• Jin-Chuan Yan, Zong-Gui Wu, Xian-Tao Kong, Ren-Qian Zong, and Lin-Zen Zhan.
• Department of Cardiology, Affiliated Zhong Da Hospital, Southeast University, Nanjing 210009, China. yanjinchuan@hotmail.com
• Clin. Chim. Acta. 2003 Nov 1;337(1-2):133-40.

BackgroundIncreasing evidence shows that CD40-CD40L interaction plays a crucial role in the pathogenesis of atherosclerosis and coronary artery disease. The mechanism of CD40-CD40L interaction might be related to signal transduction via receptor. The transduction pathway of the CD40 receptor may involve the activation of phospholipase C (PLC) which induces the production of inositol trisphosphate (IP(3)) leading to the increase of the intracellular free calcium on one hand, and of diacylglycerol (DAG) which stimulates the translocation to the membrane of protein kinase C (PKC).MethodsEndothelial cells were isolated from human umbilical vein and incubated with indicated concentrations of CD40 ligand (CD40L) for various periods. The DAG levels in HUVEC were studied with radioenzymatic assay. Quantitative measurements of 32P phosphatidic acid were performed by thin-layer chromatography and autoradiography. IP(3) was quantitatively measured by the radioreceptor binding assay. The activity of PKC and [Ca(2+)]i induced by CD40L were measured by its ability to transfer phosphate from [gamma-32P]ATP to lysine-rich histone and flow cytometric analysis loading with the Ca(2+) dye fluo3/Am, respectively.ResultsThe DAG levels were raised by CD40L in a dose-dependent, biphasic manner. The early phase was rapid and transient, peaking at 20 s; and 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. The results also showed that CD40L induced PKC activity translocation from the cytosolic to membrane. Similarly, the CD40L-induced transient IP(3) formation was coincident with the first peak of DAG formation. Moreover, CD40L also induced biphasic [Ca(2+)]i responses including the rapid initial transient phase and the sustained phase. Anti-CD40 monoclonal antibody can significantly suppress CD40L-induced DAG-PKC and IP(3)-[Ca(2+)]i signal pathway activation in HUVEC.ConclusionsCD40-CD40 ligand interaction can induce a robust stimulation of the DAG-PKC and inositol trisphosphate-Ca(2+) signal transduction pathway in HUVEC.

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