IP3‐R1 localization may determine directionality of IP3 signaling through gap junctions at the myoendothelial junction

Using a vascular smooth muscle (VSMC)‐endothelial cell (EC) co‐culture system we have demonstrated that IP 3 can move from agonist stimulated VSMC to EC but that IP 3 does not move in the reverse direction. The IP 3 signaling produces a sustained (>50 sec) increase in EC intracellular calcium concentration ([Ca 2+ ] i ). The directionality in IP 3 signaling might be due to selective permeability of the gap junctions at MEJ or to specific localization of IP 3 ‐receptors(R). When the gap junctional composition was changed from heterotypic (Cx40‐43) to homotypic Cx43 at the MEJ, unidirectional IP 3 signaling persisted. We tested the alternate hypothesis that IP 3 ‐R localization on the EC side of the MEJ results in polarized signaling by using IP 3 ‐R1 siRNA to delete IP 3 ‐R1 from EC. When the VSMC was stimulated the response in EC was converted to a transient one (<30 sec) rather than sustained. We believe this indicated that the IP 3 was being degraded before it reached other IP 3 ‐R isoforms within the EC monolayer. We loaded EC with IP 3 ‐ R1 deleted with a 5‐phosphatase inhibitor (5PI) and found the EC were again responsive to IP 3 from VSMC. When 5PI was loaded into VSMC, we also noted that VSMC was responsive to IP 3 from EC. These data indicate that IP 3 moves in either direction through gap junctions at the MEJ, but that the localization of the IP 3 ‐R isoform determines whether IP 3 is used by the cell (EC) or degraded by 5‐phosphatase (VSMC).