Putative basal lateral membrane receptors for 24,25‐dihydroxyvitamin D₃ in carp and Atlantic cod enterocytes: Characterization of binding and effects on intracellular calcium regulation

Abstract

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.

Biochemical characterization and purification of a binding protein for 24,25-dihydroxyvitamin D3 from chick intestine

“…However, one difference between the lysosomal and basal lateral binding proteins is in analyses of cooperativity. In basal lateral membranes, binding exhibits positive cooperativity (Larsson et al 1999(Larsson et al , 2001, while in lysosomal fractions binding exhibits negative cooperativity. One explanation for these observations might be that the binding proteins in the two subcellular compartments have different naturally occurring ligands, resulting in differences in cooperative binding.…”

24, 25-Dihydroxycholecalciferol but not 25-hydroxycholecalciferol suppresses apolipoprotein A-I gene expression

“…However, studies using 24-hydroxylase knockout mice have shown that chondrogenesis requires this vitamin D metabolite (St-Arnaud et al, 1996;St-Arnaud, 1999). In addition, putative basal-lateral membrane receptors for 24, 25-(OH) 2 D 3 have been identified in carp and Atlantic cod enterocytes (Larsson et al, 2001), and experiments showing that 24, 25-(OH) 2 D 3 is involved in regulating endochondral ossification (Dickson and Maher, 1985;Lidor et al, 1987;Wientroub et al, 1987;Miyahara et al, 1994), have led to the belief that 24-hydroxylated vitamin D metabolites can have important biologic effects. Since previously published studies have shown that vitamin D receptor (VDR) activation can alter apo A-I expression (Wehmeier et al, 2005(Wehmeier et al, , 2008, the effect of 25-OHD 3 and its two major metabolites, 1, 25-(OH) 2 D 3 and 24, 25-(OH) 2 D 3 , were examined.…”

A short practical approach to 24R,25-dihydroxyvitamin D3

“…1) in the liver and a second hydroxylation of the resulting 25-hydroxyvitamin D 3 [25(OH)D 3 ] either at C1 or C24 in the kidney to produce 1␣,25-dihydroxyvitamin D 3 [1␣,25(OH) 2 D 3 ] or 24R,25-dihydroxyvitamin D 3 [1, 24R,25(OH) 2 D 3 ] [1][2][3][4][5]. While 1␣,25(OH) 2 D 3 is considered the hormonally active form of vitamin D 3 responsible for the regulation of gene transcription in over 30 target organs, only a few biological actions have also been attributed to 24R,25(OH) 2 D 3 [6][7][8][9], namely activation of extracellular signal-related kinase phosphorylation [10], regulation of bone fracture healing [6,10], inhibition of rapid actions of 1␣,25(OH) 2 D 3 on stimulation of calcium transport in perfused duodena, as well as activation of protein kinases C and A [11], mediation of rapid non-genomic responses in intestinal cells [12,13], regulation of cartilage and bone via autocrine mechanisms [14], and inhibition of colon carcinogenesis [15]. 24R,25-Dihydroxyvitamin D 3 (1) are also intermediates on a catabolic pathway finally leading to excretion of calcitroic acid [4].…”