Control of ascorbic acid efflux in rat luteal cells: role of intracellular calcium and oxygen radicals

3 BioCurrents Research Center, Marine Biological Laboratory, Woods Hole, Massachusetts 02543; 1 Department of Obstetrics/Gynecology, Women and Infants Hospital of Rhode Island, Providence, Rhode Island 02905; and 2 Department of Obstetrics/Gynecology, Yale Medical School, New Haven, Connecticut 06511 Submitted 16 December 2002 ; accepted in final form 17 April 2003 In luteal cells, prostaglandin (PG)F 2a mobilizes intracellular calcium concentration ([Ca] i ), generates reactive oxygen species (ROS), depletes ascorbic acid (AA) levels, inhibits steroidogenesis, and ultimately induces cell death. We investigated the hypothesis that [Ca] i mobilization stimulates ROS, which results in depletion of cellular AA in rat luteal cells. We used a self-referencing AA-selective electrode that noninvasively measures AA flux at the extended boundary layer of single cells and fluorescence microscopy with fura 2 and dichlorofluorescein diacetate (DCF-DA) to measure [Ca] i and ROS, respectively. Menadione, a generator of intracellular superoxide radical ( ), PGF 2a , and calcium ionophore were shown to increase [Ca] i and stimulate intracellular ROS. With calcium ionophore and PGF 2a , but not menadione, the generation of ROS was dependent on extracellular calcium influx. In unstimulated cells there was a net efflux of AA of 121.5 ± 20.3 fmol · cm – 1 · s – 1 (mean ± SE, n = 8), but in the absence of extracellular calcium the efflux was significantly reduced (10.3 ± 4.9 fmol · cm – 1 · s – 1 ; n = 5, P < 0.05). PGF 2a and menadione stimulated AA efflux, but calcium ionophore had no significant effect. These data suggest two AA regulatory mechanisms: Under basal conditions, AA efflux is calcium dependent and may represent recycling and maintenance of an antioxidant AA gradient at the plasma membrane. Under luteolytic hormone and/or oxidative stress, AA efflux is stimulated that is independent of extracellular calcium influx or generation of ROS. Although site-specific mobilization of calcium pools and ROS cannot be ruled out, the release of AA by PGF 2a -stimulated luteal cells may occur through other signaling pathways. luteolysis; apoptosis; self-referencing microelectrode Address for reprint requests and other correspondence: J. R. Pepperell, Dept. of Pathology, Women & Infants Hospital of Rhode Island, 101 Dudley St., Providence, RI 02905 (E-mail: jpeppere{at}wihri.org ).