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

Objective-Ca2ϩ -activated K ϩ (K Ca ) channels have been proposed to promote mitogenesis in several cell types. Here, we tested whether the intermediate-conductance K Ca channel (IKCa1) and the large-conductance K Ca channel (BK Ca ) contribute to endothelial cell (EC) proliferation and angiogenesis. Material and Results-Function and expression of IKCa1 and BK Ca /Slo were investigated by patch-clamp analysis and real-time RT-PCR in human umbilical vein ECs (HUVECs) and in dermal human microvascular ECs 1 (HMEC-1 ngiogenesis is an important process in a variety of physiological and pathophysiological conditions such as embryonic vasculogenesis, wound healing, poststenotic collateral formation, inflammation, and tumor vascularization. 1 Regarding the latter, tumor-derived angiogenic factors such as the vascular endothelial growth factor (VEGF) and the basic fibroblast growth factor (bFGF) induce endothelial cell (EC) sprouting, proliferation, migration, and finally new vessel formation. 1,2 With respect to EC proliferation, several studies have shown that EC proliferation after stimulation with angiogenic factors is initiated by a rise in intracellular calcium mediated through calcium-influx channels. [3][4][5] By keeping the membrane potential negative, chloride and potassium channels provide the driving force for this calcium entry and thus play an important role in regulating cell cycle progression 6 -9 as well as angiogenesis, as shown previously for chloride channels. 6 In particular, the intermediate- Materials and Methods Human ECs and In Vitro Proliferation StudiesHUVECs were isolated as described previously. 23 HUVECs and HMEC-1, a cell line derived from dermal HMECs 24 were cultured as described previously. 23 For proliferation and expression studies, HUVECs of second passage were used to avoid senescence.To induce growth arrest, HUVECs were kept in low serum medium (2% FCS) to allow cell survival for 48 hours before stimulation with bFGF (50 ng/mL) or VEGF-165 (50 ng/mL). HMEC-1 were kept in serum-free medium for 48 hours before stimulation with bFGF (50 ng/mL). At 5% to 10% confluence, photomicrographs of cells were taken in a fixed field before and 48 hours after stimulation and the percentage increase in cell count was calculated for each experiment. For blocker studies, cells were treated with TRAM-34 (0.1 nmol/L to 1 mol/L), 12 the inactive analogue TRAM-7 (1-tritylpyrrolidine; 1 mol/L), 12 CLT (0.1 nmol/L to 1 mol/L), or iberiotoxin (IbTx; 100 nmol/L). ReagentsPD98059, SB203580, and SP600125 were obtained from Tocris. CLT, IbTx, charydotoxin (ChTx), and apamin were obtained from Sigma. bFGF and VEGF-165 were obtained from Biochrom. All other chemicals were obtained from Sigma.

Experimental CRF leads to a loss of EDHF-type vasodilation which was caused at least in part by an impaired functional expression of endothelial hyperpolarizing K(Ca). The loss of EDHF-type vasodilation may contribute to endothelial dysfunction and abnormal arterial tone in CRF.