New assay systems for the development of inhibitory drugs against activated mast cell function

（京都大学）我们分离了小鼠组氨酸脱羧酶（histidine decarboxylase，HDC），并克隆了其cDNA和部分基因组DNA，在杆状病毒-SF 9细胞中表达的小鼠重组HDC大小为74 kDa，主要存在于颗粒组分中，酶活性较低，而C端缺失的突变型重组HDC是54 kDa亚基的同源二聚体，是一种完全活性的酶，存在于可溶性组分中。颗粒状重组74 kDa HDC经猪胰弹性蛋白酶处理后转化为具有增强活性的可溶性形式。我们进一步发现，地塞米松和TPA、Ca^<2+>和cAMP的联合作用可特异性诱导肥大细胞瘤细胞中的小鼠HDC，并且在小鼠HDC基因的基因组DHA克隆的启动子区确定了与地塞米松/TPA或Ca^<2+>/cAMP的HDC基因转录增加有关的调控元件。这些结果有助于研究开发一种新型药物，该药物对肥大细胞等组胺产生细胞中组胺合成的调节起作用。我们在大鼠和小鼠肥大细胞中检测到至少两种磷脂酶A_2。其中的c-磷脂酶A_2在细胞刺激后被磷酸化，这一过程可能对细胞活化过程中花生四烯酸的释放起重要作用。Ⅱ型分泌型磷脂酶A_2在肥大细胞受到刺激时分泌并结合于细胞膜表面。这表明，酶可能参与导致组胺释放的过程。c型或分泌型磷脂酶A_2的抑制剂可用于抑制肥大细胞的活化。

(Kyoto University)We have isolated mouse histidine decarboxylase (HDC), and cloned its cDNA and partial genomic DNA.Mouserecombinant HDC expressed in Baculovirus-SF9 cells has a 74 kDa in size mostly present in the particulate fraction with a low enzyme activity, whereas a C-termial delated mutant recombinant HDC which is a homodimer of 54 kDa subunit is a fullly active enzyme Present in soluble fraction. Particulate recombinant 74 kDa HDC was interconvertedinto the soluble form having intensified activity by porcine pancreatic elastase treatment. We further obtained that mouse HDC in mastocytoma cells was specifically induced by treatment with a combination of dexamethasone and TPA,of Ca^<2+> and cAMP.Furthermore, the regulatory elements involved in the increased transcription of the HDC gene with dexamethasone/TPA of Ca^<2+>/cAMP were determined in the promoter regions of the genomic DHA clone of the mouse HDC gene. These results contribute the study for developing a new type drug which acts on the regulation of histamine synthesis in histamine-producing cells such as mast cells.(Tokyo Univercity)We detect at least two kinda of phospholipase A_2 in rat and mouse mast cells. One of the enzyme, c-phospholipase A_2 was phospholylated shorty after cell stimulation, and this process may play an importaut role for releasing arachidonate during cell activation. Type II secretory phospholipase A_2 was secreted and bound to the surface membrane when mast cells were stimulated. It was suggested that the enzyme may be involved in the process leading to release of histamine. Inhibitors of either c-or secretory phospholipases A_2 may be useful for suppression of mast cell activation.