ACh- and caffeine-induced Ca2+ mobilization and current activation in rabbit arterial endothelial cells
Fura 2 microfluorometry and perforated-patch whole cell recording were carried out simultaneously to investigate the relationship between intracellular free Ca2+ concentration ([Ca2+](i)) and membrane current activation in response to ACh and caffeine in freshly dissociated arterial endothelial cells. ACh and caffeine evoked transient increases in [Ca2+](i). The initial increase in [Ca2+](i) was accompanied by a transient outward current, which caused membrane hyperpolarization. The amplitudes of the [Ca2+](i) transient and outward current were dependent on caffeine concentration (EC50 ~ 1 mM). Cyclopiazonic acid raised resting [Ca2+](i) levels by ≥50 nM and failed to completely block caffeine- or ACh-induced [Ca2+](i) transients but slowed [Ca2+](i) recovery fourfold. The reversal potential of caffeine-induced currents was dependent on external K+ and Cl- concentrations. Caffeine-induced current amplitudes, but not [Ca2+](i) responses, were attenuated by external tetraethylammonium, Zn2+, and La3+. A consistent temporal relationship between agonist- activated membrane current and [Ca2+](i) increases was not observed, and, in some cases, time differences were greater than expected for simple diffusion of Ca2+ throughout the cell. These results suggest that Ca2+- dependent current activation monitors local [Ca2+](i) changes adjacent to the plasmalemma, whereas single-cell photometry provides a measure of global changes in [Ca2+](i).