calcimycin and 3-deazaadenosine

The studies reported here were designed to examine the chemotactic potential, arachidonic acid (AA) metabolism and phospholipid transmethylation in peripheral blood neutrophils from patients with rheumatoid arthritis (RA): a) prior to treatment with BW 91Y (3-deazaadenosine), b) after 4 weeks when half the patients were on active medication and half were on placebo, and c) after 4 weeks at which time all patients were on active medication. The authors demonstrate that BW 91Y in vitro at 600 pg/ml caused a decrease in chemotactic potential as measured by the leading front (LF) assay in neutrophils from both normal volunteers (p less than 0.025) and RA patients. They also demonstrate that BW 91Y caused a significant increase in production of [3H]LTB4 (LTB = leukotriene B) in ionophore-stimulated neutrophils from both normal (p less than 0.025) and RA patients (p less than 0.050) as compared to initial values. BW 91Y caused decreased incorporation and percent distribution of [3H]AA into phosphatidylcholine (PC), with a resultant increase in percent distribution into phosphatidylethanolamine (PE). There was also an increased release of [3H]AA from the PE fraction in BW 91Y-treated cells in response to ionophore stimulation. BW 91Y was found to exhibit a dose-dependent (10(-7) to 10(-4) g/ml) inhibition of the uptake and incorporation of L-[methyl-3H]methionine into the cellular lipids, while at low doses (10(-9) to 10(-5) g/ml) it stimulated the significant uptake and incorporation of [methyl-14C]choline chloride into PC. Although the total cellular content and percent composition of PC remained unchanged, it was found that BW 91Y caused a slight decrease in PC plasmalogens and an apparent increase in the 1,2-diacyl-glycerophosphatidylcholine (-GPC). BW 91Y was found, however, to have no effect on the amount or stimulated metabolism of the ether-linked 1-O-alkyl-2-acyl-GPC. As further evidence for this, the authors demonstrate that BW 91Y has no effect on the ionophore-stimulated production of [14C]acetate-labelled 1-O-alkyl-2-acetyl-GPC, or [14C]PAF.

Topics: Arachidonic Acids; Arthritis, Rheumatoid; Calcimycin; Chemotaxis, Leukocyte; Chromatography, Thin Layer; Dose-Response Relationship, Drug; Double-Blind Method; Humans; Leukotriene B4; Lipid Metabolism; Lipids; Neutrophils; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Platelet Activating Factor; Tubercidin

Enhanced phospholipid methylation has been suggested to be an obligatory process in IgE-dependent stimulus-secretion coupling in human lung mast cells. Our studies with mast cell-enriched lung preparations do not support this hypothesis, demonstrating no increased 3H-methyl radiolabeling of chloroform/methanol-extracted lipids or chromatographically separated phospholipids accompanying anti-IgE-dependent histamine secretion. Inhibitors of transmethylation, 3-deazaadenosine, and homocysteine thiolactone inhibited histamine secretion by both anti-IgE and calcium ionophore A23187, reflecting a requirement of secretion for overall integrity of cellular transmethylation. These agents induced small increases in cAMP concentration which are considered to make at most a minor contribution to this inhibition. The inability of methylation inhibitors to diminish anti-IgE-dependent increases in lung mast cell cAMP levels would suggest that not only does phospholipid methylation have no role in histamine secretion but also it does not participate in the activation of adenylate cyclase by this stimulus.

Topics: Antibodies, Anti-Idiotypic; Calcimycin; Cyclic AMP; Histamine Release; Homocysteine; Humans; Immunoglobulin E; Lung; Mast Cells; Methylation; Phospholipids; Tubercidin

Inhibitors of adenosylmethionine (AdoMet)-dependent methyltransferases reduce histamine release from enzymatically dispersed human lung mast cells activated with either anti-human IgE or calcium ionophore A23187. The IC25 values for adenosine and 3-deazaadenosine (DZA) inhibiting anti-IgE-induced histamine release were 395 microM and 301 microM respectively. The addition of homocysteine thiolactone (Hcy) potentiated the effects of adenosine and DZA, reducing their IC25 values to 32 microM and 10.5 microM respectively. The adenosine deaminase (adenosine aminohydrolase EC 3.5.4.4) inhibitors erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) inhibited anti-IgE-induced histamine release with an IC50 of 162 microM. This inhibition was not potentiated by Hcy. The combination of DZA and Hcy effectively inhibited histamine release induced by concentrations of A23187 which released a similar amount of histamine to anti-IgE. However the combination was 17 times less potent against A23187-compared with anti-IgE-induced release. These observations suggest that AdoMet-dependent methyltransferases play an important role in IgE-dependent histamine release from human lung mast cells but their role in A23187-induced release is less clear.

Topics: Adenine; Adenosine; Antibodies, Anti-Idiotypic; Calcimycin; Histamine Release; Humans; Immunoglobulin E; Mast Cells; Membrane Lipids; Methylation; Methyltransferases; Phospholipids; Ribonucleosides; S-Adenosylmethionine; Tubercidin

The biochemical events that lead to thrombin-stimulated release of von Willebrand factor and prostacyclin synthesis in cultured endothelial cells are examined. Treatment of human umbilical vein endothelial cells with thrombin results in an instantaneous increase in phospholipid methylation which can be blocked by 3-deazaadenosine, a methyltransferase inhibitor. 3-Deazaadenosine also blocks the thrombin-induced Ca2+ influx into endothelial cells and the release of von Willebrand factor, indicating that these processes are coupled. The phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) and the Ca2+ ionophore A23187 both bypass the phospholipid methylation and directly stimulate Ca2+ influx and von Willebrand factor release. In contrast to the stimulus-induced von Willebrand factor release, the thrombin-induced prostacyclin synthesis cannot be blocked by 3-deazaadenosine. Similarly, incubation of endothelial cells with EDTA has no influence on the thrombin-induced prostacyclin synthesis, and PMA has no stimulatory effect on prostacyclin synthesis. These observations indicate that thrombin induces different metabolic responses in endothelial cells: phospholipid methylation followed by a Ca2+ influx, which subsequently leads to release of von Willebrand factor, and liberation of arachidonic acid from phospholipids for prostacyclin formation, which is independent of phospholipid methylation and Ca2+ influx.

Topics: Blood Coagulation Factors; Calcimycin; Calcium; Cells, Cultured; Edetic Acid; Endothelium; Epoprostenol; Female; Humans; Isomerism; Kinetics; Methylation; Phospholipids; Pregnancy; Tetradecanoylphorbol Acetate; Thrombin; Tubercidin; Umbilical Veins; von Willebrand Factor

Rabbit neutrophils were stimulated with the chemotactic peptide fMet-Leu-Phe in the presence of the methyltransferase inhibitors homocysteine (HCYS) and 3-deazaadenosine (3-DZA). HCYS and 3-DZA inhibited chemotaxis, phospholipid methylation, and protein carboxymethylation in a dose-dependent manner. The chemotactic peptide-stimulated release of [14C]arachidonic acid previously incorporated into phospholipid was also partially blocked by the methyltransferase inhibitors. Stimulation by fMet-Leu-Phe or the calcium ionophore A23187 caused release of arachidonic acid but not of previously incorporated [14C]-labeled linoleic, oleic, or stearic acids. Unlike the arachidonic acid release caused by fMet-Leu-Phe, release stimulated by the ionophore could not be inhibited by HCYS and 3-DZA, suggesting that the release was caused by a different mechanism or by stimulating a step after methylation in the pathway from receptor activation to arachidonic acid release. Extracellular calcium was required for arachidonic acid release, and methyltransferase inhibitors were found to partially inhibit chemotactic peptide-stimulated calcium influx. These results suggest that methylation pathways may be associated with the chemotactic peptide receptor stimulation of calcium influx and activation of a phospholipase A2 specific for cleaving arachidonic acid from phospholipids.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Chemotaxis; Fatty Acids; Homocysteine; N-Formylmethionine; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oligopeptides; Phospholipids; Rabbits; Receptors, Cell Surface; Receptors, Formyl Peptide; Tubercidin

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Calcimycin; Collagen; Homocysteine; Isomerism; Kinetics; Male; Methyltransferases; Rats; Rats, Inbred Strains; Ribonucleosides; Thromboxane B2; Thromboxanes; Tubercidin

Topics: Animals; Calcimycin; Collagen; Crotalid Venoms; Homocysteine; Lectins, C-Type; Male; Methylation; Phospholipases; Phospholipases A; Phospholipases A2; Phospholipids; Platelet Aggregation; Rats; Ribonucleosides; Tubercidin

Topics: Adenine; Adenosine; Basophils; Calcimycin; Histamine Release; Homocysteine; Humans; Immunoglobulin E; In Vitro Techniques; Methylation; Ribonucleosides; S-Adenosylmethionine; Tubercidin

Topics: Adenosine Diphosphate; Animals; Calcimycin; Collagen; Crotalid Venoms; Homocysteine; Immunoglobulin E; Kinetics; Lectins, C-Type; Male; Membrane Lipids; Methylation; Phosphatidylcholines; Phosphatidylethanolamines; Platelet Aggregation; Rats; Rats, Inbred Strains; Tubercidin