Effect of acetylcholine on membrane potential of cultured human nonpigmented ciliary epithelial cells

Human nonpigmented ciliary epithelial cells (NPE) were grown in tissue culture after transformation with an origin-defective mutant of SV-40 DNA. In these cells membrane potentials (V) were measured using the microelectrode technique. Addition of 10(-4) M acetylcholine led to a bisphasic voltage response. An immediate, transient hyperpolarization was followed by a sustained depolarization below the steady state level. These responses were irreversibly blocked by 10(-5) M atropine. In Ca2+-free media the initial addition of acetylcholine resulted in an unchanged voltage response. A second application of acetylcholine in Ca2+-free solution evoked only an abortive response of V, and further addition had no effect on V. In the presence of Ca2+ channel blockers (10(-5) M verapamil, 1 mM Co2+) the acetylcholine-induced response of the membrane potential was not changed. The initial hyperpolarization induced by acetylcholine was reduced by 33 +/- 3% (n = 6) in the presence of 2 mM Ba2+ and by 79 +/- 6% (n = 6) in the presence of 1 mM quinidine. Moreover, the amplitude of the hyperpolarization was dependent on the extracellular K+ concentration. With increasing extracellular K+ concentration (and decreasing transmembrane K+ gradient) the acetylcholine-induced hyperpolarization was reduced. To further elucidate the role of Ca2+ in the acetylcholine-induced responses, we measured cytoplasmic Ca2+ activity using the fluorescence of intracellularly trapped Fura-2. Cytoplasmic Ca2+ activity increased immediately and transiently upon addition of acetylcholine. We conclude that acetylcholine transiently hyperpolarizes V in cultured human NPE by activation of K+ channels mediated by mobilization of Ca2+ from intracellular stores.