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 mu m ACh stimulus evoked Ca2+ responses (9.8 +/- 0.8/min, F/F-0 = 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/F-0 = 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.