Maturation of chloride-bicarbonate exchange in rat ileal brush border membrane vesicles

The present studies were designed to examine the ontogeny of the Cl⁻HC03⁻ exchange process in the ileum of suckling (2 wk old), weanling (3 wk old), and adult (6 wk old) rats using well-validated brush border membrane vesicle techniques. The purity of the vesicle preparation was verified by demonstrating that the activity of brush border marker enzyme activity was enriched 30-fold compared to the cell homogenate. Glucose uptake experiments demonstrated an overshoot phenomenon under Na+ gradient conditions, but not in the absence of Na+. Chloride uptake varied inversely with the extravesicular osmolarity, demonstrating that transport was into an os-motically sensitive space. Membrane binding accounted for only 20% of total uptake. An inwardly-directed pH gradient (pHout/pHi„ = 5.2/7.5) produced overshoot Cl⁻ uptake in all age groups. The magnitude of overshoot was greatest in suckling rats and declined with advancing age. Addition of an outwardly-directed HCO3- gradient yielded further stimulation of CI" uptake in suckling and weanling animals but could not be demonstrated in the adult. The majority of Cl⁻ uptake proceeded via an electroneutral exchange process. However, a conductive component was present, as demonstrated by enhancement of uptake when the vesicles were rendered positive inside by a K+ gradient and valino-mycin, as compared with voltage clamp conditions. At 5 mM concentration, 4,4'-diisothiocyanostilbene-2,2'-disul-fonic acid, an inhibitor of anion exchange, inhibited Cl⁻ uptake by 62, 44, and 33%, respectively in suckling, weanling, and adult rats. The initial rate of uptake was linear for 8 s. Kinetic studies at 5 s demonstrated a Km for 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive Cl⁻ uptake of 21.4 ± 10.7,11.5 ± 7.6, and 7.9 ± 8.3 mM, and Vmax of 72.9 ± 14.4, 19.5 ± 3.7 and 4.5 ± 1.1 nmol/mg protein/min in the suckling, weanling, and adult periods respectively (mean ± SEM). These studies demonstrate for the first time the presence of a Cr-HCC3 exchanger in the suckling rat ileum, which declines in activity with advancing age.