Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of the new cardiotonic agent sulmazole on the guanine nucleotide regulatory protein-adenylate cyclase system was studied in rat adipocyte membranes. The inotrope enhanced basal adenylate cyclase activity by 46%. This stimulation occurred only at GTP concentrations (5 microM) sufficient to activate Gi. This stimulatory effect of sulmazole was abolished after functional inactivation of Gi, either by pertussis toxin or by using 10 nM GTP in the assay mixture, suggesting an important role of an active Gi in this process. Similarly, sulmazole enhanced isoproterenol-, forskolin-, and fluoride-stimulated adenylate cyclase activity by 33, 34, and 45%, respectively. However, when these latter experiments were performed after inactivation of Gi, sulmazole actually inhibited by approximately 25% adenylate cyclase activity stimulated by 1 and 10 microM isoproterenol. Under similar treatment conditions, enhancement of forskolin- and fluoride-stimulated activity by sulmazole was abolished. Sulmazole inhibited in a dose-dependent manner pertussis toxin- and cholera toxin-catalyzed labeling of Gi and Gs, respectively, with the respective inhibition observed at 100 microM of the inotrope being 29% and 56% of control. In addition, sulmazole inhibited PGE1 and isoproterenol-stimulated [3H]GDP release from Gi and Gs to 32% and 64% of control, respectively. Finally, the inotrope completely abolished PGE1-stimulated [3H]Gpp(NH)p binding with IC50 in the low micromolar range. These findings suggest that, whereas sulmazole inhibits the functioning of Gi and (to a lesser extent) Gs at low micromolar concentrations, expression of these effects on adenylate cyclase activity requires high micromolar to low millimolar concentrations of the drug. Thus, it appears sulmazole inhibits the function of Gi by decreasing its activation process, i.e., GTP-GDP exchange. Effects on Gs are manifested (at least in terms of adenylate cyclase activity) only after inactivation of Gi.
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PMID:The new positive inotrope sulmazole inhibits the function of guanine nucleotide regulatory proteins by affecting GTP turnover. 284 44

1. In the intact guinea-pig myometrium, carbachol and oxytocin stimulated a specific receptor-mediated phospholipase C activation, catalysing the breakdown of PtdIns(4,5)P2 with the sequential generation of InsP3, InsP2 and InsP. Stimulation of muscarinic receptors also triggered an inhibition of cyclic AMP accumulation caused by prostacyclin. 2. NaF plus AlCl3 mimicked the effects of carbachol and oxytocin by inducing, in a dose-dependent manner, the generation of all three inositol phosphates as well as uterine contractions. AlCl3 enhanced the fluoride effect, supporting the concept that A1F4- was the active species. Under similar conditions, fluoroaluminates activated the guanine nucleotide regulatory protein Gi, reproducing the inhibitory effect of carbachol on cyclic AMP concentrations. 3. Both carbachol- and oxytocin-mediated increases in inositol phosphates, as well as contractions, were insensitive to pertussis toxin, under conditions where the expression of Gi was totally prevented. Cholera toxin, which activates Gs and enhances cyclic AMP accumulation, failed to affect basal or oxytocin-evoked inositol phosphate generation, but induced a slight, though consistent, attenuation of the muscarinic inositol phosphate response, which was similarly evoked by forskolin. 4. The data provide evidence that, in the myometrium, (a) a G protein mediates the generation of inositol phosphates and the Ca2+-dependent contractile event, (b) the relevant G protein that most probably couples muscarinic and oxytocin receptors to phospholipase C is different from Gi and Gs, the proteins that couple receptors to adenylate cyclase, and (c) cyclic AMP does not seem to control the phosphoinositide cycle, but rather exerts a negative regulation at the muscarinic-receptor level.
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PMID:Fluoroaluminates mimic muscarinic- and oxytocin-receptor-mediated generation of inositol phosphates and contraction in the intact guinea-pig myometrium. Role for a pertussis/cholera-toxin-insensitive G protein. 284 25

Beta-adrenergic receptor-coupled adenylate cyclase is regulated by both amplification and desensitization processes. Desensitization of adenylate cyclase is divided into two major categories. Homologous desensitization is initiated by phosphorylation of the receptors by a beta-adrenergic receptor kinase. This reaction serves to functionally uncouple the receptors and trigger their sequestration away from the cell surface. These sequestered receptors can rapidly recycle to the cell surface or, with time, become down regulated, being destroyed within the cell. Dephosphorylation of the receptors is accomplished in the sequestered compartment of the cell, which may functionally regenerate the receptors and allow their return to the cell surface. In heterologous desensitization, receptor function is also regulated by phosphorylation, but in the absence of receptor sequestration or down regulation. In this case, phosphorylation serves only to functionally uncouple the receptors, that is, to impair their interactions with the guanine nucleotide regulatory protein Ns. Several protein kinases are capable of promoting this phosphorylation, including the cAMP-dependent kinase and protein kinase C. In addition to the receptor phosphorylation, heterologous desensitization is associated with modifications at the level of the nucleotide regulatory protein Ns and perhaps Ni. Adenylate cyclase systems are also subject to amplification that involves a protein kinase C-mediated phosphorylation of the catalytic unit of the enzyme. Phosphorylation of the catalytic unit enhances its catalytic activity and results in amplified stimulation by the regulatory protein Ns. Other receptor/effector systems exhibit qualitatively similar regulatory phenomena, suggesting that covalent modification (phosphorylation) may represent a general mechanism for regulating receptor function.
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PMID:Beta-adrenergic receptor-coupled adenylate cyclase. Biochemical mechanisms of regulation. 285 89

Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased 45Ca2+ uptake into the cell monolayer, and (f) increased 86Rb+ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca2+ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca2+-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca2+ gating.
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PMID:Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin. 286 Jan 11

Cyclic AMP accumulation in rat parotid slices is only transiently stimulated by isoproterenol (Harper, J.F. and Brooker, G. Molec. Pharmacol. 13:1048-1059, 1977); the progressive loss of isoproterenol effect is termed desensitization. In this report we show that desensitized cyclic AMP accumulation is associated with desensitization of adenylate cyclase in subsequently prepared membranes and in adenylate cyclase that has been detergent-solubilized from desensitized membranes. Adenylate cyclase in membranes made from isoproterenol-desensitized tissue is desensitized to both the stimulating effects of isoproterenol with 6 mM MgCl2 and of forskolin with 30 mM MnCl2. We have previously determined (Harper, J.F. J. Cyclic Nucleo. Prot. Phosphoryl. Res. 9:401-414, 1984) that cyclic AMP accumulation desensitized to isoproterenol is rapidly counteracted by 1 microM forskolin but not 0.1 microM forskolin. Similarly, if 1 microM forskolin was included in the desensitizing incubation with isoproterenol then adenylate cyclase subsequently prepared was not desensitized. Development of desensitized adenylate cyclase was only partially affected by 0.1 microM forskolin. Desensitization is counteracted by forskolin only on intact cells. Once tissue is homogenized, desensitized adenylate cyclase does not respond as well to forskolin as does control adenylate cyclase. The site of desensitization appears to be at or near the adenylate cyclase catalytic unit. Desensitization of adenylate cyclase catalytic activity remains demonstrable after membranes are solubilized with CHAPS. The adenylate cyclase activity remaining in the supernatant following solubilization of desensitized membranes is depressed to nearly the same extent as found in the membranes. Further, desensitized adenylate cyclase in membrane preparations and after solubilization is desensitized to stimulatory effects of forskolin with 30 mM MnCl2, a condition under which forskolin is probably acting directly on the adenylate cyclase catalytic unit. Desensitization appears not to be dependent on activity of the inhibitory guanine nucleotide regulatory protein (Gi), since pertussis toxin is without effect on desensitization of cyclic AMP accumulation to isoproterenol.
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PMID:Desensitization in rat parotid to beta-adrenergic agonists and counteracting effects of forskolin are conserved in membrane and detergent-solubilized adenylate cyclase catalyst activity. 287 14

Mammalian beta-adrenergic receptors are glycoproteins consisting of a single polypeptide chain of Mr approximately 64,000. Treatment of purified [125I]-labeled hamster lung beta-adrenergic receptor with alpha-mannosidase reveals two discrete populations of receptor consistent with previous studies using membrane bound photoaffinity-labeled receptor. Treatment of the [125I]-labeled receptor with endoglycosidase F results initially in the formation of a Mr approximately 57,000 peptide which is further converted to Mr approximately 49,000 suggesting that there are two N-linked carbohydrate chains per receptor polypeptide. Exoglycosidase treatments and lectin chromatography of the [125I]-labeled receptor reveals the presence of two complex type carbohydrate chains (approximately 10% of which are fucosylated) on approximately 45% of the receptors. The remaining approximately 55% of the receptors appear to contain a mixture of carbohydrate chains (possibly high mannose, hybrid and complex type chains). Deglycosylation of the receptor by endoglycosidase F does not appear to alter the binding affinity of the receptor for a variety of beta-adrenergic agonists and antagonists. Moreover, the ability of control, alpha-mannosidase sensitive or insensitive (fractionated on immobilized wheat germ agglutinin) and neuraminidase, alpha-mannosidase or endoglycosidase F treated receptors to interact with the stimulatory guanine nucleotide regulatory protein in a reconstituted system were virtually identical. The deglycosylated receptor was also unaltered in its heat lability as well as its susceptibility to a variety of proteases. These findings demonstrate that the carbohydrate portion of the beta-receptor does not contribute to determining either its specificity of ligand binding or coupling to the adenylate cyclase system.
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PMID:The mammalian beta-adrenergic receptor: structural and functional characterization of the carbohydrate moiety. 288 51

Regulation of adenylate cyclase by alpha 2 adrenergic receptors requires the inhibitory guanine nucleotide binding protein Ni. A role for this protein has also been suggested in the high-affinity binding of agonists to the alpha 2 receptor. We recently reported that alkaline treatment can selectively inactivate alpha 2 agonist binding and Ni in human platelet plasma membranes [Kim, M.H. & Neubig, R.R. (1985) FEBS Lett. 192, 321-325]. Binding of the full alpha 2 agonists epinephrine and 5-bromo-6-[N-(4,5-dihydroimidazol-2-yl)amino]quinoxaline (UK 14,304) to these membranes was determined by competition and direct radioligand binding, respectively. The high-affinity GTP-sensitive binding of the agonists is lost after alkaline treatment. Binding of [3H]UK 14,304 was reconstituted by poly(ethylene glycol)-induced fusion of alkaline-treated platelet membranes with cell membranes containing Ni but no alpha 2 receptor or with lipid vesicles containing purified guanine nucleotide binding proteins (N-proteins) from bovine brain. The reconstituted binding was of high affinity (Kd = 0.4 +/- 0.1 nM), accounted for a substantial fraction of the total alpha 2 receptors (Bmax for [3H]UK 14,304 was 78 +/- 23% of the Bmax for [3H]yohimbine), and was abolished in the presence of guanosine 5'-(beta, gamma-imidotriphosphate) (GppNHp). The brain-specific protein No (predominant guanine nucleotide regulatory protein from bovine brain) was also effective in reconstituting high-affinity alpha 2 agonist binding. The results presented here show that a guanine nucleotide regulatory protein of the No or Ni type is necessary for high-affinity alpha 2 agonist binding. These methods should also prove useful for future studies of receptor N-protein interactions.
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PMID:Membrane reconstitution of high-affinity alpha 2 adrenergic agonist binding with guanine nucleotide regulatory proteins. 288 80

Somatostatin binding and cross-linking to its receptors on rat cerebrocortical membranes were characterized with [125I-Tyr1]somatostatin-14 and [125I-Leu8, D-Trp22, Tyr25]somatostatin-28. When [125I-Tyr1]somatostatin-14 was cross-linked to its receptors with the photoreactive cross-linker, N-(5-azido-2-nitrobenzoyloxy)succinimide, the hormone was specifically associated with a Mr = 72,000 protein band in the presence or absence of reducing agents. Affinity labeling of the Mr = 72,000 protein band was decreased with increasing concentrations of unlabeled somatostatin-14 and nonhydrolyzable guanine nucleotide analog, guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Pretreatment of cerebrocortical membranes with islet-activating protein resulted in a decrease in subsequent labeled somatostatin-14 binding and affinity-labeling of the protein and abolished an inhibitory effect of somatostatin-14 on vasoactive intestinal peptide-stimulated increase in adenylate cyclase activity. When the affinity-labeled protein was solubilized with Zwittergent 3-12 and adsorbed to wheat germ agglutinin-agarose, it was eluted by N-acetylglucosamine. [125I-Leu8, D-Trp22, Tyr25]somatostatin-28 cross-linking to cerebrocortical and pancreatic membranes with the same photoreactive agent revealed specifically labeled protein bands of a Mr = 74,000 in cerebrocortical membranes and a Mr = 94,000 in pancreatic membranes, respectively. These results suggest that: 1) somatostatin receptor on cerebrocortical membranes is a monomeric glycoprotein with a Mr = 70,000 binding subunit, coupled to guanine nucleotide regulatory protein, and 2) the Mr = 70,000 protein may be a common receptor for somatostatin-28 and somatostatin-14 and is distinct from a common pancreatic type receptor.
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PMID:Somatostatin receptors on rat cerebrocortical membranes. Structural characterization of somatostatin-14 and somatostatin-28 receptors and comparison with pancreatic type receptors. 290 82

Rat substantia nigra was injected with 1 microgram of purified pertussis toxin, a substance which inactivates the inhibitory guanine nucleotide regulatory unit of adenylate cyclase. In comparison to saline-injected animals, the pertussis toxin-treated animals showed almost no inhibition of dopaminergic neurons in response to dopamine applied iontophoretically or the dopamine agonist (-)-apomorphine given intravenously. These results provide evidence that a guanine nucleotide regulatory protein is involved as a transducer in mediating the physiological actions of dopamine autoreceptors.
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PMID:Pertussis toxin blocks autoreceptor-mediated inhibition of dopaminergic neurons in rat substantia nigra. 295 48

Activation of muscarinic cholinergic receptors of 1321N1 human astrocytoma cells attenuates cyclic AMP accumulation. This effect results from an activation of phosphodiesterase with no direct inhibition of adenylate cyclase activity. In spite of this lack of coupling of muscarinic receptors to adenylate cyclase, guanine nucleotides reduce the apparent binding affinity of the agonist carbachol in a washed membrane preparation of 1321N1 cells. The order of potency for this effect is guanosine 5'-O-(3-thiotriphosphate) greater than 5'-guanylyl-imidodiphosphate = GTP = GDP; ATP has no effect. The occurrence of a Mr = 41,000 protein labeled in the presence of [32P]NAD and pertussis toxin as well as the occurrence of guanine nucleotide-mediated inhibition of forskolin-stimulated adenylate cyclase activity indicate that the functional inhibitory guanine nucleotide regulatory component of adenylate cyclase (Ni) is present in 1321N1 cells. Pertussis toxin pretreatment of NG108-15 neuroblastoma X glioma cells, which express muscarinic receptors that link through Ni to inhibit adenylate cyclase, blocked the GTP-sensitive, high affinity binding of carbachol. In contrast, pretreatment of 1321N1 cells with a concentration of pertussis toxin that blocked [32P]ADP ribosylation of the Mr = 41,000 substrate and GTP-mediated inhibition of forskolin-stimulated adenylate cyclase activity had no effect on GTP-sensitive high affinity binding of carbachol. These results suggest that muscarinic cholinergic receptors of 1321N1 cells couple to a guanine nucleotide regulatory protein that is distinct from Ni.
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PMID:Guanine nucleotide-sensitive, high affinity binding of carbachol to muscarinic cholinergic receptors of 1321N1 astrocytoma cells is insensitive to pertussis toxin. 298


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