ACH- AND CAFFEINE-INDUCED CA2-CELLS( MOBILIZATION AND CURRENT ACTIVATION IN RABBIT ARTERIAL ENDOTHELIAL)

Fura 2 microfluorometry and perforated-patch whole cell recording were carried out simultaneously to investigate the relationship between in tracellular free Ca2+ concentration ([Ca2+](i)) and membrane current a ctivation in response to ACh and caffeine in freshly dissociated arter ial endothelial cells. ACh and caffeine evoked transient increases in [Ca2+](i). The initial increase in [Ca2+](i) was accompanied by a tran sient outward current, which caused membrane hyperpolarization. The am plitudes of the [Ca2+](i) transient; and outward current were dependen t on caffeine concentration (EC50 similar to 1 mM). Cyclopiazonic acid raised resting [Ca2+](i) levels by greater than or equal to 50 nM and failed to completely block caffeine- or ACh-induced [Ca2+](i) transie nts but slowed [Ca2+](i) recovery fourfold. The reversal potential of caffeine-induced currents was dependent on external K+ and Cl- concent rations. Caffeine-induced current amplitudes, but not [Ca2+](i) respon ses, were attenuated by external tetraethylammonium, Zn2+ and La3+. A consistent temporal relationship between agonist-activated membrane cu rrent and [Ca2+](i) increases was not observed, and, in some cases, ti me differences were greater than expected for simple diffusion of Ca2 throughout the cell. These results suggest that Ca2+-dependent curren t activation monitors local [Ca2+](i) changes adjacent to the plasmale mma, whereas single-cell photometry provides a measure of global chang es in [Ca2+](i).