p38 mitogen-activated protein kinase contributes to cell cycle regulation by cAMP in FRTL-5 thyroid cells.

OBJECTIVE: Thyrotropin activates the cAMP pathway in thyroid cells, and stimulates cell cycle progression in cooperation with insulin or insulin-like growth factor-I. Because p38 mitogen-activated protein kinases (p38 MAPKs) were stimulated by cAMP in the FRTL-5 rat thyroid cell line, we investigated (i) the effect of the specific inhibition of p38 MAPKs on FRTL-5 cell proliferation and (ii) the mechanism of action of p38 MAPKs on cell cycle control, by studying the expression and/or the activity of several cell cycle regulatory proteins in FRTL-5 cells. METHODS: DNA synthesis was monitored by incorporation of [(3)H]thymidine into DNA and the cell cycle distribution was assessed by fluorescence-activated cell sorter analysis. Expression of cell cycle regulatory proteins was determined by Western blot analysis. Cyclin-dependent kinase 2 (Cdk2) activity associated to cyclin E was immunoprecipitated and was measured by an in vitro kinase assay. RESULTS: SB203580, an inhibitor of alpha and beta isoforms of p38 MAPKs, but not its inactive analog SB202474, inhibited DNA synthesis and the G1-S transition induced by forskolin plus insulin. SB203580 inhibited specifically p38 MAPK activity but not other kinase activities such as Akt and p70-S6 kinase. Treatment of FRTL-5 cells with SB203580 decreased total and cyclin E-associated Cdk2 kinase activity stimulated with forskolin and insulin. However, inhibition of p38 MAPKs by SB203580 was without effect on total cyclin E and Cdk2 levels. The decrease in Cdk2 kinase activity caused by SB203580 treatment was not due to an increased expression of p21(Cip1) or p27(Kip1) inhibitory proteins. In addition, SB203580 affected neither Cdc25A phosphatase expression nor Cdk2 Tyr-15 phosphorylation. Inhibition of p38 MAPKs decreased Cdk2-cyclin E activation by regulating the subcellular localization of Cdk2 and its phosphorylation on Thr-160. CONCLUSIONS: These results indicate that p38 MAPK activity is involved in the regulation of cell cycle progression in FRTL-5 thyroid cells, at least in part by increasing nuclear Cdk2 activity.

Accumulation of cyclic AMP in Swiss 3T3 cells adhering to a cellulose biomaterial substratum through interaction with adenylyl cyclase.

Controlling cell shape induced by cell-substrata interaction appears of prime importance to influence subsequent biological processes such as cell migration, proliferation, differentiation or apoptosis. Studies on Swiss 3T3 fibroblasts have recently provided evidence that cell spreading is mediated by integrins and RhoA. Our previous studies showed that on Cuprophan, a cellulose membrane (CU) to which vitronectin adhesive protein is poorly adsorbed, Swiss 3T3 cells are rounded and undergo cAMP-dependent aggregation. In contrast, on a polyacrylonitrile membrane (AN69) that favours the adsorption of vitronectin and fibronectin, cells spread out and contain low concentrations of cAM P. We have now examined the parts played by the three components in the cAMP pathway (receptors, G-proteins and adenylyl cyclase itself) in cAM P-dependent cell aggregation on CU. Experiments with intact cells showed no interaction between the CU and receptors, or between the CU and G-proteins. Assays on membrane preparations plus the Mn-ATP substrate, which uncouples G-proteins and adenylyl cyclase, demonstrated that activation of the cAMP pathway by CU depends primarily on the catalytic activity of the adenylyl cyclase. These investigations provide essential data for the development of biomaterials that favour cell functionality.

The thyrotropin receptor is not involved in the activation of p42/p44 mitogen-activated protein kinases by thyrotropin preparations in Chinese hamster ovary cells expressing the human thyrotropin receptor.

We studied whether bovine pituitary thyrotropin (bTSH) or human recombinant thyrotropin (rhTSH) stimulated p42/p44 mitogen-activated protein kinases (MAPKs) in Chinese hamster ovary cells expressing human thyrotropin receptor (CHO-hTSHR cells). We show that p42/p44 MAPK phosphorylation was induced by both TSH preparations at similar levels in CHO-hTSHR cells and in wild-type CHO cells. In contrast, cyclic adenosine monophosphate (cAMP) production was stimulated by TSH only in CHO-hTSHR cells, demonstrating that p42/p44 MAPK stimulation was independent of the TSH receptor. Moreover, similar results were obtained with two other cell lines: the FRTL-5 thyroid cell line and the CCL39 fibroblast cell line. Maximal stimulation of p42/p44 MAPK phosphorylation was observed after a 5- to 10-minute incubation with bTSH and rhTSH preparations. At this time, the phosphorylation of GST-Elk1 was also increased in a time- and concentration-dependent manner by bTSH preparations. The phosphorylation of p42/p44 MAPKs was abolished by PD 98059 and GF 109203X, indicating the involvement of MAPK kinases (MEK 1/2) and protein kinase C. In contrast, the activation of p42/p44 MAPKs was insensitive to H89, to cholera toxin and to pertussis toxin. These data suggest that the protein kinase A pathway was not implicated in p42/p44 MAPK activation by TSH preparations. Moreover, Gs or Gi/Go proteins do not appear to participate in p42/p44 MAPK activation. We also showed that these TSH preparations failed to induce activation of c-Jun NH2 terminal kinase. We therefore conclude that the commercial TSH preparations used in this study contained factor(s) responsible for the specific activation of p42/p44 MAPKs by a TSH receptor-independent mechanism.

Thyroid-stimulating hormone and cyclic AMP activate p38 mitogen-activated protein kinase cascade. Involvement of protein kinase A, rac1, and reactive oxygen species.

p38 mitogen-activated protein kinases (p38-MAPKs) are activated by cytokines, cellular stresses, growth factors, and hormones. We show here that p38-MAPKs are activated upon stimulation by thyroid-stimulating hormone (TSH) or cAMP. TSH caused the phosphorylation of p38-MAPK in Chinese hamster ovary cells stably transfected with the human TSH receptor but not in wild-type Chinese hamster ovary cells. The effect of TSH was fully mimicked by the adenylyl cyclase activator, forskolin, and by a permeant analog of cAMP. The effect of forskolin was reproduced in FRTL5 rat thyroid cells. TSH also stimulated the phosphorylation of MAPK kinase 3 or 6, over the same time scale as that of p38-MAPKs. TSH and forskolin stimulated the activity of the alpha-isoform of p38-MAPK assayed by phosphorylation of the transcription factor ATF2. The activity of MAPK-activated protein kinase-2 was stimulated by TSH and forskolin. This stimulation was abolished by SB203580, a specific inhibitor of p38-MAPKs. The protein kinase A inhibitor H89 inhibited the stimulation of phosphorylation of p38-MAPKs by forskolin, whereas inhibitors of protein kinase C, p70(S6k), and phosphatidylinositol 3-kinase were ineffective. Expression of the dominant negative form of Rac1, but not that of Ras, blocked forskolin-induced p38-MAPK activation. Diphenylene iodonium, a potent inhibitor of NADPH oxidase(s), and ascorbic acid, an effective free radical scavenger, suppressed TSH- or forskolin-stimulated p38-MAPK phosphorylation, indicating that the generation of reactive oxygen species plays a key role in signaling from cAMP to p38-MAPKs. Inhibition of the p38-MAPK pathway with SB203580 partially but significantly, attenuates cAMP- and TSH-induced expression of the sodium iodide symporter in FRTL-5 cells. These results point to a new signaling pathway for the G(s)-coupled TSH receptor, involving cAMP, protein kinase A, Rac1, and reactive oxygen species and resulting in the activation of a signaling kinase cascade that includes MAPK kinase 3 or 6, p38-MAPK, and MAPK-activated protein kinase-2.

Modification of adipocyte membrane adenylyl cyclase activity by NAD: evidence against NAD-induced endogenous ADP-ribosylation of Gsalpha protein.

Treatment of saponin-permeabilized adipocytes with NAD enhanced adenylyl cyclase activity stimulated by GTgammaS, [Al/F(4)](-), isoproterenol, and forskolin in membrane fractions and potentiated isoproterenol-induced cAMP accumulation in whole cells. In parallel, when permeabilized adipocytes were incubated with [(32)P]NAD, there was significant incorporation of [(32)P]ADP-ribose in a 44-kDa acceptor membrane protein. This reaction was inhibited by l-arginine and was enhanced by the addition of GTPgammaS. Surprisingly, this 44-kDa protein could not be identified as Gs protein: (1) It was not recognized by Gsalpha specific antibody; (2) it did not comigrate with the major cholera toxin substrates in either 10% SDS-PAGE or two-dimensional electrophoresis; (3) a pretreatment of adipocytes with NAD did not decrease cholera toxin-mediated ADP-ribosylation of Gsalpha proteins on membrane fractions. Our results indicate that NAD did not induce endogenous ADP-ribosylation of Gsalpha in permeabilized rat adipocytes but nonetheless modified the adenylyl cyclase response.

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)

Modification of the amounts of G proteins and of the activity of adenylyl cyclase in human benign thyroid tumours.

The possibility that a TSH post-receptor-binding defect is responsible for the pathogenesis of benign thyroid tumours was studied. Thus, we attempted to determine in hyperfunctioning (hot) nodules and non-functioning (cold) nodules whether the functional activity or the amount of G proteins were modified in comparison with surrounding normal tissues. The adenylyl cyclase response to agonists that bypass the TSH-receptor complex (forskolin, guanosine 5'- (beta.gamma-imido)triphosphate (Gpp(NH)p) or [AIF4]-) was studied on membranes from tumorous and adjacent normal thyroid tissues. We also examined the ability of G proteins to be ADP-ribosylated by cholera toxin (CT) or pertussis toxin (PT), and quantified G proteins by Western blot analysis with specific antisera directed against Gs alpha and Gi alpha subunits. Basal adenylyl cyclase activity was unchanged in hot tumours compared with normal tissue whereas the stimulation of adenylyl cyclase by Gpp(NH)p or [A1F4]- (which act directly on Gs) as well as by forskolin (which acts on the catalyst) was significantly (P less than 0.05) decreased in five of seven nodules studied. Two types of response were found in cold nodules, depending upon whether they were microfollicular or macrofollicular tumours. Basal as well as stimulated adenylyl cyclase activity was increased (0.02 less than P less than 0.05) in microfollicular tumours. In contrast, in macrofollicular tumours basal adenylyl cyclase was unchanged whereas stimulated adenylyl cyclase activity was decreased (0.02 less than P less than 0.05). The ability of Gs or Gi to be ADP-ribosylated by CT or PT respectively was maintained in tumorous tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of glycosyl inositol-phosphate on cAMP production in isolated rat fat-cells is transduced by a pertussis toxin sensitive G-protein.

In fat-cells, insulin increases synergistically the inhibitory effects of adenosine and prostaglandin E2 on adenylyl cyclase activity. When the endogenous production of these feedback inhibitors is suppressed, insulin is no more active in conditions where glycosyl inositol-phosphate which is a putative mediator of its action, is always efficient. Moreover, glycosyl inositol-phosphate signal is transduced by a G protein sensitive to IAP intoxication.

Alteration of the functional activity of Gs protein in thyrotropin-desensitized pig thyroid cells.

Changes in the sensitivity of adenylyl cyclase observed in pig thyroid cells cultured 2 days in the presence of thyroid-stimulating hormone (TSH) or forskolin were assessed by examining the properties of Gs protein. Chronic treatment of thyroid cells with various concentrations of TSH (0.01-1 mU/ml) or forskolin (0.1-10 microM) increased the response of adenylyl cyclase to a further stimulation by forskolin or NaF + AlCl3 ([AlF4]-). In contrast, the enzyme activation promoted by guanosine 5'-(beta,gamma-imido) triphosphate (Gpp(NH)p) was markedly affected. There was a significant increase in adenylyl cyclase activation by Gpp(NH)p in membranes from cells treated with low concentrations of TSH (less than or equal to 0.1 mU/ml) or forskolin (less than or equal to 1 microM) but a significant decrease in membranes from cells cultured with a higher concentration of TSH (1 mU/ml) or forskolin (10 microM). This decrease in Gpp(NH)p-stimulated adenylyl cyclase activity was mimicked by 8-bromo-cAMP but not by 1,9-dideoxyforskolin, a forskolin analogue which has lost its ability to activate adenylyl cyclase. There was a good correlation with the ability of Gs protein to be ADP-ribosylated by cholera toxin: labeling of Gs protein decreased following chronic treatment of thyroid cells with TSH (1 mU/ml) or forskolin (10 microM). In contrast, under the same experimental culture conditions a slight but significant increase in the quantity of Gs subunits was observed by immunoblotting analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of pertussis toxin on the erythropoietin-stimulated proliferation and differentiation of erythroid-responsive cells.

Erythropoietin (Ep) stimulates the proliferation and differentiation of erythroid progenitor cells. We have investigated the possibility that guanyl nucleotide regulatory (G) proteins may be involved in Ep signal transduction. Pertussis toxin (PT) was found to inhibit Ep-stimulated [3H]-thymidine incorporation and large erythroid colony formation in a dose-dependent fashion, but had no effect on small colony formation or on Ep-induced differentiation. PT specifically adenosine diphosphate-ribosylated a major protein with an apparent molecular mass of 41 kD in erythroid progenitor cell membranes. These findings indicate that the transduction of the Ep signal for proliferation could be mediated in the early steps by a PT-sensitive G protein.

Cyclic AMP regulation of Gs protein. Thyrotropin and forskolin increase the quantity of stimulatory guanine nucleotide-binding proteins in cultured thyroid follicles.

This study was carried out to clarify the way in which thyrotropin (TSH) and forskolin regulate the adenylylcyclase complex in thyroid follicle cells. We examined the effects of chronic treatment of pig thyroid follicles with TSH or forskolin on the state of G proteins by (a) assaying adenylylcyclase activity, (b) analyzing the ADP-ribosylation of stimulatory G protein (Gs) by cholera toxin, and (c) quantifying the Gs subunits by Western blotting with antipeptide antibodies. Chronic exposure (18 h) of thyroid follicles to a low concentration of TSH (0.01-0.1 milliunit/ml) enhanced the subsequent response of adenylylcyclase to TSH. Higher concentration of TSH (1 milliunit/ml) induced a homologous desensitization of this response. In cells pretreated with forskolin, the TSH-stimulated adenylylcyclase activity was higher than in control cells. The forskolin-or guanosine 5'-(beta, gamma-imido) triphosphate (Gpp(NH)p)-stimulated adenylylcyclase activity was always significantly increased after chronic treatment of cells with TSH or forskolin. Treatment of cultured thyroid follicle membranes with [32P]NAD and cholera toxin resulted in labeling of the Gs alpha (45-52-kDa) component. Culturing follicles with TSH (0.001-1 milliunit/ml) or forskolin (0.01-10 microM) greatly affected the cholera toxin-mediated ADP-ribosylation of the Gs alpha subunit. Gs alpha labeling increased progressively to level off at 1 milliunit/ml TSH or 1 microM forskolin (150-200%). Gs alpha immunoreactivity was increased in parallel (200-300%). The immunoreactivity of G beta subunits in cells cultured with TSH or forskolin was also increased compared with control cells. Cycloheximide abolished the effects of TSH and forskolin on the follicles, suggesting that new protein synthesis is required. These results indicate that Gs protein subunits are up-regulated by TSH and forskolin and suggest that their synthesis in thyroid cells is mediated, at least in part, by a cyclic AMP-dependent mechanism.

Antibodies directed against transducin beta subunits interfere with the regulation of adenylate cyclase activity in brain membranes.

In an attempt to study the mechanisms of action of membrane-bound adenylate cyclase, we have applied to rat brain synaptosomal membranes antibodies raised against purified bovine transducin (T) beta gamma subunits. The antibodies recognized one 36-kDa protein in Western blots of the membranes. Adenylate cyclase activation by GTP non-hydrolyzable analogues was greatly decreased in immune, as compared to preimmune, antibody-treated membranes, whereas the enzyme basal activity was unaffected by both types of antibodies. The inhibition of forskolin-stimulated adenylate cyclase by guanine 5'-(beta, gamma-imino)triphosphate (Gpp-(NH)p) was decreased in membranes preincubated with immune, but not preimmune, antibodies. Anti-T beta antibodies moderately decreased the extent of subsequent adenylate cyclase activation by forskolin, while not affecting activation by Al3+/F-. The enzyme activation by Gpp(NH)p in untreated membranes remained the same upon further incubation in the presence of either type of antibodies. Such results were consistent with the decreased exchange of guanine nucleotides which occurred in membrane treated with immune, but not preimmune antibodies, upon addition of GTP. The blockade of the regulation of adenylate cyclase by Gpp(NH)p observed in membranes pretreated by anti-T beta antibodies thus appears to be caused by the impairment of the guanine nucleotide exchange occurring on Gs alpha subunits. The G beta subunits in the adenylate cyclase complex seem to be instrumental in the guanine nucleotide exchange on G alpha subunits, just as T beta subunits are in the transducin complex.

Endogenous ADP-ribosylation of Gs subunit and autonomous regulation of adenylate cyclase.

Although cholera toxin induces a marked stimulation of adenylate cyclase activity in rat adipocyte plasma membranes, the holotoxin induces only a slight increase of cyclic AMP accumulation in intact cells. A similar apparent anomaly is seen with pertussis toxin, which has been shown to inhibit the Gi subunit of adenylate cyclase, and has a greater effect on cAMP accumulation and lipolysis than the activation by cholera toxin of the Gs subunit. To understand better the way in which these bacterial toxins are modifying the adipocyte cells, we prepared adipocyte plasma membranes and submitted them to ADP-ribosylation by cholera and pertussis toxins. During the incubation of control cells, we found endogenous ADP-ribosylation of Gs as a result of sustained stimulation of Gi by adenosine. Our results point to a possible homoeostatic system in which the autonomous adjustment of the basal activity of Gs as a function of that of Gi, under the control of feedback inhibitory ligands, ensures a steady production of cAMP within the cell.

Autophosphorylation of the insulin receptor in rat adipocytes is modulated by thyroid hormone status.

The effect of thyroid status on the in vitro autophosphorylation of the insulin receptors was studied in triton-solubilized adipocyte plasma membranes obtained from normal and thyroidectomized rats. Thyroidectomy results in an increase (two to three times) of the in vitro insulin-dependent phosphorylation of the insulin beta-subunit receptor. Phosphorylation occurred on tyrosine residues. In vivo injection of triiodothyronine to thyroidectomized rats restored plasma membranes autophosphorylation of the beta-subunit to the values obtained for control euthyroid rats. This effect was independent of the number and affinity of the insulin receptors, which were not modified regardless of thyroid status.