Ichikawa Atsushi

Inhibitory effect of an intracellular glutathione on Δ12-prostaglandin J2-induced protein syntheses in porcine aortic endothelial cells

Abstract Δ 12 -Prostaglandin (PG) J 2 caused porcine aortic endothelial cells to synthesize a 31,000-dalton heme oxygenase and a 67,000-dalton protein (p67). Treatment of the cells with buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, depleted intracellular GSH, and enhanced the induction of heme oxygenase and p67 syntheses by Δ 12 -PGJ 2 . In contrast, treatment with GSH increased the intracellular GSH level and reduced the induction. There was a reciprocal relationship between the level of intracellular GSH, and that of the induction of heme oxygenase and p67 syntheses by Δ 12 -PGJ 2 . An increase in the intracellular GSH level caused an increase in the ethyl acetate-unextractable form of Δ 12 -PGJ 2 in the cytosol, but suppressed the accumulation of Δ 12 -PGJ 2 in the nuclei. Furthermore, GSH strongly inhibited the in vitro binding of Δ 12 -PGJ 2 to isolated nuclei, which is N -ethylmaleimide sensitive. Moreover, the induction of heme oxygenase and p67 syntheses by the thiol-reactive agents arsenite and diethylmaleate was also inhibited by GSH treatment and enhanced by BSO treatment. These results demonstrate that intracellular GSH suppresses Δ 12 -PGJ 2 -induced heme oxygenase and p67 syntheses by inhibiting the binding of Δ 12 -PGJ 2 to nuclei.

Effects of glycyrrhizin and glycyrrhetinic acid on dexamethasone-induced changes in histamine synthesis of mouse mastocytoma P-815 cells and in histamine release from rat peritoneal mast cells

Abstract The effects of glycyrrhizin and its aglycone, glycyrrhetinic acid, on dexamethasone-induced changes in the histamine synthesis of mastocytoma P-815 cells and in the histamine release from antigen-stimulated rat peritoneal mast cells were investigated. Glycyrrhetinic acid but not glycyrrhizin, at concentrations from 20 to 35 μM, almost completely inhibited the dexamethasone-induced increases in both the histamine content and histidine decarboxylase activity of cultured mastocytoma P-815 cells. Glycyrrhetinic acid, however, showed practically no inhibition of [ 3 H]dexamethasone binding to the cytoplasmic receptor. On the other hand, glycyrrhetinic acid but not glycyrrhizin markedly inhibited the release of histamine from antigen-stimulated rat mast cells, and intensified the inhibitory activity induced by dexamethasone. Glycyrrhetinic acid inhibited the antigen-induced release and incorporation of [ 3 H]arachidonic acid in immunized rat mast cells. The administration of glycyrrhizin into rats, in contrast to the in vitro treatment of the cells with glycyrrhizin, markedly inhibited histamine release from antigen-stimulated rat mast cells. These results suggest that glycyrrhetinic acid inhibited dexamethasone-induced changes in the histamine synthesis of mastocytoma P-815 cells, and in the histamine release from rat mast cells. On the other hand, glycyrrhizin may exert its effect after conversion to glycyrrhetinic acid in vivo .

Properties of protease in mast cell granules

Abstract A neutral protease partially purified from rat peritoneal mast cells closely resembled α-chymotrypsin in pH optimum and in susceptibility to several inhibitors including chymostatin, but the two enzymes were quite different in some respects. Mast cell protease was a more basic protein than α-chymotrypsin as indicated by their isoelectric points (9.3 and 8.5, respectively). In contrast to α-chymotrypsin, which consisted of three different subunits, mast cell protease was apparently composed of a single polypeptide chain of molecular weight 27,000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Mast cell heparin ionically linked to mast cell protease and suppressed the activity of the enzyme toward N- benzoyl- l -tyrosine ethyl ester and casein by 35 and 75 per cent, respectively, but it did not affect α-chymotrypsin in a medium containing 50 mM CaCl 2 . Porcine intestinal mucosa heparin also showed similar inhibitory effects. The inhibitory action of heparin was more evident in low salt medium. Serotonin (0.25 mM), but not histamine, produced an 80 per cent inhibition of mast cell protease (2 μg) activity toward casein in the presence of heparin (2.5 μg). Zn 2+ (0.008–0.2 mM) also suppressed the protease activity by 40–90 per cent in the presence of heparin.

Calcium dependency of inhibition by arachidonic acid of compound 48/80-induced histamine release from mast cells

Compound 48/80 ( compd 48/80)-induced histamine secretion from rat mast cells was inhibited almost completely by pretreatment of the cells at 37 degrees with 25 microM arachidonic acid in the presence of 1.8 mM Ca2+. As the Ca2+ concentration was reduced below 1.8 mM, 25 microM arachidonic acid became less inhibitory and, then, progressively more stimulatory for histamine release with or without compd 48/80. No additive effect on histamine release was obtained by combining compd 48/80 and arachidonic acid. Pretreatment of mast cells with lidocaine, an inhibitor of Ca2+ binding to phospholipid, or with nordihydroguaiaretic acid, an inhibitor of Ca2+ flux and lipoxygenase, stimulated arachidonic acid-induced histamine release. Arachidonic acid also inhibited a compd 48/80-induced spike increment of intracellular 45Ca2+ uptake and a decrease of total 45Ca2+ uptake by 45Ca2+-preloaded mast cells. Arachidonic acid and Ca2+ also suppressed melittin-induced histamine release and compd 48/80-induced release of radioactivity from mast cells preloaded with [3H]arachidonic acid. These results suggest that exogenous arachidonic acid or its metabolite(s) may interact with membrane-associated Ca2+, disturbing Ca2+ availability for the trigger mechanism of compd 48/80-induced histamine release or inhibiting the subsequent metabolism of arachidonic acid via the lipoxygenase pathway to form active metabolites involved in the histamine liberating mechanism.

Fibroblast growth factor-dependent metabolism of hypoxanthine via the salvage pathway for purine synthesis in porcine aortic endothelial cells

Abstract In this study we examined the metabolism of hypoxanthine in fibroblast growth factor (FGF)-stimulated porcine aortic endothelial cells (PAEC). Our previous report indicated that hypoxanthine in fetal bovine serum (FBS) was an essential component for both basal and FGF-dependent growth of PAEC (Hayashi et al., Exp Cell Res 185: 217–228, 1989). Besides hypoxanthine, the addition of various purine bases and purine nucleosides, but not xanthine, xanthosine or any pyrimidine metabolites, restored the limited growth of PAEC cultured in medium containing 10% dialyzed FBS in the presence or absence of FGF. The metabolism of [ 14 C]hypoxanthine was compared in PAEC treated with and without FGF. Treatment of PAEC with FGF for 24 hr enhanced the radioactivity incorporation from [ 14 C]hypoxanthine into both the acid-soluble and -insoluble fractions approximately 2-fold. Upon Chromatographie analyses of hypoxanthine metabolites in the acid-soluble nucleotide fraction, it was found that in control PAEC hypoxanthine was largely metabolized to IMP, adenine nucleotides and uric acid, whereas in FGF-treated cells it was converted to ATP, ADP, GTP, xanthine and uric acid. The radioactivity of IMP was lowered in FGF-stimulated cells. The addition of FGF to PAEC increased phosphoribosyl pyrophosphate (PRPP) synthetase activity by approximately 8-fold and the PRPP content by approximately 2-fold, but it did not increase hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity or hypoxanthine transport. On the other hand, methotrexate, an inhibitor of de novo synthesis of purine, did not affect the growth of PAEC. Analyses of the rate of [ 14 C]formate incorporation into total purine compounds showed that PAEC had a low capacity to synthesize purines de novo , which was not stimulated by FGF. These data indicate that FGF stimulates the synthesis of PRPP necessary for the salvage synthesis of purine nucleotides in conjunction with purine bases, e.g. hypoxanthine.

Effect of pyruvate on thyroid hormone-induced lipolysis in rat adipose tissue in vitro.

Abstract Pyruvate markedly enhanced lipolysis induced by triiodo- l -thyronine (L-T3) or methylxanthines in fat cells from fasted rats, but not from fed rats. In contrast, pyruvate only slightly increased lipolysis which had been submaximally elevated by epinephrine, adrenocorticotropic hormone or thyroid-stimulating hormone, and did not affect the lipolytic effect of lutenizing hormone, growth hormone plus dexamethasone or hydrocortisone. Insulin and prostaglandin E1 blocked this pyruvate effect on T3-induced lipolysis. In the presence of 5 mM pyruvate, a significant lipolysis was observed with 0.001 mM L-T3; a near maximal effect was seen with L-T3 at 0.1 mM. Conversely, lipolysis induced by 0.1 mM L-T3 was clearly augmented with 0.01 mM pyruvate; a maximal effect was seen with pyruvate at 5mM. Pyruvate itself had no effect on basal lipolysis, but promptly stimulated T3-induced lipolysis after its addition. Furthermore, pyruvate (0.01 to 5 mM) did not alter the ratios of fatty acid/glycerol released during lipolysis induced by 0.1 mM L-T3. Pyruvate also markedly augmented T3-induced formation of cAMP in fat cells from fasted rats. This accumulation of cAMP was blocked by AMP, a potent inhibitor of lipolysis induced by T3 plus pyruvate.