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Epidermal Growth Factor Elevates Intracellular pH in Chicken Granulosa Cells^*

MING Li, PAUL MORLEY, ELIKPLIMI K. ASEM and BENJAMIN K. TSANG

Reproductive Biology Unit, Department of Obstetrics and Gynecology, University of Ottawa, Loeb Medical Research Institute, Ottawa Civic Hospital Ottawa, Ontario, Canada K1Y 4E9
Department of Physiology and Pharmacology, School of Veterinary Medicine, Purdue University West Lafayette, Indiana 47907

Address all correspondence and requests for reprints to: Dr. Benjamin K. Tsang, Reproductive Biology Unit, Loeb Medical Research Institute, Ottawa Civic Hospital, 1053 Carling Avenue, Ottawa, Ontario, Canada K1Y 4E9.

Abstract

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⁺₀) 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- but not by insulin, insulin-like growth factor-I, or transforming growth factor-β. 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.

Footnotes

^* This study was supported by Medical Research Council of Canada Grant MT-10369 (to B.K.T.) and NIH Grant HD-27354-01 (to E.K.A.).

Visiting scholar from Fushan Veterinary College, Fushan, Peoples Republic of China

Medical Research Council of Canada Postdoctoral Fellow.

Received March 25, 1991.

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