Uptake of 75Se-Selenite by Brush Border Membrane Vesicles from Chick Duodenum Stimulated by Vitamin D

Abstract

Brush border membrane vesicles were isolated from mucosal homogenates of duodena from normal, rachitic and vitamin D-treated rachitic chicks using a discontinuous sucrose gradient, and further purified by glycerol gradient centrifugation. In vitro uptake of ⁷⁵Se-selenite by purified brush border membrane vesicles was studied using a rapid filtration technique. The time course of ⁷⁵Se uptake was non-linear; rapid initial binding was followed by a gradual decrease in the rate of uptake until an equilibrium value was reached at 60–120 min. The initial binding at 36 s was not affected by selenite concentration in the incubation buffer, while the fractional rate of uptake between the 36 s and 2 min time periods was clearly lower with 1 mM Se than with 4–100 µM Se. ⁷⁵Se uptake did not show any dependency on the external Na-gradient, nor could it be inhibited by other anions (arsenate, phosphate). Treatment of rachitic chicks either with cholecalciferol (500 IU, 72 h) or with 1,25(OH)₂-cholecalciferol (0.5 µg given 16 h prior to isolation of the vesicles) significantly enhanced ⁷⁵Se uptake. A threefold excess of mannitol in the outside buffer reduced ⁷⁵Se uptake by vesicles from vitamin D-deficient and D-treated chicks 60% and 35% respectively, but had no effect on vesicles from vitamin D-treated chicks preloaded with ⁷⁵Se. Neither saponin treatment nor excess cold selenite could release the label from the vesicles preloaded with ⁷⁵Se. These data are compatible with the hypothesis that selenite easily crosses the brush border membrane into the intravesicular space and, once inside, is tightly bound by the membrane. Vitamin D affects this process possibly by increasing the number or the availability of sites binding selenite in the vesicles.