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)

Hepatocyte membranes from both lean and obese Zucker rats exhibited adenylate cyclase activity that could be stimulated by glucagon, forskolin, NaF and elevated concentrations of p[NH]ppG. In membranes from lean animals, functional Gi was detected by the ability of low concentrations of p[NH]ppG to inhibit forskolin-activated adenylate cyclase. This activity was abolished by treatment of hepatocytes with either pertussis toxin or the phorbol ester TPA, prior to making membranes for assay of adenylate cyclase activity. In hepatocyte membranes from obese animals no functional Gi activity was detected. Quantitative immunoblotting, using an antibody able to detect the alpha subunit of Gi, showed that hepatocyte plasma membranes from both lean and obese Zucker rats had similar amounts of Gi-alpha subunit. This was 6.2 pmol/mg plasma membrane for lean and 6.5 pmol/mg plasma membrane for obese animals. Using thiol pre-activated pertussis toxin and [32P]-NAD+, similar degrees of labelling of the 40 kDa alpha subunit of Gi were found using plasma membranes of both lean and obese Zucker rats. We suggest that liver plasma membranes from obese Zucker rats express an inactive Gi alpha subunit. Thus lesions in liver Gi functioning are seen in insulin-resistant obese rats and in alloxan- and streptozotocin-induced diabetic rats which also show resistance as regards the acute actions of insulin. Liver plasma membranes of obese animals also showed an impairment in the coupling of glucagon receptors to Gs-controlled adenylate cyclase, with the Kd values for activation by glucagon being 17.3 and 126 nM for lean and obese animals respectively. Membranes from obese animals also showed a reduced ability for high concentration of p[NH]ppG to activate adenylate cyclase. The use of [32P]-NAD+ and thiol-preactivated cholera toxin to label the 43 kDa and 52 kDa forms of the alpha-subunit of Gs showed that a reduced labelling occurred using liver plasma membranes from obese animals. It is suggested that abnormalities in the levels of expression of primarily the 52 kDa form of alpha-Gs may give rise to the abnormal coupling between glucagon receptors and adenylate cyclase in liver membranes from obese (fa/fa) Zucker rats.
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PMID:Multiple defects occur in the guanine nucleotide regulatory protein system in liver plasma membranes of obese (fa/fa) but not lean (Fa/Fa) Zucker rats: loss of functional Gi and abnormal Gs function. 256 40

Adenosine receptors in a spontaneously contracting atrial myocyte culture from 14-day chick embryos were characterized by radioligand binding studies and by examining the involvement of G-protein in coupling these receptors to a high-affinity state and to the adenylate cyclase and the myocyte contractility. Binding of the antagonist radioligand [3H]-8-cyclopentyl-1,3-diproylxanthine ([3H]CPX) was rapid, reversible and saturable and was to a homogeneous population of sites with a Kd value of 2.1 +/- 0.2 nM and an apparent maximum binding of 26.2 +/- 3 fmol/mg of protein (n = 10, +/- S.E.). Guanyl-5-yl-(beta, gamma-imido)diphosphate had no effect on either the Kd or the maximum binding and CPX reversed the N6-R-phenyl-2-propyladenosine-induced inhibition of adenylate cyclase activity and contractility, indicating that [3H] CPX is an antagonist radioligand. Competition curves for [3H] CPX binding by a series of reference adenosine agonists were consistent with labeling of an A1 adenosine receptor and were better fit by a two-site model than by a one-site model. ADP-ribosylation of the G-protein by the endogenous NAD+ in the presence of pertussis toxin shifted the competition curves from bi to monophasic with Ki values similar to those of the KL observed in the absence of prior pertussis intoxication. The adenosine agonists were capable of inhibiting both the adenylate cyclase activity and myocyte contractility in either the absence or the presence of isoproterenol. The A1 adenosine receptor-selective antagonist CPX reversed these agonist effects. The order of ability of the reference adenosine receptor agonists in causing these inhibitory effects was similar to the order of potency of the same agonists in inhibiting the specific [3H]CPX binding (N6-R-phenyl-2-propyladenosine greater than N6-S-phenyl-2-propyladenosine or N-ethyladenosine-5'-uronic acid). These data indicate that the adenosine receptor coupled to inhibition of adenylate cyclase activity and to the negative inotropic effect is the A1 subtype. Pertussis treatment uncoupled the adenosine receptor from both inhibition of adenylate cyclase activity and negative inotropic effect. Taken together, the present study indicates that adenosine receptors of the A1 subtype are present on the spontaneously contracting atrial myocytes and are negatively coupled to adenylate cyclase and to the contractile state. The cultured embryonic chick atrial myocyte preparation represents a useful model system for characterizing the cardiac A1 adenosine receptor.
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PMID:Characterization of the adenosine receptor in cultured embryonic chick atrial myocytes: coupling to modulation of contractility and adenylate cyclase activity and identification by direct radioligand binding. 273 46

The properties of the adenylate cyclase from forskolin-resistant mutants of Y1 adrenocortical tumor cells was compared with the properties of the enzyme from parental Y1 cells in order to localize the site of mutation. In parental Y1 cells, forskolin stimulated adenylate cyclase activity with kinetics suggestive of an interaction at two sites; in mutant cells, forskolin resistance was characterized by a decrease in enzymatic activity at both sites. Forskolin potentiated the enzyme's responses to NaF and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) in parent and mutant clones, and the mutant enzyme showed the same requirements for Mg2+ and Mn2+ as did the parent enzyme. The adenylate cyclase associated with forskolin-resistant mutants was insensitive to ACTH and was less responsive to Gpp(NH)p than was the parent enzyme. In parental Y1 cells and in the forskolin-resistant mutants, cholera toxin catalyzed the transfer of [32P]ADP-ribose from [32P]NAD+ into three membrane proteins associated with the alpha subunit of Gs; however, the amount of labeled ADP-ribose incorporated into mutant membranes was reduced by as much as 70%. Both parent and mutant membranes were labeled by pertussis toxin to the same extent. The insensitivity of the mutant adenylate cyclase to ACTH and Gpp(NH)p and the selective resistance of the mutant membranes to cholera toxin-catalyzed ADP-ribosylation suggest that a specific defect associated with Gs is involved in the mutation to forskolin resistance in Y1 cells.
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PMID:Forskolin-resistant Y1 mutants harbor defects associated with the guanyl nucleotide-binding regulatory protein, Gs. 282 64

The bacterial toxins, choleragen and pertussis toxin, inhibit the light-stimulated GTPase activity of bovine retinal rod outer segments by catalysing the ADP-ribosylation of the alpha-subunit (T alpha) of transducin [Abood, Hurley, Pappone, Bourne & Stryer (1982) J. Biol. Chem. 257, 10540-10543; Van Dop, Yamanaka, Steinberg, Sekura, Manclark, Stryer & Bourne (1984) J. Biol. Chem. 259, 23-26]. Incubation of retinal rod outer segments with NAD+ and a purified NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes resulted in approx. 60% inhibition of GTPase activity. Inhibition was dependent on both enzyme and NAD+, and was potentiated by the non-hydrolysable GTP analogues guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5'-[beta gamma-methylene]triphosphate (p[CH2]ppG). The transferase ADP-ribosylated both the T alpha and T beta subunits of purified transducin. T alpha (39 kDa), after ADP-ribosylation, migrated as two distinct peptides with molecular masses of 42 kDa and 46 kDa on SDS/polyacrylamide-gel electrophoresis. T beta (36 kDa), after ADP-ribosylation, migrated as a 38 kDa peptide. With purified transducin subunits, it was observed that the GTPase activity of ADP-ribosylated T alpha, reconstituted with unmodified T beta gamma and photolysed rhodopsin, was decreased by 80%; conversely, reconstitution of T alpha with ADP-ribosyl-T beta gamma resulted in only a 19% inhibition of GTPase. Thus ADP-ribosylation of T alpha, the transducin subunit that contains the guanine nucleotide-binding site, has more dramatic effects on GTPase activity than does modification of the critical 'helper subunits' T beta gamma. To elucidate the mechanism of GTPase inhibition by transferase, we studied the effect of ADP-ribosylation on p[NH]pp[3H]G binding to transducin. It was shown previously that modification of transducin by choleragen, which like transferase ADP-ribosylates arginine residues, did not affect guanine nucleotide binding. ADP-ribosylation by the transferase, however, decreased p[NH]pp[3H]G binding, consistent with the hypothesis that choleragen and transferase inhibit GTPase by different mechanisms.
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PMID:Inhibition of the GTPase activity of transducin by an NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes. 282 39

This paper investigates protein mono(ADP-ribosylation) in rat liver mitochondria. In isolated inner mitochondrial membranes, in the presence of both ADP-ribose and NAD+, a protein is mono-(ADP-ribosylated) with high specificity. The reaction apparently consists of enzymatic NAD+ glycohydrolysis and subsequent binding of free ADP-ribose to the acceptor protein. In terms of chemical stability, the resulting bond is unique among the ADP-ribose linkages thus far characterized. Formation of a Schiff base adduct between free ADP-ribose and the acceptor protein is excluded. In intact mitochondria at least three classes of proteins are ADP-ribosylated in vivo. One ADP-ribose-protein linkage is of the carboxylate ester type as indicated by its lability in neutral buffer. Another class of ADP-ribosylated proteins requires hydroxylamine for release of ADP-ribose. The third class is stable in hydroxylamine but labile to alkali, similar to the ADP-ribose-cysteine linkage in transducin formed by pertussis toxin.
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PMID:Mono(ADP-ribosylation) in rat liver mitochondria. 283 67

We investigated the mechanisms of receptor-mediated stimulation of high-affinity GTPase activity in response to opioid peptides and to foetal-calf serum in membranes of the neuroblastoma X glioma hybrid cell line NG108-15. Increases in GTPase activity in response to both of these ligands was abolished by prior exposure of the cells to pertussis toxin. Pertussis toxin in the presence of [32P]NAD+ catalysed incorporation of radioactivity into a broad band of approx. 40 kDa in membranes prepared from untreated, but not from pertussis-toxin-pretreated, cells. Additivity studies indicated that the responses to opioid peptides and to foetal-calf serum were mediated by separate guanine-nucleotide-binding proteins (G-proteins). Whereas opioid peptides produced an inhibition of adenylate cyclase in membranes of untreated cells, foetal-calf serum did not. Affinity-purified antibodies which recognize the C-terminus of the inhibitory G-protein identified a 40 kDa polypeptide in membranes of NG108-15 cells. These antibodies attenuated opioid-stimulated high-affinity GTPase activity, but did not markedly affect the response to foetal-calf serum. We conclude that receptors for the opioid peptides function via the inhibitory G-protein (Gi), whereas foetal-calf serum activates a second pertussis-toxin-sensitive G-protein, which has a C-terminal sequence significantly different from that of Gi.
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PMID:Antibodies which recognize the C-terminus of the inhibitory guanine-nucleotide-binding protein (Gi) demonstrate that opioid peptides and foetal-calf serum stimulate the high-affinity GTPase activity of two separate pertussis-toxin substrates. 283 23

The cellular mechanism of action of the cannabimimetic drugs is examined using cultured cells. In membranes from N18TG2 neuroblastoma cells and the neuroblastoma X glioma hybrid cells, NG108-15, the psychoactive cannabinoid drugs and their nantradol analogs could inhibit adenylate cyclase activity. This response was not observed in either the soluble adenylate cyclase from rat sperm or membrane-bound adenylate cyclases from C6 glioma or S49 lymphoma cells. This cellular selectivity provides further evidence for the existence of specific receptors for the cannabimimetic compounds. Receptor-mediated inhibition of adenylate cyclase requires the presence of a guanine nucleotide-binding protein complex, Gi. Gi can be functionally inactivated as a result of an ADP-ribosylation modification catalyzed by pertussis toxin. The present study demonstrates that pertussis toxin treatment of cells abolished the cannabimimetic response in intact cells and in membranes derived therefrom. The action of pertussis toxin required NAD+ as substrate for in vitro modification of neuroblastoma membranes. Furthermore, pertussis toxin was able to catalyze the labeling of a neuroblastoma membrane protein in vitro using [32P] NAD+ under conditions similar to those by which attenuation of the cannabimimetic inhibition of adenylate cyclase could be demonstrated. This evidence demonstrates the requirement for a functional Gi in the action of cannabimimetic drugs.
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PMID:Involvement of Gi in the inhibition of adenylate cyclase by cannabimimetic drugs. 286 5

Hypothyroidism is associated with an enhanced sensitivity of rat fat cells to the inhibitory action of adenosine and adenosine agonists. The sensitivity of the forskolin-stimulated cyclic AMP response of rat fat cells to the adenosine agonist N6-phenylisopropyladenosine is amplified 3-fold by hypothyroidism. Forskolin-stimulated adenylate cyclase activity is more sensitive to inhibition by this adenosine agonist in membranes of fat cells isolated from hypothyroid as compared to euthyroid rats. Hypothyroidism does not significantly alter the number of affinity of binding sites for N6-cyclohexyl[3H]adenosine or N6-phenylisopropyladenosine in membranes of rat fat cells. GTP-induced inhibition of forskolin-stimulated adenylate cyclase was markedly enhanced in the hypothyroid state, suggesting an alteration in the inhibitory regulatory component (Ni)-mediated control of adenylate cyclase. Incubating membranes with [alpha-32P]NAD+ and preactivated pertussis toxin results in the radiolabeling of two peptides with Mr = 40,000 and 41,000 as visualized in autoradiograms of polyacrylamide gels run in sodium dodecyl sulfate. The amount of label incorporated by pertussis toxin into these two peptides (putative subunits of Ni) per mg of protein of membrane is increased 2-3-fold in the hypothyroid state. The amount of the stimulatory regulatory component, Ns, in fat cell membranes is not altered by hypothyroidism (Malbon, C. C., Graziano, M. P., and Johnson, G. L. (1984) J. Biol. Chem. 259, 3254-3260). The amplified response of hypothyroid rat fat cells to the inhibitory action of adenosine appears to reflect a specific increase in the activity and abundance of Ni.
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PMID:Fat cell adenylate cyclase system. Enhanced inhibition by adenosine and GTP in the hypothyroid rat. 298 25

Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with 10 nM [D-Ala2,D-Leu5] enkephalin (DADLE) results in a reduction of cell-surface opiate delta receptors. Whether opiate receptor internalization requires the activation of the guanine nucleotide-binding protein (Ni) is unclear. Hence, activation of Ni was attenuated by treating hybrid cells with 100 ng/ml pertussis toxin (PT) for 3 h, which resulted in a decrease in DADLE's ability to inhibit adenylate cyclase activity. Despite this prior treatment with PT, chronic exposure of these cells to 10 nM DADLE resulted in a time-dependent decrease in both [3H]diprenorphine and [3H]DADLE binding. This reduction in 3H-ligand binding in cells previously treated with PT represented internalization of the receptors because translocation was observed of bound [3H]DADLE from plasma membrane fractions to the lysosomal fractions in the Percoll gradients. Thus, opiate receptors internalize without activation of Ni. The internalization of opiate receptors was not accompanied by Ni. By measuring the amount of the 41-kDa alpha subunit being labeled by PT with [32P]NAD+, it was determined that plasma membrane preparations, of both the control cells and cells treated with 10 nM of DADLE for 4 h, contained equal concentrations of Ni, 2 pmol of Ni/mg of protein. Additionally, there was no measurable alteration in the amount of PT substrate in the lysosomal fractions of the DADLE-treated cells as compared to that of control cells. Chronic DADLE treatment resulted in a decrease in Km value of NAD+ in the ADP-ribosylation of 41-kDa subunit of Ni. In summary, opiate receptors internalize as agonist-receptor complexes without the guanine nucleotide-binding component.
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PMID:Effect of pertussis toxin treatment on the down-regulation of opiate receptors in neuroblastoma X glioma NG108-15 hybrid cells. 299 25

Angiotensin II can inhibit glucagon-stimulated cyclic AMP production in hepatocytes and adenylate cyclase activity in hepatic membranes. Pertussis toxin, an exotoxin produced by Bordetella pertussis, was used to investigate the role of the inhibitory guanine nucleotide-binding regulatory protein of adenylate cyclase (Ni) in coupling angiotensin receptors to the adenylate cyclase system. An assay was developed using [32P] NAD+ to quantitate the amount of Ni protein in the membrane and the extent of its ADP-ribosylation catalyzed by toxin. The ability of angiotensin to inhibit adenylate cyclase and interact with its receptor was compared with the degree of modification of Ni in membranes prepared from isolated hepatocytes. In control membranes angiotensin II inhibited basal adenylate cyclase by 35%. When all of the Ni molecules in the membrane were ADP-ribosylated, angiotensin did not inhibit adenylate cyclase. However, the attenuation of angiotensin's effect on cyclase was not linearly correlated with the degree of modification of Ni; ADP-ribosylation of greater than 80% of the Ni was required before a reduction of the angiotensin effect was observed. A possible explanation for this finding is an excess of Ni molecules in the membrane (approximately 3.4 pmol/mg of membrane protein) over angiotensin II receptors (approximately 1.2 pmol/mg of membrane protein). 125I-angiotensin bound to sites in the membrane with two affinities. Computer fitting of the binding isotherms yielded parameters of N1 = 279 fmol/mg protein, Kd1 = 0.2 nM; N2 = 904 fmol/mg protein, Kd2 = 1.4 nM. When all of the Ni molecules in the membrane were ADP-ribosylated, angiotensin bound to only one site with binding parameters of N = 349 fmol/mg protein, Kd = 0.4 nM. GTP-gamma-S caused a 7-fold increase in the Kd of this site to 2.7 nM. Overall, the data indicate that the Ni protein mediates the effect of angiotensin on adenylate cyclase. The observation that GTP-gamma-S can markedly decrease the affinity of angiotensin receptors when all Ni molecules are ADP-ribosylated suggests that angiotensin receptors may couple to other GTP-binding proteins which may mediate the effects of angiotensin in other signal transduction systems.
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PMID:Role of Ni in coupling angiotensin receptors to inhibition of adenylate cyclase in hepatocytes. 299 49

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