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-ALPHAGENE-EXPRESSION BY THE CYCLIC-NUCLEOTIDE AND PHORBOL ESTER

Expression of the genes encoding the alpha- and beta-isoforms of the c atalytic (C) and regulatory type I (RI) and type II (RII) subunits of cAMP-dependent protein kinases (PKA) in the anterior pituitary gland o f rats was examined by hybridization of specific P-32-labeled cDNA pro bes to mRNA. Calpha, Cbeta, RIalpha, RIbeta, RIIalpha, and RIIbeta mRN As 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 k ilobases (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 an d rat pituitary and testis, differences in sizes and relative abundanc e of mRNAs and/or mRNA subtypes were observed, demonstrating that grea t 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-cA MP), RIIbeta mRNA levels increased in a dose- and time-dependent manne r 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 mRN A, already at high levels, further increased, but to a lesser extent ( 1.5-2 times compared to basal abundancy). Cholera toxin (6 nm) reprodu ced the effects of 8-Br-cAMP on both Calpha and RIIbeta mRNAs. These e ffects were completely abolished in the presence of actinomycin-D, sug gesting that cAMP enhances RIIbeta and Calpha gene transcription. On t he other hand, cell exposure to the phorbol ester 12-0-tetradecanoyl 1 3-phorbol acetate, a potent activator of protein kinase-C (PKC), readi ly induced a dose-dependent actinomycin-D-inhibited increase in the RI Ibeta mRNA content to levels about 2-fold those in control unstimulate d cells, whereas it depressed levels of Calpha mRNA by 25%. In conclus ion, we have found that the six genes coding for the alpha- and beta-i soforms of PKA subunits are all expressed in the anterior pituitary, a nd that the expression of two of those genes (RIIbeta and Calpha) is r egulated by cAMP and phorbol ester in a similar or opposite manner. Th ese data suggest that cAMP and diacylglycerol have the potential to mo dulate hormone effects within the cells, in addition to their known re spective effects on PKA and PKC activation, by affecting the productio n of specific PKA subunits at the gene level.