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REGULATORY MECHANISM OF HISTAMINE RELEASE BY CA LCIUM : USING RAT BASOPHILIC LEUKEMIA CELL LINE

钙释放组胺的调节机制：使用大鼠嗜碱性白血病细胞系

基本信息

批准号：
05670081
负责人：
MAEYAMA Kazutaka
金额：
$ 1.34万
依托单位：
EHIME UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

In mast cells and basophils, the aggregation of IgE receptors initiates histamine release and consequently, causes allevgic status such as urticaria, bronchial athma and sometimes anaphylactic shock. The regulation of histamine release is important for prevention of these diseases. The roles of cytosolic calcium as a second messenger have been revealed. The increase of intracellular Ca concentration ( [Ca^<2+>] i) is introduced by two mechanisms. One is the release from the intracellular Ca stores stimulated by inositol 1,4,5-trisphosphates and another is the influx from the extracellular medium. Non-excitable cells such as blood cells have no voltage-dependent Ca channels and the mechanism of Ca influx in these cells remains unknown. Rat basophilic leukemia cells (2H3) , a tumour analogue of rat mucosal mast cells which show histamine release after stimulation with antigen are stable and provide a useful experimental model for the study of the mechanism of Ca influx and histamine release. Therefore, effects of various agents which stimulate or attenuate histamine release on [Ca^<2+>] i in 2H3 cells were investigated.1. Intracellular Ca concentration : Cytosolic calcium concentration in single 2H3 cells was measured using a Ca probe, fluo-3 with a confocal microscopy. The oscillatrory Ca signals were observed in the absence of external Ca, but quick increase of [Ca^<2+>] i occurs after addition of external Ca.2. Ca entry in 2H3 cells : Imidazole compounds such as SK&F 96365, econazole and ketoconazole suppress both Ca uptake and histamine release.3. The changes of cytoskeleton : The changes in F-actin contents were measured with NBD-phallacidin. Antigen stimulation increases F-actin assembly both in the presence and absence of external Ca^<2+>, but thapsigargin and ionomycin which increase [Ca^<2+>] i induce actin assembly only in the presence of cytosolic Ca.

在肥大细胞和嗜碱性粒细胞中，IgE受体的聚集引发组胺释放，从而引起过敏状态，如荨麻疹、支气管哮喘，有时还引起过敏性休克。组胺释放的调节对于预防这些疾病是重要的。胞浆钙离子作为第二信使的作用已被揭示。细胞内Ca浓度（[Ca^2+] i）的增加通过两种机制引入。一个是由肌醇1，4，5-三磷酸刺激的细胞内钙库的释放，另一个是由细胞外介质的流入。非兴奋性细胞如血细胞没有电压依赖性Ca通道，这些细胞中Ca内流的机制仍然未知。大鼠嗜碱性白血病细胞（2 H3）是大鼠粘膜肥大细胞的一种肿瘤类似物，经抗原刺激后可释放组胺，且细胞稳定，为研究钙内流和组胺释放机制提供了一个有用的实验模型。因此，研究了刺激或减弱组胺释放的各种试剂对2 H3细胞中[Ca^<2+>] i的影响。细胞内Ca浓度：使用Ca探针fluo-3和共聚焦显微镜测量单个2 H3细胞中的胞质钙浓度。在无外源性钙时，可观察到细胞内钙信号，但在加入外源性钙后，[Ca^<2+>] i迅速增加. 2 H3细胞中的Ca内流：咪唑类化合物如SK&F 96365、益康唑和酮康唑抑制Ca摄取和组胺释放。细胞骨架的变化：用NBD-鬼笔菌素法测定细胞骨架中F-肌动蛋白含量的变化。在有和无外部Ca^2+的情况下，抗原刺激都能增加F-肌动蛋白的组装，但增加[Ca^2+] i的毒胡萝卜素和离子霉素只在胞浆Ca存在的情况下才能诱导肌动蛋白的组装。