Electrical permeabilization of rat luteal cells: in situ phosphorylation of endogenous protein.
Progesterone synthesis in the corpus luteum is regulated primarily by luteinizing hormone which acts via the adenylate cyclase/cyclic AMP/protein kinase A signalling cascade. Protein phosphorylation therefore plays a key role in the regulation of steroidogenesis, but there are relatively few studies of the in situ phosphorylation of luteal cell substrates. This may in part reflect the difficulties inherent in measuring changes in protein phosphorylation in intact cells preloaded with 32P and difficulties in interpreting data obtained using broken cell preparations. We have now applied a method of stable permeabilization of luteal cell plasma membranes by exposure of cell populations to a high intensity electric field. Under optimum conditions (5 kV/cm, six discharges) electrical permeabilization reproducibly produced populations of luteal cells in which 70-80% of the cells were permeabilized, as assessed by Trypan blue exclusion and [14C] sucrose space measurements. Pores were stable for at least 1 h, and there were no ultrastructural changes to the cells that could be detected by transmission electron microscopy. Permeabilized cells showed rapid cyclic AMP-induced changes in phosphorylation of endogenous proteins when provided with [gamma - 32 P] ATP. Our results demonstrate that the electricity permeabilized luteal cell offers a useful model for studying intracellular events in steroidogenic stimulus-response coupling cascades.
Ford
S L
SL
Biomedical Sciences Division, King's College London, United Kingdom.
Persaud
S J
SJ
Abayasekara
D R
DR
Jones
P M
PM
D000818:Animals; D002463:Cell Membrane Permeability / Q000502:physiology*; D003338:Corpus Luteum / Q000502:physiology*; D000242:Cyclic AMP / Q000378:metabolism; D018274:Electroporation / Q000379:methods*; D005260:Female; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D011506:Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP