Iwata Heitaroh

Formation and possible role of bis(methylmercuric) selenide in rats treated with methylmercury and selenite

Abstract The metabolic fate of methylmercury after administration of [ 203 Hg]-methylmercuric chloride in combination with sodium selenite was investigated in rats. Whole body autoradiography and radioassay showed that administration of selenite decreased the mercury concentration in the liver and kidney, and increased that in the brain. The rapid changes of methylmercury concentration in the tissues after selenite injection were accompanied by increases in mercury extractable with benzene at neutral pH. The maximum levels of benzene-extractable mercury in the blood, kidney and liver were attained 30 min after selenite injection and were 30, 23 and 8 percent, respectively, of the total mercury. Thin-layer chromatography showed that the benzene-extractable mercury was a complex of methylmercury with selenium, bis(methylmercuric) selenide. These findings indicate that selenite alters the distribution of methylmercury in the tissues by formation of a diffusible complex with methylmercury, bis(methylmercuric) selenide.

Gonadectomy changes the pituitaiy-adrenocortical response in mice to S-HT1A receptor agonists

Abstract The effects of 5-HT 1A receptor agonists such as 8-hydroxy-2-(ui-n-propylamino)tetralin (8-OH-DPAT), BP 554 and buspirone on the serum corticosterone level were significantly more pronounced in female than in male mice. A similar sex difference was observed for the effect of 8-OH-DPAT on the piasma ACTH level. Pretreatment with proadifen, an inhibitor of microsomal drug-metabolizing enzymes, did not affect the sex difference in the effect of 8-OH-DPAT. The corticosteione response to 8-OH-DPAT in female mice was attenuated by ovariectomy. The effect of 8-OH-DPAT in ovariectomized mice was enhanced by chronic estradiol and the enhancement was blocked by testosterone. In male mice the corticosterone response to 8-OH-DPAT increased at 5 weeks after castration but had not changed at 2 weeks. Chronic estradiol enhanced the corticosterone response to 8-OH-DPAT in castrated mice. There was no difference between sexes in [ 3 H]8-OH-DPAT binding to membranes and in the contents of 5-HT and 5-hydroxyindoleacetic acid in the hypothalamus. Accumulation of 5-hydroxytryptophan after decarboxylase inhibition in the hypothalamus was however greater in female than in male mice.

Pharmacological significances of peptidase and proteinase in the brain (report I)—Enzymatic inactivation of bradykinin in rat brain

Abstract The peptidase activity in rat brain was investigated using bradykinin as substrate. The nature and changes during convulsion in activity of the enzymes which inactivates bradykinin (kininase) were examined. The optimum conditions for the assay of kininase were pH 7.6 and about 45°. The kininase activity in the cerebellum was higher than that in the cerebral cortex or the brain stem. On subcellular fractionation, the highest kininase activity was observed in the supernatant, with lower activities in the microsomes, mitochondoria and nuclei. The effects of metal ions (zinc and cobalt ions) and N-ethylmaleimide on the brain kininase activity were different from those on enzyme in the plasma. The activity of the kininase in the cerebellar region increased during convulsions caused by pentetrazol or picrotoxin, but not by strychnine and, of the subcellular fractions of the cerebellum of a rat after pentetrazol administration, the increase was only found in the nuclear fraction. Increased kininase activity in the cerebellar region induced by pentetrazol, was only observed during convulsions and not in the preconvulsive or intermediate states. This suggests that some alteration in the metabolism of brain protein may occur during convulsion.

Assay of phospholipase A2 activity of synaptic membranes using a phospholipid transfer protein: stimulation by depolarization

A phospholipid transfer protein from bovine liver was used to transfer exogenous ([14C]linoleoylphosphatidylethanolamine [14C]linoleoyl PE) into synaptic membranes and synaptosomes without modifying the lipid compositions in order to study the intrinsic activity of phospholipase A2 of the preparations. Results were compared with the conventional method in which the substrate was simply dispersed with the enzyme preparations. Liberation of [14C]linoleic acid from [14C]linoleoyl PE-preloaded synaptic membranes by the transfer protein continued almost linearly over 60 min of incubation in the presence of Ca2+. The dose-response curve of Ca2+ for the activity of phospholipase A2 obtained in the present method was slightly shifted to the left in comparison with the conventional method: Ca2+ even at the concentration of 100 microM significantly enhanced the enzyme activity. Requirement of Ca2+ for the reaction is more specific in the present method than in the conventional one. When synaptosomes were prelabeled with [14C]linoleoyl PE by the transfer protein, the liberation of [14C]linoleic acid during the incubation at 37 degrees C increased linearly over 2 min. The liberation of [14C]linoleic acid was significantly enhanced in the presence of 56 mM KCl, 50 microM veratridine and 50 microM calcium ionophore A23187. These agents did not stimulate the reaction in the absence of Ca2+.

Studies on the mechanism of inhibition of xanthine oxidase by 5-diazoimidazole-4-carboxamide and related thioazoimidazole carboxamides

Abstract The mechanism of inhibition of milk xanthine oxidase by 5-diazoimidazole-4-carboxamide (diazo-ICA) and by five related thioazoimidazole carboxamides (thioazo-ICAs) was studied. The extent of inhibition of xanthine oxidase by diazo-ICA and thioazo-ICAs decreased greatly when these compounds were preincubated in a buffer before the addition of substrate and enzyme. In 0.1 M Tris-HCl buffer, pH 7.5, thioazo-ICAs were converted to 2-azahypoxanthine, a cyclized product of diazo-ICA, which inhibits xanthine oxidase slightly. The inhibition of xanthine oxidase by thioazo-ICAs is probably due to this diazo-ICA. With xanthine as a variable substrate, diazo-ICA caused uncompetitive, irreversible inhibition. The inhibition of xanthine oxidase by diazo-ICA was reduced by simultaneous addition of a sulfhydryl compound, such as cysteine, cysteamine or reduced glutathione, but not other amino acids. Diazo-ICA inactivated the enzyme more significantly and rapidly than other sulfhydryl reagents. The inhibitory activity of diazo-ICA was potentiated strongly by Fe 2+ , Mn 2+ , Co 2+ and Cu 2+ , and slightly by Mo 5+ . Treatment of milk xanthine oxidase with diazo-ICA changed the absorption spectrum of the enzyme.

Potent competitive uricase inhibitors--2,8-diazahypoxanthine and related compounds.

Abstract The inhibitory activities of 2,8-diazahypoxanthine and related compounds on uricase of bovine kidney and rat liver homogenates were compared in vitro. 2,8-Diazahypoxanthine was found to be even more inhibitory than 2-azahypoxanthine, 8-azaxanthine, 8-azahypoxanthine or oxonate. From Lineweaver-Burk plots, 2,8-diazahypoxanthine and 8-azaxanthine were shown to be competitive inhibitors of uricase. 2,8-Diazahypoxanthine had no influence on the activity of milk xanthine oxidase or rat erythrocyte hypoxanthine-guanine phosphoribosyltransferase at the concentrations tested. The inhibitory activities of 2,8-diazahypoxanthine and related compounds on rat liver uricase in vivo were compared by measuring their effects on serum urate and allantoin. 2,8-Diazahypoxanthine, administered intraperitoneally, caused the greatest increase in the urate level with a concomitant decrease in the allantoin level. 8-Azaxanthine and 8-azahypoxanthine were both relatively strong inhibitors of uricase in vivo although the latter was not a strong inhibitor in vitro.

Biological activity of diazonium compounds. Studies on the mechanism of action of 4 (or 5)-diazoimidazole-5 (or 4)-carboxamide on 5-hydroxytrypt-amine release from rabbit platelets—II: Comparative studies with N-ethylmaleimide in vitro

Abstract Studies were made on the association of the SH-blocking effect of Diazo-ICA with its activity in releasing 5-HT from isolated rabbit platelets, and the releasing activity was compared with that of NEM. High concentration of NEM, like Diazo-ICA, caused release of 5-HT from platelets, but, unlike Diazo-ICA, its action was independent of the calcium ion concentration in the incubation medium. The calcium-dependent release of 5-HT by Diazo-ICA was completely blocked by a small amount of NEM. The sulfhydryl compounds, cysteine, glutathione and BAL also blocked release of 5-HT by Diazo-ICA. ATP, ADP and inorganic pyrophosphate inhibited release of 5-HT by Diazo-ICA, but AMP and creatine phosphate did not. These findings suggest that the sulfhydryl group and pyrophosphate structures are involved in the mechanism of release of 5-HT from rabbit platelets by Diazo-ICA.

Biological activity of diazonium compounds: Studies on the mechanism of action of 4(or 5)-diazoimldazole-5(or 4)-carboxamide on 5-hydroxytryptam1ne release from rabbit platelets—I: Requirement for calcium ion

Abstract Incubation of 4(or 5)-diazoimidazole-5(or 4)-carboxamide (Diazo-ICA) at 37° with isolated rabbit platelets induces release of 5-hydroxytryptamine (5-HT). This release occurs only in the presence of a trace of calcium ions and the effect is independent of the presence of other plasma components. The release is temperature-dependent, being appreciably lower at room temperature than at 37° and almost negligible at 4°. These results imply that the mechanism of release with Diazo-ICA differs from that with reserpine or bacterial pyrogenic lipopolysaccharide. 4(or 5)-Dialkyltriazenoimidazole-5 or 4)-carboxamides, prepared by coupling Diazo-ICA with corresponding dialkylamines, cause only slight release of 5-HT from platelets, and the release is independent of the presence of calcium ion. In contrast, 4(or 5)-aminoimidazole-5(or 4)-carboxamide (the parent compound of these derivatives), 2-azahypoxanthine (a cyclized product of Diazo-ICA) and S-(5(or 4)-carbamoyl-4(or 5)-imidazolyl azo)cysteine (a coupling product of Diazo-ICA and cysteine) release no 5-HT from platelets. The other diazonium compound tested, diazobenzene sulfonate and diazobenzenesulfonamide, also cause no release.

Postnatal development of thiamine metabolism in rat skeletal muscle

Abstract 1. 1. The activities of 2-oxoglutarate dehydrogenase, transketolase, thiamine pyrophosphokinase and thiamine triphosphatase and the concentrations of thiamine phosphates were almost the same between rat extensor digitorum longus and soleus muscles at 2 weeks of age. 2. 2. These enzyme activities changed after 3 weeks of age in a different way depending on the muscle phenotype. 3. 3. Thiamine diphosphate level and the activity of 2-oxoglutarate dehydrogenase increased only in soleus muscle and thiamine triphosphate level increased only in extensor digitorum longus during development.