Chloride channel inhibition by a red wine extract and a synthetic small molecule prevents rotaviral secretory diarrhoea in neonatal mice

Abstract

Background Rotavirus is the most common cause of severe secretory diarrhoea in infants and young children globally. The rotaviral enterotoxin, NSP4, has been proposed to stimulate calcium-activated chloride channels (CaCC) on the apical plasma membrane of intestinal epithelial cells. We previously identified red wine and small molecule CaCC inhibitors. Objective To investigate the efficacy of a red wine extract and a synthetic small molecule, CaCCinh-A01, in inhibiting intestinal CaCCs and rotaviral diarrhoea. Design Inhibition of CaCC-dependent current was measured in T84 cells and mouse ileum. The effectiveness of an orally administered wine extract and CaCCinh-A01 in inhibiting diarrhoea in vivo was determined in a neonatal mouse model of rotaviral infection. Results Screening of ~150 red wines revealed a Cabernet Sauvignon that inhibited CaCC current in T84 cells with IC50 at a ~1:200 dilution, and higher concentrations producing 100% inhibition. A >1 kdalton wine extract prepared by dialysis, which retained full inhibition activity, blocked CaCC current in T84 cells and mouse intestine. In rotavirus-inoculated mice, oral administration of the wine extract prevented diarrhoea by inhibition of intestinal fluid secretion without affecting rotaviral infection. The wine extract did not inhibit the cystic fibrosis chloride channel (CFTR) in cell cultures, nor did it prevent watery stools in neonatal mice administered cholera toxin, which activates CFTR-dependent fluid secretion. CaCCinh-A01 also inhibited rotaviral diarrhoea. Conclusions Our results support a pathogenic role for enterocyte CaCCs in rotaviral diarrhoea and demonstrate the antidiarrhoeal action of CaCC inhibition by an alcohol-free, red wine extract and by a synthetic small molecule.

Anoctamins support calcium-dependent chloride secretion by facilitating calcium signaling in adult mouse intestine

“…As mice grow older, expression of Ano1 appears to shift from the luminal toward the basolateral membrane, and cholinergic Ca 2+ -dependent Cl − secretion was found to be reduced in the older animals [27]. A recent report analyzed rotavirus-induced diarrhea in vivo and in vitro and nicely demonstrates inhibition of Cl − secretion and diarrhea by different blockers of anoctamins [22]. These studies demonstrate the large medical relevance of intestinal anoctamins and may trigger subsequent clinical trials.…”

“…Cl − secretion though anoctamins and CFTR The available data clearly indicate the role of anoctamins for intestinal Ca 2+ -dependent Cl − secretion and rotavirus-induced diarrhea in younger animals [22,27,38]. It was suggested from experiments in Xenopus oocytes coexpressing CFTR and P2Y 2 receptors, and also from measurements in human airway epithelial cells, that Ca 2+ -dependent Cl − secretion elicited through stimulation of purinergic P2Y receptors is due to activation of CFTR rather than anoctamin 1 [13,34,42].…”

“…Although the cAMP- regulated Cl − channel cystic fibrosis transmembrane conductance regulator (CFTR) is central to bacterial diarrhea [52], Ca 2+ -activated Cl − channels seem to play a central role during rotaviral diarrhea [2,9,22,38]. Secretory diarrhea is a major health problem worldwide with rotavirus being the most common cause for severe secretory diarrhea in infants and young children [22]. We reported earlier the expression of Ano1 in both apical and basolateral membranes of colonic epithelial cells of young mice [38].…”

“…Electrolyte secretion is controlled by a number of hormones leading to increase in either intracellular cAMP or Ca 2+ , with consecutive activation of cystic fibrosis transmembrane conductance regulator (CFTR) and Ca 2+ -activated Cl − channels (CaCC), respectively [14,24]. While the contribution of CFTR to intestinal Cl − secretion is well defined, controversial results have been reported for CaCC, which has been identified recently as anoctamin 1 (TMEM16A; Ano1) and which was detected in the basolateral rather than in the apical membrane of adult intestinal epithelial cells [6,16,22,38,44,55]. The role of Ano1 for intestinal Cl − secretion, and also whether Ano1 is the only relevant anoctamin in the intestine, is therefore unclear.…”

Vaccines against human diarrheal pathogens

“…16 Rotavirus infects intestinal epithelial cells, within it produces an enterotoxin called NSP4, which interacts with calcium-activated chloride channels. 17 Furthermore, it reduces the effectiveness of digestive enzymes, leading to nutritional deficiencies. 18 The disease is self-limiting in otherwise healthy Figure 1.…”