Differential regulation of cholera toxin-inhibited Na-H exchange isoforms by butyrate in rat ileum

Abstract

Electroneutral Na absorption occurs in the intestine via sodium-hydrogen exchanger (NHE) isoforms NHE2 and NHE3. Bicarbonate and butyrate both stimulate electroneutral Na absorption through NHE. Bicarbonate- but not butyrate-dependent Na absorption is inhibited by cholera toxin (CT). Long-term exposure to butyrate also influences expression of apical membrane proteins in epithelial cells. These studies investigated the effects of short- and long-term in vivo exposure to butyrate on apical membrane NHE and mRNA, protein expression, and activity in rat ileal epithelium that had been exposed to CT. Ileal loops were exposed to CT in vivo for 5 h and apical membrane vesicles were isolated. 22Na uptake was measured by using the inhibitor HOE694 to identify NHE2 and NHE3 activity, and Western blot analyses were performed. CT reduced total NHE activity by 70% in apical membrane vesicles with inhibition of both NHE2 and NHE3. Reduced NHE3 activity and protein expression remained low following removal of CT but increased to control values following incubation of the ileal loop with butyrate for 2 h. In parallel there was a 40% decrease in CT-induced increase in cAMP content. In contrast, NHE2 activity partially increased following removal of CT and was further increased to control levels by butyrate. NHE2 protein expression did not parallel its activity. Neither NHE2 nor NHE3 mRNA content were affected by CT or butyrate. These results indicate that CT has varying effects on the two apical NHE isoforms, inhibiting NHE2 activity without altering its protein expression and reducing both NHE3 activity and protein expression. Butyrate restores both CT-inhibited NHE2 and NHE3 activities to normal levels but via different mechanisms.

Na-H Exchanger Isoform-2 (NHE2) Mediates Butyrate-dependent Na+ Absorption in Dextran Sulfate Sodium (DSS)-induced Colitis

“…Thus, Hecht et al (37) have concluded that increased NHE2 may not compensate for the fluid losses caused by defective NHE3 function during EPEC infection. In recent studies, however, we have shown that butyrate stimulates NHE2-mediated Na ϩ absorption, whereas NHE3 is inhibited by dibutyryl-cAMP in colon and cholera toxin in ileum (17,26). The present study demonstrates that butyrate stimulates water absorption by activating NHE2-mediated Na ϩ absorption in the DSS-inflamed rat distal colon.…”

“…Thus, an EPEC effector protein EspF inhibits NHE3 activity, whereas EPEC-mediated protein kinase C (PKC) regulates the up-regulation of NHE2 function (39,40). Similarly, cAMP and cholera toxin have also been shown to differentially regulate NHE isoforms in the rat colon and ileum, respectively (17,26). Although NHE2 function is increased in cell lines, diarrhea is present in EPEC-infected patients.…”

“…fluid absorption and secretion) was measured in distal colon loops, as previously described for ileum (26). At laparotomy the distal colon loop (5-6 cm) of the anesthetized rat was constructed between 1 cm distal to the proximal colon and the rectum.…”

“…Apical Membrane Isolation and Western Blot AnalysesWestern blot analyses of protein extracted from apical membranes (APM) of normal and DSS-induced inflamed colonic epithelial cells were performed using NHE2 and NHE3 specific antibodies, as described previously (26). The APM were purified from isolated colonic epithelial cells by the method of Stieger et al (27), as described previously (28).…”

“…Butyrate has been shown to induce NHE3 expression and increase Na ϩ absorption in rat colon, as well as blocking cholera toxin-induced fluid secretion by enhancing apical NHE3 expression in rat ileum (26,35). Thus, studies were designed to identify whether butyrate incubation altered NHE3 function and/or expression in inflamed colon.…”