Abstract
The hormonal regulation of adenylate cyclase, cAMP-dependent protein kinase activation, and adrenocorticotropic hormone (ACTH) secretion was studied in AtT20 mouse pituitary tumor cells. Corticotropin releasing factor (CRF) stimulated cAMP accumulation and ACTH release in these cells. Maximal ACTH release was seen with 30 nM CRF and was accompanied by a 2-fold rise in intracellular cAMP. When cells were incubated with both 30 nM CRF and 0.5 mM 3-methylisobutylxanthine (MIX) cAMP levels were increased 20-fold, however, ACTH release was not substantially increased beyond release seen with CRF alone. The activation profiles of cAMP-dependent protein kinases I and II were studied by measuring residual cAMP-dependent phosphotransferase activity associated with immunoprecipitated regulatory subunits of the kinases. Cells incubated with CRF in the absence of MIX showed concentration-dependent activation of protein kinase I which paralleled stimulation of ACTH release. Protein kinase II was minimally activated. When cells were exposed to CRF in the presence of 0.5 mM MIX there was still a preferential activation of protein kinase I, although 50% of the cytosolic protein kinase II was activated. Complete activation of both protein kinases I and II was seen when cells were incubated with 0.5 mM MIX and 10 microM forskolin. Under these conditions cAMP levels were elevated 80-fold. CRF, isoproterenol, and forskolin stimulated adenylate cyclase activity in isolated membranes prepared from AtT20 cells. CRF and isoproterenol stimulated cyclase activity up to 5-fold while forskolin stimulated cyclase activity up to 15-fold. Our data demonstrate that ACTH secretion from AtT20 cells is mediated by small changes in intracellular levels of cAMP and activation of only a small fraction of the total cytosolic cAMP-dependent protein kinase in these cells is required for maximal ACTH secretion.
Highlights
CAMP-dependent protein kinase activation, and adre- by calcium and CAMP.Initial evidence for a role of cAMP as nocorticotropic hormone (ACTH)secretion was studied a second messenger in ACTH secretion was provided by the in AtT2Omouse pituitary tumor cells
In thestudies presented itnhis report we have characterized the effects of CRF on ACTH release, adenylate cyclase activation,intracellular cAMP levels, and activation of the CAMP-dependent protein kinase isoenzymes in AtT20 cells
Our results demonstrate that CRF-stimulated ACTH secretion from AtT20 cells is mediated by small changes in cellular levels of cAMP althoughlarge increases in cellular cAMP can be achieved when the cyclic nucleotide phosphodiesterase inhibitor MIX is added to cells incubated with CRF
Summary
The cells (6 X lo cells/ml) were resusfetal calf serum, 10%nonessential amino acids, and 2 mM glutamine pended in fresh prewarmed medium containing 0.1% bovine serum at 37 "C in humidified air and 10%Cot. Thecells maintained under albumin in the presence or absence of 0.5 mM MIX. Thecells maintained under albumin in the presence or absence of 0.5 mM MIX The cells were these conditions grew in suspension and were fed or subcultured 1-2 incubated with various concentrations of CRF, isoproterenol, or fordays before use. Aliquots (0.1 ml) of of DMEM, 1%bovine serum albumin, and various concentrations of the cytosol were incubated with anti-R-I, anti-R-11, or normal rabbit. Cells were incubated serum and immunoprecipitated CAMP-dependentprotein kinase acat 37 "C for 30 min in humidified air and10%COZ. In each experiment the of the incubation, cells were pelleted by centrifugation a t 400 X g and CAMP-dependent protein kinase activity found in immunoprecipi-. Rabbit antisera to succinylated cAMP was generously provided by Dr J
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