Biomedical subjects

H Sommermeyer

Publications and source records attributed to H Sommermeyer.

9 recordsLinked to original sources

PubMed1992

Cleavage of phosphatidylcholine: an additional mechanism for stimulation of macrophage eicosanoid synthesis?

Cleavage of phosphatidylcholine (PtdChol) as putative mechanism leading to enhanced eicosanoid synthesis was investigated in mouse peritoneal macrophages. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) to intact [3H]choline-labelled cells did not enhance radioactivity release above control levels. Membrane-bound PtdChol-specific phospholipase (PL) activity was then measured in a cell-free assay using [3H]choline-labelled membranes as endogenous substrate. Preincubation of macrophages with TPA increased PL activity in a time course resembling the one observed for protein kinase C (PKC) activation. Diacylglycerol (DAG) species derived from PtdChol might therefore serve as additional stimulator of rapid macrophage eicosanoid synthesis via PKC.

Animals↗

PubMed1991

Activation of human thymocytes by antibodies to the CD3/T-cell receptor complex: triggering of different epitopes results in different signals.

Monoclonal antibodies (mAb) against the CD3/T cell receptor (TcR) complex were analyzed for their ability to activate human thymocytes. In addition to mAb detecting epitopes on the CD3 complex (OKT3, BMA 030) the activation potential of recently developed mAb against common epitopes on the alpha/beta T-cell receptor (anti-TcR mAb: BMA 031, BMA 032) was evaluated. Several differences were observed between the two types of mAb: (a) Binding of the tested anti-CD3 mAb to thymocytes resulted in a rapid increase in the level of cytoplasmic free calcium ions [Ca2+]i, whereas no significant changes in [Ca2+]i were detected in thymocytes stimulated with BMA 031 or BMA 032. (b) Induction of effective proliferation induced by mAb OKT3 depended on exogenous IL-2 and in addition on the presence of accessory cells or phorbol-ester. Proliferation induced by BMA 031 only required exogenous IL-2. (c) OKT3 but not BMA 031 inhibited proliferation of thymocytes induced via the CD2 molecule. These studies indicate that anti-CD3 and anti-TcR mAb transduce different signals in thymocytes. Since the two types of mAb are directed to the same molecular complex the observed differences also support the idea that there are functionally different compartments in the CD3/TcR complex which may activate different signaling pathways.

Antibodies, Monoclonal↗

PubMed1991

Cholera toxin-mediated inhibition of signalling in Jurkat cells is followed by, but not due to a loss of T cell receptor complex.

Cholera toxin treatment of the human T cell lymphoma Jurkat resulted in inhibition of signalling via the T cell antigen receptor complex (TcR/CD3-complex). Cholera toxin specifically ADP-ribosylated the alpha-subunit of the stimulatory G-protein of the adenylate cyclase (Gs alpha), no other proteins were modified in the intact cells. ADP-ribosylation of Gs alpha and its subsequent activation led to an increase of the cyclic AMP level and in addition, to a drastic reduction of the cell-surface density of the TcR/CD3-complex. Recently, we demonstrated that the effect of cholera toxin at the receptor level is not due to an increased cAMP level (4). As inhibition of signalling is also not cAMP-mediated (8), we examined whether the modulation of the TcR/CD3-complex could be the reason for the interruption of the signalling cascade. Analyzing the time courses of the multiple cholera toxin effects in Jurkat cells at 37 degrees C, the following sequence was found: ADP-ribosylation of Gs alpha--increase of cyclic AMP level--inhibition of signalling via the TcR/CD3-complex--decrease of cell-surface density of the TcR/CD3-complex. Treatment of Jurkat cells at 20 degrees C with cholera toxin resulted in an increase of cyclic AMP and inhibition of signal transduction, while no decrease of TcR/CD3-complex density could be observed. These data imply that receptor loss from the cell-surface is not causative for the inhibition of signalling. More likely, activation of Gs uncouples signal transduction in Jurkat cells via the TcR, which by a so far unknown mechanism is followed by a loss of the receptor from the cell surface.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1990

The G protein coupling T cell antigen receptor/CD3-complex and phospholipase C in the human T cell lymphoma Jurkat is not a target for cholera toxin.

Intact Jurkat cells could be stimulated by monoclonal antibodies against the Tcell antigen receptor complex (OKT3 directed against the CD3 complex, BMA031 directed against constant framework epitopes in the alpha/beta heterodimer). The accumulation of inositol phosphates was inhibited by prior incubation of the cells with cholera holotoxin. The inhibitory effect of cholera toxin (CT) was not cAMP mediated because forskolin (a direct activator of adenylate cyclase) did not mimic the inhibitory effect. When measuring phospholipase C (PLC) in a cell-free assay system by using [3H]inositol-labeled membranes, the enzyme could be stimulated by the poorly hydrolyzable GTP analogue guanosine 5'-O-(thiotriphosphate (GTP gamma S). Both anti-receptor antibodies augmented the GTP gamma S stimulatory effect, while the antibodies alone had no stimulatory capacity. In membranes from CT-pretreated cells, whereas the antibodies lost their stimulatory effect on PLC as in untreated cells, whereas the antibodies lost their stimulatory capacity in the presence of GTP gamma S. These data imply that CT exerts its inhibitory effect on signaling by acting at the receptor level while the PLC regulating G protein is not a target for CT-mediated alterations. This assumption is supported by the finding that in intact Jurkat cells CT, which ADP ribosylated only the alpha-subunit of the stimulatory G protein of the adenylate cyclase, led to a loss of the T cell antigen receptor complex from the cell surface as demonstrated by a decrease of receptor density using flow cytometry analysis. Receptor loss could not be achieved by forskolin treatment or incubation of the cells with the binding subunit of the toxin alone.

Adenosine Diphosphate Ribose↗

PubMed1990

Pertussis toxin B-subunit-induced Ca2(+)-fluxes in Jurkat human lymphoma cells: the action of long-term pre-treatment with cholera and pertussis holotoxins.

The exotoxins of Bordetella pertussis and Vibrio cholera have been used to investigate signal transduction in the human T-cell lymphoma Jurkat. Stimulation of the cells, leading to an increase in cytoplasmic free calcium, could be achieved by the anti-T-cell receptor complex antibody OKT3 and by pertussis holotoxin (PTHT), or its B-subunit (PTB), but not by cholera holotoxin (CTHT) or its B-subunit (CTB). Both holotoxins ADP-ribosylated specifically G-proteins in the plasma membrane of intact cells, while their B-subunits had no ADP-ribosyltransferase activity. Incubation of the cells with CTHT led to a state of unresponsiveness to all stimulants. CTB was without any effect, indicating that the ADP-ribosyltransferase activity of cholera toxin (located in the A-subunit of the holotoxin) was necessary for the inhibition of cellular signalling. The inhibitory effect of cholera toxin on the pertussis toxin action was not due to a blockade of pertussis toxin interaction with the cell surface, because pertussis toxin was still able to ADP-ribosylate membrane proteins in cholera toxin treated intact cells. In addition, the cholera toxin mediated inhibition was not due to elevated levels of cyclic-AMP, as forskolin (a direct activator of the adenylate cyclase) and no inhibitory effect. The stimulating effect of PTHT was independent of its ADP-ribosyltransferase activity, because it could also be obtained by the B-subunit alone. In addition, the increase of cytoplasmic free calcium after stimulation by PTHT clearly preceded the ADP-ribosylation. Pre-treatment with PTHT, PTB or OKT3, led to a long lasting increase in the level of intracellular Ca2+ in Jurkat cells, which could not, therefore, be stimulated further. Inhibition by cholera holotoxin of the stimulation by OKT3 and pertussis toxin (PTHT and PTB) imply that the mitogenic effect of pertussis toxin is perhaps mediated via the T-cell antigen receptor signalling cascade. The presented data do not support the idea that a pertussis toxin-sensitive G-protein is involved in coupling the T-cell antigen receptor to the phospholipase C.

Adenosine Diphosphate Ribose↗

PubMed1989

Transforming growth factors beta 1 and beta 2 as well as milk growth factor decrease anti-CD3-induced proliferation of human lymphocytes without inhibiting the anti-CD3-mediated increase of [Ca2+]i and the activation of protein kinase C.

Porcine transforming growth factor 1 and 2 (pTGF-beta 1 and -beta 2) and milk growth factor (MGF) at 1 ng/ml significantly inhibited the proliferation of human lymphocytes induced by anti-CD3 antibodies. In contrast, the anti-CD3-mediated increase of intracellular Ca2+ and the activation and translocation of protein kinase C were not affected by the transforming growth factors.

Animals↗

PubMed1989

Effects of nucleotides on the activity of phospholipase C in rabbit thymus lymphocytes. Differences in assays using endogenous [3H]inositol-prelabeled membranes or exogenous [3H]phosphatidylinositol 4,5-bisphosphate as substrate.

The influence of nucleotides and pyrophosphate on phospholipase C from rabbit thymocytes was investigated by using two different methods for the determination of phospholipase C activity. In a first approach the release of radiolabeled inositol phosphates from [3H]inositol-labeled membranes was examined. By a second type of experiment the cleavage of exogenously added radiolabeled phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) was measured. Using internally labeled membranes only guanosine 5'-O-(thiotriphosphate) exhibited a stimulatory effect on the phospholipase C suggesting the involvement of a G-protein. When exogenous [3H]PtdIns-4,5-P2 was used as substrate, cleavage of PtdIns-4,5-P2 was stimulated by all nucleotides investigated; in addition pyrophosphate showed a stimulatory effect. From these data we conclude that the increased cleavage of exogenous PtdIns-4,5-P2 induced by GTP analogues is not conclusive in terms of the involvement of a G-protein. Rather than induced by a G-protein this activation may be caused by an increased substrate accessibility. Our experiments with endogenous substrate clearly established the regulatory role of G-proteins for membrane-bound phospholipase C.

Adenosine Triphosphate↗

PubMed1989

Cholera toxin modulates the T cell antigen receptor/CD3 complex but not the CD2 molecule and inhibits signaling via both receptor structures in the human T cell lymphoma Jurkat.

The human T cell lymphoma Jurkat can be activated by stimuli directed either against the T cell antigen receptor-CD3 antigen complex (TcR/CD3) or the CD2 molecule. Stimulation of cells via the TcR/CD3-complex or via the CD2 molecule increases inositol phosphates and cytoplasmic free calcium. Pretreatment of Jurkat cells with cholera toxin leads to a decrease of TcR/CD3 expression on the surface of the cells, while the expression of CD2 is unaffected. In contrast to this distinct effect on the receptor expression, signaling via both pathways is inhibited by cholera toxin. The most convincing explanation for the cholera toxin-mediated inhibition of signaling is that cholera toxin interrupts the signaling pathways at a point where both, stimulation via TcR/CD3 and via CD2, use the same route. The earliest common point of the two signaling pathways, at least in the Jurkat cell line, seems to be the CD3 complex because after its down-regulation (and functional inactivation) both pathways of activation are interrupted.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1988

Phospholipase C in rabbit thymocytes: subcellular distribution and influences of calcium and GTP gamma S on the substrate dependence of cytosolic and plasma membrane-associated phospholipase C.

The subcellular distribution of phospholipase C (PLC) activity in rabbit thymocytes was examined by measuring the enzyme's activity in different subcellular fractions. PLC activity was determined using exogenously added [3H]PIP2 as substrate. Approx. 80% of the activity of the cell homogenate was found in the cytosolic fraction. A minor portion of PLC activity was attached to the particulate fraction. This membrane-associated PLC activity was found to be predominantly bound to the plasma membrane. Both PIP2-cleaving PLCs (the PLC associated with the plasma membrane and the PLC in the cytosol) exhibited maximum activity at pH 5. GTP gamma S stimulated the cytosolic and the membrane-bound PLC. As revealed by computer analysis of the substrate dependence of both basal and GTP gamma S-stimulated PLC activity, GTP gamma S enhanced the Vmax of the enzymes. Calcium, at a concentration of 1 mM, decreased PLC activity, as compared to a calcium concentration of 100 nM. The characteristic increase in Vmax induced by GTP gamma S was observed at a concentration of 1 mM calcium and was similar to that at 100 nM. These data suggest that the stimulatory effect of GTP gamma S is not due to an increased affinity of PLCs to calcium.

Animals↗