Mechanisms of guanine nucleotide-mediated regulation of adenylate cyclase activity.
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Adenylate Cyclase Toxin↗
Biomedical subjects
M Ui
Publications and source records attributed to M Ui.
Suppression of passive cutaneous anaphylaxis by pertussis toxin, an islet-activating protein, as a result of inhibition of histamine release from mast cells.
Passive cutaneous anaphylaxis (PCA) produced by antigen challenge to antibody-sensitized rats were interfered with by prior treatment with pertussis toxin, an islet-activating protein (IAP). The degree of interference was dependent on the dose and injection time of IAP; the effect of IAP developed slowly, with a maximal effect being observed 3 days later. Inhibition of PCA by IAP was associated with a decrease in histamine release from peritoneal mast cells, making it very likely that the process affected was mast cell secretion. Much less histamine was discharged in vitro, in response to certain membrane receptor (e.g. IgE receptor) stimulation, from mast cells that had been exposed to IAP than from the cells not exposed. Such an inhibitory effect of IAP was not observed when histamine release was provoked by a calcium ionophore without mediation of membrane receptors. IAP was a stronger inhibitor of histamine release than beta-adrenergic agonists. Further inhibition was produced when a beta-agonist was added to IAP-treated mast cells. The increase in the cellular content of cyclic AMP was associated with beta-agonist-induced, but not with IAP-induced, inhibition of histamine release. Thus, IAP inhibited histamine release by a mechanism in which metabolism of cyclic AMP was not directly involved.
Adrenergic beta-Agonists↗
The A protomer of islet-activating protein, pertussis toxin, as an active peptide catalyzing ADP-ribosylation of a membrane protein.
Islet-activating protein (IAP), pertussis toxin, is an oligomeric protein composed of as A protomer and a B oligomer. IAP and its A protomer were equipotent, on a molar basis, in enhancing GTP-dependent adenylate cyclase activity and in causing ADP-ribosylation of the 41,000 Mr protein when directly added to the cell-free membrane preparation from rat C6 glioma cells. Similar actions of IAP observed upon its addition to intact C6 cells were not mimicked by its A protomer, indicating that the A protomer had to be associated with the B oligomer to become accessible to its site of action on the inner surface of the membrane of intact cells. The A protomer, but not IAP, exhibited NAD-glycohydrolase activity in the reaction mixture lacking cellular components but containing dithiothreitol. Their actions on membranes were not accelerated by dithiothreitol, but markedly suppressed by oxidized glutathione. Thus, C6 cell membranes may possess certain "processing" enzyme(s) responsible for releasing the A protomer from the IAP molecule and for reductive cleavage of an intrachain disulfide bond in the released protomer, thereby producing an active peptide which functions to cause ADP-ribosylation of one of the subunits of guanine nucleotide regulatory protein in the receptor-adenylate cyclase system.
Adenosine Diphosphate Ribose↗
A role of the B-oligomer moiety of islet-activating protein, pertussis toxin, in development of the biological effects on intact cells.
Islet-activating protein (IAP), pertussis toxin, is an oligomeric protein (Tamura, M., Nogimori, K., Murai, S., Yajima, M., Ito, K., Katada, T., Ui, M., and Ishii, S. (1982) Biochemistry 21, 5516-5522), the biggest subunit (Mr = 28,000, referred to as the A-protomer) of which catalyzes transfer of the ADP-ribose moiety of NAD to the membrane Mr = 41,000 protein. The pentamer, termed the B-oligomer, consisting of the residual subunits was the moiety of IAP that was responsible for binding to the cell surface, as revealed by competitive inhibition of the development of the IAP actions on intact rat C6 glioma cells and rat adipocytes. The binding of the B-oligomer to its receptor proteins was divalent via the constituent two dimers; it stimulated mitosis of lymphocytes and caused an insulin-like action to enhance glucose oxidation in adipocytes, just as did concanavalin A, presumably as a result of cross-linking or aggregation of the membrane proteins. The A-promoter displayed its biological action on adipocytes only when the B-oligomer had been bound to the cells. Thus, IAP is a typical A-B toxin in which the B-oligomer is first bound to the cell surface proteins to enable the A-protomer to reach to the site of its action within the cell. Diverse biological actions of pertussis toxin may be accounted for by the mitogenic action of the B-oligomer as well as ADP-ribosyltransferase activity of the A-promoter.
Adenosine Diphosphate Ribose↗
Specific uncoupling by islet-activating protein, pertussis toxin, of negative signal transduction via alpha-adrenergic, cholinergic, and opiate receptors in neuroblastoma x glioma hybrid cells.
Exposure of NG108-15 hybrid cells to islet-activating protein (IAP), pertussis toxin, caused strong ADP-ribosylation of one of the membrane proteins with a molecular weight of 41,000. This ADP-ribosylation was paralleled by decreases in the inhibition of cAMP accumulation in intact cells or associated with reversal of the inhibition of GTP-dependent membrane adenylate cyclase, via alpha-adrenergic, cholinergic muscarinic, or opiate receptors. The affinity of these receptors for agonists was lowered by guanyl-5'-yl beta-gamma-imidodiphosphate (Gpp(NH)p) reflecting their coupling to the guanine nucleotide regulatory protein in this cell line. This effect of Gpp(NH)p was lost in membranes of IAP-treated cells; in the absence of Gpp(NH)p, the affinity for agonist was lower in treated than in nontreated cells. In contrast, the function of these receptors to bind antagonists remained unaltered in IAP-treated cells. Thus, IAP treatment of NG108-15 cells caused specific uncoupling of negative signal transduction from inhibitory receptors to the adenylate cyclase catalytic unit via the guanine nucleotide regulatory protein, as a result of ADP-ribosylation of one of the subunits of the regulatory protein.
Adenosine Diphosphate Ribose↗
Loss of the inhibitory function of the guanine nucleotide regulatory component of adenylate cyclase due to its ADP ribosylation by islet-activating protein, pertussis toxin, in adipocyte membranes.
Adenylate cyclase of rat adipocyte membranes exhibited dual responses in a strictly GTP-dependent manner; an activation took place in the presence of certain receptor agonists such as isoproterenol or secretin, whereas an inhibitory phase was observed with other agonists such as prostaglandin E1 or purine-modified adenosine as well as with the stimulatory agonists at higher GTP concentrations. Treatment of membrane donor cells with islet-activating protein (IAP), pertussis toxin, abolished the inhibitory phase while preserving the activatory phase. This unique action of IAP was associated with ADP-ribosylation of a membrane Mr = 41,000 protein. In contrast, the inhibitory phase was preserved in membranes from cholera toxin-treated cells. Monophasic and persistent activation of the cyclase was provoked by guanyl-5'-yl beta,gamma-imidodiphosphate. The time lag normally observed for the guanyl-5'-yl beta,gamma-imidodiphosphate activation was decreased by isoproterenol or cholera toxin but was not altered by IAP treatment. Our conclusion is that the sole site of IAP action is the guanine nucleotide regulatory protein (Ni) that is required for transmission of inhibitory signals from receptors to the catalytic unit of adenylate cyclase; the function of Ni is lost upon IAP-catalyzed ADP ribosylation of the Mr = 41,000 protein which appears to be an active subunit of Ni. A possibility is discussed that rather diverse effects of IAP so far reported with various cell types are accounted for in terms of such interference with the function of Ni.
Adenosine Diphosphate Ribose↗
Guanine nucleotide activation and inhibition of adenylate cyclase as modified by islet-activating protein, pertussis toxin, in mouse 3T3 fibroblasts.
Guanine nucleotide regulation of membrane adenylate cyclase activity was uniquely modified after exposure of 3T3 mouse fibroblasts to low concentrations of islet-activating protein (IAP), pertussis toxin. The action of IAP, which occurred after a lag time, was durable and irreversible, and was associated with ADP-ribosylation of a membrane Mr = 41,000 protein. GTP, but not Gpp(NH)p, was more efficient and persistent in activating adenylate cyclase in membranes from IAP-treated cells than membranes from control cells. GTP and Gpp(NH)p caused marked inhibition of adenylate cyclase when the enzyme system was converted to its highly activated state by cholera toxin treatment or fluoride addition, presumably as a result of their interaction with the specific binding protein which is responsible for inhibition of adenylate cyclase. This inhibition was totally abolished by IAP treatment of cells, making it very likely that IAP preferentially modulates GTP inhibitory responses, thereby increasing GTP-dependent activation and negating GTP-mediated inhibition of adenylate cyclase.
Adenylate Cyclase Toxin↗
Plasma cyclic nucleotide responses to psychological stress in normal and neurotic subjects.
The mirror drawing test (MDT) was performed to induce acute psychological stress in 10 normal volunteers and 23 neurotic patients. Plasma cAMP and cyclic guanosine 3',5'-monophosphate (cGMP) were determined serially before, during, and after the test. In controls, the MDT caused a significant increase in the plasma cAMP level, whereas no change was observed in plasma cGMP. This increase was suppressed by simultaneous administration of propranolol, although it was not affected by simultaneous injection of phentolamine. In neurotic patients, the instruction for the MDT itself resulted in increased cAMP and cGMP levels, although there were no further significant increases during and after the MDT. The results indicate: 1) the increase in plasma cAMP during the MDT reflects a beta-adrenergic stimulation; 2) in neurotics, the response of cAMP and cGMP to the MDT is different from the controls. This difference may be a potential parameter in the diagnosis and discrimination of neurotic disorders.
Adolescent↗
Species differences in actions of islet-activating protein, pertussis toxin.
Islet-activating protein (IAP) is one of the pertussis toxins. The ability of IAP to cause potentiation of insulin secretory responses and promotion of leukocytosis was studied in six animal species (hamsters, rats, guinea pigs, rabbits, dogs and monkeys). The action of IAP on insulin secretion in the animals was estimated by three kinds of tests: effects on epinephrine hyperglycemia, plasma insulin and blood glucose concentrations following the injection of stimuli, and glucose tolerance. Of all animals tested, IAP was most effective in hamsters. Marked hyperinsulinemia was also shown in IAP-treated dogs, rats and monkeys in response to insulin secretagogues, but their sensitivity to IAP was inferior to that of hamsters. In rabbits, IAP was markedly toxic, and the effect on insulin secretion was observed only slightly at a dose close to its minimal lethal dose. In addition, no significant effects of IAP were shown in guinea pigs in the present experiment. On the other hand, leukocytosis promoting activity of IAP appeared in a dose-dependent manner in all animal species; rabbits were the most sensitive to IAP in this regard. It is concluded that both actions of IAP appear differently in different animal species, and the species difference of the effect on insulin secretory responses is in agreement with that on histamine sensitizing activity.
Animals↗
Plasma cyclic AMP responses to adrenaline administration in patients with spinocerebellar degeneration, bronchial asthma, and diabetes mellitus--a preliminary report of a clinical test for detecting beta-adrenergic dysfunctions.
Plasma cyclic AMP responses to adrenaline administration in normal volunteers, patients with spinocerebellar degeneration, bronchial asthma, pulmonary emphysema, and diabetes mellitus were studied. Intramuscular administration of low doses (0.1--0.4 mg/person) of adrenaline caused a dose-dependent increase in plasma cyclic AMP. The increase in cyclic AMP was completely prevented by propranolol, while it was not affected by phentolamine or atropine. In patients with spinocerebellar degeneration, the concentrations of plasma cyclic AMP both before and after adrenaline administration were lower than in normal subjects. In asthmatic patients, the plasma cyclic AMP increase after adrenaline administration was smaller than that of the healthy controls. The plasma concentration of cyclic AMP in patients with insulin-dependent diabetes reached the peak level more slowly than in diabetic patients with dietary control alone. Examining changes in the plasma cyclic AMP level after adrenaline administration appears to be a useful means for assessing the degree of beta-adrenergic dysfunction.
Adult↗
Functional uncoupling of muscarinic receptors from adenylate cyclase in rat cardiac membranes by the active component of islet-activating protein, pertussis toxin.
Direct effects of islet-activating protein (IAP), pertussis toxin, on membrane preparations from rat heart tissues were studied. The native IAP was without effect, but its A-protomer, an active subunit, was effective after reduction of disulfide bonds in the peptide chain; it catalyzed ADP-ribosylation of the membrane Mr = 41,000 protein. Simultaneously, muscarinic receptor-mediated inhibition of adenylate cyclase was abolished. Carbachol, an agonist of muscarinic receptors, bound to membranes with the Hill coefficient smaller than unity. The affinity for the carbachol binding was lowered and the Hill coefficient was increased by guanylylimidodiphosphate (Gpp(NH)p), reflecting the muscarinic receptor coupling to the guanine nucleotide regulatory protein (N). Carbachol bound to the A-promoter-treated membranes with a lower affinity and a higher Hill coefficient, and these kinetic values were not altered by Gpp(NH)p, indicating that treatment of membranes with the A-protomer of IAP uncoupled muscarinic receptors from N. This IAP-sensitive N is Ni involved in the cyclase inhibition. Neither beta-adrenergic activation of adenylate cyclase nor beta-agonist binding to membranes was affected by the A-protomer of IAP. Thus, N (Ns) coupled to beta-receptors is not the site of its action. Although the affinity and the Hill coefficient for beta-agonist binding was not affected by preactivated cholera toxin either, the effect of Gpp(NH)p to alter these kinetic parameters was much smaller in the cholera toxin-treated membranes than in non-treated membranes. Thus, cholera toxin modified beta-receptor coupling to Ns in a manner quite different from IAP-induced modification of muscarinic receptor coupling to Ni.
Adenosine Diphosphate Ribose↗
Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model.
The subunit structure of islet-activating protein (IAP), pertussis toxin, has been analyzed to study a possibility that this protein is one of the A-B toxins [Gill, D. M. (1978) in Bacterial Toxins and Cell Membranes (Jeljaszewicz, J., & Wadstrom, T., Eds.) pp 291-332, Academic Press, New York]. Heating IAP with 1% sodium dodecyl sulfate caused its dissociation into five dissimilar subunits named S-1 (with a molecular weight of 28 000), S-2 (23 000), S-3 (22 000), S-4 (11 700), and S-5 (9300), as revealed by polyacrylamide gel electrophoresis; their molar ratio in the native IAP was 1:1:1:2:1. The molecular weight of IAP estimated by equilibrium ultracentrifugation was 117 000 which was not at variance with the value obtained by summing up molecular weights of the constituent subunits. The preparative separation of these IAP subunits was next undertaken; exposure of IAP to 5 M ice-cold urea for 4 days followed by column chromatography with carboxymethyl-Sepharose caused sharp separation of S-1 and S-5, leaving the other subunits as two dimers. These dimers were then dissociated into their constituent subunits, i.e., S-2 and S-4 for one dimer and S-3 and S-4 for the other, after 16-h exposure to 8 M urea; these subunits were obtained individually upon further chromatography on a diethylaminoethyl-Sepharose column. Subunits other than S-1 were adsorbed as a pentamer by a column using haptoglobin as an affinity adsorbent. The same pentamer was obtained by adding S-5 to the mixture of two dimers. Neither this pentamer nor other oligomers (or protomers) exhibited biological activity in vivo. Recombination of S-1 with the pentamer at the 1:1 molar ratio yielded a hexamer which was identical with the native IAP in electrophoretic mobility and biological activity to enhance glucose-induced insulin secretion when injected into rats. In the broken-cell preparation, S-1 was biologically as effective as the native IAP; both catalyzed ADP-ribosylation of a protein in membrane preparations from rat C6 glioma cells. In conclusion, IAP is an oligomeric protein consisting of an A (active) protomer (the biggest subunit) and a B (binding) oligomer which is produced by connecting two dimers by the smallest subunit in a noncovalent manner. Rationale for this terminology is discussed based on the A-B model.
Animals↗
ADP ribosylation of the specific membrane protein of C6 cells by islet-activating protein associated with modification of adenylate cyclase activity.
Islet-activating protein (IAP), one of the pertussis toxins, exerted dual actions on crude membrane preparations from rat C6 glioma cells; an Mr = 41,000 membrane protein was ADP-ribosylated while GTP (and GTP-dependent isoproterenol) activation of membrane adenylate cyclase was enhanced when membranes were incubated with IaP. Both actions of IaP were dependent on the incubation time and the concentrations of NAD and IAP, and were inhibited by nicotinamide; the one action was strictly paralleled by the other in magnitude. Tryptic digestion of the Mr = 41,000 protein was markedly influenced by the presence of guanyl-5'-yl beta-gamma-imidodiphosphate or NaF, the specific ligands of the regulatory component of the adenylate cyclase system. No ADP ribosylation occurred in the membranes prepared from intact C6 cells that had been incubated with IAP, suggesting that the IAP substrate had already been ADP-ribosylated by the intracellular NAD during incubation of the intact cells. Cholera toxin catalyzed ADP ribosylation of other proteins with Mr = 45,000 and 48,000/49,000 (doublet). It is concluded that IAP, added to intact cells or isolated membranes, causes unique modification of the receptor-adenylate cyclase coupling mechanism as a result of ADP ribosylation of the Mr = 41,000 protein which is presumably one of the subunits, other than the cholera toxin substrates, of the guanine nucleotide regulatory component of the cyclase system.
Adenosine Diphosphate Ribose↗
Modulation by islet-activating protein of adenylate cyclase activity in C6 glioma cells.
The cAMP content of intact cells as well as adenylate cyclase of the membrane-rich particulate fractions was studied with C6 glioma cells that had been exposed to the culture medium supplemented with islet-activating protein (IAP), one of the pertussis toxins. Both the increase in the cellular cAMP content in response to a beta-adrenergic agonist and the stimulation of membrane adenylate cyclase by the beta-agonist and/or GTP were markedly enhanced by the IAP treatment of C6 cells, but no change was induced in affinities of the agonist (or an antagonist) or GTP for their respective sites of action (or binding). The concentration of IAP required for the half-maximal enhancement was as low as 1 pg/ml, when the time of cell exposure to the toxin was prolonged to 18 h. No enhancement was observed for the basal cAMP content or basal enzyme activity, nor was activation of adenylate cyclase by Gpp(NH)p (or NaF) affected by IAP treatment. The Vmax value of a specific and low Km GTPase was significantly smaller in the membranes of IAP-treated cells than in those of control cells. Cholera toxin treatment of cells activated adenylate cyclase without exerting any influence on these IAP actions. Thus, IAP would appear to enhance beta-receptor-coupled stimulation of adenylate cyclase, in a manner distinct from cholera toxin, by rendering more GTP available to the GTP sites on the regulatory subunit of the receptor-enzyme system.
Adenine Nucleotides↗
A sensitive radioimmunoassay for adenosine in biological samples.
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Adenosine↗
Conversion of adrenergic regulation of glycogen phosphorylase and synthase from an alpha to a beta type during primary culture of rat hepatocytes.
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Animals↗
Plasma cyclic nucleotide responses to methacholine in patients with Adie's syndrome.
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Adie Syndrome↗
Phosphofructokinase from Ehrlich ascites tumor.
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Animals↗