Biomedical subjects

D Feldman

Publications and source records attributed to D Feldman.

At least 163 records · Page 9Linked to original sources

PubMed1989

Characterization of an estrogen-binding protein in the yeast Candida albicans.

An estrogen-binding protein (EBP) has been identified and characterized in the cytosol of the pathogenic yeast Candida albicans. Binding of [3H]estradiol was found to be optimal at pH 7.4 in the presence of 0.3 M KCl and was linearly related to protein concentration. Binding was very rapid, reaching maximal levels in about 30 min, and was reversible with a dissociation rate constant of 13.2 +/- 1.7 x 10(-4) sec-1. EBP binding was destroyed by treatment with proteolytic enzymes and by high temperatures. Scatchard analysis of the [3H]estradiol equilibrium binding data of C. albicans (strain stn-1) yielded an apparent dissociation constant of 12.3 +/- 2.1 nM and a maximal binding capacity of 753 +/- 145 fmol/mg protein. Binding competition experiments showed very high specificity and stereoselectivity of EBP, demonstrating the following order of potency in displacing [3H]estradiol: 17 beta-estradiol greater than estrone greater than estriol greater than 17 alpha-estradiol. Negligible competitive potency was found for other mammalian steroid hormones, diethylstilbestrol, tamoxifen, or fungal hormones. The abundance of EBP was 4- to 10-fold higher during the early logarithmic growth phase of yeast cells than during the stationary phase. The molecular size of EBP, measured by Sephacryl S-200 gel exclusion chromatography, yielded a Stokes radius of approximately 29 A. Sucrose density gradient sedimentation showed a sedimentation coefficient (S2020,W) of 4, with no ionic dependent aggregation of the [3H]estradiol-EBP complex. The apparent mol wt of the EBP is approximately 46,000, with an axial ratio of 1, indicating the symmetrical shape of the molecule. In summary, in addition to the previously described corticosterone-binding protein, a separate high affinity, stereospecific, estrogen-selective binder has been demonstrated in the cytosol of C. albicans.

Candida albicans↗

PubMed1989

Epidermal growth factor increases intestinal calbindin-D9k and 1,25-dihydroxyvitamin D receptors in neonatal rats.

Epidermal growth factor (EGF) has been reported to increase intestinal calcium absorption in suckling rats. The mechanism of this effect is unknown, as are the roles of vitamin D-dependent and independent pathways. The present studies were undertaken to investigate the ability of EGF to accelerate the postnatal induction of the vitamin D-dependent intestinal calcium-binding protein, calbindin-D9k. Subcutaneous administration of EGF increased duodenal calbindin-D9k in suckling rats by more than 100% (P less than 0.001). The effect of EGF was not seen in older weaned animals or when EGF was given to suckling rats by gavage. Administration of EGF simulated the changes of normal development. 1) It increased calbindin-D9k, and the effect was greater in proximal than distal duodenum. 2) EGF increased alkaline phosphatase activity to the same extent in proximal and distal duodenum. 3) EGF increased sucrase more markedly in distal than in proximal epithelium. Maximal and half-maximal effects of EGF on each of these proteins were observed at twice daily doses of 0.1 and 0.04 microgram/g BW, respectively. 4) EGF at the maximally effective dose produced a small (30%) but statistically significant (P less than 0.005) increase in serum 1,25-dihydroxyvitamin D. 5) Most importantly, EGF treatment resulted in a 2-fold increase in intestinal 1,25-dihydroxyvitamin D receptors (VDR) in the proximal segments of the small intestine (P less than 0.001). EGF effects on calbindin-D9k and VDR were specific for the intestine, as EGF did not change kidney calbindin-D9k or kidney VDR. Thus, EGF was able to prematurely initiate a complex series of molecular changes that occur during normal development. The mechanism of EGF's action to stimulate calcium absorption appears to involve a maturation effect on the vitamin D-dependent pathway.

Alkaline Phosphatase↗

PubMed1989

Abnormal binding of vitamin D receptors to deoxyribonucleic acid in a kindred with vitamin D-dependent rickets, type II.

Vitamin D-dependent rickets, type II, is a hereditary disease that results from target organ resistance to the action of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. We describe here a family (designated G) with a defect in the DNA-binding domain of the 1,25-(OH)2D receptor (VDR) manifested by normal steroid binding and decreased VDR affinity for DNA. The phenotypically normal parents are heterozygous, expressing both normal and defective forms of VDR. The affected children in this family had early-onset rickets, alopecia, hypocalcemia, and elevated serum 1,25-(OH)2D3 levels. The VDR of cultured skin fibroblasts of the affected children (G1 and G2) as well as the parents (G3 and G4) bound [3H]1,25-(OH)2D3 normally (Kd = 2-3 X 10(-11) mol/L; maximal number of binding sites = 20-40 fmol/mg protein). The cells from G1 and G2 were resistant to 1,25-(OH)2D3 action, as measured by induction of 24-hydroxylase activity, while the cells from G3 and G4 responded normally. Western blot analysis using the anti-VDR monoclonal antibody 9A7 showed that hypertonic extracts of fibroblasts from both affected children (G1 and G2) and their parents (G3 and G4) had immunoreactive bands of 48K, identical to the size of the VDR in normal cells. The VDR from G1 and G2 eluted as a single peak from DNA-cellulose columns at a lower salt concentration (0.1 mol/L) than that from normal subjects (0.2 mol/L), indicating an apparent decreased affinity for DNA. Fibroblast VDR from G3 and G4 each eluted from DNA-cellulose columns as two peaks, the normal peak (0.2 mol/L) and the abnormal peak (0.1 mol/L), which was found in G1 and G2. Western blot analysis of the 0.1 and 0.2 mol/L KCl peak fractions also confirmed that VDR was present in only the 0.1 mol/L fraction and not the 0.2 mol/L fraction from G1. However, VDR was present in both fractions from G3. In summary, this vitamin D-dependent rickets, type II, kindred has a defect in the DNA-binding domain of VDR. The parents are phenotypically normal and express both the normal and defective VDR alleles, as demonstrated by both DNA-cellulose chromatography and Western blot analysis. The affected children are resistant to 1,25-(OH)2D3 action and are homozygous for the defective VDR.

Blotting, Western↗

PubMed1989

C-type virus particles in placentas of rhesus monkeys after maternal treatment with recombinant leukocyte A interferon.

Interferon inhibits the normal replication of C-type virus particles grown in vitro. The ability of interferon to produce a similar effect in vivo was tested in rhesus monkey placentas which are known to contain C-type virus particles. Samples of placenta from 6 monkeys treated with recombinant leukocyte A interferon (25 x 10(6) units/kg) for 7 to 21 days and from 6 control monkeys were examined in the electron microscope. Budding and immature C-type virus particles were present in every placenta examined from both interferon-treated and control monkeys. Mature particles were found in placentas from 3 of 6 monkeys in both treated and control groups. Possible explanations for the absence of detectable effects of interferon in C-type virus particle formation in these placentas are unresponsiveness of the trophoblast to interferon or inadequate exposure time to interferon of placental sites from which C-type particles are produced.

Animals↗

PubMed1989

Rheumatoid factor in patients with systemic lupus erythematosus.

One hundred sixty-six patients with systemic lupus erythematosus (SLE) were surveyed and their sera tested for the presence of rheumatoid factor (RF). Fifteen were positive whenever tested, with titers of at least 1:40; the remainder were either positive only in titers of 1:20, were inconsistently positive or were persistently negative. The former group were less likely to have severe manifestations of SLE, less likely to have required treatment with "high-dose" steroids or a cytotoxic drug, and less likely to have had an episode of herpes zoster. We conclude that in patients with SLE, RF may be a reflection of relative immune competence so that they are partially protected against the more serious manifestations of SLE and the development of herpes zoster.

Adult↗

PubMed1988

Point mutations in the human vitamin D receptor gene associated with hypocalcemic rickets.

Hypocalcemic vitamin D-resistant rickets is a human genetic disease resulting from target organ resistance to the action of 1,25-dihydroxyvitamin D3. Two families with affected children homozygous for this autosomal recessive disorder were studied for abnormalities in the intracellular vitamin D receptor (VDR) and its gene. Although the receptor displays normal binding of 1,25-dihydroxyvitamin D3 hormone, VDR from affected family members has a decreased affinity for DNA. Genomic DNA isolated from these families was subjected to oligonucleotide-primed DNA amplification, and each of the nine exons encoding the receptor protein was sequenced for a genetic mutation. In each family, a different single nucleotide mutation was found in the DNA binding domain of the protein; one family near the tip of the first zinc finger (Gly----Asp) and one at the tip of the second zinc finger (Arg----Gly). The mutant residues were created in vitro by oligonucleotide directed point mutagenesis of wild-type VDR complementary DNA and this cDNA was transfected into COS-1 cells. The produced protein is biochemically indistinguishable from the receptor isolated from patients.

Amino Acid Sequence↗

PubMed1988

Amphiphile-induced heart muscle-cell (myocyte) injury: effects of intracellular fatty acid overload.

Lipid amphiphile toxicity may be an important contributor to myocardial injury, especially during ischemia/reperfusion. In order to investigate directly the potential biochemical and metabolic effects of amphiphile overload on the functioning heart muscle cell (myocyte), a novel model of nonesterified fatty acid (NEFA)-induced myocyte damage has been defined. The model uses intact, beating neonatal rat myocytes in primary monolayer culture as a study object and 5-(tetradecyloxy)-2-furoic acid (TOFA) as a nonmetabolizable fatty acid. Myocytes incubated with TOFA accumulated it as NEFA, and the consequent NEFA amphiphile overload elicited a variety of cellular defects (including decreased beating rate, depletion of high-energy stores and glycogen pools, and breakdown of myocyte membrane phospholipid) and culminated in cell death. The amphiphile-induced cellular pathology could be reversed by removing TOFA from the culture medium, which resulted in intracellular TOFA "wash-out." Although the development and severity of amphiphile-induced myocyte injury could be correlated with both the intracellular TOFA/NEFA content (i.e., the level of TOFA to which the cells were exposed) and the duration of this exposure, removal of amphiphile overload did not inevitably lead to myocyte recovery. TOFA had adverse effects on myocyte mitochondrial function in situ (decoupling of oxidative phosphorylation, impairing respiratory control) and on myocyte oxidative catabolism (transiently increasing fatty acid beta oxidation, citric acid cycle flux, and glucose oxidation). The amphiphile-induced bioenergetic abnormalities appeared to constitute a state of "metabolic anoxia" underlying the progression of myocyte injury to cell death. This anoxic state could be ameliorated to some extent, but not prevented, by carbohydrate catabolism.

Adenosine Triphosphate↗

PubMed1988

Dermatophyte-hormone relationships: characterization of progesterone-binding specificity and growth inhibition in the genera Trichophyton and Microsporum.

We reported previously that Trichophyton mentagrophytes contains a cytoplasmic macromolecule which specifically binds progesterone. Progesterone is also an effective inhibitor of growth of the fungus. We report here studies which characterize more fully the specific binding properties and the functional responses of T. mentagrophytes and taxonomically related fungi to a series of mammalian steroid hormones. Scatchard analysis of [3H]progesterone binding in both the + and - mating types of Arthroderma benhamiae and in Microsporum canis revealed a single class of binding sites with approximately the same affinity as that in T. mentagrophytes (Kd, 1 X 10(-7) to 2 X 10(-7) M). Trichophyton rubrum had a protein with a higher binding affinity (Kd, 1.6 X 10(-8) M). Characterization of the [3H]progesterone-binding sites in T. mentagrophytes showed the binder to be a protein which was destroyed by trypsin and heating to 56 degrees C. Previous examination of the steroid-binding specificity in T. mentagrophytes had demonstrated that deoxycorticosterone (DOC) and dihydrotestosterone (DHT) were effective competitors for [3H]progesterone binding. Expansion of this study to include other competitors revealed that R5020 (a synthetic progestin), androstenedione, and dehydroepiandosterone possessed relative binding affinities which were 20, 11, and 9% of that of progesterone, respectively. Other ligands tested were less effective. Competition studies for the binder in M. canis resulted in similar findings: DOC and DHT were effective competitors for [3H]progesterone binding. The growth of A. benhamiae + and -, M. canis, and T. rubrum were all inhibited by progesterone in a dose-responsive manner, with 50% inhibition achieved at concentrations of 9.8 x 10(-6), 1.2 x 10(-5), 1.5 x 10(-5), and 2.7 x 10(-6) M. respectively,.

Binding, Competitive↗

PubMed1988

Cultured human endothelial cells treated with recombinant leukocyte A interferon. Tubuloreticular inclusion formation, antiproliferative effect, and 2',5' oligoadenylate synthetase induction.

Cultured human umbilical vein endothelial cells and human thoracic aorta endothelial cells were treated with 500, 1,000, and 2,000 IU/ml of recombinant leukocyte alpha-interferon for 2, 4, and 7 days. Tubuloreticular inclusions developed in treated human umbilical vein endothelial cells and human thoracic aorta endothelial cells. The size and number of tubuloreticular inclusions observed correlated with the dose and duration of treatment. Interferon treatment inhibited the rate of proliferation of both human umbilical vein endothelial cells and human thoracic aorta endothelial cells in a dose- and duration-dependent manner. Induction of the interferon-associated enzyme, 2'-5' oligoadenylate synthetase, also occurred. Since the endothelium is exposed to endogenous interferon present in certain pathologic conditions and to exogenous interferon administered in the treatment of several neoplastic or viral diseases, there is an increased need to understand the various effects of interferon on the endothelial cell.

2',5'-Oligoadenylate Synthetase↗

PubMed1988

Tubuloreticular reorganization of cytomembranes in cells treated with human alpha interferons--a review.

Human alpha interferons (IFN-a) cause a reorganization of internal cell membranes into tubuloreticular inclusions (TRI). Morphogenesis and cytochemistry indicate a pre-Golgi intracisternal origin from the endoplasmic reticulum. Clinically, TRI formation in human blood mononuclear cells correlates with systemic IFN-a treatment or with endogenous overproduction of IFN-a in viral or autoimmune diseases (e.g., rubella syndrome, AIDS, systemic lupus erythematosus). In vitro, TRI formation can be produced by treatment of Daudi lymphoblasts or vascular endothelial cells with IFN-a, and is blocked by actinomycin-D. In Daudi lymphoblasts or vascular endothelial cell cultures, TRI formation parallels induction of 2'-5' A synthetase, inhibition of thymidine kinase and growth inhibition; however, heavy water treatment of Daudi cells prevented TRI formation while induction of 2'-5' A synthetase and growth inhibition persisted. TRI formation was dissociated from IFN-a antiproliferative activity in a mutant clone of Daudi lymphoblasts. Decreased glycoprotein biosynthesis and increased phospholipid biosynthesis may accompany progressive TRI accumulation.

Endothelium, Vascular↗

PubMed1987

Salt-induced activation of 1,25-dihydroxyvitamin D3 receptors to a DNA binding form.

In this report we examine the DNA-cellulose binding and sedimentation properties of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptors from rat intestine and cultured human mammary cancer cells (MCF-7) extracted in nonactivating (low salt) buffers. Receptors prepared in hypotonic buffer had low DNA binding (13%) compared to receptors extracted with 0.3 M KCl (50%). Treatment of low salt receptor preparations with KCl significantly increased (approximately 3-fold) DNA-binding (activation), demonstrating that receptors can be "activated" in vitro. Activated receptors eluted from DNA-cellulose at 0.18 M KCl. Sedimentation analysis followed by DNA-cellulose binding indicated that activated receptors are approximately 3.2 S and unactivated receptors 5.5 S in size. These results suggest that dissociation of an aggregated moiety may lead to receptor activation. Treatment of unactivated receptor with RNase did not alter DNA binding or sedimentation properties of the aggregated receptor. Treatment of unactivated receptor complexes with heat did not increase DNA binding, and molybdate did not block subsequent salt activation. In summary these results suggest that 1,25(OH)2D3 receptors undergo a salt-induced activation step similar to that described for other steroid receptor systems. However, 1,25(OH)2D3 receptors differ from other steroid receptors in not exhibiting heat activation nor having salt activation blocked by molybdate.

Animals↗

PubMed1987

Modulation of 1,25-dihydroxyvitamin D3 receptor binding and action by sodium butyrate in cultured pig kidney cells (LLC-PK1).

We have studied the effects of sodium butyrate (SB) on 1,25(OH)2D3 receptors in the pig kidney cell line (LLC-PK1). In this system, we have shown that 1,25(OH)2D3 induction of 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) activity is dependent on the level of 1,25(OH)2D3 receptors. Treatment of confluent cells with SB (5 mM/48 h) caused an approximately 50% decrease in total receptors, whereas the affinity for 1,25(OH)2D3 was unchanged. At 5 mm, the action of SB on these receptors required more than 24 h to be detected. The effect of the decrease in receptors on the functional response to the hormone was studied by measuring the 1,25(OH)2D3 induction of 24-hydroxylase activity after treatment with SB. The induction of 24-hydroxylase activity at higher doses of hormone paralleled the reduction in receptors and was diminished by 25-50%. At low doses of hormone, the cells appear to be more sensitive to 1,25(OH)2D3 induction, exhibiting an unexplained increase in 24-hydroxylase activity compared to cells not exposed to SB. An additional effect of SB was also noted: SB decreased cell proliferation and inhibited [3H]thymidine incorporation by 75% when added to cells prior to confluence. At confluence, SB caused a less drastic effect on protein and DNA synthesis. Therefore, most binding experiments were conducted at confluence when the SB effect on cell proliferation was less. Other short chain fatty acids in addition to SB were also tested. The action to decrease 1,25(OH)2D3 receptors was more specific upon exposure to SB. We have previously demonstrated up-regulation of 1,25(OH)2D3 receptors in LLC-PK1 cells after treatment with various vitamin D metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Biological activity of 19-nor, 10-keto, 25-hydroxyvitamin D3.

19 nor, 10 keto, 25-hydroxyvitamin D3 (19/10-25OHD3) is a metabolite of 25-OHD3 produced in vitro by various phagocytes including normal human blood monocytes and transformed cell lines, U937 and HL-60. We recently reported that 19/10-25OHD3, induced differentiation of U937 cells. In these studies, 19/10-25OHD3 alone produced no detectable effect on the growth rates, surface adherence, and oxidative metabolism of U937 and HL-60 cells. When combined with lymphocyte-conditioned medium (LCM), 19/10-25OHD3 reduced proliferation, increased surface adherence and stimulated luminol-dependent luminescence (LDL) of the U937 cells. In contrast, the combination of 19/10-25OHD3 and LCM had no effect on the growth of HL-60 cells but did increase the surface adherence and the expression of a complement receptor component. 19/10-25OHD3 competed for tritium-labeled 1,25(OH)2D3 binding to receptors extracted from cultured human skin fibroblasts. This displacement capacity was 600 times weaker than that of unlabeled 1,25(OH)2D3. Incubation of human skin fibroblasts for 24 hr with 19/10-25OHD3 induced 25OHD3-24-hydroxylase activity in the fibroblasts. The inductive potency of 19/10-25OHD3 was 1/50 that of 1,25(OH)2D3. These results demonstrate bioactivity of 19/10-25OHD3 in several systems. At least one of these responses, the induction of 25OHD3-24-hydroxylase, is a receptor-mediated event. Some of the other responses may be independent of the cellular receptor for 1,25(OH)2D3. Interestingly, the potency of 19/10-25OHD3 was highest in the receptor-mediated response (1:50) and lower in the other parameters, ranging from 1:100 to 1:600 compared to 1,25(OH)2D3. This range of bioactivity in phagocytes and fibroblasts is presently explained.

Calcifediol↗

PubMed1987

Measurement of 1,25-dihydroxyvitamin D3 receptor turnover by dense amino acid labeling: changes during receptor up-regulation by vitamin D metabolites.

We have previously demonstrated that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors are up-regulated by exposure to 24,25-(OH)2D3 and other vitamin D metabolites in several mammalian cell cultures systems as well as in vivo in rats. The goal of the current study was to determine how changes in receptor turnover could give rise to increased receptor levels after treatment with 24,25-(OH)2D3, whether by an increased rate of synthesis or a decreased rate of degradation. Receptor turnover was studied by the dense amino acid shift technique in cultured pig kidney cells (LLC-PK1). In control cells, the half-life of the 1,25-(OH)2D3 receptor was estimated to be 4.3 +/- 0.4 h, with a degradation rate constant (kD) of 0.16 +/- 0.01 h-1. In parallel experiments, cells were treated with 24,25-(OH)2D3 for about 48 h to reach a new up-regulated steady state before the measurement of receptor turnover. In up-regulated cells, the receptor half-life was prolonged to 8.9 +/- 1.0 h and the kD was 0.07 +/- 0.01 h-1. Assuming a steady state, the values obtained for total receptor levels and the degradation rate allow calculation of the synthesis rate (kS) at equilibrium. Control cells synthesized receptors at the rate of 3.4 +/- 0.4 vs. up-regulated cells at 4.9 +/- 0.5 fmol mg protein-1 h-1. In conclusion, the data indicate that up-regulation of receptors after exposure of cells to vitamin D metabolites results predominantly from a prolongation of receptor half-life in addition to a relatively smaller increase in the rate of receptor synthesis.

24,25-Dihydroxyvitamin D 3↗

PubMed1987

Estrogen down-regulation of androgen receptors in cultured human mammary cancer cells (MCF-7).

Estrogens and androgens are well known to exert opposing effects in several tissues. In this study, we explored the possibility that there might be a direct antiandrogenic effect of estradiol on human breast cancer cells (MCF-7). Since the biological activity of androgens is mediated by specific androgen receptors, and because the abundance of androgen receptors in target tissues is thought to be rate limiting for androgen action, we examined whether estradiol regulates the quantity of androgen receptors in MCF-7 cells. Cells treated with 2.6 nM estradiol exhibited markedly lower levels of cytoplasmic androgen receptors, measured by [3H]5 alpha-dihydrotestosterone ([3H]DHT) binding, compared to levels in cells receiving ethanol vehicle alone. The effect was time dependent, and 6-day treatment of cells with estradiol resulted in an 80% reduction in [3H]DHT binding. Occupancy of androgen receptors by estradiol did not account for this difference. Cytosol competition studies demonstrated that the androgen receptor in MCF-7 cells possesses an approximately 125-fold lower affinity for estradiol than for DHT. In addition, tamoxifen, a nonsteroidal estrogen antagonist, blocked the estradiol effects on [3H]DHT binding. These latter studies support the hypothesis that this estradiol action is mediated by the estrogen receptor. Equilibrium binding studies indicated that the observed decrease in [3H]DHT binding after estradiol treatment was due to an absolute decrease in the number of cytoplasmic androgen receptors per cell. The estradiol-mediated reduction in androgen receptor content was dose dependent; a 50% reduction in androgen receptor number was observed after 6 days of treatment with 2.6 X 10(-11) M estradiol. Additional experiments revealed that MCF-7 cells exhibited a time-dependent increase in androgen receptor content when estradiol was withdrawn; continued estradiol treatment prevented this rise in receptor content. Moreover, androgen receptor levels began to decrease from the point when the ethanol vehicle added to the medium was replaced with 2.6 nM estradiol. In summary, estradiol treatment caused a reduction in androgen receptors, and estradiol withdrawal lead to a rise in androgen receptors. We believe that these results provide a mechanism whereby estradiol may directly antagonize androgen action. Conversely, estradiol withdrawal may potentiate androgen action by allowing androgen receptor levels to rise. This hypothesis may help explain the basis of the estrogen/androgen ratio as a predictor of sex steroid response.

Binding, Competitive↗

PubMed1986

Replacement of liver function in rats by transplantation of microcarrier-attached hepatocytes.

Isolated hepatocytes, harvested from normal rat livers by portal vein collagenase perfusion, can be attached to collagen-coated dextran microcarriers and transplanted by intraperitoneal injection into rats. Survival and function of the transplanted hepatocytes have been demonstrated in mutant rats lacking bilirubin-uridine diphosphate glucuronosyltransferase activity (Gunn strain) and rats with inherited lack of plasma albumin (Nagase analbuminemia rat strain). This simple technique promises to be useful in the treatment of acute liver failure in humans.

Animals↗

PubMed1986

Homologous up-regulation of the 1,25 (OH)2 vitamin D3 receptor in rats.

This study investigates the ability of vitamin D-metabolites to regulate 1,25(OH)2D3 receptors in vivo. Rats made vitamin D-deficient were treated with 1,25(OH)2D3 or vehicle for 1-5 days. In treated animals, receptors for 1,25(OH)2D3 in kidney increased dramatically compared with control levels. An increase in specific binding to 220% of control was seen after 2 doses of hormone, which reached to 336% after 5 days of treatment. Intestinal receptors increased to only 130% of control levels after 5 days of treatment. In vitamin D-replete animals, the difference between control and treated groups was slightly greater when endogenously occupied sites were measured by exchange (TPCK). However, significant changes were observed only after 4 days of hormone treatment. The data indicate that homologous up-regulation of the 1,25(OH)2D3 receptor occurs in vivo. The difference in response in kidney and in intestine suggests differential importance of up-regulation in various organs.

Animals↗

PubMed1986

Hormonal responses to 1,25-dihydroxyvitamin D3 in cultured mouse osteoblast-like cells--modulation by changes in receptor level.

The level of 1,25(OH)2D3 receptors in cultured mouse osteoblast-like (OB) cells is modulated by the rate of cell proliferation. We have studied two 1,25(OH)2D3-induced bioresponses to ascertain whether the changes in receptor levels during growth in culture alter cell responsiveness. Nuclear receptor levels were high (127 fmol/100 micrograms DNA) in rapidly dividing (log) cells and low (25 fmol/100 micrograms DNA) in quiescent (confluent) cells. The bioresponses we studied were induction of 25(OH)D3-24-hydroxylase activity (24-hydroxylase) and inhibition of collagen synthesis. The basal levels of 24-hydroxylase were low and similar in cells at log growth phase and confluence. At a maximal induction dose of 13 nM, 1,25(OH)2D3 induced a three-fold rise in enzyme activity at long growth phase, but only caused less than two-fold rise at confluence. The half-maximal dose (ED50) was slightly shifted from 0.6 nM to 0.8 nM. Daily measurement of 1,25(OH)2D3 receptor levels and maximal induction of 24-hydroxylase activity throughout the culture cycle showed a strong correlation between receptor abundance and enzyme induction. The basal level of collagen synthesized by cells in log growth phase was approximately 5% and increased to approximately 8% at confluence. Maximal inhibition of collagen synthesis by 1,25(OH)2D3 reached 80% of control levels in log cells, but was only 40% of control in confluent cells. The ED50 was approximately 0.1 nM in the log cells and increased to approximately 1 nM at confluence. Daily assay of 1,25(OH)2D3 receptor levels and 1,25(OH)2D3 responses during the culture cycle indicated a correlation between changes in receptor level and the extent of inhibition of collagen synthesis. These changes in bioresponse at various growth phases did not occur in rat OB cells where the 1,25(OH)2D3 receptor levels were independent of cell proliferation. The results indicate that cell proliferation rate, via change in receptor levels, determines the magnitude and sensitivity of the cellular responses to 1,25(OH)2D3.

Animals↗