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 ...