Protein kinase C mediates the effect of vasopressin in pituitary corticotrophs.
The role of protein kinase C (PKC) on vasopressin (VP) action was investigated by inhibition of endogenous PKC using prolonged incubation of the cells with phorbol ester, and by direct measurement of PKC activity in pituitary cells. Preincubation of the cells for 6 h with 100 nM TPA at 37 C resulted in a 90% decrease in total PKC activity. In the PKC-depleted cells, cAMP responses to stimulation with 100 nM CRF for 30 min were normal, but the potentiating effects of VP and PMA on CRF-stimulated cAMP production were abolished. The stimulation of ACTH secretion by VP and PMA alone was also abolished in PKC- depleted cells. PKC activity in cytosolic and detergent-solubilized membrane fractions from enriched pituitary corticotrophs obtained by centrifugal elutriation, was directly measured by enzymatic assays and by immunoblotting techniques. Basal PKC activity was higher in the cytosol than in the membranes (8.43 +/- 0.47 and 1.93 +/- 0.11 pmol 32P incorporated/10 min, respectively). After incubation of the cells with VP for 15 min or [3H] phorbol-12-myristate-13-acetate (PMA) for 30 min, PKC activity in cytosol was decreased by 40% and 89%, respectively, while the activity in the membrane was increased by 138% and 405%, respectively. Such VP- and PMA-induced translocation of PKC was also observed when the enzyme content in the cytosol and the membranes was measured by immunoblotting using a specific anti-PKC antibody and [125I]protein A. Autoradiographic analysis of immunoblots revealed an 80 kilodalton band characteristic of PKC, with OD higher in the cytosolic than in the membrane fractions.(ABSTRACT TRUNCATED AT 250 WORDS)
Carvallo
P
P
Section of Endocrine Physiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Aguilera
G
G
D000818:Animals; D002478:Cells, Cultured; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D010703:Phorbol Esters / Q000494:pharmacology; D010902:Pituitary Gland / Q000187:drug effects / Q000201:enzymology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D051381:Rats; D011919:Rats, Inbred Strains; D014667:Vasopressins / Q000494:pharmacology*
D010703:Phorbol Esters; D014667:Vasopressins; D003346:Corticotropin-Releasing Hormone; D000242:Cyclic AMP; D011493:Protein Kinase C