Studies on the mode of action of calciferol. XVIII. Evidence for a specific high affinity binding protein for 1,25 dihydroxyvitamin D3 in chick kidney and pancreas.

Cytosol fractions prepared from rachitic chick kidney and pancreas were analyzed for binding of vitamin D₃ metabolites by sucrose density gradient centrifugation. Both cytosol fractions were found to contain a 3.6S macromolecule which specifically binds 1,25-dihydroxy[³H] vitamin D₃ and in addition a 5 to 6S macromolecule which binds 25-hydroxy[³H]vitamin D₃. Sucrose gradient analysis of a KCl extract prepared from kidney or pancreas chromatin resulted in a peak (3.6S) of bound 1,25-dihydroxyvitamin D₃ which could not be distinguished from the cytoplasmic binding component. The interaction of 1,25-dihydroxy[³H]vitamin D₃ with the cytoplasmic binding component of both tissues occurred at low concentrations of hormone with high affinity.     

Intestinal cytosol binders of 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3

The binding of 25-hydroxy-[26,27-³H]vitamin D₃ and 1,25-dihydroxy-[26,27-³H]vitamin D₃ to the cytosol of intestinal mucosa of chicks and rats has been studied by sucrose gradient analysis. The cytosol from chick mucosa showed variable binding of 1,25-dihydroxyvitamin D₃ to a 3.0S macromolecule which has high affinity and low capacity for this metabolite. However, when the mucosa was washed extensively before homogenization, a 3.7S macromolecule was consistently observed which showed considerable specificity and affinity for 1,25-dihydroxyvitamin D₃. Although 3.7S binders for 1,25-dihydroxyvitamin D₃ could also be located in other organs, competition experiments with excess nonradioactive 1,25-dihydroxyvitamin D₃ suggested that they were not identical to the 3.7S macromolecule from intestinal mucosal cytosol. As the 3.7S macromolecule was allowed to stand at 4 °C with bound 1,25-dihydroxy-[³H]vitamin D₃, the 1,25-dihydroxy-[³H]vitamin D₃ became increasingly resistant to displacement by non-radioactive 1,25-dihydroxyvitamin D₃. The 1,25-dihydroxy-[³H]vitamin D₃ remained unchanged and easily extractable with lipid solvents through this change, making unlikely the establishment of a covalent bond. Unlike the chick, mucosa from rats yielded cytosol in which no specific binding of 1,25-dihydroxy-[³H]vitamin D₃ was detected. Instead, a 5-6S macromolecule which binds both 1,25-dihydroxyvitamin D₃ and 25-hydroxyvitamin D₃ was found. This protein which was also found in chick mucosa shows preferential binding for 25-hydroxyvitamin D₃. It could be removed by washing the mucosa with buffer prior to homogenization which suggests that it may not be a cytosolic protein. Although the 3.7S protein from chick mucosa has properties consistent with its possible role as a receptor, the 5-6S macromolecule does not appear to have “receptor”-like properties.     

Vitamin D in lactation. I. The localization, specific binding and biological effect of 1,25-dihydroxyvitamin D3 in mammary tissue of lactating rats

Radiolabelled 1, 25-dihydroxyvitamin D₃ was found to accumulate in mammary tissue of lactating rats, to bind to a specific high affinity binding component in mammary cytosol and to associate with chromatin in vivo. 1,25-dihydroxyvitamin D₃ was also shown to have a direct effect on milk calcium concentration in vivo. The cytosolic binding protein was found to sediment at 3.2S on sucrose gradients and to have a dissociation constant of 2.5 × 10^?10 M. Localization of 1, 25-dihydroxyvitamin D₃ in mammary gland and other tissues of lactating rats was compared. These results provide evidence that the lactating mammary gland is a target tissue for 1,25-dihydroxyvitamin D₃.     

An equilibrium and kinetic study of 1,25-dihydroxyvitamin D3 binding to chicken intestinal cytosol employing high specific activity 1,25-dehydroxy[3H-26, 27] vitamin D3

A reexamination of the equilibrium and the kinetics of 1,25-dihydroxy vitamin D₃ binding with its receptor in chick intestinal cytosol was performed because of the recent availability in our laboratory of high specific activity 1,25-dihydroxy[${}^3\text{H-26,27}$]vitamin D₃ (160 Ci/mmol). Under saturating conditions at 25 °C, Scatchard analysis revealed an equilibrium dissociation constant (K_d) of 7.1 × 10^?11m which is several fold lower than previously reported for this binding reaction. Furthermore, an estimate of 1.8 × 10³ receptor sites per cell was obtained from the intercept of the line with the abscissa of the Scatchard plot. From a kinetic analysis of 1,25-dihydroxy vitamin D₃ binding with chick intestinal cytosol, association and dissociation rate constants were determined. Values that were obtained at 25 °C for these processes were 9.5 × 10⁸m^? min^? and 7.1 × 10^?3 min^?, respectively. Although these studies, such as for other steroid hormones, were carried out using a crude native cytosol preparation, we have been able to demonstrate unequivocally through the use of high specific activity 1,25-dihydroxy[${}^3\text{H-26,27}$] vitamin D₃ a truly high affinity binding site.     

25,26-dihydroxyvitamin D3 is not a major intermediate in 25-hydroxyvitamin D3-26,23-lactone formation

Structural similarities between 25S,26-dihydroxyvitamin D₃ and 25-hydroxyvitamin D₃-26,23-lactone and their concomitant multifold increase in the plasma of animals treated with pharmacological doses of vitamin D₃ suggest a precursor-product relationship. However, a single dose of 25S,26-[³H]dihydroxyvitamin D₃ given to rats treated chronically with pharmacological amounts of vitamin D₃ did not result in detectable plasma 25-[³H]hydroxyvitamin D₃-26,23-lactone. Multiple doses of synthetic 25S,26-dihydroxyvitamin D₃ given to vitamin D₃-deficient rats treated chronically with pharmacological amounts of vitamin D₂ also did not result in detectable plasma 25-hydroxyvitamin D₃-26,23-lactone. Furthermore, homogenates prepared from vitamin d-deficient chickens, dosed with 1,25-dihydroxyvitamin D₃, converted 25-[³H]hydroxyvitamin D₃ to 25-[³H]hydroxyvitamin D₃-26,23-lactone. But these same homogenates did not convert 25S,26-[³H]dihydroxyvitamin D₃ to 25-[³H]hydroxyvitamin D₃-26,23-lactone. These data indicate that 25,26-dihydroxyvitamin D₃ is not an intermediate in 25-hydroxyvitamin D₃26, 23-lactone formation.     

Metabolism and binding properties of 24,24-difluoro-25-hydroxyvitamin D3

To evaluate possible functional roles for 24,25-dihydroxyvitamin D₃, 24,24-difluoro-25-hydroxyvitamin D₃ has been synthesized and shown to be equally as active as 25-hydroxyvitamin D₃ in all known functions of vitamin D. The use of the difluoro compound for this purpose is based on the assumption that the C-F bonds are stable in vivo and that the fluorine atom does not act as hydroxyl in biological systems. No 24,25-dihydroxyvitamin D₃ was detected in the serum obtained from vitamin D-deficient rats that had been given 24,24-difluoro-25-hydroxyvitamin D₃, while large amounts were found when 25-hydroxyvitamin D₃ was given. Incubation of the 24,24-difluoro compound with kidney homogenate prepared from vitamin D-replete chickens failed to produce 24,25-dihydroxyvitamin D₃, while the same preparations produced large amounts of 24,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃. Kidney homogenate prepared from vitamin D-deficient chickens produced 24,24-difluoro-1,25-dihydroxyvitamin D₃ from 24,24-difluoro-25-hydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃. In binding to the plasma transport protein for vitamin D compounds, 24,24-difluoro-25-hydroxyvitamin D₃ is less active than 25-hydroxyvitamin D₃ and 24R,25-dihydroxyvitamin D₃. In binding to the chick intestinal cytosol receptor, 24,24-difluoro-25-hydroxyvitamin D₃ is more active than 25-hydroxyvitamin D₃ which is itself more active than 24R,25-dihydroxyvitamin D₃. The 24,24-difluoro-1,25-dihydroxyvitamin D₃ is equal to 1,25-dihydroxyvitamin D₃, and both are 10 times more active than 1,24R,25-trihydroxyvitamin D₃ in this system. These results provide strong evidence that the C-24 carbon of 24,24-difluoro-25-hydroxyvitamin D₃ cannot be hydroxylated in vivo, and, further, the 24-F substitution acts similar to H and not to OH in discriminating binding systems for vitamin D compounds.     

Parathyroid hormone is not involved in 1,25-dihydroxyvitamin D regulation of 25-hydroxyvitamin D metabolism in vivo

1,25-Dihydroxyvitamin D₃ administration to vitamin D-deficient rats suppresses accumulation of 1,25-dihydroxy-[3α-³H]vitamin D₃ and stimulates accumulation of 24,25-dihydroxy-[3α-³3H]vitamin D₃ from 25-hydroxy-[3α-³H]vitamin D₃ equally well in the presence and absence of parathyroid glands. These results demonstrate that this regulatory action is not mediated by the parathyroid glands and support conclusions from $\text{in}\text{vitro}$ studies that this represents a direct action of 1,25-dihydroxyvitamin D₃.     

The chick intestinal cytosol binding protein for 1,25-dihydroxyvitamin D3: A study of analog binding

The structural features of 1,25-dihydroxyvitamin D₃ that permit its high affinity binding to a 3.7 S protein from chick intestinal cytosol were determined in a series of binding and competition experiments analyzed by sucrose density gradient centrifugation. Optimal binding to the 3.7 S protein was achieved when both 1α- and 25-hydroxyls were present in the vitamin D₃ molecule. Modification of the side chain by the introduction of a methyl on C-24 and a double bond on C-22,23 (1,25-dihydroxyvitamin D₂) did not alter the binding of 1,25-dihydroxyvitamin D₃, but significantly diminished the binding of 25-hydroxyvitamin D₃. However, introduction of a hydroxyl on C-24 decreased the ability of either 1,25-dihydroxyvitamin D₃ or 25-hydroxyvitamin D₃ to compete, especially when the 24-hydroxyl was in the S configuration. These results reveal that the 3.7 S protein requires specific ligand structural features for binding and suggest that metabolite discrimination by the chick intestinal receptor system is likely located in the 3.7 S cytosol protein.     

Localization of 1,25-dihydroxyvitamin D3 in intestinal nuclei in vivo

Autoradiography of frozen sections of intestinal tissue taken from rachitic chickens given a single intravenous dose of 1,25-dihydroxy[23,24³H]vitamin D₃ (650 pmol, 78 Ci/ mmol) has been carried out. Specific localization of label in the nuclei of intestinal villi and crypt cells could be demonstrated at 2.5 to 6 h postinjection. In contrast, no concentration or localization of radioactivity could be detected in intestinal muscle, liver, and skeletal muscle. These results strongly support the concept that the function of 1,25-dihydroxyvitamin D₃ in intestine is mediated by a nuclear mechanism.     

Intestinal 1,25-dihydroxyvitamin D3 binding protein: Specificity of binding

The binding of metabolites of vitamin D and their analogs to the 3.7S chick intestinal cytosol receptor protein has been specifically studied by competitive binding techniques and polyethylene glycol precipitation of the complex. The structural requirements for the interaction between the vitamin D molecule and the receptor could be assessed without the nuclear chromatin binding step. These measurements have shown that 1,25-dihydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₂ are equally competitive and are the most active. Of the structural features of the compounds, the 1α-hydroxyl is most important followed by the 25-hydroxyl and the 3β-hydroxyl. The addition of a second hydroxyl near carbon 25 markedly reduces binding whether on the 26 carbon or the 24 carbon. A hydroxyl on C-24 could substitute to some degree for the 25-hydroxyl inasmuch as 24-hydroxyvitamin D₃ was much more effective than vitamin D₃ but less effective than 25-hydroxyvitamin D₃. In general the patterns of binding affinities correlated well with the biological activity of the various analogs strongly supporting a physiological role for the 1,25-dihydroxyvitamin D₃ binding protein. It also suggests that of the two-step receptor mechanism, the structural specificity is located in the initial interaction of the 1,25-dihydroxyvitamin D₃ and the cytosol receptor.     

Calcitroic acid: biological activity and tissue distribution studies

Calcitroic acid was recently identified as a major metabolite of 1,25-dihydroxyvitamin D₃ (Esvelt, Schnoes, and DeLuca, Biochemistry 18, 3977, 1979). The metabolite was found to have little, although significant, activity in healing rickets, and causing bone mineral mobilization but elicited no significant elevation in intestinal calcium transport. The compound showed little affinity for either the serum 25-hydroxyvitamin D binding protein or the intestinal cytosol receptor for 1,25-dihydroxyvitamin D₃. Various tissues of the rat were examined for the presence of calcitroic acid following a 120-ng dose of 1,25-dihydroxy-[3α-³H]vitamin D₃. The metabolite was detected in liver, intestinal mucosa, kidneys, and blood with livers and mucosa containing the highest concentrations. In each of these tissues the calcitroic acid content increased during the period between 4 and 12 h after the dose. The presence of calcitroic acid in femurs was indicated but could not be confirmed. Bile duct cannulation reduced but did not abolish the intestinal calcitroic acid content. In addition to calcitroic acid, other polar metabolites of 1,25-dihydroxyvitamin D₃ were detected in these experiments.     

An estrogen-stimulated 1,25-dihydroxyvitamin D3 receptor in rat uterus

Sucrose density gradient analysis was utilized to determine whether 1,25-dihydroxyvitamin D₃ receptors are present in the rat uterus. A distinct 3.6S [³H]1,25-dihydroxyvitamin D₃ binding component was observed in chromatin extracts of estrogen-primed, ovariectomized rat uteri. Binding to this putative 1,25-dihydroxyvitamin D₃ receptor was inhibited by excess 1,25-dihydroxyvitamin D₃, but not by 25-hydroxyvitamin D₃, estradiol-17β, promegestone, or cortisol. Low levels of the receptor seemed to be present in the unprimed uterus. Estrogen injection significantly increased the number of 1,25-dihydroxyvitamin D₃ receptors and progesterone co-administration reduced, but did not abolish, this effect.     

Stabilization of 1,25-dihydroxyvitamin D-3 receptor in the human leukemia cell line, HL-60, with diisopropylfluorophosphate

We have investigated the reason for the lack of specific 1,25-dihydroxyvitamin D-3 binding activity in extracts of ATCC HL-60 cells. Although intact ATCC HL-60 cells specifically and saturably take up 1,25-dihydroxy[3H]vitamin D-3, whole cell extracts have little or no specific binding of 1,25-dihydroxyvitamin D-3. The absence of specific binding can now be explained by the action of a serine proteinase in these cells. When diisopropylfluorophosphate (DFP), a potent inhibitor of serine proteinase, is added to the buffer used for extraction, specific binding of 1,25-dihydroxy[3H]vitamin D-3 in the extract is observed. The loss of specific binding could not be prevented by hydrolyzed DFP or other serine proteinase inhibitors, such as phenylmethylsulfonylfluoride, benzamidine and aprotinin. The proteolytic activity from ATCC cells also destroyed specific 1,25-dihydroxy[3H]vitamin D-3 binding in high-salt extracts from pig intestinal nuclei or from another HL-60 cell line (LG HL-60 cells). However, the proteinase did not affect the levels of the specific binding in these preparations if the receptor was occupied with 1,25-dihydroxy[3H]vitamin D-3 prior to exposure to the proteinase. The binding and sedimentation characteristics of the receptors from various sources were not changed by the presence of DFP. The Kd of the receptor in ATCC HL-60 cells is 1.2.10(-10) M, which is identical to that in the LG HL-60 cells. The 1,25-dihydroxy[3H]vitamin D-3 receptor complex from the ATCC cells sediments as a single 3.5 S component and elutes from DNA-Sephadex column in two peaks at 0.09 and 0.15 M KCl. The material eluting at 0.15 M KCl has the same DNA-binding activity as preparations from pig intestine or LG HL-60 cells. Immunoprecipitation studies demonstrated that monoclonal antibodies to the pig receptor, IVG8C11, quantitatively precipitate the 1,25-dihydroxy[3H]vitamin D-3-binding activity from ATCC HL-60 cells as well as that from LG HL-60 cells or pig intestinal nuclei. Therefore, the previous failure to demonstrate the 1,25-dihydroxyvitamin D-3 receptor in ATCC HL-60 cells is because of the presence of a potent serine proteinase and not because of an abnormal or absent receptor.     

Studies on the role of 1,25-dihydroxyvitamin D in chick embryonic development

Vitamin D-deficient laying hens were repleted with 25-hydroxy[26,27-3H]vitamin D3 or 1,25-dihydroxy[26,27-3H]vitamin D3. Egg production returned to normal for both groups of hens by the third week. Eggs from hens fed either 25-hydroxy[26,27-3H]vitamin D3 or 1,25-dihydroxy[26,27-3H]vitamin D3 contained 1,25-dihydroxy[26,27-3H]vitamin D3. Eggs from hens fed 25-hydroxy[26,27-3H]vitamin D3 contained substantial amounts of 25-hydroxy[26,27-3H]vitamin D3, while those from hens fed 1,25-dihydroxy[26,27-3H]vitamin D3 contained none. Plasma from 18-day embryos from hens fed 1,25-dihydroxy[26,27-3H]vitamin D3 contained little or no 1,25-dihydroxy[26,27-3H]vitamin D3, while that from 18-day embryos from hens given 25-hydroxy[26,27-3H]vitamin D3 had normal levels of 1,25-dihydroxy[26,27-3H]vitamin D3. No eggs from hens fed 1,25-dihydroxy[26,27-3H]vitamin D3 hatched, while eggs from hens fed 25-hydroxy[26,27-3H]vitamin D3 achieved a hatchability of 90%. It appears that embryos from hens maintained on 1,25-dihydroxyvitamin D3 as their sole source of vitamin D are essentially vitamin D deficient.     

An improved radioreceptor assay for 1,25-dihydroxyvitamin D in human plasma

We describe a modified assay technique for quantitating 1,25-dihydroxyvitamin D in plasma. The method involves a rapid extraction of the hormone using minicolumn (made of granular diatomaceous earth) chromatography followed by single-step purification on high-performance liquid chromatography. Quantitation of plasma 1,25-dihydroxyvitamin D is achieved by a radioligand receptor assay employing lyophilized cytosolic receptor protein from chick intestine and high-specific-activity 1,25-dihydroxy[³H]vitamin D₃ (166 Ci/mmol). A new incubation medium including an ethanol extract of vitamin D-deficient chick serum yields high specific binding and improves the precision of the radioassay. Bound and free hormone are separated with dextran-coated charcoal of equivalent particle size. The method is sensitive to 0.5 pg/tube with a practical detection range of 1–20 pg/tube, permitting duplicate assay of endogenous 1,25-dihydroxyvitamin D in plasma volumes as small as 0.5 ml. The intra- and interassay coefficient of variation are 5 and 9%, respectively, and the method is valid over a wide-range sample dilution. This assay technique was applied to the measurement of plasma 1,25-dihydroxyvitamin D hormone concentration in normal young adults (55.2 ± 13.6 pg/ml; n = 20) and in patients with chronic renal failure (13.5 ± 5.2 pg/ml; n = 9) and primary hyperparathyroidism (83.3 ± 18 pg/ml; n = 10).     

Normal rabbit intestinal cytosol as a source of binding protein for the 1,25-dihydroxyvitamin D3 assay

Cytosol prepared from small intestine of vitamin D-sufficient rabbits contains a specific high-affinity binding protein for 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). This binding protein sediments at 3.0–3.5 S in sucrose density gradients containing 0.3 m KCl. Scatchard analysis using intestinal cytosol demonstrated a K_d of 0.05 nm and a maximum binding capacity of 92 fmol/mg cytosol protein for 1,25(OH)₂D₃ at 4°C. Competitive binding studies with various metabolites of vitamin D showed a relative binding affinity of this protein for 1,25(OH)₂D₃ > 25-hydroxy-vitamin D₃ > vitamin D₃. With 200 μg of rabbit intestinal cytosol protein, as little as 1.0–2.5 pg of 1,25(OH)₂D₃ reproducibly displaced the tracer sterol from the binding protein. Analyses of human plasma 1,25(OH)₂D₃ content yielded values consistent with published results. The vitamin D-replete rabbit provides a convenient, plentiful, and inexpensive source of binding protein for 1,25(OH)₂D₃ assays.     

Synthesis of 25-hydroxy[26,27-3h]vitamin D3 with high specific activity.

A synthesis of radiochemically pure 25-hydroxy[26,27-³H]vitamin D₃ with a specific activity of 160 Ci/mmol is reported. The structure and biological activity of the radiolabeled compound was verified by comigration on high-pressure liquid chromatography with synthetic 25-hydroxyvitamin D₃ to constant specific activity, and by conversion in vitro to 1α,25-dihydroxy[26,27-³H]vitamin D₃ with the chick kidney 1α-hydroxylase.     

Metabolism of 25-hydroxy-vitamin D3 by primary cultures of chick kidney cells

The primary culture of kidney cells from vitamin D deficient chicks is described. After four days in culture the cells reach confluency and retain their ability to metabolize 25-hydroxyvitamin D₃ to 1,25-dihydroxyvitamin D₃. Addition of one unit of bovine parathyroid hormone to the culture medium for 48 hours prior to assay had no effect on the cells' ability to produce 1,25-dihydroxy vitamin D₃, whereas after 24 hours in the presence of 5×10^?8$\text{M}$ 1,25-dihydroxyvitamin D₃ the cells produced not this metabolite, but 24,25-dihydroxyvitamin D₃. This cell culture system will allow the investigation of the regulation of renal 25-hydroxyvitamin D₃ metabolism under controlled $\text{in vitro}$ conditions.     

Amplification of the action of subthreshold doses of aldosterone by 19-hydroxyandrost-4-ene-3, 17-dione.

Various 1α-hydroxylated side chain analogs of vitamin D₃ have been studied for their ability to compete with 1α,25-dihydroxy[³H]vitamin D₃ for binding to the chick intestinal receptor. Of the analogs examined, 1α,24R-dihydroxyvitamin D₃ was found to be nearly equivalent to 1α,25-dihydroxyvitamin D₃ in its ability to compete for receptor binding. However, this near equivalence was not shared by its stereoisomer, 1α,24S-dihydroxyvitamin D₃, which was only 10% as effective a competitor. It is proposed that the ability of a 24R-hydroxyl group to mimic the 25-hydroxyl group is not due to a lack of side chain specificity on the part of the receptor, but is instead due to the similar orientation of the 25-hydroxyl and the 24R-hydroxyl such that they can be accommodated equivalently by the receptor.     

Characteristics of [3H]1 alpha, 25-(OH)2D3 binding to nuclear fractions from rat pituitary adenoma GH3 cells

Specific high affinity binding sites for [³H]1α, 25-dihydroxy-vitamin D₃ were observed in nuclear fractions of rat pituitary adenoma GH₃ cells. Crude nuclear (P1) sites demonstrated a pharmacological specificity for vitamin D₃ metabolites and analogues that was in accord with the characteristics of 1α, 25-dihydroxyvitamin D₃ receptors in recognized target organs. GH₃ cells grown in serum-containing medium contained significant amounts of 1α, 25-dihydroxy-vitamin D₃ in a P1 extract, whereas no 1α, 25-dihydroxyvitamin D₃ was detectable in P1 extracts from cells cultured in the absence of serum. Binding of [³H]1α, 25-dihydroxyvitamin D₃ to the P1 fraction was unaffected by prior depletion of intracellular 1α, 25-dihydroxyvitamin D₃, suggesting that association of [³H]1α, 25-dihydroxyvitamin D₃ to nuclear sites is not attributable to translocation of a cytosolic hormone-receptor complex and molecular exchange. The results support the concept that 1α, 25-dihydroxyvitamin D₃ has a physiological role in mediating pituitary hormone secretion.