Cyclic AMP regulates G(omicronalpha) protein and mRNA levels by modulating the transcriptional rate of G(omicronalpha) gene.

In rat astroglial cells, four G(omicronalpha) transcripts were found: G(omicron2alpha) mRNA (5.7 kb) and three G(omicron1alpha) mRNAs (4.0, 3.0 and 2.3 kb). However, G(omicron2alpha) but little G(omicronalpha1) proteins were present in membrane-enriched fractions. Culturing astroglial cells with forskolin (10 microM) or isoproterenol (10 microM) a beta-adrenergic agonist increased transiently in a time-dependent manner the levels of G(omicronalpha) proteins. The degradation rate of G(omicronalpha) proteins was slightly decreased by the cAMP treatment. In parallel, forskolin (10 microM) treatment increased transiently the amounts of both G(omicron1alpha) and G(omicron2alpha) mRNAs. The relative transcription rate of G(omicronalpha) gene was increased by 1.7-fold in forskolin-treated cells whereas the half-lives of G(omicron1alpha) and G(omicron2alpha) mRNAs were not significantly changed. These results suggest that cAMP regulates the transcription rate of G(omicronalpha) gene and this is compatible with the existence of a cAMP responsive element in the promoter of the G(omicronalpha) gene.

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

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.

Sodium saccharin inhibits adenylyl cyclase activity in non-taste cells.

We have studied the in vitro effect of sodium saccharin (NaSacch) on the rat adipocyte adenylyl cyclase complex. NaSacch (2.5-50 mM) inhibited significantly in a dose-dependent manner basal and isoproterenol-stimulated cAMP accumulation on isolated rat adipocytes. Similarly, NaSacch (2.5-50 mM) inhibited forskolin-stimulated adenylyl cyclase activity measured in the presence of Mg(2+)-ATP on adipocyte, astrocyte and thyrocyte membrane fractions. In contrast, NaSacch did not inhibit but slightly increased the forskolin-stimulated adenylyl cyclase activity measured in the presence of Mn(2+)-ATP and GDP beta S, a stable GDP analogue. The effect of NaSacch was not mediated through either the A1-adenosine receptor (A1R) or the alpha 2-adrenergic receptor (alpha 2AR). The inhibitory effect of NaSacch was additive to that of A1R agonist and was not blocked by the addition of the alpha 2AR antagonist RX 821002. Pretreatment of adipocytes with pertussis toxin slightly attenuated but did not abolish the inhibitory effect of NaSacch on forskolin-stimulated adenylyl cyclase activity on membrane fractions. These data suggest that the inhibitory effect of NaSacch on forskolin stimulated-adenylyl cyclase in adipocytes does not imply only Gi protein but also other direct or indirect inhibitory pathway(s) which remain to be determined.

Long-term effect of forskolin on the activation of adenylyl cyclase in astrocytes.

Long-term (48-h) forskolin treatment of rat astroglial cells led to a slight decrease (30-40%) in the response to isoproterenol, vasoactive-intestinal peptide, guanyl 5'-(beta gamma-imido)diphosphate, guanosine 5'-O-(3-thiotriphosphate) [GTP(S)], and AIF4- in crude membrane fractions. In contrast, the acute stimulatory effect of forskolin was increased by 1.25-1.5-fold. These two opposite effects of forskolin were mediated by a cyclic AMP-dependent mechanism. No changes in Gs alpha, Gi alpha, or G beta protein levels could be determined by immunoblotting using specific antisera. No significant differences were observed in the ability of G proteins extracted from control and forskolin-treated cells to reconstitute a full adenylyl cyclase activity in membranes from S49 cyc- cells, lacking Gs alpha protein. Gs alpha proteins were detected in two pools of membranes, one in the heavy sucrose fractions and the other in light sucrose fractions. Forskolin treatment of the cells shifted Gs alpha protein toward the light-density membranes. We did not find any significant change in the distribution of adenylyl cyclase. In contrast to the decreased stimulation of adenylyl cyclase activity by agonists acting via Gs alpha, observed in the crude membrane fraction, the responses of adenylyl cyclase to forskolin as well as to GTP(S) were increased in the purified plasma membrane fractions. These results may indicate that sensitization of the catalyst appears to be the dominant component in the astroglial cell response to long-term treatment by forskolin.

Effects of sodium saccharin diet on fat-cell lipolysis: evidence for increased function of the adenylyl cyclase catalyst.

OBJECTIVE: To evaluate the effect of a 14 days' sodium saccharin (NaS) diet on lipolysis and cyclic-AMP accumulation in isolated rat white epididymal adipocytes. ANIMALS: Male Wistar rats (3 weeks old) were fed, for 14-days, ad libitum with a regular diet supplemented with or without 1-5% NaS dissolved in drinking water. MEASUREMENTS: Lipolysis and cAMP accumulation were assessed on isolated adipocytes. Adenylyl cyclase activities were measured on membrane fractions prepared from isolated adipocytes. The levels of Gs and Gi proteins were determined by Western blot analysis using specific antisera. RESULTS: Only high dietary NaS (5%) affected significantly the body growth and food consumption. Feeding rats with 2.5% of NaS increased both basal and isoproterenol-stimulated lipolysis and cAMP production. A 14-days diet of rats with 2.5% aspartame did not reproduce the effects of NaS on lipolysis and cAMP production. In fat-cell membranes of 2.5% NaS-treated rats, basal and stimulated-adenylyl cyclase activities were increased by 200% whatever the agonists used: GTP, GTP[S], [AIF4]-, isoproterenol or forskolin in the presence of Mg2+ or Mn2+ with or without GDP[S]. These effects cannot be explained by modifications of the expression of Gs and Gi proteins. The level of Gs alpha subunits was not affected by NaS treatment while the level of Gi alpha 1/2 was slightly increased. The stimulatory effect of NaS on adenylyl cyclase activity appears to be specific to adipocyte when compared with thyroid, brain or heart membrane fractions. CONCLUSION: Based on these data, and on the fact that cAMP regulates the lipolytic rate, we conclude that NaS diet increases lipolysis and cAMP formation in fat-cells by modifying the activity of the adenylyl cyclase catalyst(s).

12-O-tetradecanoyl-phorbol-13-acetate (TPA) counteracts the cAMP up-regulation of the expression of the stimulatory guanine nucleotide binding protein (Gs alpha) and Gs alpha messenger RNA in cultured pig thyroid cells.

In this study, we examined whether the protein kinase C (PKC) pathway could interfere with the regulation of Gs protein in porcine thyroid cells. The two days culture of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) (0.1 microM) alone neither affected adenylyl cyclase activity, nor the level of Gs alpha protein in membranes when compared with control cells. The co-addition of TPA with thyrotropin (TSH) (1 mU/ml) or forskolin (fk) (10 microM) in the culture medium, abolished the stimulatory effects of either agonists on the activation of adenylyl cyclase by fk or [AlF4]- and on the increase of Gs alpha protein. By contrast, TPA had effects neither on the Gi-dependent inhibition of adenylyl cyclase nor on Gi alpha proteins levels. The level of Gs alpha mRNA measured by Northern blot analysis was increased (200%) in TSH- or fk-treated cells and this increase was counteracted by TPA. The effects of TPA observed after 6-9 h of contact with cells were mimicked by mezerezin, a non-phorbol protein kinase C activator and blocked by bisindolylmaleimide a specific protein kinase C inhibitor (GF 109203X). These results suggest that the activation of the PKC pathway prevents the cAMP-dependent up-regulation of Gs alpha protein and Gs alpha mRNA expression.

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 mRNA 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 (Gs) in a well characterized astroglial cells culture was established. The culture of astroglial cells for 3-6 h with isoproterenol (10 microM) or forskolin (10 microM) (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 adenylycyclase activity decreased to the value observed in control cells. The same biphasic response of adenylylcyclase to isoproterenol (10 microM) plus GTP (10 microM) occurred in membrane fractions from cells cultured with forskolin, whereas a diminished 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 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 Gs alpha and the Gi alpha protein levels, while the level of G beta subunits was unaffected. We also identified Gs alpha 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 Gs alpha 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 Gs alpha mRNA level was threefold increased when cells were cultured for 2-6 h with 8-bromoadenosine 3':5'-cyclic monophosphate (10 microM), a permeant analogue of cAMP. These results indicate that cAMP induces a time-dependent increase of Gs alpha mRNA. The half-life of Gs alpha protein and Gs alpha mRNA were determined. Pulse-chase studies revealed that the decay of Gs alpha 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 Gs alpha protein.(ABSTRACT TRUNCATED AT 400 WORDS)