Role of cyclic adenosine 3',5'-monophosphate-dependent protein kinase in hormone-stimulated beta-endorphin secretion in AtT20 cells.
Secretion of beta-endorphin from mouse pituitary AtT20 cells is stimulated by a variety of compounds that raise intracellular cAMP and Ca2+. To investigate the role of cAMP-dependent protein kinases in secretion, AtT20 cells were transfected with an expression vector coding for a regulatory (R) subunit of cAMP-dependent protein kinase containing mutations in both cAMP-binding sites. Expression of the mutant regulatory subunit in stable transformants (RAB cells) results in a dominant inhibition of cAMP-dependent protein kinase activity. Isoproterenol (1 microM) or analogs of cAMP stimulated beta-endorphin secretion from AtT20 cells, but failed to stimulate secretion in RAB cells expressing the mutant R subunit. Secretion in response to CRF (100 nM) was inhibited by 80% in these mutant clones, whereas the secretory response to vasoactive intestinal peptide (VIP; 100 nM) or phorbol ester (100 nM phorbol myristate acetate) was not inhibited by the R subunit mutation. Intracellular cAMP was elevated in response to CRF (11- to 15-fold), isoproterenol (5- to 10-fold), and VIP (4- to 8-fold) in RAB cells. Similar concentrations of VIP were required to evoke beta-endorphin secretion in either RAB cells or AtT20 cells. As with most secretagogues, VIP-induced secretion was inhibited in the presence of either EGTA or a voltage-sensitive Ca2+ channel antagonist, PN200-110. The secretory response to VIP was unaffected by down-regulation of protein kinase-C. These results suggest that CRF and isoproterenol work via cAMP-dependent protein kinase to activate beta-endorphin secretion, whereas VIP can act by a different mechanism that does not involve cAMP-dependent protein kinase or protein kinase-C.
Schecterson
L C
LC
Department of Pharmacology, University of Washington, Seattle 98195.
McKnight
G S
GS
D000818:Animals; D015220:Calcium Channels / Q000502:physiology; D002461:Cell Line, Transformed; D002772:Cholera Toxin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology*; D015870:Gene Expression; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D016296:Mutagenesis; D010911:Pituitary Neoplasms; D011493:Protein Kinase C / Q000502:physiology; D011494:Protein Kinases / Q000235:genetics / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology; D001615:beta-Endorphin / Q000378:metabolism*
D015220:Calcium Channels; D013873:Thionucleotides; D014660:Vasoactive Intestinal Peptide; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D001615:beta-Endorphin; D002772:Cholera Toxin; D000242:Cyclic AMP; D011494:Protein Kinases; D011493:Protein Kinase C; D007545:Isoproterenol; D013755:Tetradecanoylphorbol Acetate