Evidence for a Na+-H+ exchange across human colonic basolateral plasma membranes purified from organ donor colons

The mechanism(s) of electrolyte transport across the human colonic contralu minal domain is not well understood. Current studies were undertaken to dev elop a technique for the isolation and purification of the human colonic ba solateral membrane vesicles (BLMV) and to examine the presence of a Na+-Hexchange process in these membranes. BLMV were purified from mucosal scrapi ngs of organ donor proximal colons utilizing a Percoll density gradient cen trifugation technique, and Na+ transport was examined utilizing a rapid fil tration, technique. Our data demonstrate that purified basolateral membrane s were enriched 10- to 11-fold in Na+, K+-ATPase activity compared to crude homogenate. Results consistent with the Na+-H+ exchange in BLMV are as fol lows: (1) an outwardly directed H+ gradient stimulated Na-22 uptake; (2) Na -22 uptake was markedly inhibited by EIPA and amiloride; (3) H+-gradient-st imulated Na-22 uptake was not inhibited by bumetanide, SITS, DIDS, acetazol amide, phenamil and benzamil; (4) Na-22 uptake was voltage insensitive; (5) Na-22 uptake demonstrated saturation kinetics; (6) Na-22 uptake was marked ly inhibited by Na+ and Li+ but was unaffected by N-methyl glucamine(+), ch oline(+), and NH4+ Immunoblotting studies demonstrated this Na+-H+ exchange r isoform to be represented by NHE1. In conclusion, a technique has been es tablished for the purification of functional human proximal colonic BLMV, a nd an electroneutral Na+-H+ exchange process has been demonstrated in these membranes.