The present study used isometric tension recording to investigate the vasorelaxant effect of limonene (LM), carveol (CV), and perillyl alcohol (POH) on contractility parameters of the rat aorta, focusing in particular on the structure-activity relationship. LM, CV, and POH showed a reversible inhibitory effect on the contraction induced by electromechanical and pharmacomechanical coupling. In the case of LM, but not CV and POH, this effect was influenced by preservation of the endothelium. POH and CV but not LM exhibited greater pharmacological potency on BayK-8644-induced contraction and on electromechanical coupling than on pharmacomechanical coupling. In endothelium-denuded preparations, the order of pharmacological potency on electrochemical coupling was LM < CV < POH. These compounds inhibited also, with grossly similar pharmacological potency, the contraction induced by phorbol ester dibutyrate. The present results suggest that LM, CV and POH induced relaxant effect on vascular smooth muscle by means of different mechanisms likely to include inhibition of PKC and IP3 pathway. For CV and POH, hydroxylated compounds, it was in electromechanical coupling that the greater pharmacological potency was observed, thus suggesting a relative specificity for a mechanism likely to be important in electromechanical coupling, for example, blockade of voltage-dependent calcium channel.

Endothelial cells (ECs) line the wall of blood vessels and regulate arterial contractility to tune regional organ blood flow and systemic pressure. Chloride (Cl ⁻ ) is the most abundant anion in ECs and the Cl ⁻ sensitive With-No-Lysine (WNK) kinase is expressed in this cell type. Whether intracellular Cl ⁻ signaling and WNK kinase regulate EC function to alter arterial contractility is unclear. Here, we tested the hypothesis that intracellular Cl ⁻ signaling in ECs regulates arterial contractility and examined the signaling mechanisms involved, including the participation of WNK kinase. Our data obtained using two-photon microscopy and cell-specific inducible knockout mice indicated that acetylcholine, a prototypical vasodilator, stimulated a rapid reduction in intracellular Cl ⁻ concentration ([Cl ⁻ ] _i ) due to the activation of TMEM16A, a Cl ⁻ channel, in ECs of resistance-size arteries. TMEM16A channel-mediated Cl ⁻ signaling activated WNK kinase, which phosphorylated its substrate proteins SPAK and OSR1 in ECs. OSR1 potentiated transient receptor potential vanilloid 4 (TRPV4) currents in a kinase-dependent manner and required a conserved binding motif located in the channel C terminus. Intracellular Ca ²⁺ signaling was measured in four dimensions in ECs using a high-speed lightsheet microscope. WNK kinase-dependent activation of TRPV4 channels increased local intracellular Ca ²⁺ signaling in ECs and produced vasodilation. In summary, we show that TMEM16A channel activation reduces [Cl ⁻ ] _i , which activates WNK kinase in ECs. WNK kinase phosphorylates OSR1 which then stimulates TRPV4 channels to produce vasodilation. Thus, TMEM16A channels regulate intracellular Cl ⁻ signaling and WNK kinase activity in ECs to control arterial contractility.

Systemic blood pressure is acutely controlled by total peripheral resistance as determined by the diameter of small arteries and arterioles, the contractility of which is regulated by endothelial cells lining the lumen of blood vessels. We investigated the physiological functions of the chloride (Cl ⁻ ) channel TMEM16A in endothelial cells. TMEM16A channels generated calcium (Ca ²⁺ )–activated Cl ⁻ currents in endothelial cells from control ( TMEM16A ^fl/fl ) mice that were absent in those from mice with tamoxifen-inducible, endothelial cell–specific knockout of TMEM16A ( TMEM16A ecKO). TMEM16A currents in endothelial cells were activated by the muscarinic receptor agonist acetylcholine and an agonist of the Ca ²⁺ channel TRPV4, which localized in nanoscale proximity with TMEM16A as assessed by single-molecule localization imaging of endothelial cells. Acetylcholine stimulated TMEM16A currents by activating Ca ²⁺ influx through surface TRPV4 channels without altering the nanoscale properties of TMEM16A and TRPV4 surface clusters or their colocalization. In pressurized arteries, activation of TMEM16A channels in endothelial cells induced by acetylcholine; TRPV4 channel stimulation; or intraluminal ATP, another vasodilator, produced hyperpolarization and dilation. Furthermore, deficiency of TMEM16A channels in endothelial cells resulted in increased systemic blood pressure in conscious mice. These data indicate that vasodilators stimulate TRPV4 channels, leading to Ca ²⁺ -dependent activation of nearby TMEM16A channels in endothelial cells to produce arterial hyperpolarization, vasodilation, and reduced blood pressure. Thus, TMEM16A is an anion channel in endothelial cells that regulates arterial contractility and blood pressure.

Endothelial cells (ECs) line the lumen of all blood vessels and regulate functions, including contractility. Physiological stimuli, such as acetylcholine (ACh) and intravascular flow, activate transient receptor potential vanilloid 4 (TRPV4) channels, which stimulate small (SK3)- and intermediate (IK)-conductance Ca ²⁺ -activated potassium channels in ECs to produce vasodilation. Whether physiological vasodilators also modulate the surface abundance of these ion channels in ECs to elicit functional responses is unclear. Here, we show that ACh and intravascular flow stimulate rapid anterograde trafficking of an intracellular pool of SK3 channels in ECs of resistance-size arteries, which increases surface SK3 protein more than two-fold. In contrast, ACh and flow do not alter the surface abundance of IK or TRPV4 channels. ACh triggers SK3 channel trafficking by activating TRPV4-mediated Ca ²⁺ influx, which stimulates Rab11A, a Rab GTPase associated with recycling endosomes. Superresolution microscopy data demonstrate that SK3 trafficking specifically increases the size of surface SK3 clusters which overlap with TRPV4 clusters. We also show that Rab11A-dependent trafficking of SK3 channels is an essential contributor to vasodilator-induced SK current activation in ECs and vasorelaxation. In summary, our data demonstrate that vasodilators activate Rab11A, which rapidly delivers an intracellular pool of SK3 channels to the vicinity of surface TRPV4 channels in ECs. This trafficking mechanism increases surface SK3 cluster size, elevates SK3 current density, and produces vasodilation. These data also demonstrate that SK3 and IK channels are differentially regulated by trafficking-dependent and -independent signaling mechanisms in endothelial cells.

Polycystin 1 (PKD1) is a large transmembrane protein expressed by the Pkd1 gene. Previous data obtained using germline non‐specific knockout mice suggested that PKD1 regulates arterial smooth muscle cell contractility. Here, we generated tamoxifen‐inducible, smooth muscle cell‐specific ( Myh11 ‐ CreER ^T2 ) Pkd1 knockout ( Pkd1  smKO) mice to further investigate the functional significance of PKD1 in arterial myocytes. Reverse‐transcription polymerase chain reaction, western blotting and immunofluorescence imaging demonstrated that PKD1 message and protein were expressed in arterial myocytes of male control ( Pkd1 ^fl/fl ) mice but absent in arterial myocytes of Pkd1  smKO mice. Polycystin 2 (PKD2), a transient receptor potential channel which can couple to PKD1, was similarly expressed in arteries of Pkd1 ^fl/fl and Pkd1  smKO mice. Kidney anatomy and glomerular and proximal tubule sizes were unaltered in Pkd1  smKO mice. Intravascular pressures of between 10 and 125 mmHg similarly constricted resistance‐size mesenteric and gastrocnemius muscle arteries of Pkd1 ^fl/fl and Pkd1 smKO mice, regardless of whether experiments were performed 14 or 28 days after tamoxifen administration. Membrane depolarization (60 m m K ⁺ ), phenylephrine and angiotensin II also comparably constricted arteries of Pkd1 ^fl/fl and Pkd1  smKO mice. Acetylcholine, an endothelium‐dependent vasodilator, stimulated equivalent dilatation in Pkd1 ^fl/fl and Pkd1  smKO mouse arteries. Consistent with these results, blood pressure and heart rate were similar in Pkd1 ^fl/fl and Pkd1  smKO mice. In summary, these data indicate that PKD1 is expressed in systemic artery myocytes, but it does not contribute to pressure‐, depolarization‐ or agonist‐induced vasoconstriction, or regulate blood pressure. image Key points: Polycystin 1 (PKD1) is a large membrane protein expressed in several cell types, including vascular smooth muscle cells. Previous data obtained using germline non‐specific knockout mice proposed that PKD1 regulates arterial smooth muscle cell contractility. We generated tamoxifen‐inducible smooth muscle cell‐specific PKD1 knockout mice to further investigate the regulation of arterial smooth muscle cell contractility by PKD1. Our data show that PKD1 is expressed in smooth muscle cells, but it does not contribute to intravascular pressure‐, agonist‐ or depolarization‐induced constriction, nor does its knockout alter endothelial cell‐dependent vasodilatation in resistance‐size systemic arteries of male mice. Smooth muscle cell PKD1 does not regulate blood pressure or heart rate. We conclude that PKD1 is dispensable for physiological regulation of arterial smooth muscle cell contractility.