ABSTRACT
The inhibitory effect of cromakalim on the mediator release from mast cells caused by antigenantibody reactions was in controversy with the specific antigen used. However, it has recently been observed that cromakalim inhibits the release of mediators from superfused tracheal and parenchymal strips or lung mast cells after passive sensitization with the IgG1 antibody. An attempt, therefore, was made to determine the inhibitory mechanisms of cromakalim on the release of mediators such as histamine and leukotriene released by the activation of enzymes during mast cell activation. Guinea pig lung mast cells were purified through enzyme digestion, rough percoll and continuous percoll density gradients. The purified mast cells were prelabeled with [3H]palmitic acid. PLD activity was assessed more directly by the production of labeled phosphatidylethanol by PLD-mediated transphosphatidylation in the presence of ethanol. In the cells labelled with [3H]myristic acid, [3H] DAG production was measured. The methyltransferase activity was assessed by measuring the incorporation of [3H]methyl moiety into phospholipids in sensitized mast cells labelled with L-[3H] methylmethionine. cAMP level was measured by radioimmunoassay. Cromakalim resulted in a decrease in the amount of histamine and leukotrienes releases by 30% in the ovalumin-induced mast cell. Cromakalim had little effect on phospholipase D activity enhanced by the activated mast cell. Cromakalim inhibited the initial increase of diacylglycerol production during mast cell activations. Cromakalim inhibited the phospholipid methylation increased in the activated mast cell. These results show that cromakalim decreases histamine release by inhibiting the initial increase of 1,2-diacylglycerol during the mast cell activation, which is mediated via the phosphatidylinositide-phospholipase C system rather than the phosphatidylcholine-phospholipase D system. Furthermore, cromakalim reduces phosphatidylcholine production by inhibiting the methyltransferase, which decreases the conversion of phosphatidylcholine into arachidonic acid and inhibits the production of leukotrienes.
Subject(s)
Female , Adenylyl Cyclases/metabolism , Animals , Antigen-Antibody Reactions , Benzopyrans/pharmacology , Cromakalim , Diglycerides/biosynthesis , Guinea Pigs , Histamine Release/drug effects , Leukotrienes/metabolism , Lung/drug effects , Mast Cells/drug effects , Methylation , Phospholipase D/metabolism , Phospholipids/metabolism , Potassium Channels/drug effects , Pyrroles/pharmacologyABSTRACT
The inhibitory effect of cromakalim on the mediator release from mast cells caused by antigenantibody reactions was in controversy with the specific antigen used. However, it has recently been observed that cromakalim inhibits the release of mediators from superfused tracheal and parenchymal strips or lung mast cells after passive sensitization with the IgG1 antibody. An attempt, therefore, was made to determine the inhibitory mechanisms of cromakalim on the release of mediators such as histamine and leukotriene released by the activation of enzymes during mast cell activation. Guinea pig lung mast cells were purified through enzyme digestion, rough percoll and continuous percoll density gradients. The purified mast cells were prelabeled with [3H]palmitic acid. PLD activity was assessed more directly by the production of labeled phosphatidylethanol by PLD-mediated transphosphatidylation in the presence of ethanol. In the cells labelled with [3H]myristic acid, [3H] DAG production was measured. The methyltransferase activity was assessed by measuring the incorporation of [3H]methyl moiety into phospholipids in sensitized mast cells labelled with L-[3H] methylmethionine. cAMP level was measured by radioimmunoassay. Cromakalim resulted in a decrease in the amount of histamine and leukotrienes releases by 30% in the ovalumin-induced mast cell. Cromakalim had little effect on phospholipase D activity enhanced by the activated mast cell. Cromakalim inhibited the initial increase of diacylglycerol production during mast cell activations. Cromakalim inhibited the phospholipid methylation increased in the activated mast cell. These results show that cromakalim decreases histamine release by inhibiting the initial increase of 1,2-diacylglycerol during the mast cell activation, which is mediated via the phosphatidylinositide-phospholipase C system rather than the phosphatidylcholine-phospholipase D system. Furthermore, cromakalim reduces phosphatidylcholine production by inhibiting the methyltransferase, which decreases the conversion of phosphatidylcholine into arachidonic acid and inhibits the production of leukotrienes.
Subject(s)
Female , Adenylyl Cyclases/metabolism , Animals , Antigen-Antibody Reactions , Benzopyrans/pharmacology , Cromakalim , Diglycerides/biosynthesis , Guinea Pigs , Histamine Release/drug effects , Leukotrienes/metabolism , Lung/drug effects , Mast Cells/drug effects , Methylation , Phospholipase D/metabolism , Phospholipids/metabolism , Potassium Channels/drug effects , Pyrroles/pharmacologyABSTRACT
Systemic lupus erythematosus (SLE) is a disease affecting blood vessels and connective tissue, which are damaged by deposition of pathogenic autoantibodies and immune complexes. Although a complex disease, SLE provides a number of insights into autoimmune pathogenesis. Autoimmune disease, in general, is characterized by B cell hyperactivity which results in hypergammaglobulinemia and production of a variety of autoantibodies reactive to organ-nonspecific antigens such as DNA, RNA, and cell membrane structures. SLE patients usually show a marked increase in the number of activated and immunoglobulin-producing circulating B cells. Recently, cytokines with specific effects on immune regulation have been detected and extensively studied. One of them, Interleukin-6 (IL-6), is an activated monocyte derived factor which stimulates B cell growth and differentiation. We investigated the serum IL-6 levels of SLE patients in an attempt to demonstrate their relationship with the patients' clinical manifestation, and the serum levels of C-reactive protein (CRP), circulating immune complexes (CICs,) and soluble interleukin-2 receptor (sIL-2R). The study subjects consisted of 22 patients with SLE who had visited Severance Hospital from July 1986 to September 1987 and 10 normal controls. The patients' sera were stored at -70degrees C and later analyzed. The serum levels of IL-6 were measured by ELISA method with Inter Test-6X Human IL-6 ELISA kit; the serum CRP levels by fluorescence polarization immunoassay; the serum CIC levels by solid phase Clp binding assay; and the serum sIL-2R levels ELISA method. The results were as follows: The mean serum IL-6 level of SLE patients (1,366 pg/ml) was higher that of the controls (98pg/ml) (p<0.05). Among the SLE patients studied. the mean serum IL-6 level was higher in those with vasculitis than those without. ln the SLE patients studied. a linear correlation was present between the measured serum IL-6 and CIC levels; however no correlation was present either between IL-6 and CRP levels, or between IL-6 level and platelet count. The mean sIL-2R level of the SLE patients studied (1,864 U/ml) was higher than that of the controls (300 U/ml). However, in the SLE patients studied, no correlation was present between the serum IL-6 and sIL-2R levels measured. The high serum IL-6 level might play an important role in the pathogenesis of SLE.