Title Page / Table of Contents / Abstracts

Microdomain signalling at sites of close vascular endothelial and smooth muscle cell associations (less than 30nm distance) is integral for endothelium-dependent relaxation and thus for the control of blood flow and pressure. At such specialized myoendothelial sites, transfer of endothelial hyperpolarizing current and/or localized K+ activity occurs, with the net effect being hyperpolarization of the smooth muscle, voltage-dependent calcium channel closure and relaxation. In rat mesenteric artery localized gap junction connexins, endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptors, and intermediate conductance calciumactivated potassium channels (IKCa) are present in close proximity. Similar sites of close association are also present in an artificial co-culture system of mouse aortic endothelial and smooth muscle cells, where transient receptor potential canonical type 3 channels (TRPC3) have been suggested to be present [1]. Together with previous data, such close spatial associations are consistent with potential for functional interaction. The aim of this study was to identify the prospective channel responsible for ER calcium refilling at the highly discrete myoendothelial microdomain signalling sites in intact 20-22 week old male SD rat mesenteric artery. Specificity and expression of an antibody directed against C’ amino acids of mouse 822-835 TRPC3 (Alomone ACC-016; batches AN-02, 03, 07; an antibody with ~93% (13/14) sequence homology to rat TRPC3; noting that AN-06 was non-specific), was characterized in fresh rat liver and HEK cells stably transfected with TRPC3 mouse cDNA using Western blotting and cell transfection, respectively. PCR amplification and sequencing verified the presence of transfected mouse TRPC3 gene transcript in HEK cells. Western blotting and confocal and ultrastructural immunohistochemistry determined the expression and distribution of TRPC3 in rat mesenteric artery. Western blotting in liver confirmed antibody specificity with the presence of a faint ~98kDa band that was partially blocked by peptide, and an apparent monoglycosylated band at ~120kDa, which has been previously recognized as the functional form of the channel [2]; labelling for which was blocked by peptide. Antibody specificity was further confirmed by cell labelling in transfected HEK cells, where untransfected cells failed to label. Western blotting confirmed expression of monoglycosylated TRPC3 in rat mesenteric artery. Confocal immunohistochemistry demonstrated TRPC3 localization at the endothelial-smooth muscle interface as discrete bright spots, corresponding to ~80% of internal elastic lamina (IEL) holes. In comparison, the density of myoendothelial gap junctions in this bed was significantly less than that of TRPC3- IEL hole localization (being at ~15% of IEL holes) whilst and IKCa was at ~77% of IEL holes. Ultrastructural immunohistochemistry demonstrated TRPC3 localization at regions of endothelial-smooth muscle cell association. These data suggest that TRPC3 is in close spatial association with IKCa and with myoendothelial gap junctions. Functional studies are stymied by the lack of selective TRP blockers. However, subsequent studies will characterize the functional properties of TRPC3 at myoendothelial associations using TRPC3 directed antibodies, as potential selective targets for control of endothelial and vascular function