Jürgen Merke

Demonstration of 1,25(OH)2 vitamin D3 receptors and actions in vascular smooth muscle cells in vitro

SummaryBinding of [3H] 1,25 (OH)2D3 and effects of 1,25 (OH)2D3 on cell ultrastructure were evaluated in vascular smooth muscle cells (VSMC) primary cultures (aortic media). Specific reversible binding of [3H] 1,25 (OH)2D3 by a 3.5 S macromolecule with DNA binding, KD 6.2×10−10M and Nmax 16 fmol/mg protein was demonstrated. Incubation of VSMC with 10−8 M 1,25 (OH)2D3, but not 25 (OH)D3, in the presence of 10% FCS for up to three weeks caused rapid reversible appearance in the cytoplasm of membrane-bounded electron-dense lysosomal particles which on electronspectroscopic imaging contained Ca and Pi. VSMC are targets for vitamin D.

1,25(OH)2D3 receptor regulation and 1,25(OH)2D3 effects in primary cultures of growth cartilage cells of the rat

SummaryVitamin D deficiency leads to disturbed calcification of growth cartilage and enlargement of growth plate, illustrating that chondrocytes are a target for vitamin D. This observation prompted an investigation of 1,25(OH)2D3 receptor expression and action of vitamin D metabolites on chondrocyte proliferation. In primary cultures of tibial growth cartilage of male SD rats (80 g), specific binding of [3H]-1,25(OH)2D3 is noted in both the logarithmic growth phase and at confluence (Nmax 12780 molecules/cell versus 4368 molecules/cell). Scatchard analysis revealed the presence of a single class of noninteracting binding sites. KD was 10−11 M irrespective of growth phase. The binding macromolecule had a sedimentation coefficient of 3.5 S. Interaction with DNA was demonstrated by DNA cellulose affinity chromatography. In immunohistology, growth cartilage cells (rabbit tibia) expressed nuclear 1,25(OH)2D3 receptors most prominently in the proliferative and hypertrophic zone. This corresponds to binding data which showed highest Nmax in the proliferating cartilage. 1,25(OH)2D3 in the presence of delipidated fetal calf serum (FCS) had a biphasic effect on cell proliferation and density, i.e., stimulation at 10−12 M and dose-dependent inhibition at 10−10 M and below. Inhibition was specific and not seen with 24,25(OH)2D3 or dexamethasone. Growth phase-dependent 1,25(OH)2D3 receptor expression and effects of 1,25(OH)2D3 on chondrocyte proliferation point to a role of vitamin D in the homeostasis of growth cartilage.

Nuclear testicular 1,25-dihydroxyvitamin D3 receptors in Sertoli cells and seminiferous tubules of adult rodents.

1,25(OH)2D3 receptors were studied in whole testes, Sertoli cells, seminiferous tubules, Leydig cells and spermatogonia of adult NMRI mice and SD rats. Specific reversible high affinity binding (KD 1.4 x 10(-10)M; Nmax 72 fmol/mg protein) by a 3.5 S macromolecule was demonstrated in whole testes, Sertoli cells and seminiferous tubules. With identical techniques, no receptors were found in Leydig cells despite previous reports of 1,25(OH)2D3 actions on Leydig cell function.

Demonstration and characterization of 1,25-dihydroxyvitamin D3 receptors in basal cells of epidermis of neonatal and adult mice

SummaryNuclear and cytosolic receptors for 1,25-dihydroxycholecalciferol [1,25(OH)2D3] were demonstrated in the epidermis of neonatal and adult mice. The macromolecular binding protein sedimented at 3.5 S (sucrose density gradient) and was distinct from the 6.0 S binding protein for 25-hydroxycholecalciferol [25(OH)D3]. Analysis at different ionic strengths suggested the presence of unoccupied nuclear receptors. Digestion with proteases or nucleases, respectively, and inactivation with alkylating agents demonstrated that the binding macromolecule is a protein with SH groups at the active site.Binding of 1,25(OH)2D3 was specific and reversible. In neonatal mice KD was 1.6 × 10−10 M for both cytosolic and nuclear fractions, binding capacity was 54 fmol/mg protein in the cytosolic and 108 in the nuclear fractions, respectively. The phenotypic expression of the 1,25(OH)2D3 receptor (dissociation constant, binding capacity) was identical in neonatal and adult epidermis. Half maximal displacement of 1,25(OH)2D3 was achieved with an 80-fold and 200-fold molar excess of 25(OH)D3 and 1-alpha-hydroxycholecalciferol [1(OH)D3], respectively. Using Percoll density gradient centrifugation, 1,25(OH)2D3 receptors could be localized in the basal cell fraction. DNA cellulose chromatography with 1.25(OH)2D3 receptor elution from DNA at 0.25 M KCl (linear gradient) points to a possible role in gene transcription. In mouse primary epidermal cell cultures, 1,25(OH)2D3, but not 25(OH)D3, 24,25(OH)2D3, and 1(OH)D3 influenced [3H]thymidine incorporation (at physiological concentrations); the magnitude of change depending on the concentration of 1,25(OH)2D3 and the time of incubation.These data demonstrate that skin is a target organ for the active vitamin D secosterol.

Actions of 1,25-Dihydroxyvitamin D3 on Human Mesangial Cells

In the present study, we examined specific binding of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] by an effects of 1,25(OH)2D3 on human mesangial cells (hMC), obtained from healthy portions of tumor-bearing kidneys. Receptors for 1,25(OH)2D3 were characterized by (1) sucrose density gradient analysis, (2) Scatchard analysis, and (3) DNA affinity of the receptor molecule. Specific binding occurred by a single class of macromolecules, sedimenting with 3.5 S in sucrose density gradients (5% to 20%). Receptors showed high affinity (Kd, 1.39 x 10(-10)), and specific binding capacity (Nmax) of 821 binding sites per cell. 1,25(OH)2D3 (10(-6) to 10(-10)) reduced both DNA synthesis (by [3H]thymidine incorporation) and cell growth (by cell counting) throughout the log-phase and confluence. Further evidence of functional effects of 1,25(OH)2D3 on hMC is provided by ultrastructural studies, which showed rapid increase of electron-dense lysosomal particles in hMC exposed to 1,25(OH)2D3. The data identify actions of 1,25(OH)2D3, a molecule with recently recognized immunoregulatory roles, on hMC. The results are consistent with a role of 1,25(OH)2D3 in control of mesangial cell function.

No decrease of 1,25(OH)2D3 receptors and duodenal calbindin-D9k in uraemic rats

Abstract. In parathyroids of uraemic patients or animals, decreased specific binding of 1,25(OH)2D3 has been observed and implicated in the genesis of secondary hyperparathyroidism of renal failure. We re‐examined binding of 1,25(OH)2D3 using chromatin preparations for receptor characterization which differed from previous studies (a) by inclusion of protease inhibitors (PMSF, aprotinin) and molybdate in the extraction buffer and (b) by omitting the K‐extraction step. With this method, the Nmax in the intestinal mucosa and parathyroids of uraemic animals was significantly higher, while the receptor sedimentation constant (S), DNA affinity and KD were all unchanged. The ratio of occupied to total receptors was not significantly altered. The regulation of 1,25(OH)2D3 receptors in response to acute injection of 1,25(OH)2D3 was abnormal. Calbindin‐D9k concentration in the intestines of uraemic and control rats was comparable both before and after administration of 1,25(OH)2D3.

Evidence for in vivo upregulation of 1,25(OH)2 vitamin D3 receptor in human monocytes.

SummaryMammalian cells increase net expression of 1,25(OH)2D3 receptors after exposure to physiological concentrations of 1,25(OH)2D3in vitro. we examined specific binding of 1,25(OH)2D3 by human monocytes before and after daily administration of 1.5–2 ug 1,25(OH)2D3 p.o. for 3 days in 5 healthy normal D-replete probands. Median specific binding (Nmax) at baseline was 793 molecules/cell and 2052 or 2828 at 24h and 72h of 1,25(OH)2D3 treatment respectively. The results suggest (a) up-regulation of 1,25(OH)2D3 receptors occurs in man and (b) monocyte preparations can be used to assess receptor regulationin vivo.

Hyperparathyroidism and abnormal 1,25(OH)2vitamin D3 metabolism in experimental lead intoxication

Abstract. Clinical evidence points to disturbed calcium metabolism in lead (Pb) intoxication. To further clarify the mechanisms involved, serum levels of 1,25(OH)2D3, receptors for 1,25(OH)2D3 as well as size and ultrastructure of parathyroid glands were examined in Wistar Kyoto rats exposed to 1% lead (Pb) acetate in drinking water for 10 weeks (short‐term study) or 0·001 – 1 % Pb acetate for 24 weeks (long‐term study). After administration of Pb for 10 weeks, bone Pb was significantly increased (641±66·9 (SD) vs. 0·648±0·39 mg kg‐1 ash in controls). Total serum calcium and ionized Ca2+ (1·15±0·031 vs. 1·25±0·03 mmol 1‐1) were significantly decreased. Renal function (Ccr) was unchanged, but urinary cAMP excretion and circulating 1,25(OH)2D3 (177±10·9 vs. 232±18·9 pmol 1‐1) were diminished. Specific binding of 1,25(OH)2D3 was increased in parathyroids (Bmax 128±4·7 vs. 108±0·6 fmol mg‐1 protein) and intestinal mucosa; Bmax failed to adequately rise in response to pretreatment with 1,25(OH)2D3 (2·10 ng day‐1 for 4 d) in Pb‐exposed animals. Receptor characteristics (sedimentation constant, KD, DNA affinity) were unchanged. Parathyroid weight was significantly increased (178·25 vs. 96·34 μg) with no change of estimated nuclear volume, cell volume or cell ultrastructure. After 24 weeks of Pb exposure, a dose‐dependent but non‐linear increase of parathyroid weight was noted between 0·001% and 1% Pb in drinking fluid.