing that the inhibitor did not affect the Akt pathway. 8-Bromo-cAMP alone phosphorylated eNOS-S(1179) within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S(1179) in a PKA-dependent, but Aktindependent manner, whereas the NO(*) production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction between Akt and PKA may be an important mechanism by which eNOS activity is regulated in response to physiological stimuli such as shear stress.     

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.     

Endothelial cell superoxide anion radical generation is not dependent on endothelial nitric oxide synthase-serine 1179 phosphorylation and endothelial nitric oxide synthase dimer/monomer distribution

Tetrahydrobiopterin (BH4) and heat shock protein 90 (hsp90) have been anticipated to regulate endothelial nitric oxide synthase (eNOS)-dependent superoxide anion radical (O2*-) generation in endothelial cells. It is not known, however, whether hsp90 and BH4 increase O2*- in a synergistic manner, or whether this increase is a consequence of downstream changes in eNOS phosphorylation on serine 1179 (eNOS-S1179) and changes in dimer/monomer distribution. Here O2*- production from purified BH4 -free eNOS and eNOS:hsp90 complexes determined by spin-trapping methodology showed that hsp90 neither inhibits O2*- nor alters the requirement of BH4 to inhibit radical release from eNOS. In endothelial cells, O2*- detection with the novel high-performance liquid chromatography assay of 2-hydroxyethidium showed that inhibition of hsp90 did not increase O2*-, while a significant increase in O2*- was detected in BH4 -depleted cells. Radicicol, a hsp90 inhibitor, disrupted eNOS:hsp90 association, decreased eNOS-S1179, but increased biopterin production in a dose-dependent fashion. These changes were followed by an increase in eNOS activity, demonstrating that high biopterin levels offset inhibition of eNOS phosphorylation and diminished interaction with hsp90. In contrast, depletion of biopterin did not affect hsp90 levels or interaction with eNOS or eNOS dimer/monomer ratio in bovine aorta endothelial cells (BAECs). We conclude that low BH4 but not inhibition of hsp90 increases O2*- in BAECs by mechanism(s) that unlikely involve phosphorylation to eNOS-S1179 or eNOS monomerization.