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

Abstract

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.

Two types of non‐selective cation channel opened by muscarinic stimulation with carbachol in bovine ciliary muscle cells

“…In the dog ciliary muscle, high K + ‐induced contraction is also strongly suppressed by atropine although there is a small component which is resistant to atropine and is inhibited by organic Ca 2+ antagonists (Ito & Yoshitomi, 1986). In a human ciliary muscle cell line, Stahl et al (1992) measured the change of [Ca 2+ ] i by the fura‐2 method. They showed that application of muscarinic agonists resulted in an initial [Ca 2+ ] i peak followed by a plateau.…”

“…The initial peak was still clearly observed in the absence of extracellular Ca 2+ and in the presence of verapamil, whereas the [Ca 2+ ] i plateau was reversibly abrogated by removal of extracellular Ca 2+ but was only slightly suppressed by verapamil. Stahl et al (1992) attributed the initial peak to Ca 2+ release from intracellular stores and the plateau to Ca 2+ entry through ‘receptor‐operated’ calcium channels.…”

Characteristics of palytoxin-induced cation currents and Ca2+ mobilization in smooth muscle cells of rabbit portal vein

“…The PTX-induced channel is similar to receptor-coupled non-selective cation channels (Inoue 1991;Stahl et al 1992) in its sensitivity to Ni 2+ and La 3+ , but differs in that the PTX-induced channel does not allow Ca 2+ to permeate whereas the latter channels permit the passage of Ca 2+ (Stahl et al 1992). Ni 2+ , La 3+ and DCB are known to inhibit Na + -Ca 2+ exchange (Kimura et al 1986;Slaughter et al 1988;Beuckelmann and Wier 1989).…”

Calcium Signalling in Ocular Tissues: Functional Activity of G-protein and Tyrosine–Kinase Coupled Receptors

“…In a similar study, adenosine and adrenalin synergistically potentiated the mobilization of stored Ca 2 by acetylcholine through a pertussis-toxin-sensitive mechanism such as Gi (Farahbakhsh and Cilluffo, 1997). Furthermore, in cultured human ciliary muscle cells, acetylcholine elicited a typical biphasic effect on intracellular Ca 2 concentration, which produced a concomitant transient hyperpolarization followed by a sustained depolarization of membrane voltage (Stahl et al, 1992). The hyperpolarization coincided with the initial store-release Ca 2 transient, while the depolarization was dependent on Ca 2 in¯ux from the extracellular medium.…”

“…Therefore, a functional contractile response in ciliary muscle and trabecular meshwork is likely to be mediated mainly by M3 receptor sub-type (Wiederholt et al, 1996;Masuda et al, 1998). However, other G-protein coupled receptor systems linked to Ca 2 mobilization have been identi®ed in human NPE cells, including the H1 histamine receptor (Crook et al, 1991), and endothelin-1 receptor (Stahl et al, 1992;Tao et al, 1998). The ET-1 receptor has also been found to stimulate Ca 2 release in human ciliary smooth muscle (Matsumoto et al, 1996;Prasanna et al, 2000) and trabecular meshwork cells (Tao et al, 1998), but at present there is limited experimental evidence to support a clear role for either of these receptor systems in contraction of ciliary muscle cells.…”