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)), was tested for its ability to specifically bind to basal lateral membranes isolated from intestinal epithelium of Atlantic cod (a seawater fish), carp (a freshwater fish), and chicken. Specific saturable binding was demonstrated in membranes from all three species. Membranes from Atlantic cod, carp, and chicken revealed K(d)'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 175 +/- 0.8 fmol/mg protein, respectively. Scatchard analyses indicated a convex curvature and Hill analyses revealed apparent Hill coefficients of 1.84 +/- 0.28, 1.80 +/- 0.29, and 1.78 +/- 0.27 for Atlantic cod, carp and chicken, suggesting a positive cooperative binding in all three species. Basal lateral membranes from Atlantic cod and carp were used to further characterize the binding moiety. In competition studies, basal lateral membranes from Atlantic cod or carp did not discriminate between 24R,25(OH)(2)D(3) and the 24S,25(OH)(2)D(3) isomer, whereas, 1,25(OH)(2)D(3) and 25(OH)D(3), were less effective in competing with [(3)H]24R,25(OH)(2)D(3) for binding to basal lateral membranes in Atlantic cod and carp. In both the Atlantic cod and carp enterocyte basal lateral membranes, the binding activity could be extracted equally well with high salt as with detergent, indicating a peripheral membrane protein rather than an integral membrane binding protein. Finally, isolated Atlantic cod and carp enterocytes were chosen for analyses of signal transduction events mediated by the putative receptor. In both species, 24R,25(OH)(2)D(3) but not 24S,25(OH)(2)D(3), suppressed CA(2+)-uptake by enterocytes in a dose-dependent manner. Enterocytes from Atlantic cod and carp, acclimated to CA(2+)-free media, responded by an intracellular CA(2+)-release within seconds after addition of 24R,25(OH)(2)D(3) or 24S,25(OH)(2)D(3). The effects on intracellular CA(2+)-release were dose-dependent for both metabolites. 24S,25(OH)(2)D(3) was effective at lower concentrations and triggered a higher response compared to 24R,25(OH)(2)D(3). These results suggest that the binding molecule(s) for 24R,25(OH)(2)D(3) and 24S,25(OH)(2)D(3) is/are capable of acting as a receptor, mediating rapid, non-genomic responses in intestinal cells.