Effect of CD40-CD40 ligand interaction on diacylglycerol-protein kinase C signal transduction pathway and intracellular calcium in cultured human monocytes  

CD40-CD40配体相互作用对培养的人单核细胞二酰基甘油-蛋白激酶C信号通路及胞内Ca^(2+)的影响(英文)

作　　者：严金川[1] 吴宗贵[2] 孔宪涛[2] 仲人前[2] 张玲珍[2] 

机构地区：[1]东南大学附属中大医院心内科,南京210009 [2]第二军医大学长征医院心内科

出　　处：《Acta Pharmacologica Sinica》2003年第7期687-691,726,共6页中国药理学报（英文版）

基　　金：Project supported by the National Basic Research Program of China (No G2000056903);Shanghai Medical Development(2000IZD002).

摘　　要：AIM: To investigate whether CD40-CD40 ligand interaction can activate diacylglycerol (DAG)-protein kinase C (PKC) signaling pathway and intracellular free calcium ([Ca^(2+)]_i) in cultured human peripheral blood monocytes (PBMC). METHODS: The DAG levels in PBMC were studied with radio-enzymatic assay. Quantitative measurements of ^(32)P-phosphatidic acid were performed by thin-layer chromatography and autoradiography. The activity of PKC and [Ca^(2+)]_i induced by CD40 ligand (CD40L) in PBMC were measured by its ability to transfer phosphate from [γ-^(32)p]ATP to lysine-rich histone and flow cytometric analysis loading with the Ca^(2+) dye Fluo-3/Am, respectively. RESULTS: The DAG levels in PBMC were increased by CD40L in a dose-dependent, biphasic manner. The early phase was rapid and transient, peaking at 20 s; the late phase reached the maximal level at 10 min and then decayed slowly. CD40L increased the PKC total activity in a dose-dependent manner with phase peaking at 12 min, then decreased slowly and maintained for at least 20 min. CD40L induced PKC activity translocation from the cytosol to membrane. Moreover, CD40L also induced biphasic [Ca^(2+)]_i responses including the rapid initial transient phase and the sustained phase. Removal of extracellular Ca^(2+) did not inhibit the rapid phase of CD40L-induced rise in [Ca^(2+)]_i, but abolished the sustained phase of [Ca^(2+)]_i response to CD40L. Anti-CD40 monoclone antibody 10 mg/L significantly suppressed CD40L-induced DAG-PKC signal transduction pathway activation and [Ca^(2+)]_i changes in PBMC. CONCLUSION: CD40-CD40 ligand interaction induced a robust stimulation of the DAG-PKC pathway and calcium mobilization from intracellular pool in PBMC.目的:探讨CD40-CD40配体(CD40L)相互作用是否能激活人外周血单核细胞(PBMC)内二酰基甘油(DAG)-蛋白激酶C(PKC)信号通路。方法:细胞内DAG含量采用放射酶标记、薄层层析和放射自显影方法检测,细胞PKC活性及胞内游离钙分别采用[Υ^(32)P]ATP磷酸转移法和Fluo-3荧光负载流式细胞术检测。结果:CD40L以剂量依赖方式刺激人外周血单核细胞合成DAG,并具有双时限性变化,第一峰值在20s,第二峰值在10min时出现,然后DAG水平缓慢下降,至少持续20-30min,单核细胞蛋白激酶C总活性受CD40L刺激后明显增加,峰值在12min,持续20min以上,并且这种作用主要是胞浆PKC活性向胞膜PKC活性转位所致,CD40L能刺激胞内游离Ca^(2+)出现短暂的快速升高,继之为持续阶段,移去细胞外Ca^(2+),胞内快速阶段无影响,而持续阶段明显受到抑制,抗CD40抗体能显著抑制CD40L引起的胞内DAGPKC信号通路激活及[Ca^(2+)]_i的动态变化。结论:CD40-CD40L相互作用能激活人外周血单核细胞[Ca^(2+)]_i动态变化及二酰基甘油-蛋白激酶C信号通路。

关 键 词：CD40 antigens CD40 ligand protein kinase C DIGLYCERIDES MONOCYTES signal transduction calcium 

分 类 号：R33[医药卫生—人体生理学]