Effect Of Gpp Research Articles

A Possible Mechanism in Interaction of a Partial Agonist with β-Adrenoceptor in Guinea-Pig Taenia Caecum: Effects of Gpp(NH)p on Its Two Different Binding Sites

The mechanisms of the actions of the β-adrenergic partial agonist (befunolol) were studied in isolated guinea-pig taenia caecum. Befunolol, 2-acetyl-7-(2-hydroxy-3-isopropylaminopropoxy)benzofuran hydrochloride was found to be a typical partial agonist in guinea-pig taenia caecum. The pD2-value of befunolol was in agreement with its pKA-value obtained with photoaffinity labeling, but was different from its pA2-value against isoprenaline and pKi-value obtained from the inhibition of specific [3H]-dihydroalprenolol binding. The Scatchard plot of the specific [3H]-befunolol binding showed two affinity sites of the receptor in the absence of Gpp(NH)p, but the low affinity site was reduced while the high affinity site was not affected in the presence of Gpp(NH)p. The pKD-value of the high affinity site of befunolol was in agreement with its pA2-value, and the pKD-value of the low affinity site was in agreement with its pD2-value or pKA-value. These results suggest that the β-adrenergic partial agonist may interact with two different sites: an agonist binding site and an antagonist binding site.

Forskolin as a probe of adenylate cyclase of adipocytes

New information regarding the relationship between the agonist receptor and the regulatory and catalytic components of adenylate cyclase has been obtained by applying forskolin in combination with several different activators of adenylate cyclase. Adipocyte adenylate cyclase cannot be fully activated by forskolin; maximal stimulation by forskolin can be further enhanced by Gpp(NH)p. The effect of Gpp(NH)p on membrane enzyme differs between rat and rabbit and between enzymes of membrane-bound and solubilized preparations of rat adipocytes. Conditions that produce maximal activation of the membrane adenylate cyclase have been found. Gpp(NH)p and NaF are both activators of adenylate cyclase but mutually oppose each other. These findings indicate multiple activatable forms of catalytic components and multiple activatable forms of regulatory components of adenylate cyclase.

Effects of guanine nucleotides on vasopressin-induced water flow and sodium transport of the frog bladder.

In the present study, we examined the effects of guanine nucleotides on vasopressin-induced osmotic water flow and sodium transport in the 14-h preincubated frog bladder. We also examined the effects of the adenylate cyclase-cyclic AMP and cyclic AMP-dependent protein kinase system in the bladder's epithelial cells. Gpp(NH)p significantly enhanced vasopressin-induced water flow while it did not affect cyclic AMP-induced water flow. However, Gpp(NH)p did not enhance the vasopressin-induced increment of sodium transport across the frog bladder. The adenylate cyclase activity of the crude homogenate was enhanced by vasopressin, Gpp(NH)p and NaF. The effects of Gpp(NH)p and vasopressin, at their maximum doses, on the enzyme activities were additive, while other combinations were not. Specific Gpp(NH)p binding sites were found in the pellet fraction after 2,400 X g centrifugation. No direct effect on the protein kinase activity was observed in the presence of 10(-6) M nucleotides, such as GTP, GDP, GMP, CTP, UTP, ITP and Gpp(NH)p. Cyclic AMP stimulated the phosphorylation of discrete protein bands, however, Gpp(NH)p did not influence cyclic AMP-dependent protein phosphorylation of crude homogenate of the bladder's epithelial cells. These results suggest the guanine nucleotides stimulate the vasopressin-induced osmotic water flow in frog bladder by enhancing the vasopressin-mediated adenylate cyclase activity, so that accumulated cyclic AMP might activate cyclic AMP-dependent protein kinase.

Specific uncoupling by islet-activating protein, pertussis toxin, of negative signal transduction via alpha-adrenergic, cholinergic, and opiate receptors in neuroblastoma x glioma hybrid cells.

Exposure of NG108-15 hybrid cells to islet-activating protein (IAP), pertussis toxin, caused strong ADP-ribosylation of one of the membrane proteins with a molecular weight of 41,000. This ADP-ribosylation was paralleled by decreases in the inhibition of cAMP accumulation in intact cells or associated with reversal of the inhibition of GTP-dependent membrane adenylate cyclase, via alpha-adrenergic, cholinergic muscarinic, or opiate receptors. The affinity of these receptors for agonists was lowered by guanyl-5'-yl beta-gamma-imidodiphosphate (Gpp(NH)p) reflecting their coupling to the guanine nucleotide regulatory protein in this cell line. This effect of Gpp(NH)p was lost in membranes of IAP-treated cells; in the absence of Gpp(NH)p, the affinity for agonist was lower in treated than in nontreated cells. In contrast, the function of these receptors to bind antagonists remained unaltered in IAP-treated cells. Thus, IAP treatment of NG108-15 cells caused specific uncoupling of negative signal transduction from inhibitory receptors to the adenylate cyclase catalytic unit via the guanine nucleotide regulatory protein, as a result of ADP-ribosylation of one of the subunits of the regulatory protein.

Potentiation of monovalent cation effects on ligand binding to cardiac muscarinic receptors in N-ethylmaleimide treated membranes

Guanine nucleotides and monovalent cations decrease the affinity of cardiac muscarinic receptors for agonists and are required for muscarinic receptor mediated inhibition of adenylate cyclase. N-ethylmaleimide abolished the effects of Gpp(NH)p on the ability of the agonist oxotremorine to inhibit the binding of the antagonist [3H]quinuclidinyl benzilate to purified chick heart membranes. However, the effects of NH4+ to decrease the IC50 for oxotremorine were retained in N-ethylmaleimide treated membranes. The N-ethylmaleimide treatment mimicked the effects of Gpp(NH)p and the oxotremorine inhibition curves obtained with treated membranes in the presence of NH4+ were identical to those obtained in control membranes in the presence of NH4+ and Gpp(NH)p. The results suggest that monovalent cation effects on muscarinic receptors are mediated at a site distinct from effects produced by guanine nucleotides and are greater on free receptors than on receptors coupled to guanine nucleotide binding proteins.

Inhibitory and stimulatory effects of guanyl-5'-yl imidodiphosphate on the adenylate cyclase activity of rat synaptosomal fractions.

Guanyl-5'-yl imidodiphosphate (Gpp(NH)p), a nucleotide phosphohydrolase-resistant analog of GTP, caused inhibitory and stimulatory effects on the basal adenylate cyclase activity of rat synaptosomal fractions when manganese was present in the assay mixture, whereas the nucleotide caused only a stimulatory effect when magnesium was employed. In the presence of manganese, the inhibitory and stimulatory effects of Gpp(NH)p could be seen at around concentrations of 10(-7) M and 10(-4) M Gpp(NH)p, respectively. The inhibitory and stimulatory effects of Gpp(NH)p were both antagonized competitively by GTP; these effects of the analog were the opposite of those observed with GTP, which was stimulatory and inhibitory for fat call adenylate cyclase at 10(-7) M and 10(-4) M, respectively (Yamamura, H., Lad, P.M., and Rodbell, M. (1977) J. Biol. Chem. 252, 7964--7966). The degree of inhibition by Gpp(NH)p did not depend on the concentration of manganese nor on the addition of ethylene glycol bis(beta-aminoethyl ether)-N, N, N', N'-tetraacetic acid.

States of activation of chick kidney adenylate cyclase induced by parathyroid hormone and guanyl nucleotides.

Several aspects of the activation of adenylate cyclase by guanosine 5'-triphosphate (GTP), 5'-guanylylimidodiphosphate (Gpp(NH)p) and bovine parathyroid hormone (bPTH) have been studied in chick kidney plasma membrane preparations. GTP (10(-4) mol/l), Gpp(NH)p (10(-4) mol/l) and bPTH (10 i.u./ml) activated adenylate cyclase without any significant time lag. However a 2 min delay was observed before the activity of the enzyme increased after the addition of bPTH (-6 leads to +34) to incubations. The early (0-3 min) effects of GTP and Gpp(NH)p upon chick kidney adenylate cyclase activity were antagonized by the addition of the alternative guanyl nucleotide. After 5 min of incubation with kidney plasma membranes, Gpp(NH)p induced a stable state of activation of adenylate cyclase which was not reversible by subsequent addition of GTP. GTP did not induce an irreversible state of enzyme activation. In pre-incubation studies, GTP did not produce a persistent enzyme activation and did not modify the effect of Gpp(NH)p added subsequently at the incubation stage. Gpp(NH)p produced a stable state of activation of adenylate cyclase which was not inhibited by addition of GTP at the incubation stage. Bovine PTH (2-34) inhibited the effect of bPTH upon adenylate cyclase activity when the native hormone (10 i.u./ml) had been incubated with plasma membranes for up to 8 min before addition of the analogue (5 mug/ml). Incubation of plasma membranes with bPTH (2-34) for as little as 10 s prevented activation of adenylate cyclase by subsequent addition of bPTH. This pattern was confirmed in pre-incubation studies. After pre-incubation of kidney membranes with bPTH and bPTH (2-34), followed by washing, an acid extract of the membranes contained immunoreactive bPTH. Gpp(NH)p produced a greater increase in adenylate cyclase activity in membranes pre-incubated with bPTH or bPTH (2-34) than in membranes pre-incubated with buffer alone, suggesting that the hormone and analogue facilitated the interaction of Gpp(NH)p with adenylate cyclase.

Regulation of beta-adrenergic receptors by guanyl-5'-yl imidodiphosphate and other purine nucleotides.

Guanyl-5'-yl imidodiphosphate (Gpp(NH)p), GTP, and other purine nucleotides selectively decrease the binding affinity of the beta-adrenergic receptors of frog erythrocyte membranes for beta-adrenergic agonists but not antagonists. Shifts in binding affinity were assessed by determining the ability of unlabeled ligands to compete with (-)-[3H]dihydroalprenolol for the membrane-bound receptors. The magnitude of the"right" shift in the binding displacement curve for any of 13 ligands tested was directly related to the intrinsic activity (maximal stimulatory capacity) of that agent for stimulation of the frog erythrocyte membrane adenylate cyclase. Thus, Gpp(NH)p-induced shifts in binding affinity were greatest for full agonists such as isoproterenol, intermediate for partial agonists such as soterenol, and no shifts were observed for antagonists such as propranolol. Shifts in binding affinity were observed only in preparations where agonist binding to the receptors leads to "coupling" of the receptors with adenylate cyclase. In solubilized preparations where the beta-adrenergic receptors and adenylate cyclase are functionally "uncoupled", Gpp(NH)p did not cause right shifts in agonist receptor binding displacement curves. In particulate preparations the Km of Gpp(NH)p for stimulation of adenylate cyclase was identical with that for its effect on beta-adrenergic agonist binding affinity, 1 to 2 muM. Moreover, the ability of several other nucleotides to cause shifts in receptor binding affinity directly paralleled their previously determined affinities for the nucleotide regulatory sites on adenylate cyclase. Gpp(NH)p also shifted agonist dose-response curves for stimulation of adenylate cyclase, but to the left. As with the effects on the receptor binding curves, the effects of Gpp(NH)p on the "apparent affinities" of agonists for enzyme stimulation were directly related to their intrinsic activities. Gpp(NH)p also markedly increased the intrinsic activity of partial agonists. These results appear to indicate that conformational alterations in adenylate cyclase caused by occupation of nucleotide regulatory sites by Gpp(NH)p are capable of inducing alterations in the beta-adrenergic receptors. These receptor alterations are induced only when the receptors are "coupled" to the enzyme by virtue of agonist binding. The nucleotide-altered conformation of the beta-adrenergic receptors is characterized by decreased binding affinity for agonist but increased functional efficacy in stimulating the enzyme.