135 CHARACTERISTICS OF PROTEIN KINASE C MEDIATED PITUITARY ACTH RELEASE

Abstract

Protein kinase C is a proposed mediator of pituitary ACTH secretion. To investigate this role of protein kinase C, we studied the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of protein kinase C activation, on in vitro pituitary ACTH release. Normal rat anterior pituitaries were epzymatically dispersed to monolayer culture. Dishes with 3.5 × 105 cells were incubated 3 days, washed, and incubated in test media for 3.5 hours. TPA stimulated ACTH(RIA) release in a dose related fashion with an ED50 of 0.7 ng/ml and a maximal stimulation of 900% over unstimulafed cells. Increasing the concentration of culture media calcium from 0 to 1 mM, 3mM and 6 mM led to a significant increase of TPA induced ACTH release from 4.80 to 6.79, 8.41, and 8.62 ng/dish respectively. Nifedipine (10 μM), a membrane calcium channel blocker, and penfluridol (10 μM), a calcium calmodulin antagonist, inhibited TPA (1 ng/ml) mediated ACTH release by 21% and 31% respectively (p < 0.01). The addition of PGE2 (0.1 μM), an inhibitor of CRF mediated ACTH release, caused a 33% decrease of TPA stimulated ACTH release. TPA (100 μg/ml) significantly increased intracellular PGE2 (RIA) and PGE2 released into culture media by 178% and 172% respectively, over unstimulated cells. In conclusion, TPA stimulated ACTH release in vitro appears to be dependent on extracellular calcium, membrane calcium influx, and calcium calmodulin activation. Also, TPA stimulates pituitary PGE2 synthesis and exogenously administrated PGE2 inhibits TPA induced ACTH release.