Localized expression of an Ins(1,4,5)P3 receptor at the myoendothelial junction selectively regulates heterocellular Ca2+ communication

Abstract

Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] originating in the vascular smooth-muscle cells (VSMCs) has been shown to modulate the Ca2+ stores in endothelial cells (ECs). However, the reverse is not found, suggesting that Ins(1,4,5)P3 movement might be unidirectional across gap junctions at the myoendothelial junction (MEJ), or that distribution of the Ins(1,4,5)P3 receptor [Ins(1,4,5)P3-R] is different between the two cell types. To study trans-junctional communication at the MEJ, we used a vascular-cell co-culture model system and selectively modified the connexin composition in gap junctions in the two cell types. We found no correlation between modification of connexin expression and Ins(1,4,5)P3 signaling between ECs and VSMCs. We next explored the distribution of Ins(1,4,5)P3-R isoforms in the two cell types and found that Ins(1,4,5)P3-R1 was selectively localized to the EC side of the MEJ. Using siRNA, selective knockdown of Ins(1,4,5)P3-R1 in ECs eliminated the secondary Ins(1,4,5)P3-induced response in these cells. By contrast, siRNA knockdown of Ins(1,4,5)P3-R2 or Ins(1,4,5)P3-R3 in ECs did not alter the EC response to VSMC stimulation. The addition of 5-phosphatase inhibitor (5-PI) to ECs that were transfected with Ins(1,4,5)P3-R1 siRNA rescued the Ins(1,4,5)P3 response, indicating that metabolic degradation of Ins(1,4,5)P3 is an important part of EC-VSMC coupling. To test this concept, VSMCs were loaded with 5-PI and BAPTA-loaded ECs were stimulated, inducing an Ins(1,4,5)P3-mediated response in VSMCs; this indicated that Ins(1,4,5)P3 is bidirectional across the gap junction at the MEJ. Therefore, localization of Ins(1,4,5)P3-R1 on the EC side of the MEJ allows the ECs to respond to Ins(1,4,5)P3 from VSMCs, whereas Ins(1,4,5)P3 moving from ECs to VSMCs is probably metabolized before binding to a receptor. This data implicates the MEJ as being a unique cell-signaling domain in the vasculature.