Identification and characterization of an ascorbic acid transporter in human granulosa–lutein cells

Abstract

Ascorbic acid serves a vital role as a pre-eminent antioxidant. In animals, it has been shown to be concentrated in granulosa and theca cells of the follicle, in luteal cells of the corpus luteum, and in the peripheral cytoplasm of the oocyte. We have previously identified hormonally-regulated ascorbic acid transporters in rat granulosa and luteal cells, and herein present preliminary evidence for the presence of a transporter for ascorbic acid in human granulosa–lutein cells. Granulosa–lutein cells were obtained from the follicular fluid of patients undergoing in-vitro fertilization. Following an overnight incubation, the cells were incubated with [14C]-ascorbic acid (0.15 μCi; 150 μMM) and ascorbic acid uptake was determined. The uptake of ascorbic acid was saturable with a Michaeli's constant (Km) and maximum velocity (Vmax) of 21 μM and 3 pmol/106 cells/min respectively. Ouabain, low Na+ medium, and dinitrophenol significantly inhibited ascorbic acid uptake (P < 0.05). Neither the presence of insulin, human chorionic gonadotrophin (HCG), insulin-like growth factor (IGF)-I, nor IGF-II affected the uptake of ascorbic acid in a statistically significant fashion. Following saturation of cellular uptake, the ascorbic acid level was estimated to be 1.04 pmoles/106 cells or ~1 mM, a high concentration similar to that seen in rat luteal cells. Active ascorbic acid transport in human granulosa–lutein cells appears to occur via a Na+- and energy-dependent transporter, with high levels of ascorbic acid being accumulated in these cells.