TRPC3 Channels underlie endothelium-mediated S/IKCa-dependent vasodilator activity in rat mesentric artery

In rat mesenteric artery, myoendothelial microdomain signalling sites are important for control of vascular tone and blood flow. This study determined the distribution and role of TRPC3 in rat mesenteric artery; with the hypothesis that TRPC3 are critical for endothelium-derived hyperpolarization (EDH) activity. TRPC3 antibody specificity, expression and distribution was determined using rat tissue, TRPC3 transfected HEK cells, TRPC3 knock-out mouse tissue using Western blotting, and confocal and ultrastructural immunohistochemistry. The putative TRPC3 channel blocker Pyr3 was used to examine functional role of TRPC3. Pyr3 specificity was examined in patch clamped TRPC3 transfected HEK cells, TRPC3 knock-out mouse aortic endothelial cells, and in mesenteric artery of wild-type and TRPC3 KO mouse using pressure myography and endothelial cell membrane potential recordings. Confocal immunohistochemistry demonstrated TRPC3 expression in the endothelium and not in the smooth muscle, with diffuse expression in endothelium surface, and high expression at myoendothelial microdomain sites, with ~5-fold higher fluorescence density. Immunoelectron microscopy and Western blotting further confirmed TRPC3 expression at these sites, with endothelial removal (verified by vWF labeling), reducing expression >2-fold, supporting primary endothelial expression. TRPC3 inhibition with Pyr3 blocked current changes in patch clamped TRPC3 transfected HEK cells and reduced the calcium flux in wild-type mouse aortic endothelial cells. In isolated and pressurized wild-type mouse mesenteric artery Pyr3 mimicked the response in rat mesenteric, whilst in KO, it had no effect, supporting the agents specificity for TRPC3. In rat small mesenteric arteries Pyr3 inhibited ACh induced vasodilation in the presence of nitric oxide inhibitors, L-NAME, ODQ and prostacyclin inhibitor, Indomethacin. Additional presence of IKCa and SKCa channel inhibitors, TRAM-34 and Apamin, respectively, differentially blocked the residual EDH mediated relaxation, with endothelial cell membrane potential recordings supporting these results. These data demonstrate TRPC3 channel function and localization is consistent with a critical role in control of rat small mesenteric artery tone.