CYCLIC-AMP REGULATION OF MESSENGER-RNA LEVEL OF THE STIMULATORY GTP-BINDING PROTEIN GS-ALPHA - ISOPROTERENOL, FORSKOLIN AND 8-BROMOADENOSINE 3' 5'-CYCLIC MONOPHOSPHATE INCREASE THE LEVEL OF GS-ALPHA MESSENGER-RNA IN CULTURED ASTROGLIAL CELLS/

The existence of a cAMP-dependent regulation of the expression of the alpha-subunit of the stimulatory guanine nucleotide-binding protein (G s) in a well characterized astroglial cells culture was established. T he culture of astroglial cells for 3-6 h with isoproterenol (10 mu M) or forskolin (10 mu M) (a cAMP-inducing agent) increased (200-400%) th e response of adenylylcyclase to agents which bypass the receptor; GTP , GTP[SI or forskolin. For prolonged exposure times (15 h or more) to isoproterenol or forskolin, the adenylylcyclase activity decreased to the value observed in control cells. The same biphasic response of ade nylylcyclase to isoproterenol (10 mu M) plus GTP (10 mu M) occurred in membrane fractions from cells cultured with forskolin, whereas a dimi nished response to isoproterenol was observed in isoproterenol-treated cells, indicating that the beta-adrenergic receptor was desensitized. To understand the molecular mechanism of these phenomena, we measured the levels of the alpha subunits of the guanine-nucleotide binding pr otein (Gs and Gi) by Western-blot analysis. The culture of astroglial cells with isoproterenol or forskolin (3-24 h) resulted in a transient increase of both the Gs alpha and the Gi alpha protein levels, while the level of G beta subunits was unaffected. We also identified Gs alp ha protein (about 40% of the total cellular protein) in the supernatan t fraction of astroglial cells but its level was not modified by the s timulation of cells by forskolin. The level of Gs alpha mRNA measured by Northern-blot analysis was transiently increased (200%) after stimu lation of astroglial cells with isoproterenol or forskolin for an incu bation period of 6-9 h, then returned to that of control cells for lon ger period of time. In addition, the Gs alpha mRNA level was threefold increased when cells were cultured for 2-6h with X-bromoadenosine 3': 5'-cyclic monophosphate (10 mu M), a permeant analogue of cAMP. These results indicate that cAMP induces a time-dependent increase of Gs alp ha mRNA. The half-life of Gs alpha protein and Gs alpha mRNA were dete rmined. Pulse-chase studies revealed that the decay of Gs alpha protei n was clearly biphasic with an early phase (5-6 h) and a slower second phase (20-25 h) but the treatment of cells with forskolin did not acc elerate or slow down the turnover of Gs alpha protein. The use of acti nomycin D to estimate the stability of Gs alpha mRNA showed that the h alf-life of Gs alpha mRNA was approximately 7-8 h and was not differen t in cells treated with g-bromoadenosine 3':5'-cyclic monophosphate an d in non-treated cells. In contrast, treatment of cells with cyclohexi mide led to a significant increase of Gs alpha mRNA but did not abolis h the effect of forskolin. These studies suggest that the expression o f Gs alpha mRNA is regulated at the transcriptional level and does not require protein synthesis.