Abstract
Introduction: In rat mesenteric artery, myoendothelial microdomain-dependent signalling sites are important for control of vascular tone and blood flow. Aim: To determine the distribution and role of TRPC3 in rat mesenteric artery; with the hypothesis that TRPC3 are critical for EDH-mediated vaosodilator activity. Methods: TRPC3 antibody specificity, expression and distribution was determined using rat tissue, TRPC3 transfected HEK cells, and 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; with characterization in TRPC3 transfected HEK cells with patch clamp, TRPC3 knock-out mouse aortic endothelial cells with calcium imaging, and in mesenteric artery of rat and wild-type and TRPC3 KO mouse using pressure myography and endothelial cell membrane potential recordings. Results: Confocal immunohistochemistry and Western blotting demonstrated TRPC3 expression in the endothelium and not in the smooth muscle of mesenteric artery, with diffuse expression on the endothelial surface, and high expression at putative myoendothelial microdomain sites, where fluorescence density was ~5-fold higher. Immunoelectron microscopy confirmed TRPC3 expression at these sites, with endothelial removal (verified by vWF labeling), reducing expression >2-fold, supporting primary endothelial expression. In isolated and pressurized rat small mesenteric arteries Pyr3 inhibited ACh-induced dilation in the presence of the nitric oxide synthase and sGC 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.Discussion: TRPC3 channel function and localization is consistent with their role in endothelial and myoendothelial microdomain-dependent signalling. Thus, TRPC3 has a critical role in endothelial function and control of tone.