Control of ascorbic acid efflux in rat luteal cells: role of intracellular calcium and oxygen radicals.
In luteal cells, prostaglandin (PG)F2a 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 (O2-), PGF2a, and calcium ionophore were shown to increase [Ca]i and stimulate intracellular ROS. With calcium ionophore and PGF2a, 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 x cm-1 x s-1 (mean +/- SE, n = 8), but in the absence of extracellular calcium the efflux was significantly reduced (10.3 +/- 4.9 fmol x cm-1 x s-1; n = 5, P < 0.05). PGF2a 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 PGF2a-stimulated luteal cells may occur through other signaling pathways.
Department of Obstetrics and Gynecology, Women and Infants Hospital of Rhode Island, 101 Dudley St., Providence, RI 02905, USA. jpeppere@wihri.org
John R
JR
Pepperell
D Marshall
DM
Porterfield
David L
DL
Keefe
Harold R
HR
Behrman
Peter J S
PJ
Smith
D000818:Animals; D000933:Antifibrinolytic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology; D001205:Ascorbic Acid / Q000493:pharmacokinetics*; D002118:Calcium / Q000378:metabolism*; D003338:Corpus Luteum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015237:Dinoprost / Q000378:metabolism; D005260:Female; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D003341:Luteolysis / Q000502:physiology*; D008839:Microelectrodes; D051381:Rats; D011919:Rats, Inbred Strains; D017382:Reactive Oxygen Species / Q000378:metabolism; D013256:Steroids / Q000096:biosynthesis; D024483:Vitamin K 3 / Q000494:pharmacology
D000933:Antifibrinolytic Agents; D007476:Ionophores; D017382:Reactive Oxygen Species; D013256:Steroids; D015759:Ionomycin; D024483:Vitamin K 3; D015237:Dinoprost; D001205:Ascorbic Acid; D002118:Calcium