guanosine-triphosphate and Hypothyroidism

Hypothyroidism profoundly reduces the capacity of brown adipose tissue (BAT) to generate cAMP in response to adrenergic stimulation. Evidence obtained with isolated brown adipocytes suggests a postreceptor defect that offsets the hypothyroidism-induced increase in beta3-adrenergic receptors. The goal of the present studies was to identify the defect in the cAMP generation pathway for which we studied cAMP generation in isolated cells and purified BAT membranes from normal and hypothyroid rats. Studies with adenosine deaminase and the adenosine receptor-1 agonist r-phenyl isopropyl adenosine (R-PIA) show that hypothyroid cells are not more sensitive to adenosine (same EC50) but more inhibited by high concentrations of R-PIA. Pretreatment with pertussis toxin reduced the gap in cAMP generation between eu- and hypothyroid cells and the inhibition mediated by R-PIA, but did not normalize the cAMP response to forskolin in hypothyroid cells. Although purified euthyroid BAT membranes increased cAMP production with GTP concentrations up to submillimolar range, to plateau or slightly decrease at higher levels, hypothyroid membranes were weakly stimulated by low concentrations of GTP and markedly inhibited (>50%) at concentrations > or = 10(-4) M. When assayed at 0.3 mM ATP and 1 microM GTP, hypothyroid membranes actually generated more cAMP in response to forskolin, but this was reversed when GTP concentration was 1 mM. Immunoblotting studies showed no significant effects of hypothyroidism on the abundance of G(alpha)i or Gbeta subunits, and ADP ribosylation of G(alpha)i was only 45% increased in hypothyroidism in contrast to a 2.5-fold increase in hypothyroid white adipose tissue membranes from the same rats. Hypothyroid membranes also exhibited different kinetics regarding ATP, with higher cAMP generation at submillimolar concentrations but less at >1 mM ATP. Actually, at ATP concentrations >0.6 mM, cAMP generation was markedly inhibited in hypothyroid membranes. Fixing the concentration of free Mg++ in these experiments indicates that most of the inhibition seen in hypothyroid membranes is caused by ATP, whereas euthyroid membranes are more sensitive to changes in free Mg++. Ca++ +/- calmodulin did not stimulate adenylyl cyclase (AC) activity. On the contrary, AC activity was inhibited by Ca++ in a concentration-dependent manner, by as low as 100 nM free Ca++, and to greater extent in hypo- than in euthyroid membranes (maximal inhibition 60 vs. 25-30%). Our res

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclases; Adipose Tissue, Brown; Animals; Cell Membrane; Cyclic AMP; GTP-Binding Proteins; Guanosine Triphosphate; Hypothyroidism; Osmolar Concentration; Pertussis Toxin; Phenylisopropyladenosine; Rats; Rats, Sprague-Dawley; Reference Values; Virulence Factors, Bordetella

The effect of GTP concentration of forskolin-stimulated adenylyl cyclase activity was examined in synaptosomal membranes from 15-day-old rats that were hypothyroid owing to administration of propylthiouracil and a low-iodine diet to the mothers during pregnancy and suckling. In membranes from the forebrain hypothyroidism abolished the overall stimulatory effect of GTP, which was seen in the euthyroid case. In membranes from the hindbrain hypothyroidism had the opposite effect in that there was an enhancement of an overall stimulatory effect of GTP. It is suggested that these findings reflect changes during early development of the brain in the expression of various G-proteins and/or the expression of different isoforms of adenylyl cyclase.

Topics: Adenylyl Cyclases; Animals; Colforsin; Critical Period, Psychological; Female; Guanosine Triphosphate; Hypothyroidism; Pregnancy; Prosencephalon; Rats; Rats, Sprague-Dawley; Rhombencephalon; Synaptosomes

1. Synaptosomal membranes were isolated from rats made hypothyroid by treatment with propylthiouracil and a low iodine diet. 2. When assayed in the presence of 100 mM-Na+, inhibition of forskolin-stimulated adenylate cyclase by GTP was enhanced in membranes from hypothyroid animals. 3. Hypothyroidism also enhanced inhibition of adenylate cyclase by phenylisopropyladenosine (with 100 mM-Na+ and 10 microM-GTP present). 4. Hypothyroidism did not increase binding of the A1 adenosine receptor agonist phenylisopropyladenosine to synaptosomal membranes; rather, the maximum binding was slightly decreased without any change in the KD. 5. The effect of GTP in modifying the displacement of the antagonist [3H]diethylphenylxanthine from synaptosomal membranes by unlabelled phenylisopropyladenosine was more pronounced in the hypothyroid state. 6. These findings are consistent with hypothyroidism causing modification of the brain adenylate cyclase system at the level of the coupling protein Gi.

Topics: Adenosine; Adenylyl Cyclase Inhibitors; Animals; Brain; Colforsin; Enzyme Activation; GTP-Binding Proteins; Guanosine Triphosphate; Hypothyroidism; Phenylisopropyladenosine; Rats; Receptors, Purinergic; Sodium; Synaptosomes; Xanthines

In hepatocytes obtained from hypothyroid rats, phorbol myristate acetate (PMA) and vasopressin diminished the accumulation of cyclic AMP and the stimulation of ureagenesis induced by isoprenaline or glucagon without altering significantly the accumulation of cyclic AMP induced by forskolin. Pretreatment with PMA markedly reduced the stimulation of ureagenesis and the accumulation of cyclic AMP induced by isoprenaline or glucagon. In membranes from cells pretreated with PMA, the stimulation of adenylate cyclase induced by isoprenaline + GTP, glucagon + GTP or by Gpp[NH]p were clearly diminished as compared to the control, whereas forskolin-stimulated activity was not affected. The data indicate heterologous desensitization of adenylate cyclase. It was also observed that the homologous (García-Sáinz J.A. and Michel, B. (1987) Biochem. J. 246, 331-336) and this heterologous beta-adrenergic desensitizations were additive. Pertussis toxin treatment markedly reduced the heterologous desensitization of adenylate cyclase but not the homologous beta-adrenergic desensitization. It is concluded that the homologous and heterologous desensitizations involve different mechanisms. The homologous desensitization seems to occur at the receptor level, whereas the heterologous probably involves the guanine nucleotide-binding regulatory protein, Ns.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Colforsin; Cyclic AMP; Drug Interactions; Enzyme Activation; Female; Glucagon; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hypothyroidism; Isoproterenol; Liver; Pertussis Toxin; Propranolol; Protein Kinase C; Rats; Rats, Inbred Strains; Receptors, Adrenergic, beta; Tetradecanoylphorbol Acetate; Urea; Vasopressins; Virulence Factors, Bordetella

Adipocytes from hypothyroid rats have a decreased responsiveness to agents that activate adenylate cyclase, whereas cells from hyperthyroid rats have an increased responsiveness as compared to the controls. This is reflected in cyclic AMP accumulation as well as lipolysis. Administration of pertussis toxin to rats or its in vitro addition to adipocytes increased basal lipolysis and cyclic AMP accumulation as well as the response to norepinephrine or forskolin. The effects of thyroid status was not abolished by toxin treatment. Pertussis toxin-catalyzed ADP ribosylation of Ni was increased in adipocyte membranes from hypothyroid rats as compared to those from euthyroid rats. However, no change in sensitivity to N6-(phenylisopropyl)adenosine was observed. The data suggest that the amount of Ni might not be rate-limiting for the inhibitory action of adenosine. A consistent decrease in maximal lipolysis was observed in freshly isolated adipocytes from hypothyroid animals as compared to those from the controls. Such defective maximal lipolysis was not corrected by adenosine deaminase or in vivo administration of pertussis toxin. The relationship between cyclic AMP levels and lipolysis suggests that in fat cells from hypothyroid rats either the cyclic AMP-dependent protein kinase or the lipase activity itself may limit maximal lipolysis. There appears to be multiple effects of thyroid status on lipolysis involving factors other than those affecting adenylate cyclase activation.

Topics: Adenosine Deaminase; Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclases; Adipose Tissue; Animals; Colforsin; Cyclic AMP; Female; Glycerol; Guanosine Triphosphate; Hyperthyroidism; Hypothyroidism; Lipolysis; Norepinephrine; Pertussis Toxin; Phenylisopropyladenosine; Rats; Rats, Inbred Strains; Time Factors; Virulence Factors, Bordetella

Hypothyroidism is associated with an enhanced sensitivity of rat fat cells to the inhibitory action of adenosine and adenosine agonists. The sensitivity of the forskolin-stimulated cyclic AMP response of rat fat cells to the adenosine agonist N6-phenylisopropyladenosine is amplified 3-fold by hypothyroidism. Forskolin-stimulated adenylate cyclase activity is more sensitive to inhibition by this adenosine agonist in membranes of fat cells isolated from hypothyroid as compared to euthyroid rats. Hypothyroidism does not significantly alter the number of affinity of binding sites for N6-cyclohexyl[3H]adenosine or N6-phenylisopropyladenosine in membranes of rat fat cells. GTP-induced inhibition of forskolin-stimulated adenylate cyclase was markedly enhanced in the hypothyroid state, suggesting an alteration in the inhibitory regulatory component (Ni)-mediated control of adenylate cyclase. Incubating membranes with [alpha-32P]NAD+ and preactivated pertussis toxin results in the radiolabeling of two peptides with Mr = 40,000 and 41,000 as visualized in autoradiograms of polyacrylamide gels run in sodium dodecyl sulfate. The amount of label incorporated by pertussis toxin into these two peptides (putative subunits of Ni) per mg of protein of membrane is increased 2-3-fold in the hypothyroid state. The amount of the stimulatory regulatory component, Ns, in fat cell membranes is not altered by hypothyroidism (Malbon, C. C., Graziano, M. P., and Johnson, G. L. (1984) J. Biol. Chem. 259, 3254-3260). The amplified response of hypothyroid rat fat cells to the inhibitory action of adenosine appears to reflect a specific increase in the activity and abundance of Ni.

Topics: Adenosine; Adenylyl Cyclase Inhibitors; Adipose Tissue; Animals; Cell Membrane; Colforsin; Cyclic AMP; Diterpenes; Female; GTP-Binding Proteins; Guanosine Triphosphate; Hypothyroidism; Phenylisopropyladenosine; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Purinergic

Incubation of fat cell ghosts with activated cholera toxin, nucleoside triphosphate, cytosol, and NAD results in increased adenylate cyclase activity and the transfer of ADP-ribose to membrane proteins. The major ADP-ribose protein comigrates on sodium dodecyl sulfate-polyacrylamide gels with the putative GTP-binding protein of pigeon erythrocyte membranes (Mr 42 000), which is also ADP-ribosylated by cholera toxin. The treatment with cholera toxin enhances the stimulation of the fat cell membrane adenylate cyclase by GTP, but the stimulation by guanyl-5'-yl imidodiphosphate is unaltered. Subsequent stimulation of fat cell adenylate cyclase by 10 micrometers epinephrine is not particularly affected. These changes were qualititatively the same for membranes isolated from fat cells of hypothyroid rats. Although the cyclase of these membranes has a reduced response to epinephrine, guanyl-5'-yl imidodiphosphate or GTP, as compared to euthyroid rat fat cell membranes, the defect is not rectified by toxin treatment and cannot be explained by a deficiency in the cholera toxin target.

Topics: Adenosine Diphosphate Ribose; Adenylyl Cyclases; Adipose Tissue; Animals; Cell Membrane; Cholera Toxin; Enzyme Activation; Epinephrine; Female; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hypothyroidism; Membrane Proteins; Nucleoside Diphosphate Sugars; Rats