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

The role of the cAMP-dependent kinase (AK) in neurotransmission was in vestigated by genetic alteration of AK subunit expression in AtT-20 cl onal pituitary cells. We characterized and compared wildtype [AK(wt)] cells and two clones with different AK activities. The first stably ex presses a gene for a mutant AK regulatory subunit (RI) that does not b ind 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 A K(-) cells relative to AK(wt), with the transfection-induced expressio n of one subunit producing a compensatory increase in the expression o f the other. The basal AK activities varied among the cell types, with AK(+) cells possessing 3-fold higher basal AK activity than AK(wt) ce lls, and AK(-) cells possessing half the AK activity of AK(wt) cells. Preincubation of cultures with 300 mu M 8-(4-chlorophenylthio)-cAMP in creased AK activity approximately 4-fold in AK(wt) and AK(+) cells, bu t was without effect in AK(-) cells. Subsequent addition of 1 mu M cAM P in vitro increased AK activity an additional 2- to 3-fold in all cel l types. The higher basal AK activity found in AK(wt) and AK(+) cells was associated with larger whole cell calcium currents (similar to 43% and similar to 75% larger than in AK(-) cells, respectively) and fast er rates of current rundown. The currents from each cell line had simi lar voltage-dependent and pharmacological properties, however, and [H- 3]PN200-110 binding was similar among the cell types. Maximal currents were evoked at clamp potentials of 0-10 mV; currents were inactivated similar to 30% in the steady state at holding potentials of -40 mV co mpared to -80 mV, and currents were reduced similar to 45% in the pres ence 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 similar to 50% greater, but stimulated rates were similar to 400% greater compared to those i n AK(-) cells. We conclude that a greater number of calcium channels w ere activated by depolarization in the phosphorylated state, that curr ent rundown was largely due to dephosphorylation, and that activation of calcium channels was coupled to the release of beta-endorphin. Ther e was a positive correlation of AK activity, calcium current magnitude , and rates of beta-endorphin release, although these relationships we re not simple or linear. These studies also demonstrate the power of u sing molecular biological techniques to alter second messenger systems and thereby to determine their roles in regulating calcium channel ac tivity and secretion.