Abstract
Secretory diarrhea, which primarily originates through intestinal pathogens and viruses, is a health burden in many regions worldwide. Enterocyte Cl− channels, as the final step in enterotoxin-induced fluid secretion, constitute an attractive class of targets for diarrhea therapy. Chloride channel inhibitors have become a new class of candidates for antisecretion and anti-intestinal motility agents. In the present study, we identified plumbagin as a transmembrane protein 16A (TMEM16A) inhibitor in a cell-based fluorescence-quenching assay, and the IC50 value was ∼12.46 µM. Short-circuit current measurements showed that plumbagin reversibly inhibited the Eact-induced Cl− current on the apical side of TMEM16A-transfected Fischer rat thyroid (FRT) cells with no significant effect on cytoplasmic Ca2+ signaling. Notably, plumbagin also inhibited the activity of intestinal epithelial calcium-activated chloride channel (CaCC) and cystic fibrosis transmembrane conductance regulator (CFTR) in both HT-29 cells and mouse colons, but had no effects on the activity of the Na+-K+ ATPase or K+ channels. In in vivo experiments, the administration of plumbagin reduced both Escherichia coli heat-stable enterotoxin (STa)- and cholera toxin (CT)-induced intestinal fluid secretion. In neonatal mouse models of CT- and rotavirus infection-induced diarrhea, 0.4 µg plumbagin inhibited secretory diarrhea by >40% and 50%, respectively, without affecting intestinal epithelial integrity or the rotaviral infection. In addition, plumbagin exerted inhibitory effects on the vasoactive intestinal peptide (VIP)-, prostaglandin E2 (PGE2)-, and 5-hydroxytryptamine (5-HT)-stimulated Cl− currents. In the evaluations of intestinal motility, plumbagin significantly delayed intestinal motility and inhibited intestinal smooth muscle contractility without an evident impact on contractive frequency. Collectively, our results indicate that plumbagin inhibits both Ca2+- and cAMP-activated Cl− channels, accounting for the mechanisms of plumbagin inhibition of chloride secretion and intestinal motility. Thus, plumbagin can be a lead compound in the treatment of CT-induced, Traveler’s, and rotaviral diarrhea, as well as other types of secretory diarrhea that result from excessive intestinal fluid secretion and increased intestinal peristalsis.
Highlights
Secretory diarrhea has a severe impact on both the mortality and morbidity of patients in all age groups and from different geographical locations, and this malady remains a leading cause of death in children under 5 years old worldwide (Walker et al, 2013)
There is increasing evidence that Transmembrane protein 16A (TMEM16A) plays an important role in the regulation of intestinal motility (Sanders et al, 2012), which is based on the following results: (a) the interstitial cells of Cajal (ICC) highly express TMEM16A (Isozaki et al, 1997; He et al, 2001); (b) the major function of the ICC is to participate in intestinal electric slow-wave generation and transmission and the regulation of intestinal smooth muscle contractions (Sanders and Ward, 2006); (c) pharmacological inhibition of TMEM16A function blocks the slow wave (Hwang et al, 2009); and (d) knockout of the TMEM16A gene leads to the disappearance of the slow wave in intestinal smooth muscle (Hwang et al, 2009)
The inhibition of TMEM16A activity by 20 μM plumbagin reached its maximum after 10 min which seemed slower compared with T16Ainh-A01 (Namkung et al, 2011)
Summary
Secretory diarrhea has a severe impact on both the mortality and morbidity of patients in all age groups and from different geographical locations, and this malady remains a leading cause of death in children under 5 years old worldwide (Walker et al, 2013). Secretory diarrhea is the result of increased intestinal fluid secretion, which is mainly due to chloride secretion from the intestinal epithelial cells in the lumen and future water exudation along the paracellular spaces (Schiller, 1999). CFTR is a cAMP-dependent chloride channel that is expressed in the epithelial cells of all tissues related to fluid secretion and absorption (Kunzelmann, 1999). There is increasing evidence that TMEM16A plays an important role in the regulation of intestinal motility (Sanders et al, 2012), which is based on the following results: (a) the interstitial cells of Cajal (ICC) highly express TMEM16A (Isozaki et al, 1997; He et al, 2001); (b) the major function of the ICC is to participate in intestinal electric slow-wave generation and transmission and the regulation of intestinal smooth muscle contractions (Sanders and Ward, 2006); (c) pharmacological inhibition of TMEM16A function blocks the slow wave (Hwang et al, 2009); and (d) knockout of the TMEM16A gene leads to the disappearance of the slow wave in intestinal smooth muscle (Hwang et al, 2009)
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