Cyclic AMP regulation of G(i alpha2) and G(i alpha3) mRNAs and proteins in astroglial cells.

摘要

Culture of rat astrocytes in the presence of 10 microM forskolin, isoproterenol, a nonselective beta-adrenergic agonist, or 8-bromo-cyclic AMP increased transiently in a time-dependent manner the levels of G(i alpha2) and G(i alpha3) mRNAs as measured by northern blot analysis. G(i alpha1) mRNA was not expressed in these cells. After 6-9 h of culture with forskolin or isoproterenol the amounts of G(i alpha2) and G(i alpha3) mRNAs were maximal (150-300% over control). In cells challenged with 8-bromo-cyclic AMP, the level of G(i alpha2) mRNA level was maximal after 1 h of culture, while the maximal for G(i alpha3) mRNA was reached after 6 h of culture. For prolonged exposure times (24 h) to these agents the levels of G(i alpha2) and G(i alpha3) mRNAs decreased to the values observed in control cells. The forskolin-induced increase in G(i alpha2) protein expression measured by western blot analysis was similar to the increase in G(i alpha2) mRNA amount. In contrast, forskolin induced only a modest increase in the quantity of G(i alpha3) protein (150% over control) despite a large increase of G(i alpha3) mRNA content. Transcription rates and RNA stability were measured simultaneously after isolation of newly synthesized mRNA was performed. The half-lives of G(i alpha) mRNAs were approximately 0.7 and 3 h for G(i alpha2) and G(i alpha3), respectively. Culturing astrocytes in the presence of forskolin resulted in an increase in the half-lives of G(i alpha2) mRNA and G(i alpha3) mRNA by five- and twofold, respectively. The relative transcription rate of the G(i alpha3) gene was slightly increased by approximately 1.3-1.5-fold in forskolin-treated cells but not that of G(i alpha2) gene. These results suggest that cyclic AMP enhanced G(i alpha2) and G(i alpha3) mRNA expression by both transcriptional and posttranscriptional mechanisms, with an increase of G(i alpha) mRNA stability as one of the major checkpoints.