The mechanism regulating the luteolytic release of superoxide radical (SOR) was examined in prostaglandin F-2 alpha (PGF(2 alpha))-treated rats. Tail vein injection of PGF(2 alpha), caused a rapid increase in SOR in mitochondria and plasma membrane samples prepared from luteinized rat ovaries. The peak in the mitochondria preceded that in the plasma membrane, and both occurred before progesterone concentrations decreased in the blood. The amount of SOR produced was greater when samples from the plasma membrane, mitochondria, and cytosol were combined. In plasma membrane samples, SOR generation was lowered by inhibitors of intracellular signaling pathways, but not by cyanide, which blocks electron transport in respiratory enzymes. In mitochondria samples, however, SOR was blocked by cyanide, but not by inhibitors of intracellular signaling enzymes. The addition of phospholipase-A(2), phorbol myristate acetate (protein kinase-C activator), or arachidonic acid stimulated SOR production in plasma membrane samples from ovaries of control rats, and phorbol myristate acetate and arachidonic acid inhibited LH-stimulated progesterone secretion in dispersed rat luteal cells. Also, when mitochondria prepared from ovaries of PGF(2 alpha)-treated rats were added to dispersed corpus luteum cells, there was an increase in SOR generation and an inhibition of LH-stimulated cAMP formation and progesterone secretion. These results indicate that SOR production in the corpus luteum after PGF(2 alpha), treatment is generated by several subcellular components. Formation in the plasma membrane may be initiated by SOR generation from the mitochondria and regulated by intracellular signaling pathways. Our results indicate that formation of SOR may lead to the disruption of LH stimulation of progesterone secretion.
