Muscarinic cholinergic stimulation elevates intracellular pH in chicken granulosa cells by a Ca(2+)-dependent,...

Various hormones and growth factors act at least in part by raising the cytosolic pH of the target cell. In the present studies we examined the influence of the muscarinic cholinergic agonist carbachol on intracellular pH (pHi) in chicken granulosa cells. The pHi in granulosa cells from the two largest preovulatory follicles of hens was determined spectrofluorometrically using the pH-sensitive dye 2',7'-bis-(carboxyethyl)- 5(6)-carboxyfluorescein (BCECF). Carbachol (0.1-2 mM) induced a concentration-dependent increase in pHi, which reached a maximum of 0.23 +/- 0.02 pH units at a concentration of 1 mM. Cytosolic alkalinization was observed within 10 min of the addition of carbachol and lasted over the 60-min observation period. The effect of carbachol was mimicked by acetylcholine (1 mM) and muscarine (1 mM), but not by nicotine (0.1 mM). The carbachol-induced alkalinization was blocked by pretreating the cells with the muscarinic cholinergic receptor antagonists atropine (1 and 10 microM) and pirenzepine (0.01 and 0.1 mM). The increase in pHi did not appear to be mediated by a conventional mechanism involving the Na+/H+ antiporter, because it was unaffected by replacement of extracellular Na+ with the nonpermeant choline chloride or the presence of antiporter inhibitors, such as amiloride, dimethylamiloride, or ethylisopropylamiloride. However, the mechanism required external Ca2+, but did not involve Ca2+ channels, because it was unaffected by the general Ca2+ channel blocker methoxyverapamil (50 microM). Thus, Ca(2+)-dependent and Na(+)-independent intracellular alkalinization may be a part of the signalling mechanism by which muscarinic receptor activation regulates chicken granulosa cell function.