Cyclic AMP Regulation of Giα2 and Giα3 mRNAs and Proteins in Astroglial Cells

Abstract: Culture of rat astrocytes in the presence of 10 µM forskolin, isoproterenol, a nonselective β-adrenergic agonist, or 8-bromo-cyclic AMP increased transiently in a time-dependent manner the levels of G_iα2 and G_iα3 mRNAs as measured by northern blot analysis. G_iα1 mRNA was not expressed in these cells. After 6–9 h of culture with forskolin or isoproterenol the amounts of G_iα2 and G_iα3 mRNAs were maximal (150–300% over control). In cells challenged with 8-bromo-cyclic AMP, the level of G_iα2 mRNA level was maximal after 1 h of culture, while the maximal for G_iα3 mRNA was reached after 6 h of culture. For prolonged exposure times (24 h) to these agents the levels of G_iα2 and G_iα3 mRNAs decreased to the values observed in control cells. The forskolin-induced increase in G_iα2 protein expression measured by western blot analysis was similar to the increase in G_iα2 mRNA amount. In contrast, forskolin induced only a modest increase in the quantity of G_iα3 protein (150% over control) despite a large increase of G_iα3 mRNA content. Transcription rates and RNA stability were measured simultaneously after isolation of newly synthesized mRNA was performed. The half-lives of G_iα mRNAs were ～0.7 and 3 h for G_iα2 and G_iα3, respectively. Culturing astrocytes in the presence of forskolin resulted in an increase in the half-lives of G_iα2 mRNA and G_iα3 mRNA by five- and twofold, respectively. The relative transcription rate of the G_iα3 gene was slightly increased by ～1.3–1.5-fold in forskolin-treated cells but not that of G_iα2 gene. These results suggest that cyclic AMP enhanced G_iα2 and G_iα3 mRNA expression by both transcriptional and posttranscriptional mechanisms, with an increase of G_iα mRNA stability as one of the major checkpoints.