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.

Casein kinase II-like ectokinase activity on RBL-2H3 cells.

We studied the properties of the ectokinase activity on the outer cell surfaces of RBL-2H3 cells and examined the phosphorylation of exogenous substrates to clarify the substrate specificity of the ectokinases on RBL-2H3 cells. Among the several protein substrates tested, casein was the most strongly phosphorylated with [gamma-32P]ATP, and the net incorporation of 32P into casein was 0.65 pmol P/50 microg/10(6) cells. Casein kinase II peptide was also phosphorylated with [gamma-32P]ATP. The phosphorylation of casein and casein kinase II peptide was almost completely inhibited by the addition of 3 microg/ml of cell-impermeable K252b. Phosphorylation of casein and casein kinase II peptide was also observed by [gamma-32P]GTP. Western blot analysis using anti-casein kinase II antibody revealed a 44-kDa casein kinase band in the membrane fraction and Fc epsilonRI complexes. The immunofluorescence microscopic analysis using anti-casein kinase II antibody showed the existence of casein kinase II on the surface of the cells. This is the first report about the existence of ectokinase on mast cells.

Suppression of calcium oscillation by tri-n-butyltin chloride in cultured rat hepatocytes.

The effects of tri-n-butyltin chloride (TBT), an environmental pollutant, on cytoplasmic free calcium ion concentration ([Ca2+]i) were investigated in primary cultured rat hepatocytes. A high concentration (4.0 microM) of TBT increased resting levels of [Ca2+]i and then induced cell blebs resulting in cell death within 2 h. The increase in [Ca2+]i, but not the cell death, depended on the presence of extracellular Ca2+, suggesting that the increase in [Ca2+]i is not critical for the cytotoxicity of TBT. A low concentration (0.1 microM) of TBT did not have any toxic effect (decrease in ATP content, decrease in viability, and shape change) on cultured hepatocytes and did not change [Ca2+]i. However, the calcium responses induced by phenylephrine, [Arg8]-vasopressin, and ATP were suppressed in the cells pretreated with 0.1 microM TBT for 30 min. The suppression was not observed in the cells pretreated with 0.1 microM TBT for only 1 min. Pretreatment with 0.1 microM TBT for 30 min had no effect on the inositol 1,4,5-triphosphate content or its increase in response to hormonal stimulation. These results suggest that TBT suppresses hormone-induced calcium responses at nontoxic low concentrations.

Ca2+-ATPase inhibitors and PKC activation synergistically stimulate TNF-alpha production in RBL-2H3 cells.

OBJECTIVE AND DESIGN: To investigate the effect of Ca2+-ATPase inhibitors on the production of TNF-alpha in rat basophilic leukemia (RBL-2H3) cells. MATERIAL: Two Ca2+-ATPase inhibitors, thapsigargin (TG) and cyclopiazonic acid (CPA), and three hydroquinone-antioxidants, 2,5-di-(tert-butyl)-1,4-hydroquinone (DTBHQ), 2,5-di-(tert/amyl)-1,4-hydroquinone (DTAHQ), 2-(tertbutyl)-1,4-hydroquinone (MTBHQ) were used. TREATMENT: Cells were treated with TG, CPA, DTBHQ, DTAHQ and MTBHQ for 3 h in the presence of 12-Otetradecanoylphorbol-13-acetate (TPA) and released TNF-alpha from the cells was measured (n > or = 4). RESULTS: All Ca2--ATPase inhibitors (TG, CPA, DTBHQ and DTAHQ) induced TNF-alpha release in a dose-dependent manner. TNF-alpha release was inhibited by treatment with protein kinase C inhibitors (staurosporine, Ro31-8220, calophostin C) (p < or = 0.05). In contrast, MTBHQ, which does not induce increases in [Ca2+]i, did not induce the release of TNF-alpha. TNF-alpha release induced by DTBHQ and CPA was inhibited by treatment with actinomycin-D, the immunosuppressant FK506 and the glucocorticoid dexamethasone (p < or = 0.01). CONCLUSIONS: These results suggest 1) that [Ca2+]i increase and subsequent activation of protein kinase C is necessary for the release of TNF-alpha, and they work synergistically, 2) that the TNF-alpha release induced by Ca2+-ATPase inhibitors can be regulated at the transcriptional level.

Production of antiserum for sensitive enzyme-linked immunosorbent assay of 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid by chemiluminescence.

To obtain a specific antiserum for use in enzyme-linked immunosorbent assay (ELISA) of 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), we prepared a hapten-carrier conjugate in which the CMPF hapten was linked to a carrier protein through the 5-(1-hydrazopropyl) group. The antisera raised against this antigen in guinea pigs had excellent specificity for CMPF, showing little cross-reactivity with closely related compounds and no significant cross-reactivities with other furan compounds. The results indicated that a specific antiserum to CMPF could be produced by an antigen whose CMPF moiety is linked to a carrier protein through a position remote from the inherent functional groups. A standard curve of CMPF by ELISA using a chemiluminescence system showed a high sensitivity and a linearity in the range of 5-100 ng/mL.

Activation of protein kinase C alpha enhances human growth hormone-binding protein release.

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, PKCalpha, delta, mu and iota were detected in IM-9 cells by immunoblotting. Of these isoforms, PKCalpha, delta and mu were present in the membrane fraction, which is a known activation marker of PKC. Furthermore, when several PKC-specific inhibitors (Gö 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 PKCalpha but not in PKCdelta and mu. These results suggest that activation of PKCalpha is involved in PDBu-enhanced hGH-BP release.

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 monocolonal 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.

Effect of an ectokinase inhibitor, K252b, on degranulation and Ca2+ signals of RBL-2H3 cells and human basophils.

We examined the effects of K252b, an ectoprotein kinase inhibitor of microbial origin, on the activation process of RBL-2H3 cells by cross-linking of IgE receptors by the endoplasmic reticulum Ca2+-ATPase inhibitor 2,5-di(tert-butyl)-1,4-hydroquinone or by the Ca2+ ionophore A23187. Analysis of phosphorylation of ectoproteins following IgE receptor cross-linking revealed that K252b mainly inhibited the phosphorylation of a 130-kDa protein. The inhibitor simultaneously inhibited degranulation and the sustained increase in the cytosolic calcium ion concentration even after addition of Ag. In contrast, K252b did not inhibit the increase in degranulation and cytosolic calcium ion concentration caused by stimulation with 2,5-di(tert-butyl)-1,4-hydroquinone and A23187. Permeation of K252b into RBL-2H3 cells, assessed by fluorescence intensity, was very low. K252b also inhibited degranulation caused by IgE receptor cross-linking in human basophils, but did not inhibit the degranulation caused by A23187. Thus, our findings suggest that the effects of K252b may be mediated by outer surface-bound or -anchored K252b-sensitive molecules on RBL-2H3 cells and human basophils, and that the phosphorylation of ectoprotein may involve a transmembrane influx of Ca2+ by IgE receptor cross-linking.

Effects of three different Ca(2+)-ATPase inhibitors on Ca2+ response and leukotriene release in RBL-2H3 cells.

The effects of three Ca(2+)-ATPase inhibitors, thapsigargin (TG), cyclopiazonic acid (CPA), and 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ), on the Ca2+ response, degranulation, and leukotriene C4 (LTC4) release in RBL-2H3 cells were investigated. All three compounds elevated the intracellular free Ca2+ concentration ([Ca2+]i), and caused degranulation in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator. The dose-dependency of each compound in the Ca2+ response was in good agreement with that in degranulation. TG and CPA also caused the release of LTC4 in a dose-dependent manner, and this effect was unaffected by TPA or calphostin C, a selective PKC inhibitor. DTBHQ, however, did not induce LTC4 release, and rather inhibited the antigen-induced release of LTC4. These results suggest [1] that both degranulation and LTC4 release caused by these compounds are dependent on their [Ca2+]i increasing effect, [2] that degranulation and LTC4 release are mediated via independent pathways following the Ca2+ response, and [3] that DTBHQ additionally prevents the synthesis of LTC4 possibly by inhibition of 5-lipoxygenase.

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.

Easy enzyme-linked immunosorbent assay for spectinomycin in chicken plasma.

An easy, sensitive, competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) for spectinomycin in chicken plasma was developed. Preparation of a spectinomycin-bovine serum albumin conjugate in which the hapten is linked to the carrier protein through the C-4 position is described. Antibodies raised against antigens in rabbits had excellent specificity for spectinomycin, exhibiting a cross-reactivity of 44.0% with dihydrospectinomycin and 13.8% with tetrahydrospectinomycin. No cross-reactivity was observed with other antibiotics. The detection limit of the CI-ELISA was 2 ng/mL (equivalent into 40 ng/mL undiluted chicken plasma) spectinomycin. Known amounts (0.1-100 micrograms/mL) of spectinomycin were added to chicken plasma and then analyzed. Average recoveries were 97-110%. This procedure may be used without prior extraction of samples.

Release of a soluble form of growth hormone receptors (growth hormone-binding proteins) from human IM-9 cells by proteolytic cleavage.

Soluble forms of growth hormone receptors, growth hormone-binding proteins (GH-BPs), with molecular weights of 60 and 55kDa were found to be constitutively released from human IM-9 cells. The release of the GH-BPs was not inhibited by inhibitors of protein synthesis (cycloheximide) and transport (brefeldin A). Down-regulation by human growth hormone or trypsin pretreatment of surface growth hormone receptors abolished the GH-BP release, suggesting that cell-surface human growth hormone receptors are involved in the GH-BP release. Several inhibitors of serine-, thiol-, and acid-proteases did not affect the GH-BP release. EDTA efficiently blocked the GH-BP release. This inhibition by EDTA was restored by addition of Mg(2+) and Co(2+). These results suggest that human GH-BPs are constitutively released by proteolytic cleavage of cell-surface growth hormone receptors by a metalloprotease.

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.

Stimulatory effect of pervanadate on calcium signals and histamine secretion of RBL-2H3 cells.

We examined the effect of pervanadate on the activation of rat basophilic leukaemia (RBL-2H3) cells. The pervanadate, generated from a combination of H2O2 and vanadate (Vi), induced concomitantly protein tyrosine phosphorylation, formation of inositol 1,4,5-trisphosphate (IP3), an increase in [Ca2+]i, and histamine secretion in RBL-2H3 cells. These effects were clearly dependent on the ratio of H2O2/Vi. The secretion of histamine, IP3 formation, and sustained increase in [Ca2+]i were effectively induced by treatment of the cells with the pervanadate produced from 1 mM H2O2 and 1 mM Vi. These effects mimic the stimulatory effects of an antigen (dinitrophenylated BSA) on Ca2+ signals, histamine secretion and morphological changes. Protein tyrosine phosphorylation, formation of IP3 and transient increase in [Ca2+]i were markedly induced by the pervanadate produced from 3 mM H2O2 and 1 mM Vi. However, histamine secretion induced by the pervanadate was very low. After the pervanadate from 3 mM H2O2 and 1 mM Vi was treated with catalase, it was able to induce the [Ca2+]i increase and histamine secretion as much as the antigen did. This indicates that pervanadate from a lower H2O2 concentration (1 mM H2O2/1 mM Vi) and catalase-treated pervanadate from a higher H2O2 concentration (3 mM H2O2/1 mM Vi) are able to mimic the activity that was caused by cross-linking of IgE receptors with antigen. The present results also demonstrate that protein tyrosine phosphorylation seems to have a crucial role in Ca2+ entry from the external medium, and that a sustained [Ca2+]i increase is an important step for histamine secretion in RBL-2H3 cells.

Preparation of monoclonal antibodies for immunoblotting human growth hormone receptor and growth hormone-binding protein.

Monoclonal anti-peptide antibodies against the extracellular domain of human growth hormone receptor (hGHR) were prepared. Four monoclonal antibodies (mAbs) reacted with an extracellular domain protein produced by genetic engineering. Among them, GHRP2-88 was the most reactive against hGHRs from human IM-9 cells. The lower limit of detection for immunoblotting using this mAb was about 200 pg hGHR. The GHRP2-88 antibody also reacted with deglycosylated hGHRs from tunicamycin-treated IM-9 cells and with the growth hormone-binding protein (GH-BP) in human plasma.

Effects of herbimycin A and ST638 on Fc epsilon 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.

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.

Sensitive determination of zearalenone and alpha-zearalenol in barley and Job's-tears by liquid chromatography with fluorescence detection.

A sensitive and reliable method for liquid chromatographic (LC) determination of zearalenone and alpha-zearalenol in barley and Job's-tears was investigated. The method by which these toxins were determined involves addition of an internal standard (zearalenone 6'-oxime) to barley and Job's-tears samples. Extracts from grain samples were cleaned up by passage through chromatography on piperidinohydroxypropyl Sephadex LH-20 as a lipophilic gel. Individual toxins were resolved by LC on a reversed-phase (ODS) column with fluorescence detection. The detection limit is estimated to be 0.2 ng for zearalenone and alpha-zearalenol standards. Known amounts of zearalenone and alpha-zearalenol (25-1250 ng) were added to a barley sample (5 g). Average recoveries for alpha-zearalenol and zearalenone, respectively, ranged from 96 to 102% (mean CV, 3.6%) and from 96 to 103% (mean CV, 3.3%). This method is applicable to determination of alpha-zearalenol and zearalenone in barley and Job's tears with satisfactory sensitivity and accuracy.

Identification of metallothionein in cultured cells by immunoblotting and immunofluorescence using a new monoclonal antibody.

A hybridoma clone (MT45-5-3) producing an IgG-class monoclonal antibody specific for metallothioneins (MTs) was established. The monoclonal antibody (MT45) cross-reacted with mouse, rat and rabbit Cd(2+)-induced MTs 1 and 2 and Zn(2+)-induced MT 2 as assessed by enzyme-linked immunosorbent assay. When the antibody was used to detect the MTs transferred to nylon membranes after SDS-polyacrylamide gel electrophoresis, the antibody reacted with cultured human cell MTs as well as rat, mouse and rabbit MTs 1 and 2 even after carboxymethylation. The antibody could be used for the indirect immunofluorescence test for Cd(2+)-induced MTs in cultured human and mouse cells.