Objective— Ca ²⁺ -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 ²⁺ measurements, and pressure myography in carotid artery (CA) and Arteria gracilis . In RCAECs in situ, TRPV4 currents were activated by the selective TRPV4 opener 4α-phorbol-12,13-didecanoate (4αPDD), 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 ²⁺ measurements, 4αPDD increased [Ca ²⁺ ] _i by &140 nmol/L above basal levels. In pressure myograph experiments in CAs and A gracilis , 4αPDD caused robust endothelium-dependent and strictly endothelium-dependent vasodilatations by &80% (K _D 0.3 μmol/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 ). 4αPDD 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— Ca ²⁺ entry through endothelial TRPV4 channels triggers NO- and EDHF-dependent vasodilatation. Moreover, TRPV4 appears to be mechanistically important in endothelial mechanosensing of shear stress.

In 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. In 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. Genetically 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.

Background— It has been proposed that activation of endothelial SK3 (K _Ca 2.3) and IK1 (K _Ca 3.1) K ⁺ channels plays a role in the arteriolar dilation attributed to an endothelium-derived hyperpolarizing factor (EDHF). However, our understanding of the precise function of SK3 and IK1 in the EDHF dilator response and in blood pressure control remains incomplete. To clarify the roles of SK3 and IK1 channels in the EDHF dilator response and their contribution to blood pressure control in vivo, we generated mice deficient for both channels. Methods and Results— Expression and function of endothelial SK3 and IK1 in IK1 ^−/− /SK3 ^T/T mice was characterized by patch-clamp, membrane potential measurements, pressure myography, and intravital microscopy. Blood pressure was measured in conscious mice by telemetry. Combined IK1/SK3 deficiency in IK1 ^−/− /SK3 ^T/T (+doxycycline) mice abolished endothelial K _Ca currents and impaired acetylcholine-induced smooth muscle hyperpolarization and EDHF-mediated dilation in conduit arteries and in resistance arterioles in vivo. IK1 deficiency had a severe impact on acetylcholine-induced EDHF-mediated vasodilation, whereas SK3 deficiency impaired NO-mediated dilation to acetylcholine and to shear stress stimulation. As a consequence, SK3/IK1-deficient mice exhibited an elevated arterial blood pressure, which was most prominent during physical activity. Overexpression of SK3 in IK1 ^−/− /SK3 ^T/T mice partially restored EDHF- and nitric oxide-mediated vasodilation and lowered elevated blood pressure. The IK1-opener SKA-31 enhanced EDHF-mediated vasodilation and lowered blood pressure in SK3-deficient IK1 ^+/+ /SK3 ^T/T (+doxycycline) mice to normotensive levels. Conclusions— Our study demonstrates that endothelial SK3 and IK1 channels have distinct stimulus-dependent functions, are major players in the EDHF pathway, and significantly contribute to arterial blood pressure regulation. Endothelial K _Ca channels may represent novel therapeutic targets for the treatment of hypertension.

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 Ca²⁺-activated K⁺channels (K_Ca), leading to hyperpolarization of the endothelium and subsequently to hyperpolarization and relaxation of vascular smooth muscle. In the present study, we tested the functional role of the endothelial intermediate-conductance K_Ca(IK_Ca/K_Ca3.1) in endothelial hyperpolarization, in EDHF-mediated dilation, and in the control of arterial pressure by targeted deletion of K_Ca3.1. K_Ca3.1-deficient mice (K_Ca3.1^−/−) were generated by conventional gene-targeting strategies. Endothelial K_Cacurrents and EDHF-mediated dilations were characterized by patch-clamp analysis, myography and intravital microscopy. Disruption of the K_Ca3.1 gene abolished endothelial K_Ca3.1 currents and significantly diminished overall current through K_Cachannels. As a consequence, endothelial and smooth muscle hyperpolarization in response to acetylcholine was reduced in K_Ca3.1^−/−mice. Acetylcholine-induced dilations were impaired in the carotid artery and in resistance vessels because of a substantial reduction of EDHF-mediated dilation in K_Ca3.1^−/−mice. Moreover, the loss of K_Ca3.1 led to a significant increase in arterial blood pressure and to mild left ventricular hypertrophy. These results indicate that the endothelial K_Ca3.1 is a fundamental determinant of endothelial hyperpolarization and EDHF signaling and, thereby, a crucial determinant in the control of vascular tone and overall circulatory regulation.

Background— Angioplasty stimulates proliferation and migration of vascular smooth muscle cells (VSMC), leading to neointimal thickening and vascular restenosis. In a rat model of balloon catheter injury (BCI), we investigated whether alterations in expression of Ca ²⁺ -activated K ⁺ channels (K _Ca ) contribute to intimal hyperplasia and vascular restenosis. Methods and Results— Function and expression of K _Ca in mature medial and neointimal VSMC were characterized in situ by combined single-cell RT-PCR and patch-clamp analysis. Mature medial VSMC exclusively expressed large-conductance K _Ca (BK _Ca ) channels. Two weeks after BCI, expression of BK _Ca was significantly reduced in neointimal VSMC, whereas expression of intermediate-conductance K _Ca (IKCa1) channels was upregulated. In the aortic VSMC cell line, A7r5 epidermal growth factor (EGF) induced IKCa1 upregulation and EGF-stimulated proliferation was suppressed by the selective IKCa1 blocker TRAM-34. Daily in vivo administration of TRAM-34 to rats significantly reduced intimal hyperplasia by ≈40% at 1, 2, and 6 weeks after BCI. Two weeks of treatment with the related compound clotrimazole was equally effective. Reduction of intimal hyperplasia was accompanied by decreased neointimal cell content, with no change in the rate of apoptosis or collagen content. Conclusions— The switch toward IKCa1 expression may promote excessive neointimal VSMC proliferation. Blockade of IKCa1 could therefore represent a new therapeutic strategy to prevent restenosis after angioplasty.

Small-conductance (KCa2.1-2.3) and intermediate-conductance (KCa3.1) calcium-activated K⁺ channels are critically involved in modulating calcium-signaling cascades and membrane potential in both excitable and nonexcitable cells. Activators of these channels constitute useful pharmacological tools and potential new drugs for the treatment of ataxia, epilepsy, and hypertension. Here, we used the neuroprotectant riluzole as a template for the design of KCa2/3 channel activators that are potent enough for in vivo studies. Of a library of 41 benzothiazoles, we identified 2 compounds, anthra[2,1-d]thiazol-2-ylamine (SKA-20) and naphtho[1,2-d]thiazol-2-ylamine (SKA-31), which are 10 to 20 times more potent than riluzole and activate KCa2.1 with EC₅₀ values of 430 nM and 2.9 μM, KCa2.2 with an EC₅₀ value of 1.9 μM, KCa2.3 with EC₅₀ values of 1.2 and 2.9 μM, and KCa3.1 with EC₅₀ values of 115 and 260 nM. Likewise, SKA-20 and SKA-31 activated native KCa2.3 and KCa3.1 channels in murine endothelial cells, and the more “drug-like” SKA-31 (half-life of 12 h) potentiated endothelium-derived hyperpolarizing factor-mediated dilations of carotid arteries from KCa3.1(+/+) mice but not from KCa3.1(-/-) mice. Administration of 10 and 30 mg/kg SKA-31 lowered mean arterial blood pressure by 4 and 6 mm Hg in normotensive mice and by 12 mm Hg in angiotensin-II-induced hypertension. These effects were absent in KCa3.1-deficient mice. In conclusion, with SKA-31, we have designed a new pharmacological tool to define the functional role of the KCa2/3 channel activation in vivo. The blood pressure-lowering effect of SKA-31 suggests KCa3.1 channel activation as a new therapeutic principle for the treatment of hypertension.