Adenylate cyclase inhibition by hormones

Adenosine Ri receptors and inhibitory guanine-nucleotide-regulatory components were solubilized from rat cerebral-cortical membranes with sodium cholate. (-)-N6-Phenylisopropyl[2,8-3H]adenosine [( 3H]PIA) binds with high affinity to the soluble receptors, which retain the pharmacological specificity of adenosine Ri receptors observed in membranes. The binding is regulated by bivalent cations and guanine nucleotides. Bivalent cations increase [3H]PIA binding by increasing both the affinity and the apparent number of receptors. Guanine nucleotides decrease agonist binding by increasing the dissociation of the ligand-receptor complex. Adenosine agonists stabilize the high-affinity form of the soluble receptor. The hydrodynamic properties of the adenosine receptor were determined with cholate extracts of membranes that were treated with [3H]PIA. Sucrose-gradient-centrifugation analysis indicates that the receptor has a sedimentation coefficient of 7.7 S. The receptor is eluted from Sepharose 6B columns with an apparent Stokes radius of 7.2 nm. Labelling of either sucrose-gradient or gel-filtration-column fractions with pertussis toxin and [32P]-NAD+ reveals that both the 41,000- and 39,000-Mr substrates overlap with the receptor activity. These studies suggest that the high-affinity adenosine-receptor-binding activity in the cholate extract represents a stable R1-N complex.

Section: Introductionmentioning

confidence: 99%

Hydrodynamic properties of adenosine Ri receptors solubilized from rat cerebral-cortical membranes

Yeung

Perez‐Reyes

Cooper

1987

“…It has also been known for a considerable time that bivalent cations, most effectively Mn2+, but also Mg2+ at higher concentrations, uncouple hormonal inhibition of adenylate cyclase (Cooper et al, 1979;Hoffman et al, 1981). Bockaert et al (1984) have proposed that inhibitory ligands act to decrease the affinity of the catalytic unit for Mg2+, whereas stimulatory ligands increase this affinity. It is unlikely that all of the reported effects of Mg2+ are mediated through one site.…”

mentioning

confidence: 99%

Magnesium ion exerts a central role in the regulation of inhibitory adenosine receptors

Yeung¹,

Fossom²,

Gill³

et al. 1985

Guanine nucleotides and Mg2+ differentially regulate agonist binding to adenosine (Ri) receptors in fat-cell plasma membranes. GTP alone decreases binding of the agonist ligand [3H]N6-cyclohexyladenosine (CHA) by increasing the dissociation constant (Kd). Mg2+ alone also decreases [3H]CHA binding, which is associated with a decrease in the number of receptors and in the dissociation constant. In the presence of Mg2+, the effect of GTP is to increase [3H]CHA binding by increasing the total number of receptors. It thus appears that Mg2+ acts specifically at a bivalent-cation site which, with GTP, regulates agonist binding. This putative Mg site is highly sensitive to alkylating agents. Mild treatment with N-ethylmaleimide (NEM) abolishes the characteristic GTP effect on agonist binding in the presence of Mg2+. In addition, the effect of Mg2+ alone is also eliminated. The effect of GTP alone is largely unaltered. Studies of the adenylate cyclase activity indicate that this NEM treatment also abolishes the inhibition of basal activity by adenosine analogues, whereas guanylyl imidodiphosphate inhibition of forskolin-stimulated activity is only slightly impaired at this NEM concentration. These observations indicate that a Mg2+ 'site' or 'component' is required for the integration of receptor (Ri) occupancy with regulation of catalytic activity (C). The regulatory role of Mg2+ is more demonstrable in receptor-GTP-regulatory-protein (Ri-Ni) interactions than in GTP-regulatory-protein-catalytic-unit (Ni-C) interactions.

“…The hormone-sensitive adenylate cyclase system is composed of several interacting subunits embedded in the lipid bilayer of the plasma membrane: stimulatory and inhibitory hormone receptors, acting as external discriminators, the adenylate cyclase catalytic moiety, catalysing the formation of cyclic AMP from MgATP, and two guanine nucleotide-binding regulatory components, N. and Ni, transducing the signal from the hormone-activated stimulatory and inhibitory receptors respectively to the adenylate cyclase (Gilman, 1984;Birnbaumer et al, 1985). Modulation of adenylate cyclase activity by hormone-and guanine nucleotideactivated Ns and Ni is associated with changes in the affinity of the adenylate cyclase for its activating cation Mg2+ (Cech et al, 1980;Iyengar & Birnbaumer, 1982;Jakobs et al, 1983;Bockaert et al, 1984). However, a change in the affinity of the adenylate cyclase for its substrate, MgATP, by hormone-and guanine nucleotideactivated Ns and Ni has not been described so far.…”

Section: Introductionmentioning

confidence: 99%

Forskolin sensitizes human platelet adenylate cyclase to modulation of substrate (MgATP) affinity by hormones

Watanabe¹,

Jakobs²

1986

Stimulation of human platelet adenylate cyclase by the diterpene forskolin is associated with a decrease in the apparent substrate (MgATP) affinity of the enzyme. Addition of the stimulatory hormone prostaglandin E1 not only further increased the Vmax. of the forskolin-stimulated platelet adenylate cyclase but also caused a further increase in the Km value for MgATP, by up to 20-fold compared with basal conditions. On the other hand, the inhibitory hormone adrenaline decreased not only the Vmax. but also the Km value of the platelet adenylate cyclase stimulated by forskolin, with or without prostaglandin E1 present. The data indicate that forskolin sensitizes human platelet adenylate cyclase to modulation of substrate (MgATP) affinity by hormones, but there is no such effect in the absence of the diterpene.