title: PKCα mediates acetylcholine-induced activation of TRPV4-dependent calcium influx in endothelial cells.
abstract: 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.
authors.affiliation: Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, Ohio 44272, USA.
authors.forename: Ravi K
authors.initials: RK
authors.lastname: Adapala
authors.forename: Phani K
authors.initials: PK
authors.lastname: Talasila
authors.forename: Ian N
authors.initials: IN
authors.lastname: Bratz
authors.forename: David X
authors.initials: DX
authors.lastname: Zhang
authors.forename: Makoto
authors.initials: M
authors.lastname: Suzuki
authors.forename: J Gary
authors.initials: JG
authors.lastname: Meszaros
authors.forename: Charles K
authors.initials: CK
authors.lastname: Thodeti
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects*; D002227:Carbazoles / Q000494:pharmacology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology; D004789:Enzyme Activation; D008297:Male; D008638:Mesenteric Arteries / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009154:Mutation; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D050916:TRPV Cation Channels / Q000819:agonists* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014664:Vasodilation / Q000187:drug effects*; D014665:Vasodilator Agents / Q000494:pharmacology*
chemical_list: D002227:Carbazoles; D047428:Protein Kinase Inhibitors; D050916:TRPV Cation Channels; C486576:Trpv4 protein, mouse; D014665:Vasodilator Agents; C081021:Go 6976; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; C496630:Prkca protein, mouse; D051571:Protein Kinase C-alpha; D000109:Acetylcholine; D013755:Tetradecanoylphorbol Acetate