LIN Jian Yin and LIN Jing
Dept. of Molecular Medicine, Fujian Medical University, Fuzhou,
China
Objective: To explore whether NO is capable of inducing apoptosis in Toxoplasma gondii tachyzoite and its calcium signal transduction mechanism.
Methods: Apoptosis induced by NO in Toxoplasma gondii tachyzoite was investigated by TUNEL method, electron microscopy and agarose gel electrophoresis. Cytoplasmic free calcium concentration in toxoplasmatic tachyzoite was determined by Fura-2 fluorescein load technique.
Results: NO donor, sodium nitroprusside (SNP) induced apoptosis in toxoplasmatic tachyzoite in a time- and dose- dependent manner by TUNEL. N-acetylcystein, a NO scavenger, greatly inhibited SNP-induced tachyzoite apoptosis, but potassium ferricyanide, an analogue of SNP but devoid of NO, alone can't induce tachyzoite apoptosis. SNP-treated Toxoplasmatic tachyzoite have shown characteristic morphological features of apoptosis, including aggregation of chromatin at the nuclear membrane, condensation of nucleus and apoptotic body by electron microscopy and characteristic biochemical features, including DNA fragmentation and the appearance of the "DNA ladder" by agarose gel electrophoresis. The cytoplasmic free [Ca2+]i in resting tachyzoite were 108.49°¿4.87nmol/L(n=15).[Ca2+]i in the process of SNP-induced Toxoplasmatic tachyzoite apoptosis was significantly higher than that in control groups in time- and dose- dependent manner, which was greatly inhibited by N-acetylcystein, but K3Fe(CN)6 alone can not induce increase in [Ca2+]i. Chelation of extracellular calcium with EGTA entirely inhibited the increase in and voltage-dependent calcium channel blockers, verapamil only partially inhibited the increase in [Ca2+]i induced by SNP .However, EGTA and verapamil inhibited SNP-induced apoptosis in T.gondii tachyzoite.
Conclusion: It has been shown that NO donor, SNP play a role in antitoxoplasmatic tachyzoite infection through inducing tachyzoite apoptosis according to characteristic morphological and biochemical features and SNP-induced apoptosis in T.gondii tachzoite is mediated by elevating cytoplasmic free calcium concentration, which is mainly resulted from the entry of extracellular calcium. These findings indicate potential chemotherapeutic importance of NO.
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created 21/12/00
