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)

The purpose of our study was to investigate the interactions of allosteric antagonists at the individual m1, m2 and m3 muscarinic receptor subtypes. This was achieved through the use of transformed Chinese hamster ovary cells stably expressing the rat m1 or m3 receptor genes. A homogeneous population of the m2 subtype was obtained from rat heart tissue. Our data indicate that the cardioselective antagonists (gallamine, methoctramine, AF-DX 116 and himbacine) display the following rank order of potency for both displacing ligand binding to the primary site on the receptor and allosterically decelerating ligand dissociation: m2 greater than m1 greater than m3. Schild analysis showed the following rank order of the magnitude of gallamine's cooperative interactions with the three receptor subtypes: m3 greater than m1 greater than m2. By comparison, the ion-channel blockers (verapamil, phencyclidine and quinidine) exhibited a rank order of potency for cooperative effects similar to that of cardioselective antagonists; however, these blockers did not show appreciable specificity in their interaction with the receptor primary binding site. There was a lack of correlation between the displacement of ligand binding and the allosteric potencies of the allosteric antagonists at each of the three muscarinic receptor subtypes, thus revealing the complex nature of interaction (both competitive and allosteric) between many of these compounds with the muscarinic receptor. Despite the fact that the majority of allosteric muscarinic antagonists are also K+ channel blockers, the use of pertussis toxin did not support the notion that this channel represents the allosteric site coupled to the receptor.
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PMID:Allosteric interactions at the m1, m2 and m3 muscarinic receptor subtypes. 199 91

We have studied Ca2+ mobilization mediated by the constitutively expressed muscarinic receptor on a subclone of PC-12 cells. The subclone, ACH2, was isolated with a flow cytometer by selection of single cells that exhibited a strong intracellular Ca2+ response to acetylcholine (ACh). Cell to cell heterogeneity of resting Ca2+ levels was markedly reduced in the subclone and homogeneity of the population response was also dramatically improved. ACH2 cells were highly sensitive to ACh and the Ca2+ response in all cells was blocked by muscarinic antagonists. Membranes from ACH2 exhibited muscarinic binding affinities which were not typical of M1, M2, or M3 receptors but were consistent with the profile of the putative m4 receptor. The same percentage of cells responded to ACh whether or not extracellular Ca2+ was reduced with EGTA, but the response was eliminated in all cells by preincubation with pertussis toxin. Thus, the constitutive m4 receptor on ACH2 cells is efficiently coupled to intracellular Ca2+ release by a pertussis toxin-sensitive mechanism. Stimulation of the ACH2 cells by bradykinin (BK) evoked a Ca2+ response in 90% of the cells. Prestimulation with BK diminished the magnitude of the muscarinic Ca2+ response but did not reduce the number of cells which responded to ACh. Inhibition was partially attributed to inhibition of a Ca2+ influx pathway in resting cells. Thus, the signaling mechanism coupled to the m4 muscarinic receptor can be inhibited by signals initiated by the BK receptor.
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PMID:Characterization of the m4 muscarinic receptor Ca2+ response in a subclone of PC-12 cells by single cell flow cytometry. Inhibition of the response by bradykinin. 205 Jun 75

Carbachol produces both negative and positive inotropy in rat left atria. It is not clear whether these two effects are mediated by two separate cell surface muscarinic receptors or a single receptor interacting with two coupling proteins in the cell membrane. Pirenzepine, known to selectively block some biochemical muscarinic responses, was used in this study to block the biphasic response to carbachol in rat left atria. The negative inotropy to carbachol was blocked by pirenzepine, and Schild analysis indicated a -log dissociation constant (pKb) for the pirenzepine-receptor complex of 6.2. However, the Schild analysis may have been complicated by positive inotropy observed with pirenzepine. This positive inotropic effect was sensitive to blockade by other muscarinic antagonists. In atria from rats pretreated with pertussis toxin, carbachol produced a positive inotropic effect. Schild analysis with pirenzepine for antagonism of this response indicated a -log equilibrium dissociation constant for the pirenzepine-receptor complex of 6.7, significantly different from that for antagonism of negative inotropy. This ostensibly suggested a difference in the receptors mediating these responses. In view of the possible complicating effects of the positive inotropic effects of pirenzepine in this assay, an alternative method for the measurement of pirenzepine affinity was utilized. Resultant analysis was used to measure the pKb for pirenzepine antagonism of negative inotropy to carbachol. This method had the advantage of cancelling the positive inotropy to pirenzepine. Under these circumstances, pirenzepine had a pKb of 6.9, a value not significantly different from for antagonism of the positive inotropy to carbachol. The relevance of these findings is discussed in terms of a single promiscuous muscarinic receptor or heterogeneous receptors in this tissue. These data do not support the hypothesis that two separate receptors mediate these two effects.
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PMID:Promiscuous or heterogeneous muscarinic receptors in rat atria? II. Antagonism of responses to carbachol by pirenzepine. 209

Receptors stimulating phospholipase C do so through heterotrimeric GTP-binding proteins to produce two second messengers, inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol. In spite of the detailed understanding of phospholipase C structure and phosphatidyl inositol signalling, the identity of the GTP-binding protein involved is so far unknown. To address this issue, we have used the Xenopus oocyte in which muscarinic receptors couple to phospholipase C through a pertussis toxin-sensitive GTP-binding protein. In this cell, InsP3 mobilizes intracellular Ca2+ to evoke a Cl- current. The magnitude of this Cl- current is proportional to the amount of InsP3 in the cell, and therefore can be used as an assay for InsP3 production. We report here that the activated alpha-subunit of the GTP-binding protein GO, when directly injected into oocytes, evokes a Cl- current by mobilizing Ca2+ from intracellular InsP3-sensitive stores. We also show that holo-GO, when injected into oocytes, can specifically enhance the muscarinic receptor-stimulated Cl- current. These data indicate that GO can serve as the signal transducer of the receptor-regulated phospholipase C in Xenopus oocytes.
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PMID:Go protein as signal transducer in the pertussis toxin-sensitive phosphatidylinositol pathway. 210 59

These studies demonstrate a novel mechanism for the coupling of the muscarinic receptor to phospholipase C activity in embryonic chick atrial cells. In monolayer cultures of atrial cells from hearts of embryonic chicks at 14 days in ovo, carbamylcholine stimulated the sequential appearance of InsP3, InsP2 and InsP1 with an EC50 (concn. causing 50% of maximal stimulation) of 30 microM. In the presence of 15 mM-Li, a 5 min exposure to carbamylcholine (0.1 mM) increased InsP3 levels to a maximum of 47 +/- 12% over basal, InsP2 to 108 +/- 13% over basal and InsP1 to 42 +/- 5% over basal. This effect was blocked by 5 microM-atropine. Incubation of these cells with pertussis toxin (15 h; 0.5 ng/ml) inhibited carbamylcholine-stimulated InsP3, InsP2 and InsP1 formation by 42 +/- 7%, 30 +/- 3% and 48 +/- 7% respectively. The IC50 (concn. causing 50% inhibition) for pertussis toxin inhibition of all three inositol phosphates was 0.01 ng/ml, with a half-time of 6 h at 0.5 ng/ml. This partial sensitivity to pertussis toxin was not due to incomplete ADP-ribosylation of the guanine-nucleotide-binding protein (G-protein), since autoradiography of polyacrylamide gels of cell homogenates incubated with [32P]NAD+ in the presence of pertussis toxin demonstrated that incubation of cells with 0.5 ng of pertussis toxin/ml for 15 h resulted in complete ADP-ribosylation of pertussis toxin substrates by endogenous NAD+. In cells permeabilized with saponin (10 micrograms/ml), 0.1 mM-GTP[S] (guanosine 5'-[gamma-thio]triphosphate) stimulated InsP1 by 102 +/- 15% (mean +/- S.E.M., n = 4), InsP2 by 421 +/- 67% and InsP3 by 124 +/- 33% above basal. Incubation of cells for 15 h with 0.5 ng of pertussis toxin/ml decreased GTP[S]-stimulated InsP1 production in saponin-treated cells by 30 +/- 10% (n = 3), InsP2 production by 45 +/- 7% (n = 4) and InsP3 production by 49 +/- 6% (n = 4). These data demonstrate that in embryonic chick atrial cells at least two independent G-proteins, a pertussis toxin-sensitive G-protein and a pertussis toxin-insensitive G-protein, play a role in coupling muscarinic agonist binding to phospholipase C activation and to inositol phosphate production.
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PMID:Muscarinic cholinergic stimulation of inositol phosphate production in cultured embryonic chick atrial cells. Evidence for a role of two guanine-nucleotide-binding proteins. 212 87

We have demonstrated that muscarinic stimulation of inositol phosphate production in cultured atrial cells from chicks at 14 days in ovo is partially sensitive to inhibition by pertussis toxin. In these cells, muscarinic agonist binding is coupled to phospholipase C activity via at least two guanine-nucleotide-binding proteins (G-proteins), one sensitive to pertussis toxin and the other (Gp) insensitive to pertussis toxin [Barnett, Shamah, Lassegue, Griendling & Galper (1990) Biochem. J. 271, 437-442]. In the current study we demonstrate that during embryonic development of the chick heart, muscarinic stimulation of inositol phosphate production decreases by 50% between days 5 and 14 in ovo in cells cultured from both atrium and ventricle. In atrial cells, however, pertussis toxin-sensitive muscarinic stimulation of inositol phosphate production increased from undetectable levels at day 5 in ovo to 40% of total stimulation at day 12 in ovo. Muscarinic stimulation of inositol phosphate production in the ventricle did not become sensitive to pertussis toxin at any age studied. In permeabilized atrial cells from embryonic chicks at 5 days in ovo, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated InsP1 levels by 40 +/- 10% (mean +/- S.E.M., n = 3), InsP2 levels by 117 +/- 18% and InsP3 levels by 51 +/- 8%, suggesting that at day 5 in ovo all of the muscarinic-stimulated inositol phosphate production was coupled to phospholipase C via Gp. H.p.l.c. analysis demonstrated that, in spite of these changes in coupling of phospholipase C to different G-proteins, no changes could be demonstrated in the isomers of InsP3 produced in response to carbamylcholine at both days 5 and 14 in ovo. These data demonstrate that embryonic development of the chick atrium is associated with a switch in coupling of muscarinic receptors to phospholipase C from Gp to a pertussis toxin substrate. This developmental switch in coupling of G-proteins may be related to possible developmental switches in levels of muscarinic receptor isoforms or switches in the subtype of phospholipase C.
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PMID:Development of muscarinic-cholinergic stimulation of inositol phosphate production in cultured embryonic chick atrial cells. Evidence for a switch in guanine-nucleotide-binding protein coupling. 212 88

Guanine nucleotide binding proteins (G proteins) sensitive to pertussis toxin (PTX) mediate the muscarinic receptor responses in several tissues. Therefore, the present study sought to investigate whether smooth muscle contractions and/or endothelium-dependent relaxations in response to acetylcholine (ACh) and other agonists were sensitive to PTX. In endothelium-denuded rabbit pulmonary artery rings, ACh, clonidine and serotonin produced concentration-dependent contractions which were markedly inhibited in nominally Ca+(+)-free medium and abolished in the presence of ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (0.2 mM). In endothelium-denuded arterial rings obtained from rabbits treated in vivo with PTX (5 micrograms/kg i.v., 5 days before sacrifice) maximum contractions to ACh, clonidine and serotonin were inhibited by 77, 67 and 35%, respectively. Contractions induced with KCl (10-40 mM) were also abolished in Ca+(+)-free medium, but they were not affected by PTX. Endothelium-dependent relaxations of phenylephrine-contracted pulmonary arteries in response to ACh adenosine triphosphate and substance P were also reduced or abolished upon removal of extracellular Ca++. However, the endothelium-dependent relaxations were not affected by PTX. These data demonstrate that contractions of pulmonary arterial smooth muscle cells after stimulation through muscarinic receptors, alpha adrenoceptors and serotonin receptors require the influx of extracellular Ca++. This receptor-stimulated Ca++ influx is likely to be regulated by a PTX-sensitive G protein. Also, the induction of release of relaxing factor from endothelial cells of the pulmonary artery via muscarinic, purinergic or substance P receptors requires extracellular Ca++. However, in these cells, a different mode of signal transduction, insensitive to PTX, seems to be involved.
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PMID:Pertussis toxin inhibits contractions but not endothelium-dependent relaxations of rabbit pulmonary artery in response to acetylcholine and other agonists. 215 2

The mechanism of phospholipase C regulation by inhibitory receptors was analyzed both in intact and in permeabilized rat thyroid cells (FRTL5). In this system, the muscarinic agonist carbachol inhibited phospholipase C, as indicated by the decrease in the basal levels of inositol 1,4,5-trisphosphate as well as by the reduced adrenergic stimulation of phosphoinositol accumulation, which was paralleled by a fall in the cytosolic Ca2+ levels. This inhibition involved an M2 muscarinic receptor because it was abolished by atropine but not by the M1 antagonist pirenzepine. Cells pretreated with pertussis toxin were not responsive to carbachol, indicating the involvement of a guanine nucleotide-binding protein in this inhibitory process. This possibility was further evaluated in permeabilized cells, where the carbachol inhibition was shown to be completely dependent on GTP. Known second messengers were not involved in this inhibitory process since Ca2+, cAMP, and activators of protein kinases were not able to mimic or prevent the carbachol effect either in intact or in permeabilized FRTL5 cells. In this system, the phospholipases C and A2 are coupled to two classes of muscarinic receptors that display a different sensitivity to pertussis toxin. The carbachol inhibitory effect occurred under conditions that prevented activation of phospholipase A2, excluding a role of the arachidonic acid metabolism in this process. Taken together these data provide the strongest support to date that an inhibitory guanine nucleotide-binding protein sensitive to pertussis toxin can directly mediate receptor-induced inhibition of phospholipase C.
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PMID:Evidence that a guanine nucleotide-binding protein linked to a muscarinic receptor inhibits directly phospholipase C. 216 60

Although adenosine is known to activate K+ conduction in atrial tissue, there is still debate as to the involvement of cAMP-dependent mechanisms. In isolated adult guinea pig atrial myocytes, we demonstrate that the highly A1-selective adenosine receptor agonist 2-chloro-N6-cyclopentyladenosine reduced basal cAMP levels by 30-40% in the absence and presence of the nonxanthine phosphodiesterase inhibitor Ro 20-1724. Isoprenaline caused a concentration-dependent increase in cAMP levels, which was more pronounced in the presence of the phosphodiesterase inhibitor. Several adenosine derivatives suppressed the isoprenaline-induced cAMP increase by approximately 80%. The rank order of potency was 2-chloro-N6-cyclopentyladenosine (IC50, 93 nM) greater than (R)-N6-phenylisopropyladenosine (IC50, 309 nM) greater than 5'-N-ethylcarboxamidoadenosine (IC50, 813 nM) much greater than (S)-N6-phenylisopropyladenosine (IC50, 26,300 nM). A similar but complete suppression of the isoprenaline-induced cAMP increase was produced by the muscarinic receptor agonist carbachol (IC50, 398 nM), which like adenosine is known to activate atrial K+ channels. The A1-adenosine receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine antagonized the effect of 2-chloro-N6-cyclopentyladenosine concentration-dependently, with a KB value of 9.6 nM. In atrial myocytes isolated from guinea pigs pretreated with pertussis toxin, the inhibitory effects of adenosine analogs on basal and isoprenaline-stimulated cAMP accumulation were markedly attenuated. It is concluded that the adenosine receptor in guinea pig atrial myocytes, which is known to be linked to K+ channels, is also coupled to adenylate cyclase via a pertussis toxin-sensitive guanine nucleotide-binding protein and shows the characteristics of the A1-adenosine receptor subtype.
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PMID:Pharmacological characterization of the adenylate cyclase-coupled adenosine receptor in isolated guinea pig atrial myocytes. 216 17

A selective amplification of the coding sequence of the rat M2 muscarinic receptor gene was achieved by the polymerase chain reaction. The error rate of this amplification system under conditions specified was 1 nucleotide substitution in 841 base pairs. In vitro expression of this gene in murine fibroblasts (B82) via the eukaryotic expression vector, pH beta APr-1-neo, resulted in high level expression of specific [3H] (-)MQNB binding in transfected B82 cell lines. One of these clones, M2LKB2-2, showed a stable expression of [3H] (-)MQNB binding with a Kd value of 265 pM and a Bmax value of 411 +/- 50 fmol/10(6) cells. Cardiac selective muscarinic antagonists such as himbacine and AF-DX 116 show high affinities for this binding site in the M2LKB2-2 cells. The rank order of potency of several antagonists in inhibiting [3H] (-)MQNB binding in these cells conformed to the characteristics of an M2 type muscarinic receptor. Carbachol showed a single affinity state for the receptors in the M2LKB2-2 cells with a Ki value of 2.0 microM. This receptor appeared to be inversely coupled to adenylate cyclase via a pertussis toxin sensitive G-protein. Carbachol also had a slight stimulatory effect on the hydrolysis of inositol lipids. The polymerase chain reaction proves highly effective in cloning genes from genomic material, as demonstrated by the first in vitro functional expression of the rat M2 type muscarinic receptor.
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PMID:Amplification of the rat M2 muscarinic receptor gene by the polymerase chain reaction: functional expression of the M2 muscarinic receptor. 217 74


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