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 contrast, reactive oxygen species block the action of FSH and induce DNA damage 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 and whether uptake is under hormone control. Granulosa cells mere isolated 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 48 h increased ascorbic acid uptake by 2.7- and 1.9-fold (P < 0.05), respectively, and the response to FSH plus IGF-I was additive (4.5-fold; P < 0.05). The interval for maximum induction of ascorbic acid transport 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-myristate 13-acetate; 1 mu M), and 8-bromo-cAMP (8Br-cAMP; 1 mM) also induced ascorbic acid transport by 1.7-, 1.9-, and 2.3-fold (P < 0.05) within 24 h, and the response to maximal levels of phorbol ester and 8Br-cAMP was synergistic (4.8-fold; P < 0.05). Kinetic analysis showed a similar Michaelis constant (K-m; 50.8 +/- 5.3 mu M) and max imum velocity (3.3 +/- 0.4 pmol/10(6) cells . min) for ascorbic acid transport in FSH-, 8Br-cAMP-, or phorbol ester-treated cells. Ouabain (100 mu M) or removal of extracellular Na+ significantly inhibited ascorbic acid uptake, as did dinitrophenol (1 mM), an inhibitor of mitochondrial production 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 transporters in granulosa cells occurs through multiple hormones that ultimately influencetyrosine-specific protein kinases. The hormone-dependent induction of ascorbic acid accumulation in granulosa cells appears to be an essential process for the development and maintenance of a viable follicle.