Protein kinase C- and Ca2+ ionophore-stimulated production of reactive oxygen species in mechanically dispersed isolated bovine luteal cells

We measured the production of reactive oxygen species (ROS) using luminol-horseradish peroxidase-induced chemiluminescence in mechanically dispersed cell suspensions from bovine corpus luteum (CL). Since other cell types besides luteal cells were present in crude cell suspensions from CL, cell preparations were purified by centrifugation on Percoll. Only cell suspensions that gave no significant response when stimulated with formyl-methionyl-leucyl-phenylalanine, a potent stimulator of ROS production by phagocytes, were used routinely. Basal ROS production by purified bovine luteal cell preparations was low but could be stimulated rapidly and in a dose-dependent manner by nanomolar concentrations of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA), though only in cells purified by fractionation on Percoll. Luteal ROS responses to PMA were quenched by returning bovine erythrocytes to purified luteal cells, or by exogenous catalase or superoxide dismutase. The magnitude of the response to PMA varied markedly from one luteal cell preparation to another but appeared to be unrelated to the stage of the luteal phase of the CL from which the cells were prepared. The luteal ROS response to PMA was blocked by staurosporine, an inhibitor of PKC. Although the inactive phorbol ester (4 alpha-phorbol didecanoate; 4 alpha PDD) alone had little or no effect on luteal ROS production, 4 alpha PDD significantly potentiated the effects of submaximal concentrations of PMA in a dose-dependent manner. ROS production could also be stimulated by the Ca2+ ionophore A23187. This response was rapidly abolished by treatment with EDTA or EGTA. A23187 also augmented the response to submaximal PMA levels: however, pretreatment with 4 alpha PDD did not significantly enhance the ROS response to A23187. In conclusion, we have shown that isolated bovine luteal cell suspensions are capable of generating a marked acute ROS response triggered by activation of PKC and/or elevation of cytosolic calcium.