Nitric oxide and protein kinase G act on TRPC1 to inhibit 11,12-EET-induced vascular relaxation

Vascular endothelial cells synthesize and release vasodilators such as nitric oxide (NO) and epoxyeicosatrienoic acids (EETs). NO is known to inhibit EET-induced smooth muscle hyperpolarization and relaxation. This study investigates the underlying mechanism of this inhibition.

Through measurements of membrane potential and arterial tension, we show that 11,12-EET induced membrane hyperpolarization and vascular relaxation in endothelium-denuded porcine coronary arteries. These responses were suppressed by S-nitroso-N-acetylpenicillamine (SNAP) and 8-Br-cGMP, an NO donor and a membrane-permeant analogue of cGMP, respectively. The inhibitory actions of SNAP and 8-Br-cGMP on 11,12-EET-induced membrane hyperpolarization and vascular relaxation were reversed by hydroxocobalamin, an NO scavenger; ODQ, a guanylyl cyclase inhibitor; and KT5823, a protein kinase G (PKG) inhibitor. The inhibitory actions of SNAP and 8-bromo cyclic GMP (8-Br-cGMP) on the EET responses were also abrogated by shielding TRPC1-PKG phosphorylation sites with an excessive supply of exogenous PKG substrates, TAT-TRPC1(S172) and TAT-TRPC1(T313). Furthermore, a phosphorylation assay demonstrated that PKG could directly phosphorylate TRPC1 at Ser(172) and Thr(313). In addition, 11,12-EET failed to induce membrane hyperpolarization and vascular relaxation when TRPV4, TRPC1, or KCa1.1 was selectively inhibited. Co-immunoprecipitation studies demonstrated that TRPV4, TRPC1, and KCa1.1 physically associated with each other in smooth muscle cells.

Our findings demonstrate a novel role of the NO-cGMP-PKG pathway in the inhibition of 11,12-EET-induced smooth muscle hyperpolarization and relaxation via PKG-mediated phosphorylation of TRPC1.