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¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1992 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1991 : ¥1,300,000 (Direct Cost : ¥1,300,000)
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
+>], 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