NSP4 elicits age-dependent diarrhea and Ca2+-mediated I- influx into intestinal crypts of CF mice

Homologous disruption of the murine gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to the loss of cAMP-mediat ed ion transport. Mice carrying this gene defect exhibit meconium ileus at birth and gastrointestinal plugging during the neonatal period, both contri buting to high rates of mortality. We investigated whether infectious mamma lian rotavirus, the recently characterized rotaviral enterotoxin protein NS P4, or its active NSP4(114-135) peptide, can overcome these gastrointestina l complications in CF (CFTRm3Bay null mutation) mice. All three agents elic ited diarrhea when administered to wildtype (CFTR+/+), heterozygous (CFTR+/ -), or homozygous (CFTR-/-) 7- to 14-day-old mouse pups but were ineffectiv e when given to older mice. The diarrheal response was accompanied by non-a ge-dependent intracellular Ca2+ mobilization within both small and large in testinal crypt epithelia. Significantly, NSP4 elicited cellular I- inf-lux into intestinal epithelial cells from all three genotypes, whereas both car bachol and the cAMP-mobilizing agonist forskolin failed to evoke influx in the CFTR-/- background. This unique plasma membrane halide permeability pat hway was age dependent, being observed only in mouse pup crypts, and was ab olished by either the removal of bath Ca2+ or the transport inhibitor DIDS. These findings indicate that NSP4 or its active peptide may induce diarrhe a in neonatal mice through the activation of an age- and Ca2+-dependent pla sma membrane anion permeability distinct from CFTR. Furthermore, these resu lts highlight the potential for developing synthetic analogs of NSP4(114-13 5) to counteract chronic constipation/obstructive bowel syndrome in CF pati ents.