Cyclic AMP regulation of messenger RNA level of the stimulatory GTP‐binding protein Gsα

Abstract

The existence of a CAMP-dependent regulation of the expression of the a-subunit of the stimulatory guanine nucleotide-binding protein (Gs) in a well characterized astroglial cells culture was established. The culture of astroglial cells for 3-6 h with isoproterenol (10 pM) or forskolin (10 pM) (a CAMP-inducing agent) increased (200-400%) the response of adenylylcyclase 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 pM) plus GTP (10 pM) occurred in membrane fractions from cells cultured with forskolin, whereas a diminished response to isoproterenol was observed in isoproterenol-treated cells, indicating that the P-adrenergic receptor was desensitized. To understand the molecular mechanism of these phenomena, we measured the levels of the a subunits 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 Gsa and the Gia protein levels, while the level of Gp subunits was unaffected.We also identified Gsa 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 Gsa 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 Gsa mRNA level was threefold increased when cells were cultured for 2-6 h with 8-bromoadenosine 3':5'-cyclic monophosphate (10 pM), a permeant analogue of CAMP. These results indicate that CAMP induces a time-dependent increase of Gsa mRNA.The half-life of Gsa protein and Gsa mRNA were determined. Pulse-chase studies revealed that the decay of Gsa 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 Gsa protein.The use of actinomycin D to estimate the stability of Gsa mRNA showed that the half-life of Gsa 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 Gsa mRNA but did not abolish the effect of forskolin.These studies suggest that the expression of Gsa mRNA is regulated at the transcriptional level and does not require protein synthesis.In a previous study, we have demonstrated that thyrotropin, via a CAMP-dependent mechanism, regulates the expression of the stirnulatory guanine-nucleotide-binding regulatory component of adenylylcyclase (Gs)

Estudo da distribuição da proteína S100b em encéfalo de ratos.

“…Moléculas específicas presentes na superfície dos astrócitos ligam-se a receptores extracelulares serotonérgicos e glutamatérgicos-metabotrópicos em neurônios. Por sua vez, esta ligação induz ao desacoplamento e à ativação de proteínas G existentes no interior da membrana plasmática destas células, levando à propagação de diversos sinais intracelulares associados ao Ca 2+ (Dib et al, 1994;Glowinski et al, 1994).…”

“…Existe a hipótese de que esse mecanismo possa estar relacionado com o Ca 2+ , e que isso resulte na exposição de um domínio hidrofóbico, permitindo interação com a membrana ou com proteínas de membrana, como as anexinas. (Schafer et al, 1996) Outro mecanismo está associado com ação de estruturas presentes na superfície dos astrócitos, receptores extracelulares serotonérgicos, que após ligação com moléculas específicas, induz ao desacoplamento e à ativação de proteínas G existentes no interior da membrana plasmática destas células, levando à propagação de diversos sinais intracelulares, (Dib et al, 1994;Glowinski et al, 1994). Como exemplo, a ativação destas vias de segundosmensageiros em receptores neuronais serotonérgicos e glutamatérgicos metabotrópicos, iniciada por mecanismo glial, induz a aumento concomitante da expressão dos receptores 5HT1A.…”

Estudo da imunorreatividade da proteína S100b no Hipocampo e Núcleo do Trato Solitário de ratos neonatos submetido à anóxia.

“…Existe a hipótese de um domínio hidrofóbico, permitindo interação com a membrana ou com proteínas de membrana, como as anexinas (SCHAFER e HEIZMANN, 1996). Outro mecanismo está associado com ação de estruturas presentes na superfície dos astrócitos, receptores extracelulares serotonérgicos, que após ligação com moléculas específicas, induz ao desacoplamento e à ativação de proteínas G existentes no interior da membrana plasmática destas células, ocasionando propagação de diversos sinais intracelulares, (DIB et al, 1994;GLOWINSKI et al, 1994).…”