The rotavirus enterotoxin NSP4 mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase C-mediated inositol 1,4,5-trisphosphate production

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.