Cellular localization of NKCC2 and its possible role in the Cl− absorption in the rat and human distal colonic epithelia

Recently, we demonstrated the expression of NKCC2, an absorptive isoform of NKCC specifically expressed in the kidney, in the rat gastrointestinal tract including the distal colonic mucosa. This study aims to investigate its localization in colonic epithelia and possible role in the colonic ion transport.Reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry were used to investigate the expression and localization of NKCC2. The role of NKCC2 on the colonic ion transport was examined by mean of short-circuit current (ISC) monitoring. The results indicated that NKCC2 was expressed in the apical region of the epithelia in rat distal colon and human sigmoid colon. NKCC1, which is a secretive NKCC isoform, was localized predominantly in the basolateral membrane, which has been well documented. Serosal (basolateral) administration of bumetanide, an inhibitor of both NKCC1 and NKCC2, inhibited serosal forskolin-induced ISC increase by 66% but enhanced the luminal (apical) forskolin-induced ISC response by 63%. Furthermore, the blocking of epithelial Na+ channels by apical addition of amiloride (10 μmol/L), K+ channels by tetraethylammoniumion (TEA) (5 mmol/L), or glibenclimide (0.1 mmol/L) did not affect apical forskolin-induced ISC increase, excluding the involvement of cations, Na+ and K+, in the ISC response. The luminal forskolin-induced ISC increase was enhanced markedly by the apical pretreatment with bumetanide or the reduction of apical Cl− concentration by 114% and 198%, respectively, which were inhibited by apical addition of glibenclimide (1 mmol/L) by more than 60%. This finding suggests the involvement of an anion. Furthermore, the removal of basolateral HCO3− reduced apical forskolin-induced ISC by more than 75% indicated that the apical forskolin-induced ISC increase in rat distal colon was mediated by Cl− absorption and HCO3− secretion. In conclusion, NKCC2 is expressed widely in the colonic epithelium in rat distal colon and human sigmoid colon, especially in the apical membrane. It involves the process of colonic Cl− absorption coupled with HCO3− secretion.