Kinetics and stoichiometry of the human red cell Na+/H+ exchanger

Publisher Website
Google Scholar
Abstract
We have investigated the kinetic properties of the human red blood cell Na+/H+ exchanger to provide a tool to study the role of genetic, hormonal and environmental factors in its expression as well as its functional properties in several clinical conditions. The present study reports its stoichiometry and the kinetic effects of internal H+ (H i ) and external Na+ (Na o ) in red blood cells of normal subjects. Red blood cells with different cell Na+ (Na i ) and pH (pH i ) were prepared by nystatin and DIDS treatment of acid-loaded cells. Unidirectional and net Na+ influx were measured by varying pH i (from 5.7 to 7.4), external pH (pH o ), Na i and Na o and by incubating the cells in media containing ouabain, bumetanide and methazolamide. Net Na+ influx (Na i o = 150mm) increased sigmoidally (Hill coefficient 2.5) when pH i fell below 7.0 and the external pH o was 8.0, but increased linearly at pH o 6.0. The net Na+ influx driven by an outward H+ gradient was estimated from the difference of Na+ influx at the two pH o levels (pH o 8 and pH o 6). The H+-driven Na+ influx reached saturation between pH i 5.9 and 6.1. TheV max had a wide interindividual variation (6 to 63 mmol/liter cell · hr, 31.0±3, mean±sem,n=20). TheK m for H i to activate H+-driven Na+ influx was 347±30nm (n=7). Amiloride (1mm) or DMA (20 μm) partially (59±10%) inhibited red cell Na+/H+ exchange. The stoichiometric ratio between H+-driven Na+ influx and Na+-driven H+ efflux was 1∶1. The dependence of Na+ influx from Na o was studied at pH i 6.0, and Na i lower than 2 mmol/liter cell at pH o 6.0 and 8.0. The meanK m for Na o of the H+-gradient-driven Na+ influx was 55±7mm. An increase in Na i from 2 to 20 mmol/liter cell did not change significantly H+-driven net Na+ influx as estimated from the difference between unidirectional22Na influx and efflux. Na+/Na+ exchange was negligible in acid-loaded, DIDS-treated cells. Na+ and H+ efflux from acid-loaded cells were inhibited by amiloride analogs in the absence of external Na+ indicating that they may represent nonspecific effects of these compounds and/or uncoupled transport modes of the Na+/H+ exchanger. It is concluded that human red cell Na+/H+ exchange performs 1∶1 exchange of external Na+ for internal protons, which is partially amiloride sensitive. Its kinetic dependence from internal H+ and external Na+ is similar to other cells, but it displays a larger variability in theV max between individuals.