EPIDERMAL GROWTH-FACTOR ELEVATES INTRACELLULAR PH IN CHICKEN GRANULOSA-CELLS

Many bioregulators, such as epidermal growth factor (EGF), induce intracellular alkalinization by activating a membrane bound Na+/H+ antiporter. The present studies were designed to examine the influence of EGF on intracellular pH (pH(i)) in chicken granulosa cells. pH(i) in granulosa cells from the two largest preovulatory follicles of hens was determined spectrofluorometrically using the dye 2',7'-bis (carboxyethyl-5(6)-carboxyfluorescein. The resting pH(i) was 6.81 +/- 0.006 (n = 30) when the extracellular pH and sodium concentration (Na+o) were 7.3 and 144 mM, respectively. EGF (5-100 ng/ml) induced a concentration-dependent increase in pH(i), which reached a maximum of 0.217 +/- 0.009 pH units at a concentration of 100 ng/ml EGF. Cytosolic alkalinization was observed within 10 min of the addition of EGF and lasted over the 60 min observation period. The increase in pH(i) was dependent upon the presence of Na+o, since the EGF effect was attenuated when Na+o was substituted with equimolar concentrations of nonpermeant choline chloride. The EGF-induced pH(i) change was also inhibited by amiloride, dimethyl amiloride, and ethylisopropyl amiloride, inhibitors of the Na+/H+ antiporter. The alkalinization effect of EGF was mimicked by transforming growth factor-alpha but not by insulin, insulin-like growth factor-I, or transforming growth factor-beta. These studies suggest for the first time that intracellular alkalinization resulting from activation of the Na+/H+ antiporter may be a part of the transmembrane signaling pathway in the action of EGF on chicken granulosa cells.