The existence of a cAMP‐dependent regulation of the expression of the α‐subunit of the stimulatory guanine nucleotide‐binding protein (Gs) in a well characterized astroglial cells culture was established. The culture of astroglial cells for 3–6h with isoproterenol (10 μM) or forskolin (10 μM) (a cAMP‐inducing agent) increased (200–400%) the response of adenyllcyclase to agents which bypass the receptor; GTP, GTP[S] 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 adenylylcyclase to isoproterenol (10 μM) plus GTP (10 μM) occurred in membrane fractions from cells cultured with forskolin, whereas a diminished response to isoproterenol was observed in isoproterenol‐treated cells, indicating that the β‐adrenergic receptor was desensitized. To understand the molecular mechanism of these phenomena, we measured the levels of the αsubunits of the guanine‐nucleotide binding protein (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 Gsα and the Giα protein levels, while the level of Gβ subunits was unaffected. We also identified Gsα protein (about 40% of the total cellular protein) in the supernatant fraction of astroglial cells but its level was not modified by the stimulation of cells by forskolin. The level of Gsα mRNA measured by Northern‐blot analysis was transiently increased (200%) after stimulation of astroglial cells with isoproterenol or forskolin for an incubation period of 6–9 h, then returned to that of control cells for longer period of time. In addition, the Gsα mRNA level was threefold increased when cells were cultured for 2–6 h with 8‐bromoadenosine 3′:5′‐cyclic monophosphate (10 μM), a permeant analogue of cAMP. These results indicate that cAMP induces a time‐dependent increase of Gsα mRNA. The half‐life of Gsα protein and Gsα mRNA were determined. Pulse‐chase studies revealed that the decay of Gsα protein 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 accelerate or slow down the turnover of Gsα protein. The use of actinomycin D to estimate the stability of Gsα mRNA showed that the half‐life of Gsα mRNA was approximately 7–8 h and was not different in cells treated with 8‐bromoadenosine 3′:5′‐cyclic monophosphate and in non‐treated cells. In contrast, treatment of cells with cycloheximide led to a significant increase of Gsα mRNA but did not abolish the effect of forskolin. These studies suggest that the expression of Gsα mRNA is regulated at the transcriptional level and does not require protein synthesis.
|Original language||English (US)|
|Number of pages||9|
|Journal||European Journal of Biochemistry|
|State||Published - Jan 1994|
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