Journal
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
Volume 285, Issue 3, Pages C642-C651Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpcell.00587.2002
Keywords
luteolysis; apoptosis; self-referencing microelectrode
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Funding
- NICHD NIH HHS [HD-10718] Funding Source: Medline
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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 (O-2(-)), 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.
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