Nitric oxide (NO) produced by the endothelial NO synthase (eNOS) is a fundamental determinant of cardiovascular homesotasis: it regulates systemic blood pressure, vascular remodelling and angiogenesis. Physiologically, the most important stimulus for the continuous formation of NO is the viscous drag (shear stress) generated by the streaming blood on the endothelial layer. Although shear-stress-mediated phosphorylation of eNOS is thought to regulate enzyme activity, the mechanism of activation of eNOS is not yet known. Here we demonstrate that the serine/threonine protein kinase Akt/PKB mediates the activation of eNOS, leading to increased NO production. Inhibition of the phosphatidylinositol-3-OH kinase/Akt pathway or mutation of the Akt site on eNOS protein (at serine 1177) attenuates the serine phosphorylation and prevents the activation of eNOS. Mimicking the phosphorylation of Ser 1177 directly enhances enzyme activity and alters the sensitivity of the enzyme to Ca2+, rendering its activity maximal at sub-physiological concentrations of Ca2+. Thus, phosphorylation of eNOS by Akt represents a novel Ca2+-independent regulatory mechanism for activation of eNOS.

Abstract-The activity of the endothelial nitric oxide synthase (eNOS) can be regulated independently of an increase in Ca 2ϩ by the phosphorylation of Ser 1177 but results only in a low nitric oxide (NO) output. In the present study, we assessed whether the agonist-induced (Ca 2ϩ -dependent, high-output) activation of eNOS is associated with changes in the phosphorylation of

In most arterial beds a significant endothelium-dependent dilation to various stimuli persists even after inhibition of nitric oxide synthase and cyclo-oxygenase. This dilator response is preceded by an endothelium-dependent hyperpolarization of vascular smooth muscle cells, which is sensitive to a combination of the calcium-dependent potassium-channel inhibitors charybdotoxin and apamin, and is assumed to be mediated by an unidentified endothelium-derived hyperpolarizing factor (EDHF). Here we show that the induction of cytochrome P450 (CYP) 2C8/34 in native porcine coronary artery endothelial cells by beta-naphthoflavone enhances the formation of 11,12-epoxyeicosatrienoic acid, as well as EDHF-mediated hyperpolarization and relaxation. Transfection of coronary arteries with CYP 2C8/34 antisense oligonucleotides results in decreased levels of CYP 2C and attenuates EDHF-mediated vascular responses. Thus, a CYP-epoxygenase product is an essential component of EDHF-mediated relaxation in the porcine coronary artery, and CYP 2C8/34 fulfils the criteria for the coronary EDHF synthase.

Abstract-TRPV4 is a broadly expressed Ca 2ϩ -permeable cation channel in the vanilloid subfamily of transient receptor potential channels. TRPV4 gates in response to a large variety of stimuli, including cell swelling, warm temperatures, the synthetic phorbol ester 4␣-phorbol 12,13-didecanoate (4␣-PDD), and the endogenous lipid arachidonic acid (AA). Activation by cell swelling and AA requires cytochrome P450 (CYP) epoxygenase activity to convert AA to epoxyeicosatrienoic acids (EETs) such as 5,6-EET, 8,9-EET, which both act as direct TRPV4 agonists. To evaluate the role of TRPV4 and its modulation by the CYP pathway in vascular endothelial cells, we performed Ca 2ϩ imaging and patch-clamp measurements on mouse aortic endothelial cells (MAECs) isolated from wild-type and TRPV4 Ϫ/Ϫ mice. All TRPV4-activating stimuli induced robust Ca 2ϩ responses in wild-type MAECs but not in MAECs isolated from TRPV4 Ϫ/Ϫ mice. Upregulation of CYP2C expression by preincubation with nifedipine enhanced the responses to AA and cell swelling in wild-type MAECs, whereas responses to other stimuli remained unaffected. Conversely, inhibition of CYP2C9 activity with sulfaphenazole abolished the responses to AA and hypotonic solution (HTS). Moreover, suppression of EET hydrolysis using 1-adamantyl-3-cyclo-hexylurea or indomethacin, inhibitors of soluble epoxide hydrolases (sEHs), and cyclooxygenases, respectively, enhanced the TRPV4-dependent responses to AA, HTS, and EETs but not those to 4␣-PDD or heat. Together, our data establish that CYP-derived EETs modulate the activity of TRPV4 channels in endothelial cells and shows the unraveling of novel modulatory pathways via CYP2C modulation and sEH inhibition. Key Words: TRP channels Ⅲ endothelium Ⅲ epoxygenases Ⅲ epoxide hydrolases T RPV4 is a Ca 2ϩ entry channel belonging to the vanilloid subfamily of the transient receptor potential (TRP) channels. 1,2 It is expressed in a broad range of tissues, including lung, spleen, kidney, testis, fat, brain, cochlea, skin, smooth muscle, liver, and vascular endothelium. [3][4][5] The physiological role of TRPV4 in these tissues may be highly divergent because TRPV4 is able to respond to a wide variety of physical, thermal, and chemical stimuli.Initially, TRPV4 was put forward as a mechanosensor or osmosensor. This was based on the finding that the channel opens in response to hypotonic cell swelling 3-8 as well as shear stress. 9 Indeed, mice lacking the TRPV4 gene show a disturbance of osmoregulation and an increased mechanical nociceptive threshold. 10,11 Furthermore, TRPV4 functions as a transducer of hypo-osmotic stimuli in primary afferent nociceptors 7 and plays an essential role in taxol-induced nociceptive behavioral responses to mechanical and hypotonic stimulation of the hind paw. 12 More recently, it was found that TRPV4 can also be activated by heating, 13,14 and that it is required for normal thermal responsiveness in vivo. The TRPV4 Ϫ/Ϫ mice "select" a warmer floor temperature in a gradient apparatus than their wild-type littermates ...

Abstract-In the porcine coronary artery, a cytochrome P450 (CYP) isozyme homologous to CYP 2C8/9 has been identified as an endothelium-derived hyperpolarizing factor (EDHF) synthase. As some CYP enzymes are reported to generate reactive oxygen species (ROS), we hypothesized that the coronary EDHF synthase may modulate vascular homeostasis by the simultaneous production of ROS and epoxyeicosatrienoic acids. In bradykinin-stimulated coronary arteries, antisense oligonucleotides against CYP 2C almost abolished EDHF-mediated responses but potentiated nitric oxide (NO)-mediated relaxation. The selective CYP 2C9 inhibitor sulfaphenazole and the superoxide anion (O 2 Ϫ ) scavengers Tiron and nordihydroguaretic acid also induced a leftward shift in the NO-mediated concentration-relaxation curve to bradykinin. CYP activity and O 2 Ϫ production, determined in microsomes prepared from cells overexpressing CYP 2C9, were almost completely inhibited by sulfaphenazole. Sulfaphenazole did not alter the activity of either CYP 2C8, the leukocyte NADPH oxidase, or xanthine oxidase. ROS generation in coronary artery rings, visualized using either ethidium or dichlorofluorescein fluorescence, was detected under basal conditions. The endothelial signal was attenuated by CYP 2C antisense treatment as well as by sulfaphenazole. In isolated coronary endothelial cells, bradykinin elicited a sulfaphenazole-sensitive increase in ROS production. Although 11,12 epoxyeicosatrienoic acid attenuated the activity of nuclear factor-B in cultured human endothelial cells, nuclear factor-B activity was enhanced after the induction or overexpression of CYP 2C9, as was the expression of vascular cell adhesion molecule-1. These results suggest that a CYP isozyme homologous to CYP 2C9 is a physiologically relevant generator of ROS in coronary endothelial cells and modulates both vascular tone and homeostasis. Key Words: coronary artery Ⅲ cytochrome P450 Ⅲ endothelium-derived hyperpolarizing factor Ⅲ NADPH oxidase Ⅲ reactive oxygen species T he bioavailability of nitric oxide (NO) within the vascular wall and, as a consequence, NO-mediated relaxation is attenuated by an elevation in superoxide anion (O 2 Ϫ ) production. 1 Enzymes capable of generating reactive oxygen species (ROS) within the vasculature are, for example, NO synthases (NOS), cyclooxygenases, lipoxygenases, xanthine oxidase, and NADPH oxidase, all of which are reported to be functional in endothelial cells. 2 Diphenyleneiodonium has been used extensively to characterize ROS-generating enzymatic systems; however, this compound inactivates flavoproteins during electron-transfer reactions 3 and completely inhibits the activity of all isoforms of NOS, 3-6 xanthine oxidase, 3,7 and NADPH oxidase. 8 Thus, although pharmacological studies using isolated arteries have demonstrated that the production of ROS is markedly increased in animal models of hypertension, atherosclerosis, and heart failure, 9 the relative contribution of each of the potential O 2 Ϫ -producing enzymes to the overall ROS prod...

Abstract-The AMP-activated protein kinase (AMPK) was initially identified as the kinase that phosphorylates the 3-hydroxy 3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme for cholesterol biosynthesis. As the name suggests, the AMPK is activated by increased intracellular concentrations of AMP, and is generally described as a "metabolite-sensing kinase" and when activated initiates steps to conserve cellular energy. Although there is a strong link between the activity of the AMPK and metabolic control in muscle cells, the activity of the AMPK in endothelial cells can be regulated by stimuli that affect cellular ATP levels, such as hypoxia as well as by fluid shear stress, Ca 2ϩ -elevating agonists, and hormones such as adiponectin. To date the AMPK in endothelial cells has been implicated in the regulation of fatty acid oxidation, small G protein activity and nitric oxide production as well as inflammation and angiogenesis. Moreover, there is evidence indicating that the activation of the AMPK may help to prevent the vascular complications associated with the metabolic syndrome. Key Words: angiogenesis Ⅲ nitric oxide synthase Ⅲ atherosclerosis Ⅲ 3-hydroxy-3-methylglutaryl coenzyme A Ⅲ energy metabolism T he AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase consisting of the catalytic subunit (␣) and 2 regulatory subunits (␤ and ␥) that exist as multiple isoforms and splice variants, resulting in the generation of 12 possible heterotrimeric combinations. As its name suggests, the AMPK is activated in many different cell types by increased intracellular concentrations of AMP and is generally referred to as a "metabolite-sensing kinase." Indeed, the AMPK is activated following heat shock, vigorous exercise, hypoxia/ischemia, and starvation and appears to be a metabolic master switch, phosphorylating key target proteins that control flux through metabolic pathways of hepatic ketogenesis, cholesterol synthesis, lipogenesis, triglyceride synthesis, adipocyte lipolysis, skeletal muscle fatty acid oxidation, and protein synthesis (reviewed recently 1,2 ). Although the complex picture regarding the regulation of AMPK activity is far from complete, it seems safe to state that the AMPK determines whole body insulin sensitivity and may prevent insulin resistance, in part, by inhibiting pathways that antagonize insulin signaling. Through its effects on signaling, metabolism, and gene expression, the AMPK enhances insulin sensitivity and may reduce the risk of type 2 diabetes. 3 The 2 previous review articles in this series have focused on the role of the AMPK in metabolic control and insulin signaling 4 and in the heart. 5 This review focuses on the role of the AMPK in vascular cells and the data implicating defects in AMPK signaling in the development of vascular disease. Activation of the AMPKEach subunit within the heterotrimeric AMPK complex has a distinct structure and function and their interaction is necessary for the modulation of kinase activity. The ␣ subunits Ori...

PP1 the phosphorylation of AMPK was unaffected. Downregulation of PECAM-1 using a siRNA approach attenuated the shear-stress-induced phosphorylation of Akt and eNOS, as well as the shear-stress-induced accumulation of cyclic GMP levels while the shear-stressinduced phosphorylation of AMPK remained intact. A comparable attenuation of Akt and eNOS (but not AMPK) phosphorylation and NO production was also observed in endothelial cells generated from PECAM-1-deficient mice.These data indicate that the shear-stress-induced activation of Akt and eNOS in endothelial cells is modulated by the tyrosine phosphorylation of PECAM-1 whereas the shear-stress-induced phosphorylation of AMPK is controlled by an alternative signaling pathway.

Abstract-Fluid shear stress enhances NO formation via a Ca 2ϩ -independent tyrosine kinase inhibitor-sensitive pathway. In the present study, we investigated the effects of the protein tyrosine phosphatase inhibitor phenylarsine oxide and of fluid shear stress on endothelial NO production as well as on the membrane association and phosphorylation of the NO synthase (NOS) III. Phenylarsine oxide (10 mol/L) induced an immediate and maintained NO-mediated relaxation of isolated rabbit carotid arteries, which was insensitive to the removal of extracellular Ca 2ϩ and the calmodulin antagonist calmidazolium. This phenylarsine oxide-induced vasodilatation was unaffected by genistein but abrogated by the tyrosine kinase inhibitor erbstatin A. Incubation of native or cultured endothelial cells with phenylarsine oxide resulted in a time-dependent tyrosine phosphorylation of mainly Triton X-100 -insoluble (cytoskeletal) proteins, along with a parallel change in the detergent solubility of NOS III, such that the enzyme was recovered in the cytoskeletal fraction. A similar, though slightly delayed, phenomenon was also observed after the application of fluid shear stress but not in response to any receptor-dependent agonist. Although Ca 2ϩ -independent NO formation was sensitive to erbstatin A, phenylarsine oxide treatment was associated with the tyrosine dephosphorylation of NOS III rather than its hyperphosphorylation. Proteins that also underwent redistribution in response to the tyrosine phosphatase inhibitor included paxillin, phospholipase C-␥ 1 , mitogen-activated protein kinase, and the tyrosine kinases Src and Fyn. We envisage that fluid shear stress and tyrosine phosphatase inhibitors may alter the conformation and/or protein coupling of NOS III, facilitating its interaction with specific phospholipids, proteins, and/or protein kinases that enhance/maintain its Ca 2ϩ -independent activation. (Circ Res. 1998;82:686-695.)