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

The binding affinities of muscarinic antagonists were compared with their abilities to block carbachol (CCh)-mediated stimulation of Ca2+ mobilization and inhibition of isoproterenol-elicited adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in rat parotid cells. The binding of [3H]quinuclidinyl benzilate (QNB) to membranes was inhibited by antagonists with the following potencies (dissociation constant, nM): atropine (1.1) approximately 4-diphenylacetoxy-N-methylpiperidine methbromide (4-DAMP) (1.6) much greater than pirenzepine (136) greater than 11-[[2-[(diethylamino)methyl-1-piperidinyl]-acetyl]acetyl]-5,11- dihydro-6H-pyrido[2,3-b][1,4]-benzodiazepine-6-one (AF-DX 116) (5,293). AF-DX 116 blocked Ca2+ mobilization and inhibition of cAMP accumulation with low affinities [inhibitory concentration at 50% (IC50) = 3150 and 6,528 nM, respectively], whereas 4-DAMP blocked these responses with considerably higher affinities (IC50 = 4.3 and 11.4 nM, respectively). Schild plots of 4-DAMP and AF-DX 116 antagonism of CCh-stimulated inositol trisphosphate accumulation showed inhibitor constant (Ki) values of 0.85 and 1,585 nM, respectively, whereas Schild plots of 4-DAMP, AF-DX 116, and methoctramine antagonism of CCh-induced inhibition of cAMP accumulation showed Ki values of 1.3, 1,585, and 2,754 nM, respectively. Preincubation of cells with 0.1 mM 3-isobutyl-1-methylxanthine did not prevent the capacity of CCh to inhibit cAMP accumulation. Pertussis toxin blocked the CCh-elicited and Gi-mediated inhibition of cAMP formation. Northern blot analysis showed the presence of mRNA for the M3, but not for the M2, subtype in parotid gland. An immunochemical procedure using m1-m5 specific antibodies was performed in parotid membranes and showed that the m3 receptor accounts for 93% of precipitable receptors. These data suggest that M3 receptors in the rat parotid are coupled to both the stimulation of Ca2+ mobilization and the inhibition of cAMP accumulation.
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PMID:Evidence that M3 muscarinic receptors in rat parotid gland couple to two second messenger systems. 172 44

Arecoline produces a biphasic response in rat left atria, i.e., a depression of basal inotropy at low doses and a positive inotropic effect at higher doses. These present studies were designed to determine whether it can be shown that the two separate responses to arecoline are mediated by two distinct cell surface muscarinic receptors. The antagonists scopolamine, 4-DAMP and AF-DX 116 produced apparent simple competitive antagonism of the negative responses to arecoline. Schild analysis was used to measure the equilibrium dissociation constant of the antagonist-receptor complex for antagonism of this response to arecoline by these antagonists. In atria from rats treated with pertussis toxin, the negative inotropy to arecoline was abolished and only the positive inotropic effects were observed. The antagonism of the positive inotropic response to arecoline by these antagonists was studied separately in atria from rats treated with pertussis toxin by the Schild technique. The pKB estimates made from the Schild regressions indicated no evidence to suggest that the two responses to arecoline (negative and positive inotropy) were mediated by two separate receptors in rat left atria. These data are discussed in terms of a single muscarinic receptor in this tissue mediating these two responses by interaction with two G-proteins in the same cell membrane. These data also are discussed in terms of the use of agonist potency ratios for the classification of receptors.
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PMID:Biphasic dose-response curves to arecoline in rat atria-mediation by a single promiscuous receptor or two receptor subtypes? 194 13

Ligand binding properties of muscarinic receptors were examined in membranes and isolated cells prepared from bovine trachea. The binding of the muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) to both membranes and isolated cells was saturable, reversible, and of high affinity [dissociation constant (KD) = 100-200 pM]. The binding constants of three selective antagonists, pirenzepine, AF-DX 116, and 4-DAMP, were examined, and the results indicate that the smooth muscle cells contain at least two receptor subtypes. The majority of receptors exhibit binding constants for these selective antagonists similar to those of the M2-subtype. AF-DX 116 binding curves indicated the presence of a small population of receptors with binding constants similar to those reported for the M3-subtype. These findings suggest that the smooth muscle cells may contain both M2- and M3-receptors and are in agreement with evidence of the presence of mRNAs coding for these two subtypes in tracheal extracts (A. Maeda, T. Kubo, M. Mishina, and S. Numa. FEBS Lett. 239: 339-342, 1988). [3H]QNB displacement curves of the muscarinic agonist oxotremorine were best described as a sum of binding to high- and low-affinity sites with KD values of 3.8 nM and 2.2 microM. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) shifted the high-affinity sites to low affinity, suggesting that the high-affinity sites may represent receptors coupled to G proteins. Pertussis toxin catalyzed the ADP ribosylation of a 40- to 41-kDa protein band present in the membranes but had no significant effect on high-affinity agonist binding, suggesting that most of the receptors are coupled to G proteins in a toxin-insensitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ligand binding and G protein coupling of muscarinic receptors in airway smooth muscle. 211 Apr 18

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

Gentamicin nucleotidyltransferase-catalyzed reaction of (Sp)-[alpha-17O]dATP with tobramycin produced 2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin. The configuration at phosphorus in this product was shown to be Rp by chemical degradation to chiral [17O, 18O]dAMP using a stereochemically defined procedure, and determination of the configuration at phosphorus in this product. Periodate-base treatment of 2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin followed by NaBH4 reduction produced (2-glyceryl)-[17O]dAMP, which upon snake venom phosphodiesterase-catalyzed hydrolysis in H(2)18O produced [17O,18O] dAMP. The configuration at phosphorus in this product was shown to be S by enzymatic phosphorylation to [17O,18O]dATP, adenylylcyclase (Bordetella pertussis)-catalyzed cyclization to 3',5'-cyclic [17O,18O]dAMP, and 31P NMR analysis of the ethyl esters. Since snake venom phosphodiesterase-catalyzed hydrolyses proceed with retention of configuration at phosphorus, (Sp)-[17O,18O]dAMP must have been produced from (Rp)-(2-glyceryl)-[17O]dAMP; and since the chemical degradation to the latter compound did not involve cleavage of any bonds to phosphorus, the initial enzymatic product must have been (Rp)-2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin. Therefore, nucleotidyl transfer catalyzed by gentamicin nucleotidyl-transferase proceeds with inversion of configuration at phosphorus, and the reaction mechanism involves an uneven number of phosphotransfer steps. Inasmuch as this is an uncomplicated two-substrate group transfer reaction, the mechanism probably involves direct nucleotidyl transfer from the nucleoside triphosphate to the aminoglycoside. The B. pertussis adenylylcyclase reaction was shown to proceed with inversion at phosphorus, as has been established for other adenylylcyclases.
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PMID:Gentamicin nucleotidyltransferase. Stereochemical inversion at phosphorus in enzymatic 2'-deoxyadenylyl transfer to tobramycin. 287 83

1. The acetylcholine (ACh)-induced currents (IACh) in dissociated outer hair cells (OHCs) of guinea-pig cochlea were investigated using the whole-cell patch-clamp technique, in both conventional and nystatin perforated-patch configurations. 2. ACh and carbamylcholine (CCh) induced outward currents at a holding potential (VH) of -60 mV in the perforated-patch configuration. The IACh increased in a sigmoidal fashion over the concentration range between 3 x 10(-6) and 10(-3) M. The dissociation constant (KD) was 1.7 x 10(-5) M and the Hill coefficient (n) was 2.7. The KD and n for CCh were 8.7 x 10(-5) M and 2.2, respectively. Neither nicotine nor muscarine induced any detectable current up to a concentration of 10(-3) M. 3. Various muscarinic agonists such as oxotremorine-M, McN-A-343 and oxotremorine could also induce the outward currents, although these current amplitudes were about one-third that of ACh, indicating that they were partial agonists. 4. The muscarinic antagonists atropine, 4-DAMP, AF-DX 116 and pirenzepine inhibited the IACh in a concentration-dependent manner. The half-inhibitory concentrations (IC50) for atropine, 4-DAMP, AF-DX 116 and pirenzepine were 4.8 x 10(-6), 6.2 x 10(-6), 2.1 x 10(-5) and 2.9 x 10(-4) M, respectively. 5. When the extracellular Ca2+ concentration ([Ca2+])o) was reduced to lower than 1 mM, the amplitude of IACh, abruptly decreased. In a nominally Ca(2+)-free external solution ACh did not induce any current. The increase of [Ca2+]o beyond 1 mM did not change the IACh. 6. When OHCs were perfused intracellularly with a pipette solution containing 10 mM BAPTA in the conventional whole-cell mode, ACh could not induce outward K+ currents. The Ca2+ ionophore A23187 induced an outward current. These results indicate that intracellular Ca2+ is involved in the ACh response. 7. Calmodulin inhibitors such as chlorpromazine, W-7 and trifluoperazine inhibited the IACh in a concentration-dependent manner. 8. When OHCs were dialysed with either 100 microM GDP beta S or 1 micrograms/ml pertussis toxin (PTX) through the patch pipette at a VH of -60 mV, the IACh diminished within 10 min, whereas the IACh of the control remained steady for over 20 min, suggesting that a PTX-sensitive G-protein is involved in the ACh response.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Cellular mechanism of acetylcholine-induced response in dissociated outer hair cells of guinea-pig cochlea. 750 5

The goal of this study was to characterize the receptors and coupling mechanisms mediating muscarinic inhibition of adenylyl cyclase activity in bovine tracheal smooth muscle. In radioligand binding experiments, methoctramine and AF-DX 116 competed for approximately 85% of the 3H-quinuclidynyl benzilate (3H-QNB) binding sites on intact cells with high affinities (-log KI of 7.73 +/- 0.16 and 6.67 +/- 0.31, respectively) characteristic of binding to M2 receptors. The antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) bound the receptors on intact cells with an affinity (-log KI = 7.76 +/- 0.21) characteristic of binding at M2 receptors. In experiments measuring 3',5'-cyclic adenosine monophosphate (cAMP) accumulation, methoctramine, AF-DX 116, and 4-DAMP antagonized the inhibitory effect of carbachol on isoproterenol-stimulated cAMP accumulation with potencies consistent with mediation by M2 muscarinic receptors (-log Kb of 8.01 +/- 0.22 to 7.58 +/- 0.25 for methoctramine; 7.43 +/- 0.36 to 7.02 +/- 0.30 for AF-DX 116; and 7.60 +/- 0.21 for 4-DAMP). In other experiments, 24 +/- 3% of the inhibitory effect of carbachol was not reversed by 60 min exposure to atropine. Moreover, pertussis toxin (10, 250, and 1,000 ng/ml) decreased only a portion of the inhibitory effect of carbachol (8 +/- 19%, 32 +/- 10%, and 33 +/- 8%, respectively) on cAMP accumulation. These findings indicated that M2 receptors were coupled to adenylyl cyclase in trachealis cells, but that coupling mechanisms in addition to those of pertussis toxin-sensitive guanine nucleotide binding proteins were involved. Since the inhibitory effect of carbachol (10(-8) M) on isoproterenol-stimulated cAMP accumulation was decreased from 20 +/- 4% to -1 +/- 5% (n = 6) by okadaic acid (1 microM), protein phosphatases may regulate the processes coupling muscarinic receptors to adenylyl cyclase.
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PMID:Muscarinic regulation of cyclic AMP in bovine trachealis cells. 762 89

In the present studies, the pharmacology and regulation of the functional muscarinic receptors on HSDM1C1 cells were probed using phosphoinositide (PI) turnover assays. In addition, the receptor binding of the putative M3-selective radioligand, [3H]4-DAMP, to cell homogenates was characterized. Carbachol (EC50 = 9 microM), (+)muscarine (EC50 = 4.5 microM) and cis-dioxolane (EC50 = 0.72 microM) were full agonists which stimulated PI turnover by 13.3 +/- 1.0 fold above basal values. The potencies of numerous agonists in this assay system were relatively similar to their affinities in receptor binding assays. Exposure of HSDM1C1 cells to 10 nM-10 microM muscarine during the last 24h of [3H]myo-inositol-labeling resulted in a concentration-dependent reduction in the cis-dioxolane affinity and maximal PI response induced by subsequent treatment with cis-dioxolane. Pertussis toxin (5-2000 ng/ml) caused a partial reduction in the cis-dioxolane-induced PI turnover. Likewise, exposure of the HSDM1C1 cells to an active phorbol ester (TPA) resulted in a partial inhibition of the cis-dioxolane-induced (100 microM) PI turnover. The half-maximal effect of TPA was produced at 1.8 +/- 0.3 nM. [3H]4-DAMP binding to cell homogenates was of high affinity (Kd = 0.19 +/- 0.04 nM) and moderate capacity (Bmax = 201 +/- 22 fmol/mg protein). The pharmacological specificity (4-DAMP > p-FHHSiD > dicyclomine > pirenzepine > methoctramine > AFDX-116 > gallamine) resembled that for [3H]NMS binding and correlated well with that observed for inhibition of PI turnover. These studies further support the identification of M3 receptors on HSDM1C1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:M3 muscarinic receptors on murine HSDM1C1 cells: further functional, regulatory, and receptor binding studies. 773 61

1. The effects of pertussis toxin (PTX) on contraction and/or relaxation induced by agonists or transmural nerve stimulation (TNS) were examined in the rat iris dilator and sphincter muscles. 2. TNS in the presence of phentolamine induced an atropine-sensitive biphasic response: initial contraction followed by relaxation in dilator muscles. Exogenously applied acetylcholine (ACh) elicited a large relaxation at low doses (3 microM or less) and a concentration at high doses. 3. Only the ACh-induced relaxation was affected by injection of PTX (10 ng) into the anterior eye chamber. Relaxation was decreased 12 h after injection and had completely disappeared after 24 h. Relaxation recovered in part 3 weeks and almost completely 8 weeks after PTX treatment. A gradual decrease in muscarinic relaxation in a dilator muscle was also observed in vitro after addition of PTX to the bathing solution. 4. The pA2 values of muscarinic blockers, pirenzepine, AF-DX 116, 4-DAMP, and himbacine for competitive antagonism to ACh-induced contraction were 7.14, 6.53, 9.03, and 6.80, respectively, in PTX-pretreated dilator muscles. These values are comparable to those obtained in parasympathectomized dilator muscles and may indicate involvement of M3 or M3-like receptors in muscle contraction. 5. Pretreatment with PTX did not significantly affect contraction induced by noradrenaline or 5-hydroxytryptamine or the relaxation induced by isoprenaline in dilator muscles. 6. In conclusion, among several agonist-induced responses in the rat iris dilator and sphincter muscles, only muscarinic relaxation in dilator muscle occurs via activation of PTX-sensitive GTP binding proteins.
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PMID:Pertussis toxin-sensitive muscarinic relaxation in the rat iris dilator muscle. 777 37


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