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 involvement of GTP binding proteins in muscarinic acetylcholine receptor (mAChR) mediated responses of cultured chick embryonic cardiac muscle cells was studied by using islet activating protein (IAP) from Bordetella pertussis. Incubation of cells for 24 h with IAP resulted in inhibition of subsequent IAP-catalyzed incorporation of [alpha-32P]ADP-ribose into membrane proteins of Mr 39 000 (No alpha) and 41 000 (Ni alpha); treatment of cultures with 5 ng/mL IAP was sufficient to ADP-ribosylate all available No alpha and Ni alpha. Inhibition of forskolin-stimulated cAMP accumulation by the muscarinic agonist carbachol was abolished in cultures pretreated with IAP. The affinity of carbachol for the mAChR in membranes from IAP-treated cells was considerably decreased compared to control membranes and was not further decreased by addition of guanyl-5'-yl imidodiphosphate. In contrast, the affinity of carbachol for the mAChR on intact cells was not affected by pretreatment with IAP. To investigate the involvement of No and/or Ni in mAChR-mediated increases in K+ permeability, the effect of IAP treatment on mAChR stimulation of 86Rb+ efflux was determined. Treatment of cultures with 5 ng/mL IAP for 24 h completely blocked the stimulation of 86Rb+ efflux evoked by carbachol. Because previous work has shown that mAChR regulation of K+ permeability is independent of changes in cAMP levels, these results suggest a role for No and/or Ni in coupling the mAChR directly to K+ channels in the heart.
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PMID:Islet activating protein inhibits physiological responses evoked by cardiac muscarinic acetylcholine receptors. Role of guanosine triphosphate binding proteins in regulation of potassium permeability. 241 67

The effect of short-term cholinergic desensitization on muscarinic acetylcholine receptor (mAChR)-mediated activation of phospholipase C was investigated in membranes isolated from the bovine iris sphincter smooth muscle. Membranes prepared from normal or desensitized muscles, prelabeled with either [3H]myo-inositol or 32P from [gamma-32P]ATP, were incubated with a hydrolysis-resistant analogue of GTP, GTP gamma S, or GTP gamma S plus carbachol (CCh), and the production of [3H]myo-inositol 1,4,5-trisphosphate (IP3) and the breakdown of polyphosphoinositides were assessed. In normal membranes, GTP (greater than or equal to 1 mM), GTP gamma S (greater than 10 microM) and GTP gamma S (1 microM) plus CCh (10 microM), but not GDP or GDP beta S, increased phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and IP3 production. GTP gamma S increased IP3 accumulation in a time- and dose-dependent manner, and CCh, which had no effect on phospholipase C activity in the absence of GTP gamma S, potentiated the effects of GTP gamma S. The effect of CCh plus GTP gamma S on IP3 production was inhibited by atropine, had an absolute requirement for nM amounts of Ca2+ and was not affected by pertussis toxin. At higher concentrations (greater than 1 microM), Ca2+ alone induced PIP2 hydrolysis. Short-term exposure (less than 60 min) of the muscle to CCh (100 microM) did not affect the total number (Bmax) of mAChRs nor their affinity (KD) for [3H]-N-methylscopolamine. Desensitization did, however, result in: (1) a loss of the CCh-high affinity binding state of the sphincter mAChRs in a manner analogous to that produced by GTP gamma S; (2) a loss of the ability of GTP gamma S to affect CCh binding to the receptors; and (3) an attenuation of the GTP gamma S plus CCh-stimulated PIP2 hydrolysis. In conclusion, the data presented suggest that, in the iris smooth muscle, G-proteins are involved in the coupling of mAChRs to phospholipase C and that short-term cholinergic desensitization results in (1) the uncoupling of the receptor-G-protein complex and (2) the attenuation of mAChR-activation of phospholipase C.
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PMID:Muscarinic-agonist and guanine nucleotide stimulation of myo-inositol trisphosphate formation in membranes isolated from bovine iris sphincter smooth muscle: effects of short-term cholinergic desensitization. 255 97

1. Smooth muscle fragments from the longitudinal layer of the small intestine of the guinea-pig were permeabilized with Staphylococcus aureus alpha toxin (alpha-toxin) and used to investigate the role of G-protein activation in the regulation of muscarinic acetylcholine receptor (AChR)-stimulated inositol phospholipid hydrolysis. 2. The efficiency of alpha-toxin permeabilization was estimated by the release of [3H]-2-deoxyglucose ([3H]-2DG) after prior loading or lactate dehydrogenase (LDH) enzyme release from the smooth muscle fragments. 3. In alpha-toxin-permeabilized smooth muscle, but not in non-permeabilized muscle, GTP gamma S induced time- and concentration-dependent increases in labelled inositol phosphates. Carbachol (CCh) increased labelled inositol phosphates in both permeabilized and non-permeabilized muscle, although the increases were greater in non-permeabilized smooth muscle. The response to 100 microM CCh was severely reduced by 0.5 microM atropine. 4. In permeabilized muscle the effects of GTP gamma S or CCh on inositol phosphate levels were reduced by treatment with pertussis toxin (PTX) and completely inhibited by GDP beta S. 5. GTP gamma S caused a concentration-dependent inhibition of the CCh-induced increases in the levels of labelled inositol phosphates. Dibutyryl cyclic AMP or Sp-cAMPs (adenosine-3',5'-cyclic phosphorothiolate-Sp) reduced the effects of CCh on inositol phosphate levels. 6. The results suggest that muscarinic AChR activation induces inositol phospholipid hydrolysis via more than one G-protein in this smooth muscle and that several mechanisms may contribute to the modulation of both stimulatory and inhibitory responses observed.
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PMID:Effects of GTP gamma S on muscarinic receptor-stimulated inositol phospholipid hydrolysis in permeabilized smooth muscle from the small intestine. 764 69

1. Aluminium fluoride (AlF), pertussis toxin (PTX) and cholera toxin (ChTX) have been used to examine the involvement of G-proteins during muscarinic acetylcholine receptor (AChR) stimulation of inositol phospholipid hydrolysis in fragments of longitudinal smooth muscle from the small intestine of the guinea-pig. 2. Carbachol (CCh) induced time- and concentration-dependent increases in [3H]-inositol monophosphates, [3H]-inositol (1,4) bisphosphate, [3H]-inositol (1,3,4) trisphosphate, [3H]-inositol (1,4,5) trisphosphate ([3H]-Ins (1,4,5)P3) and [3H]-inositol tetrakisphosphates measured by h.p.l.c. These increases were inhibited > 95% in the presence of the muscarinic AChR antagonist atropine (0.5 microM). 3. AlF transiently increased the basal levels of [3H]-Ins (1,4,5)P3 but increases in the levels of the other [3H]-inositol phosphates occurred more slowly. CCh-induced increases in the levels of all the [3H]-inositol phosphates were strongly inhibited in the presence of AlF. 4. PTX had no effect on basal levels of any of the [3H]-inositol phosphates but reduced the effects of CCh on these; ChTX had no effects on either basal or CCh-stimulated levels. 5. It was concluded that muscarinic AChR-stimulated increases in the levels of [3H]-inositol phosphates occur via both a PTX-sensitive G-protein and a PTX-insensitive mechanism. The actions of AlF may suggest the involvement of an inhibitory G-protein in the regulation of muscarinic AChR-stimulated inositol phospholipid turnover.
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PMID:G-protein involvement in muscarinic receptor-stimulation of inositol phosphates in longitudinal smooth muscle from the small intestine of the guinea-pig. 771 7

Intracellular free Ca2+ concentrations ([Ca2+]i) were measured in subclones of NL308 neuroblastoma x fibroblast hybrid cells expressing each of the individual muscarinic acetylcholine receptor (mAChR) subtypes m1, m2, m3 and m4. Application of 100 microM acetylcholine (ACh) increased [Ca2+]i in all four subclones. The increased [Ca2+]i levels were significantly higher in m1- and m3-transformed cells than those in m2- and m4-transformed cells. In more than 95% of m2- and m4-transformed cells, [Ca2+]i showed sinusoidal oscillations. ACh-induced increases in [Ca2+]i were not observed in cells treated with an intracellular Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Removal of extracellular Ca2+ with ethylene-glycol-bis-(beta- aminoethyl)-N,N,N',N'-tetraacetate (EGTA) did not affect the initial [Ca2+]i increases, but reduced the late phases of delta [Ca2+]i in ml- and m3-transformed cells by 20-30%. Oscillations in m2- and m4-transformed cells persisted in EGTA solution (though sometimes slowed in frequency), suggesting that they were of intracellular origin. ACh-induced delta [Ca2+]i and inositol 1,4,5-trisphosphate formation was completely suppressed by pre-treatment with 50-100 ng ml-1 Pertussis toxin (PTX) for 12 h in m2- and m4-transformed cells, but not in m1- and m3-transformed cells. In all cells, extracellular application of caffeine and ryanodine, or intracellular application of cyclic adenosine diphosphate ribose (cAD-PR) produced a rise in [Ca2+]i. ACh-induced [Ca2+]i oscillations were not observed in ryanodine-treated m2-transformed cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inositol 1,4,5-trisphosphate formation and ryanodine-sensitive oscillations of cytosolic free Ca2+ concentrations in neuroblastoma x fibroblast hybrid NL308 cells expressing m2 and m4 muscarinic acetylcholine receptor subtypes. 776 Dec 66

Pleckstrin homology (PH) domains are 90-110 amino acid regions of protein sequence homology that are found in a variety of proteins involved in signal transduction and growth control. We have previously reported that the PH domains of several proteins, including beta ARK1, PLC gamma, IRS-1, Ras-GRF, and Ras-GAP, expressed as glutathione S-transferase fusion proteins, can reversibly bind purified bovine brain G beta gamma subunits in vitro with varying affinity. To determine whether PH domain peptides would behave as antagonists of G beta gamma subunit-mediated signal transduction in intact cells, plasmid minigene constructs encoding these PH domains were prepared, which permit transient cellular expression of the peptides. Pertussis toxin-sensitive, G beta gamma subunit-mediated inositol phosphate (IP) production was significantly inhibited in COS-7 cells transiently coexpressing the alpha 2-C10 adrenergic receptor (AR) and each of the PH domain peptides. Pertussis toxin-insensitive, Gq alpha subunit-mediated IP production via coexpressed M1 muscarinic acetylcholine receptor (M1 AChR) was attenuated only by the PLC gamma PH domain peptide, suggesting that the inhibitory effect of most of the PH domain peptides was G beta gamma subunit-specific. Stimulation of the mitogen-activated protein (MAP) kinase pathway by Gi-coupled receptors in COS-7 cells has been reported to require activation of p21ras and to be independent of protein kinase C. Since several proteins involved in activation contain PH domains, the effect of PH domain peptide expression on alpha 2-C10 AR-mediated p21ras-GTP exchange and MAP kinase activation as well as direct G beta gamma subunit-mediated activation of MAP kinase was determined. In each assay, coexpression of the PH domain peptides resulted in significant inhibition. Increasing G beta gamma subunit expression surmounted PH domain peptide-mediated inhibition of MAP kinase activation. These data suggest that the PH domain peptides behave as specific antagonists of G beta gamma-mediated signaling in intact cells and that interactions between PH domains and G beta gamma subunits or structurally related proteins may play a role in the activation of mitogenic signaling pathways by G protein-coupled receptors.
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PMID:Effect of cellular expression of pleckstrin homology domains on Gi-coupled receptor signaling. 776 89

The effects of muscarinic acetylcholine receptor stimulation on phosphoinositides breakdown and adenylate cyclase activity were examined in the circular smooth muscle of the rabbit caecum. In Myo-[3H]inositol-labeled circular smooth muscle cells, carbachol caused a concentration-dependent increase in [3H]inositol phosphates ([3H]IPs) accumulation (EC50 of 3 +/- 1 microM). The M1-selective antagonist pirenzepine (PRZ), the M2-selective AF-DX 116 (11-2[[2-[(diethyl-amino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6Hypyrido[2,3-b][1,4]benzodiazepin-6-one) and the M3-selective para-fluoro-hexahydrosiladifenidol (p-F-HHSiD) inhibited the carbachol-induced [3H]inositol phosphates accumulation with the following order of potency; p-F-HHSiD > PRZ > AF-DX 116. In saponin-permeabilized circular smooth muscle cells, carbachol and GTP gamma [S] elicited a concentration-dependent increase in [3H]inositol phosphates accumulation. The concentration-response curve for GTP gamma [S] was shifted to the left when cells were incubated with 1 microM carbachol. The [3H]inositol phosphates accumulation elicited by simultaneous addition of 0.1 microM GTP gamma [S] and 1 microM carbachol to permeabilized cells was significantly decreased (78.28 +/- 18.23% inhibition) when cells were preincubated for 5 min with 0.1 mM GDP beta [S]. In nonpermeabilized cells, pertussis toxin did not alter the carbachol-induced increase in [3H]inositol phosphates accumulation. On the other hand, the 0.1 mM carbachol-induced inhibition of forskolin-stimulated adenylate cyclase activity in circular smooth muscle homogenates was significantly reversed by atropine and AF-DX 116, whereas PRZ and p-F-HHSiD were ineffective (muscarinic antagonists were used at 1 microM final concentration).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Signal transduction pathways of muscarinic receptors in circular smooth muscle from the rabbit caecum. 787 99

Previous studies have shown that a single type of transmembrane receptor is able to regulate multiple effectors through the activation of heterotrimeric G proteins. For example, the m2 muscarinic acetylcholine receptor (mAChR) expressed in Chinese hamster ovary (CHO) cells inhibits adenylyl cyclase, stimulates phospholipase C-dependent intracellular Ca2+ release, and activates phospholipase A2 through pertussis toxin-sensitive G proteins. However, it is unclear whether multiple effector enzymes can be regulated by one type of heterotrimeric G protein within a single cell. To investigate this question, we constructed a derivative of G alpha i3 (termed G alpha i3 C > S) in which the carboxyl-terminal cysteine residue, the site for pertussis toxin modification, was changed to a serine. Following pertussis toxin treatment of transfected CHO cells expressing the m2 mAChR, we found that the G alpha i3 C > S protein underwent guanine nucleotide exchange in response to the muscarinic agonist carbachol, while the m2 mAChR failed to activate the endogenous G alpha i2 and G alpha i3 proteins. Moreover, coupling of heterotrimeric G proteins containing G alpha i3 C > S to the m2 mAChR resulted in pertussis toxin-resistant inhibition of adenylyl cyclase, stimulation of phospholipase C-induced intracellular Ca2+ release, and phospholipase A2-mediated arachidonic acid release. Therefore, these studies provide conclusive evidence that heterotrimeric G proteins containing just G alpha i3 can regulate multiple effector enzymes within the same cell type.
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PMID:Heterotrimeric G proteins containing G alpha i3 regulate multiple effector enzymes in the same cell. Activation of phospholipases C and A2 and inhibition of adenylyl cyclase. 796 42

The modulatory effects of pertussis toxin pretreatment on responses mediated via beta-adrenoceptors and muscarinic acetylcholine receptors were investigated in isolated rat hearts and aortic rings 4 days after in vivo administration of pertussis toxin. In isolated hearts, pertussis toxin increased heart weight and baseline coronary flow values but did not effect baseline left ventricular pressure values. In unpaced hearts, pertussis toxin inhibited the arecoline-induced cardiac standstill, while in paced hearts, the beta 2-adrenoceptor agonist salbutamol produced a dose-dependent vasodilation with similar characteristics in pertussis toxin and control preparations. Pertussis toxin had no effect on myocardial or aortic cyclic nucleotide levels and the myocardial beta-adrenoceptor density (Bmax) and dissociation constant (Kd). In precontracted aortic rings, pertussis toxin had no effect on the salbutamol or arecoline induced vasorelaxation. In summary, we demonstrated a reduced cholinergic responsiveness in isolated hearts but an intact beta 2-adrenoceptor pathway in isolated hearts as well as in isolated aortic rings after pertussis toxin pretreatment. In aortic rings no change in muscarinic acetylcholine receptor responsiveness occurred.
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PMID:Modulation by pertussis toxin of salbutamol- and arecoline-induced effects in the isolated heart and aorta of the rat. 811 1

Regulation of effector functions by muscarinic acetylcholine receptor subtypes is mediated by pertussis toxin-sensitive and -insensitive G proteins. In membranes from human embryonic kidney 293 cells transfected with m1, m2, and m3 muscarinic acetycholine receptors, we detected the pertussis toxin-sensitive G proteins Gi1, Gi2, and Gi3 and the pertussis toxin-insensitive G proteins Gq/11 and Gs. Subtype-specific immunoprecipitation of G protein alpha subunits photolabeled with [alpha-32P] GTP azidoanilide, in the absence and presence of carbachol, revealed the selective coupling of activated muscarinic receptors to G protein subtypes. Gq/11 was activated via m1 and m3 receptors and Gi2 was activated via m2 receptors. All three receptors subtypes mediated the activation of Gi1 and Gi3. Effective activation of Gi1 and Gi3 via m1 and m3 receptors occurred only at high carbachol concentrations (EC50 about 10-20 microM), whereas carbachol with higher potency (EC50 about 1 microM) induced activation of all G1 subtypes via m2 receptors. Thus, coupling of muscarinic receptors and G protein subtypes was principally selective; however, activation of distinct G protein subtypes by different muscarinic receptors occurred with different efficacies.
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PMID:Transfected muscarinic acetylcholine receptors selectively couple to Gi-type G proteins and Gq/11. 819 Jan 5


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