Localization and vasopressin regulation of the Na~+-K~+-2Cl~- cotransporter in the distal colonic epithelium

AIM:To investigate whether Na+-K+-2Cl-cotransporter(NKCC2)is expressed in the mouse distal colonic epithelia and whether it is regulated by vasopressin in the colon.METHODS:The mRNA expression of NKCC2 in the mouse colonic mucosa was examined by reverse transcription-polymerase chain reaction.NKCC trafficking in the colon stimulated by 1-D-amino(8-D-arginine)-vasopressin(dDAVP)infusion(10 ng/mouse,intraperitoneal injection)within 15 min,30 min and 1h was investigated by laser confocal scanning microscopy.Total and membrane NKCC2 expression in the colonic mucosa from control and dDAVP-treated mice was detected by Western blotting.Short circuit current method was performed to determine regulation of NKCC2 by vasopressin in the colon.RESULTS:NKCC2 was predominantly located in the apical region of the surface of the distal colonic epithelia;by comparison,a large amount of NKCC1 was distributed in the basolateral membrane of the lower crypt epithelia of the mouse distal colon.Short-term treatment with dDAVP,a V2-type receptor-specific vasopressin analog,induced NKCC2 re-distribution,i.e.,NKCC2traffics to the apical membrane after dDAVP stimulation.In contrast,no obvious NKCC1 membrane translocation was observed.Western blotting results confirmed that membrane NKCC2 had significantly higher abundance in the dDAVP-treated mouse colonic mucosa relative to that in the untreated control,which is consistent with our immunostaining data.Moreover,the short-circuit current method combined with a NKCC2 inhibitor demonstrated that NKCC2 was also activated by serosal vasopressin in isolated distal colonic mucosa.CONCLUSION:Our results provide direct evidence that vasopressin also plays an important role in the colonic epithelia by stimulating NKCC2 trafficking to the apical membrane and inducing NKCC2-mediated ion transport.