GONADOTROPIN-RELEASING-HORMONE AND PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE AFFECT LEVELS OF CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE-DEPENDENT PROTEIN-KINASE-A (PKA) SUBUNITS IN THE CLONAL GONADOTROPE ALPHA-T3-1 CELLS - EVIDENCE FOR CROSS-TALK BETWEEN PKA AND PROTEIN-KINASE-C PATHWAYS

We have shown previously that protein kinase A (PKA) subunit levels ar e regulated by activation of PKA or protein kinase C (PKC) in anterior pituitary cells. GnRH also influenced PKA subunit levels, suggesting that hormonal regulation occurs in gonadotrophs, and therefore, we hav e reexamined this question using the clonal gonadotrope-derived cell l ine (alpha T3-1 cells). Western blot analysis, using specific immunoaf finity purified immunoglobulins, revealed expression of catalytic (Cat ) and regulatory type I (RI) and type II (RII) subunits of PKA in thes e cells. Activation of adenylyl cyclase (AC) with forskolin, or of PKC with tetradecanoyl phorbol acetate (TPA), caused a rapid (detectable at 0.5-1 h) and concentration-dependent loss of all PKA subunits. Fors kolin (10-100 mu M) reduced Cat and RI by 60% and RII by 30%, whereas TPA (0.1-1 mu M) reduced Cat and RII by 50% and RI by 40%. Simultaneou s activation of PKA and PKC caused the expected dose-dependent reducti ons in Cat, and the effects of forskolin or TPA were nearly additive. RI and RII were reduced similarly by 10 nM TPA, whereas 100 nM TPA ten ded to prevent the reduction of RI or RII caused by forskolin. GnRH, w hich activates phosphoinositidase C and not AC in these cells, caused a clear loss of Cat or RII at all concentrations tested and of RI at 0 .1 nM. Pituitary adenylate cyclase-activating polypeptide 38, which ac ts via PVR-1 receptors to stimulate both phosphoinositidase C and AC i n these cells, also caused a clear dose-dependent decrease in Cat, RI, and RII, although higher concentrations were needed for the latter ef fects. Together, the data demonstrate that catalytic and regulatory su bunits of PKA are subject to both hormonal and receptor-independent re gulation in alpha T3-1 cells, reinforcing the possibility that such ef fects occur in nonimmortalized gonadotropes. Whereas the effects of PK A activators very likely involve proteolytic degradation of the dissoc iated PKA holoenzyme, the effects of TPA and GnRH occur in the absence of cAMP elevation by unknown mechanisms. Whatever the mechanisms invo lved, the data reveal a mechanism for cross-talk between phosphoinosit idase C and AC-mediated hormonal signals, in which PKC activation seem s to play a pivotal role.