Coordination of Intercellular Ca²⁺ Signaling in Endothelial Cell Tubes of Mouse Resistance Arteries

Objective: To test the hypothesis that Ca²⁺ responses to GPCR activation are coordinated between neighboring ECs of resistance arteries. Methods: EC tubes were freshly isolated from superior epigastric arteries of C57BL/6 mice. Intercellular coupling was tested using microinjection of propidium iodide. Following loading with fluo-4 dye, intracellular Ca²⁺ responses to ACh were imaged with confocal microscopy. Results: Cell-to-cell transfer of propidium iodide confirmed functional GJCs. A 1 μm ACh stimulus evoked Ca²⁺ responses (9.8 ± 0.8/min, F/F₀ = 3.11 ± 0.2) which pseudo-line-scan analysis revealed as composed of Ca²⁺ waves and spatially restricted Ca²⁺ release events. A 100 nm ACh stimulus induced Ca²⁺ responses of lower frequency (4.5 ± 0.7/min) and amplitude (F/F₀ = 1.95 ± 0.11) composed primarily of spatially restricted events. The time interval between Ca²⁺ waves in adjacent cells (0.79 ± 0.12 s) was shorter (p < 0.05) than that between nonadjacent cells (1.56 ± 0.25 s). Spatially restricted Ca²⁺ release events had similar frequencies and latencies between adjacent and nonadjacent cells. Inhibiting intracellular Ca²⁺ release with 2-APB, Xestospongin C or thapsigargin eliminated Ca²⁺ responses. Conclusions: With moderate GPCR stimulation, localized Ca²⁺ release events predominate among cells. Greater GPCR stimulation evokes coordinated intercellular Ca²⁺ waves via the ER. Calcium signaling during GPCR activation is complex among cells, varying with stimulus intensity and proximity to actively signaling cells.