Abstract
Introduction. In mouse aortic endothelial and smooth muscle coculture, transient receptor potential canonical type 3 (TRPC3) channels occur at myoendothelial contact sites (Isakson et al, 2006). In rat mesenteric artery, such sites contain colocalized IKCa and IP3R that are critical for control of vascular tone. Aim. To determine the distribution and role of TRPC3 in rat mesenteric artery; the hypothesis being that TRPC3 are integral for endothelium-mediated tone. Methods. TRPC3 antibody specificity and distribution in adult male SD rat mesenteric artery were determined using control rat tissue, HEK cells stably transfected with mouse TRPC3 cDNA and TRPC3 knock-out mouse tissue, with Western blotting, confocal and ultrastructural immunohistochemistry. TRPC3 function was examined in TRPC3-transfected HEK cells, cultured TRPC3 knockout mouse aortic endothelial cells and pressure myography in rat mesenteric artery using Pyr3 (putative TRPC3 blocker; Kiyonaka et al, 2009). Animals were anaesthetized with 100 mg/kg pentobarbitol, ip. Results. Western blotting in liver and confocal immunohistochemistry of TRPC3 transfected HEK cells confirmed antibody specificity. In rat mesenteric artery, confocal and ultrastructural TRPC3 labelling showed diffuse endothelial expression, increased expression at myoendothelial microdomain sites, and an absence in smooth muscle. Western blotting confirmed rat mesenteric artery TRPC3 expression, with endothelial removal (vWF verified) reducing expression >2-fold; supporting endothelial expression. TRPC3 labelling was present in mouse aortic smooth muscle and endothelium. Pyr3 blocked currents in patch-clamped TRPC3 transfected HEK cells and attenuated ATP-induced calcium flux in cultured aortic endothelial cells from wild-type, but not TRPC3 knock-out mice. Pyr3 blocked KCa-dependent EDH-type relaxation in rat mesenteric artery. Discussion. TRPC3 localization in rat mesenteric artery is consistent with their role in endothelial and myoendothelial microdomain-dependent signalling; and thus for their critical role in endothelial function and vascular tone.