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.

Contraction by Ca2+ Influx via the L-Type Ca2+ Channel Voltage Window in Mouse Aortic Segments is Modulated by Nitric Oxide

“…This is also consistent with old observations that the resting level of cGMP falls after removal of external Ca 2+ (White & Martin, 1989). In other studies, it has been shown that the TRPV4-mediated Ca 2+ signal was required for eNOS activation by ACh (Adapala et al, 2011;Zhang et al, 2009), also illustrating the necessity of Ca 2+ influx for endothelial NO release. Isshiki et al (Isshiki et al, 2004) suggested that agonist-stimulated eNOS activity in the endothelial cells was sensitive to external Ca 2+ -dependent acute changes in intracellular subcortical Ca 2+ signals and that basal eNOS activity was maintained and regulated by subplasmalemmal Ca 2+ equilibrated with extracellular Ca 2+ .…”

Analysis of responses to the TRPV4 agonist GSK1016790A in the pulmonary vascular bed of the intact-chest rat

“…Effect of L-NAME. It has been previously reported that the TRPV4 agonist increases intracellular Ca 2ϩ concentration and activates endothelial NOS in endothelial cells (1,22). The role of NOS and NO in mediating or modulating cardiovascular responses to the TRPV4 agonist GSK1016790A were investigated in experiments with the NOS inhibitor L-NAME; these data are shown in Fig.…”

Update on vascular endothelial Ca2+signalling: A tale of ion channels, pumps and transporters

“…TRPC3-and TRPC6-induced Ca 2+ inflow underpin the stimulatory effect of VEGF on endothelial proliferation, migration and permeability [18,231,236] , while ATP exploits TRPC3 to activate NF-κB and increase vascular cell adhesion molecule-1 expression [237] . TRPC3-driven NO synthesis and vasore- TRPV1 TRPV2 TRPV3 TRPV4 TRPV5 TRPV6 PCa/PNa and conductance (pS) [202] 10, 35-80 1-3, NM 12, 172 6, 90 > 100, 75 > 100, 40-70 Human coronary artery ECs [300] (+RT-PCR, WB, IHC) Human pulmonary artery ECs [415] +(RT-PCR) +(RT-PCR) +(RT-PCR) Human pulmonary microvascular ECs +(RT-PCR) Human cerebral microvascular ECs [272] +(RT-PCR, IC) Human cerebral arterioles ECs [305] +(RT-PCR, IHC) Human dermal microvascular ECs [319] +(RT-PCR, WB) Breast cancer derived microvascular ECs [319] +(RT-PCR, WB) Human umbilical vein ECs [227,277,296,301] +(RT-PCR) +(WB, IHC) Mouse aortic ECs [280,285,299,306] +(WB) +(RT-PCR, WB, NM, IHC) Mouse pulmonary artery ECs [313] +(WB, IHC) Mouse mesenteric artery ECs [27,280,302] +(RT-PCR, WB, IC) +(RT-PCR, IC) Mouse cerebral microvascular ECs +(RT-PCR) +(RT-PCR, WB) +(RT-PCR) Mouse dermal microvascular ECs [307] +(RT-PCR, WB) Mouse carotid artery ECs [290,296] +(IHC) Rat mesenteric artery ECs [226,275,302] +(WB) +(WB, IC, IHC) Rat femoral artery ECs [312] +(RT-PCR, IC) Rat pulmonary artery ECs [303] +(WB, IHC) Rat renal artery ECs [303] +(IHC) Rat cardiac microvascular ECs [303,…”

“…Under physiological conditions, TRPV4-mediated vasodilation, mainly via NO and EDHF production, is induced by both shear stress [27,290,291,296] and extracellular autacoids, such as Ach and UTP [297,301,305,306] . Although the cellular mechanisms linking membrane receptors to TRPV4 opening are still unclear, UTP utilizes PLA2 to gate the channel [297] , while PKCα mediates Ach-dependent activation [307] . Despite the evidence for TRPV4-evoked vasodilation, the baseline blood pressure is not affected in TRPV4 -/-mice [305,306] .…”