Unique Regulation of Coupled NaCl Absorption by Inducible Nitric Oxide in a Spontaneous SAMP1/YitFc Mouse Model of Chronic Intestinal Inflammation

In the small intestine, Na:H (NHE3) and Cl:HCO3 (DRA or PAT1) exchangers present in the brush border membrane (BBM) of absorptive villus cells are primarily responsible for the coupled absorption of NaCl, the malabsorption of which causes diarrhea, a common symptom of inflammatory bowel disease (IBD). Inducible nitric oxide (iNO), a known mediator of inflammation, is increased in the mucosa of the chronically inflamed IBD intestine. An SAMP1/YitFc (SAMP1) mouse, a spontaneous model of chronic ileitis very similar to human IBD, was used to study alterations in NaCl absorption. The SAMP1 and control AKR mice were treated with I-N(6)-(1-Iminoethyl)-lysine (L-NIL) to inhibit iNO production, and DRA/PAT1 and NHE3 activities and protein expression were studied. Though Na:H exchange activity was unaffected, Cl:HCO3 activity was significantly decreased in SAMP1 mice due to a reduction in its affinity for Cl, which was reversed by L-NIL treatment. Though DRA and PAT1 expressions were unchanged in all experimental conditions, phosphorylation studies indicated that DRA, not PAT1, is affected in SAMP1. Moreover, the altered phosphorylation levels of DRA was restored by L-NIL treatment. Inducible NO mediates the inhibition of coupled NaCl absorption by decreasing Cl:HCO3 but not Na:H exchange. Specifically, Cl:HCO3 exchanger DRA but not PAT1 is regulated at the level of its phosphorylation by iNO in the chronically inflamed intestine.

Unique Regulation of Intestinal Villus Epithelial Cl−/HCO3− Exchange by Cyclooxygenase Pathway Metabolites of Arachidonic Acid in a Mouse Model of Spontaneous Ileitis

Electrolytes (NaCl) and fluid malabsorption cause diarrhea in inflammatory bowel disease (IBD). Coupled NaCl absorption, mediated by Na+/H+ and Cl−/HCO3− exchanges on the intestinal villus cells brush border membrane (BBM), is inhibited in IBD. Arachidonic acid metabolites (AAMs) formed via cyclooxygenase (COX) or lipoxygenase (LOX) pathways are elevated in IBD. However, their effects on NaCl absorption are not known. We treated SAMP1/YitFc (SAMP1) mice, a model of spontaneous ileitis resembling human IBD, with Arachidonyl Trifluoro Methylketone (ATMK, AAM inhibitor), or with piroxicam or MK-886, to inhibit COX or LOX pathways, respectively. Cl−/HCO3− exchange, measured as DIDS-sensitive 36Cl uptake, was significantly inhibited in villus cells and BBM vesicles of SAMP1 mice compared to AKR/J controls, an effect reversed by ATMK. Piroxicam, but not MK-886, also reversed the inhibition. Kinetic studies showed that inhibition was secondary to altered Km with no effects on Vmax. Whole cell or BBM protein levels of Down-Regulated in Adenoma (SLC26A3) and putative anion transporter-1 (SLC26A6), the two key BBM Cl−/HCO3− exchangers, were unaltered. Thus, inhibition of villus cell Cl−/HCO3− exchange by COX pathway AAMs, such as prostaglandins, via reducing the affinity of the exchanger for Cl−, and thereby causing NaCl malabsorption, could significantly contribute to IBD-associated diarrhea.