1,25-Dihydroxycholecalciferol, when present at and above 10 nM in an organ-culture system of embryonic chick jejunum, approximately doubled the rate of Na+-gradient-driven D-glucose uptake by brush-border membrane vesicles, but had no effect on Na+-independent D-glucose transfer. The sterol also had no effect on Na+ influx along an outside/inside Na+ gradient ("a' ], = 100 mM; [Na+Ii = 0 mM). This renders it unlikely that in embryonic intestine, calcitriol raises Na+-dependent D-glUCOSe transport through changes in the electrochemical Natracer exchange, measured under voltage-clamp condition at Na+/D-glucose equilibrium, revealed that addition of calcitriol to the culture medium approximately doubled the activity of the Na +/D-glucose transporter in the brush-border membrane. This was also reflected by an corresponding increase in the maximal velocity of the transfer process. Increased [3H]phlorizin binding after calcitriol treatment suggests that the steroid hormone activates Na+/D-glucose transport through increasing the number of carrier molecules in the brush-border membrane.10 nM triiodothyronine, which by itself has no effect on Na+-dependent D-glucose transport, potentiated the effect of 1,25-dihydroxycholecalciferol such that in the presence of both hormones, Na'/D-glucose-carrier activity was increased fourfold above control levels.Triiodothyronine has been shown to facilitate the expression of genomic effects of 1,25-dihydroxycholecalciferol (calcitriol) on calcium, Na f -dependent inorganic phosphate and D-glucose transport in organ-cultured embryonic chick jejunum (Cross et al., 1986;Cross and Peterlik, 1988;Cross and Peterlik, 1991). We had also demonstrated previously that calcitriol can increase Na+-dependent uptake of D-glucose from the intestinal lumen in two ways: by increasing the maximal velocity of the Na+/D-glucose transport system (Peterlik et al., 1981); through appropriate actions on pathways of Na + transfer across the brush-border membrane of enterocytes (Fuchs et al., 1985). In the present study, we utilized brush-border membrane vesicles (BBMV) isolated from organ-cultured embryonic chick jejuna to further characterize the effect of calcitriol on Na+ /D-glucose transport mechanisms and to gain insight into the mode of triiodothyronine interaction.
MATERIALS AND METHODS
Organ culture of embryonic chick jejunumFertilized eggs were obtained from a local poultry farm and kept in an incubator with forced ventilation at 70% rela- modified medium (Corradino, 1973). Calcitriol or triiodothyronine were added to cultures in ethanol so that the final ethanol concentration did not exceed 0.15%. Ethanol only was added to control cultures. Culture time was 48 h. D-Glucose transport in cultured jejuna was evaluated by tissue accumulation of the radiolabelled non-metabolizable analogue, a-methyl-D-glucoside (0.5 pCi/ml incubation buffer), as described previously (Peterlik et al., 1981 ;Cross and Peterlik, 1982).
Preparation of BBMVA modification of a divalent-cation-precipitation method (Schmitz et ...