White J R, Zembryki D, Hanna N, Mong S
Department of Immunology, Smith Kline & French Laboratories, King of Prussia, PA 19406-0939.
Biochem Pharmacol. 1990 Aug 1;40(3):447-56. doi: 10.1016/0006-2952(90)90542-s.
Pretreatment of rat peritoneal mast cells with either staurosporine or an analog K-252a [(8R*,9S*,11S*)-(-)-9-hydroxyl-9-methoxycarbonyl-8-methyl-2,3, 9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11-atrizadibenzo- [a,g]cycloocta[cde]trinden-1-one] led to a concentration-related inhibition of histamine release when the cells were stimulated with anti-IgE (IC50: staurosporine = 110 nM; K-252a = 100 nM). In contrast, the two protein kinase C (PKC) inhibitors (1-1000 nM) partially (less than 15%) inhibited histamine release induced by compound 48/80 (0.5 to 1 micrograms/mL). Furthermore, prostaglandin E2 (PGE2) synthesis mediated by anti-IgE from rat peritoneal mast cells was also inhibited by staurosporine and K-252a (IC50 = 100 nM). Exposure of anti-arsenate IgE (anti-Ars-IgE) sensitized mouse bone marrow derived mast cells to arsenate-bovine serum albumin (Ars-BSA) led to the release of both histamine (510 +/- 12.6 ng/10(6) cells) and immunoreactive leukotriene C4 (LTC4) (27.0 +/- 2.6 ng/10(6) cells). Both histamine and LTC4 release was inhibited by staurosporine and K-252a with an IC50 of 50 nM for both compounds. We also characterized a 45K molecular weight protein which is phosphorylated by PKC after Ars-BSA or phorbol, 12-myristate, 13-acetate (PMA) stimulation. This protein is phosphorylated in a broken cell preparation in which PKC is activated by phosphatidylserine/Diolein and Ca2+. Peptide mapping by V8 protease of the phosphorylated 45K protein revealed that the 45K protein phosphorylation patterns induced by IgE or PMA or in the broken cell preparation are identical. Pretreatment of 32P-labeled mouse bone marrow derived mast cells with either staurosporine or K-252a led to a concentration-related inhibition of 45K protein phosphorylation induced by PMA or Ars-BSA. This inhibition of protein phosphorylation correlated well with the inhibition of histamine and leukotriene release in bone marrow derived mast cells.
用星形孢菌素或其类似物K-252a [（8R*，9S*，11S*）-（-）-9-羟基-9-甲氧基羰基-8-甲基-2,3,9,10-四氢-8,11-环氧-1H，8H，11H-2,7b,11-三氮杂二苯并[a,g]环辛[cde]三茚-1-酮]预处理大鼠腹膜肥大细胞，当用抗IgE刺激细胞时，会导致组胺释放呈浓度依赖性抑制（IC50：星形孢菌素 = 110 nM；K-252a = 100 nM）。相比之下，两种蛋白激酶C（PKC）抑制剂（1 - 1000 nM）对化合物48/80（0.5至1微克/毫升）诱导的组胺释放有部分抑制作用（小于15%）。此外，星形孢菌素和K-252a也抑制大鼠腹膜肥大细胞中由抗IgE介导的前列腺素E2（PGE2）合成（IC50 = 100 nM）。将抗砷酸盐IgE（抗-Ars-IgE）致敏的小鼠骨髓来源肥大细胞暴露于砷酸盐-牛血清白蛋白（Ars-BSA）会导致组胺（510±12.6纳克/10^6个细胞）和免疫反应性白三烯C4（LTC4）（27.0±2.6纳克/10^6个细胞）的释放。组胺和LTC4的释放均被星形孢菌素和K-252a抑制，两种化合物的IC50均为50 nM。我们还鉴定了一种45K分子量的蛋白质，在Ars-BSA或佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）刺激后被PKC磷酸化。在一种破碎细胞制剂中该蛋白质会被磷酸化，在该制剂中PKC被磷脂酰丝氨酸/二油精和Ca2+激活。用V8蛋白酶对磷酸化的45K蛋白进行肽图谱分析表明，由IgE或PMA诱导或在破碎细胞制剂中诱导的45K蛋白磷酸化模式是相同的。用星形孢菌素或K-252a预处理32P标记的小鼠骨髓来源肥大细胞会导致PMA或Ars-BSA诱导的45K蛋白磷酸化呈浓度依赖性抑制。这种蛋白质磷酸化的抑制与骨髓来源肥大细胞中组胺和白三烯释放的抑制密切相关。
