Hideharu Ikebuchi

Morphine metabolism in isolated rat hepatocytes and its implications for hepatotoxicity.

Isolated rat hepatocytes metabolized morphine to its glucuronide conjugate, morphinone-glutathione conjugate, normorphine and morphinone. Addition of morphine to the isolated hepatocytes induced a marked decrease in the level of glutathione in the cells and resulted in cell death. The formation of glutathione conjugate was correlated well with the loss of intracellular glutathione. The cytotoxicity of morphinone was higher than that of morphine. Naloxone and normorphine showed no cytotoxic effect on the cells. Naloxone inhibited the formation of morphinone-glutathione conjugate and prevented the morphine-induced cytotoxicity. Naloxone also blocked morphine-induced liver damage in vivo. In contrast, the morphinone-induced hepatotoxicity was not prevented by naloxone. It is concluded that morphine has a hepatotoxic effect, that the morphine-induced hepatotoxicity is due to its metabolic activation, and that naloxone acts as an inhibitor of an enzyme converting morphine to morphinone.

Isolation and characterization of a major allergenic component (gp55) of Aspergillus fumigatus

IgE class antibodies specific for antigens in a water-soluble extract of Aspergillus fumigatus (strain NHL-5759) were analyzed by immunoblotting with sera from patients with allergic bronchopulmonary aspergillosis. All the sera tested were reactive with a major 50 to 60 kd protein in the extract. This allergen, designated gp55, was purified by gel filtration and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigen was found to be present in the water-soluble extract in the form of a complex composed of approximately eight molecules of gp55. The carbohydrate and phosphate content of the purified antigen were 23.1% and 0.46%, respectively. The molar ratio of mannose to galactose residues was 2.76:1, and the protein was glycosylated predominantly with N-linked oligosaccharides. The serologic activity of the gp55 antigen was abolished by treatment with nonspecific protease (Pronase) but not by treatment with sodium metaperiodate or endoglycosidases. Thus the major antigenic site of the glycoprotein is located within its peptide moiety. The antigen itself displayed no chymotryptic or tryptic activity. The amino acid sequence of the 20 N-terminal residues of the antigen (ATPHEPVFFSWDAGAVTSFP) is different from that of any other protein previously reported.

A single cell observation of staurosporine effect on the Ca2+ signals in rat basophilic leukemia cells

A digital imaging fluorescence microscope was used to study the effect of a protein kinase inhibitor staurosporine on the antigen‐dependent calcium signals in an individual rat basophilic leukemia cell (RBL‐2H3). Although dose dependency of staurosporine was different from one cell to another, staurosporine inhibited, at low concentration, the calcium influx from the external medium into RBL‐2H3 cells. At high concentration, however, it inhibited both the removal of calcium ion from internal stores and the calcium influx from the external medium. These results indicated that staurosporine is necessary for the inhibition of the calcium influx from the external medium and that a protein kinase (possibly protein kinase C) is involved in the calcium influx from the external medium into the cytoplasm.

Effects of herbimycin A and ST638 on Fcϵ receptor-mediated histamine release and Ca2+ signals in rat basophilic leukemia (RBL-2H3) cells

We examined the effect of the two protein tyrosine kinase inhibitors, alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamide (ST638) and herbimycin A, on the activation processes of rat basophilic leukemia (RBL-2H3) cells by cross-linking of IgE receptors. RBL-2H3 cells sensitized with DNP-specific monoclonal IgE antibody were stimulated with multivalent antigen (DNP conjugate of bovine serum albumin). Analysis of phosphotyrosine-containing proteins in their lysates by SDS-PAGE and immunoblotting revealed that these two inhibitors efficiently inhibited the tyrosine phosphorylation of several proteins (32, 42, 56, 66, 72, 92, 150 kDa) including phospholipase C-gamma 1. The inhibitors also caused parallel inhibitions of the histamine release, the formation of inositol 1,4,5-trisphosphate, and the increase in cytosolic calcium ion concentration at the late sustained phase. A digital imaging fluorescence microscopic analysis of antigen-dependent calcium signals in individual cells showed that these two tyrosine kinase inhibitors inhibited the calcium influx from the external medium more powerfully than the mobilization of calcium ion from internal stores. In contrast, the inhibitors did not affect the increase in the cytosolic calcium ion concentration or the histamine release induced by the calcium ionophore A23187. Taken together, our results suggest that tyrosine phosphorylation following antigen stimulation regulates phosphatidylinositol hydrolysis and the influx of extracellular calcium.

Effects of hydroquinone-type and phenolic antioxidants on calcium signals and degranulation of RBL-2H3 cells.

We previously reported that a hydroquinone-type antioxidant, 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ), increases intracellular free Ca2+ concentration ([Ca2+]i), causes degranulation together with a protein kinase C activator, phorbol 12-myristate 13-acetate (TPA), and increases antigen-induced degranulation in rat basophilic leukemia (RBL-2H3) cells. In this study, the effects of five-hydroquinone-type and phenolic antioxidants (2,5-di(tert-amyl)-1,4-hydroquinone [DTAHQ], 2-tert-butyl-1,4-hydroquinone [MTBHQ], 3,5-di(tert-butyl)-4-hydroxytoluene [BHT], 3,5-di(tert-butyl)-4-hydroxyanisole [DTBHA], and 3-tert-butyl-4-hydroxyanisole [MTBHA]) on [ca2+]i and degranulation (beta-hexosaminidase release) were examined and compared with that of DTBHQ. DTAHQ (> or = 3 microM) showed effects similar to those of DTBHQ (10 microM) on [Ca2+]i elevation, induction of degranulation with TPA, and increase of antigen-induced degranulation. BHT (50 microM) and DTBHA (50 microM) caused [Ca2+]i elevation and increased degranulation in the presence of TPA or antigen, but their effects were less than those of DTBHQ and DTAHQ. MTBHQ and MTBHA had no effect on [Ca2+]i and degranulation, even at 50 microM. The degree of Ca2+ response caused by the compounds correlated well with the increase in degranulation, but not with their antioxidant activity estimated with the first oxidation potential. From these results, it is suggested that the increasing effects of six antioxidants on degranulation in the presence of TPA or antigen were dependent on [Ca2+]i increase caused by the compounds, probably through their ability to inhibit endoplasmic reticulum Ca2+-ATPase.

Ligand-induced internalization and phosphorylation-dependent degradation of growth hormone receptor in human IM-9 cells

The human growth hormone (hGH) induced a marked reduction in the number of human growth hormone receptors (hGHR) within 60 min, as assessed by immunoblotting of the crude membrane fraction from human IM-9 cells, without an increase in soluble forms of hGHR. The disappearance of hGH-induced hGHR was markedly inhibited by reagents that raise the internal pH of acidic organella and partially by protease inhibitors. These results suggest that hGH stimulation results in degradation of internalized hGHRs, where proteases in acidic compartments such as lysosomes may be involved. The relationship between the hGH concentration and the number of residual cell surface hGHRs 60 min after hGH stimulation yielded a curve with an inverted bell shape showing maximum internalization at 10 nM hGH. A similar relationship was shown in the hGHR degradation. The fact that the ligands in excess gave reduced internalization and degradation supports the idea that dimerization of hGHRs on the cell surface through the bivalent ligand hGH is required for their internalization and subsequent degradation. Following hGH stimulation, several hGHR-associated proteins including JAK2 were phosphorylated. These phosphorylations were inhibited by pretreatment with a protein kinase inhibitor, staurosporine. The hGHR internalization, however, was not markedly affected by the inhibitor. In contrast, the staurosporine inhibited the degradation of hGHR in a dose-dependent manner. These results suggest that staurosporine-sensitive phosphorylation is not required for the hGHR internalization, but the phosphorylation is involved in the degradation of hGHR.

Activation of protein kinase Cα enhances human growth hormone-binding protein release

Abstract The effect of phorbol ester on human growth hormone-binding protein (hGH-BP) release was investigated. The hGH-BP release from human IM-9 cells measured by immunoblotting was dose-dependently enhanced by a phorbol ester, phorbol 12, 13-dibutyrate (PDBu), and reached plateau at 100 nM. The increased hGH-BP release was shown after 10 min incubation with PDBu and reached a plateau at 60 min after stimulation. Similarly, a diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol, enhanced hGH-BP release. The enhancement was not inhibited by cycloheximide pretreatment, suggesting that the enhanced hGH-BP release does not require de novo protein synthesis. The PDBu-enhanced hGH-BP release was strongly inhibited by extracellular EDTA, and was dose-dependently inhibited by protein kinase C (PKC)-specific inhibitor, Ro 31-8220. These results suggest that activation of PKC mediates the PDBu-enhanced hGH-BP release. Of the 11 known PKC isoforms in human cells, PKCα, δ, μ and ι were detected in IM-9 cells by immunoblotting. Of these isoforms, PKCα, δ and μ were present in the membrane fraction, which is a known activation marker of PKC. Furthermore, when several PKC-specific inhibitors (Go 6976, GF 109203X or bisindolylmaleimide III) with different specificities for each isoform were used, there was a good correlation between inhibition of the enhancement of hGH-BP release and inhibition of the phosphorylation of PKC isoforms, another activation marker of PKC, in PKCα but not in PKCδ and μ. These results suggest that activation of PKCα is involved in PDBu-enhanced hGH-BP release.

An immunological study of a Pb-thionein like protein in rat liver

Administration of a sublethal dose of lead acetate to rats induced the simultaneous synthesis of a Pb-thionein like protein (Pb-BP) and Zn-thionein in the liver. To determine of the Pb-BP is a species of metallothionein, immunological properties of this protein were investigated. The results indicate that the Pb-BP is cross-reactive with the antibody against rat Zn-thionein II, strongly suggesting that the Pb-BP is a metallothionein.

Expression of opioid-binding cell adhesion molecule (OBCAM) and neurotrimin (NTM) in E. coli and their reactivity with monoclonal anti-OBCAM antibody.

OPIOID-BINDING cell adhesion molecule (OBCAM), neurotrimin (NTM) and limbic system-associated membrane protein (LAMP) are homologous and are the members of the IgLON family which is a subfamily within the immunoglobulin superfamily. We cloned the cDNAs for OBCAM and NTM, prepared recombinant proteins, and examined the reactivity of the previously prepared monoclonal anti-OBCAM antibody, OBC53, with the recombinant proteins by immunoblotting. These experiments revealed that OBC53 recognizes OBCAM about 1000 times as efficiently as NTM. Moreover, the NTM and LAMP peptides which have sequences homologous to the OBCAM peptide used for the preparation of OBC53 were 150 times less reactive to OBC53. Thus, the OBC53 antibody is a useful tool for specifically detecting OBCAM in immunochemical experiments.

Role of ecto-kinase in phorbol ester-enhanced growth hormone-binding protein release from human IM-9 cells.

Previously we reported that a phorbol ester, phorbol 12, 13-dibutyrate (PDBu), increased the release of human growth hormone-binding protein (hGH-BP) in IM-9 cells, and that this phorbol ester-enhanced release was mediated by protein kinase Ca (PKCalpha). In the present study, the mechanisms of the phorbol ester-enhanced hGH-BP release were further investigated. Treatment of IM-9 cells with PDBu did not increase hGH-BPs (55-60 kDa) in the intracellular soluble fraction. When the cells were treated with trypsin to remove human growth hormone receptors (hGHRs) on the cell surface after stimulation, no hGH-BPs were detected in the culture supernatants, nor did treatment with bafilomycin A1 or chloroquine affect the PDBu-enhanced hGH-BP release. These results suggest that hGH-BPs released by PDBu stimulation are derived from cell surface hGHRs and not generated within the cells. Protein kinase inhibitors with broad specificities, K-252a and K-252b, inhibited the PDBu-enhanced release with almost the same dose-dependency, although only a trace amount of K-252b was incorporated into IM-9 cells than K-252a, suggesting that K-252b probably inhibits an ecto-kinase extracellularly. PDBu actually enhanced the phosphorylation of several extracellular proteins, and this enhanced phosphorylation was completely inhibited by K-252b treatment. Moreover, the PKCalpha-specific inhibitor bisindolylmaleimide III which inhibits PDBu enhanced hGH-BP release inhibited the PDBu-enhanced phosphorylation of extracellular proteins. On the other hand, the impermeable PKC inhibitor PKC inhibitor peptide 19-31 did not inhibit PDBu-enhanced release, suggesting that the target PKCalpha for PDBu is not present on the extracellular surface. Taken together, these results suggest that, in addition to intracellular PKCalpha, activation of an undefined ecto-kinase may also be involved in the PDBu-enhanced hGH-BP release.