MESSENGER RIBONUCLEIC-ACIDS FOR ALPHA-ISOFORMS AND BETA-ISOFORMS OF CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE-DEPENDENT PROTEIN-KINASE SUBUNITS PRESENT IN THE ANTERIOR-PITUITARY - REGULATION OF RII-BETA AND C-ALPHA GENE-EXPRESSION BY THE CYCLIC-NUCLEOTIDE AND PHORBOL ESTER

Expression of the genes encoding the alpha- and beta-isoforms of the catalytic (C) and regulatory type I (RI) and type II (RII) subunits of cAMP-dependent protein kinases (PKA) in the anterior pituitary gland of rats was examined by hybridization of specific P-32-labeled cDNA probes to mRNA. Calpha, Cbeta, RIalpha, RIbeta, RIIalpha, and RIIbeta mRNAs were all present in the anterior pituitary gland and cultured cells, but in different proportions; Ca was the most abundant, and RIIalpha was the least. For Calpha, Cbeta, RIbeta, and RIIalpha, Northern blot analysis revealed single mRNAs with sizes of 2.4, 4.5, 2.8, and 6.0 kilobases (kb), respectively. For RIalpha, three distinct mRNAs (1.7, 3.2, and 3.7 kb) were identified, and for RIIbeta, two were found (1.8 and 3.5 kb). Compared to other tissues and species, including ovine and rat pituitary and testis, differences in sizes and relative abundance of mRNAs and/or mRNA subtypes were observed, demonstrating that great diversity exists, which may reflect tissue and species variation in posttranscriptional processing of mRNA transcripts. When rat anterior pituitary cells were cultured in the presence of 8-bromo-cAMP (8-Br-cAMP), RIIbeta mRNA levels increased in a dose- and time-dependent manner to a maximum of about 4- to 5-fold the basal values after 5 h in the presence of 2 mM 8-Br-cAMP. Under the same conditions, the Calpha mRNA, already at high levels, further increased, but to a lesser extent (1.5-2 times compared to basal abundancy). Cholera toxin (6 nm) reproduced the effects of 8-Br-cAMP on both Calpha and RIIbeta mRNAs. These effects were completely abolished in the presence of actinomycin-D, suggesting that cAMP enhances RIIbeta and Calpha gene transcription. On the other hand, cell exposure to the phorbol ester 12-0-tetradecanoyl 13-phorbol acetate, a potent activator of protein kinase-C (PKC), readily induced a dose-dependent actinomycin-D-inhibited increase in the RIIbeta mRNA content to levels about 2-fold those in control unstimulated cells, whereas it depressed levels of Calpha mRNA by 25%. In conclusion, we have found that the six genes coding for the alpha- and beta-isoforms of PKA subunits are all expressed in the anterior pituitary, and that the expression of two of those genes (RIIbeta and Calpha) is regulated by cAMP and phorbol ester in a similar or opposite manner. These data suggest that cAMP and diacylglycerol have the potential to modulate hormone effects within the cells, in addition to their known respective effects on PKA and PKC activation, by affecting the production of specific PKA subunits at the gene level.