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First published online October 22, 2008
doi: 10.1242/10.1242/jcs.037481

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

Robert M. Berne Cardiovascular Research Center, and Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, P.O. Box 801394, Charlottesville, VA 22908, USA

e-mail: bei6n{at}virginia.edu

Accepted 6 August 2008

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.

Key words: Inositol (1,4,5)-trisphosphate, Calcium, Connexin, 5-phosphatase, Endothelium, Smooth muscle

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Ins(1,4,5)P₃ goes both ways

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