Abstract
Using the pH indicator, seminaphtorhodafluor, we studied the effects of GnRH on intracellular pH (pHi) in single gonadotroph cells, obtained from 3-week ovariectomized rats, treated or not with estradiol (E2) (OVX + E2, OVX). In a majority of cells (77.7% for OVX cells and 93.7% for OVX + E2 cells), GnRH induced acidification. A biphasic change of pHi, acidification followed by alkalinization, was observed in about 44% of the cells tested. In E2-treated cells, amplitude of acidification and duration of alkalinization were increased. Acidification and Ca2+ mobilization were related in time with a short delay (4-5 sec.). Depolarization with KCl and ionomycin, a Ca2+ ionophore, induced acidification. Taken together these observations suggest that acidification was caused by [Ca2+]i increase. When the Na+/H+ exchanger was blocked by amiloride or in Na(+)-free medium, GnRH-induced alkalinization was inhibited. Alkalinization disappeared completely when the cells were depleted in protein kinase C (PKC). Nevertheless, acute application of phorbol myristate acetate, known to activate PKC, was not sufficient to induce alkalinization. We conclude that PKC is necessary but not sufficient for alkalinization. In contrast, the GnRH response can be mimicked by a simultaneous application of phorbol myristate acetate and KCl. To further explore the putative role of pHi in the secretory process, LH release was studied. Using Na(+)-free medium or amiloride, we show that basal LH was not dependent upon the Na+/H+ exchanger activity. Conversely, GnRH-induced LH release was significantly decreased; this decrease was greater in E2-treated cells but prevented by bicarbonate. These data show that pHi and the Na+/H+ exchanger play an important role in the stimulus secretion coupling process of gonadotrophs. E2, which is an important factor in the regulation of gonadotropic hormone release, participates also in the pHi variations.
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