Mondragon R, Frixione E
Departamento de Biología Celular, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del IPN, México.
J Eukaryot Microbiol. 1996 Mar-Apr;43(2):120-7. doi: 10.1111/j.1550-7408.1996.tb04491.x.
The role of Ca2+ in conoid extrusion was investigated in isolated Toxoplasma gondii tachyzoites by treatment with Ca(2+)-ionophores, Ca(2+)-chelating agents and an inhibitor of the Ca(2+)-ATPase at the endoplasmic reticulum. The results were evaluated by light phase-contrast microscopy and electron microscopy. Ionomycin (0.5-1 microM) caused an immediate and sustained extrusion of the conoid in up to 80% of the tachyzoites, depending on the concentrations of ionophore and Ca2+ in the medium. However, over 50% of the tachyzoites extruded the conoid when treated with ionomycin in Ca(2+)-free saline complemented with EGTA. The effect of ionomycin was reversible and could be induced a second time in about half of the responsive population. Similar results were obtained with A23187. Conoid extrusion induced by ionomycin in Ca(2+)-free medium was almost completely abolished when the tachyzoites were previously loaded with a permeable compound known to chelate intracellular Ca2+ (BAPTA/AM; 25 microM). On the other hand, exposure of tachyzoites to the Ca(2+)-ATPase inhibitor thapsigargin (0.5-1 microM) produced significant extrusion of the conoid. Tachyzoites loaded with BAPTA/AM as well as those treated with ionomycin, i.e. with conoids paralyzed in opposite positions, had a diminished capacity to invade cultured epithelial cells. A substantial reduction in the response to stimulation by ionomycin was found also in parasites treated with cytochalasin-D, a drug that depolymerizes actin-filaments. The results suggest that Ca(2+)-release from internal stores may act as a key signal to activate a mechanism of conoid extrusion probably mediated, at least in part, by actin-filaments.
通过用钙离子载体、钙离子螯合剂以及内质网钙离子ATP酶抑制剂处理分离出的刚地弓形虫速殖子，研究了钙离子在类锥体挤出中的作用。通过光学相差显微镜和电子显微镜对结果进行评估。离子霉素（0.5 - 1微摩尔）可导致高达80%的速殖子立即且持续地挤出类锥体，这取决于培养基中离子载体和钙离子的浓度。然而，在无钙盐并用乙二醇双四乙酸（EGTA）补充的情况下用离子霉素处理时，超过50%的速殖子挤出了类锥体。离子霉素的作用是可逆的，并且在大约一半的反应群体中可再次诱导。用A23187也获得了类似的结果。当速殖子预先加载一种已知可螯合细胞内钙离子的可渗透化合物（1,2 - 双（2 - 氨基苯氧基）乙烷 - N,N,N',N'-四乙酸甲酯；25微摩尔）时，离子霉素在无钙培养基中诱导的类锥体挤出几乎完全被消除。另一方面，速殖子暴露于钙离子ATP酶抑制剂毒胡萝卜素（0.5 - 1微摩尔）会导致类锥体显著挤出。加载了1,2 - 双（2 - 氨基苯氧基）乙烷 - N,N,N',N'-四乙酸甲酯的速殖子以及用离子霉素处理的速殖子，即类锥体处于相反位置而麻痹的速殖子，侵袭培养上皮细胞的能力减弱。在用细胞松弛素 - D处理的寄生虫中也发现对离子霉素刺激的反应大幅降低，细胞松弛素 - D是一种使肌动蛋白丝解聚的药物。结果表明，从内部储存释放的钙离子可能作为一个关键信号，激活一种可能至少部分由肌动蛋白丝介导的类锥体挤出机制。
