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Epidermal Growth Factor Elevates Intracellular pH in Chicken Granulosa Cells by Activating Protein Kinase C^*

MING Li, PAUL MORLEY and BENJAMIN K. TSANG

Reproductive Biology Unit, Department of Obstetrics and Gynecology and Physiology, University of Ottawa, Loeb Medical Research Institute, Ottawa Civic Hospital Ottawa, Ontario, Canada K1 Y 4E9

Address correspondence and requests for reprints to: Benjamin K. Tsang, Ph.D., Reproductive Biology Unit, Loeb Medical Research Institute, Ottawa Civic Hospital, 1053 Carling Avenue, Ottawa, Ontario, Canada KlY 4E9.

Abstract

Previous studies from our laboratory have demonstrated that epidermal growth factor (EGF), induces intracellular alkalinization in chicken granulosa cells by activating a sodium-dependent and amiloride-sensitive Na⁺/H⁺ antiporter. In the present investigation we have examined the possible involvement of protein kinase C (PKC) in the regulation of intracellular pH (pH_i) by EGF in chicken granulosa cells. Intracellular pH in granulosa cells obtained from the two largest preovulatory follicles was determined spectrofluorometrically using the dye 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein. The resting pH_i was 6.81 ± 0.01 (n = 30) when the extracellular pH and sodium concentration were 7.3 and 144 mM, respectively. 12-O-Tetradecanoyl-phorbol-13-acetate (TPA; 50β400 ng/ml) and 1-oleoyl-2-acetylglycerol (OAG; 1β75 µg/ml) mimicked the actions of EGF by inducing a concentration-dependent increase in pH_i which reached a maximum of 0.25β0.30 pH units. 4- Phorbol 12,13-didecanoate, a phorbol ester with no tumor promoting activity had no effect on pH_i. Cytosolic alkalinization was observed within 10 min of the addition of each agent and increased over the 60-min observation period. Like EGF-induced cytosolic alkalinization, the increases in pHi in response to TPA or OAG were dependent on the presence of sodium concentration and were inhibited by amiloride, an inhibitor of the Na⁺/H⁺ antiporter. The effects of EGF, TPA, and OAG were attenuated by the PKC inhibitors 5-isoquinolinylsulfonyl-2-methyl piperazine and trifluoperazine. Down-regulation of granulosa cell PKC by pretreatment with TPA (200 ng/ml) for 2.5 h inhibited EGF-, TPA-, and OAG-induced cytosolic alkalinization. The effects of maximally stimulatory concentrations of EGF and TPA on cytosolic alkalinization were not additive. The increases in pH: induced by TPA and OAG, but not by EGF, were dependent on the presence of extracellular Ca⁺⁺. These studies suggest that the EGF-induced intracellular alkalinization in chicken granulosa cells involves a PKC-mediated activation of the Na⁺/H⁺ antiporter. {Endocrinology 129: 2957-2964, 1991)

Footnotes

^* This study was supported by the Medical Research Council Canada (MRC Grant MT-10369 to B.K.T.).

Visiting Scholar from Fushan Veterinary College, Fushan, Peoples’ Republic of China.

MRC Postdoctoral fellow.

Received July 24, 1991.