The different members of the vascular endothelial growth factor (VEGF) family act as key regulators of endothelial cell function controlling vasculogenesis, angiogenesis, vascular permeability and endothelial cell survival. In this study, we have functionally characterized a novel member of the VEGF family, designated VEGF‐E. VEGF‐E sequences are encoded by the parapoxvirus Orf virus (OV). They carry the characteristic cysteine knot motif present in all mammalian VEGFs, while forming a microheterogenic group distinct from previously described members of this family. VEGF‐E was expressed as the native protein in mammalian cells or as a recombinant protein in Escherichia coli and was shown to act as a heat‐stable, secreted dimer. VEGF‐E and VEGF‐A were found to possess similar bioactivities, i.e. both factors stimulate the release of tissue factor (TF), the proliferation, chemotaxis and sprouting of cultured vascular endothelial cells in vitro and angiogenesis in vivo. Like VEGF‐A, VEGF‐E was found to bind with high affinity to VEGF receptor‐2 (KDR) resulting in receptor autophosphorylation and a biphasic rise in free intracellular Ca2+ concentration, whilst in contrast to VEGF‐A, VEGF‐E did not bind to VEGF receptor‐1 (Flt‐1). VEGF‐E is thus a potent angiogenic factor selectively binding to VEGF receptor‐2. These data strongly indicate that activation of VEGF receptor‐2 alone can efficiently stimulate angiogenesis.

CD95͞Fas͞APO-1 mediated apoptosis is an important mechanism in the regulation of the immune response. Here, we show that CD95 receptor triggering activates an outwardly rectifying chloride channel (ORCC) in Jurkat T lymphocytes. Ceramide, a lipid metabolite synthesized upon CD95 receptor triggering, also induces activation of ORCC in cell-attached patch clamp experiments. Activation is mediated by Src-like tyrosine kinases, because it is abolished by the tyrosine kinase inhibitor herbimycin A or by genetic deficiency of p56lck. In vitro incubation of excised patches with purified p56lck results in activation of ORCC, which is partially reversed upon addition of anti-phosphotyrosine antibody. Inhibition of ORCC by four different drugs correlates with a 30-65% inhibition of apoptosis. Intracellular acidification observed upon CD95 triggering is abolished by inhibition of either ORCC or p56lck. The results suggest that tyrosine kinase-mediated activation of ORCC may play a role in CD95-induced cell death in T lymphocytes.

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.

We studied monovalent permeability of Ca2+ release-activated Ca2+ channels (ICRAC) in Jurkat T lymphocytes following depletion of calcium stores. When external free Ca2+ ([Ca2+]o) was reduced to micromolar levels in the absence of Mg2+, the inward current transiently decreased and then increased approximately sixfold, accompanied by visibly enhanced current noise. The monovalent currents showed a characteristically slow deactivation (tau = 3.8 and 21.6 s). The extent of Na+ current deactivation correlated with the instantaneous Ca2+ current upon readdition of [Ca2+]o. No conductance increase was seen when [Ca2+]o was reduced before activation of ICRAC. With Na+ outside and Cs+ inside, the current rectified inwardly without apparent reversal below 40 mV. The sequence of conductance determined from the inward current at -80 mV was Na+ > Li+ = K+ > Rb+ >> Cs+. Unitary inward conductance of the Na+ current was 2.6 pS, estimated from the ratios delta sigma2/delta Imean at different voltages. External Ca2+ blocked the Na+ current reversibly with an IC50 value of 4 microM. Na+ currents were also blocked by 3 mM Mg2+ or 10 microM La3+. We conclude that ICRAC channels become permeable to monovalent cations at low levels of external divalent ions. In contrast to voltage-activated Ca2+ channels, the monovalent conductance is highly selective for Na+ over Cs+. Na+ currents through ICRAC channels provide a means to study channel characteristics in an amplified current model.

Activation of the CD95 death receptor as well as ionizing radiation induces apoptotic cell death in human lymphoma cells. The activation of caspases is a hallmark of apoptosis induction irrespective of the apoptotic trigger. In contrast to death receptor signaling, the exact mechanisms of radiation-induced caspase activation are not well understood. We provide evidence that both, radiation and CD95 stimulation, induce the rapid activation of caspase-8 and BID followed by apoptosis in Jurkat T-cells. To analyse the relative position of caspase-8 within the apoptotic cascade we studied caspase activation and apoptosis in Jurkat cells overexpressing Bcl-2 or Bcl-x L . Caspase-8 activation, proapoptotic BID cleavage and apoptosis in response to radiation were abrogated in these cells, while the responses to CD95 stimulation were only partially attenuated by overexpression of Bcl-2 family members. In parallel, the breakdown of the mitochondrial transmembrane potential (DC m ) in response to radiation was inhibited by overexpression of Bcl-2/Bcl-x L Jurkat cells genetically de®cient for caspase-8 were found to be completely resistant towards CD95. However, radiationinduced apoptotic responses in caspase-8-negative cells displayed only a modest reduction. We conclude that ionizing radiation activates caspase-8 and BID downstream of mitochondrial damage suggesting that, in contrast to CD95, both events function as executioners rather than initiators of the apoptotic process.

Osmotic cell swelling activates Cl− channels to achieve anion efflux. In this study, we find that both the tyrosine kinase inhibitor herbimycin A and genetic knockout of p56lck, a src-like tyrosine kinase, block regulatory volume decrease (RVD) in a human T cell line. Activation of a swelling-activated chloride current (ICl−swell) by osmotic swelling in whole-cell patch-clamp experiments is blocked by herbimycin A and lavendustin. Osmotic activation of ICl−swell is defective in p56lck-deficient cells. Retransfection of p56lck restores osmotic current activation. Furthermore, tyrosine kinase activity is sufficient for activation of ICl−swell. Addition of purified p56lck to excised patches activates an outwardly rectifying chloride channel with 31 pS unitary conductance. Purified p56lck washed into the cytoplasm activates ICl−swell in native and p56lck-deficient cells even when hypotonic intracellular solutions lead to cell shrinkage. When whole-cell currents are activated either by swelling or by p56lck, slow single-channel gating events can be observed revealing a unitary conductance of 25–28 pS. In accordance with our patch-clamp data, osmotic swelling increases activity of immunoprecipitated p56lck. We conclude that osmotic swelling activates ICl−swell in lymphocytes via the tyrosine kinase p56lck.

Calcium influx through store-operated calcium release-activated calcium channels (CRAC) is required for T cell activation, cytokine synthesis, and proliferation. The CD95 (Apo-1͞Fas) 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؉ The CD95-receptor induces apoptosis in lymphocytes and is critically involved in the maintenance of self-tolerance (reviewed in ref. 5). Expression of the CD95 ligand (CD95L) can create immune privileged sites (6, 7), and malignant tumors may escape from the immune attack by expressing high levels of CD95L (8,9).However, CD95 may have additional functions apart from lymphocyte apoptosis. First, freshly activated T lymphocytes express CD95 but are resistant to CD95-mediated apoptosis (10, 11). Second, in tumor patients, defective lymphocyte function rather than apoptotic deletion has been observed (12, 13). Third, mice inoculated with a CD95L-expressing tumor develop severe CD95-mediated defects in lymphocyte function (14). Fourth, the knockout of CD95-activated signaling molecules required for apoptosis does not, in contrast to CD95-knockout, result in lymphoproliferative disease (15,16). Thus, apoptosis may not represent the only function of CD95 in immune tolerance. Materials and MethodsCells. Jurkat T cells, Niemann-Pick Disease type A (NPDA), Daudi, and JY B cells were maintained in RPMI medium 1640, and LN18 and LN229 glioblastoma were maintained in DMEM (17). The stimulating monoclonal mouse anti-human CD95 (CH11; Upstate Biotechnology, Lake Placid, NY) was added at 100 ng͞ml. Apoptosis was assayed by nuclear fragmentation (Hoe33342, propidium iodide or SYTO 16; Molecular Probes) or FITC-annexin flow cytometry. Peripheral T lymphoblasts were prepared by using a Ficoll gradient, purified with nylon wool columns (Ͼ90% CD3 ϩ ), and stimulated with 1 g͞ml phythaemaglutinin (PHA) (24 h) plus 2.5 units͞ml IL-2 (4-6 days). Thymocytes were prepared from homozygous C57͞Bl6 lpr-and gld mice (ages 10-15 weeks). Transient transfection of NPDA cells was performed by electroporation with an expression vector encoding acidic sphingomyelinase (ASM) (pJK-asm, 15 g͞ml) and a Myc-tagged single chain (18). Twelve hours after electroporation viable cells were purified by Ficoll gradient centrifugation and cultured for an additional 24 h. After 36 h, Myc presented on the cell surface was stained with a monoclonal anti-Myc-antibody (9E10; American Type Culture Collection, Bethesda, MD) and a phycoerythrin-conjugated secondary antibody (18...

No abstract