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Biomedical subjects

E Gulbins

Publications and source records attributed to E Gulbins.

At least 37 records · Page 2Linked to original sources
PubMed2000

Natural ceramide reverses Fas resistance of acid sphingomyelinase(-/-) hepatocytes.

The role of the second messenger ceramide in Fas-mediated death requires clarification. To address this issue, we generated hepatocytes from paired acid sphingomyelinase (ASMase; asmase)(+/+) and asmase(-/-) mice. asmase(-/-) hepatocytes, derived from 8-week-old mice, manifested normal sphingomyelin content and normal morphological, biochemical, and biologic features. Nonetheless, ASMase-deficient hepatocytes did not display rapid ceramide elevation or apoptosis in response to Jo2 anti-Fas antibody. asmase(-/-) hepatocytes were not inherently resistant to apoptosis because staurosporine, which did not induce early ceramide elevation, stimulated a normal apoptotic response. The addition of low nanomolar quantities of natural C16-ceramide, which by itself did not induce apoptosis, completely restored the apoptotic response to anti-Fas in asmase(-/-) hepatocytes. Other sphingolipids did not replace natural ceramide and restore Fas sensitivity. Overcoming resistance to Fas in asmase(-/-) hepatocytes by natural ceramide is evidence that it is the lack of ceramide and not ASMase which determines the apoptotic phenotype. The ability of natural ceramide to rescue the phenotype without reversing the genotype provides evidence that ceramide is obligate for Fas induction of apoptosis in hepatocytes.

Animals↗
PubMed2000

CD95/CD95 ligand interactions on epithelial cells in host defense to Pseudomonas aeruginosa.

Pseudomonas aeruginosa causes severe infections, particularly of the lung, that are life threatening. Here, we show that P. aeruginosa infection induces apoptosis of lung epithelial cells by activation of the endogenous CD95/CD95 ligand system. Deficiency of CD95 or CD95 ligand on epithelial cells prevented apoptosis of lung epithelial cells in vivo as well as in vitro. The importance of CD95/CD95 ligand-mediated lung epithelial cell apoptosis was demonstrated by the rapid development of sepsis in CD95- or CD95 ligand-deficient mice, but not in normal mice, after P. aeruginosa infection.

Animals↗
PubMed2000

CD95-mediated apoptosis in vivo involves acid sphingomyelinase.

Acid sphingomyelinase (ASM) is reported to have an essential function in stress-induced apoptosis although the physiological function of ASM in receptor-triggered apoptosis is unknown. Here, we delineate a pivotal role for ASM in CD95-triggered apoptosis of peripheral lymphocytes or hepatocytes in vivo. We employed intravenous injection of anti-CD4 antibodies or phytohemagglutinin that was previously shown to result in apoptosis of peripheral blood lymphocytes or hepatocytes via the endogenous CD95/CD95 ligand system. Our results demonstrate a high susceptibility in normal mice whereas ASM knock-out mice fail to immunodeplete T cells or develop autoimmune-like hepatitis. Likewise, ASM-deficient mice or hepatocytes and splenocytes ex vivo manifest resistance to anti-CD95 treatment. These results provide in vivo evidence for an important physiological function of ASM in CD95-induced apoptosis.

Animals↗
PubMed2000

Acid sphingomyelinase is involved in CEACAM receptor-mediated phagocytosis of Neisseria gonorrhoeae.

The interaction with human phagocytes is a hallmark of symptomatic Neisseria gonorrhoeae infections. Gonococcal outer membrane proteins of the Opa family induce the opsonin-independent uptake of the bacteria that relies on CEACAM receptors and an active signaling machinery of the phagocyte. Here, we show that CEACAM receptor-mediated phagocytosis of Opa(52)-expressing N. gonorrhoeae into human cells results in a rapid activation of the acid sphingomyelinase. Inhibition of this enzyme by imipramine or SR33557 abolishes opsonin-independent internalization without affecting bacterial adherence. Reconstitution of ceramide, the product of acid sphingomyelinase activity, in imipramine- or SR33557-treated cells restores internalization of the bacteria. Furthermore, we demonstrate that CEACAM receptor-initiated stimulation of other signalling molecules, in particular Src-like tyrosine kinases and Jun N-terminal kinases, requires acid sphingomyelinase. These studies provide evidence for a crucial role of the acid sphingomyelinase for CEACAM receptor-initiated signalling events and internalization of Opa(52)-expressing N. gonorrhoeae into human neutrophils.

Antigens, Bacterial↗
PubMed2000

CD95/CD95 ligand-mediated counterattack does not block T cell cytotoxicity.

Expression of CD95 ligand on parenchymal, epithelial, or tumor cells has been suggested to downregulate the immune response and to control lymphocyte activation. Suppression might be mediated by induction of apoptosis or by inhibition of Ca(2+) channels upon CD95 triggering. We, therefore, aimed to employ this model to modify the immune response to an antigen presented to cytotoxic T cells by antigen-presenting MC57 cells. This model would be very useful to specifically downregulate the immune response to autoantigens in autoimmune situations. However, cytotoxic T cell lines tested in the present study were resistant to CD95 ligand expression on antigen-presenting MC57 cells. In addition, coincubation of the lymphocytes with antigen presenting cells failed to block cytotoxicity mediated by the T lymphocytes. We, therefore, conclude that single expression of CD95 ligand on antigen-presenting cells is insufficient to specifically downregulate an immune response by CD8(+-)triggered immune response.

Animals↗
PubMed2000

The involvement of caspases in the CD95(Fas/Apo-1)- but not swelling-induced cellular taurine release from Jurkat T-lymphocytes.

Following a delay of 45 min, stimulation of the CD95 (Fas/Apo-1)-receptor in Jurkat T-lymphocytes leads to the release of the osmolyte taurine, an event coinciding with apoptotic cell shrinkage. The present study has been performed to elucidate the cellular mechanisms involved in CD95-induced taurine release as compared to swelling-induced taurine release, and to explore whether taurine modifies apoptotic DNA fragmentation and cell shrinkage. Taurine release stimulated by osmotic cell swelling is insensitive to the tyrosine kinase inhibitor herbimycin A and the caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) but is blunted in the absence of extracellular Ca2+. Conversely, the Ca2+ ionophore ionomycin stimulates taurine release. However, the taurine release following CD95 stimulation is not paralleled by an increase of cytosolic Ca2+ and not inhibited by complexation of extracellular Ca2+. None of herbimycin A, the phosphatase inhibitor vanadate, spingomyelinase or Lck56 deficiency prevent CD95-induced taurine release. In contrast, the caspase inhibitor zVAD, but not the caspase inhibitor Ac-Tyr-Val-Ala-Asp-chloromethylketone (YVAD), almost abolishes CD95-induced taurine release. Both caspase inhibitors blunt CD95-induced cell shrinkage and DNA fragmentation, zVAD being more effective than YVAD. Preloading of the cells with 40 mM taurine but not with 40 mM mannitol significantly inhibits CD95-induced DNA fragmentation (by 28%) and apoptotic cell shrinkage (by 25%). In conclusion, CD95-receptor triggering leads to caspase-dependent stimulation of cellular taurine release, which facilitates, but is not sufficient for, the triggering of apoptotic DNA fragmentation and cell shrinkage.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Inhibition of Jurkat-T-lymphocyte Na+/H+-exchanger by CD95(Fas/Apo-1)-receptor stimulation.

Mitogenic factors are known to stimulate the Na+/H+-exchanger (NHE), leading to cytosolic alkalinization and/or cell swelling. Conversely, a hallmark of apoptosis is cell shrinkage and CD95-induced apoptosis has been reported to be paralleled by cytosolic acidification. To assess whether the CD95-receptor regulates NHE activity in Jurkat T-lymphocytes, we performed conventional BCECF fluorescence measurements and SNARF flow cytometric analysis (FACS). The recoveries from acidifications following application of butyrate or a NH3 pulse were both abolished by a specific NHE-inhibitor, HOE694, indicating that they fully depend on NHE activity. Thus they were taken as a measure of NHE activity. CD95-receptor stimulation caused a cytosolic acidification and blunted the recovery from acidification following application of butyrate or a NH3 pulse. Moreover, the NHE-dependent alkalinization following osmotic cell shrinkage was almost abolished by CD95-receptor stimulation. As apparent from the effect of osmotic cell shrinkage, inhibition of the NHE by CD95-receptor stimulation was absent in Lck56-deficient J-CaM1.6 cells and restored by retransfection of J-CaM1.6-cells with Lck56. CD95-receptor stimulation led within 4 h to a decrease of cellular ATP which could contribute to NHE inhibition. Treatment of Jurkat cells with the NHE inhibitor HOE694 accelerated CD95-induced DNA fragmentation. In conclusion, CD95-receptor stimulation inhibits NHE activity through a mechanism that depends directly or indirectly on the activation of the Src-like kinase Lck56. This effect contributes to CD95-induced cytosolic acidification, DNA fragmentation and cell shrinkage.

Adenosine Triphosphate↗
PubMed2000

Physiology of apoptosis.

Ion fluxes and volume changes of the whole cell as well as of organelles belong to the hallmarks of apoptosis; however, the molecular mechanism regulating these changes is only poorly characterized. Several ion channels in the plasma membrane, in particular the N-type K(+) channel, the chloride channel cystic fibrosis conductance regulator, and an outward rectifying chloride channel, as well as the mitochondrial permeability transition pore, have been implicated to be involved in signal transduction cascades regulating apoptosis. Furthermore, Bcl-2-like proteins have been suggested to function, at least in part, as ion channels, because they display some homology to bacterial pore-forming toxins. In contrast to the demonstration of the involvement of these different ion channels in apoptosis, the molecular consequences regulated by these ion channels, and finally triggering apoptosis, are almost completely unknown.

Animals↗
PubMed2000

Inhibition of CD95/Fas-induced DNA degradation by osmotic cell shrinkage.

Apoptosis (programmed cell death) is an active physiological mechanism from which removal of abundant or potentially harmful cells follows. Apoptosis of lymphocytes is critical for the development of the immune system and during the immune response. As we have shown previously, moderate osmotic cell shrinkage interferes with CD95(Fas/Apo-1)-induced cell death. The present study has been performed to further elucidate the underlying mechanisms. To this end, apoptosis in Jurkat T-lymphocytes was elicited by triggering the CD95-receptor with monoclonal CD95/Fas-antibody. Osmotic cell shrinkage which was induced by the addition of 100 mM NaCl, did not significantly interfere with CD95-induced phosphatidylserine exposure nor the activation of caspase 3 activity as determined by PARP cleavage, DEVD-AMC consumption, or the activation of PAK2-kinase. However, osmotic cell shrinkage almost abolished CD95-induced DNA fragmentation (as revealed by propidium iodide staining) and the activation of a DNase as evidenced from SDS-PAGE gel assay. Western blot analysis showed CD95-induced tyrosine phosphorylation of a nuclear protein of ca. 20 kD which comigrated with nuclease activity. This tyrosine phosphorylation was almost completely abolished by the addition of 100 mM NaCl. Furthermore, osmotic cell shrinkage blunted the CD95-induced activation of the Src-like kinase p56lck. It is concluded that different signaling pathways mediate FITC-Annexin-V binding and DNase activation. Only the latter is sensitive to osmotic cell shrinkage.

Annexin A5↗
PubMed2000

Tyrosine kinases open lymphocyte chloride channels.

Osmotic swelling of lymphocytes opens outwardly rectifying Cl(-) channels (ORCC) through the src-like kinase p56(lck). The central role of this tyrosine protein kinase has been shown by genetic and pharmacologic manipulation of the enzyme. Furthermore, p56(lck) activates ORCC independently of cell volume increase. ORCC in lymphocytes and epithelial cells from cystic fibrosis (CF) patients are resistant to activation by cAMP. However, osmotic swelling as well as intracellular purified p56(lck) can activate ORCC in CF lymphocytes. In non-CF lymphocytes ORCC is opened by either, intracellular cAMP, p56(lck) or by osmotic swelling. Osmotic activation of ORCC can be blocked by the tyrosine kinase inhibitor lavendustin in both cell types. Regulation of ORCC by p56(lck) thus represents an alternative pathway of stimulating membrane chloride conductance that is left functional in cystic fibrosis. In addition to osmoregulation these mechanisms could play a major role when cells actively change their volume, i.e. during proliferation and apoptosis. Activation of the tyrosine kinase p56(lck) is an important regulatory step for opening of chloride channels in lymphocytes.

Animals↗
PubMed2000

Cell volume in the regulation of cell proliferation and apoptotic cell death.

Cell proliferation must - at some time point - lead to increase of cell volume and one of the hallmarks of apoptosis is cell shrinkage. At constant extracellular osmolarity those alterations of cell volume must reflect respective changes of cellular osmolarity which are hardly possible without the participation of cell volume regulatory mechanisms. Indeed, as shown for ras oncogene expressing 3T3 fibroblasts, cell proliferation is paralleled by activation of Na(+)/H(+) exchange and Na(+),K(+),2Cl(-) cotransport, the major transport systems accomplishing regulatory cell volume increase. Conversely, as evident from CD95-induced apoptotic cell death, apoptosis is paralleled by inhibition of Na(+)/H(+) exchanger and by activation of Cl(-) channels and release of the organic osmolyte taurine, major components of regulatory cell volume decrease. However, ras oncogene activation leads to activation and CD95 receptor triggering to inhibition of K(+) channels. The effects counteract the respective cell volume changes. Presumably, they serve to regulate cell membrane potential, which is decisive for Ca(++) entry through I(CRAC) and the generation of cytosolic Ca(++) oscillations in proliferating cells. As a matter of fact I(CRAC) is activated in ras oncogene expressing cells and inhibited in CD95-triggered cells. Activation of K(+) channels and Na(+)/H(+) exchanger as well as Ca(++) oscillations have been observed in a wide variety of cells upon exposure to diverse mitogenic factors. Conversely, diverse apoptotic factors have been shown to activate Cl(-) channels and organic osmolyte release. Inhibition of K(+) channels is apparently, however, not a constant phenomenon paralleling apoptosis which in some cells may even require the operation of K(+) channels. Moreover, cell proliferation may at some point require activation of Cl(-) channels. In any case, the alterations of cell volume are obviously important for the outcome, as cell shrinkage impedes cell proliferation and apoptosis can be elicited by increase of extracellular osmolarity. At this stage little is known about the interplay of cell volume regulatory mechanisms and the cellular machinery leading to mitosis or death of the cell. Thus, considerable further experimental effort is required in this exciting area of cell physiology.

Apoptosis↗
PubMed2000

Streptolysin O-permeabilized granulocytes shed L-selectin concomitantly with ceramide generation via neutral sphingomyelinase.

Cleavage of membrane-associated L-selectin regulates leukocyte rolling on vascular endothelium at sites of inflammation. We report that rapid and massive shedding of L-selectin occurs from granulocytes attacked by the pore-forming bacterial toxin streptolysin O (SLO). Shedding was not induced by an SLO mutant that retained binding capacity but lacked pore-forming activity. Cells permeabilized with SLO exhibited a 1.5-fold increase in the activity of neutral sphingomyelinase, which was accompanied by increased ceramide formation. L-selectin cleavage was inducible by treatment of cells with bacterial sphingomyelinase, and also through exogenous application of a cell-permeable ceramide analog. Our data identify a novel path to the shedding process and show that activation of neutral sphingomyelinase with the generation of ceramide is an important event underlying enhanced sheddase function in cells permeabilized by a pore-forming toxin.

Bacterial Proteins↗
PubMed1999

Stimulation of CD95 (Fas) blocks T lymphocyte calcium channels through sphingomyelinase and sphingolipids.

Calcium influx through store-operated calcium release-activated calcium channels (CRAC) is required for T cell activation, cytokine synthesis, and proliferation. The CD95 (Apo-1/Fas) receptor plays a role in self-tolerance and tumor immune escape, and it mediates apoptosis in activated T cells. In this paper we show that CD95-stimulation blocks CRAC and Ca(2+) influx in lymphocytes through the activation of acidic sphingomyelinase (ASM) and ceramide release. The block of Ca(2+) entry is lacking in CD95-defective lpr lymphocytes as well as in ASM-defective cells and can be restored by retransfection of ASM. C2 ceramide, C6 ceramide, and sphingosine block CRAC reversibly, whereas the inactive dihydroceramide has no effect. CD95-stimulation or the addition of ceramide prevents store-operated Ca(2+) influx, activation of the transcriptional regulator NFAT, and IL-2 synthesis. The block of CRAC by sphingomyelinase metabolites adds a function to the repertoire of the CD95 receptor inhibiting T cell activation signals.

Animals↗
PubMed1999

The tyrosine kinase lck is required for CD95-independent caspase-8 activation and apoptosis in response to ionizing radiation.

Induction of apoptosis is a hallmark of cytostatic drug and radiation-induced cell death in human lymphocytes and lymphoma cells. However, the mechanisms leading to apoptosis are not well understood. We provide evidence that ionizing radiation induces a rapid activation of caspase-8 (FLICE) followed by apoptosis independently of CD95 ligand/receptor interaction. The radiation induced cleavage pattern of procaspase-8 into mature caspase-8 resembled that following CD95 crosslinking and resulted in cleavage of the proapoptotic substrate BID. Overexpression of dominant-negative caspase-8 interfered with radiation-induced apoptosis. Caspase-8 activation by ionizing radiation was not observed in cells genetically defective for the Src-like tyrosine kinase Lck. Cells lacking Lck also displayed a marked resistance towards apoptosis induction upon ionizing radiation. After retransfection of Lck, caspase-8 activation and the capability to undergo apoptosis in response to ionizing radiation was restored. We conclude that radiation activates caspase-8 via an Lck-controlled pathway independently of CD95 ligand expression. This is a novel signaling event required for radiation induced apoptosis in T lymphoma cells.

Antibodies↗
PubMed1999

Selective potentiation of drug cytotoxicity by NSAID in human glioma cells: the role of COX-1 and MRP.

Here, we report that nonsteroidal anti-inflammatory drugs (NSAID) enhance the cytotoxic effects of doxorubicin and vincristine in T98G human malignant glioma cells. The cytotoxicity of BCNU, cisplatin, VM26, camptothecin, and cytarabine is unaffected by NSAID. No free radical formation is induced by doxorubicin or vincristine in the absence or presence of NSAID. Doxorubicin and vincristine cytotoxicity in the absence or presence of NSAID are unaffected by free radical scavengers. Functional inhibitors of phospholipase A2 (PLA2), such as dexamethasone and quinacrine, do not mimick the effects of NSAID. T98G cells, but not LN-18, LN-229, LN-308, or A172 glioma cells, express cyclooxygenase (COX-1) and NSAID do not modulate drug cytotoxicity in the other cell lines, except T98G. Thus, augmentation of doxorubicin and vincristine cytotoxicity by NSAID correlates with COX-1 expression. However, ectopic expression of COX-1 in LN-229 cells does not induce the phenotype of T98G cells, indicating that COX-1 inhibition does not mediate the effects of NSAID on drug cytotoxicity. In contrast, a multidrug resistance (MDR) phenotype due to expression of the multidrug resistance-associated protein (MRP) is most prominent in T98G cells and is amenable to modulation by indomethacin, suggesting that inhibition of MRP is at least in partly responsible for the potentiation of doxorubicin and vincristine cytotoxicity by NSAID.

ATP-Binding Cassette Transporters↗
PubMed1999

Ca2+-activated K channel of the BK-type in the inner mitochondrial membrane of a human glioma cell line.

A single channel current was recorded from mitoplasts (i.e., inner mitochondrial membrane) of the human glioma cell line LN229 using patch-clamp techniques in the mitoplast-attached mode. We frequently found a 295 +/- 18 pS channel that showed a straight i-E relation in the range +/-60 mV in 150 mM KCl solutions on either side of the mitoplast. If KCl in the bath was exchanged against NaCl, outward currents were undetectable, indicating potassium selectivity. Channel activity determined as open probability increased with increasing Ca2+ concentrations (EC50 = 0.9 microM at 60 mV). Open probability was voltage dependent. An e-fold increase of time spent in the open state was induced by a depolarization of 10.5 mV. Open probability was decreased by charybdotoxin concentration and voltage dependently (EC50 = 1.4 nM). In conclusion, we show for the first time that the inner mitochondrial membrane in human glioma cells contains a calcium-dependent K channel of the BK-type.

Calcium↗
PubMed1999

Cell volume regulatory mechanisms in apoptotic cell death.

One of the hallmarks of apoptosis is cell shrinkage, which--at constant extracellular osmolarity--requires a decrease of cellular osmolarity. Moreover, apoptosis can be elicited by increase of extracellular osmolarity and the resistance of cells towards apoptosis correlates with their ability to regulate their volume in hypertonic environment. On the other hand, CD95-receptor-mediated apoptosis is blunted at moderate increases of extracellular osmolarity. Given the role of cell volume alterations it is not surprising that apoptosis is paralleled by marked alterations of cell volume regulatory mechanisms. Stimulation of the CD95-receptor, which confers apoptosis to a variety of cells, leads to activation of cell volume regulatory anion channel ORCC. However, activation of ORCC is paralleled by inhibition of cell volume regulatory K+ channel Kv1.3. It is only 40 to 60 minutes after triggering of the CD95-receptor when the cells release the organic osmolyte taurine and shrink.

Animals↗