Evidence for a Na+–H+ Exchange Across Human Colonic Basolateral Plasma Membranes Purified from Organ Donor Colons

Abstract

The mechanism(s) of electrolyte transport across the human colonic contraluminal domain is not well understood. Current studies were undertaken to develop a technique for the isolation and purification of the human colonic basolateral membrane vesicles (BLMV) and to examine the presence of a Na⁺–H⁺ exchange process in these membranes. BLMV were purified from mucosal scrapings of organ donor proximal colons utilizing a Percoll density gradient centrifugation technique, and Na⁺ transport was examined utilizing a rapid filtration, technique. Our data demonstrate that purified basolateral membranes 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 follows: (1) an outwardly directed H⁺ gradient stimulated ²²Na uptake; (2) ²²Na uptake was markedly inhibited by EIPA and amiloride; (3) H⁺-gradient-stimulated ²²Na uptake was not inhibited by bumetanide, SITS, DIDS, acetazolamide, phenamil and benzamil; (4) ²²Na uptake was voltage insensitive; (5) ²²Na uptake demonstrated saturation kinetics; (6) ²²Na uptake was markedly inhibited by Na⁺ and Li⁺ but was unaffected by N-methyl glucamine⁺, choline⁺, and NH₄ ⁺. Immunoblotting studies demonstrated this Na⁺–H⁺ exchanger isoform to be represented by NHE1. In conclusion, a technique has been established for the purification of functional human proximal colonic BLMV, and an electroneutral Na⁺–H⁺ exchange process has been demonstrated in these membranes.