Protein Kinase C alpha mediates acetylcholine‐induced TRPV4 activation in microvascular endothelial cells

Transient Receptor potential vanilloid channel 4 (TRPV4) is a non‐selective cationic channel implicated in the regulation of vasodilation and hypertension. The objective of this study is to elucidate the molecular mechanism through which acetylcholine activates TRPV4 in endothelial cells. Here, we show that acetylcholine induces rapid calcium influx and endothelial nitric oxide synthase (eNOS) activation in microvascular endothelial cells. Acetylcholine‐induced calcium influx and eNOS activation, but not calcium release from intracellular stores, was inhibited by TRPV4 inhibitors, ruthenium red and AB159908. In order to identify the possible molecular mechanism of TRPV4 activation, we focused on protein kinase C which was shown to phosphorylate and sensitize TRPV4 activation. Indeed, we found that inhibition of PKC alpha activity by a specific inhibitor Go6976 or down regulation of PKCalpha expression by chronic TPA treatment completely abolished acetylcholine‐induced calcium influx. Finally, we found that acetylcholine‐induced vasodilation was significantly inhibited by TRPV4 inhibitor AB159908 and PKC alpha inhibitor Go6976 in small mesenteric arteries from wild type mice, but not from TRPV4 knockout mice. Taken together, these findings demonstrate, for the first time, that PKC alpha mediates agonist‐induced G‐protein coupled receptor mediated TRPV4 activation in endothelial cells.