维拉帕米、噻庚啶和山莨菪碱拮抗TNFα诱导单个内皮细胞胞内游离Ca^2＋浓度的增高

目的：研究肿瘤坏死因子（TNFα）对单个内皮细胞胞内游离Ca^2 浓度（[Ca^2 ]i)的影响及维拉帕米（Ver)、噻庚啶（Cyp)和山莨菪碱（Ani)对TNFα介导休克和Cyp、Ani的抗休克的机制。方法：人脐静脉内皮细胞株（ECV304）接种于35mm含有2mL DMEM培养基的组织培养盘中培养，Fluo-3/AM负载细胞，激光扫描共聚集显微技术测定单个内皮细胞[Ca^2 ]i。结果：TNFα使单个内皮细胞[Ca^2 ]i呈剂量依赖性升高，在60s内达到峰值，然后下降并保持在基础水平之上。共聚焦扫描图像显示细胞核区[Ca^2 ]i升高比胞浆区明显，下降比胞浆区慢。维拉帕米1和2，噻庚啶30和60或山莨菪碱20和40μmol/L均能显著抑制由TNFα 1.2nmol/L诱导的单个内皮细胞[Ca^2 ]i升高。结论：TNFα介导休克的重要机制；维拉帕米、噻庚啶和山莨菪碱对TNFα诱导的[Ca^2 ]i升高有拮抗作用，可能是噻庚啶和山莨菪碱抗休克作用的机制之一。

Verapamil, cyproheptadine, and anisodamine antagonized [Ca2 ]_i elevation induced by TNFa in a single endothelial cell

AIM: To study the effect of tumor necrosis factor alpha (TNFalpha) on intracellular free Ca2+ concentration ([Ca2+]i) and the effects of verapamil (Ver), cyproheptadine (Cyp), and anisodamine (Ani) on TNFalpha-induced [Ca2+]i changes in single endothelial cell, and to explore the mechanisms of TNFalpha-mediated shock and antishock actions of Cyp and Ani. METHODS: Human umbilical vein endothelial cell strains (ECV304) were seeded in 35-mm tissue culture dish with 2 mL DMEM culture medium. The cultured cells were loaded by Fluo-3/AM. The spatial distribution and the dynamic changes of [Ca2+]i in single endothelial cell were determined by laser scanning confocal microscopy. RESULTS: After stimulation with TNFalpha, [Ca2+]i in single endothelial cell rapidly increased in a concentration-dependent manner and arrived at the peak value within 60 s, afterwards, decreased and kept above the basal level. The confocal scanning image showed that [Ca2+]i elevation was more obvious in nuclear than in cytoplasma and decreased slowly. Ver (1, 2 micromol/L), Cyp (30, 60 micromol/L), and Ani (20, 40 micromol/L) markedly inhibited TNFalpha 1.2 nmol/L-induced [Ca2+]i elevation. CONCLUSION: TNFalpha markedly induces elevation of [Ca2+]i in a single endothelial cell, it may be an important mechanism of TNFalpha-induced shock and tissue injury. That Cyp and Ani obviously suppress TNFalpha-induced [Ca2+]i elevation probably is one of the mechanisms of their antishock effects.