HORMONE INDUCTION OF ASCORBIC-ACID TRANSPORT IN IMMATURE GRANULOSA-CELLS

Ascorbic acid serves a vital role as an antioxidant, and like FSK, it inhibits apoptosis of granulosa cells in cultured follicles. In contra st, reactive oxygen species block the action of FSH and induce DNA dam age in these cells. As the uptake of ascorbic acid by granulosa cells may be a site for regulation, we examined the nature of this process a nd whether uptake is under hormone control. Granulosa cells mere isola ted from immature rats pretreated with estradiol or diethylstilbestrol for 3-4 days and placed in culture. Culture of the cells with either FSH (50 ng/ml) or insulin-like growth factor I (IGF-I; 30 ng/ml) for 4 8 h increased ascorbic acid uptake by 2.7- and 1.9-fold (P < 0.05), re spectively, and the response to FSH plus IGF-I was additive (4.5-fold; P < 0.05). The interval for maximum induction of ascorbic acid transp ort by FSH was between 4-8 h, whereas a significant response to IGF-I was not seen until 48 h. GnRH (1 mu M), phorbol ester (phorbol 12-myri state 13-acetate; 1 mu M), and 8-bromo-cAMP (8Br-cAMP; 1 mM) also indu ced ascorbic acid transport by 1.7-, 1.9-, and 2.3-fold (P < 0.05) wit hin 24 h, and the response to maximal levels of phorbol eater and 8Br- cAMP was synergistic (4.8-fold; P < 0.05). Kinetic analysis showed a s imilar Michaelis constant (K-m; 50.8 +/- 5.3 mu M) and max imum veloci ty (3.3 +/- 0.4 pmol/10(6) cells . min) for ascorbic acid transport in FSH-, 8Br-cAMP-, or phorbol eater-treated cells. Ouabain (100 mu M) o r removal of extracellular Na+ significantly inhibited ascorbic acid u ptake, as did dinitrophenol (1 mM), an inhibitor of mitochondrial prod uction of ATP. The induction of ascorbic acid transport by FSH, IGF-I, or GnRH was abolished by simultaneous incubation with tyrphostin (AG- 18; 80 mu m), a specific tyrosine kinase inhibitor, whereas induction was unaffected by an inactive, but chemically similar, compound (A-1; 80 mu M). From these results we conclude that ascorbic acid uptake is energy and Na+ dependent and that the induction of ascorbic acid trans porters in granulosa cells occurs through multiple hormones that ultim ately influencetyrosine-specific protein kinases. The hormone-dependen t induction of ascorbic acid accumulation in granulosa cells appears t o be an essential process for the development and maintenance of a via ble follicle.