The role of caveolin-1 in PCB77-induced eNOS phosphorylation in human-derived endothelial cells

Polychlorinated biphenyls (PCBs) may contribute to the pathology of atherosclerosis by activating inflammatory responses in vascular endothelial cells. Endothelial nitric oxide synthase (eNOS) is colocalized with caveolae and is a critical regulator of vascular homeostasis. PCBs may be proatherogenic by causing dysfunctional eNOS signaling. The objective of this study was to investigate the role of caveolin-1 in PCB-induced endothelial dysfunction with a focus on mechanisms associated with eNOS signaling. Cells derived from an immortalized human vascular endothelial cell line were treated with PCB77 to study nitrotyrosine formation through eNOS signaling. Phosphorylation studies of eNOS, caveolin-1, and kinases, such as Src, phosphatidylinositol 3-kinase (PI3K), and Akt, were conducted in cells containing either functional or small-interfering RNA-silenced caveolin-1 protein. We also investigated caveolin-1-regulated mechanisms associated with PCB-induced markers of peroxynitrite formation and DNA binding of NF-kappaB. Cellular exposure to PCB77 increased eNOS phosphorylation and nitric oxide production, as well as peroxynitrite levels. A subsequent PCB-induced increase in NF-kappaB DNA binding may have implications in oxidative stress-mediated inflammatory mechanisms. The activation of eNOS by PCB77 treatment was blocked by inhibitors of the Src/PI3K/Akt pathway. PCB77 also increased phosphorylation of caveolin-1, indicating caveolae-dependent endocytosis. Caveolin-1 silencing abolished both the PCB-stimulated Akt and eNOS phosphorylation, suggesting a regulatory role of caveolae in PCB-induced eNOS signaling. These findings suggest that PCB77 induces eNOS phosphorylation in endothelial cells through a Src/PI3K/Akt-dependent mechanism, events regulated by functional caveolin-1. Our data provide evidence that caveolae may play a critical role in regulating vascular endothelial cell activation and toxicity induced by persistent environmental pollutants such as coplanar PCBs.     

Effects of simulated microgravity on human umbilical vein endothelial cell angiogenesis and role of the PI3K-Akt-eNOS signal pathway

Endothelial cells are very sensitive to microgravity and the morphological and functional changes in endothelial cells are believed to be at the basis of weightlessness-induced cardiovascular deconditioning. It has been shown that the proliferation, migration, and morphological differentiation of endothelial cells play critical roles in angiogenesis. However, the influence of microgravity on the ability of endothelial cells to foster angiogenesis remains to be explored in detail. In the present study, we used a clinostat to simulate microgravity, and we observed tube formation, migration, and expression of endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVEC-C). Specific inhibitors of eNOS and phosphoinositide 3-kinase (PI3K) were added to the culture medium and gravity-induced changes in the pathways that mediate angiogenesis were investigated. After 24 h of exposure to simulated microgravity, HUVEC-C tube formation and migration were significantly promoted.This was reversed by co-incubation with the specific inhibitor of N-nitro-L-arginine methyl ester hydrochloride (eNOS). Immunofluorescence assay, RT-PCR, and Western blot analysis demonstrated that eNOS expression in the HUVEC-C was significantly elevated after simulated microgravity exhibition. Ultrastructure observation via transmission electron microscope showed the number of caveolae organelles in the membrane of HUVEC-C to be significantly reduced. This was correlated with enhanced eNOS activity. Western blot analysis then showed that phosphorylation of eNOS and serine/threonine kinase (Akt) were both up-regulated after exposure to simulated microgravity. However, the specific inhibitor of PI3K not only significantly downregulated the expression of phosphorylated Akt, but also downregulated the phosphorylation of eNOS. This suggested that the PI3K-Akt signal pathway might participate in modulating the activity of eNOS. In conclusion, the present study indicates that 24 h of exposure to simulated microgravity promote angiogenesis among HUVEC-C and that this process is mediated through the PI3K-Akt-eNOS signal pathway.     

Inhibition of insulin signaling in endothelial cells by protein kinase C-induced phosphorylation of p85 subunit of phosphatidylinositol 3-kinase (PI3K)

The regulation of endothelial function by insulin is consistently abnormal in insulin-resistant states and diabetes. Protein kinase C (PKC) activation has been reported to inhibit insulin signaling selectively in endothelial cells via the insulin receptor substrate/PI3K/Akt pathway to reduce the activation of endothelial nitric-oxide synthase (eNOS). In this study, it was observed that PKC activation differentially inhibited insulin receptor substrate 1/2 (IRS1/2) signaling of insulin's activation of PI3K/eNOS by decreasing only tyrosine phosphorylation of IRS2. In addition, PKC activation, by general activator and specifically by angiotensin II, increased the phosphorylation of p85/PI3K, which decreases its association with IRS1 and activation. Thr-86 of p85/PI3K was identified to be phosphorylated by PKC activation and confirmed to affect IRS1-mediated activation of Akt/eNOS by insulin and VEGF using a deletion mutant of the Thr-86 region of p85/PI3K. Thus, PKC and angiotensin-induced phosphorylation of Thr-86 of p85/PI3K may partially inhibit the activation of PI3K/eNOS by multiple cytokines and contribute to endothelial dysfunction in metabolic disorders.     

Fractalkine stimulates angiogenesis by activating the Raf-1/MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways

Fractalkine (FKN) has been implicated in modulation of angiogenesis and vascular inflammation, but the underlying mechanism has not been elucidated. We have investigated the molecular mechanism by which FKN regulates angiogenesis. We found that recombinant FKN increases in vitro proliferation, migration, and tube formation of human umbilical vein endothelial cells and stimulates in vivo angiogenesis. FKN-induced angiogenesis was accompanied by phosphorylation of ERK, Akt, and endothelial nitric oxide (NO) synthase (eNOS), as well as an increase in NO production. These biochemical events and angiogenesis were completely inhibited by the G protein-coupled receptor inhibitor pertussis toxin. Inhibitors of Raf-1, MEK, phosphatidylinositol 3-kinase (PI3K), and eNOS or transfection with dominant-negative forms of ERK and Akt significantly suppressed the angiogenic activity of FKN. However, inhibitors of Raf-1 and MEK or a dominant-negative ERK mutant blocked FKN-induced ERK, but not Akt and eNOS, phosphorylation. The PI3K inhibitor and a dominant-negative mutant of Akt suppressed Akt and eNOS phosphorylation and NO production. Our results demonstrated that FKN stimulated angiogenesis by activating the Raf-1/MEK/ERK and PI3K/Akt/eNOS/NO signal pathways via the G protein-coupled receptor CX3CR1, indicating that two pathways are required for full angiogenic activity of FKN. This study suggests that FKN may play an important role in the pathophysiological process of inflammatory angiogenesis.     

Forskolin increases angiogenesis through the coordinated cross-talk of PKA-dependent VEGF expression and Epac-mediated PI3K/Akt/eNOS signaling

Forskolin, a potent activator of adenylyl cyclases, has been implicated in modulating angiogenesis, but the underlying mechanism has not been clearly elucidated. We investigated the signal mechanism by which forskolin regulates angiogenesis. Forskolin stimulated angiogenesis of human endothelial cells and in vivo neovascularization, which was accompanied by phosphorylation of CREB, ERK, Akt, and endothelial nitric oxide synthase (eNOS) as well as NO production and VEGF expression. Forskolin-induced CREB phosphorylation, VEGF promoter activity, and VEGF expression were blocked by the PKA inhibitor PKI. Moreover, phosphorylation of ERK by forskolin was inhibited by the MEK inhibitor PD98059, but not PKI. The forskolin-induced Akt/eNOS/NO pathway was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, but not significantly suppressed by PKI. These inhibitors and a NOS inhibitor partially inhibited forskolin-induced angiogenesis. The exchange protein directly activated by cAMP (Epac) activator, 8CPT-2Me-cAMP, promoted the Akt/eNOS/NO pathway and ERK phosphorylation, but did not induce CREB phosphorylation and VEGF expression. The angiogenic effect of the Epac activator was diminished by the inhibition of PI3K and MEK, but not by the PKA inhibitor. Small interfering RNA-mediated knockdown of Epac1 suppressed forskolin-induced angiogenesis and phosphorylation of ERK, Akt, and eNOS, but not CREB phosphorylation and VEGF expression. These results suggest that forskolin stimulates angiogenesis through coordinated cross-talk between two distinct pathways, PKA-dependent VEGF expression and Epac-dependent ERK activation and PI3K/Akt/eNOS/NO signaling.     

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

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

Icariin stimulates angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways in human endothelial cells

We investigated the molecular effect and signal pathway of icariin, a major flavonoid of Epimedium koreanum Nakai, on angiogenesis. Icariin stimulated in vitro endothelial cell proliferation, migration, and tubulogenesis, which are typical phenomena of angiogenesis, as well as increased in vivo angiogenesis. Icariin activated the angiogenic signal modulators, ERK, phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric oxide synthase (eNOS), and increased NO production, without affecting VEGF expression, indicating that icariin may directly stimulate angiogenesis. Icariin-induced ERK activation and angiogenic events were significantly inhibited by the MEK inhibitor PD98059, without affecting Akt and eNOS phosphorylation. The PI3K inhibitor Wortmannin suppressed icariin-mediated angiogenesis and Akt and eNOS activation without affecting ERK phosphorylation. Moreover, the NOS inhibitor NMA partially reduced the angiogenic activity of icariin. These results suggest that icariin stimulated angiogenesis by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways and may be a useful drug for angiogenic therapy.     

Oxidized LDL at low concentration promotes in-vitro angiogenesis and activates nitric oxide synthase through PI3K/Akt/eNOS pathway in human coronary artery endothelial cells

It has long been considered that oxidized low-density lipoprotein (oxLDL) causes endothelial dysfunction and is remarkably related to the development of atherosclerosis. However, the effect of oxLDL at very low concentration (<10 μg/ml) on the endothelial cells remains speculative. Nitric oxide (NO) has a crucial role in the endothelial cell function. In this study, we investigated the effect of oxLDL at low concentration on NO production and proliferation, migration, tube formation of the human coronary artery endothelial cells (HCAEC). Results showed that oxLDL at 5 μg/ml enhanced HCAEC proliferation, migration and tube formation. These phenomena were accompanied by an increased intracellular NO production. l-NAME (a NOS inhibitor), LY294002 and wortmannin (PI3K inhibitors) could abolish oxLDL-induced angiogenic effects and prevent NO production in the HCAEC. The phosphorylation of Akt, PI3K and eNOS were up-regulated by oxLDL, which was attenuated by LY294002. Our results suggested that oxLDL at low concentration could promote in-vitro angiogenesis and activate nitric oxide synthesis through PI3K/Akt/eNOS pathway in HCAEC.     

Flow shear stress stimulates Gab1 tyrosine phosphorylation to mediate protein kinase B and endothelial nitric-oxide synthase activation in endothelial cells

Fluid shear stress generated by blood flow modulates endothelial cell function via specific intracellular signaling events. We showed previously that flow activated the phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) via Src kinase-dependent transactivation of vascular endothelial growth factor receptor 2 (VEGFR2). The scaffold protein Gab1 plays an important role in receptor tyrosine kinase-mediated signal transduction. We found here that laminar flow (shear stress = 12 dynes/cm2) rapidly stimulated Gab1 tyrosine phosphorylation in both bovine aortic endothelial cells and human umbilical vein endothelial cells, which correlated with activation of Akt and eNOS. Gab1 phosphorylation as well as activation of Akt and eNOS by flow was inhibited by the Src kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and VEGFR2 kinase inhibitors SU1498 and VTI, suggesting that flow-mediated Gab1 phosphorylation is Src kinase-dependent and VEGFR2-dependent. Tyrosine phosphorylation of Gab1 by flow was functionally important, because flow stimulated the association of Gab1 with the PI3K subunit p85 in a time-dependent manner. Furthermore, transfection of a Gab1 mutant lacking p85 binding sites inhibited flow-induced activation of Akt and eNOS. Finally, knockdown of endogenous Gab1 by small interference RNA abrogated flow activation of Akt and eNOS. These data demonstrate a critical role of Gab1 in flow-stimulated PI3K/Akt/eNOS signal pathway in endothelial cells.     

Glimepiride activates eNOS with a mechanism Akt but not caveolin-1 dependent

Insulin stimulates caveolin-1 and eNOS phosphorylation. The sulfonylurea glimepiride mimics several insulin actions by mechanisms that are poorly understood. Glimepiride induces caveolin-1 phosphorylation and activates PI3K and Akt in rat adipocytes. In this paper, we investigated the effect of glimepiride on eNOS activation in human endothelial cells. We found that glimepiride induces caveolin-1 and eNOS phosphorylation. To better understand the role of caveolin-1 in glimepiride action, we downregulated caveolin-1 expression by specific siRNA transfection. Caveolin-1 silencing did not change eNOS and Akt phosphorylation induced by glimepiride. On the contrary, LY294002, a specific PI3K inhibitor, blocked eNOS serine 1177 phosphorylation. These findings suggest that glimepiride induces eNOS phosphorylation in endothelial cells through an Akt-dependent mechanism, not regulated by caveolin-1.     

Insulin resistance in spontaneously hypertensive rats is associated with endothelial dysfunction characterized by imbalance between NO and ET-1 production

Insulin stimulates production of NO in vascular endothelium via activation of phosphatidylinositol (PI) 3-kinase, Akt, and endothelial NO synthase. We hypothesized that insulin resistance may cause imbalance between endothelial vasodilators and vasoconstrictors (e.g., NO and ET-1), leading to hypertension. Twelve-week-old male spontaneously hypertensive rats (SHR) were hypertensive and insulin resistant compared with control Wistar-Kyoto (WKY) rats (systolic blood pressure 202 +/- 11 vs. 132 +/- 10 mmHg; fasting plasma insulin 5 +/- 1 vs. 0.9 +/- 0.1 ng/ml; P < 0.001). In WKY rats, insulin stimulated dose-dependent relaxation of mesenteric arteries precontracted with norepinephrine (NE) ex vivo. This depended on intact endothelium and was blocked by genistein, wortmannin, or N(omega)-nitro-l-arginine methyl ester (inhibitors of tyrosine kinase, PI3-kinase, and NO synthases, respectively). Vasodilation in response to insulin (but not ACh) was impaired by 20% in SHR (vs. WKY, P < 0.005). Preincubation of arteries with insulin significantly reduced the contractile effect of NE by 20% in WKY but not SHR rats. In SHR, the effect of insulin to reduce NE-mediated vasoconstriction became evident when insulin pretreatment was accompanied by ET-1 receptor blockade (BQ-123, BQ-788). Similar results were observed during treatment with the MEK inhibitor PD-98059. In addition, insulin-stimulated secretion of ET-1 from primary endothelial cells was significantly reduced by pretreatment of cells with PD-98059 (but not wortmannin). We conclude that insulin resistance in SHR is accompanied by endothelial dysfunction in mesenteric vessels with impaired PI3-kinase-dependent NO production and enhanced MAPK-dependent ET-1 secretion. These results may reflect pathophysiology in other vascular beds that directly contribute to elevated peripheral vascular resistance and hypertension.     

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…     

Activation of the phosphatidylinositol 3-kinase/protein kinase Akt pathway mediates CD151-induced endothelial cell proliferation and cell migration

We previously reported that CD151 promotes neovascularization and improves blood perfusion in rat hind-limb ischemia model, but the precise mechanism is still unclear. Endothelial cell proliferation and cell migration play critical roles in angiogenesis. Many growth factors and hormones have been shown to regulate cell proliferation, cell migration and angiogenesis, including the activation of eNOS activity, via the PI3K/Akt signaling pathway. Whether CD151 induces cell proliferation and cell migration via PI3K/Akt signaling pathway is not known. Here we showed that CD151 promotes human umbilical vein endothelial cell (HUVEC) proliferation, migration and tube formation in vitro, accompanied by increased phosphorylation of Akt and eNOS, leading to increased eNOS activity and nitric oxide (NO) levels after rAAV-CD151 infection, whereas infection with rAAV-anti-CD151 attenuated the effects of CD151, which suggested that CD151 can activate PI3K/Akt pathway. Moreover, inhibitors of PI3K (LY294002) and eNOS (l-NAME) can attenuate CD151-induced cell proliferation and cell migration. The results suggested that activation of PI3K/Akt signaling pathway mediates CD151-induced cell proliferation and migration.     

Involvement of androgen receptor in nitric oxide production induced by icariin in human umbilical vein endothelial cells

Icariin, a flavonoid isolated from Epimedii herba, stimulated phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177, Akt (Ser473) and ERK1/2 (Thr202/Tyr204). The icariin-induced eNOS phosphorylation was abolished by an androgen receptor (AR) antagonist, nilutamide in human umbilical vein endothelial cells (HUVECs). Furthermore, it was also reduced in the cells transfected with small interfering RNA in which the expression of AR was broken down. The icariin-induced eNOS phosphorylation was inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor and partially attenuated by PD98059, an upstream inhibitor for ERK1/2. These data suggest that icariin stimulates release of NO by AR-dependent activation of eNOS in HUVECs. PI3K/Akt and MAPK-ERK kinase (MEK)/ERK1/2 pathways were involved in the phosphorylation of eNOS by icariin.     

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.     

Src kinase mediates phosphatidylinositol 3-kinase/Akt-dependent rapid endothelial nitric-oxide synthase activation by estrogen

17beta-Estradiol activates endothelial nitric oxide synthase (eNOS), enhancing nitric oxide (NO) release from endothelial cells via the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. The upstream regulators of this pathway are unknown. We now demonstrate that 17beta-estradiol rapidly activates eNOS through Src kinase in human endothelial cells. The Src family kinase specific-inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) abrogates 17beta-estradiol- but not ionomycin-stimulated NO release. Consistent with these results, PP2 blocked 17beta-estradiol-induced Akt phosphorylation but did not inhibit NO release from cells transduced with a constitutively active Akt. PP2 abrogated 17beta-estradiol-induced activation of PI3-kinase, indicating that the PP2-inhibitable kinase is upstream of PI3-kinase and Akt. A 17beta-estradiol-induced estrogen receptor/c-Src association correlated with rapid c-Src phosphorylation. Moreover, transfection of kinase-dead c-Src inhibited 17beta-estradiol-induced Akt phosphorylation, whereas constitutively active c-Src increased basal Akt phosphorylation. Estrogen stimulation of murine embryonic fibroblasts with homozygous deletions of the c-src, fyn, and yes genes failed to induce Akt phosphorylation, whereas cells maintaining c-Src expression demonstrated estrogen-induced Akt activation. Estrogen rapidly activated c-Src inducing an estrogen receptor, c-Src, and P85 (regulatory subunit of PI3-kinase) complex formation. This complex formation results in the successive activation of PI3-kinase, Akt, and eNOS with consequent enhanced NO release, implicating c-Src as a critical upstream regulator of the estrogen-stimulated PI3-kinase/Akt/eNOS pathway.     

Role of G(i/o)-Src kinase-PI3K/Akt pathway and caveolin-1 in β₂-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery

Activation of the β₂-adrenoceptor (β₂-AR) elicits an endothelial nitric oxide synthase (eNOS)-dependent relaxation in mouse pulmonary artery, which, contrary to the muscarinic receptor-dependent relaxation, is preserved in hypoxic pulmonary arterial hypertension. We therefore characterized the signaling pathways underlying the β₂-AR-mediated eNOS activation, with special focus on G_i/o proteins, protein kinases and caveolae. Functional studies (for evaluation of vasorelaxant response), Western blotting (for assessment of eNOS and caveolin-1 phosphorylation) and transmission electron microscopy (for visualization of caveolae) were conducted in pulmonary arteries from wild-type or caveolin-1 knockout mice. In wild-type isolated arteries, relaxation to the selective β₂-AR agonist procaterol was reduced by inhibitors of G_i/o proteins (pertussis toxin, PTX), phosphatidylinositol 3-kinase (PI3K; wortmannin or LY 294002), Akt (Akt inhibitor X) and Src-kinase (PP2) and by cholesterol depletion (using methyl-β-cyclodextrin). Procaterol induced eNOS phosphorylation at Ser¹¹⁷⁷, which was prevented by PTX, PP2 or Akt inhibitor. Procaterol also promoted caveolin-1 phosphorylation at Tyr¹⁴, which was decreased by PTX or PP2. Caveolin-1 gene deletion resulted in endothelial caveolae disruption in mouse pulmonary artery and in potentiation of procaterol-induced relaxation. Unlike procaterol, acetylcholine-induced relaxation was unaffected by PTX, methyl-β-cyclodextrin or caveolin-1 gene deletion. To conclude, the mouse pulmonary endothelial β₂-AR is coupled to a G_i/o-Src kinase-PI3K/Akt pathway to promote eNOS phosphorylation at Ser¹¹⁷⁷ leading to a NO-dependent vasorelaxation. Caveolin-1 exerts a negative control on this response that is abrogated by its phosphorylation at Tyr¹⁴, through a G_i/o-Src kinase pathway. Since pulmonary β₂-AR- and muscarinic receptor-mediated relaxations differentiate in their respective signaling pathways leading to eNOS activation and sensitivities during hypoxia-induced pulmonary arterial hypertension, mechanisms underlying eNOS activation might be key determinants of pulmonary endothelial dysfunction.     

Sphingosine 1-phosphate and isoform-specific activation of phosphoinositide 3-kinase beta. Evidence for divergence and convergence of receptor-regulated endothelial nitric-oxide synthase signaling pathways

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that elicits diverse biological responses, including angiogenesis, via the activation of G protein-coupled EDG receptors. S1P activates the endothelial isoform of nitric-oxide synthase (eNOS), associated with eNOS phosphorylation at Ser-1179, a site phosphorylated by protein kinase Akt. We explored the proximal signaling pathways that mediate Akt activation and eNOS regulation by S1P/EDG receptors. Akt is regulated by the lipid kinase phosphoinositide 3-kinase (PI3-K). We found that bovine aortic endothelial cells (BAEC) express both alpha and beta isoforms of PI3-K, while lacking the gamma isoform. S1P treatment led to the rapid and isoform-specific activation of PI3-Kbeta in BAEC. PI3-Kbeta can be regulated by G protein betagamma subunits (Gbetagamma). The overexpression of a peptide inhibitor of Gbetagamma attenuated S1P-induced eNOS enzyme activation, as well as S1P-induced phosphorylation of eNOS and Akt. In contrast, bradykinin, a classical eNOS agonist, neither activated any PI3-K isoform nor induced eNOS phosphorylation at Ser-1179, despite activating eNOS in BAEC. Vascular endothelial growth factor activated both PI3-Kalpha and PI3-Kbeta via tyrosine kinase pathways and promoted eNOS phosphorylation that was unaffected by Gbetagamma inhibition. These findings indicate that PI3-Kbeta (regulated by Gbetagamma) may represent a novel molecular locus for eNOS activation by EDG receptors in vascular endothelial cells. These studies also indicate that different eNOS agonists activate distinct signaling pathways that diverge proximally following receptor activation but converge distally to activate eNOS.