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T Katada

Publications and source records attributed to T Katada.

215 records · Page 12Linked to original sources
PubMed1982

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↗
PubMed1982

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↗
PubMed1982

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↗
PubMed1982

Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein.

GTP and isoproterenol activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in washed membranes prepared from C6 gliomas cells was enhanced by incubation with islet-activating protein, one of the pertussis toxins, if the incubation mixture was supplemented with NAD and ATP. The action of the protein was observed immediately after its addition and increased progressively in magnitude as the protein concentration or the incubation time increased. There was simultaneous incorporation of radioactivity from the ADP-ribose moiety of variously labeled NAD into the membrane protein with a molecular weight of 41,000. We conclude that islet-activating protein enhances receptor-mediated GTP-induced activation of membrane adenylate cyclase as a result of ADP-ribosylation of a membrane protein, probably one of the components of the receptor-adenylate cyclase system.

Adenosine Diphosphate Ribose↗
PubMed1982

Alpha 2-adrenergic inhibition of insulin secretion via interference with cyclic AMP generation in rat pancreatic islets.

Glucose-induced secretion and cyclic AMP accumulation in isolated rat pancreatic islets as well as GTP-activated adenylate cyclase of the membrane-rich preparation from the islets were strongly inhibited by some alpha-adrenergic agonists. The relative potencies of the agonists, estimated according to their dose-dependent actions, were in such an order that clonidine greater than epinephrine (congruent to norepinephrine) greater than phenylephrine congruent to methoxamine, regardless of which of the three parameters (i.e., insulin release, cyclic AMP accumulation, and adenylate cyclase activity) was used for estimation. There was a highly significant correlation between the amounts of cyclic AMP accumulation and the rate of insulin release that were changed in response to these agonists. The order of the potencies of alpha-adrenergic antagonists to reverse epinephrine inhibition of these parameters was invariably yohimbine congruent to dihydroergotamine congruent to phenylephrine greater than prazosin. In conclusion, the rat islet cell membrane is equipped with alpha 2-adrenoceptors which are linked to adenylate cyclase to cause diminution of the cellular content of cyclic AMP; insulin secretion may be inhibited consequently.

Adenylyl Cyclases↗
PubMed1981

Islet-activating protein. A modifier of receptor-mediated regulation of rat islet adenylate cyclase.

Adenylate cyclase of the membrane-rich fraction of 24-h cultured islets was inhibited by epinephrine via alpha-adrenergic receptors. Epinephrine was inhibitory only when the enzyme was activated by GTP; the degree of inhibition was highly proportional to the degree of GTP activation. Adenylate cyclase of islets cultured with islet-activating protein (IAP), one of the pertussis toxins, was less susceptible to epinephrine inhibition. The degree of the inhibition was markedly reduced without changes in potency of the catecholamine and in GTP dependence after IAP treatment. None of the other kinetic properties of the enzyme including the affinity for substrate, sensitivity to guanine nucleotide and fluoride activation, and cholera toxin-induced modification of enzymic activity were affected by treatment of islets with IAP, suggesting that neither the catalytic nor the GTP-regulatory component of the membrane adenylate cyclase complex is the site of IAP action. Slight activation of the enzyme by glucagon or adenosine tended to be enhanced by IAP treatment. Thus, a mechanism whereby membrane receptors are linked to adenylate cyclase appears to be modified by exposure of islet cells to IAP.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
PubMed1981

In vitro effects of islet-activating protein on cultured rat pancreatic islets. Enhancement of insulin secretion, adenosine 3':5'-monophosphate accumulation and 45Ca flux.

Pancreatic islets were maintained in culture with or without islet-activating protein (IAP), which is a new protein purified from culture medium of Bordetella pertussis. These cultured islets (IAP-treated or control) were then incubated for 30 min in IAP-free medium with various insulin secretagogues. During incubation, much more insulin was released from IAP-treated islets than control islets in response to glucose, arginine, glucagon, and sulfonylurea. IAP was effective in this regard when added to cultures at concentrations higher than 0.01 ng/ml; the effect was dependent on concentration up to 100 ng/ml. Enhanced insulin secretion was associated with accumulation of cyclic AMP when breakdown of the nucleotide was prevented by a methylxanthine. Epinephrine caused marked inhibitions, via alpha-adrenergic receptors, of glucose-induced insulin release, cyclic AMP accumulation and 45Ca uptake in control islets but did not in IAP-treated islets during incubation. None of these effects of IAP pretreatment were observed unless the medium for incubation was supplemented with Ca ions. 45Ca ion flux through the islet cell membrane was accelerated by the IAP treatment; conceivably, IAP was effective in causing sustained activation of native calcium ionophores on the membrane, which would be responsible for the enhanced insulin and cyclic AMP responses characteristic of IAP-treated islets.

1-Methyl-3-isobutylxanthine↗
PubMed1980

Slow interaction of islet-activating protein with pancreatic islets during primary culture to cause reversal of alpha-adrenergic inhibition of insulin secretion.

The manner in which islet-activating protein (IAP), a protein purified from the culture medium of Bordetella pertussis, interacts with the islet B-cell was studied by following the progressive development of IAP-induced reversal of alpha-adrenergic inhibition of insulin release during maintenance of islets in culture with glucose and epinephrine. This action of IAP developed in an exponential manner dependent on its concentration after a true lag period of about 1 h. The lag period was not grossly dependent on the concentration of IAP added but highly dependent on temperature of culture, and was still seen upon adding a second dose of IAP to partially stimulated cells. After 24-h culture significantly more insulin was secreted with IAP at a concentration as low as 1 pg/ml and the half-maximal effect was observed at 0.1 ng/ml. The development of IAP action occurred even in the islets that had been exposed to IAP for only 30 s, but was significantly prevented by anti-IAP serum added before the end of the lag period. IAP was effective in the presence of cycloheximide, an inhibitor of protein synthesis, or of vinblastine or cytochalasin B, microtubular-microfilamentous modifiers. It is suggested that the IAP molecule is rapidly bound to the receptor area of the islet B-cell and then is gradually inserted into the cell membrane before appearance of its action to activate native calcium ionophores. This slow interaction of IAP with the membrane may be responsible for potentiation of insulin secretory and cAMP responses of the cell to various stimuli as well as for reversal of alpha-adrenergic inhibition.

Animals↗
PubMed1979

Islet-activating protein. Enhanced insulin secretion and cyclic AMP accumulation in pancreatic islets due to activation of native calcium ionophores.

The mechanism whereby "islet-activating protein" (IAP) purified from the culture medium of Bordetella pertussis potentiates insulin secretion was studied by experiments in vitro with islets of rats once injected with IAP (0.5 micrograms/100 g body weight, 3 days before killing) or with islets that had been exposed to IAP (0.1 to 100 ng/ml) for 24 h. The IAP treatment markedly enhanced insulin secretory responses and cAMP accumulation in islets, facilitated the efflux of 45Ca through the cell membrane, and abolished the alpha-adrenergic action of epinephrine (and somatostatin) to inhibit glucose-induced insulin release, cAMP accumulation, and 45Ca uptake. These effects of the IAP treatment were reduced when islets were incubated in a low calcium medium. Based on these results, it was concluded that IAP interacts directly but slowly with the islet B cell in such a manner as to render more calcium available to the stimulus-secretion coupling mechanism as a result of sustained activation of native calcium ionophores on the cell membrane.

Animals↗
PubMed1979

Effect of in vivo pretreatment of rats with a new protein purified from Bordetella pertussis on in vitro secretion of insulin: role of calcium.

Rats were injected once iv with an islet-activating protein (IAP), a new protein purified from the culture medium of Bordetella pertussis. Three days later, their pancreases were studied in vitro for insulin secretory responses. As with pertussis vaccine, pretreatment of rats with IAP was effective in enhancing insulin release from pancreas during perfusion or from islets during incubation in response to secretagogues such as glucose and glibenclamide. The alpha-adrenergic inhibition of insulin secretion induced by epinephrine was also reversed by the pretreatment with IAP. 3-Isobutyl-1-methylxanthine caused insulin release due to accumulation of cAMP. This 3-isobutyl-1-methylxanthine-induced insulin release during perfusion was enhanced in a Ca-containing perfusate, but was conversely reduced in a Ca-free perfusate by the IAP pretreatment. Upon the addition of Ca to the Ca-free perfusate, more insulin was released from pancreases of IAP-treated rats than from those of nontreated rats.

Animals↗
PubMed1977

Spontaneous recovery from streptozotocin-induced diabetes in rats pretreated with pertussis vaccine or hydrocortisone.

Following the intravenous injection of streptozotocin into rats, postprandial hyperglycaemia was sustained from 24 hours over a subsequent period of some weeks and the rats were glucose intolerant. When streptozotocin was similarly injected into pertussis-sensitized or hydrocortisone treated rats, the postprandial hyperglycaemia observed at 24 hours did not persist, but showed a progressive decline until near normoglycaemia was obtained a week later. These animals manifested normal glucose tolerance one week after streptozotocin. Thus, a spontaneous recovery from streptozotocin-induced diabetes occurred under these conditions. This spontaneous recovery from diabetes was associated with hyperinsulinaemia in the fed state.

Animals↗
PubMed1977

Increased absorption of iodochlorhydroxyquin by rat intestine in the presence of solubilizing agents.

The intestinal absorption of iodochlorhydroxyquin (clioquinol) by the rat was studied by determining the radioactivity in the bile, blood and several organs 90 min after direct application of 125I-labeled clioquinol into the duodenum. The addition of solubilizing agents such as carboxymethyl-cellulose and lauryl sulfate to clioquinol preparation markedly enhanced the intestinal absorption of the drug in either the presence of absence of bile secretion which, by itself, increased the drug absorption. Possible significance of the enhanced absorption of clioquinol by solubilizing agents in the etiology of subacute myelo-optic neuropathy (SMON) in Japan is discussed.

Animals↗
PubMed1977

Perfusion of the pancreas isolated from pertussis-sensitized rats: potentiation of insulin secretory responses due to beta-adrenergic stimulation.

In order to study the mechanism by which pertussis-sensitized rats showed enhanced insulin secretory responses to various secretagogues (Sumi, T., and M. Ui, Endocrinology 97: 352, 1975), pancreases of rats receiving a single injection of Bordetella pertussis cells 3 days before were perfused with Krebs-Ringer solution, and release of insulin therefrom was compared with that from the pancreases of normal rats. Much more insulin was released from the pancreas of the pertussis-sensitized rat than from the pancreas of the normal rat in response to glucose, arginine, glibenclamide and 3-isobuty-l-methylxanthine. The inhibition of insulin secretion caused by epinephrine, norepinephrine or phenylephrine via alpha-adrenergic receptors in the pancreas of normal rats was no longer observable with the pancreas from pertussis-sensitized rats. Instead, the addition of epinephrine with or without phentolamine gave rise to a marked secretion of insulin from the pancreas of pertussis-sensitized rats which was prevented by propranolol. It is concluded that a single injection of B. pertussis into rats results in a sustained modification of insulin secretory processes in the pancreatic beta-cells in such a manner as to favor insulin secretory responses to beta-adrenergic stimulation and other secretagogues.

Animals↗
PubMed1976

Accelerated turnover of blood glucose in pertussis-sensitized rats due to combined actions of endogenous insulin and adrenergic beta-stimulation.

1. Epinephrine-induced hyperglycemia was attenuated by the treatment of rats with pertussis vaccine, but this attenuation was abolished when endogenous insulin was suppressed by streptozotocin or anti-insulin serum. It was concluded that epinephrine-induced hyperglycemia was counterbalanced by the hypoglycemic action of insulin, the secretion of which was markedly potentiated in pertussis-sensitized rats. 2. Without epinephrine, no hypoglycemia developed in pertussis-sensitized rats despite the higher blood level of insulin. Tracer experiments with [14C,3H] glucose or [14C]bicarbaonate showed that, in pertussis-sensitized rats, more glucose was liberated into the blood from hepatic gluconeogenesis at the expense of hepatic glycogenesis, thereby accelerating the turnover of blood glucose. 3. Since this activation of hepatic glucose production was reduced by propranolol, a beta-adrenergic blocking agent, it is very likely that adrenergic beta-stimulation is, at least partly, responsible for the metabolic alterations observed in pertussis-sensitized rats.

Animals↗
PubMedDate not supplied

Functional reconstitution of purified muscarinic receptors and inhibitory guanine nucleotide regulatory protein.

Muscarinic receptors trigger several different responses including an increase in concentration of cyclic GMP, a decrease in cyclic AMP concentration, breakdown of polyphosphoinositides and changes in ion permeability. It is not yet clear whether these reactions occur sequentially or independently and which directly coupled to the muscarinic receptor. Several lines of evidence indicate that muscarinic receptors in many, if not all, cell types are coupled to the inhibitory guanine nucleotide regulatory protein (Ni or Gi) of adenylate cyclase. To provide direct evidence for this coupling, we have reconstituted muscarinic receptors purified from porcine brain with Ni purified from rat brain in a phospholipid vesicle. Here, we report that the GTPase activity of Ni is stimulated by carbachol. This action is blocked by the simultaneous addition of atropine and is not observed when the Ni protein is ADP-ribosylated. We conclude that one function of the muscarinic receptor is the activation of Ni.

Adenosine Diphosphate Ribose↗
PubMedDate not supplied

Activation mechanism and physiological roles of stress-activated protein kinase/c-Jun NH2-terminal kinase in mammalian cells.

Stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK), which belongs to the family of mitogen-activated protein kinase (MAPK), is activated by many types of cellular stress or extracellular signals. Recent studies, including the analysis with knockout cells and mice, have led towards understanding the molecular mechanism of stress-induced SAPK/JNK activation and the physiological roles of SAPK/JNK in embryonic development and immune responses. Two SAPK/JNK activators, SEK1 and MKK7, are required for full activation of SAPK/JNK, which responds to various stimuli in an all-or-none manner in mouse embryonic stem (ES) cells. SAPK/JNK activation plays essential roles in organogenesis during mouse development by regulating cell proliferation, survival or apoptosis and in immune responses by regulating cytokine gene expression. Furthermore, SAPK/JNK is involved in regulation of mRNA stabilization, cell migration, and cytoskeletal integrity. Thus, SAPK/JNK has a wide range of functions in mammalian cells.

Animals↗