一氧化氮经钙信号转导途径诱导弓形虫速殖子凋亡

目的:为探讨胞浆Ca^2 是否为一氧化氮（Nitric Oxide,NO)诱导弓形虫速殖子凋亡的重要信号分子。方法：采用脱氧核苷酸末端转移酶（TdT)介导的缺口末端标记法（TUNEL）、琼脂糖凝胶电泳和流式细胞仪检测凋亡以及应用Fura-2荧光负载技术测定胞浆游离钙浓度Ca^2 ]i。结果：NO供体亚硝基铁氧化钠（Na2Fe(CN)5NO,SNP)诱导弓形虫速殖子凋亡过程中胞浆Ca^2 ]i明显升高。NO清除剂，N－乙酰半胱氨酸能明显抑制SNP诱导的速殖子凋亡及SNP诱导的速殖子胞浆Ca^2 ]i升高，而不含NO的SNP为似物，铁氰化钾K3Fe(CN)6]不能诱导速殖子凋亡及其胞浆Ca^2 ]i升高。胞外钙螯合剂EGTA，和L型电压依赖性钙通道阻滞剂异搏定，完全或部分抑制SNP引起的速殖子胞浆Ca^2 ]i升高，胞内钙螯合剂BAP－TA／AM及胞外钙螯合剂EGTA明显抑制SNP诱导的速殖子凋亡。结论：证明NO的供体SNP诱导弓形虫速殖子凋亡主要通过促进胞外钙内流，胞浆Ca^2 ]i升高所致。

Nitric oxide induces apoptosis in Toxoplasma gondii tachyzoite via calcium signal transduction pathway

Aim To explore whether cytoplasmic free calcium is a critical signal molecule in NO induced apoptosis in toxoplasmatic tachyzoites Methods Apoptosis induced by NO in toxoplasmatic tachyzoites was investigated by TUNEL(terminal deoxynucleotidyl transferase mediated d UTP nick end labeling )method,agarose gel electrophoresis and flow cytometry Cytoplasmic free calcium concentration(Ca 2+ ]i)in toxoplasmatic tachyzoites was determined by applying Fura 2 fluorescein load technic Results NO donor,sodium nitroprusside(Na 2Fe(CN) 5NO,SNP),induced apoptosis and increased inCa 2+ ]i in Toxoplasma gondii tachyzoites N acetylcysteine,a NO scavenger,greatly inhibited SNP induced apoptosis and SNP induced increase inCa 2+ ]i in toxoplasmatic tachyzoites Potassium ferricyanideK 3Fe(CN) 6],an analogue of SNP but devoid of NO,alone did not induce apoptosis and the increasing inCa 2+ ]i in toxoplasmatic tachyzoites Chelation of extracellular calcium with EGTA entirely inhibited the increase inCa 2+ ]i and voltage dependent calciunm channel blockers,verapamil only partially inhibited the increase inCa 2+ ]i induced by SNP SNP induced apoptosis in toxoplasmatic tachyzoites was greatly inhibited by intracellular Ca 2+ chelator(BAPTA/AM)and extracellular Ca 2+ chelator(EGTA) Conclusion SNP induced apoptosis is mediated by elevating cytoplasmic free calcium concentrationCa 2+ ]i in toxoplasmatic tachyzoites,which is mainly resulted from the entry of extra cellular calcium