UMLS:C0043167 - FACTA Search

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Query: UMLS:C0043167 (pertussis)
19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Human alpha-thrombin is known to elicit bone resorption in vitro and has been proposed as a mediator of increased bone turnover in inflammatory diseases. We used UMR 106-H5 rat osteoblast-like osteosarcoma cells to explore the signal transduction mechanism utilized by thrombin in bone. Thrombin produced a dose-dependent increase in the accumulation of [3H]inositol phosphates (IPs) in UMR 106-H5 cells prelabeled with [3H]myo-inositol (EC50 15 U/ml). In saponin-permeabilized cells, GTP gamma S increased [3H]IP production, whereas GDP beta S inhibited the response to both GTP gamma S and thrombin, indicating involvement of a G-protein in thrombin action. Thrombin produced a dose-dependent increase in intracellular free calcium (Cai2+) in UMR 106-H5 cells (EC50 1 U/ml; maximal increase 4-fold), as well as a small (20%) increase in [3H]thymidine incorporation. Treatment of UMR 106-H5 membranes with pertussis toxin (PT) and [32P]NAD+ resulted in labeling of a 40-kDa protein. However, pretreatment of cells with a dose of PT sufficient to produce maximal endogenous labeling of this protein failed to influence thrombin action on IP accumulation, Cai2+, or [3H]thymidine incorporation. In contrast, PT treatment of CCL39 hamster lung fibroblasts significantly blunted thrombin-stimulated [3H]IP accumulation and [3H]thymidine incorporation. These results suggest that thrombin raises Cai2+ in UMR 106-H5 cells by activating polyphosphoinositide-specific phospholipase C. Whereas in fibroblasts and platelets, thrombin receptors appear to couple to both PT-sensitive and PT-insensitive G-proteins, only a PT-insensitive G-protein appears to mediate thrombin action in UMR 106-H5 cells. Either these cells lack the relevant PT-sensitive G-protein or they possess thrombin receptors that selectively couple to a pertussis toxin-insensitive G-protein.
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PMID:Thrombin stimulates inositol phosphate production and intracellular free calcium by a pertussis toxin-insensitive mechanism in osteosarcoma cells. 215 36

The expression of guanine nucleotide-binding proteins (G-proteins) was compared in two clonal lines of rat Nb2 node lymphoma cells, the lactogen-dependent Nb2-11C line and the lactogen-independent Nb2-Sp (spontaneous) line. Both cell lines expressed mRNA transcripts for the G-protein species Gs alpha [1.85 kilobases (kb)], Gi2 alpha (2.35 kb), Go alpha (4.1-4.5 kb), and Gi3 alpha (3.5 kb). Gi1 alpha was not detected. ADP ribosylation in the presence of activated cholera or pertussis toxins and [32P]NAD demonstrated the presence of G-proteins in the membrane fractions of both lines. The cholera toxin substrates consisted of two proteins (mol wt, 46.5 and 43.5 kD), while a single protein (mol wt, 41.5 kD) was ADP ribosylated by pertussis toxin. Surprisingly, the cholera toxin-sensitive proteins (Gs) were at least 20-fold less abundant in the Nb2-Sp cells than in the Nb2-11C cells. Since Gs and Gi2 are associated with the adenylate cyclase system and the regulation of intracellular cAMP, the effects of the cAMP analog, (Bu)2cAMP (dbcAMP), on Nb2-11C and Nb2-Sp cell growth were examined. dbcAMP (100 microM) completely inhibited the growth of lactogen-dependent Nb2-11C cells. The inhibitory effect of dbcAMP was exerted at an early point in the cell cycle, as it also inhibited PRL-stimulated c-myc expression measured 3 h after addition of the mitogen. In contrast, dbcAMP had only minor inhibitory effects on lactogen-independent Nb2-Sp cells, increasing their doubling time from 20 to 30 h and slightly reducing their density at confluence. The inhibitory effect of dbcAMP on both cell lines was reversible. Nb2-11C cells resumed growth after a lag period of approximately 3 days. The recovered cells did not arise from selection of a cAMP-resistant subpopulation, since both they and normal untreated Nb2-11C cells remained equally sensitive to dbcAMP. Similarly, Nb2-Sp cells resumed their normal doubling time upon removal of dbcAMP. The observation that the lactogen-independent Nb2-Sp cell line contained 20-fold less cholera toxin-sensitive Gs protein provides circumstantial evidence that dysfunction of the adenylate cyclase system may be implicated in the autonomous growth of these cells. This possibility is strengthened by the observation that Nb2-Sp cells are markedly less sensitive than the Nb2-11C clone to the growth inhibitory effects of an exogenous cAMP analog.
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PMID:The role of G-proteins in the mitogenesis of rat lactogen-dependent and lactogen-independent Nb2 lymphoma cells. 215 99

Kenimer et al. (J. G. Kenimer, J. Kim, P. G. Probst, C. R. Manclark, D. G. Burstyn, and J. L. Lowell, Hybridoma 8:37-51, 1989) identified three classes of monoclonal antibodies, termed A, B, and C, that recognize the S1 subunit of pertussis toxin. This report presents data demonstrating that class A monoclonal antibodies (3CX4, 6D11C, and 3C4D), which block the ADP-ribosyltransferase activity and recognize the predominant neutralizing epitope on the S1 subunit of the toxin, do not inhibit the NAD-glycohydrolase activity of the toxin. In addition, alkylation of cysteine 41 of the S1 subunit, which may interact with NAD, inactivates the toxin but does not prevent binding by class A antibodies. Taken together, these results support the conclusion that proper alterations of amino acids that interact with NAD should allow for inactivation of the toxin without destruction of the predominant neutralizing epitope. The class A antibodies recognized control but not heat-treated pertussis toxin spotted onto nitrocellulose, indicating that class A antibodies do not recognize denatured S1 subunit. In contrast, a nonneutralizing class C antibody (X2X5) failed to bind to control toxin or S1 subunit in solution and recognized heat-treated pertussis toxin better than control toxin when spotted onto nitrocellulose. Thus, this type of analysis presents a heterogeneous mixture of fully or partially denatured and native S1 proteins and fails to distinguish between neutralizing and nonneutralizing antibodies.
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PMID:Monoclonal antibodies that inhibit ADP-ribosyltransferase but not NAD-glycohydrolase activity of pertussis toxin. 215 82

The S1 subunit (Mr 28,000) of pertussis toxin expresses thiol-dependent enzymatic ADP-ribosyltransferase and NAD-glycohydrolase activities. Site-directed mutagenesis experiments were performed on the codon for Cys-41 of this subunit to investigate the role of this residue in both enzymatic activities. Deletion of Cys-41 caused a decrease in both activities below detectable levels, whereas replacement of this residue by serine, glycine, proline, or asparagine only slightly reduced the activities. The enzymatic activities of these mutants were thiol-independent. The deletion of Ser-40, adjacent to Cys-41, again caused reduction of the enzymatic activities to undetectable levels. Steady-state kinetic experiments showed that the kcat of the mutant protein in which Cys-41 was replaced by glycine was nearly identical to the kcat of the parent version. However, the Km for NAD of the mutant was significantly higher relative to that of the wild type version. These results indicate that the side-chain of Cys-41 is not essential for enzymatic activities and that Cys-41 is not involved in the rate of catalysis but is probably located at or close to the NAD-binding site. The introduction of a negative charge at position 41 through the replacement of Cys-41 by either aspartate or glutamate reduced the enzymatic activities to very low but measurable levels, suggesting a charge-charge repulsive interaction between these residues and possibly one or both of the phosphates of NAD. Cys-41 may therefore be located close to the phosphate subsite of the NAD-binding site.
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PMID:The role of cysteine 41 in the enzymatic activities of the pertussis toxin S1 subunit as investigated by site-directed mutagenesis. 215 32

Pertussis (whooping cough) is a serious infectious disease caused by the bacterium Bordetella pertussis. One of the major virulence factors is a protein known as pertussis toxin, which is composed of six subunits, with a total molecular weight of 106,000. Enzymatic transfer of ADP-ribose from NAD to a family of GTP-binding proteins is effected by the largest subunit (S1 or the A monomer), while binding of host cells and entry of S1 to the interior is a function of the other subunits (the B oligomer). The holotoxin crystallizes in the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 98.4 A, b = 164.2 A and c = 195.2 A. The crystals are suitable for high-resolution X-ray diffraction analysis.
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PMID:Preliminary X-ray crystallographic analysis of holotoxin from Bordetella pertussis. 235 76

The S1 subunit of pertussis toxin catalyses the hydrolysis of NAD+ (NAD+ glycohydrolysis) and the NAD(+)-dependent ADP-ribosylation of guanine-nucleotide-binding proteins. Recently, the S1 subunit of pertussis toxin was shown to be photolabelled by using radiolabelled NAD+ and u.v.; the primary labelled residue was Glu-129, thereby implicating this residue in the binding of NAD+. Studies from various laboratories have shown that the N-terminal portion of the S1 subunit, which shows sequence similarity to cholera toxin and Escherichia coli heat-labile toxin, is important to the maintenance of both glycohydrolase and transferase activity. In the present study the photolabelling technique was applied to the analysis of a series of recombinant-derived S1 molecules that possessed deletions or substitutions near the N-terminus of the S1 molecule. The results revealed a positive correlation between the extent of photolabelling with NAD+ and the magnitude of specific NAD+ glycohydrolase activity exhibited by the mutants. Enzyme kinetic analyses of the N-terminal mutants also identified a mutant with substantially reduced activity, a depressed photolabelling efficiency and a markedly increased Km for NAD+. The results support a direct role for the N-terminal region of the S1 subunit in the binding of NAD+, thereby providing a rationale for the effect of mutations in this region on enzymic activity.
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PMID:Photolabelling of mutant forms of the S1 subunit of pertussis toxin with NAD+. 236 91

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.
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PMID:Agonist-induced down-regulation of muscarinic cholinergic and alpha 2-adrenergic receptors after inactivation of Ni by pertussis toxin. 242 98

The activation mechanisms of K+ channels by muscarinic acetylcholine (m-ACh) receptors were examined in isolated atrial cells by use of patch-recording technique. In "cell-attached" patch recordings, ACh, present in the pipette, activated an inwardly rectifying K+ channel. In "inside-out" patches, activation of the K+ channel by ACh diminished with time following excision of the patch, but it resumed when GTP was present in the solution bathing the intracellular side of the membrane. The A protomer of pertussis toxin, together with NAD, inhibited the channel activation in the presence of GTP. Since pertussis toxin specifically ADP-ribosylates GTP-binding proteins Ni and No, which can interact with m-ACh receptors, and inhibits their functions, it was concluded that m-ACh receptors communicate with the K+ channel via GTP-binding proteins, probably Ni and/or No, in atrial cell membrane.
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PMID:Acetylcholine activation of K+ channels in cell-free membrane of atrial cells. 242 58

The molecular mechanisms underlying activation of a K+ channel by adenosine (Ado) and acetylcholine (ACh) were examined in single atrial cells of guinea-pig. Whole cell clamp and patch clamp techniques were used to characterize the K+ channel. In the whole cell clamp conditions, Ado and ACh increased the K+ channel current in a dose-dependent manner. The maximum responses and the apparent dissociation constants were different for Ado and ACh activations of the current. Theophylline blocked activation of the K+ current by Ado, while atropine blocked ACh-activation, indicating that two different membrane receptors were involved. Measurements of the conductance and kinetic properties of both whole cell and single channel currents indicate that Ado and ACh regulate the same K+ channels. In "inside-out" patch conditions, GTP was required in the intracellular side of the membrane for activation of the K+ channel by agonists (present in the patch electrode). The A promoter of pertussis toxin inhibited the channel activation only when NAD was also present. Furthermore, GTP-gamma S, a non-hydrolyzable GTP analogue, gradually caused activation of the K+ channel in the absence of agonists. Therefore, it was concluded that Ado and m-ACh receptors link with the same population of K+ channels via GTP-binding proteins Ni and/or No in the atrial cell membrane.
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PMID:On the mechanism of activation of muscarinic K+ channels by adenosine in isolated atrial cells: involvement of GTP-binding proteins. 242 51

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