Li M, Morley P, Tsang B K
Department of Obstetrics and Gynecology, University of Ottawa, Loeb Medical Research Institute, Ottawa Civic Hospital, Ontario, Canada.
Endocrinology. 1991 Dec;129(6):2957-64. doi: 10.1210/endo-129-6-2957.
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 (pHi) 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 pHi 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; 50-400 ng/ml) and 1-oleoyl-2-acetylglycerol (OAG; 1-75 micrograms/ml) mimicked the actions of EGF by inducing a concentration-dependent increase in pHi which reached a maximum of 0.25-0.30 pH units. 4 alpha-Phorbol 12,13-didecanoate, a phorbol ester with no tumor promoting activity had no effect on pHi. 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 pHi 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.
我们实验室之前的研究表明，表皮生长因子（EGF）通过激活一种钠依赖性且对氨氯地平敏感的Na⁺/H⁺反向转运蛋白，诱导鸡颗粒细胞内碱化。在本研究中，我们检测了蛋白激酶C（PKC）在EGF调节鸡颗粒细胞内pH（pHi）过程中可能的作用。使用染料2',7'-双（羧乙基）-5（6）-羧基荧光素，通过荧光分光光度法测定从两个最大的排卵前卵泡获得的颗粒细胞内pH。当细胞外pH和钠浓度分别为7.3和144 mM时，静息pHi为6.81±0.01（n = 30）。12-O-十四烷酰佛波醇-13-乙酸酯（TPA；50 - 400 ng/ml）和1-油酰-2-乙酰甘油（OAG；1 - 75μg/ml）通过诱导pHi浓度依赖性增加来模拟EGF的作用，pHi最大增加0.25 - 0.30个pH单位。4α-佛波醇12,13-二癸酸酯，一种无肿瘤促进活性的佛波醇酯，对pHi无影响。在添加每种试剂后10分钟内观察到胞质碱化，并在60分钟观察期内增加。与EGF诱导的胞质碱化一样，对TPA或OAG反应的pHi增加依赖于钠浓度的存在，并被Na⁺/H⁺反向转运蛋白的抑制剂氨氯地平抑制。EGF、TPA和OAG的作用被PKC抑制剂5-异喹啉基磺酰基-2-甲基哌嗪和三氟拉嗪减弱。用TPA（200 ng/ml）预处理2.5小时使颗粒细胞PKC下调，抑制了EGF、TPA和OAG诱导的胞质碱化。最大刺激浓度的EGF和TPA对胞质碱化的作用不是相加的。TPA和OAG诱导的pHi增加，但EGF诱导的不增加，依赖于细胞外Ca²⁺的存在。这些研究表明，EGF诱导鸡颗粒细胞内碱化涉及PKC介导的Na⁺/H⁺反向转运蛋白的激活。
