leukotriene-d4 and Neuroblastoma

To determine whether cysteinyl leukotriene receptor agonist LTD(4) and cysteinyl leukotriene receptor 1 (CysLT(1)) antagonist pranlukast affect the differentiation of human neuroblastoma SK-N-SH cells.. SK-N-SH cell morphological changes induced by LTD(4), pranlukast and LTD(4) + pranlukast were observed with retinoid acid (RA) as the positive control. The expressions of CysLT(1) and CysLT(2) receptors were detected by immunoblotting analysis, and the expression of microtubule-associated protein-2 (MAP-2), a neuron marker, was detected by fluorescent immunostaining.. The immunoblotting results showed that SK-N-SH cells expressed CysLT(1) receptor moderately, and CysLT(2) receptor highly. The morphological results showed that RA, pranlukast and LTD(4) + pranlukast induced the compaction of the cell bodies and the outgrowth of neurites, while LTD(4) had no significant effect. The immunostaining results showed that MAP-2 was distributed in the cell bodies in control or pranlukast-treated cells; it was distributed in cell bodies and neuritis in RA-treated cells. Pranlukast increased the numbers of MAP-2-positive cells.. The CysLT(1)receptor antagonist pranlukast modulates the differentiation of SK-N-SH cells.

Topics: Cell Differentiation; Cell Line, Tumor; Chromones; Humans; Immunoblotting; Immunohistochemistry; Leukotriene Antagonists; Leukotriene D4; Membrane Proteins; Microtubule-Associated Proteins; Neuroblastoma; Receptors, Leukotriene

To explore whether the proinflammatory products of the 5-lipoxygenase (5-LOX) pathway are involved in microglia-mediated toxicity toward neuronal cells, we evaluated the effects of 5-LOX inhibitors using an in vitro assay system where human neuronal SH-SY5Y cells are exposed to toxic secretions from THP-1 monocytic cells or human microglia. The specific 5-LOX inhibitors, REV 5901, zileuton, and 5-hydroxyeicosatetraenoic acid lactone; the nonselective LOX inhibitors, phenidone and dapsone; the dual 5-LOX/cyclooxygenase inhibitor, tepoxalin; and the selective inhibitor of the 5-LOX-activating protein (FLAP), MK-886, inhibited such toxicity. The toxicity was enhanced by the 5-LOX product leukotriene (LT)D(4) and reduced by the selective cysteinyl LT receptor (CysLT(1)) antagonist MK-571. The mRNAs for 5-LOX and FLAP were detected in THP-1 cells and human microglia but not in SH-SY5Y cells. The data suggest that inhibition of proinflammatory LT production by 5-LOX inhibition could selectively reduce toxicity of microglial cells and thus be beneficial in neuroinflammatory diseases.

Topics: 5-Lipoxygenase-Activating Proteins; Arachidonate 5-Lipoxygenase; Carrier Proteins; Cell Communication; Cell Survival; Enzyme Inhibitors; Humans; Immunohistochemistry; Leukotriene D4; Lipoxygenase Inhibitors; Membrane Proteins; Microglia; Monocytes; Neuroblastoma; Neurons; RNA, Messenger; Tumor Cells, Cultured

The effects of LY-171883, an orally active leukotriene antagonist, on membrane currents were examined in pituitary GH(3) and in neuroblastoma IMR-32 cells. In GH(3) cells, LY-171883 (1-300 microM) reversibly increased the amplitude of Ca(2+)-activated K(+) current in a concentration-dependent manner with an EC(50) value of 15 microM. In excised inside-out patches recorded from GH(3) cells, the application of LY-171883 into cytosolic face did not modify single channel conductance of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels; however, it did increase the channel activity. The LY-171883-stimulated activity of BK(Ca) channels is dependent on membrane potential, and results mainly from an increase in mean open time and a decrease in mean closed time. However, REV-5901 (30 microM) suppressed the activity of BK(Ca) channels and MK-571 (30 microM) did not have any effect on it. Under the current-clamp condition, LY-171883 (30 microM) caused membrane hyperpolarization as well as decreased the firing rate of action potentials in GH(3) cells. In neuroblastoma IMR-32 cells, the application of LY-171883 (30 microM) also stimulated BK(Ca) channel activity in a voltage-dependent manner. However, neither clofibrate (30 microM) nor leukotriene D(4) (10 microM) affected the channel activity in IMR-32 cells. Troglitazone (30 microM) decreased the channel activity, but ciglitazone (30 microM) enhanced it. This study clearly demonstrates that LY-171883 stimulates the activity of BK(Ca) channels in a manner unlikely to be linked to its blockade of leukotriene receptors or stimulation of peroxisome proliferator-activated receptors. The stimulatory effects on these channels may, at least in part, contribute to the underlying cellular mechanisms by which LY-171883 affects neuronal or neuroendocrine function.

Topics: Acetophenones; Action Potentials; Animals; Cell Membrane; Electrophysiology; Humans; Large-Conductance Calcium-Activated Potassium Channels; Leukotriene Antagonists; Leukotriene D4; Membrane Potentials; Neuroblastoma; Patch-Clamp Techniques; Pituitary Neoplasms; Potassium Channels, Calcium-Activated; Propionates; Quinolines; Rats; Tetrazoles; Trypsin; Tumor Cells, Cultured

Recent studies suggest that bone marrow (BM)-derived chemotactic mediators such as chemokines play key roles in hematopoietic stem cell trafficking. Lipid mediators, particularly leukotrienes, are involved in leukocyte chemotaxis during inflammation but have also been detected in the normal BM. Therefore, the effects of leukotrienes on hematopoietic progenitor cells were analyzed. Cysteinyl leukotrienes, particularly leukotriene D4 (LTD4), induced strong intracellular calcium fluxes and actin polymerization in mobilized and BM CD34(+) progenitors. Chemotaxis and in vitro transendothelial migration of CD34(+) and more primitive CD34(+)/CD38(-) cells were 2-fold increased by LTD4 at an optimum concentration of 25 to 50 nM. Accordingly, CD34(+) cells expressed the 7-transmembrane LTD4 receptor CysLT1 by reverse transcriptase-polymerase chain reaction and Western blot. Effects of LTD4 were suppressed by the CysLT1 receptor antagonist MK-571 and reduced by pertussis toxin. In contrast, LTB4 induced strong responses only in mature granulocytes. LTD4-induced calcium fluxes were also observed in granulocytes but were not reduced by MK-571, suggesting that these effects were mediated by other receptors (eg, CysLT2) rather than by CysLT1. In addition, expression of 5-lipoxygenase, the key enzyme of leukotriene biosynthesis, was detected in both hematopoietic progenitor cells and mature leukocytes. The study concludes that the functionally active LTD4 receptor CysLT1 is preferentially expressed in immature hematopoietic progenitor cells. LTD4 released in the BM might regulate progenitor cell trafficking and could also act as an autocrine mediator of hematopoiesis. This would be a first physiologic effect of cysteinyl leukotrienes apart from the many known pathophysiologic actions related to allergy and inflammation. (Blood. 2001;97:3433-3440)

Topics: Antigens, CD34; Arachidonate 5-Lipoxygenase; Blotting, Western; Bone Marrow Cells; Calcium; Chemotaxis; Endothelium; Gene Expression; Granulocytes; Hematopoietic Stem Cells; HL-60 Cells; Humans; Leukotriene Antagonists; Leukotriene D4; Membrane Proteins; Neuroblastoma; Pertussis Toxin; Propionates; Quinolines; Receptors, Leukotriene; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stromal Cells; Tumor Cells, Cultured; Virulence Factors, Bordetella

Arachidonic acid or its metabolites have been implicated in the regulatory volume decrease (RVD) response after hypotonic cell swelling in some mammalian cells. The present study investigated the role of arachidonic acid (AA) during RVD in the human neuroblastoma cell line CHP-100. During the first nine minutes of hypo-osmotic exposure the rate of 3H-arachidonic acid (3H-AA) release increased to 250 +/- 19% (mean +/- SE, n = 22) as compared with cells under iso-osmotic conditions. This release was significantly inhibited after preincubation with AACOCF3, an inhibitor of the 85-kDa cytosolic phospholipase A2 (cPLA2). This indicates that a PLA2, most likely the 85-kDa cPLA2 is activated during cell swelling. In contrast, preincubation with U73122, an inhibitor of phospholipase C, did not affect the swelling-induced release of 3H-AA. Swelling-activated efflux of 36Cl and 3H-taurine were inhibited after preincubation with AACOCF3. Thus the swelling-induced activation of cPLA2 may be essential for stimulation of both 36Cl and 3H-taurine efflux during RVD. As the above observation could result from a direct effect of AA or its metabolite leukotriene D4 (LTD4), the effects of these agents were investigated on swelling-induced 36Cl and 3H-taurine effluxes. In the presence of high concentrations of extracellular AA, the swelling-induced efflux of 36Cl and 3H-taurine were inhibited significantly. In contrast, addition of exogenous LTD4 had no significant effect on the swelling-activated 36Cl efflux. Furthermore, exogenous AA increased cytosolic calcium levels as measured in single cells loaded with the calcium sensitive dye Fura-2. On the basis of these results we propose that cell swelling activates phospholipase A2 and that this activation via an increased production of AA or some AA metabolite(s) other than LTD4 is essential for RVD.

Topics: Arachidonic Acid; Arachidonic Acids; Chlorides; Cytosol; Egtazic Acid; Enzyme Inhibitors; Estrenes; Humans; Hypotonic Solutions; Kinetics; Leukotriene D4; Molecular Weight; Neuroblastoma; Osmolar Concentration; Phosphodiesterase Inhibitors; Phospholipases A; Phospholipases A2; Pyrrolidinones; Taurine; Tumor Cells, Cultured