Characterization of acetylcholine- and endothelin-induced calcium entry in cultured human ciliary muscle cells.

We characterized the effects of acetylcholine and endothelin on cultured human ciliary muscle cells, using the calcium-sensitive dye fura-2 to measure intracellular calcium and intracellular microelectrodes to measure the membrane potential. Both agonists, endothelin and acetylcholine, had a typical biphasic effect on the intracellular calcium concentration. Calcium peaked initially, because of its release from intracellular stores, and then reached a plateau, owing to entry of extracellular calcium. Endothelin-induced calcium entry was almost completely blocked by addition of extracellular La3+ (50 mumol/l) and Ni2+ (1 mmol/l). Acetylcholine-induced calcium entry was likewise almost completely abolished by La3+ and Ni2+. Both endothelin and acetylcholine led to an initial transient hyperpolarization with a subsequent depolarization. The hyperpolarization of the membrane potential had a time course similar to the initial calcium peak, while the depolarization occurred parallel to the calcium plateau. The depolarization induced by both agonists was reduced in the presence of La3+ and Ni2+. Verapamil (10 mumol/l) had no effect on either the calcium entry or the depolarization. Acetylcholine did not induce a [Ca2+]i peak when it was applied during the endothelin-induced [Ca2+]i plateau and vice versa. The [Ca2+]i plateau was not higher with concomitant than with single application of acetylcholine or endothelin. Thus, calcium entry and membrane depolarization induced by acetylcholine and endothelin seem to be mediated by a common La(3+)- and Ni(2+)-sensitive but verapamil-insensitive mechanism.