Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0043167 (pertussis)
 19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cells of the TE671/RD human clonal line express a finite number (Bmax) of about 350 fmol/mg of membrane protein) of apparently noninteracting, high-affinity binding sites (KD of 0.07 nM and a Hill coefficient close to unity, nH = 0.94) for the muscarinic acetylcholine receptor (mAChR) radio antagonist, tritium-labeled quinuclidinyl benzilate [( 3H]QNB). The rank order potency of selective antagonists that inhibit specific [3H]QNB binding is: atropine greater than 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) greater than pirenzepine greater than methoctramine greater than AFDX-116 (11-2[[2-[(diethylamino)methyl]-1-[piperidinyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one). Functional studies indicate that phosphoinositide (PIns) hydrolysis in TE671/RD cells is increased by carbachol (EC50 of 10 microM), but not by nicotine (to concentrations as high as 1 mM). Agonist-stimulated PIns metabolism is inhibited by antagonists with the same rank order potency as for inhibition of [3H]QNB binding. Functional responses are augmented in the presence of a nonhydrolyzable GTP analog, are strongly inhibited after 24-hr exposure to cholera toxin, but are only slightly inhibited after long-term exposure to pertussis toxin or forskolin. These studies identify a pharmacologically-defined M3-subtype of mAChR strongly coupled via a cholera toxin-sensitive mechanism to PIns hydrolysis in these cells. Within 1 hr of treatment of TE671/RD cells with 1 mM dibutyryl cyclic AMP or with 10 microM phorbol-12-myristate-13-acetate (PMA), there is a 30 to 50% decrease in carbachol-stimulated PIns responsiveness that recovers to control values after 5 days of continued drug treatment. However, a comparable and more persistent inhibition of mAChR function is observed on cell treatment with 20 nM PMA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ligand binding and functional characterization of muscarinic acetylcholine receptors on the TE671/RD human cell line. 164 30

Allosteric regulation of [3H]N-methylscopolamine [( 3H]NMS) and [3H]quinuclidinyl benzilate [( 3H]QNB) dissociation from the m1-m5 muscarinic receptor subtypes was examined in transfected CHO-K1 cells. Half-times of dissociation of [3H]NMS from cell membranes (at 23 degrees) ranged from less than 5 min for the m2 subtype to more than 60 min for the m5 subtype. For [3H]QNB, half-times (at 37 degrees) ranged from 1 hr (m2) to almost 4 hr (m3). The presence of gallamine slowed the dissociation of [3H]NMS from all of the subtypes, with an order of potency of m2 greater than m4 greater than m1 greater than m3 greater than m5. Dissociation of [3H]QNB from m1 and m2 receptors was modulated by gallamine in the biphasic manner that we have described previously for cardiac receptors; that is, low concentrations (1-10 microM) of gallamine accelerated dissociation, while 1 mM gallamine slowed it. Verapamil slowed the dissociation of [3H]-QNB from the m2 receptor in a monophasic manner, while the action of d-tubocurarine was qualitatively similar to that of gallamine. The potency of gallamine in allosterically regulating the m2 receptor was inversely related to ionic strength. Inactivation of pertussis toxin-sensitive G proteins abolished the ability of guanine nucleotides to regulate agonist affinity at the m2 receptor, but had no effect on allosteric regulation of the m2 receptor. These findings indicate that susceptibility to allosteric regulation varies in a complex way across muscarinic receptor subtypes and according to the choice of ligand.
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PMID:Allosteric regulation of cloned m1-m5 muscarinic receptor subtypes. 174 70

Direct interactions of the bispyridinium oxime HGG-12 with muscarinic acetylcholine receptors were investigated in porcine cardiac atrial membranes. Competition binding experiments using the radiolabeled muscarinic receptor antagonist (3H)QNB revealed specific binding of HGG-12 to muscarinic acetylcholine receptors of porcine atrial membranes with a dissociation constant of 3.8 x 10(-7) mol/l. Muscarinic acetylcholine receptor-stimulated binding of the radiolabeled GTP analog (35S)GTP[S] to guanine nucleotide binding proteins (G-proteins) was used to study antagonistic and possible agonistic effects of HGG-12 at muscarinic acetylcholine receptors. HGG-12 completely inhibited carbachol- and oxotremorine-stimulated (35S)GTP[S] binding to pertussis toxin sensitive and insensitive G-proteins in a competitive manner. Inhibition constants (K1) of HGG-12 for blockade of carbachol- and oxotremorine-stimulated GTP[S]-binding (9.7 x 10(-7) mol/l and 1.7 x 10(-6) mol/l, respectively) were higher by about a factor of 100 than those of the muscarinic acetylcholine receptor antagonist atropine. In the absence of muscarinic acetylcholine receptor agonists. HGG-12 by itself had no stimulatory effect on (35S)GTP[S] binding in porcine atrial membranes. The results of this study show that the oxime HGG-12 is a competitive antagonist without intrinsic activity at porcine atrial muscarinic acetylcholine receptors. The stimulatory action of HGG-12 on muscarinic acetylcholine receptors which has been described by several authors is, therefore, suggested to be due to partial inhibition of acetylcholinesterase by the oxime rather than to direct agonism at muscarinic acetylcholine receptors.
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PMID:The oxime HGG-12 as a muscarinic acetylcholine receptor antagonist without intrinsic activity in cardiac membranes. 192 74

Muscarinic acetylcholine receptors were identified by the specific binding of [H](-)quinuclidinylbenzilate [( 3H](-)QNB) and [3H]oxotremorine-M [( 3H]Oxo-M), to membranes isolated from the sino-atrial (SA) node and right atrium (RA) of bovine heart. The density of [3H](-)QNB binding sites was greater in the SA node compared to the RA. Specific [3H](-)QNB binding was saturable and occurred to a single population of binding sites in both regions. The binding of antagonists, as assessed by competition with [3H](-)QNB, also occurred to a single population of sites; the binding affinities of all antagonists were similar in either region. Agonist competition curves, except for McN-A-343, were complex and computer analyses indicated that agonists bound to at least two populations of binding sites that differed in affinity. The proportion of high-affinity agonist binding sites was consistently greater in the SA nodal, relative to the RA membranes, while the affinity of the high-affinity agonist binding sites to a given agonist was essentially similar in either region. The high-affinity binding of [3H]Oxo-M was saturable and occurred to a single population of sites. The maximal binding of [3H]Oxo-M in the SA nodal membranes was higher than in the RA membranes. Guanine nucleotides and N-ethylmaleimide (NEM) markedly decreased [3H]Oxo-M binding; NEM did not appear to influence guanine nucleotide-dependent decrease in [3H]Oxo-M binding. Phospholipase A2 decreased both [3H](-)QNB and [3H]Oxo-M specific binding, the latter being affected to a greater extent. Phospholipase C also decreased [3H](-)QNB and [3H]Oxo-M binding, although to a lesser degree compared to phospholipase A2. Either lipase, however, increased the guanine nucleotide-sensitive agonist binding. Analysis of [3H](-)QNB binding to microsomal subfractions showed that binding sites were enriched in the light plasma membrane fractions that were also enriched in pertussis toxin sensitive guanine nucleotide binding proteins.
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PMID:Muscarinic acetylcholine receptors in the sino-atrial node and right atrium of bovine heart. 225 3

The interaction of cholinergic agonists and antagonists with smooth muscle muscarinic receptors has been investigated by measurement of displacement of the muscarinic antagonist [3H]QNB (quinuclidinyl benzilate) in membranes prepared from toad stomach. The binding of [3H]QNB was saturable, reversible and of high affinity (KD = 423 pM). The muscarinic receptor subtypes present in gastric smooth muscle were classified by determining the relative affinities for the selective antagonists pirenzepine (M1), AF-DX 116 (M2) and 4-DAMP (M3). The results from these studies indicate the presence of a heterogeneous population of muscarinic receptor subtypes, with a majority (88%) exhibiting characteristics of M3 receptors and a much smaller population (12%) exhibiting characteristics of M2 receptors. The binding curve for the displacement of [3H]QNB binding by the agonist oxotremorine was complex and was consistent with presence of two affinity states: 24% of the receptors had a high affinity (KD = 4.7 nM) for oxotremorine and 76% displayed nearly a 1,000-fold lower affinity (KD = 4.4 microM). When oxotremorine displacement of [3H]QNB binding was determined in the presence GTP gamma S, high affinity binding was abolished, indicating that high affinity agonist binding may represent receptors coupled to G proteins. Moreover, pertussis toxin pretreatment of membranes also abolished high affinity agonist binding, indicating that the muscarinic receptors are coupled to pertussis toxin-sensitive G proteins. Reaction of smooth muscle membranes with pertussis toxin in the presence [32P]NAD caused the [32P]-labelling of a 40 dD protein that may represent the alpha subunit(s) of G proteins that are known to by NAD-ribosylated by the toxin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interaction of agonists and selective antagonists with gastric smooth muscle muscarinic receptors. 249 69

The muscarinic receptors in a B82 cell line which were transfected with the rat m1 muscarinic receptor gene (cTB10 cells) were studied by using radioligand binding assays. Their possible coupling to the hydrolysis of inositol lipids and cyclic AMP formation were also investigated. [(-)-[3H]Quinuclidinyl benzilate [(-)-[3H]QNB] binding to the intact cTB10 cells was saturable and displaceable by 1 microM atropine sulfate. The Kd and maximum binding values of (-)-[3H]QNB from saturation studies were 12 pM and 17 fmol/10(6) cells, respectively. Inhibition studies of (-)-[3H]QNB binding to intact cTB10 cells suggested that these muscarinic receptors are of the M1 type defined by their high affinity for pirenzepine and low affinity for AF-DX 116 [11-[2-diethylamino methyl-1-piperidinylacetyl]-5,11-dihydro-6H-pyrido(2,3-b) (1,4)benzodiazepine-6-one]. The muscarinic agonist carbachol stimulated [3H]inositol monophosphate accumulation in the cTB10 cells, which could be reversed by the muscarinic antagonists atropine, pirenzepine or AF-DX 116. The rank order of potency of the muscarinic antagonists in inhibiting carbachol-stimulated [3H]inositol monophosphate accumulation was atropine greater than pirenzepine greater than AF-DX 116, in agreement with that from ligand/(-)-[3H]QNB competition experiments. Pertussis toxin and 4 beta-phorbol, 12-beta-myristate, 13-alpha-acetate reduced carbachol-stimulated [3H]inositol monophosphate accumulation. Prostaglandin E1 stimulated cyclic AMP formation in the cTB10 cells. Carbachol at the concentration of 10 mM exhibited no stimulatory or inhibitor effect on the basal or prostaglandin E1-stimulated cyclic AMP formation. These results suggest that the muscarinic receptors encoded by the transfected m1 gene in the cTB10 cells are of the M1 type and are coupled to the hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein.
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PMID:Pharmacological characterization of the M1 muscarinic receptors expressed in murine fibroblast B82 cells. 253 6

In previous studies, we showed that cardiac muscarinic receptors (M2) are composed of two subgroups, M2 alpha and M2 beta, with different affinities for agonists and that the M2 alpha subgroup is coupled with inhibition of adenylate cyclase. We now studied which subgroup was responsible for the formation of inositol mono- (IP), bis- (IP2), tris- (IP3) and tetrakis- (IP4) phosphates in guinea pig heart. Carbachol (1 mM) significantly stimulated the formation of all four IPs in [3H]myoinositol-preloaded slices of guinea-pig ventricles. Acetylcholine (1 mM) also stimulated the formation of IP2, IP3 and IP4. However, oxotremorine (1 mM) only slightly stimulated the formation of IP2, and pilocarpine did not stimulate the formation of any IP. The pED50 values of carbachol for IP2 and IP3 formation were 3.76 and 4.23, respectively, which coincided with the pKd values of the low-affinity agonist binding site (L site) measured by competition of carbachol with [3H]quinuclidinyl benzilate [( 3H]QNB) binding while the pKd value for inhibition of adenylate cyclase coincided with the pKd value of the high-affinity agonist binding site (H site). Treatment of animals with pertussis toxin decreased the formation of IP2 and IP3 by carbachol to 66 and 54%, respectively, but resulted in complete inhibition of adenylate cyclase. These results suggested that muscarinic stimulation of the formation of IPs was manifested through a different receptor subgroup (M2 beta) and GTP binding protein different from those for inhibition of adenylate cyclase.
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PMID:The H-L subgroup of guinea-pig cardiac M2 receptors (M2 beta) regulates inositol phosphate formation. 258 43

The injection of Bordetella pertussis, inactivated by merthiolate, causes a 2-fold increase in the IC50 of carbamylcholine (carbachol) in displacing [3H];-L(-) quinuclidinyl benzilate binding ([3H]QNB) to the receptor. In control animals, 50 microM Gpp(NH)p causes a 6-fold decrease in the affinity of carbachol binding, whereas after vaccination the reduction is only 1.6-fold. After pertussis treatment there is no alteration in the affinity and number of [3H]QNB binding sites of to the muscarinic receptor.
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PMID:Pertussis vaccine reduces agonist binding to the rat heart muscarinic receptor and its guanine nucleotide modulation. 372 69

The effect of pertussis toxin on the affinity for agonists and antagonists of the heart muscarine acetylcholine receptor was studied using the radiolabeled antagonist [3H]quinuclidinyl benzylate ([3H]QNB). In cardiac membranes from control rats the displacement of [3H]QNB by carbachol was consistent with two classes of binding sites, kDH 25 +/- 10 nM and kDL 3,006 +/- 869 nM. The proportion of sites in the high and low affinity state for agonists was 55 and 45% respectively. In the presence of 100 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p), only the low affinity state for agonists was observed (kDL 3,804 +/- 759 nM). In cardiac membranes from pertussis toxin-treated rats, two classes of binding sites with affinities similar to those seen in the controls were also observed in the absence of guanine nucleotide (kDs 39 +/- 12 and 3,315 +/- 845 nM) but the proportion of sites were 20 and 80% for high and low affinity respectively. Gpp(NH)p shifted the remaining 20% of sites from the high affinity to the low affinity state (KD 4,093 +/- 744 nM). The receptor KD for antagonists was decreased by pretreatment with pertussis toxin from 83 +/- 7 to 56 +/- 5 pM (P less than 0.01); Gpp(NH)p induced a further change in the affinity for the antagonist in membranes from both control and pertussis toxin-treated rats. The change suggested positive cooperativity. The total number of sites was not modified significantly by either pertussis toxin treatment or guanine nucleotides. These results are consistent with a possible reciprocal modulation of the affinity for agonists and antagonists of the cardiac muscarine receptor.
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PMID:Effect of pertussis toxin on the heart acetylcholine muscarinic receptor affinity. 375 77

Although the neurotoxicity of organophosphorus compounds is generally attributed to inhibition of acetylcholinesterase, recent reports have indicated that direct interactions with muscarinic receptors and signal transduction may be an additional mechanism of neurotoxicity. We have previously shown that the organophosphorus insecticide O,O-diethyl O-3,5,6-trichloro-2-pyridinyl phosphorothioate (chlorpyrifos) binds directly to muscarinic receptors and inhibits adenylate cyclase of rat striatum. We have further pursued those results in this study by investigating the effect of chlorpyrifos oxon in NG108-15 neuroblastoma-glioma cells and Chinese hamster ovary cells transfected with cDNA for human m2 or m4 muscarinic receptor subtypes. At millimolar concentrations, chlorpyrifos oxon inhibited [3H]QNB binding in all cell lines. Likewise, [3H]CD binding was inhibited in NG108-15 and CHO-Hm2 cells. When the effect of chlorpyrifos oxon on adenylate cyclase was examined, the oxon was found to inhibit adenylate cyclase at millimolar concentrations. Though this effect on cyclase required greater concentrations of oxon than the comparable effect in striatal cells, it displayed the common characteristic of being atropine-insensitive, suggesting that the effect on cyclase was not muscarinic receptor dependent. The inhibition of adenylate cyclase produced by chlorpyrifos oxon was not eliminated in pertussis toxin treated cells, lending further support to the idea that it is not a receptor-mediated event, and suggesting a potential direct interaction of chlorpyrifos oxon with the adenylate cyclase molecule.
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PMID:In vitro effect of chlorpyrifos oxon on muscarinic receptors and adenylate cyclase. 756 87


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