PKCα mediates acetylcholine-induced activation of TRPV4-dependent calcium influx in endothelial cells

• Published in: American journal of physiology. Heart and circulatory physiology 2011-09, Vol.301 (3), p.H757-H765
• Main Authors: Adapala, Ravi K, Talasila, Phani K, Bratz, Ian N, Zhang, David X, Suzuki, Makoto, Meszaros, J Gary, Thodeti, Charles K
• Format: Article
• Language: English
• Subjects: Acetylcholine - pharmacology; Animals; Calcium Signaling - drug effects; Carbazoles - pharmacology; Cells, Cultured; Endothelial Cells - drug effects; Endothelial Cells - enzymology; Enzyme Activation; Male; Mesenteric Arteries - drug effects; Mesenteric Arteries - enzymology; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Nitric Oxide Synthase Type III - metabolism; Phosphorylation; Protein Kinase C-alpha - genetics; Protein Kinase C-alpha - metabolism; Protein Kinase Inhibitors - pharmacology; Tetradecanoylphorbol Acetate - pharmacology; Time Factors; Transfection; TRPV Cation Channels - agonists; TRPV Cation Channels - deficiency; TRPV Cation Channels - genetics; TRPV Cation Channels - metabolism; Vascular Biology and Microcirculation; Vasodilation - drug effects; Vasodilator Agents - pharmacology
• ISSN: 0363-6135; 1522-1539
• DOI: 10.1152/ajpheart.00142.2011

Transient receptor potential vanilloid channel 4 (TRPV4) is a polymodally activated nonselective cationic channel implicated in the regulation of vasodilation and hypertension. We and others have recently shown that cyclic stretch and shear stress activate TRPV4-mediated calcium influx in endothelial cells (EC). In addition to the mechanical forces, acetylcholine (ACh) was shown to activate TRPV4-mediated calcium influx in endothelial cells, which is important for nitric oxide-dependent vasodilation. However, the molecular mechanism through which ACh activates TRPV4 is not known. Here, we show that ACh-induced calcium influx and endothelial nitric oxide synthase (eNOS) phosphorylation but not calcium release from intracellular stores is inhibited by a specific TRPV4 antagonist, AB-159908. Importantly, activation of store-operated calcium influx was not altered in the TRPV4 null EC, suggesting that TRPV4-dependent calcium influx is mediated through a receptor-operated pathway. Furthermore, we found that ACh treatment activated protein kinase C (PKC) α, and inhibition of PKCα activity by the specific inhibitor Go-6976, or expression of a kinase-dead mutant of PKCα but not PKCε or downregulation of PKCα expression by chronic 12-O-tetradecanoylphorbol-13-acetate treatment, completely abolished ACh-induced calcium influx. Finally, we found that ACh-induced vasodilation was inhibited by the PKCα inhibitor Go-6976 in small mesenteric arteries from wild-type mice, but not in TRPV4 null mice. Taken together, these findings demonstrate, for the first time, that a specific isoform of PKC, PKCα, mediates agonist-induced receptor-mediated TRPV4 activation in endothelial cells.