ELECTRICAL PERMEABILIZATION OF RAT LUTEAL CELLS - IN-SITU PHOSPHORYLATION OF ENDOGENOUS PROTEINS

Progesterone synthesis in the corpus luteum is regulated primarily by luteinizing hormone which acts via the adenylate cyclase/cyclic AMP/pr otein kinase A signalling cascade. Protein phosphorylation therefore p lays a key role in the regulation of steroidogenesis, but there are re latively few studies of the in situ phosphorylation of luteal cell sub strates. This may in part reflect the difficulties inherent in measuri ng changes in protein phosphorylation in intact cells preloaded with P -32 and difficulties in interpreting data obtained using broken cell p reparations. We have now applied a method of stable permeabilization o f luteal cell plasma membranes by exposure of cell populations to a hi gh intensity electric field. Under optimum conditions (5 kV/cm, six di scharges) electrical permeabilization reproducibly produced population s of luteal cells, in which 70-80% of the cells were permeabilized, as assessed by Trypan blue exclusion and [C-14]sucrose space measurement s. Pores were stable for at least 1 h, and there were no ultrastructur al changes to the cells that could be detected by transmission electro n microscopy. Permeabilized cells showed rapid cyclic AMP-induced chan ges in phosphorylation of endogenous proteins when provided with [gamm a-P-32]ATP. Our results demonstrate that the electrically permeabilize d luteal cell offers a useful model for studying intracellular events in steroidogenic stimulus-response coupling cascades. (C) 1997 by Else vier Science Inc.