REGULATION OF HISTAMINE RELEASE AND SYNTHESIS; USING BASOPHILIC LEUKEMIA CELL LINES

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，其导致荨麻疹、哮喘和有时过敏性休克。我们使用培养的嗜碱性白血病细胞系研究了第二信使系统（即胞浆Ca）的变化与组胺释放之间的关系。并运用分子生物学技术对组胺合成的调控进行了研究.细胞内Ca浓度的测定：将大鼠嗜碱性白血病细胞（2 H3）在盖玻片上单层化，并用刺激剂如抗原和毒胡萝卜素（一种Ca-ATP酶抑制剂）灌流。使用Ca探针fura-2监测2 H3细胞中细胞内Ca浓度（[Ca^<2+>]i）的变化。同时通过HPLC-荧光法测定灌流液中释放的组胺。在存在外部Ca的情况下，兴奋剂诱导持续的Ca信号和组胺释放。然而，在缺乏外部Ca的情况下，[Ca^<2 ...更多信息 +>]，但无组胺释放。单个细胞中的Ca信号：使用具有计算机成像系统的显微镜测定，研究单个2 H3细胞中的Ca信号。在外源性钙存在的情况下，抗原刺激诱导每个细胞内钙信号振荡的时间间隔小于60秒. 2 H3细胞的钙内流机制：包括2 H3细胞在内的非兴奋性细胞没有电压依赖性钙通道，钙内流机制尚不清楚。SK&F 96365，咪唑衍生物抑制Ca信号和组胺分泌。其他咪唑衍生物，如益康唑和酮康唑，它们是细胞色素P-450的抑制剂，抑制Ca摄取和组胺分泌。这表明，钙内流是由P-450.4产生的代谢产物调节。组氨酸脱羧酶（HDC）的诱导：用抗原和佛波酯刺激引起HDC活性增加。星形孢菌素，蛋白激酶C的抑制剂，抑制这种增加。这些结果表明，HDC的诱导受蛋白激酶C的调节。在北方印迹分析中，在与佛波酯孵育1小时期间，人嗜碱性白血病细胞（KU-812）中的2.4 Kb HDCmRNA增加了1.5倍。少

In mast cells and basophils, the aggregation of lgE receptors initiates histamine release which results in urticaria, asthma and sometimes anaphylactic shock. We investigated the relationship between the changes in second messenger systems(i.e. cytosolic Ca) and histamine release using cultured basophilic leukemia cell lines. Moreover, the regulation of histamine syntehsis was studied with molecular biological techniques.1. Determination of intracellular Ca concentration: Rat basophilic leukemia cells(2H3) were monolayered on a coverglass and superfused with stimulants such as an antigen and thapsigargin, an inhibitor of Ca-ATPase. The changes of intracellular Ca concentration([Ca^<2+>]i) in 2H3 cells were monitored using a Ca probe, fura-2. Histamine released in superfusates was measured simultaneously by HPLC-fluorometry. In the presence of external Ca, stimulants induced sustained Ca signal and histamine release. Whereas, in the absence of external Ca, a transient increase in [Ca^<2 … More +>], but no histamine release was observed.2. Ca signal in a single cell: Using a microscopic determination with a computer imaging system, Ca signal in a single 2H3 cells was investigated. In the presence of external Ca, antigen stimulation induced the oscillation of Ca signal at intervals of less than 60 sec in each cell.3. Mechanism of Ca entry in 2H3 cells: Non-excitable cells including 2H3 cells have no voltage-dependent Ca channels and the mechanism of Ca entry remains unknown. SK&F 96365, an imidazole derivative suppressed Ca signal and histamine secretion. Other imidazole derivatives such as econazole and ketoconazole, which are inhibitors of cytochrome P-450, inhibit Ca uptake and histamine secretion. It is suggested that Ca entry is regulated by metabolites produced by P-450.4. Induction of histidine decarboxylase(HDC): Stimulation with an antigen and phorbol ester caused increase of HDC activity. Staurosporine, an inhibitor of protein kinase C, suppressed this increase. These results suggest that the induction of HDC is regulated by protein kinase C. In northern blot analysis, 2.4-Kb HDCmRNA in human basophilic leukemia cells(KU-812) increased by 1.5-fold during the 1h incubation with phorbol ester. Less