Solubilization and partial purification of a rat intestinal 1,25-dihydroxyvitamin D3 binding protein.

A protein containing fraction that will bind 1,25-dihydroxyvitamin D₃ both $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$ has been solubilized from the nuclear-debris fraction of rat intestinal mucosa and purified 15-fold.     

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.     

1Alpha,25-dihydroxyvitamin D3 receptor: competitive binding of vitamin D analogs

The intestinal nuclear receptor for lα,25-dihydroxyvitamin D₃ has been utilized to determine the ability of vitamin D-active sterols to compete with this hormone at the molecular level. 25-Hydroxyvitamin D₃ and lα-hydroxyvitamin D₃ must be present in 150 and 450 times the concentration respectively of lα,25-dihydroxyvitamin D₃, $\text{in}$$\text{vitro}$, to displace the physiologic hormone. These data indicate that: i) superphysiologic levels of 25-hydroxyvitamin D₃ may simulate lα,25-dihydroxyvitamin D₃ and act directly on isolated target organs and ii) the biologic potency observed for low doses of lα-hydroxyvitamin D₃, $\text{in}$$\text{vivo}$, is probably the result of 25-hidroxylation of the lα-derivative to form lα,25-dihydroxyvitamin D₃.     

Cytosol Preparations are Inadequate for Quantitating Unoccupied Receptors for 1,25-Dihydroxyvitamin D3

Abstract

Recent studies in this laboratory have indicated that 90% of the unoccupied receptors for 1,25-dihydroxyvitamin D₃ [1,25(OH)₂-D₃] are associated with nuclear components when chick intestinal mucosa is homogenized in low salt buffer (TED: 10 mM Tris, 1.5 mM EDTA, 1.0 mM dithiothreitol, pH 7.4). This observation suggested that previously reported cytosol 1,25(OH)₂D₃ receptors could result instead from salt extraction of nuclear receptors. The studies herein indicate that tissue 1,25(OH)₂D₃ receptor recovery is 30–50% lower in cytosol prepared from KTED (0.3 M KC1 + TED) or STKM (0.25 M sucrose, 50 mM Tris, 25 mM KC1, 5 mM MgCl₂, pH 7.4) than in TED-prepared chromatin. Thus tissue concentrations of unoccupied 1,25(OH)₂D₃ receptors can be closely estimated in TED-chromatin; full quantitation can be achieved by summing the number of receptors in TED-chromatin plus TED-cytosol. Incubation at different temperatures for varying times yielded maximal receptor recovery (6.1 pmol/g mucosal wet weight) at 4°C for 4–24 h or at 23° for 30 min. Scatchard analyses confirmed that only a single class of high affinity (K_d 0.4 nM) binding sites was present under all incubation conditions. Dithiothreitol significantly improved receptor recovery both in cytosol and in chromatin preparations. Conversely, inclusion of 20% glycerol caused an artificial increase in specific H-1,25(OH)₂D₃ binding due to a second class of chromatin binding sites with ten-fold higher K_d (8.1 nM) and a greater number of binding sites than the 1,25(OF)₂D₃ receptor. In conclusion, the TED-chromatin assay procedure provides better quantitation of the tissue content of unoccupied 1,25(OH)₂D₃ receptors than do previously described techniques. The presence of unoccupied nuclear-associated 1,25(OH)₂D₃ receptors in other target tissues emphasizes the potential for erroneous physiological conclusions if these chromatin-associated receptors are overlooked.     

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.     

Further evidence for the 1,25-dihydroxyvitamin D-like activity of Solanum malacoxylon

Administration of an aqueous extract of the calcinogenic plant $\text{Solanum}\text{malacoxylon}$ (S.m.) to vitamin D-deficient or strontium fed chicks produces significant plasma 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) activity within 6 hr. (via radioreceptor assay) and subsequently elicits the appearance of immunoreactive intestinal calcium binding protein. Studies of a purified aqueous extract of S.m. show that it does not compete effectively with radioactive 1,25-(OH)₂D₃ for binding to the sterol's intestinal receptor. However, treatment of the extract with β-glucosidase releases a biologically active substance which is soluble in organic solvents and efficiently competes with labeled sterol for the receptor. This factor migrates exactly with tritiated 1,25-(OH)₂D₃ on high resolution Celite liquid-liquid partition columns. Thus, S.m. contains a molecule very similar or identical to 1,25-(OH)₂D₃ which is combined with one or more carbohydrate moieties in the native plant. This glycoside is probably cleaved $\text{in}\text{vivo}$ before biological activity is attained.     

Isolation and identification of 23,25-dihydroxy-24-oxo-vitamin D3: A metabolite of vitamin D3

A new metabolite of vitamin D₃ has been isolated in pure form from incubations of rat kidney homogenates with 25-hydroxyvitamin D₃ [25-OH-D₃]. It was identified as 23,25-dihydroxy-24-oxo-vitamin D₃ [23,25(OH)₂-24-oxo-D₃] by means of ultraviolet absorption spectrophotometry and mass spectrometry. Also, 25-OH-D₃-26,23-lactone and 24R,25-dihydroxyvitamin D₃ were obtained from the same incubation mixtures. The enzyme activity responsible for the conversion of 25-OH-D₃ to 23,25(OH)₂-24-oxo-D₃ was induced by perfusion of the kidneys $\text{in}\text{vitro}$ with 50 nM 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃].     

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.     

Nuclear and cytoplasmic binding components for vitamin D metabolites.

Specific binding of 1α,25-dihydroxyvitamin D₃ to macromolecular components of small intestinal nuclei and cytosol is demonstrated. The nuclear 1α,25-dihydroxyvitamin D₃ complex can be extracted from chromatin by 0.3 M KCl and sediments at 3.7S in sucrose density gradients. The cytoplasmic 1α,25-dihydroxyvitamin D₃-binding components also sediment at 3.7S, identically to the nuclear complex under the ultracentrifugation procedures employed.Macromolecular binding components with a high affinity for 25-hydroxyvitamin D₃ (K_d = 4.5 × 10⁻⁹ M) were also identified in intestinal cytosol which differ from the 1α,25-hydroxyvitamin D₃ receptor in that: 1) they sediment at 5–6S in sucrose gradients, 2) they are observed in organs other than the intestine, and 3) while they do bind 1α,25-dihydroxyvitamin D₃ at higher concentrations than 25-hydroxyvitamin D₃, they are not observed to transfer either 25-hydroxyvitamin D₃ or 1α,25-dihydroxyvitamin D₃ to the nucleus, $\text{in vitro}$.     

1,25-Dihydroxyvitamin D3-glycoside: Identification of a calcinogenic principle of solanum malacoxylon

The water soluble calcinogenic factor present in the plant $\text{Solanum}$$\text{malacoxylon}$ is partially purified by selective extraction and chromatography on silicic acid and then hydrolyzed with a mixed preparation of glycosidases from the sea worm, $\text{Charonia}$$\text{lampus}$. Hydrolysis produces a chloroform soluble factor with biologic characteristics of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃), the hormonal form of vitamin D. Purification of this factor is accomplished by chromatography on Sephadex LH-20, silicic acid, and Celite columns, yielding 3 μg of active material. During the isolation, bioactivity (as assessed by the ability of fractions to compete with labeled 1,25-(OH)₂D₃ for binding to a specific intestinal receptor protein) migrates exactly with authentic tritiated 1,25-(OH)₂D₃. The purified factor has an ultraviolet absorption spectrum identical to that of 1,25-(OH)₂D₃ and analysis via direct probe mass spectrometry yields a parent molecular ion of m/e 416 and a fragmentation pattern indistinguishable from synthetic 1,25-(OH)₂D₃ hormone. We therefore conclude that the vitamin D-like principle in $\text{Solanum}$$\text{malacoxylon}$ is a sterol-glycoside which contains the 1,25-(OH)₂D₃ molecule as its active sterol component.     

Solubilization and partial purification of a rat intestinal 1,25-dihydroxyvitamin d3 binding protein

A protein containing fraction that will bind 1,25-dihydroxyvitamin D₃ both $\text{in vivo}$ and $\text{in vitro}$ has been solubilized from the nuclear-debris fraction of rat intestinal mucosa and purified 15-fold.     

Nuclear and cytoplasmic receptors for 1,25-dihydroxycholecalciferol in intestinal mucosa

The apparent hormonal form of cholecalciferol, 1,25-dihydroxycholecalciferol (1,25-(OH)₂-CC), was incubated with intestinal mucosa homogenates and whole intestinal tissue, $\text{in}\text{vitro}$. After 40–70 min, 1,25-(OH)₂-CC was specifically associated with the nuclear chromatin fraction. This sterol remains bound to the cytosol fraction at 0°C and a dramatic movement to the nuclear chromatin occurs at 37°C indicating that the subcellular localization of the sterol is temperature dependent. Isolated intestinal cytosol, previously incubated with 1,25-(OH)₂-CC, is required for transportation of the hormone to the intestinal chromatin fraction; cytosol fractions from other tissues are ineffective mediators of this sterol migration. It is concluded that the intestinal cytosol contains a specific receptor that functions to transport 1,25-(OH)₂-CC to the nucleus, its probable site of action.     

Regulation of 24,25-dihydroxyvitamin D-3 production by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1–34 in a cloned monkey kidney cell line (JTC-12)

Regulation of 25-hydroxyvitamin D-3 24-hydroxylase by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1–34 (PTH_1–34) was investigated using a cloned monkey kidney cell line, JTC-12. Treatment of the cells with 1,25-dihydroxyvitamin D-3 markedly enhanced the conversion of [³H]-25-hydroxyvitamin D-3 into a more polar metabolite. The metabolite was identified as 24,25-dihydroxyvitamin D-3 by normal phase and reverse phase high-performance liquid chromatography and periodate oxidation. The 24-hydroxylae activity appeared to follow Michaelis-Menten kintics, and 1,25-dihydroxyvitamin D-3 treatment increased the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 24-hydroxylase from 33 to 95 pmol/h per 10⁶ cells without affecting the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of the enzyme (220 nM in control vs. 205 nM in 1,25-dihydroxyvitamin D-3 treated cells). The enzyme activity reached a maximum between 4 and 8 h of treatment with 1,25-dihydroxyvitamin D-3. The dose of 1,25-dihydroxyvitamin D-3 required to cause a half-maximal stimulation was about 3 · 10^?10 M. The 1,25-dihydroxyvitamin D-3-induced increase in 24-hydroxylase was almost completely inhibited by the presence of 1 μM cycloheximide. Treatment of the cells with PTH_1–34 caused a dose-dependent increase in cyclic AMP production. Half-maximal stimulation of cyclic AMP production was obtained at about 5 · 10^?9 M PTH_1–34. When 2.4 · 10^?9 M PTH_1–34 was added after 1,25-dihydroxyvitamin D-3 treatment, the 1,25-dihydroxyvitamin D-3-stimulated 24-hydroxylase was inhibited to $\text{70.7 ± 2.9\%}$ of control. Higher concentrations of PTH_1–34 caused less inhibition of the enzyme activity. When cyclic AMP was added instead of PTH_1–34, the enzyme activity was also suppressed significantly. These results indicate that, in JTC-12 cells, 1,25-dihydroxyvitamin D-3 stimulates 24-hydroxylase in a dose- and time-dependent manner by increasing the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the enzyme through a mechanism dependent upon new protein synthesis, and suggest that PTH_1–34 inhibits the 1,25-dihydroxyvitamin D-3-induced stimulation of 24-hydroxylase through its effect on cyclic AMP production.     

Binding properties of 23S,25-dihydroxyvitamin D3: an in vivo metabolite of vitamin D3

23$\text{S}$,25-Dihydroxyvitamin D₃ was isolated from the plasma of vitamin D₃-toxic pigs. An ultraviolet absorbance spectrum confirmed its purity. The configuration of the 23-hydroxyl group was determined to be S by comparison of the natural product with synthetic 23$\text{R}$,25- and 23$\text{S}$,25-dihydroxyvitamin D₃ by high-pressure liquid chromatography. The affinity of both 23$\text{S}$,25- and 23$\text{R}$,25-dihydroxyvitamin D₃ for the plasma vitamin D binding protein was similar to vitamin D₃. Thus, with respect to the plasma vitamin D binding protein, 23$\text{S}$,25-dihydroxyvitamin D₃ is the least potent, naturally-occurring, dihydroxylated vitamin D₃ metabolite known.     

Preparation of metaphase chromatin of Physarumpolycephalum without the loss of repressed RNA synthesis

A novel method for the preparation of intact chromatin from the slime mold $\text{Physarum}\text{polycephalum>}$ which retains the $\text{in}\text{vivo}$ property of RNA synthesis is described. Preparations from G₂-cells were highly active, while those from metaphase-cells were inactive. The plasmodial cells were disrupted by gentle homogenization on a polyethylene sieve in a neutral isotonic sucrose medium containing Mg⁺⁺, deoxycholate and EGTA, a Ca⁺⁺-chelating agent. The nuclei were lysed in a hypotonic buffer without use of EDTA and chromatin was precipitated by centrifugation after addition of Mg⁺⁺.     

Studies on the mode of action of calciferol: Subcellular localization of 1,25-dihydroxyvitamin D3 in chicken parathyroid glands

As a further means of evaluating 1,25-dihydroxyvitamin D₃-parathyroid gland interaction and its relation to calcium homeostasis, a comparative study of the subcellular localization of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]in the parathyroid glands, intestinal mucosa, kidney, and liver of rachitic chickens has been carried out. Only in the chromatin fraction from parathyroids and intestinal mucosa could there be demonstrated selective and specific localization of the 1,25(OH)₂D₃. The chromatin-bound picomoles of 1,25(OH)₂D₃ (per gram of tissue) was in the ratio (mucosa:parathyroids:kidney:liver) of 1.0:0.23:0.11:0.17 2 h after an intracardial injection of 290 pmol of [³H]1,25(OH)₂D₃. This same ratio after a 30-min (23 °C) homogenate incubation with 1 × 10^?8m [³H]1,25(OH)₂D₃ was 1.0:1.0:0.10:0.03. Analogous results were obtained when reconstituted chromatin and cytosol fractions from the different tissues were compared for chromatin localization efficiency. This chromatin localization of 1,25(OH)₂D₃ in the parathyroid glands was temperature dependent. In addition, parathyroid glands were found to contain 3.0–3.5 S cytoplasmic and KCl-extractable chromatin receptors specific for 1,25(OH)₂D₃.     

The adrenal: A new target organ of the calciotropic hormone 1,25-dihydroxyvitamin D3

Summary Target cells for 1,25-dihydroxyvitamin D₃ were demonstrated in the adrenal medulla by frozen-section autoradiography. The appearance of these target cells was age-dependent in neonatal mice. Immunocytochemical staining for phenylethanolamine-N-methyltransferase revealed that both epinephrine and non-epinephrine cells concentrate 1,25-dihydroxyvitamin D₃ in their nuclei. In contrast, immunocytochemical staining for vitamin D-dependent calcium-binding protein (D-CaBP) demonstrated that D-CaBP immunoreactivity is localized in only a small percentage of adrenomedullary cells, in mice and rats. Comparison of PNMT and D-CaBP immunoreactivities in sequential sections showed that epinephrine-producing cells do not contain D-CaBP. These results indicate that adrenal medullary cells have receptors for 1,25-dihydroxyvitamin D₃ and that 1,25-dihydroxyvitamin D₃ may directly affect certain functions of these endocrine cells.Supported by US PHS grant PCM8200569 and postdoctoral training grant AM07240-02     

Nuclear 115cadmium: uptake and disappearance correlated with cadmium-binding protein synthesis.

The intracellular distribution of ¹¹⁵cadmium was determined following a pulsed exposure to the metal. The uptake and disappearance of label from rat liver nuclei was correlated with the appearance of a cytoplasmic Cd-binding protein. By coupling $\text{in}\text{vivo}$ - $\text{in}\text{vitro}$ experiments it was shown that unspecifically bound cadmium is free to enter the nucleus while specifically bound cadmium remains in the cytoplasm.     

Sarcoid granulomas metabolize 25-hydroxyvitamin D3invitro

Sarcoid granulomas metabolized 25-hydroxyvitamin D₃ to two unidentified metabolites during $\text{in}\text{vitro}$ incubation. A two-step high pressure liquid chromatography system revealed two unique elution positions of these sarcoid-derived metabolites that exactly comigrated with the elution positions of 5(Z)-19-nor-10-oxo-25(OH)D₃ and 5(E)-19-nor-10-oxo-25(OH)D₃, respectively. These unique metabolites did not bind specifically to a protein receptor for 1,25(OH)₂D₃.