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R U Simpson

Publications and source records attributed to R U Simpson.

At least 55 records · Page 3Linked to original sources
PubMed1986

Isolation, stages of differentiation, and long term maintenance of adult rat myocytes.

Although a variety of techniques have been developed to isolate myocytes from adult hearts, the long term viability of such cells has only recently been investigated. In addition, relatively little is known about the stages of differentiation such cells proceed through following isolation. In the present study myocytes were isolated using two techniques, one involving retrograde perfusion via the aorta, and the other involving mechanical "shearing." In addition, several modifications were made to minimize the trauma normal associated with isolating myocytes from adult hearts. Both techniques yielded a high percentage of rod-shaped, quiescent myocytes, although myocytes isolated using the "shearing" method were less likely to remain viable for more than 24 hours. With both techniques those cells which remained viable for more than 24 hours proceeded through an identical pattern of differentiation leading to stable, attached cells which remained viable for up to four weeks. These results demonstrate that with the appropriate isolation techniques it is possible to maintain adult myocardial cells in culture for lengthy periods of time.

Animals↗
PubMed1986

Calcium antagonizes 1,25-dihydroxyvitamin D3 inhibition of breast cancer cell proliferation.

The hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited proliferation of two breast cancer cell lines (HT-39 and MCF-7) in a dose-dependent manner. We showed that 10 nM 1,25-(OH)2D3 inhibited HT-39 cell growth by 26.3 +/- 3.2% and MCF-7 proliferation by 19.5 +/- 8.6% (mean +/- SE). When both cell lines were cultured in the presence of medium containing varying concentrations of calcium, analysis of cell growth revealed that lowering the medium Ca2+ concentration from 1.3 to 0.1 mM inhibited cell proliferation of HT-39 cells by 20.0 +/- 2.3% and that of MCF-7 cells by 13.4 +/- 3.4% (mean +/- SE). Raising medium Ca2+ from 1.3 to 2.6 mM stimulated cell proliferation of HT-39 cultures by 51.8 +/- 2.3% and that of MCF-7 cells by 13.3 +/- 3.3% (mean +/- SE). When HT-39 cells were grown in medium containing different calcium levels and dosed with 10 nM 1,25-(OH)2D3, we found that the inhibitory action of 1,25-(OH)2D3 was modified. HT-39 cells in medium containing 0.1 mM calcium were more potently inhibited by 10 nM 1,25-(OH)2D3 (increased by 74% relative to that in cells in 1.3 mM calcium). Moreover, the inhibitory action of 1,25-(OH)2D3 (10 nM) on HT-39 cells grown in 2.6 mM calcium media was decreased by 68% relative to that in cells in 1.3 mM extracellular calcium. The phorbol ester 4 beta-phorbol 12,13-dibutyrate (PDB) stimulated HT-39 proliferation by 30.1 +/- 4.1%. Cells treated with PDB showed increased cell mitotic index, but unlike extracellular calcium, PDB had no effect on the dose response of 1,25-(OH)2D3 to inhibit HT-39 cell growth. We show that 1,25-(OH)2D3 action on malignant cell proliferation is dependent on the extracellular calcium concentration. The data suggest that the effect of calcium to antagonize 1,25-(OH)2D3 on HT-39 cells is not shared by all agents (e.g. PDB) that increase HT-39 cell proliferation.

Breast Neoplasms↗
PubMed1986

High performance liquid chromatography analysis of 1,25-dihydroxyvitamin D3 receptor in malignant cells. Correlation of effects on cell proliferation and receptor concentration.

Malignant cells were assayed for 1,25(OH)2D3 receptors and for the effects of 1,25(OH)2D3 on cell proliferation. The established lines studied were human promyelocytic leukemia (HL-60), T-cell lymphocytic leukemias (Molt-4, RPMI-8402, CEM), mouse leukemia (L1210), breast cancers (HT-39 and MCF-7) and a glioma (C-6) cultures. A TSK 3000 SW (0.75 X 60 cm) HPLC size exclusion column was used to characterize specific 1,25(OH)2D3 binding. We show for the first time that this column is capable of resolving the 3.2-3.5S 1,25(OH)2D3 mammalian receptor (Rs = 32 A) from the 5.5-6.0S form of the mammalian serum 25(OH)D3 transport receptor (Rs = 40 A). The molecular size of the 1,25(OH)2D3 receptors from these cancer cell lines was identical to that from rabbit intestine. HT-39, HL-60, MCF-7, Molt-4, C-6, RPMI-8402 and L1210 cells demonstrated specific 1,25(OH)2[3H]D3 binding (120, 90, 80, 45, 30 and 18 fmoles of sites/mg protein, respectively). Receptors were not detected in the CEM line. 1,25(OH)2D3 inhibited cell proliferation of HT-39, HL-60, MCF-7 and Molt-4 cells by 20% to 70%. In contrast, mouse leukemia (L1210) cells were stimulated to proliferate by this hormone. Proliferation of RPMI and CEM cells was not affected by 1,25(OH)2D3. We demonstrate that size-exclusion HPLC of 1,25(OH)2D3 binding proteins from mammalian intestine and cancer cells provided a rapid method for identification of specific 1,25(OH)2D3 receptors. Furthermore, in the cells studied, the presence and concentration of 1,25(OH)2D3 receptors qualitatively predicted the potency of this hormone to alter cell proliferation. We believe this assay will be useful for rapid analysis of human tumor receptor concentrations.

Animals↗
PubMed1986

Effects of calcitriol on nuclear transcription during human HL-60 promyelocytic leukemia cell differentiation.

An HL-60 in vitro transcription assay was developed and used to monitor HL-60 human promyelocytic leukemia cell transcription during calcitriol-induced monocytic differentiation. Treatment of cells with calcitriol (50 nM) for 96 h produced a 48% reduction in total DNA-dependent RNA polymerase activity with a corresponding reduction in alpha-amanitin-sensitive (RNA polymerase II) activity. Nuclei isolated from cells treated with the less efficacious vitamin D analogues, 24,25-dihydroxyvitamin D3, and 1 alpha-hydroxyvitamin D3, did not exhibit nuclear transcription that was significantly different from control nuclei. Transcription in nuclei isolated from calcitriol-treated cells was decreased by 40 to 50% concomitant with monocytic differentiation, as assayed by acquisition of nitroblue tetrazolium reducing activity. Maximal decrease in transcription was achieved by 45 h postinduction, whereas expression of nitroblue tetrazolium reducing activity peaked at approximately 70 h, and decreased DNA synthesis was evident by 48 h. These observations suggest that calcitriol induction of HL-60 differentiation results in monocytes with reduced requirements for gene transcription and that transcription changes accompany expression of nitroblue tetrazolium reducing activity.

Calcitriol↗
PubMed1985

Identification of 1,25-dihydroxyvitamin D3 receptors and activities in muscle.

Cytosols from cultured myoblast cells (G-8 and H9c2) prepared in high salt (0.3 M KCl) possesses receptor like proteins for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) that sediment in the 3.2 S region of sucrose gradients. These receptors were characterized as having high affinity (Kd less than 0.1 nM) for 1,25-(OH)2D3 and are in low capacity (less than 80 fmol/mg of cytosol protein). Analog competition for receptor binding revealed that 1,25-(OH)2D3 was more potent than 24,25-(OH)2D3, or 25-(OH)2D3 for displacement of 1,25-(OH)2[3H]D3 from these 3.2 S region sedimenting receptors. Furthermore, the receptor proteins had affinity for DNA and eluted from Sephacryl S-200 as a macromolecule with Stokes radius (Rs) of 32 A. High salt cytosol from collagenase-dispersed skeletal muscle cells was also found to possess a 3.2 S 1,25-(OH)2D3 receptor-like protein. The 1,25-(OH)2D3 receptor concentration in both G-8 and H9c2 myoblast lines was found to down-regulate by 50-70% when cells were stimulated to differentiate to myotubes by lowering fetal calf serum to 5% of the medium. Moreover, we demonstrated that 1,25-(OH)2D3 can inhibit DNA synthesis and cell proliferation of the G-8 myoblast cells in a dose-dependent manner. 1,25-(OH)2D3 was more potent at inhibiting cell proliferation in cells grown in 5% serum than in 20% serum. The data suggest that 1,25-(OH)2D3 can act directly on muscle myoblast via a 1,25-(OH)2D3 receptor that is similar to those found in intestine and bone. The data support the possibility that muscle is a target tissue for 1,25-(OH)2D3 and the hormone may act to initiate terminal differentiation of myoblast cells.

Animals↗
PubMed1985

Molybdate and the 1,25-dihydroxyvitamin D3 receptor from chick intestine.

The nature of the 1,25-dihydroxyvitamin D3 receptor from chick intestine was examined in regard to its response to sodium molybdate. Sodium molybdate (10 mM) stabilized the receptor from crude nuclear extract but not that from the supernatant or cytoplasmic fraction, suggesting the molybdate may act by binding to the DNA binding region of the receptor. At a concentration of 50 mM, sodium molybdate prevented aggregation of the nuclear receptor. This concentration of sodium molybdate also inhibited the receptor from binding to DNA cellulose while the same ionic strength KCl (90 mM) did not. These properties also suggest that molybdate interacts with the DNA binding region. Purification of the receptor using DNA cellulose chromatography has also been improved by using a sodium molybdate gradient (0-0.2 M) instead of the KCl gradient used previously.

Animals↗
PubMed1984

Metabolism of 25-hydroxyvitamin D3 by rat kidney cells in culture: isolation and identification of cis- and trans-19-nor-10-oxo-25-hydroxyvitamin D3.

A primary confluent culture of epithelial cells from rat kidney has been developed. These cells possess a 3.2-3.4 S high-affinity, low-capacity binding protein for 1,25-dihydroxyvitamin D3. They metabolize 25-hydroxyvitamin D3 to at least five metabolites. Two have been identified as 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. Two others have been identified by means of physical data and cochromatography as trans 19-nor-10-oxo-25-hydroxyvitamin D3 and the other as its cis isomer. These two "metabolites" have not been observed in vivo, but one of them (cis) comigrates with 1,25-dihydroxyvitamin D3 on straight-phase high-performance liquid chromatography. Thus, mere cochromatography on high-performance liquid chromatography is not sufficient to identify critical vitamin D metabolites.

Animals↗
PubMed1984

Subcellular distribution of DNA-binding and non-DNA-binding 1,25-dihydroxyvitamin D receptors in chicken intestine.

A comparison of 1,25-dihydroxyvitamin D3 receptor concentration and equilibrium dissociation constants of whole tissue, nuclear, and cytosol extracts of vitamin D-deficient chicken intestine has been carried out. The extracts had the following order of receptor concentration: nuclei greater than whole tissue greater than cytosol. The receptors in each preparation had identical equilibrium dissociation constants (Kd) for 1,25-dihydroxyvitamin D3. However, the receptor in the cytosol fraction did not bind to DNA-cellulose, while a large fraction of the receptor population in crude nuclear extract and whole tissue extract bound to DNA-cellulose and could be eluted with 0.22 M KCl, suggesting that the cytosolic form of the receptor does not possess a DNA binding site.

Animals↗
PubMed1984

Angiotensin and prostaglandin interactions in cultured kidney tubules.

Angiotensin and prostaglandin interactions in cultured kidney tubules were studied in tissue from fetal calves. Methods were developed for the isolation and culture of renal proximal tubule cells. Tubule cells survived three generations in culture. They had microvilli and flagellae characteristic of proximal tubule cells. Binding of 125I-angiotensin II to receptor-like sites in cells and homogenates was partially saturable, and the saturable binding was reversed by excess unlabeled hormone. Two types of binding sites were identified by Scatchard analysis. The higher-affinity site had a dissociation constant of 5 X 10(-10)M. PGE2 and PGA2 inhibited angiotensin binding. PGF2 alpha had no effect. Cultured tubule cells were loaded with 22Na+ by incubation in hypoxic medium free of potassium and glucose. Cells extruded the sodium when oxygen, glucose, and potassium were added. The rate of extrusion was accelerated by angiotensin II at concentrations of 10(-10)M and 10(-9)M. Higher concentrations had less effect. The primary prostaglandins PGA2 and PGF2 alpha inhibited 22Na+ efflux at 6 X 10(-7)M. Angiotensin had no detectable effect on sodium efflux in the presence of PGA2. Angiotensin apparently reversed inhibition of efflux by PGF2 alpha. Our findings suggest that prostaglandins affect angiotensin receptors in renal tubule cells. Prostaglandins also have direct effects on sodium efflux in these cells under the experimental conditions described.

Angiotensin II↗
PubMed1983

Purification of the receptor for 1 alpha,25-dihydroxyvitamin D3 from chicken intestine.

Methods were investigated for use in the purification of the chicken intestinal receptor for 1 alpha,25-dihydroxyvitamin D3. The techniques investigated include column isoelectric focusing, gel exclusion, polyacrylamide gel electrophoresis, and DNA-cellulose, DEAE-cellulose, and hydroxylapatite chromatography. For the starting receptor preparation, a nuclear extract of chicken intestinal mucosa was found to be enriched above cytosol preparations and a plentiful source of receptor. A five-step purification scheme that resulted in the purification of the receptor protein by 5800-fold with 8% yield has been described. Analysis of the purified proteins on polyacrylamide gel electrophoresis containing sodium dodecyl sulfate suggests homogeneity. Analysis using two-dimensional polyacrylamide electrophoresis characterized the purified protein as having a molecular weight of approximately 63 000 and a pI of 6.0-6.2. Furthermore, assessment of protein purity by 125I iodination followed by sucrose gradient analysis revealed that approximately 90% of the iodinated macromolecules have the same sedimentation coefficient as the titrated 1 alpha,25-dihydroxyvitamin D3 receptor complex. The final purified receptor that bound tritiated 1 alpha,25-dihydroxyvitamin D3 retained affinity for DNA-cellulose and possesses a 3.7S sedimentation coefficient. The receptor has an estimated Stokes radius of 37 A.

Animals↗
PubMed1983

Quantitation of endogenously occupied and unoccupied binding sites for 1,25-dihydroxyvitamin D3 in rat intestine.

The quantitative reversible dissociation of the 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-receptor complex by the mercurial reagent mersalyl was used to develop an assay for endogenously occupied and unoccupied 1,25-(OH)2D3 binding sites. Incubation of intestinal cytosol preparations in buffer containing 50 mM Tris . HCl, 300 mM KCl, and 1.5 mM EDTA, pH 7.4, with 1 mM mersalyl for 60 min was effective in inhibiting 98% of 1,25-(OH)2D3 specific binding activity. Dissociation of bound 1,25-(OH)2[26,27-3H]D3 from the hormone-receptor complex approached completion by 180 min. In cytosol incubated with saturating levels of nonradioactive hormone, 96% of total binding activity was measurable with the hormone binding assay after displacement of bound nonradioactive ligand with 1 mM mersalyl. Endogenously occupied 1,25-(OH)2D3 binding sites contributed 0, 9, and 19% of total binding activity in rats with plasma 1,25-(OH)2D3 levels averaging 2, 121 +/- 36 and 516 +/- 92 pg/ml, respectively. Therefore, the major fraction of cytosolic 1,25-(OH)2D3 specific binding activity is unoccupied in rat intestine. The results suggest that only a small proportion of the measurable receptors are in the bound form to provide maximal 1,25-(OH)2D3-induced calcium transport.

Animals↗
PubMed1983

Glucocorticoids and appearance of 1,25-dihydroxyvitamin D3 receptor in rat intestine.

The ontogenesis of the 1,25-dihydroxyvitamin D3 specific binding activity in intestine was examined in vitamin D-deficient and replete rats. The absence of binding activity in intestines during the first two postnatal weeks was not influenced by vitamin D supplementation. The concentration of binding sites peaked on day 18 in vitamin D-replete rats and preceded that in the deficient group by approximately 1 wk. The influence of glucocorticoids on 1,25-dihydroxyvitamin D3-binding protein levels was examined by sequential hydrocortisone administration and adrenalectomy. Subcutaneous hydrocortisone administration before day 14 postpartum did not induce binding activity. The concentration of binding sites was significantly increased to 369 +/- 60 fmol/mg of protein by hydrocortisone injections from days 15 to 17 postpartum when compared with an average of 182 +/- 16 fmol/mg of protein in littermate controls. Hydrocortisone administration did not further increase receptor levels in rats injected from days 19 to 21. Bilateral adrenalectomy on day 17 postpartum significantly decreased the concentration of binding sites. It is concluded that adrenal glucocorticoids play an important role in the developmental appearance of 1,25-dihydroxyvitamin D3 specific binding activity in the postnatal rat intestine.

Adrenalectomy↗
PubMed1982

Purification of chicken intestinal receptor for 1 alpha, 25-dihydroxyvitamin D3 to apparent homogeneity.

The chicken intestinal 1 alpha, 25-dihydroxyvitamin D3 receptor-like protein has been purified to apparent homogeneity as determined by sodium dodecyl sulfate gel electrophoresis. The techniques employed for the purification include selective precipitation of the receptor by Polymin P (polyethyleneimine) and (NH4)2SO4 and batch adsorption to and selective elution from hydroxylapatite, followed by gel exclusion and DEAE-cellulose chromatography. Finally, the labeled receptor was eluted at a pH of approximately 6.0 on a chromatofocusing column. The protein was purified 6100-fold and the receptor was obtained in 9% yield.

Animals↗
PubMed1981

Angiotensin receptors in bovine umbilical artery and their inhibition by nonsteroidal anti-inflammatory drugs.

1 The contractile effect of angiotensin II on bovine isolated umbilical arteries was compared to [125I]-angiotensin II binding by a subcellular fraction of that tissue. The ED50 of angiotensin was 3.1 +/- 2.8 x 10(-8) M, while the apparent dissociation constant was 4.9 +/- 1.6 x 10(-9) M. 2 Indomethacin, meclofenamate, and eicosatetraynoic acid inhibited angiotensin-induced contraction of the isolated artery and binding to a particulate fraction at comparable doses. Phenylbutazone inhibited [125I]-angiotensin binding more potently than the response. Inhibition by the first three agents was noncompetitive, whereas phenylbutazone inhibited competitively. 3 Inhibition of angiotensin activity by the nonsteroidal anti-inflammatory agents was not specific. These agents also inhibited 5-hydroxytryptamine-induced contraction, but not the contraction induced by KCl. 4 The data suggest that the angiotensin binding sites studied include receptors that mediate contraction of the isolated umbilical artery. Our data also indicate that indomethacin, meclofenamate, eicosatetraynoic acid and phenylbutazone are capable of direct inhibitory effects on receptors, as well as their well-known synthetase actions. The net effect of these activities will determine the change these agents cause in tissue responses to hormones.

Angiotensin II↗
PubMed1980

Characterization of a specific, high affinity binding macromolecule for 1 alpha, 25-dihydroxyvitamin D3 in cultured chick kidney cells.

Cytosol prepared from vitamin D3-deficient kidney cells in culture contains a 3.7 S protein that specifically binds 1,25-dihydroxyvitamin D3 with high affinity and low capacity. Whole kidney homogenate cytosol preparations are shown to possess two 1,25-dihydroxyvitamin D3 binding macromolecules. One of the binding proteins sediments at 3.5 to 3.7 S while the second sediments at 6.0 S. The 6.0 S component has a greater affinity for 25-dihydroxyvitamin D3 than for 1,25-dihydroxyvitamin D3. Cultured cell cytosol was found to have little 6.0 S 25-hydroxyvitamin D3 binding protein. Scatchard analysis of the cultured cell cytosol reveals an equilibrium binding constant (KD) of 5.6 x 10 (-11) with 57 fmol of sites/mg of protein. The receptor-like protein has a Mr = 72,000 and as with other steroid receptors it aggregates in the presence of low potassium concentrations. Analog competition for receptor binding reveals the following potency order: 1,25-dihydroxyvitamin D3 > 25-hydroxyvitamin D3 > 1 alpha-hydroxyvitamin D3 > 24(R),25-dihydroxyvitamin D3; the receptor had no detectable affinity for vitamin D3. The kidney cells respond to 1,25-dihydroxyvitamin D3 by diminishing 25-hydroxyvitamin D3 1 alpha-hydroxylation and increasing 24R-hydroxylation. Cultured cells provide a preparation of cytosol which has allowed extensive characterization of the renal 1,25-dihydroxyvitamin D3 receptor and should facilitate investigations into the role this receptor plays in renal control of vitamin D3 metabolism.

Animals↗