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 approximately 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.