Pathogenic Roles of Ion Channels and Transporters: Dextran sulfate sodium-induced chronic colitis attenuates Ca2+-activated Cl− secretion in murine colon by downregulating TMEM16A

摘要

Attenuated Ca²⁺-activated Cl⁻ secretion has previously been observed in the model of dextran sulfate sodium (DSS)-induced colitis. Prior studies have implicated dysfunctional muscarinic signaling from basolateral membranes as the potential perpetrator leading to decreased Ca²⁺-activated Cl⁻ secretion. However, in our chronic model of DSS-colitis, cholinergic receptor muscarinic 3 (Chrm3) transcript (1.028 ± 0.12 vs. 1.029 ± 0.27, P > 0.05) and CHRM3 protein expression (1.021 ± 0.24 vs. 0.928 ± 0.09, P > 0.05) were unchanged. Therefore, we hypothesized that decreased carbachol (CCH)-stimulated Cl⁻ secretion in DSS-induced colitis could be attributed to a loss of Ca²⁺-activated Cl⁻ channels (CaCC) in apical membranes of colonic epithelium. To establish this chemically-induced colitis, Balb/C mice were exposed to 4% DSS for five alternating weeks to stimulate a more moderate, chronic colitis. Upon completion of the protocol, whole thickness sections of colon were mounted in an Ussing chamber under voltage-clamp conditions. DSS-induced colitis demonstrated a complete inhibition of basolateral administration of CCH-stimulated Cl⁻ secretion that actually displayed a reversal in polarity (15.40 ± 2.22 μA/cm² vs. −2.47 ± 0.25 μA/cm²). Western blotting of potential CaCCs, quantified by densitometric analysis, demonstrated no change in bestrophin-2 and cystic fibrosis transmembrane regulator, whereas anoctamin-1 [ANO1, transmembrane protein 16A (TMEM16A)] was significantly downregulated (1.001 ± 0.13 vs. 0.510 ± 0.12, P < 0.05). Our findings indicate that decreased expression of TMEM16A in DSS-induced colitis contributes to the decreased Ca²⁺-activated Cl⁻ secretion in murine colon.