title: Genetic alteration of cyclic adenosine 3',5'-monophosphate-dependent protein kinase subunit expression affects calcium currents and beta-endorphin release in AtT-20 clonal pituitary cells.
abstract: The role of the cAMP-dependent kinase (AK) in neurotransmission was investigated by genetic alteration of AK subunit expression in AtT-20 clonal pituitary cells. We characterized and compared wild-type [AK(wt)] cells and two clones with different AK activities. The first stably expresses a gene for a mutant AK regulatory subunit (RI) that does not bind cAMP [AK(-)]; the second stably expresses a gene for the catalytic subunit (C) of AK [AK(+)]. Western blot analysis of RI and C subunit expression showed increased expression of both subunits in AK(+) and AK(-) cells relative to AK(wt), with the transfection-induced expression of one subunit producing a compensatory increase in the expression of the other. The basal AK activities varied among the cell types, with AK(+) cells possessing 3-fold higher basal AK activity than AK(wt) cells, and AK(-) cells possessing half the AK activity of AK(wt) cells. Preincubation of cultures with 300 microM 8-(4-chlorophenylthio)-cAMP increased AK activity approximately 4-fold in AK(wt) and AK(+) cells, but was without effect in AK(-) cells. Subsequent addition of 1 microM cAMP in vitro increased AK activity an additional 2- to 3-fold in all cell types. The higher basal AK activity found in AK(wt) and AK(+) cells was associated with larger whole cell calcium currents (approximately 43% and approximately 75% larger than in AK(-) cells, respectively) and faster rates of current rundown. The currents from each cell line had similar voltage-dependent and pharmacological properties, however, and [3H]PN200-110 binding was similar among the cell types. Maximal currents were evoked at clamp potentials of 0-10 mV; currents were inactivated approximately 30% in the steady state at holding potentials of -40 mV compared to -80 mV, and currents were reduced approximately 45% in the presence of nifedipine at -40 mV, but were insensitive to omega-conotoxin GVIA. AK(wt) and AK(+) cells also had higher basal and cAMP-stimulated release of beta-endorphin; control rates were approximately 50% greater, but stimulated rates were approximately 400% greater compared to those in AK(-) cells. We conclude that a greater number of calcium channels were activated by depolarization in the phosphorylated state, that current rundown was largely due to dephosphorylation, and that activation of calcium channels was coupled to the release of beta-endorphin.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Neurology, University of Minnesota Medical School, Minneapolis 55455.
authors.forename: R A
authors.initials: RA
authors.lastname: Gross
authors.forename: J
authors.initials: J
authors.lastname: Huggenvik
authors.forename: E
authors.initials: E
authors.lastname: Massa
authors.forename: R G
authors.initials: RG
authors.lastname: Allen
authors.forename: M D
authors.initials: MD
authors.lastname: Uhler
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000495:Allosteric Site; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D051379:Mice; D009543:Nifedipine / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation; D010911:Pituitary Neoplasms / Q000473:pathology*; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction / Q000187:drug effects; D014407:Tumor Cells, Cultured; D001615:beta-Endorphin / Q000378:metabolism*; D020866:omega-Conotoxin GVIA
chemical_list: D015220:Calcium Channels; D010455:Peptides; D001615:beta-Endorphin; D000324:Adrenocorticotropic Hormone; D020866:omega-Conotoxin GVIA; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D009543:Nifedipine; D002118:Calcium