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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 …