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)

The pharmacological properties of alpha-2 adrenoceptors and the existence of nonadrenergic idazoxan-binding sites (NAIBS) were investigated in the insulin-secreting cell-line, RINm5F, using [3H]RX821002 and [3H]idazoxan. Analysis of [3H]RX821002 saturation isotherms revealed the presence of a single class of binding sites (Bmax = 47.5 +/- 3.5 fmol/mg protein) having high affinity (Kd = 1.26 +/- 0.18 nM). Inhibition of [3H]RX821002 binding by adrenergic compounds showed that the labeled sites displayed the properties expected for an alpha-2 adrenoceptor. Based on competition data with drugs having alpha-2 adrenoceptor subtype selectivity, the receptor from RINm5F is neither an alpha-2B nor an alpha-2C. It resembles the alpha-2A, but deviates from this subtype because of a weak affinity for yohimbine and rauwolscine. In this respect, RINm5F alpha-2 adrenoceptor is identical to the receptor previously described in rat intestinal mucosa and corresponds to a fourth subtype: alpha-2D. Agonist inhibition curves were better fitted by a two-site model and indicated that about half of the receptor population was under a high-affinity state corresponding to G protein-coupled receptors. [32P]ADP-ribosylation with pertussis toxin and immunodetection with specific antibodies permitted the identification of three distinct G proteins: Gi2, Gi3 and G0. Binding experiments with [3H]idazoxan showed that this imidazoline labeled two types of sites corresponding to alpha-2 adrenoceptors and NAIBS. Analysis of saturation isotherms under binding conditions allowing to discriminate between the two site populations indicated that the density of NAIBS (44 +/- 2 fmol/mg protein) was fairly identical to that of alpha-2 adrenoceptors. The pharmacological properties of NAIBS, as assessed by determining the relative affinity of imidazolinic and nonimidazolinic compounds, reasonably matched that reported in other tissues. Taken together, these data make the RINm5F cell-line 1) the first model in permanent culture known as expressing an alpha-2 adrenoceptor of the alpha-2D subtype; 2) a good system for studying in vitro the respective role of alpha-2 adrenoceptors and NAIBS in the regulation of insulin secretion by beta cells.
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PMID:The insulin-secreting cell line, RINm5F, expresses an alpha-2D adrenoceptor and nonadrenergic idazoxan-binding sites. 134 66

The alpha 2-C10 adrenergic receptor from human platelets was expressed permanently in Rat-1 fibroblasts. A series of clones that varied in expression of the receptor from 0 to 3.5 pmol/mg of membrane protein were isolated. We have demonstrated recently in cells of one of these clones (1C) that the alpha 2-C10 receptor interacts directly with two distinct pertussis toxin-sensitive G-proteins, Gi2 and Gi3 (Milligan, G., Carr, C., Gould, G. W., Mullaney, I., and Lavan, B.E. (1991) J. Biol. Chem. 266, 6447-6455). High affinity GTPase activity in membranes of cells from the various clones was stimulated by the addition of the alpha 2-adrenergic agonist UK14304, defining that the receptor coupled productively to the G-protein signaling system. Maximal stimulation of high affinity GTPase activity correlated with the levels of receptor expressed. Clones expressing the receptor also demonstrated agonist-mediated inhibition of adenylylcyclase. Futhermore, the alpha 2-C10 receptor in one clone (1C), but not other clones, promoted a marked stimulation in the generation of water-soluble products derived from phosphatidylcholine. The concentration of UK14304 required to produce half-maximal regulation of GTPase activity (20-30 nM), of forskolin-amplified adenylylcyclase activity (30-40 nM), and of choline generation (30-40 nM) were similar. Transphosphatidylation experiments with cells of clone 1C indicated that the receptor-mediated hydrolysis of phosphatidylcholine was via the action of a phospholipase D. All of these effects were attenuated by pretreatment of the cells with pertussis toxin. Dose-effect curves of pertussis toxin-treatment demonstrated similar effective concentrations of the toxin in causing endogenous ADP-ribosylation of both Gi2 and Gi3, inhibition of receptor-stimulated GTPase activity, and phospholipase D activity. Receptor activation of phospholipase D activity was not dependent upon prior phospholipase C-dependent activation of protein kinase C, as alpha 2-adrenergic stimulation of inositol phosphate production was negligible and the presence of the selective protein kinase C inhibitor RO-31-8220, at concentrations up to 10 microM, had no effect on UK14304-mediated production of phosphatidylbutanol. These results demonstrate that expression of the alpha 2-C10 receptor in a heterologous system can result in receptor regulation of signaling elements that appear not to be primary targets for the receptor in vivo. Such results are important in respect to recent observations that transfection of a single defined receptor into separate cell lines can lead to the regulation of distinct effector systems (Vallar, L., Muca, C., Magni, M., Albert, P., Bunzow, J., Meldolesi, J. and Civelli, O. (1990) J. Biol. Chem. 265, 10320-10326).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Alpha 2-C10 adrenergic receptors expressed in rat 1 fibroblasts can regulate both adenylylcyclase and phospholipase D-mediated hydrolysis of phosphatidylcholine by interacting with pertussis toxin-sensitive guanine nucleotide-binding proteins. 134 92

Inhibitory regulation of dopamine neurons is mediated by dopamine autoreceptor and gamma-aminobutyric acidB receptor opening of potassium channels. Increased potassium conductance by either receptor is G protein dependent. To evaluate the role of G proteins in vivo, pertussis toxin (PTX) was microinjected into the A10 dopamine region and changes in dopamine metabolism and synthesis measured. PTX produced an elevation in dopamine metabolism and synthesis in the A10 region and nucleus accumbens for up to 4 days after injection. By day 7 the levels of the dopamine precursor and metabolites had returned to normal. A less consistent increase was also measured in the A9 dopamine region and the prefrontal cortex. Although dopamine synthesis and metabolism had returned to normal by day 7, the in vitro ADP-ribosylation of G proteins in the A10 region by PTX remained depressed by approximately 50% from day 1 to day 14 after administration, returning to normal by day 30. The data suggest that in vivo ribosylation of G proteins may lead to a short-term attenuation of the tonic inhibitory control of dopamine neurons, which can be compensated for by PTX-insensitive mechanisms.
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PMID:Pertussis toxin in the A10 region increases dopamine synthesis and metabolism. 134 27

Whole-cell patch-clamp recordings were obtained from light-responsive on-bipolar cells in retinal slices of the dogfish. Inclusion of the A-subunit of pertussis toxin in the patch-pipette solution resulted in an increase in inward current and membrane conductance, and a block of light-evoked currents of on-bipolar cells. The opposite effect was obtained with the A-subunit of cholera toxin, which blocked light responses, and induced an outward current and a decrease in membrane conductance. These actions were NAD+ dependent. The results show that the G-protein(s) linking glutamate receptors to a cGMP cascade in on-bipolar cells possess sites which are ADP-ribosylated by pertussis and cholera toxins, with no homology to the adenylate cyclase system but possibly with a homology to transducin. Furthermore, inclusion of H-7, a kinase inhibitor in the patch-pipette solution, or of a non-hydrolysable ATP analogue (AMP-PNP) had no effect on light responses, membrane conductance or dark current of on-bipolar cells, suggesting that the components of this cGMP cascade are unlikely to be regulated by protein kinases.
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PMID:The glutamate-receptor linked cGMP cascade of retinal on-bipolar cells is pertussis and cholera toxin-sensitive. 134 16

Nitric oxide-releasing compounds were shown to activate an ADP-ribosyltransferase activity in the cytosol of Dictyostelium discoideum. The enzyme ADP-ribosylated a cytosolic protein of approximately 41 kDa, p41. Neither cGMP nor GTP and its analogues affected this ADP-ribosylation. p41 differs from other substrates ADP-ribosylated by cholera, pertussis, or diphtheria toxins. Treatment of ADP-ribosylated p41 with snake venom phosphodiesterase released adenosine 5'-monophosphate, indicating a mono-ADP-ribose-protein linkage. This linkage was stable to neutral hydroxylamine but was sensitive to mercury ions and iodomethane, suggesting an attachment to a cysteine residue. Treatment of intact cells with nitric oxide-releasing compounds appeared to stimulate the ADP-ribosylation of p41 and this modification was reversible.
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PMID:Nitric oxide stimulates the ADP-ribosylation of a 41-kDa cytosolic protein in Dictyostelium discoideum. 135 80

A somatostatin (SRIF) receptor and its associated Gi regulatory proteins was purified from GH4C1 rat pituitary cells by: 1) saturation of the membrane-bound receptor with biotinyl-NH-[Leu8,D-Trp22,Tyr25] SRIF28 (bio-S28); 2) solubilization of receptor-ligand (R.L) complex with deoxycholate-lysophosphatidylcholine (D.L); 3) adsorption of solubilized receptor-ligand complex to immobilized streptavidin; and 4) elution of receptor and G-protein by GTP. The receptor, a glycoprotein with an average M(r) of 85,000, was then purified to substantial homogeneity on immobilized wheat germ agglutinin. The 85-kDa glycoprotein was identified as a SRIF receptor by several criteria. (a) It had the same size as the chemically cross-linked R.[125I]L complex. (b) Yield of the purified protein increased and plateaued in the same range of bio-S28 concentrations where specific high affinity binding reached saturation. (c) It was copurified with appropriate G-protein subunits. The 85-kDa receptor and two other proteins with M(r) values of 35,000 and 40,000, the sizes of G beta and G alpha, did not appear in eluates from control streptavidin columns done with SRIF receptors loaded with nonbiotinylated S14. The 40-kDa protein was identified as a Gi alpha by ADP-ribosylation from [32P]NAD catalyzed by pertussis toxin. (d) Both the chemically cross-linked R.[125I]L complex and SRIF receptor purified from [35S]methionine-labeled GH4C1 cells were reduced in size to about 38 kDa by endoglycosidase F. (e) Amino acid sequence from the purified receptor was nearly identical with that of a recently cloned SRIF receptor subtype.
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PMID:Purification of a pituitary receptor for somatostatin. The utility of biotinylated somatostatin analogs. 135 97

In enzymatically dispersed enriched rat parietal cells we studied the effect of pertussis toxin on prostaglandin E2 (PGE2)- or somatostatin-induced inhibition of H(+)-production. Parietal cells were incubated in parallel in the absence (control cells) and presence of pertussis toxin (250 ng/ml; 4 h). [14C]Aminopyrine accumulation by both pertussis toxin-treated and control cells was used as an indirect measure of H(+)-production after stimulation with either histamine, forskolin or dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) alone and in the presence of PGE2 (10(-9)-10(-7) M) or somatostatin (10(-9)-10(-6) M). PGE2 inhibited histamine- and forskolin-stimulated [14C]aminopyrine accumulation but failed to alter the response to dbcAMP. Somatostatin was less effective and less potent than PGE2 in inhibiting stimulation by histamine or forskolin and reduced the response to dbcAMP. Pertussis toxin completely reversed inhibition by both PGE2 and somatostatin on histamine- and forskolin-stimulated H(+)-production but failed to affect inhibition by somatostatin of the response to dbcAMP. After incubation of crude control cell membranes with [32P]NAD+, pertussis toxin catalysed the incorporation of [32P]adenosine diphosphate (ADP)-ribose into a membrane protein of molecular weight of 41,000, the known molecular weight of the inhibitory subunit of adenylate cyclase (Gi alpha). Pertussis toxin treatment of parietal cells prior to the preparation of crude membranes almost completely prevented subsequent pertussis toxin-catalysed [32P]ADP ribosylation of the 41,000 molecular weight protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Pertussis toxin reverses prostaglandin E2- and somatostatin-induced inhibition of rat parietal cell H(+)-production. 135 83

Exposure of cardiomyocytes from chicken embryos for 3 days to the beta-adrenoceptor agonist, isoproterenol, lead to a down-regulation of beta-adrenoceptors by about 70% and to a decrease in isoproterenol-stimulated adenylyl cyclase activity by about 40% (homologous desensitization). In addition, the isoproterenol treatment induced an increase in the level of muscarinic acetylcholine receptors by about 30% and an increase in pertussis toxin-catalyzed ADP-ribosylation of two about 40 kDa proteins, most probably alpha-subunits of the inhibitory G-protein (Gi), by about a factor of two (heterologous desensitization). The purpose of the present study was to characterize the role of beta-adrenoceptor-dependent and -independent mechanisms in heterologous desensitization of adenylyl cyclase. Therefore, the effect of pretreatment with the beta-adrenoceptor antagonist, propranolol, with the partial agonists, celiprolol and xamoterol, and with the beta-adrenoceptor-independent adenylyl cyclase activators, prostaglandin E1 and forskolin, on beta-adrenoceptors, muscarinic acetylcholine receptors and pertussis-toxin-catalyzed ADP-ribosylation of G-protein alpha-subunits was studied. Pretreatment of the cardiomyocytes for 3 days with xamoterol or celiprolol, but not with propranolol, induced a small decrease in beta-adrenoceptor number and in isoproterenol-stimulated adenylyl cyclase activity by about 15-20%. Exposure to prostaglandin E1 and forskolin lead to a more pronounced decrease in beta-adrenoceptor binding and in isoproterenol-mediated adenylyl cyclase stimulation by about 40-60% (heterologous desensitization). An increase in the level of muscarinic acetylcholine receptors, similar to that induced by isoproterenol exposure, was only observed after pretreatment with the partial agonists, celiprolol and xamoterol, but not after pretreatment with the beta-adrenoceptor-independent agonists, prostaglandin E1 and forskolin, nor after pretreatment with propranolol. In contrast, prostaglandin E1 and forskolin exposure lead to a similar increase in pertussis toxin-catalyzed ADP-ribosylation of about 40 kDa G-proteins as isoproterenol exposure whereas treatment with propranolol, celiprolol and xamoterol had no or only a very small effect on pertussis toxin substrates. In summary, the data suggest that, similar as shown for homologous desensitization, cyclic AMP-dependent and -independent mechanisms are also involved in heterologous desensitization of adenylyl cyclase stimulation. The beta-adrenoceptor-induced upregulation of muscarinic acetylcholine receptors and of the alpha-subunits of pertussis toxin-sensitive G-proteins, most probably of Gi, seem to be mediated via distinct pathways.
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PMID:Distinct pathways for beta-adrenoceptor-induced up-regulation of muscarinic acetylcholine receptors and inhibitory G-protein alpha-subunits in chicken cardiomyocytes. 135 32

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

1. The functional antagonism that exists between muscarinic and beta-adrenoceptor function in guinea-pig tracheal smooth muscle was investigated by assessing Gs and Gi regulated adenylyl cyclase activity in isolated membranes. 2. Membranes from guinea-pig tracheal smooth muscle contain both Gi alpha and Gs alpha as assessed by Western blots with anti-G-protein antibodies. 3. GppNHp, a non-hydrolysable analogue of guanosine 5'-triphosphate (GTP), was shown to stimulate adenylyl cyclase activity at high concentrations (10(-6)-10(-4) M). GppNHp also produced a concentration-dependent reduction in pertussis toxin-catalysed adenosine diphosphate (ADP)-ribosylation of Gi alpha. 4. Pretreatment of tracheal smooth muscle slices with methacholine (10(-6) M) provoked a blockade of isoprenaline plus GTP, GppNHp- and GTP-stimulated adenylyl cyclase. 5. Addition of methacholine to membranes did not trigger inhibition of GTP-stimulated adenylyl cyclase activity but did block the isoprenaline-mediated augmentation of GTP-stimulated adenylyl cyclase activity. 6. Pretreatment of tracheal smooth muscle with methacholine (10(-6) M) provoked a blockade of cholera toxin-catalysed NAD(+)-dependent ADP-ribosylation of Gs alpha. 7. Phorbol-12-myristate 13-acetate (PMA)-treatment of tracheal smooth muscle slices actually enhanced GppNHp-stimulated adenylyl cyclase activity in subsequently prepared membranes. 8. We suggest that methacholine in addition to inhibiting adenylyl cyclase via a Gi-dependent mechanism induces a functional inactivation of Gs activity. These results together may explain the functional antagonism that exists between increased muscarinic tone and the ability of beta-adrenoceptor agonists to provoke excitation-contraction uncoupling.
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PMID:Muscarinic blockade of beta-adrenoceptor-stimulated adenylyl cyclase: the role of stimulatory and inhibitory guanine-nucleotide binding regulatory proteins (Gs and Gi). 136 73

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