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)

Bovine liver cytosol contains a phosphoinositide phospholipase C (PLCcyt) that is activated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S)-activated G-proteins from liver plasma membranes. Heparin-Sepharose chromatography indicated that PLCcyt was immunologically distinct from PLC-beta 1, PLC-gamma 1, or PLC-delta 1 from brain. Initial purification of the GTP gamma S-activated G-proteins that stimulated PLCcyt indicated that the beta gamma complex was responsible. G-proteins were subsequently extracted from liver membranes as heterotrimers and purified in the presence of AlCl3, MgCl2, and NaF to allow reversible activation. Immunoblot analysis with an antiserum selective for the beta subunit showed that the stimulatory activity corresponded with the presence of this protein at every chromatographic step. When liver beta gamma complex was purified and separated from all detectable alpha subunits, as shown by immunoblotting and silver staining, it strongly stimulated PLCcyt after removal of the activating ligand [AlF4]- by gel filtration. beta gamma prepared from brain was approximately equipotent with that from liver. beta gamma was half-maximally effective at 33 nM and produced a maximal 50-fold activation of the PLC. Under identical conditions, beta gamma had no effect on brain PLC-gamma 1 or PLC-delta 1 and produced a 2-fold stimulation of PLC-beta 1 activity. Addition of purified GDP-bound alpha o, which had no effect by itself, completely reversed the beta gamma activation of PLCcyt, confirming that beta gamma was the active species. These data provide evidence for a novel mechanism by which beta gamma subunits of pertussis toxin-sensitive or -insensitive G-proteins activate phospholipase C.
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PMID:Activation of cytosolic phosphoinositide phospholipase C by G-protein beta gamma subunits. 133 Oct 76

The mitogenic effect of extracellular ATP on porcine aortic smooth muscle cells (SMC) was examined. Stimulation of [3H]thymidine incorporation by ATP was dose-dependent; the maximal effect was obtained at 100 microM. ATP acted synergistically with insulin, IGF-1, EGF, PDGF, and various other mitogens. Incorporation of [3H]thymidine was correlated with the fraction of [3H]thymidine-labeled nuclei and changes in cell counts. The stimulation of proliferation was also determined by measurement of cellular DNA using bisbenzamide and by following the increase of mitochondrial dehydrogenase protein. The effect of ATP was not due to hydrolysis to adenosine, which shows synergism with ATP. ATP acted as a competence factor. The mitogenic effect of ATP, but not adenosine, was further increased by lysophosphatidate, phosphatidic acid, or norepinephrine. The inhibitor of adenosine deaminase, EHNA, stimulated the effect of adenosine but not ATP. The adenosine receptor antagonist theophylline depressed adenosine-induced mitogenesis. ADP and the non-hydrolyzable analogue adenosine 5'-[beta, gamma-imido]triphosphate (AMP-PNP) were equally mitogenic. Thus extracellular ATP stimulated mitogenesis of SMC via P2Y purinoceptors. The mechanism of ATP acting as a mitogen in SMC was further explored. Extracellular ATP stimulated the release of [3H]arachidonic acid (AA) and prostaglandin E2 (PGE2) into the medium, and enhanced cAMP accumulation in a dose-dependent fashion similar to ATP-induced [3H]thymidine incorporation. Inhibitors of the arachidonic acid metabolism pathway, quinacrine and indomethacin, partially inhibited the mitogenic effect of ATP but not of adenosine. Pertussis toxin inhibited ATP-stimulated DNA synthesis, AA release, PGE2 formation, and cAMP accumulation. Down-regulation of protein kinase C (PKC) by long-term exposure to phorbol dibutyrate (PDBu) partially prevented stimulation of DNA synthesis and activation of the AA pathway by ATP. The PKC inhibitor, staurosporine, antagonized mitogenesis stimulated by ATP. No synergistic effect was found when PDBu and ATP were added together. Therefore, a dual mechanism, including both arachidonic acid metabolism and PKC, is involved in ATP-mediated mitogenesis in SMC. In addition, ATP acted synergistically with angiotensin II, phospholipase C, serotonin, or carbachol to stimulate DNA synthesis. Finally, the possible physiological significance of ATP as a mitogen in SMC was further studied. The effect of endothelin and heparin, which are released from endothelial cells, on ATP-dependent mitogenesis was investigated. Extracellular ATP acted synergistically with endothelin to stimulate a greater extent of [3H]thymidine incorporation than was seen with PDGF plus endothelin. Heparin, believed to have a regulatory role, partially inhibited the stimulation of DNA synthesis caused both by ATP and PDGF.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Extracellular ATP and ADP stimulate proliferation of porcine aortic smooth muscle cells. 135 98

Heparin, a glycosaminoglycan synthesized in connective tissue-mast cells, appeared to inhibit the hemagglutination of rabbit erythrocytes induced by the filamentous hemagglutinin (FHA), a major adhesin of Bordetella pertussis. This inhibition suggested an interaction of heparin with the FHA region responsible for the hemagglutination activity. FHA-heparin interactions may play a role in bacterial attachment and persistence in the lungs during human pertussis. To confirm a direct FHA-heparin interaction, heparin was used as ligand in an affinity chromatography procedure. This technique allowed to purify FHA directly from the bacterial culture medium in a single-step using heparin-Sepharose CL-6B or Zetaffinity heparin 60 disks. The purified FHA was highly immunoreactive with anti-FHA monoclonal antibodies and showed no signs of degradation after 15 successive cycles of freezing-thawing. The described purification method is simple, and suitable for the rapid preparation of FHA.
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PMID:Interaction of the Bordetella pertussis filamentous hemagglutinin with heparin. 203 24

The influence of heparin was studied on the inhibitory regulation of adenylate cyclase in human platelet membranes. Heparin blocked the adrenaline-induced inhibition of adenylate cyclase and the stimulation of GTP hydrolysis with half-maximal and maximal efficiency at 0.3 and 1-3 micrograms/ml, respectively. The effect of heparin was reversed by washing the membranes. Heparin did not change the number of alpha-adrenoceptors. In contrast, the affinity of the alpha-adrenoceptor for adrenaline was decreased in the presence of heparin. The pertussis toxin-catalysed ADP-ribosylation of the inhibitory guanine nucleotide-binding Gi-protein was not altered by heparin. Heparin also abolished the inhibition of adenylate cyclase caused by GTP itself. The data indicate that heparin can impair the hormone-induced inhibition of adenylate cyclase and the stimulation of GTP hydrolysis and suggest that the effects of heparin are caused by an action at the Gi-protein of the adenylate cyclase system.
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PMID:Heparin uncouples alpha 2-adrenoceptors from the Gi-protein in membranes of human platelets. 283 29

Using recombinantly expressed proteins and synthetic peptides, we examined the structural/functional features of the platelet chemokines, neutrophil-activating peptide-2 (NAP-2) and platelet factor 4 (PF4); that were important in their activation of neutrophils. Previous studies with the chemokine interleukin-8 (IL-8) had shown that the N-terminal region preceding the first cysteine residue was critical in defining neutrophil-activating properties. We examined whether NAP-2 and PF4 had similar structural requirements. In the Ale-glu-leu-arg (AELR) N-terminus of NAP-2, substitution of E or R abolished Ca2+ mobilization and elastase secretion. Unlike the parent molecule PF4, AELR/PF4, the hybrid formed by replacing the N-terminal sequence of PF4 before the first cysteine residue with the homologous sequence of NAP-2, stimulated Ca2+ mobilization and elastase secretion. Furthermore, the effect of amino acid substitutions in the ELR motif differed from those seen with NAP-2 in that conserved substitutions of E or R in NAP-2 abolished activity, but only reduced neutrophil activation in the hybrid. These studies show that just as with IL-8, the N-termini of NAP-2 and PF4 are critical for high-level neutrophil-activating function. Desensitization studies provided information on receptor binding. NAP-2, which binds almost exclusively to the type 2 IL-8 receptor (IL-8R), did not desensitize neutrophils to activation by IL-8 because IL-8 could bind to and activate via both type 1 and 2 IL-8R. AELR/PF4 appears to bind to both types of receptors because it desensitized neutrophils to NAP-2 activation; but was not desensitized by NAP-2, and because it desensitized to and was desensitized by IL-8. Thus, although NAP-2 and AELR/PF4 share approximately 60% amino acid homology, they have different receptor affinities. Studies were performed to define the role of the C-termini of these platelet chemokines in receptor binding. Heparin and a monoclonal antibody specific for the heparin-binding domain of PF4 both inhibited Ca2+ mobilization and elastase release, further suggesting that the C-terminus of these chemokines is important in receptor binding. Synthetic NAP-2(51-70) failed to mobilize Ca2+, whereas PF4(47-70) and PF4(58-70) induced Ca2+ mobilization and secretion of elastase at high concentrations. Pertussis toxin inhibited neutrophil activation by 40% to 50%, establishing a role for G-protein-coupled receptors such as the IL-8Rs in activation by the PF4 C-terminal peptides.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Structural requirements of platelet chemokines for neutrophil activation. 791 50

The high-affinity receptor for melatonin is coupled to a pertussis toxin-sensitive, inhibitory guanine nucleotide regulatory protein, Gi, which mediates inhibition of adenylate cyclase activity in the chick and other species. Heparin has been found to uncouple alpha 2-adrenoceptors from a similar Gi; therefore it was of interest to examine the effect of this agent on melatonin binding and signal transduction in chick brain. In competition studies, melatonin inhibited the binding of 2-[125I]iodo-melatonin with high- and low-affinities of KH = 20 pM and KL = 15 nM, respectively. In the presence of heparin (100 units/ml), a single site with an affinity of about 6 nM was detected. The monovalent cations, Na+ and Li+, produced a greater rightward shift of the agonist competition curve than heparin and converted all high-affinity sites to a low affinity of approximately 15-18 nM. In saturation studies, heparin reduced the affinity of high-affinity sites and caused a significant decrease in the density of low-affinity sites. In keeping with its ability to reduce high-affinity binding, heparin blocked melatonin's inhibitory effect on forskolin-stimulated adenylate cyclase activity in chick brain synaptosomal membranes. These findings indicate that heparin impairs the coupling between the melatonin receptor and Gi, with a consequent decrease in binding affinity and a loss of receptor-mediated signalling.
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PMID:Heparin inhibits melatonin binding and signal transduction in chick brain. 792 Sep 73

The roles of heterotrimeric GTP-binding regulatory proteins (G-proteins) and inositol polyphosphates in the mechanism by which vasopressin stimulates Ca2+ inflow in hepatocytes were investigated by using single cells loaded with fura2 by microinjection. Vasopressin-stimulated Ca2+ inflow was mimicked by microinjection of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) or guanosine 5'-[beta gamma-imido]triphosphate to the cells, but not adenosine 5'-[gamma-thio]triphosphate (ATP[S]) or guanosine 5'-[beta-thio]diphosphate (GDP[S]). Extracellular Gd3+ (5 microM) inhibited both vasopressin- and GTP[S]-stimulated Ca2+ inflow. GDP[S], but not GMP, administered to hepatocytes by microinjection, completely inhibited vasopressin-stimulated Ca2+ inflow and partially inhibited vasopressin-induced release of Ca2+ from intracellular stores. The microinjection of pertussis toxin had no effect either on the release of Ca2+ from intracellular stores or on Ca2+ inflow induced by vasopressin, but completely inhibited changes in these processes induced by epidermal growth factor (EGF). Hepatocytes isolated from rats treated with pertussis toxin for 24 h exhibited no vasopressin- or GTP[S]-stimulated Ca2+ inflow, whereas the vasopressin-stimulated release of Ca2+ from intracellular stores was similar to that observed for control cells. Heparin or ATP[S] inhibited, or delayed the onset of, both vasopressin-induced release of Ca2+ from intracellular stores and vasopressin-stimulated Ca2+ inflow. Vasopressin-induced oscillations in intracellular [Ca2+] were observed in some heparin-treated cells. It is concluded that the stimulation by vasopressin of Ca2+ inflow to hepatocytes requires inositol 1,4,5-trisphosphate (InsP3) and, by implication, the pertussis-toxin-insensitive G-protein required for the activation of phospholipase C beta [Taylor, Chae, Rhee and Exton (1991) Nature (London) 350, 516-518], and another G-protein which is slowly ADP-ribosylated by pertussis toxin and acts between InsP3 and the putative plasma-membrane Ca2+ channel. EGF-stimulated Ca2+ inflow involves at least one G-protein which is rapidly ADP-ribosylated and is most likely required for InsP3 formation.
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PMID:A slowly ADP-ribosylated pertussis-toxin-sensitive GTP-binding regulatory protein is required for vasopressin-stimulated Ca2+ inflow in hepatocytes. 817

We have previously shown the possibility that endogenous type II phospholipase A2 (PLA2) might participate in degranulation in mast cells (MC) (Murakami, M., et al. 1992. Eur. J. Biochem. 209:257). Now we have examined whether or not exogenously added type II PLA2 triggers MC degranulation. When rat peritoneal connective tissue MC (CTMC) were exposed to purified rat type II PLA2 at concentrations of more than 10 micrograms/ml, significant release of histamine was observed, whereas PGD2 was not generated under the same conditions. Mouse peritoneal CTMC as well as bone marrow-derived immature MC also responded to PLA2. Preincubation of CTMC with tyrosine kinase inhibitors, genistein, and herbimycin A, but not with pertussis toxin, resulted in abolition of the sensitivity to PLA2. The ability of type II PLA2 to induce histamine release was inhibited by an antibody or chemicals, both of which blocked the catalytic activity of type II PLA2. Heparin or an antibody recognizing the heparin-binding domain of type II PLA2 also suppressed the MC-degranulating activity, probably due to inhibition of binding of PLA2 to the cells. The interaction between heparan sulfate on cell surface and the heparin-binding domain of type II PLA2 may be important for the induction of exocytosis. The catalytic domain of the enzyme is also crucially important for the degranulation induction. Furthermore, we found that nerve growth factor, one of the potent regulators of MC function, significantly potentiated type II PLA2-induced histamine release from rat CTMC. These results suggest the possible role of extracellular type II PLA2 in activation of CTMC primed with nerve growth factor at inflamed sites.
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PMID:Triggering of degranulation in mast cells by exogenous type II phospholipase A2. 822 55

1. We investigated the effects of histamine on membrane currents and contractile state of isolated guinea-pig tracheal myocytes using perforated patch and whole-cell recording techniques. The effects of histamine were compared to those of acetylcholine (ACh) and caffeine. 2. During voltage clamp (Vhold = -60 mV), histamine elicited contraction and an inward current (Ihist) which was often followed by current oscillations. Ihist had a reversal potential (Vrev) of -9 +/- 3 mV. 3. Ihist was dependent on the Cl- gradient and was antagonized by the Cl- channel blocker niflumic acid. Vrev was more positive (+2 +/- 1 mV) when K(+)-selective currents were blocked by Cs+ and TEA. When all external Na+ was replaced with N-methyl-D-glucamine, there was a small reduction in the amplitude of Ihist. 4. The histamine-induced current was similar to that elicited by ACh and by caffeine with respect to time course, amplitude, and current-voltage relationship. Responses to histamine and to ACh were non-additive, consistent with a convergence of histaminergic and cholinergic signalling pathways. Ihist was antagonized by the H1 histaminergic receptor antagonist astemizole, but not by atropine. 5. When recorded using the perforated patch configuration, Ihist could be elicited repeatedly for more than 30 min. When cells were studied in the whole-cell configuration using a pipette solution containing 0.025 mM EGTA, the amplitude of Ihist was initially the same as that obtained using perforated patch but then decreased; the time required for the responses to decrease to 50% (t1/2) was 8.2 +/- 1.0 min. When 1 mM EGTA was included in the pipette solution (whole-cell configuration), the initial response to histamine was significantly decreased in size and t1/2 was reduced to 3.3 +/- 0.7 min. 6. The characteristics of the signalling pathway were examined in cells studied using the whole-cell configuration with 0.025 mM EGTA in the recording pipette. Heparin significantly reduced t1/2 to 4.3 +/- 0.8 min. GTP gamma S elicited inward current and oscillations; both effects were enhanced by histamine. GTP gamma S also reduced t1/2 to 1.4 +/- 0.1 min. Pertussis toxin did not alter the amplitude or time course of Ihist. 7. We conclude that in guinea-pig tracheal myocytes, binding of histamine to H1 receptors leads to release of Ca2+ from intracellular stores and subsequent activation of Cl- and K+ conductances as well as contraction. Furthermore, we demonstrate that ACh elicits similar physiological responses due to a convergence of the histaminergic and muscarinic signalling pathways.
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PMID:Histamine activates Cl- and K+ currents in guinea-pig tracheal myocytes: convergence with muscarinic signalling pathway. 822 56

The selectivity of heparin in inducing potentiation of binding of antagonist ligands to muscarinic receptors was investigated at the five known subtypes of muscarinic receptors. The effects of heparin on binding of [3H]N-methylscopolamine at equilibrium was studied in Chinese hamster ovary (CHO) cells which express each of the individual muscarinic receptor subtypes and in membranes prepared from these cells. Heparin markedly increased equilibrium binding of subsaturating concentrations of the ligand only in membranes of CHO cells which express muscarinic M2 receptors. These effects of heparin were qualitatively similar to those obtained in heart membranes. In contrast, heparin did not influence ligand binding to muscarinic M2 receptors in intact cells. The positive cooperative effects of heparin at muscarinic receptors were abolished following treatment of cells with pertussis toxin. The latter treatment by itself resulted in a significant increase in [3H]N-methylscopolamine binding. Taken together with previous reports of heparin-induced uncoupling of receptors and G-proteins, these data suggest that the effects of heparin on ligand binding to muscarinic M2 receptors might be due to disruption of receptor-G-protein interactions which results in enhancement of binding of antagonist ligands to the receptor.
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PMID:Selective enhancement of antagonist ligand binding at muscarinic M2 receptors by heparin due to receptor uncoupling. 872 Apr 84


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