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Ca²⁺ and Inositol 1,4,5-Trisphosphate–Mediated Signaling Across the Myoendothelial Junction

From the Department of Molecular Physiology and Biological Physics (B.E.I., B.R.D.), Robert M. Berne Cardiovascular Research Center (B.E.I., B.R.D.), and Cardiology Division of Internal Medicine (S.I.R.), University of Virginia School of Medicine, Charlottesville.

Correspondence to Brant E. Isakson, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, PO Box 801394, Charlottesville, VA 22908. E-mail bei6n{at}virginia.edu

Second messenger signaling between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) is poorly understood, but intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in the 2 cells are coordinated, possibly through gap junctions at the myoendothelial junction. To study heterocellular calcium signaling, we used a vascular cell coculture model composed of monolayers of ECs and VSMCs. Stimulation of either cell type leads to an increase in [Ca²⁺]_i in the stimulated cell and a secondary increase in [Ca²⁺]_i in the other cell type that was blocked by gap junction inhibitors. To determine which second messengers are involved, we initially depleted Ca²⁺ stores in the endoplasmic reticulum Ca²⁺ with thapsigargin in ECs or VSMCs, but this had no effect on heterocellular calcium signaling. Alternatively, we loaded ECs or VSMCs with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) to buffer changes in [Ca²⁺]_i. BAPTA loading of ECs inhibited agonist-induced increases in intracellular calcium concentration ([Ca²⁺]_i), in both ECs and VSMCs. In contrast, BAPTA loading of the VSMCs blunted the VSMC response but did not alter the secondary increase in EC [Ca²⁺]_i. Xestospongin C (an inositol 1,4,5-trisphosphate receptor inhibitor) had no effect on the secondary Ca²⁺ response, but when xestospongin C or thapsigargin was loaded into ECs and BAPTA into VSMCs, intercellular Ca²⁺ signaling was completely blocked. We conclude that 1,4,5-trisphosphate and Ca²⁺ originating in the VSMCs induces the secondary increase in EC [Ca²⁺]_i but stimulation of the ECs generates a Ca²⁺ dependent response in the VSMCs.

Key Words: calcium signaling • inositol-1,4,5-trisphosphate • connexins • gap junctions • myoendothelial junctions • heterocellular

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