Protein kinase C stimulates adenylate cyclase activity in prolactin-secreting rat adenoma (GH4C1) pituicytes by inactivating the inhibitory GTP-binding protein Gi.
The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and thyroliberin exerted additive stimulatory effects on prolactin release and synthesis in rat adenoma GH4C1 pituicytes in culture. Both TPA and thyroliberin activated the adenylate cyclase in broken cell membranes. When combined, the secretagogues displayed additive effects. TPA did not alter the time course (time lag) of adenylate cyclase activation by hormones, guanosine 5'-[beta,gamma-imino]triphosphate or forskolin, nor did it affect the enzyme's apparent affinity (basal, 7.2 mM; thyroliberin-enhanced, 2.2 mM) for free Mg2+. The TPA-mediated adenylate cyclase activation was entirely dependent on exogenously added guanosine triphosphate. ED50 (dose yielding half-maximal activation) was 60 microM. Access to free Ca2+ was necessary to express TPA activation of the enzyme, however, the presence of calmodulin was not mandatory. TPA-stimulated adenylate cyclase activity was abolished by the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, by the protein kinase C inhibitor polymyxin B and by pertussis toxin, while thyroliberin-sensitive adenylate cyclase remained unaffected. Experimental conditions known to translocate protein kinase C to the plasma membrane and without inducing adenylate cyclase desensitization, increased both basal and thyroliberin-stimulated enzyme activities, while absolute TPA-enhanced adenylate cyclase was maintained. Association of extracted GTP-binding inhibitory protein, Gi, from S49 cyc- murine lymphoma cells with GH4C1 cell membranes yielded a reduction of basal and hormone-stimulated adenylate cyclase activities, while net inhibition of the cyclase of somatostatin was dramatically enhanced. However, TPA restored completely basal and hormone-elicited adenylate cyclase activities in the Gi-enriched membranes. Finally, TPA completely abolished the somatostatin-induced inhibition of adenylate cyclase in both hybrid and non-hybrid membranes. These data suggest that, in GH4C1 cells, protein kinase C stimulation by phorbol esters completely inactivates the n alpha i subunit of the inhibitory GTP-binding protein, leaving the n beta subunit functionally intact. It can also be inferred that thyroliberin conveys its main effect on the adenylate cyclase through activation of the stimulatory GTP-binding protein, Gs.
Gordeladze
J O
JO
Institute for Surgical Research, National Hospital, Oslo, Norway.
Björo
T
T
Torjesen
P A
PA
Ostberg
B C
BC
Haug
E
E
Gautvik
K M
KM
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000478:Alkylation; D000818:Animals; D002118:Calcium / Q000502:physiology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000096:biosynthesis; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007700:Kinetics; D037342:Pertussis Toxin; D010911:Pituitary Neoplasms / Q000201:enzymology* / Q000378:metabolism; D011112:Polymyxin B / Q000494:pharmacology; D011388:Prolactin / Q000378:metabolism*; D011493:Protein Kinase C / Q000502:physiology*; D051381:Rats; D013004:Somatostatin / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D010566:Virulence Factors, Bordetella; D005576:Colforsin; D006165:Guanylyl Imidodiphosphate; D013004:Somatostatin; D013973:Thyrotropin-Releasing Hormone; D006160:Guanosine Triphosphate; D011388:Prolactin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D011112:Polymyxin B; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium