The effect of muscarinic cholinergic agonists on intracellular calcium and progesterone production by chicken granulosa cells.

ABSTRACT

The effects of the muscarinic cholinergic receptor agonist carbachol on intracellular calcium ion concentration ([Ca2+]i) and progesterone production was determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the calcium-responsive fluorescent dye fura-2. Resting [Ca2+]i was 96 +/- 5 nM (n = 13). There was a 4- to 8-fold increase in [Ca2+]i in 85% (n = 80) of the cells within 10 sec after the addition of a maximally stimulating concentration (2 mM) of carbachol. The initial [Ca2+]i spike was followed by a sustained, but lower, calcium elevation, with superimposed oscillations which returned to resting level after several minutes. Both phases of the calcium transient were inhibited by pretreating the cells with atropine (1 microM), pirenzepine (2 microM) or 4-diphenylacetoxy-N-methylpiperidine methiodide (1 microM). The sustained phase of the response with its superimposed oscillations, but not the initial spike, was inhibited by pretreating the cells with the calcium channel blockers lanthanum (1 mM), cobalt (5 mM), or methoxyverapamil (50 microM), or by incubating the cells in calcium-free medium. Nifedipine (0.5-20 microM) did not affect the carbachol-induced calcium transient. 8-(N,N-Diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (50 microM) blocked the sustained-oscillatory phase of the carbachol-induced [Ca2+]i transient, but did not affect the initial spike when added before carbachol (2 mM). Despite its ability to stimulate [Ca2+]i surges, carbachol (10(-6)-10(-3) M) did not affect basal or LH-stimulated cAMP or progesterone production in 24-h cultures. These studies demonstrate that activation of chicken granulosa cell muscarinic receptors causes a rapid increase in [Ca2+]i through the release of Ca2+ from intracellular stores, followed by a sustained elevation of Ca2+ with superimposed oscillations caused by the influx of extracellular Ca2+. These results also indicate that an increase in [Ca2+]i in chicken granulosa cells does not alone stimulate steroidogenesis, since the carbachol-induced increases in [Ca2+]i were not accompanied by increased progesterone production.