Metabolic patterns (NAD(P)H) in rat basophilic leukemia (RBL-2H3) cells and human hepatocellular carcinoma (Hep G2) cells with autofluorescence imaging

Although the spatial and temporal distributions of cellular NAD(P)H concentrations have been theoretically predicted as typical patterns of the metabolism in living cells, so far such a pattern was observed only in neutrophils. In this work, the dynamic NAD(P)H distributions in rat basophilic leukemia (RBL-2H3) and human hepatocellular carcinoma (Hep G2) cells were studied by imaging the autofluorescence of cellular NAD(P)H with a sensitive CCD detector in a confocal microscope. The typical pattern of the cytoplasmic NAD(P)H wave traveling along the long axis of the elongated cell with a velocity of 2.2+/-0.6 mircom/s was detected in RBL-2H3 cells. While in the case of Hep G2 cells, only the oscillation of the mitochondrial NAD(P)H was observed because the NAD(P)H mainly localized in mitochondria of Hep G2 cells. These results confirm the metabolic pattern of NAD(P)H in living cells and suggest that the expression of the metabolic pattern probably differs in different cell lines.     

羧胺三唑对RBL-2H3细胞活化脱颗粒的影响

目的研究羧胺三唑(CAI)对RBL-2H3肥大细胞增殖、凋亡及活化脱颗粒的影响,探索CAI的抗感染作用机制。方法以C48/80诱导RBL-2H3细胞活化脱颗粒模型,中性红染色法观察细胞脱颗粒的形态学,分别用ELISA法和底物显色法检测细胞培养上清中组胺和β-氨基己糖苷酶的释放水平,CCK-8法测定细胞活力,Hoechst 33342荧光染色法检测细胞凋亡。结果与对照组相比,10、20和40μmol/L CAI能够不同程度抑制C48/80诱导的RBL-2H3细胞脱颗粒反应,20和40μmol/L CAI能够降低C48/80诱导的组胺释放(P0.01),40μmol/L CAI能够降低β-氨基己糖苷酶的释放(P0.01)。另外,所用各浓度的CAI对细胞增殖和凋亡均无明显影响。结论 CAI能有效抑制RBL-2H3肥大细胞的活化脱颗粒,此作用并不是通过细胞毒发挥作用的。CAI可能部分通过下调肥大细胞的功能活化,发挥其抗感染作用。     

The characterization of tyrosine phosphorylation of 78 and 92 kDa proteins in rat basophilic RBL-2H3 cells

Previously, we have demonstrated that tyrosine phosphorylation of 78 and 92 kDa proteins in rat basophilic leukemia cells (RBL-2H3) is involved in a signal transduction system for high-affinity IgE receptor (FcRI)-mediated histamine secretion. However, it is not clarified whether the tyrosine phosphorylation of 78 and 92 kDa proteins in RBL-2H3 cells is regulated by activation of protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI3-kinase). In this study, therefore, the effect of depletion of PKC in RBL-2H3 cells, or the influence of PKC, PI3-kinase and tyrosine kinase inhibitors on histamine release from RBL-2H3 cells was examined. The elimination of PKC in RBL-2H3 cells induced significant suppression of histamine release, although the tyrosine phosphorylation of 78 and 92 kDa proteins was not inhibited. The inhibition of histamine release was also observed by the treatment with a PKC inhibitor such as H-7, calphostin C, a PI3-kinase inhibitor such as wortmannin or a tyrosine kinase inhibitor such as ST638, genistein, hervimycin A, although the tyrosine phosphorylation of both proteins was inhibited by only ST638. These results suggest that the 78 kDa protein in RBL-2H3 cells is not identical to the protein-tyrosine kinase PTK72 and the tyrosine phosphorylation of 78 and 92 kDa proteins in RBL-2H3 cells occurs upstream of PKC and PI3-kinase activation or is regulated independently of the PKC- and PI3-kinase-dependent signaling pathway.accepted by M. Katori     

Inhibition of the Antigen-Induced Activation of RBL-2H3 Cells by Gab2 siRNA

Gab2 plays an important role in FcεRI mediated signal events which lead to degranulation from mast cells. The present study was designed to investigate the effect of the synthetic Gab2 （scaffolding adapter Grb2-associated binder 2） siRNA on the antigen-induced activation of RBL-2H3 cells. A double stranded siRNA against Gab2- mRNA was synthesized and transfected into RBL-2H3 cells. After 6 h, cells were then sensitized with dinitrophenyl （DNP）-specific IgE overnight and challenged with dinitrophenyl-human serum albumin （DNP-HSA） to induce mast cell degranulation before supernatants were collected. Effects of Gab2 siRNA on antigen-induced release of β-hexosaminidase and histamine, cytokine production and regulation of the proteins in the pathway were measured by enzymatic assay, EIA, ELISA and Western blotting. Treatment with Gab2 siRNA significantly decreased Gab2 expression, inhibited the FcεRI-mediated mast cell release of β-hexosaminidase and histamine, reduced the production of IL-4 and TNF-α and inhibited the phosphorylation of Akt, PKC8 and p38 mitogen-activated protein kinase （MAPK）. Data showed that Gab2 siRNA could suppress the antigen-induced activation of RBL-2H3 cells and suggested a possible mechanism through inhibition of signaling molecules downstream of Gab2 in the 2＋ FcεRI-mediated Ca -independent pathway. Furthermore, potential usefulness of Gab2 knock-down as a method for inhibition of mast cell-mediated allergic reactions was demonstrated. Cellular ＆ Molecular Immunology. 2008; 5（6）：433-438.     

青藤碱对RBL-2H3肥大细胞增殖凋亡以及活化脱颗粒的影响

目的 通过研究青藤碱对肥大细胞增殖凋亡以及活化脱颗粒的影响,深入探讨青藤碱的抗炎作用机理.方法 应用细胞培养,流式细胞术等方法,观察青藤碱等药物对体外培养的RBL-2H3肥大细胞增殖凋亡以及活化脱颗粒的影响.结果 青藤碱对肥大细胞增殖有明显的抑制作用,其IG50约为1 mmol/L,并能促进肥大细胞凋亡,且对RBL-2H3肥大细胞活化脱颗粒有较强的抑制作用.结论青藤碱可能部分通过下调肥大细胞功能发挥抗炎抗风湿作用.     

Ethanolic Extract of Alternanthera sessilis (AS-1) Inhibits IgE-mediated Allergic Response in RBL-2H3 Cells

The present study was designed to investigate the anti-allergic effects of ethanolic extract of Alternanthera sessilis (AS-1) in rat basophilic leukemia (RBL-2H3) cells. It significantly reduced the β-hexosaminidase release from anti-DNP-IgE sensitized RBL-2H3 cells. AS-1also inhibited the IgE antibody-induced increase in Interleukin-6 (IL-6), TNF-α, IL-13 and IL-4 production in these cells. The inhibitory effect of AS-1 on these cytokine was found to be nuclear factor-KB (NF-kB) dependent, as it attenuated the degradation of IKBa and nuclear translocation of NFkB. In addition, AS-1 significantly attenuated the DNP HAS-induced intracellular Ca²⁺ release from these cells, which makes us speculate strongly that the decreased intracellular Ca²⁺ is involved in the inhibitory effect of AS-1 on β-hexoaminidase release. Taken together, anti-allergic effects of AS-1 suggest possible therapeutic application of this extract in allergic diseases.     

Clustering the mast cell function-associated antigen (MAFA) leads to tyrosine phosphorylation of p62Dok and SHIP and affects RBL-2H3 cell cycle

The mast cell function-associated antigen (MAFA) is a type II membranal glycoprotein expressed by rat mast cells and basophils. MAFA clustering by its specific monoclonal antibody, (mAb) G63, efficiently inhibits the FcvarepsilonRI induced secretory response of mucosal-type mast cells of the RBL-2H3 line, as well as bone marrow-derived mast cells. Here we present results which suggest that MAFA has also a capacity of modulating the cell cycle of the RBL-2H3 line. We found that MAFA clustering, by mAb G63 or by its F(ab')2 fragments, reduces the cell proliferation rate. Cell cycle analysis by flow cytometry revealed that the number of cells in sub-G phase is considerably higher for cells on which MAFA was clustered. Results of biochemical experiments established that MAFA clustering leads to a marked increase in the transient tyrosine phosphorylation of the adaptor protein p62(Dok) and the inositol phosphatase SHIP. Concomitantly, their respective binding to RasGAP and Shc was increased. Furthermore, the GTP binding protein Sos1 was found to dissociate from Shc upon MAFA clustering, suggesting that SHIP and Sos1 compete for Shc binding. We therefore suggest that MAFA has also a role in regulating RBL-2H3 cell proliferation rate by inhibiting RasGTP formation in the Ras signaling pathway.     

Possible involvement of phosphorylation of a 36,000-dalton protein of rat basophilic leukemia (RBL-2H3) cell membranes in serotonin release

Phosphorylation of a 36,000-dalton (36k-Da) protein of rat basophilic leukemia (RBL-2H3) cell membranes was investigated. This phosphoprotein has been suggested to be the beta-subunit protein of the immunogloblin E (IgE) receptor of RBL-2H3 cells [Teshima et al., Biochem. biophys. Res. Commun. 125, 867-874 (1984)]. Phospholipids such as phosphatidyl serine, phosphatidyl inositol and phosphatidyl ethanolamine, which are known to be activators of protein kinase C, enhanced the phosphorylation of the 36K-Da protein. In contrast, 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7) which has been identified as a potent inhibitor of protein kinase C in vitro decreased incorporation of radioactive phosphate from [gamma-32P]ATP into this protein. These results indicate that the phosphorylation of the 36K-Da protein of RBL-2H3 cell membranes is catalyzed by protein kinase C. H-7 also inhibited the release of serotonin from RBL-2H3 cells stimulated with an antigen or calcium ionophore A23187 and 12-O-tetradecanoyl phorbol 13-acetate (TPA). Treatment of the antigen-stimulated cells with TPA caused a synergistic effect on the serotonin release. A similar effect was obtained by treatment of A23187-stimulated cells with TPA or 1-oleoyl-2-acetyl glycerol.     

Antibodies against human CD63 activate transfected rat basophilic leukemia (RBL-2H3) cells

CD63 is a widely expressed glycoprotein member of the transmembrane 4 superfamily (TM4SF) that is present on activated platelets, monocytes and macrophages and many non-lymphoid cells. It has been proposed that CD63 and other members of the TM4SF couple to intracellular signal transduction pathways and may have a role in cellular adhesion, proliferation and activation. We have investigated the functions of human CD63 by expression in the rat basophilic leukemia cell line, RBL-2H3, which has previously been reported to respond to antibodies against the rat homolog of CD63. Using a panel of antibodies against human CD63 we have shown that high levels of granular secretion from transfected RBL cells can be stimulated by some, but not all, of the antibodies. The specificity of this response suggests that these activating antibodies may be mimicking a natural ligand for CD63. The secretory response to crosslinking of the high affinity IgE receptor and also that to non-receptor stimuli (phorbol ester and calcium ionophore) is inhibited by an antibody that appears to recognise both human and rat homologs of CD63. These results suggest that stimulus-secretion coupling can occur through human CD63 and that RBL cells transfected with this protein will constitute a valuable tool in elucidating its function.     

丹参酮对RBL-2H3肥大细胞增殖凋亡以及活化脱颗粒的影响

研究丹参酮对肥大细胞增殖凋亡以及活化脱颗粒的影响,深入探讨丹参酮的抗炎药理机制。应用细胞培养、流式细胞术等方法,观察丹参酮等药物对体外培养的RBL-2H3肥大细胞增殖凋亡以及活化脱颗粒的影响。结果:丹参酮I、IIA对肥大细胞增殖有明显的抑制作用,其IC50约为20μmol/L,并能明显促进细胞凋亡,且对RBL-2H3细胞活化脱颗粒有较强的抑制作用。结论:丹参酮对肥大细胞功能具有明显的抑制作用,可能是其发挥抗炎作用的一个重要方面。     

Investigation of NAD（P） H Fluorescence Decay in Living Cardiomyocytes with Spectrally-resolved Fluorescence Lifetime Spectroscopy

Objective：To study the mitochondrial redox state in experimental animals to sensitively detect early signs of mitochondrial function in pathophysiologieal conditions, such as isehemia. Methods： Fluorescence of nieotinamide adenine dinucleotide （phosphate） , or NAD（P）H, the principal electron donor in mitochondrial respiration responsible for vital ATP supply of cardiomyocytes, is studied for non-invasive fluorescent probing of the mitochondrial function. Examination of NAD （P）H fluorescence in living cardiomyocytes following excitation by UV-pulsed laser diode and detection by spectrally-resolved time-correlated single photon counting （TCSPC） , is based on the simultaneous measurement of the fluorescence spectra and lifetime. Results ： The dynamic characteristics of NAD （P） H fluorescence decay in living rat cardiomyocytes show that at least a 3-exponential decay model, with 0.4 - 0.7 ns, 1.2 - 1.9 ns and 8.0 - 13.0 ns lifetimes, is necessary to describe cardiomyocyte autofluorescenee （AF）. Decay-associated spectra （DSA） revealed the presence of 4 spectrally-distinct populations of NADH molecules in eardiomyocytes with spectral maximum at 470 nm for short-lifetime pool for the first time, and emission peaks at 450 nm, 470 nm and 490 nm for intermediate and long-lifetime pools. Increased mitochondrial NADH content ratio by ketone bodies enhanced the AF intensity, without the significant change in fluorescent lifetimes. Rotenone, the inhibitor of Complex I of the mitochondrial respiratory chain, increased AF and shortened the average fluorescence lifetime. Dinitrophenol （DNP）, an uncoupling agent of the mitochondrial oxidative phosphorylation, lowered AF,broadened the spectral shoulder at 520 nm and increased the average lifetime. These effects, comparable to the changes in the concentration and in the rate of dehydrogenation of NADH in vitro, were also examined under ischemia-mimetic conditions. Conclusion： Our findings anticipate a contribution of both conformational NADH changes and energy transfer from NADH to lipoamide dehydrogenase （LipDH）-bound flavins, to explain observed fluorescence kinetics. Presented spectrally resolved fluorescence lifetime approach provides promising new tool for analysis of mitochondrial NAD （P） H in living cardiomyocytes, and hence for investigation of energy metabolism and mitoehondrial dysfunction at a cellular level.     

大鼠RBL-2H3细胞瞬时感受器电位M7通道参与细胞存活及凋亡**☆

背景：瞬时感受器电位M7是肥大细胞上重要的钙离子通道,但其在肥大细胞存活及凋亡过程中的作用仍未明确。 目的：观察瞬时感受器电位M7通道对大鼠RBL-2H3细胞存活及凋亡的影响。 方法：取对数生长期RBL-2H3细胞,①分别以50,100,200 μmol/L瞬时感受器电位通道阻断剂2-APB进行干预,并以0.1%DMSO干预的细胞及正常培养的细胞作对照。②以瞬时感受器电位M7-siRNA反转录病毒载体转染RBL-2H3细胞,并设立空载体组和正常对照组。 结果与结论：经过100,200 μmol/L的2-APB作用72 h后,RBL-2H3细胞数减少,吸光度值降低(P < 0.05),细胞凋亡增多,早期凋亡率和总凋亡率增加(P < 0.05);RBL-2H3细胞转染si-瞬时感受器电位M7 72 h 后,吸光度值降低(P < 0.05),细胞凋亡增多,早期凋亡率和总凋亡率增加(P < 0.05)。说明瞬时感受器电位M7通道参与了RBL-2H3细胞存活和凋亡过程。       

CD45-Deficient RBL-2H3 Cells Cellular Response to FCER -and Ionophore-Induced Stimulation

The CD45 protein is a transmembrane tyrosine phosphatase that is required for normal T and B cell receptor-mediated signaling. In order to study the function of this phosphatase in mast cells, we have isolated a CD45-deficient variant from the rat basophilic leukemia cell line (RBL-2H3), a tumor analog of mucosal mast cells. The secretory response as well as the inositol 1,4,5-triphosphate (InsP₃) formation to FcRI and ionophore stimuli were similar in the RBL-2H3 cell line and its derived CD45-deficient sub-population. However, pretreatment with the phorbol ester TPA, which directly activates protein kinase C (PKC), caused a marked increase in mediator release and InsP₃ production in the CD45-deficient variant compared to the parental RBL-2H3 cells. These findings suggest that CD45 might directly or indirectly modify the activity of PKC or the InsP₃-dephosphorylating phosphatase.     

Gene expression and production of tumour necrosis factor by a rat basophilic leukaemia cell line (RBL-2H3) with IgE receptor triggering

This study evaluated the gene expression of tumour necrosis factor (TNF) and the molecular weight of the cytotoxic factor in a subline of a rat basophilic leukaemia cell line, RBL-2H3. After IgE receptor triggering with a specific antigen that was associated with histamine release, cytotoxic activity in the cell lysates and supernatants increased for 2 hr during the culture of RBL-2H3 cells. Furthermore, calcium ionophore A23187 could induce release of histamine and cytotoxic activity from RBL-2H3 cells. However, compound 48/80, lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) were unable to induce the release of either histamine or cytotoxic activity from the cells. These data suggested that, at least in part, there was a common pathway in histamine release and production of cytotoxic activity. A protein synthesis inhibitor, cycloheximide, did not affect histamine release, but inhibited the induction of cytotoxic activity. This cytotoxic activity from RBL-2H3 cells was completely neutralized by anti-mouse TNF rabbit serum. With Northern blot analysis, mouse TNF cDNA probe could hybridize with RNA isolated from RBL-2H3 cells. TNF mRNA was induced as early as 1 hr after stimulation with specific antigen and decreased by 4 hr. Moreover, the molecular weight (MW) of the released cytotoxic factor from RBL-2H3 cells triggered with IgE receptors was approximately 17,000 by SDS-PAGE, which was compatible to that of TNF. Thus, it is concluded that the gene expression and production of TNF occurred in RBL-2H3 cells after IgE receptor triggering in association with histamine release, suggesting that TNF produced by basophils and mast cells may play an important role in allergic reaction through its wide range of biological activity.     

Stimulation of Ca(2+)-dependent exocytosis and release of arachidonic acid in cultured mast cells (RBL-2H3) by quercetin

Basic secretagogues, such as compound 48/80, stimulate secretion in rat peritoneal mast cells by directly activating the heterotrimeric G-protein Gi(3) (Aridor M, et al. Science 1993;262:1569-72). Cultured RBL-2H3 mast cells do not normally respond to basic secretagogues, but acquire such responsiveness upon prolonged exposure to the kinase inhibitor, quercetin, which also increases the cellular level of Gi(3) (Senyshyn J, Baumgartner RA, Beaven MA. J Immunol 1998;160:5136-44). Expression of a GTPase-deficient mutant of Galphai(3) in RBL-2H3 cells results in the stimulation of Ca(2+)-triggered exocytosis and release of arachidonic acid (AA) (Zussman A, Hermuet S, Sagi-Eisenberg R. Eur J Biochem 1998;258:144-6). Here we show that long-term incubation with quercetin markedly stimulates Ca(2+)-triggered exocytosis and release of AA from the RBL-2H3 cells. We further show that membranes derived from such quercetin-treated cells display a reduced GTPase, but not ATPase, activity. Taken together with our previous observations, these results further implicate Gi(3) as one of the cellular targets through which quercetin confers responsiveness towards the family of basic secretagogues.     

CD45-Deficient RBL-2H3 Cells Cellular Response to FCER -and Ionophore-Induced Stimulation

The CD45 protein is a transmembrane tyrosine phosphatase that is required for normal T and B cell receptor-mediated signaling. In order to study the function of this phosphatase in mast cells, we have isolated a CD45-deficient variant from the rat basophilic leukemia cell line (RBL-2H3), a tumor analog of mucosal mast cells. The secretory response as well as the inositol 1,4,5-triphosphate (InsP₃) formation to FcRI and ionophore stimuli were similar in the RBL-2H3 cell line and its derived CD45-deficient sub-population. However, pretreatment with the phorbol ester TPA, which directly activates protein kinase C (PKC), caused a marked increase in mediator release and InsP₃ production in the CD45-deficient variant compared to the parental RBL-2H3 cells. These findings suggest that CD45 might directly or indirectly modify the activity of PKC or the InsP₃-dephosphorylating phosphatase.     

The role of phosphatidylinositol 4-kinase type IIα in degranulation of RBL-2H3 cells

Objective and Design We have studied the role of phosphatidylinositol 4-kinase IIα (PI4KIIα) in activation of rat basophilic leukemia (RBL-2H3) cells. Materials and Methods Antigen-mediated intracellular Ca²⁺ concentration ([Ca²⁺]_i) increase and β-hexosaminidase secretion were measured using RBL-2H3 cells stably expressing PI4KIIα-yellow fluorescent protein (YFP) or its kinase-deficient mutant PI4KIIα (K151A)-YFP. Results Neither PI4KIIα-YFP nor PI4KIIα (K151A)-YFP were distributed on the plasma membranes but on the exocytotic vesicles. The RBL-2H3 cells stably expressing PI4KIIα-YFP showed significantly enhanced β-hexosaminidase secretion but not an increase in [Ca²⁺]_i after antigen stimulation. The cells with PI4KIIα (K151A)-YFP showed no change in the [Ca²⁺]_i increase nor degranulation. The promotion of secretion by PI4KIIα-YFP was not observed using co-stimulation with Ca²⁺ ionophore and the protein kinase C activator, phorbol myristate acetate. Conclusions These results suggest that PI4KIIα plays a role in the exocytotic process downstream of Ca²⁺ signaling in antigen-mediated mast cell activation. Received 29 September 2005; returned for revision 31 October 2005; accepted by A. Falus 23 May 2006     

T_(H1)/T_(H2)细胞平衡与病毒性肝炎

TH 细胞是机体重要的免疫调节细胞 ,分 TH1 、TH2 、TH0 3种 ,分别产生不同的细胞因子调节细胞和体液免疫。TH1 和 TH2 细胞可通过所产生的细胞因子发挥相互调节和制约作用 ,TH1 / TH2 细胞的调节对维持机体正常的免疫功能非常重要 ,现已知许多免疫性和感染性疾病与 TH1 / TH2 细胞的失衡有关 ,本文主要阐述 HBV和 HCV这 2种肝炎病毒感染与 TH1 / TH2细胞平衡的关系。     

Differential contribution of poly(ADP-ribose)polymerase-1 and -2 (PARP-1 and -2) to the poly(ADP-ribosyl)ation reaction in rat primary spermatocytes

Poly(ADP-ribose)polymerases (PARP-1 and -2) are activated by DNA strand breaks to synthesize protein-bound ADP-ribose polymers from NAD+. The two enzymes are overexpressed in rat spermatocytes and are likely to play a role in meiosis. Indeed parp-2-/- mice, but not parp-1 knockouts, show hypofertility. Aside, PARP-1 and PARP-2 are both involved in DNA damage repair and signalling, but their relative contributions to such processes remain as yet unknown, largely because of the lack of PARP isoform-specific inhibitors that has precluded in vivo studies. Here, we used permeabilized rat primary spermatocytes or isolated spermatocyte nuclei and radiolabelled NAD+ to investigate potential isoform-specific effects on basic features of the poly(ADP-ribosyl)ation reaction, including size of ADP-ribose polymers at different NAD+ concentrations, extent of auto- versus etheromodification, and modulation of such reactions by the PARP inhibitor, PJ34. We found that PARP-1 automodification prevailed over PARP-2 modification. In addition, over 50% of cellular poly(ADP-ribose) was covalently bound to histones H1 and H2. The inhibitory effect of PJ34 appeared to be targeted mainly to the elongation step of the reaction. We propose that a different propensity of PARP-1 and PARP-2 to undergo automodification and/or catalyze etheromodification, both in terms of number of enzyme molecules being involved and amount of bound poly(ADP-ribose), may underlie distinct roles in the regulation of spermatocyte functions.