Na+-H+ antiporter of rat colonic basolateral membrane vesicles

The present experiments were conducted, using acridine orange and 22Na uptake techniques, to demonstrate the presence of an electroneutral Na+-H+ exchange process in rat colonic basolateral membrane vesicles. Results consistent with the existence of a distinct Na+-H+ antiporter in these vesicles include the following: 1) an outwardly directed Na+ gradient stimulated proton influx (Na+in, 100 mM; pHin 7.5/pHout 7.5) and an inwardly directed sodium gradient (Na+out, 5-50 mM; pHin 6.0/pHout 7.5) stimulated proton efflux; 2) sodium-stimulated proton influx was minimally decreased (approximately 10-25%) under voltage clamp conditions (addition of valinomycin in the presence of K+ on both sides of vesicles), indicating that Na+ for H+ exchange in these vesicles could not be explained solely on the basis of a membrane potential; 3) an outwardly directed proton gradient (pHin 5.5/pHout 7.5) stimulated 22Na uptake into these vesicles and a threefold "over-shoot" was observed; 4) 22Na uptake and sodium-stimulated proton efflux were saturable with a Km for Na+ of 5.8 +/- 0.9 and 7.0 +/- 0.3 mM, respectively; 5) amiloride (1 mM) significantly inhibited both sodium-stimulated proton efflux (approximately 69%) and 22Na uptake (approximately 89%), but other transport inhibitors (acetazolamide, 4-acetamido-4'isothiocyanostilbene-2,2'-disulfonic acid, and bumetanide) had no effect on 22Na uptake; 6) N-methylglucamine+ (a nonpermeant cation) did not affect pH gradient-stimulated 22Na uptake, whereas Li+ inhibited Na+ uptake; 7) an inwardly directed Li+ gradient stimulated proton efflux and the Km for Li+ was 12.5 +/- 1.5 mM. These findings establish the existence of an electroneutral Na+-H+ exchange mechanism in rat colonic basolateral membrane vesicles.