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

在肥大细胞和嗜碱性粒细胞中，IgE受体的聚集启动组胺的释放，从而导致过敏状态，如荨麻疹、支气管炎，有时还会引起过敏性休克。组胺释放的调控对于预防这些疾病具有重要意义。细胞内钙作为第二信使的作用已被揭示。细胞内钙离子浓度([Ca^&lt；2+&gt；]i)的增加有两种机制。一种是由肌醇1，4，5-三磷酸刺激的细胞内钙库的释放，另一种是细胞外介质的内流。非兴奋性细胞，如血细胞，没有电压依赖性的钙通道，这些细胞内钙内流的机制尚不清楚。大鼠嗜碱性白血病细胞(2H3)是大鼠粘膜肥大细胞的肿瘤类似物，在抗原刺激后表现出组胺释放，是研究钙内流和组胺释放机制的一个有用的实验模型。因此，我们研究了各种刺激或减弱组胺释放的药物对2H3细胞[Ca~(2+)&gt；]i的影响。细胞内钙离子浓度：用钙探针Fluo-3在共聚焦显微镜下测定单个2H3细胞的胞内钙离子浓度。在没有外加钙的情况下，观察到钙信号的振荡，但加入外加钙后，[Ca^&lt；2+&gt；]i迅速增加。2H3细胞钙内流：咪唑类化合物SK&F 96365、益康唑、酮康唑均可抑制钙摄取和组胺释放。细胞骨架变化：用NBD-Pallacidin法测定F-肌动蛋白含量的变化。无论有无外在的钙离子，抗原刺激都能增加F-肌动蛋白的组装，但塔普西林和离子霉素只在胞内有钙存在时才能诱导肌动蛋白的组装。

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.