guanosine-triphosphate and 1-3-diethyl-8-phenylxanthine

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

Extracellular ATP and other purinergic agonists were found to inhibit cAMP accumulation by depressing adenylate cyclase as an "inhibitory action" and/or to stimulate arachidonate release in association with phospholipase C or A2 activation and Ca2+ mobilization as "stimulatory actions" in FRTL-5 cells. The stimulatory actions of a group of P2-agonists represented by ATP were partially inhibited by the pretreatment of the cells with islet-activating protein (IAP), pertussis toxin, even when an about 41-kDa membrane protein(s) was completely ADP-ribosylated. Only the IAP-sensitive part of the stimulatory actions was antagonized by 1,3-diethyl-8-phenylxanthine (DPX), an adenosine antagonist. GTP and 8-bromoadenosine 5'-triphosphate (Br-ATP) at two to three orders of higher concentrations than ATP also exerted the stimulatory actions, although they were entirely insensitive to both IAP and DPX. Ligand binding experiments with, [35S]ATP gamma S and [3H]DPX showed that ATP occupies both DPX-sensitive and insensitive receptor sites, whereas GTP does only ATP-displaceable DPX-insensitive sites. Thus, lack of sensitivity of GTP action to DPX was associated with its inability to occupy the DPX-sensitive sites. Adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S), adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) and P1-agonists such as AMP and N6-(L-2-phenylisopropyl-adenosine (PIA) did not show any stimulatory action. Nevertheless, the agonists remarkably enhanced the stimulatory actions of GTP or Br-ATP. Such permissive actions of PIA and others were sensitive to both IAP and DPX, as were shown for a part of the stimulatory actions of ATP as well as the "inhibitory actions" of both PIA and ATP. We conclude that an IAP substrate G-protein(s) which mediates the inhibitory action of purinergic agonists via a DPX-sensitive purinergic receptor(s) may not directly link to the phospholipase C or A2 system but enhance the system which links to a DPX-insensitive P2-receptor, in an indirect or permissive manner.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Adenylate Cyclase Toxin; Animals; Arachidonic Acid; Arachidonic Acids; Calcium; Cell Line; Cyclic AMP; GTP-Binding Proteins; Guanosine Triphosphate; Pertussis Toxin; Phenylisopropyladenosine; Phosphatidylinositols; Rats; Receptors, Purinergic; Signal Transduction; Thyroid Gland; Thyrotropin; Virulence Factors, Bordetella; Xanthines

A1 adenosine receptors from rat brain membranes were solubilized with the zwitterionic detergent 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized receptors retained all the characteristics of membrane-bound A1 adenosine receptors. A high and a low agonist affinity state for the radiolabelled agonist (R)-N6-[3H]phenylisopropyladenosine([3H]PIA) with KD values of 0.3 and 12 nM, respectively, were detected. High-affinity agonist binding was regulated by guanine nucleotides. In addition agonist binding was still modulated by divalent cations. The solubilized A1 adenosine receptors could be labelled not only with the agonist [3H]PIA but also with the antagonist 1,3-diethyl-8-[3H]phenylxanthine. Guanine nucleotides did not affect antagonist binding as reported for membrane-bound receptors. These results suggest that the solubilized receptors are still coupled to the guanine nucleotide binding protein Ni and that all regulatory functions are retained on solubilization.

Topics: Animals; Binding, Competitive; Brain Chemistry; Calcium; Cations, Divalent; Cell Membrane; GTP Phosphohydrolases; Guanosine Triphosphate; Kinetics; Magnesium; Manganese; Phenylisopropyladenosine; Rats; Receptors, Cell Surface; Receptors, Purinergic; Xanthines

The binding of agonists and antagonists to Ri adenosine receptors of synaptosomal membranes from rat and bovine brain was studied. The effects of guanine nucleotides and temperature were analyzed with the aid of computerized curve fitting. Evidence is presented for two different states of the receptor: one of high and one of low affinity for agonists. Antagonists bind to both states with the same affinity. The two states are characterized by saturation, competition, and kinetic experiments with very similar results. Guanine nucleotides cause transition of the high- to the low-affinity state. The ratio of the KD values for the two affinity states is 90-150 in rat brain but only 10 in bovine brain. The proportions of the two affinity states are the same for all agonists tested; in the absence of exogenous guanine nucleotides, 75% of the total receptor population is in the high-affinity state, whereas in the presence of guanine nucleotides only 5% remain in the high-affinity state. Binding of antagonists to the receptor is enthalpy-driven whereas binding of the agonist (-)-N6-phenylisopropyladenosine to the high-affinity state of the receptor is entropy-driven. Binding of the agonist to the low-affinity state is enthalpy-driven and thus similar to the binding of antagonists. Our data indicate that guanine nucleotides convert the Ri adenosine receptor from a high- to a low-agonist affinity state and that agonist binding shows thermodynamic differences from antagonist binding only when it is to the high-affinity state of the receptor.

Topics: Adenosine; Animals; Brain; Cattle; Guanosine Triphosphate; Intracellular Membranes; Kinetics; Male; Phenylisopropyladenosine; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Purinergic; Species Specificity; Synaptosomes; Thermodynamics; Xanthines

The effects of barbiturates on radioligand binding to inhibitory Ri adenosine receptors of rat brain membranes were investigated. Binding of the adenosine receptor agonist (-)N6-phenylisopropyl[3H]adenosine and the antagonist 1,3-diethyl-8-[3H]phenylxanthine was inhibited by several barbiturates. This inhibition was concentration-dependent and occurred in the range of pharmacologically effective concentrations. Pentobarbital was the most potent of the barbiturates tested with a Ki of 92 mumol/l. The (+)isomers of hexobarbital and mephobarbital were more potent than the respective (-)isomers. Barbituric acid itself did not displace either radioligand in concentrations up to 1 mmol/l. The inhibitory effect of pentobarbital was reversed by a single wash of membranes preincubated with the barbiturate. The presence of pentobarbital caused a decrease of the affinity of the receptor for the antagonist radioligand but did not alter the number of binding sites, suggesting a competitive antagonism. The effects of pentobarbital on radioligand binding to the receptor were not changed by the presence of picrotoxinin nor by the absence of chloride ions. This indicates that they are not mediated via the picrotoxinin binding site. The barbiturates could not be classified as either agonists or antagonists at the Ri adenosine receptor. The presence of GTP did not influence the inhibition of radioligand binding by pentobarbital; this is also observed for antagonists, whereas the affinity of agonists is markedly reduced by GTP. Binding of antagonists to the receptor is enthalpy-driven; the interaction of pentobarbital with the receptor was entropy-driven and the same was true for agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Animals; Barbiturates; Binding, Competitive; Brain Chemistry; Drug Interactions; Guanosine Triphosphate; In Vitro Techniques; Kinetics; Male; Phenylisopropyladenosine; Picrotoxin; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Purinergic; Sesterterpenes; Synaptosomes; Xanthines

Previous work from this laboratory showed that rat hippocampal membranes contain adenosine receptors that mediate GTP-dependent inhibition of adenylate cyclase activity (Yeung, S. -M. H., and R. D. Green (1983) J. Biol. Chem. 258: 2334-2339). Furthermore, we reported that guanine nucleotides decrease agonist and increase antagonist binding to these adenosine receptors. The present study examines the effects of monovalent cations and guanine nucleotides, alone and in combination, on the binding of agonist [( 3H]N6(L-phenylisopropyl)adenosine) and antagonist [( 3H]diethylphenylxanthine) radioligands to adenosine receptors in rat hippocampal membranes. Low concentrations of monovalent cations (less than or equal to 100 mM) did not affect agonist binding. 5'-Guanylylimidodiphosphate (Gpp(NH)p) alone increased the KD of the agonist without affecting the maximal number of sites labeled by the agonist (Bmax); in the presence of monovalent cations, Gpp(NH)p both increased the KD and decreased the number of sites labeled by the agonist. In contradistinction, Gpp(NH)p increased the maximal number of sites to which the antagonist bound without affecting its KD, while monovalent cations decreased the KD of the antagonist both in the absence and the presence of Gpp(NH)p. It is proposed that both agonist and antagonist-receptor complexes exist in three distinct affinity states and that the transitions between these states are modulated by guanine nucleotides and monovalent cations.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Animals; Cations, Monovalent; Female; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hippocampus; Male; Phenylisopropyladenosine; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Purinergic; Xanthines