Determination of vitamins D,A, and E in sera and vitamin D in milk from captive and free-ranging polar bears (Ursus maritimus), and 7-dehydrocholesterol levels in skin from captive polar bears

The difference between serum levels from 36 captive and 56 free-ranging polar bears (Ursus maritimus) for 25-hydroxyvitamin D (25-OH-D) was found not to be significant (mean ± SD = 348 ± 215 nmol/L [captive], 360 ± 135 nmol/L [free-ranging], t = 0.30, df = 52.8, P = 0.76), whereas the difference for retinol and α-tocopherol was significant (retinol, 1.37 ± 0.67 μmol/L [captive] 1.89 ± 0.63 μmol/L [free-ranging], t = 3.88, df = 72.4, P <0.001, α-tocopherol, 18.56 ± 18.56 μmol/L [captive], 48.76 ± 13.92 μmol/L [free-ranging], t = 7.85, df = 61.9, P < 0.001). Due to the high fat content in the polar bear diet, seal blubber may be the source of these fat-soluble vitamins. Six skin biopsies were analyzed from captive polar bears at the Denver Zoological Gardens for 7-dehydrocholesterol levels and found to contain 0.11 ± 0.03 nmol/cm². This finding also helps to support the contention that the source of vitamin D for polar bears may be ingestion and not cutaneous production. Vitamin D content in the milk from one captive sow in the den (0.14 nmol/g) and 10 free-ranging sows with cubs of the year out on the ice pack (0.0042 ± 0.0073 nmol/g) were also evaluated. It would be helpful to evaluate additional milk samples from denning and non-denning sows with cubs to see whether vitamin D content varies according to the stage of lactation. Zoo Biol 17:285–293, 1998. © 1998 Wiley- Liss, Inc.     

Effects of the vitamin D3 analog 1α,25-dihydroxyvitamin D3-3β-bromoacetate on rat osteosarcoma cells: Comparison with 1α,25-dihydroxyvitamin D3

The actions of the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D₃ [1α,25-(OH)₂D₃], are mediated by both genomic and nongenomic mechanisms. Several vitamin D synthetic analogs have been developed in order to identify and characterize the site(s) of action of 1α,25-(OH)₂D₃ in many cell types including osteoblastic cells. We have compared the effects of 1α,25-(OH)₂D₃ and a novel 1α,25-(OH)₂D₃ bromoester analog (1,25-(OH)₂-BE) that covalently binds to vitamin D receptors. Rat osteosarcoma cells that possess (ROS 17/2.8) or lack (ROS 24/1) the classic intracellular vitamin D receptor were studied to investigate genomic and nongenomic actions. In ROS 17/2.8 cells plated at low density, the two vitamin D compounds (1 × 10⁻⁸ M) caused increased cell proliferation, as assessed by DNA synthesis and total cell counts. Northern blot analysis revealed that the mitogenic effect of both agents was accompanied by an increase in steady-state osteocalcin mRNA levels, but neither agent altered alkaline phosphatase mRNA levels in ROS 17/2.8 cells. ROS 17/2.8 cells responded to 1,25-(OH)₂-BE but not the natural ligand with a significant increase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatment. Treatment of ROS 17/2.8 cells with the bromoester analog also resulted in a significant decrease in alkaline phosphatase-specific activity. To compare the nongenomic effects of 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE, intracellular calcium was measured in ROS 24/1 cells loaded with the fluorescent calcium indicator Quin 2. At 2 × 10⁻⁸ M, both 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE increased intracellular calcium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH)₂-BE are similar to those of 1α,25-(OH)₂D₃, and since 1,25-(OH)₂-BE has more potent effects on osteoblast function than the naturally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of cellular vitamin D receptors. © 1996 Wiley-Liss, Inc.     

Evidence that annexin II is not a putative membrane receptor for 1alpha,25(OH)2-vitamin D3

The seco-steroid hormone 1alpha,25(OH)(2)-vitamin D(3) (1,25-D(3)) is known to generate biological responses via both genomic and non-genomic rapid signal transduction pathways. The calcium regulated annexin II/p11 heterotetramer (AII(2)/p11(2)] was proposed by Baran and co-authors to be the membrane receptor responsible for mediating non-genomic, rapid actions of 1,25-D(3), based on ligand affinity labeling, competition, and saturation analysis experiments. Given the cytosolic presence of both the monomeric and heterotetrameric form of AII and their functional regulation by intracellular calcium concentrations, which are known to be affected by 1,25-D(3) rapid, non-genomic activities, we investigated in vitro the affinity of [(3)H]1,25-D(3) for the AII monomer and AII(2)/p11(2) in the absence and presence of calcium using saturation analysis and gel-filtration chromatography. Using two different techniques for separating bound from free ligand (perchlorate and hydroxylapatite (HAP)) over a series of 30 experiments, no evidence for specific binding of [(3)H]1,25-D(3) was obtained with or without the presence of 700 nM exogenous calcium, using either the AII monomer or AII(2)/p11(2). However saturable binding of [(3)H]1,25-D(3) to the lipid raft/caveolae enriched rat intestinal fraction was consistently observed (K(d) = 3.0 nM; B(max) = 45 fmols/mg total protein). AII was detected in lipid raft/caveolae enriched fractions from rat and mouse intestine and ROS 17/2.8 and NB4 cells by Western blot, but incubation in the presence of exogenous calcium did not ablate 1,25-D(3) binding as reported by Baran et al. Our results suggest that AII does not bind 1,25-D(3) in a physiologically relevant manner; however, recent studies linking AII(2)/p11(2) phosphorylation to vesicle fusion and its calcium regulated localization may make AII a possible down-stream substrate for 1,25-D(3) induced rapid cellular effects.     

Binding of 1α,25‐dihydroxyvitamin D3 to annexin II: Effect of vitamin D metabolites and calcium

We have recently reported that annexin II serves as a membrane receptor for 1α,25‐(OH)₂D₃ and mediates the rapid effect of the hormone on intracellular calcium. The purpose of these studies was to characterize the binding of the hormone to annexin II, determine the specificity of binding, and assess the effect of calcium on binding. The binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to purified annexin II was inhibited by 1α,25‐(OH)₂D₃ in a concentration‐dependent manner. Binding of the radiolabeled ligand to annexin II was markedly diminished by 1α,25‐(OH)₂D₃ at 24 μM, 18 μM, and 12 μM and blunted by 6 μM and 3 μM. At a concentration of 12 μM, 1β,25‐(OH)₂D₃ also diminished the binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to annexin II, but cholecalciferol, 25‐(OH)D₃, and 24,25‐(OH)₂D₃ did not. Saturation analyses of the binding of [³H]‐1α,25‐(OH)₂D₃ to purified annexin II showed a K_D of 5.5 × 10⁻⁹ M, whereas [³H]‐1β,25‐(OH)₂D₃ exhibited a K_D of 6.0 × 10⁻⁹ M. Calcium, which binds to the carboxy terminal domain of annexin II, had a concentration‐dependent effect on [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate binding to annexin II, with 600 nM calcium being able to inhibit binding of the radiolabeled analog. The inhibitory effect of calcium was prevented by EDTA. Homocysteine, which binds to the amino terminal domain of annexin II, had no effect on the binding of the bromoacetate analog to the protein. The data indicate that 1α,25‐(OH)₂D₃ binding to annexin II is specific and suggest that the binding site may be located on the carboxy terminal domain of the protein. The ability of 1β,25‐(OH)₂D₃ to inhibit the binding of [¹⁴C]‐1α,25(OH)₂D₃ bromoacetate to annexin II provides a biochemical explanation for the ability of the 1β‐epimer to inhibit the rapid actions of the hormone in vitro. J. Cell. Biochem. 80:259–265, 2000. © 2000 Wiley‐Liss, Inc.