PROSTAGLANDIN-F2-ALPHA TREATMENT IN-VIVO, BUT NOT IN-VITRO, STIMULATES PROTEIN-KINASE C-ACTIVATED SUPEROXIDE PRODUCTION BY NONSTEROIDOGENICCELLS OF THE RAT CORPUS-LUTEUM

Luteal regression is associated with the generation of reactive oxygen species (ROS). To determine the nature of the ROS generator, cells is olated from luteinized rat ovaries were examined for ROS production us ing luminol-amplified chemiluminescence (LCL). Cells cultured for 2-48 h exhibited minimal LCL, but there was a significant (30- to 50-fold) , rapid (maximum at 3-5 min), and dose-dependent increase in LCL in re sponse to phorbol ester (phorbol 12-myristate 13-acetate; TPA; ED50 = 0.03 mu M) and diacylglycerol (1,2-dioctanoyl-glycerol; ED50 = 30 mu M ). The TPA-induced response was cell number dependent and was virtuall y abolished by superoxide dismutase, freezing, or heating (95 degrees C for 5 min). Zymosan, known to induce a phagocytic response in leukoc ytes, stimulated a superoxide (O-2(-.)) response with a slow onset (ma ximum at 40 to 60 min) and a maximum about one third of that observed for TPA. The response to TPA and zymosan was inhibited by the NADPH/NA DH-oxidase inhibitor, diphenylene iodonium (ID50 = 5 mu M for TPA), bu t not by the mitochondrial inhibitors, potassium cyanide, rotenone, or sodium azide. Fractionation of cells by centrifugal elutriation showe d that TPA-stimulated O-2(-.) production coeluted with the nonsteroido genic cells and that little, if any, O-2(-.) generation coeluted with the steroidogenic cells. Cells isolated 1, 2, and 4 h after in vivo tr eatment with a luteolytic dose of prostaglandin F-2 alpha (PGF(2 alpha )) showed a significant increase in TPA-stimulated O-2(-.) production at 2 h, whereas luteal cells or corpora lutea incubated directly with 1 mu M PGF(2 alpha) did not show any increase in response. Corpora lut ea isolated from naturally regressed ovaries (18 days after ovulation) showed a significantly elevated level of TPA-stimulated O-2(-.) produ ction. In conclusion, there is a superoxide generator in luteinized ov aries that is activated through a protein kinase C pathway, localized in nonsteroidogenic cells, transiently increased during PGF(2 alpha)-i nduced luteolysis in vivo, and elevated during natural luteal regressi on.