Organization of IP3‐R1 and TRPC3 at the Myoendothelial Junction May Influence Polarized Calcium Signaling

We recently developed a vascular cell co‐culture (VCC) system consisting of a single layer each of endothelial cells (EC) and smooth muscle cells (SMC) separated by a porous membrane. A functional MEJ is established expressing heterotypic gap junctions composed of Cx40 and Cx43. We hypothesized that this unique organization of gap junctions may regulate calcium signaling. Stimulation of either EC or SMC induced an increase in [Ca 2+ ] i in the stimulated cell, and a secondary increase in the unstimulated cell that was blocked by peptide inhibitors (i.e. gap junctionally mediated). We used 20 μM BAPTA‐AM (to block a rise in [Ca 2+ ] i in stimulated cells) or 20 μM Xestospongin C (to block IP 3 receptors in the responding cell) of the VCC. The subsequent responses indicated that, following stimulation, IP 3 and Ca 2+ move from SMC to EC, but only Ca 2+ moves from EC to SMC. We used Cx40‐null EC in the VCC to transform the MEJ into a homotypic Cx43 junction. This did not alter the polarized calcium signaling between the two cells. As an alternative explanation we hypothesized a differential distribution of IP3‐R or TRPC channels across the MEJ. IP3‐R1 was found in EC and SMC monolayers, but only in the EC extensions into the MEJ and not the SMC extensions. TRPC3 showed an identical pattern of expression. Thus, polarized calcium signaling across the MEJ may reflect the restriction of calcium channels to distinct cellular domains, not polarized gap junctions.