Putative basal lateral membrane receptors for 24,25-dihydroxyvitamin D-3 in carp and Atlantic cod enterocytes: Characterization of binding and effects on intracellular calcium regulation

The vitamin D metabolite, 24R,25-dihydroxyvitamin D-3 (24R,25(OH)(2)D-3), w as tested for its ability to specifically bind to basal lateral membranes i solated from intestinal epithelium of Atlantic cod (a seawater fish), carp (a freshwater fish), and chicken. Specific saturable binding was demonstrat ed in membranes from all three species. Membranes from Atlantic cod, carp, and chicken revealed Kd's of 7.3 +/- 0.9, 12.5 +/- 0.9 and 7.8 +/- 0.1 nM, and a B-max for each species estimated to 57.9 +/- 2.9, 195.1 +/- 8.4 and 1 75 +/- 0.8 fmol/mg protein, respectively. Scatchard analyses indicated a co nvex curvature and Hill analyses revealed apparent Hill coefficients of 1.8 4 +/- 0.28, 1.80 +/- 0.29, and 1.78 +/- 0.27 for Atlantic cod, carp and chi cken, suggesting a positive cooperative binding in all three species. Basal lateral membranes from Atlantic cod and carp were used to further characte rize the binding moiety. In competition studies, basal lateral membranes fr om Atlantic cod or carp did not discriminate between 24R,25(OH)2D3 and the 24S,25(OH)(2)D-3 isomer, whereas, 1,25(OH)(2)D-3 and 25(OH)D-3, were less e ffective in competing with [H-3]24R,25(OH)(2)D-3 for binding to basal later al membranes in Atlantic cod and carp. In both the Atlantic cod and carp en terocyte basal lateral membranes, the binding activity could be extracted e qually well with high salt as with detergent, indicating a peripheral membr ane protein rather than an integral membrane binding protein. Finally, isol ated Atlantic cod and carp enterocytes were chosen for analyses of signal t ransduction events mediated by the putative receptor. In both species, 24R, 25(OH)(2)D-3 but not 24S,25(OH)(2)D-3, suppressed Ca2+-uptake by enterocyte s in a dose-dependent manner. Enterocytes from Atlantic cod and carp, accli mated to Ca2+-free media, responded by an intracellular Ca2+-release within seconds after addition of 24R,25(OH)(2)D-3 or 24S,25(OH)(2)D-3. The effect s on intracellular Ca2+-release were dose-dependent for both metabolites. 2 4S,25(OH)(2)D-3 was effective at lower concentrations and triggered a highe r response compared to 24R,2S(OH)(2)D-3. These results suggest that the bin ding molecule(s) for 24R,25(OH)(2)D-3 and 24S,25(OH)(2)D-3 is/are capable o f acting as a receptor, mediating rapid, nongenomic responses in intestinal cells. (C) 2001 Wiley-Liss, Inc.