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Uptake of ⁷⁵Se-Selenite by Brush Border Membrane Vesicles from Chick Duodenum Stimulated by Vitamin D^{1, 2,}

Department of Physiology, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 14853

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.

KEY WORDS: • ⁷⁵Se-selenite • duodenum • chick • brush border membrane vesicles • cholecalciferol • vitamin D deficiency

¹ This work was funded by a grant from the National Institutes of Health, Bethesda, MD (DK04652). H. M. M. was supported by grants from the Finnish Academy of Sciences and the Finnish Cultural Foundation.

² Parts of this work have been presented at the Federation of American Societies for Experimental Biology, 72nd Annual Meeting, May 1–5, 1988, Las Vegas, NV. Abstract #7693.

³ Permanent address: Department of Nutrition, University of Helsinki, 00710 Helsinki, Finland.

⁴ To whom requests for reprints should be sent.

Manuscript received 20 June 1988. Revision accepted 8 September 1988.