The rotavirus enterotoxin NSP4 mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase C-mediated inositol 1,4,5-trisphosphate production
The rotavirus enterotoxin NSP4 mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase C-mediated inositol 1,4,5-trisphosphate production
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DOI：
10.1073/pnas.94.8.3960
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发表时间：
1997-04-15
影响因子：
11.1
通讯作者：
Morris, AP
中科院分区：
文献类型：
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作者：
Dong, YJ;Zeng, CQY;Morris, AP
轮状病毒感染是全球婴幼儿严重腹泻的主要原因。轮状病毒非结构蛋白NSP4是一种病毒肠毒素，可引起幼年小鼠腹泻，并引起钙依赖的跨上皮氯离子分泌。这一现象的细胞学基础是在人体肠道的体外细胞系模型中进行的。用显微镜荧光成像法检测Fura-2负载的HT-29细胞内钙离子浓度([Ca~(2+)](I))。NSP4(1 NM~5 mU M)既可引起细胞内钙释放，又可引起质膜钙内流。在NSP4诱导的[Ca~(2+)](I)动员过程中，[Na~+](I)的动态平衡没有被破坏，表明NSP4选择性地调节细胞外Ca~(2+)进入这些细胞。NSP4对峰值[Ca~(2+)](I)动员的ED50为4.6+/-0.8 nM。用2.3×10~(-3)单位/毫升胰酶或4.4×10~(-2)单位/毫升胰凝乳酶处理细胞1-10分钟后，NSP_4诱导的[Ca~(2+)](I)动员消失。用磷脂酶C的抑制剂U-73122对细胞进行超灌注会消融NSP4的反应。在1，4，5-三磷酸(IP3)特异性放射受体分析中，NSP4诱导IP3产生的快速起效和瞬时刺激。综上所述，这些结果表明，NSP4通过受体介导的磷脂酶C激活和IP3的产生来动员人肠道细胞内的[Ca~(2+)](I)。
Rotavirus infection is the leading cause of severe diarrhea in infants and young children worldwide. The rotavirus nonstructural protein NSP4 acts as a viral enterotoxin to induce diarrhea and causes Ca2+-dependent transepithelial Cl- secretion in young mice. The cellular basis of this phenomenon was investigated in an in vitro cell line model for the human intestine. Intracellular calcium concentration ([Ca2+](i)) was monitored in fura-2-loaded HT-29 cells using microscope-based fluorescence imaging. NSP4 (1 nM to 5 mu M) induced both Ca2+ release from intracellular stores and plasmalemma Ca2+ influx. During NSP4-induced [Ca2+](i) mobilization, [Na+](i) homeostasis was not disrupted, demonstrating that NSP4 selectively regulated extracellular Ca2+ entry into these cells. The ED50 of the NSP4 effect on peak [Ca2+](i) mobilization was 4.6 +/- 0.8 nM. Pretreatment of cells with either 2.3 x 10(-3) units/ml trypsin or 4.4 x 10(-2) units/ml chymotrypsin for 1-10 min abolished the NSP4-induced [Ca2+](i) mobilization. Superfusing cells with U-73122, an inhibitor of phospholipase C, ablated the NSP4 response. NSP4 induced a rapid onset and transient stimulation of inositol 1,4,5-trisphosphate (IP3) production in an IP3-specific radioreceptor assay. Taken together, these results suggest that NSP4 mobilizes [Ca2+](i) in human intestinal cells through receptor-mediated phospholipase C activation and IP3 production.