Objective-Ca2ϩ -influx through transient receptor potential (TRP) channels was proposed to be important in endothelial function, although the precise role of specific TRP channels is unknown. Here, we investigated the role of the putatively mechanosensitive TRPV4 channel in the mechanisms of endothelium-dependent vasodilatation. Methods and Results-Expression and function of TRPV4 was investigated in rat carotid artery endothelial cells (RCAECs) by using in situ patch-clamp techniques, single-cell RT-PCR, Ca 2ϩ measurements, and pressure myography in carotid artery (CA) and Arteria gracilis. In RCAECs in situ, TRPV4 currents were activated by the selective TRPV4 opener 4␣-phorbol-12,13-didecanoate (4␣PDD), arachidonic acid, moderate warmth, and mechanically by hypotonic cell swelling. Single-cell RT-PCR in endothelial cells demonstrated mRNA expression of TRPV4. In FURA-2 Ca 2ϩ measurements, 4␣PDD increased [Ca 2ϩ ] i by Ϸ140 nmol/L above basal levels. In pressure myograph experiments in CAs and A gracilis, 4␣PDD caused robust endothelium-dependent and strictly endothelium-dependent vasodilatations by Ϸ80% (K D 0.3 mol/L), which were suppressed by the TRPV4 blocker ruthenium red (RuR). Shear stress-induced vasodilatation was similarly blocked by RuR and also by the phospholipase A 2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF 3 ). 4␣PDD produced endothelium-derived hyperpolarizing factor (EDHF)-type responses in A gracilis but not in rat carotid artery. Shear stress did not produce EDHF-type vasodilatation in either vessel type. Conclusions-Ca2ϩ entry through endothelial TRPV4 channels triggers NO-and EDHF-dependent vasodilatation. Moreover, TRPV4 appears to be mechanistically important in endothelial mechanosensing of shear stress. Key Words: endothelium-dependent vasodilatation Ⅲ transient receptor potential Ⅲ TRPV4 Ⅲ calcium Ⅲ shear stress Ⅲ nitric oxide Ⅲ 4␣PDD Ⅲ rat carotid artery C a 2ϩ -influx in response to mechanical or humoral stimulation plays a significant role in a variety of endothelial functions and especially in the Ca 2ϩ -dependent synthesis of endothelium-derived vasodilators such as NO, prostacyclin, or the endothelium-derived hyperpolarizing factor (EDHF). 1

Abstract-The endothelium plays a key role in the control of vascular tone and alteration in endothelial cell function contributes to several cardiovascular disease states. Endothelium-dependent dilation is mediated by NO, prostacyclin, and an endothelium-derived hyperpolarizing factor (EDHF). EDHF signaling is thought to be initiated by activation of endothelial Ca 2ϩ

BackgroundIn blood vessels, the endothelium is a crucial signal transduction interface in control of vascular tone and blood pressure to ensure energy and oxygen supply according to the organs' needs. In response to vasoactive factors and to shear stress elicited by blood flow, the endothelium secretes vasodilating or vasocontracting autacoids, which adjust the contractile state of the smooth muscle. In endothelial sensing of shear stress, the osmo- and mechanosensitive Ca2+-permeable TRPV4 channel has been proposed to be candidate mechanosensor. Using TRPV4−/− mice, we now investigated whether the absence of endothelial TRPV4 alters shear-stress-induced arterial vasodilation.Methodology/Principal FindingsIn TRPV4−/− mice, loss of the TRPV4 protein was confirmed by Western blot, immunohistochemistry and by in situ-patch–clamp techniques in carotid artery endothelial cells (CAEC). Endothelium-dependent vasodilation was determined by pressure myography in carotid arteries (CA) from TRPV4−/− mice and wild-type littermates (WT). In WT CAEC, TRPV4 currents could be elicited by TRPV4 activators 4α-phorbol-12,13-didecanoate (4αPDD), arachidonic acid (AA), and by hypotonic cell swelling (HTS). In striking contrast, in TRPV4−/− mice, 4αPDD did not produce currents and currents elicited by AA and HTS were significantly reduced. 4αPDD caused a robust and endothelium-dependent vasodilation in WT mice, again conspicuously absent in TRPV4−/− mice. Shear stress-induced vasodilation could readily be evoked in WT, but was completely eliminated in TRPV4−/− mice. In addition, flow/reperfusion-induced vasodilation was significantly reduced in TRPV4−/− vs. WT mice. Vasodilation in response to acetylcholine, vasoconstriction in response to phenylephrine, and passive mechanical compliance did not differ between genotypes, greatly underscoring the specificity of the above trpv4-dependent phenotype for physiologically relevant shear stress.Conclusions/SignificanceGenetically encoded loss-of-function of trpv4 results in a loss of shear stress-induced vasodilation, a response pattern critically dependent on endothelial TRPV4 expression. Thus, Ca2+-influx through endothelial TRPV4 channels is a molecular mechanism contributing significantly to endothelial mechanotransduction.