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

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.
 ...
PMID:Promiscuous or heterogeneous muscarinic receptors in rat atria? II. Antagonism of responses to carbachol by pirenzepine. 209

On separation of rat pancreatic plasma membrane proteins by two-dimensional gel electrophoresis, 15 GTP-binding protein (G-protein) alpha-subunits could be detected immunochemically using an alpha common antibody. These consisted of five 48 kDa proteins (pI 5.70, 5.80, 5.90, 6.10 and 6.25) and five 45 kDa proteins (pI 5.90, 6.05, 6.25, 6.30 and 6.70), presumably corresponding to low- and high-molecular mass forms of the Gs-protein, as well as three 40/41 kDa proteins (pI 5.50, 5.70 and 6.00) and two 39 kDa proteins (pI 5.50 and 6.00). All of these proteins except for the more acidic 39 kDa protein were ADP-ribosylated by cholera toxin (CT). In addition, the three 40/41 kDa proteins and the more alkaline 39 kDa protein were also ADP-ribosylated by pertussis toxin (PT). CT- and PT-induced ADP-ribosylation changed the pI values of G-protein alpha-subunits by 0.2 pI units to more acidic values. Preincubation of isolated pancreatic membranes with cholecystokinin octapeptide (CCK-OP), which stimulates phospholipase C in acinar cells, decreased CT-induced as well as PT-induced ADP-ribosylation of the three 40/41 kDa proteins, whereas CT-induced ADP-ribosylation of one 45 kDa (pI 5.80) and all 48 kDa proteins was enhanced in the presence of CCK. Carbachol, another stimulant of phospholipase C, had no effect. The three 40/41 kDa proteins and one 48 kDa protein could be labelled with the GTP analogue [alpha-32P]GTP-gamma-azidoanilide. CCK, but not carbachol, stimulated incorporation of the GTP analogue into all of these four proteins. Using different anti-peptide antisera specific for alpha-subunits of G-proteins we identified the three 40/41 kDa Gi-proteins as Gi1 (pI 6.00), Gi2 (pI 5.50) and Gi3 (pI 5.70). The Gi3-protein was found to be the major Gi-protein of pancreatic plasma membranes. One of the 39 kDa proteins (pI 6.0) was identified as Go. These results indicate that CCK receptors functionally interact with six Gs-proteins and with Gi1, Gi2 and Gi3-proteins. Since evidence suggests that a 40/41 kDa CT substrate is involved in the stimulation of phospholipase C in pancreatic acinar cells, it is likely that one, two or all three 40/41 kDa Gi-proteins are involved in the coupling of CCK receptors with phospholipase C.
 ...
PMID:Cholecystokinin activates Gi1-, Gi2-, Gi3- and several Gs-proteins in rat pancreatic acinar cells. 211 41

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.
 ...
PMID:Amplification of the rat M2 muscarinic receptor gene by the polymerase chain reaction: functional expression of the M2 muscarinic receptor. 217 74

The cloning and functional expression of five mammalian muscarinic acetylcholine receptor genes (m1-m5) has revealed that m1, m3, and m5 primarily couple to stimulation of phosphoinositide (PI) turnover, whereas m2 and m4 are strongly linked to inhibition of adenylate cyclase, albeit not exclusively. To identify the structural domains responsible for this functional specificity, cDNAs encoding chimeric m2/m3 receptors were constructed. The abilities of these receptors to mediate stimulation of PI hydrolysis and inhibition of prostaglandin E2-stimulated cAMP accumulation, as well as the pertussis toxin (PTX) sensitivity of these responses, were examined after stable expression in mouse A9 L cells. Substitution of the putative third cytoplasmic loop (i3) of m2 with the corresponding m3 sequence resulted in a chimeric receptor that, similar to m3, stimulated PI breakdown by a PTX-insensitive mechanism but did not inhibit adenylate cyclase. Conversely, a chimeric m3 receptor containing the i3 domain of m2 showed the same functional profile as m2 (i.e., inhibition of adenylate cyclase and weak stimulation of PI turnover by a PTX-sensitive mechanism), indicating that the i3 loop is sufficient to determine coupling selectivity. Similarly, exchange of a short N-terminal segment of i3 (16 or 17 amino acids) between m2 and m3 resulted in chimeric receptors that gained the ability to mediate the functional responses of the wild-type receptor from which the segment was derived, although with substantially reduced efficiency. However, the chimeric m2 receptor containing the 17-amino acid m3 sequence in the N-terminal portion of i3 retained its ability to inhibit adenylate cyclase. Carbachol binding studies involving the use of the GTP analog 5'-guanylyl imidodiphosphate and PTX-pretreated cells generally correlated well with the functional findings. Our data indicate that the N-terminal portion of i3 is a sufficient but not the exclusive determinant of coupling selectivity displayed by the various muscarinic receptors.
 ...
PMID:Delineation of muscarinic receptor domains conferring selectivity of coupling to guanine nucleotide-binding proteins and second messengers. 217 67

The present study reports the differential effects of pertussis toxin on muscarinic regulation of intracellular Ca2+ and inositol phosphate generation and alpha 2-adrenoceptor-mediated inhibition of cAMP formation in SH-SY5Y human neuroblastoma cells. Carbachol caused a biphasic increase in intracellular Ca2+ (release of internal stores and Ca2+ entry) and a dose-dependent increase in inositol phosphate formation. Pertussis toxin pretreatment did not affect either of these components of the signal transduction pathway but did completely reverse the alpha 2-adrenoceptor-mediated inhibition of forskolin-stimulated cAMP formation. These data indicate that muscarinic regulation of inositol phosphate generation occurs via a pertussis toxin-insensitive G-protein and that Ca2+ entry in these cells may not occur via a G-protein.
 ...
PMID:Pertussis toxin inhibits alpha 2-adrenoceptor-mediated inhibition of adenylate cyclase without affecting muscarinic regulation of [Ca2+]i or inositol phosphate generation in SH-SY5Y human neuroblastoma cells. 217 26

The effects of the cholinergic agonist carbachol on ouabain-sensitive K(+)-activated 4-nitrophenylphosphatase (K(+)-O2NPhPase) activity of rabbit and pig ventricular sarcolemma were examined. Carbachol (0.01-1000 microM) alone had no effect on K(+)-O2NPase. However, in the presence of GTP (100 microM) or its analog guanosine 5'-[gamma-thio]triphosphate (GTP[S], 1 microM) the agonist reduced this enzymatic activity (IC50 = 0.3 microM) by about 45% in a concentration-dependent manner. The GTP[S]-dependent effect of carbachol was blocked by 10 microM atropine, an antagonist of muscarinic acetylcholine receptor (mAcChoR). In the presence of micromolar concentrations of ATP or the GDP analog guanosine 5'-[beta-thio]diphosphate, carbachol did not change sarcolemmal K(+)-O2NPhPase activity. GTP[S] alone reduced this activity (IC50 = 2 microM) by about 40% in a concentration-dependent manner with a lag period of about 3 min. This lag disappeared in the presence of carbachol. Treatment of sarcolemmal membranes with 20 micrograms/ml pertussis toxin, which catalyzed ADP-ribosylation of the 40-41-kDa alpha-subunits of inhibitory GTP-binding protein (Gi), abolished the GTP[S]-promoted inhibitory effect of carbachol. Immunochemically, these alpha-subunits were identified as alpha 12- and alpha i3-subunits. It is suggested that the carbachol-induced inhibition of ouabain-sensitive K(+)-O2NPhPase activity of mammalian myocardial sarcolemma is a result of a negative coupling between mAcChoR and Na+/K(+)-ATPase via Gi protein.
 ...
PMID:Involvement of pertussis-toxin-sensitive G protein in muscarinic-receptor-mediated inhibition of K(+)-activated 4-nitrophenylphosphatase activity of cardiac sarcolemma. 217 73

STa, the heat-stable enterotoxin of Escherichia coli, stimulates membrane-bound guanylate cyclase in enterocytes, elevates cyclic GMP, and results in intestinal secretion of ions and fluid. Using the T84 colon carcinoma cell line as a model. Weikel et al. reported that phorbol esters enhance STa-stimulated cyclic GMP production by 60-140% [(1990) Infect. Immun. 58, 1402-1407]. In the present report we demonstrate that the acetylcholine analog carbachol enhanced toxin-stimulated cyclic GMP accumulation in intact T84 cells by 50-100% and that this effect was blocked by 10 microM atropine and 10 microM sphingosine. Pertussis toxin treatment of the T84 cells did not affect the subsequent response to carbachol. Carbachol, which elevates intracellular calcium in these cells, may act through protein kinase C to enhance cyclic GMP production.
 ...
PMID:Carbachol mimics phorbol esters in its ability to enhance cyclic GMP production by STa, the heat-stable toxin of Escherichia coli. 217 3

Atrial and ventricular adenylate cyclase activity and atrial membrane potentials were measured in hearts from hatched chicks at 2-3 days after intravenous administration of pertussis toxin (0.5-1.0 micrograms, total) or saline. Both in atrium and ventricle, treatment with pertussis toxin antagonized inhibition by carbachol of basal and isoproterenol-stimulated adenylate cyclase activity without changing either basal or isoproterenol-stimulated adenylate cyclase. In atria from pertussis toxin-treated animals (5.4 mM potassium), carbachol hyperpolarized the resting membrane by 0.3 +/- 0.3 mV (n = 9) and did not increase resting potassium conductance. In contrast, carbachol hyperpolarized the resting membrane by 4.5 +/- 0.8 mV (n = 11) and increased resting potassium conductance more than 4-fold in saline-treated animals. Carbachol did not significantly affect the atrial action potential peak or duration at 50% repolarization of pertussis toxin-treated animals. This muscarinic agonist reduced action potential peak by 7.8 +/- 1.2 mV and the duration at 50% repolarization by 22.1 +/- 3.0 msec in atria from saline-treated animals. Pertussis toxin treatment also prevented the negative inotropic effect and the inhibition of calcium-dependent action potentials caused by carbachol in atrial muscle. Neither the affinity nor the maximal specific binding of [3H]quinuclidinyl benzilate in ventricular homogenates was changed by pertussis toxin treatment. The apparent affinity of carbachol for muscarinic receptor was slightly (approximately 2-fold) diminished in pertussis toxin-treated animals. The inhibition of carbachol-induced hyperpolarization by pertussis toxin treatment implicates a guanosine 5'-triphosphate-dependent protein (Ni or a similar protein) as an essential link that permits muscarinic receptor to regulate atrial potassium channels.
 ...
PMID:Pertussis toxin treatment blocks hyperpolarization by muscarinic agonists in chick atrium. 241 29

Desensitization of the responsiveness to hormones or drugs is often mediated by down-regulation of receptors. The stimulatory coupling protein (Ns) of adenylate cyclase has been shown to be involved in the down-regulation of stimulatory beta-adrenergic receptors. Whether the inhibitory coupling protein (Ni) is involved in the down-regulation of receptors that inhibit adenylate cyclase is not known. We wished to determine whether down-regulation of inhibitory muscarinic cholinergic and alpha 2-adrenergic receptors occurs in neuroblastoma X glioma hybrid cells after the ability of Ni to inhibit adenylate cyclase is inactivated by pertussis toxin. After treatment of cells with pertussis toxin, the ability of carbachol or epinephrine to inhibit prostaglandin E1-stimulated cAMP accumulation in intact cells was either completely prevented or markedly attenuated, respectively, indicating functional inactivation of Ni. Furthermore, pertussis toxin treatment of membrane fragments from these cells did not result in labeling of the 41,000-dalton alpha-subunit of Ni with ADP ribose from [32P] NAD, indicating maximal ADP ribosylation of Ni by prior treatment of cells with pertussis toxin. Carbachol treatment of cells resulted in down-regulation of muscarinic cholinergic receptors to 45.7 +/- 12.5% and 52.5 +/- 13.5% of control values for toxin-untreated and toxin-treated cells, respectively. Epinephrine treatment of cells caused homologous desensitization of alpha 2-receptor-mediated inhibition of cAMP accumulation and down-regulation of alpha 2-adrenergic receptors to 42.9 +/- 11.4% and 53.2 +/- 5.3% of control values for toxin-untreated and toxin-treated cells, respectively. Down-regulation of muscarinic cholinergic receptors by carbachol and of alpha 2-adrenergic receptors by epinephrine was not due to the effect of retained agonist and was agonist specific, since it could be prevented by the antagonists atropine and yohimbine, respectively. We conclude that agonist-mediated down-regulation of both the muscarinic cholinergic receptor and the alpha 2-adrenergic receptor does not require functional inhibitory coupling.
 ...
PMID:Agonist-induced down-regulation of muscarinic cholinergic and alpha 2-adrenergic receptors after inactivation of Ni by pertussis toxin. 242 98

Using a recently developed canine primary enteric endocrine cell culture system, we have investigated the role of adenosine 3',5'-cyclic monophosphate (cAMP) in mediating the release of neurotensin and enteroglucagon. Epinephrine-stimulated peptide release was concomitant with an increase in cAMP accumulation. Carbachol and somatostatin (SRIF) markedly inhibited the epinephrine effect on both peptide release and cAMP content. The addition of 3-isobutyl-1-methylxanthine potentiated epinephrine-stimulated peptide release without altering the relative inhibition by carbachol and SRIF, suggesting that these agents did not inhibit endocrine cell function by increasing phosphodiesterase activity. To determine the role of cAMP production in mediating inhibition of peptide release, cells were incubated with the bacterial toxin, pertussis toxin (PT). In cultures pretreated with PT, carbachol inhibition of both peptide release and cAMP accumulation was completely reversed. In contrast, SRIF inhibition of cAMP content was completely reversed after PT treatment, but inhibition of peptide release was only partially reversed. Additionally, toxin treatment only partially reversed SRIF inhibition of forskolin- and calcium ionophore-stimulated peptide release. These data suggest that muscarinic cholinergic inhibition of neurotensin and enteroglucagon release is mediated entirely through the guanine nucleotide-binding protein (Ni) or a similar toxin-sensitive, GTP-binding protein. SRIF-inhibited peptide release is mediated partially through a toxin-sensitive substrate, as evidenced by PT reversal of reduced cAMP levels. SRIF may also inhibit neurotensin and enteroglucagon release by a cAMP-independent pathway that is not coupled to Ni or a similar PT-sensitive, GTP-binding protein.
 ...
PMID:Somatostatin and muscarinic inhibition of canine enteric endocrine cells: cellular mechanisms. 244 8


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>