Regulation of the glucocorticoid receptor by glucocorticoids in human mononuclear leukocytes

We analyzed glucocorticoid receptor binding in peripheral blood mononuclear leukocytes from normal adult males and from females at the follicular and luteal phases. Healthy controls were analyzed before and after 17 days of treatment with two synthetic glucocorticoids: prednisone and an oxazoline derivative of prednisolone (deflazacort). We also studied for comparison 4 patients with adrenocortical insufficiency, two of them on long-term corticoid replacement, and 7 patients with Cushing's syndrome. Using a whole-cell competitive binding assay and 3H-dexamethasone as tracer, normal human mononuclear leukocytes (19 males, 6 females) were found to have 4,529 +/- 1,532 (mean +/- SD) binding sites per cell and a dissociation constant (Kd) of 9.5 +/- 2.3 nM. In Cushing's syndrome the receptor parameters were within the normal range. Cells from patients with untreated Addison's disease had low levels of sites per cell. The number of binding sites increased to normal after long-term glucocorticoid replacement. All the adrenal insufficiency cases had a normal Kd. Finally, following treatment with the synthetic glucocorticoid, deflazacort, the sites per cell were reduced but the Kd remained unchanged. Prednisone had no effects.     

The effect of diet on ouabain binding to erythrocytes from obese subjects

Ouabain binding to erythrocyte membranes is increased in obese subjects. Three study groups are compared: 14 reference subjects, 102 +/- 16% of ideal weight; 9 obese on unrestricted diets, 207 +/- 16% of ideal weight; 11 obese on restricted diets, 202 +/- 35% of ideal weight. A reproducible (CV = 11.3%) ouabain-binding assay is used to measure Na+-K+ ATPase sites in erythrocyte membranes. The number of binding sites per red blood cell for obese subjects on unrestricted diets, 431 +/- 30, is greater than for the reference group, 346 +/- 66 (p less than 0.01), or for obese subjects on restricted diets, 371 +/- 68 (p less than 0.05). These data suggest that caloric intake influences the number of Na+-K+ ATPase sites. Scatchard plots indicate only one type of binding site for ouabain with an affinity constant of about 3 X 10(8) M-1.     

Binding of Escherichia coli protein synthesis initiation factor IF1 to 30S ribosomal subunits measured by fluorescence polarization

The interaction of initiation factor IF1 with 30S ribosomal subunits was measured quantitatively by fluorescence polarization. Purified IF1 was treated with 2-iminothiolane and N-[[(iodoacetyl)-amino]ethyl]-5-naphthylamine-1-sulfonic acid in order to prepare a covalent fluorescent derivative without eliminating positive charges on the protein required for biochemical activity. The fluorescent-labeled IF1 binds to 30S subunits and promotes the formation of N-formylmethionyl-tRNA complexes with 70S ribosomes. Analyses of mixtures of fluorescent-labeled IF1 and 30S ribosomal subunits with an SLM 4800 spectrofluorometer showed little change in fluorescence spectra or lifetimes upon binding, but a difference in polarization between free and bound forms is measurable. Bound to free ratios were calculated from polarization data and used in Scatchard plots to determine equilibrium binding constants and number of binding sites per ribosomal subunit. Competition between derivatized and nonderivatized forms of IF1 was quantified, and association constants for the native factor were determined: (5 +/- 1) X 10(5) M-1 with IF1 alone; (3.6 +/- 0.4) X 10(7) M-1 with IF3; (1.1 +/- 0.2) X 10(8) M-1 with IF2; (2.5 +/- 0.5) X 10(8) M-1 with both IF2 and IF3. In all cases, 0.9-1.1 binding sites per 30S subunit were detected. Divalent cations have little effect on affinities, whereas increasing monovalent cations inhibit binding. On the basis of the association constants, we predict that greater than 90% of native 30S subunits are complexed with all three initiation factors in intact bacterial cells.     

Endogenous Tumor necrosis factor alpha unmasks the binding sites for N-acetylglucosaminyl-(beta1-4)-N-acetylmuramyl-alanyl-D-isoglutamine on the surface of melanoma cells

The surface of the melanoma BRO cells was shown to contain binding sites for N-acetylglucosaminyl-(beta 1-4)-N-acetylmuramyl-alanyl-D-isoglutamine (GMDP). Their number (1500 +/- 200 per cell) and affinity (Kd = 10 +/- +/- 1.2 nM) were determined. The occurrence of these sites was found to correlate with the ability of the melanoma cells to react in vitro with GMDP by increasing the expression of melanoma-associated antigens (MAA). An increased number of the GMDP binding sites (5200 +/- 500 per cell) was observed upon treating the melanoma BRO cells with tumor necrosis factor alpha (TNF-alpha). The mechanism of the TNF-alpha action most likely involves the unmasking of GMDP binding sites, initially expressed on the cell surface, by activating the endogenous protease that hydrolyzes surface proteins, in particular, highly glycosylated LAMP-2 protein exposed on the melanoma cell surface.     

Differential binding of dexamethasone to ammonium sulfate precipitates of human adipose tissue cytosols

Saturation analysis of the binding of [3H]dexamethasone [( 3H]DEX) to ammonium sulfate precipitates (ASPs) confirmed the presence of a limited-capacity, high-affinity binder in human adipose tissue cytosols. Various non-radioactive steroids competed with [3H]DEX for binding to the ASPs in the following sequence: dexamethasone (DEX) approximately equal to triamcinolone acetonide (TA) greater than progesterone (P) much greater than estradiol (E2). The steroid specificity of the binder precipitated by AS was consistent with the specificities reported for glucocorticoid receptors in a number of systems. In order to investigate possible regional differences, glucocorticoid binding to ASPs derived from adipose tissues removed from two different sites in the same subject was quantitated. ASPs of human omental adipose tissue bound significantly more [3H]DEX than did similar preparations of subcutaneous adipose tissue from the abdominal wall (116 +/- 32 vs. 50 +/- 22 fmol/mg protein; mean +/- SD; p less than 0.02). The findings are consistent with reports from other laboratories suggesting that intra-abdominal fat is more responsive to glucocorticoids than is subcutaneous adipose tissue.     

Inhibitory effect of physiological concentrations of cortisol but not estradiol on interleukin (IL)-6 production by human osteoblast-like cell lines with different constitutive IL-6 expression

Estrogen deficiency and glucocorticoid excess are two well-known conditions that account for osteoporosis. Interleukin (IL)-6 plays an important role in bone resorption; both estrogens and glucocorticoids are credited with an inhibitory effect on osteoblast production of IL-6. The aim of the study was to investigate whether endogenous hormones, which lead to opposite changes in bone mass, have a common inhibitory effect upon constitutive and inducible IL-6 production by human osteoblast-like cells. We used two human osteosarcoma cell lines (MG-63 and Saos-2) with a different degree of differentiation and constitutive production of IL-6 [2587+/-536 (mean+/-SE) and 3.65+/-0.06 pg/10(6) cells, respectively]. We examined the effects of physiological and supraphysiological concentrations of 17beta-estradiol (E2) and cortisol on basal and IL-1beta-induced IL-6 release in the medium. In all experimental conditions, cellular estrogen receptors (ERs) and glucocorticoid receptors (GRs) were measured by binding assay. Both MG-63 and Saos-2 cell lines had measurable GRs (106 300+/-24 996 and 18 100+/-3215 binding sites/cell, respectively) and ERs (2197+/-377 and 1261+/-66.5 binding sites/cell, respectively). In MG-63 cells, cortisol treatment for 20 h decreased both basal and IL-1beta-induced IL-6 release in a dose-dependent manner; in Saos-2 cells the same effect was apparent for IL-1beta-induced release. Mifepristone (RU-486) did function as partial agonist and antagonist of cortisol. At variance with cortisol, E2 did not exert any effect on IL-6 secretion. Treatment with 1,25(OH)2D3 increased by 100-200% ER concentrations, but did not change ineffectiveness of E2 in modifying IL-6 production; furthermore, when E2 was combined with cortisol, there was no additive effect on cortisol-induced inhibition. The dissociation between glucocorticoid and estrogen effects observed in these human cell lines is a sufficiently robust phenomenon to raise questions about the pathogenetic role of IL-6 in osteoporosis associated with estrogen deficiency. Conversely inhibition of osteoblast production of IL-6 may offer an explanation why bone resorption is not the dominant factor in the pathogenesis of glucocorticoid-induced osteoporosis.     

Glucocorticoid receptor expression in human bronchial epithelial cells: effects of smoking and COPD.

Previously, we found that inflammatory mediators modulated the number and binding affinity of glucocorticoid receptors (GR) in human bronchial epithelial cell lines. In this study we investigated whether smoking and chronic obstructive pulmonary disease (COPD), both characterized by airway inflammation with increased levels of inflammatory mediators, affect GR characteristics in cultured human bronchial epithelial cells (HBEC). A statistically significant difference was found between the dissociation constant (Kd) values in HBEC from smoking (Kd = 0.98+/-0.08 nM; n = 6) and nonsmoking controls (Kd = 0.76+/-0.10 nM, P = 0.03; n = 5), but no significant difference was found between the mean number of binding sites. Our results are the first indication that cultured HBEC from smokers possess GR with a lower binding affinity. This may result from the inflammation found in the airways from smokers. Furthermore, these results provide further evidence that the bronchial epithelium may be an actual target for inhaled glucocorticoid therapy.     

Circadian and seasonal variations of glucocorticoid receptors in normal human lymphocytes

Circulating human lymphocytes are known to contain specific glucocorticoid receptors. Using a competitive binding whole cell assay, we have examined the binding of [3H] dexamethasone to peripheral lymphocytes of normal male subjects. Lymphocytes were found to contain 2000-10000 glucocorticoid receptor sites/cell and the Kd value was in the range of 0.5-9 X 10(-9) M. The number and affinity of glucocorticoid receptors did not change throughout a 1-year observation time. In contrast, there was a significant diurnal variation in receptor content (38% higher at 11 p.m. than at 8 a.m.), while receptor affinity did not change.     

IGFs, IGFBPs, IGF-binding sites and biochemical markers of bone metabolism during differentiation in human pulp fibroblasts

OBJECTIVE: To investigate the role of the insulin-like growth factors (IGF) system during the differentiation of human pulp-derived fibroblasts (HPF). METHODS: Primary HPF were cultured for 24 days in DMEM medium with IGF-I or IGF-II (50 ng/ml each). Cell growth and morphology, alkaline phosphatase (ALP) activity, the concentration of free deoxypyridinoline (DPD), IGF-I, -II, IGFBP-2 and -3 were studied. The number of (125)I-IGF-I binding sites was estimated by Scatchard analysis. RESULTS: Light-microscopically visible nodules emerged during differentiation. Simultaneously, the ALP activity increased steadily between days 8 and 24, while the DPD concentration decreased by about 50%. The HPF produced high concentrations of IGF-II (2.00-1.30 microg/10(6) cells) but low IGF-I, IGFBP-2. IGFBP-2 was not changed, IGFBP-3 increased by 65% during differentiation. The number of IGF binding sites increased from 8,500 +/- 55 per cell (day 8) up to 22,000 +/- 570 (day 24). CONCLUSION: The increasing number of IGF-binding sites accompanied by alterations in the biochemical bone markers during the differentiation of HPF suggests an autocrine/paracrine role for the IGFs in the formation of dentinal hard tissue.     

19-hydroxyandrostenedione does not modulate [3H]aldosterone binding to human mononuclear leucocytes and rat renal cytosol

To verify the aldosterone amplifying action of 19-hydroxyandrostenedione (19-OH-AD), we investigated [3H]aldosterone and [3H]19-OH-AD binding to type I (mineralocorticoid) receptor in the renal cytosol of adrenalectomized and ovariectomized rat, and human mononuclear leucocytes (MNL). In the [3H]aldosterone binding study, the cytosol was incubated with [3H]aldosterone and 200-fold RU28362 (11 beta,17 beta-dihydroxy-6-methyl,17 alpha-(1-propynyl)-androsta-1,4,6- trien-3-one), a pure glucocorticoid, with or without 19-OH-AD. Scatchard plots of [3H]aldosterone binding to cytosol with 0.2 or 20 nM 19-OH-AD or without 19-OH-AD were linear. Dissociation constants (Kd) and maximum bindings (Bmax) without 19-OH-AD, and with 0.2 and 20 nM 19-OH-AD were: 0.71 +/- 0.03 nM and 23.0 +/- 3.4 fmol/mg protein (mean +/- SD, n = 3), 0.72 +/- 0.05 nM and 23.1 +/- 2.3 fmol/mg protein (n = 3), and 0.77 +/- 0.04 nM and 22.9 +/- 4.8 fmol/mg protein (n = 3), respectively. 19-OH-AD did not significantly change the Kd and Bmax of [3H]aldosterone binding. A high concentration of 19-OH-AD slightly displaced 0.2 or 5 nM [3H]aldosterone bound to cytosol. In human MNL, Scatchard plots of [3H]aldosterone binding with both 0.2 and 20 nM 19-OH-AD and without 19-OH-AD were linear. Kd and Bmax were, respectively, 1.00 nM and 780 sites/cell in the absence of 19-OH-AD, and 1.07 nM and 774 sites/cell in the presence of 0.2 nM 19-OH-AD. Without 19-OH-AD they were, respectively, 0.95 nM and 551 sites/cell, and 1.10 nM and 560 sites/cell with 20 nM 19-OH-AD. A high concentration of 19-OH-AD slightly displaced 0.2 or 5 nM of [3H]aldosterone bound to MNL. In both tissues, there was no obvious specific binding of [3H]19-OH-AD within the range of 1-60 nM. The above results suggest that the amplifying effect of 19-OH-AD on aldosterone mineralocorticoid action may not occur at the binding site of aldosterone to type I receptor, and that 19-OH-AD itself may not have any direct or indirect mineralocorticoid actions on the steroid receptor-mediated process in the rat kidney and human MNL.     

EGF binding site number of rat anterior pituitary is increased by chronic administration with dopaminergic agonist, CV 205-502]

In order to assess whether a chronic treatment with a dopamine agonist, CV205-502, could modulate anterior pituitary epidermal growth factor (EGF) binding sites, female Wistar rats were treated or not (controls) with CV205-502 0.25 mg/kg/day sc for 8 days. This treatment significantly reduced rats' pituitary weight and plasma prolactin levels when compared to controls (weight: 10.4 +/- 0.1 vs 11.1 +/- 0.1 mg, p less than 0.01; prolactin: 1.2 +/- 0.2 vs 4.9 +/- 0.5 ng/ml, p less than 0.01). These decreases were associated with a significant stimulation of the number of pituitary EGF binding sites Bmax: 16.7 +/- 2.3 vs 11.3 +/- 1.9 fmoles/mg proteins, p less than 0.01) with no significant effect on their affinity (Kd: 0.94 +/- 0.17 vs 0.95 +/- 0.14 nM). Therefore, the modulation of pituitary EGF binding sites might be one of the mechanisms by which the dopamine agonist, CV205-502, exerts its pharmacological effects on hormonal secretions and/or cell multiplication in the pituitary.     

Distribution of insulin receptors in human erythrocyte membranes. Insulin binding to sealed right-side-out and inside-out human erythrocyte vesicles

Analyses of insulin binding to human erythrocytes and to resealed right-side-out and inside-out erythrocyte membrane vesicles have revealed that high affinity insulin binding receptors are present on both sides of the erythrocyte membranes. Insulin binding to human erythrocytes was examined with the use of a binding assay designed to minimize the potential errors arising from the low binding capacity of this cell type and from non-specific binding in the assay. Scatchard analysis of equilibrium binding to the cells revealed a class of high affinity sites with a dissociation constant (Kd) of (1.5 +/- 0.5) X 10(-8) M and a maximum binding capacity of 50 +/- 5 sites per cell. Interestingly, both resealed right-side-out and inside-out membrane vesicles exhibited nearly identical specific sites for insulin binding. At the high affinity binding sites, for both right-side-out and inside-out vesicles, the dissociation constant (Kd) was (1.5 +/- 0.5) X 10(-8) M, and the maximum binding capacity was 17 +/- 3 sites per cell equivalent. These findings suggest that insulin receptors are present on both sides of the plasma membrane and are consistent with the participation of the erythrocyte insulin receptors in an endocytic/recycling pathway which mediates receptor-ligand internalization/externalization.     

Lung vascular permeability changes in lambs with hyaline membrane disease

To investigate the mechanism of pulmonary edema in hyaline membrane disease (HMD), lymph from the efferent duct of the mediastinal lymph node was collected in premature lambs before and after delivery by cesarean section. Mean lymph flow in 7 lambs with histologically verified HMD increased progressively over 4 h after delivery to 3 times the fetal value, while lymph flow in 7 lambs without HMD increased to 3.5 times at 1 h and decreased thereafter. At 4 h after birth, lung lymph flow was significantly higher in lambs with HMD than in lambs without HMD (0.70 +/- 0.15 (SEM) vs 0.43 +/- 0.07 ml.h-1.kg-1). Lymph/plasma concentration ratio for small endogenous protein fractions (effective molecular radius, 3.6 and 3.8 nm) was significantly higher in lambs with HMD than in lambs without HMD at 2-4 h. Postmortem extravascular lung water was significantly higher in lambs with HMD (6.1 +/- 0.5 vs 4.3 +/- 0.3 ml/g dry lung weight). It is concluded that lung water is high in lambs with HMD, which appears to be a result both of delayed absorption of fetal lung liquid and increased permeability of the pulmonary exchange vessels.     

Histamine type I (H1) receptor radioligand binding studies on normal T cell subsets, B cells, and monocytes

We have documented a single, specific binding site for [3H]pyrilamine on normal human T helper, T suppressor, B cells, and monocytes. The binding of the radioligand to its receptor is reversible with cold H1 antagonist, saturates at 40 to 60 nM, and binding equilibrium is achieved in 2 to 4 min. Using a computer program (Ligand), we calculated the dissociation constants, binding capacities, and numbers of receptors per cell for each of the different cell types. Monocytes were found to have the highest affinity (mean KD +/- SD; 3.8 +/- 4.8 nM) for [3H]pyrilamine, followed by T helper cells (KD = 5.0 +/- 6.6 nM), B cells (KD = 14.2 +/- 2.0 nM), and T suppressor cells (KD = 44.6 +/- 49.4 nM). T suppressor cells were found to express the higher number of H1 receptors per cell (35,697 +/- 15,468), followed by B cells (10,732 +/- 9060), T helper cells (6838 +/- 8167), and monocytes (5589 +/- 2266). The kinetics of binding for this radioligand was carried out in resting and mitogen-stimulated T cells over a 48-hr period. We found that the binding affinity for [3H]pyrilamine increased over the 48-hr period, whereas the number of receptors per T cell was essentially unchanged. In contrast, T cells stimulated with Con A or PHA were shown to have a greater than fourfold increase in the number of receptors per cell, whereas the binding affinity for [3H]pyrilamine decreased over the 48-hr period. Preincubation of T cells with unlabeled histamine before carrying out the radioligand binding assay resulted in a decrease in the binding affinity of the receptors to [3H]pyrilamine, but the number of receptors per cell did not change significantly. Although the function of H1 receptors on T cells, B cells, and monocytes has not been completely defined, this receptor has the potential of playing an important role in modulating the immune response.     

Thrombin increases proliferation and decreases fibrinolytic activity of kidney glomerular epithelial cells.

Human glomerular epithelial cells (GECs) in culture synthesize single-chain, urokinase-type plasminogen activator (SC-uPA), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor 1 (PAI-1) and possess specific membrane-binding sites for u-PA. Using purified 125I-alpha thrombin, we demonstrate here the presence of two populations of specific binding sites for thrombin on GECs (1.Kd = 4.3 +/- 1.0 x 10(-10) M, 5.4 +/- 1.4 x 10(4) M sites per cell, 2. Kd = 1.6 +/- 0.5 x 10(-8) M, 7.9 +/- 1.8 x 10(5) sites per cell). Purified human alpha thrombin promoted the proliferation of GECs and induced a time- and dose-dependent increase of SC-uPA, t-PA, and PAI-1 antigens released by GECs. Thrombin-mediated increase in antigen was paralleled by an increase in the levels of corresponding u-PA and PAI-1 messenger RNA. In contrast, thrombin decreased u-PA activity in conditioned medium. This discrepancy between u-PA antigen and u-PA activity was explained by a limited proteolysis of SC-uPA by thrombin, leading to a two-chain form detected by immunoblotting and that could not be activated by plasmin. Thrombin also decreased the number of u-PA binding sites on GECs (p less than 0.05) without changing receptor affinity. Hirudin inhibited the binding and the cellular effects of thrombin, whereas thrombin inactivated by diisopropylfluorophosphate had no effect, indicating that both membrane binding and catalytic activity of thrombin were required. We conclude that thrombin, through specific membrane receptors, stimulates proliferation of GECs and decreases the fibrinolytic activity of GECs both at the cell surface and in the conditioned medium. These results suggest that thrombin could be involved in the pathogenesis of extracapillary proliferation and persistency of fibrin deposits in crescentic glomerulonephritis.     

A characterization of beta-adrenergic receptors on cellular and perigranular membranes of rat peritoneal mast cells

Beta-adrenergic receptors were characterized by measuring the specific binding of 3H-dihydroalprenolol (DHA) on intact isolated rat peritoneal mast cells (RPMC) and on perigranular membranes derived from purified RPMC granules. The specific binding of 3H-DHA reaches an equilibrium within 30 min at 5 degrees C and is linear with cell number. Scatchard analysis reveals two populations of binding sites on intact cells: with KD = 10.6 +/- 2.6 and 129 +/- 4.7 nM and Bmax of 186 +/- 38 and 1200 +/- 415 fmol/10(6) cells, respectively. Each cell contains 120 X 10(3) high-affinity binding sites and 720 X 10(3) low-affinity binding sites. There appears to be neither alpha-adrenergic nor muscarinic cholinergic receptors on the RPMC. Specific binding of 3H-DHA also occurred to isolated granules with perigranular membranes. The binding was saturable with a single population of binding sites with an affinity (KD) of 7.0 +/- 0.45 nM. Maximum binding (Bmax) was calculated at 56.6 +/- 1.9 fmol/10(9) granules. Subfractionation of granule components demonstrated that the specific binding sites appear to be localized exclusively on the perigranular membrane.     

Modulation of cell proliferation and senescence of WI-38 cells by hydrocortisone.

The specific binding of glucocorticoid hormones has been studied in the normal diploid human cell line WI-38. These cells were found to contain high affinity glucocorticoid binding sites whose molecular specificity showed a high correlation to that required for the stimulation of cell growth. When hydrocortisone (HC) was selectively added to or removed from parasynchronously dividing cultures, we observed that HC -enhanced stimulation of cell growth was associated with the hormone's presence in the pre-DNA synthetic period of the cell cycle. Similarly, the specific binding of [3H]dexamethasone in stimulated quiescent cells was found to increase significantly in the pre-DNA synthetic period. The concentration of specific binding sites per cell achieved in stimulated cell cultures was found to decrease with increasing in vitro age. These results suggest that the stimulation of WI-38 cell proliferation by HC involves specific glucocorticoid receptors whose concentration per cell is under cell cycle control. The age-associated decrease in specific glucocorticoid binding sites may explain, in part, our previously observed loss of responsiveness to HC in aging cell cultures.     

Different states of glucocorticoid receptors in intact cells and cytosol preparations

Binding of the glucocorticoid dexamethasone was studied in intact cells of the mouse lymphoma lines S49. 1, WEHI-7, WEHI-22, and WEHI-112. The number of binding sites per cell varied from 13 000 to 130 000 depending on the cell line. The equilibrium dissociation constant at 37° was in the range of 10 nM. When dexamethasone binding was investigated at 0° in cytosol preparations of the same cell lines significantly lower receptor levels were found and the dissociation constants were about one order of magnitude lower than those determined in whole cells. The data suggest that glucocorticoid receptors exist in different states in intact cells and cell extracts.     

Long time incubation of monocytic U 937 cells with LDL increases specific paf-acether binding and the cellular acetylhydrolase activity.

Besides the well established role of low density lipoproteins (LDL), the phospholipid PAF-acether (paf) seems to be involved in atherogenesis. The effect of LDL (10 micrograms/ml for 24 h, n = 3) on paf binding characteristics of monocyte/macrophage-like U 937 cells was investigated using the radioligand [3H]paf, unlabeled paf and the paf receptor antagonist WEB 2086. The specific [3H]paf binding significantly increased at 1.4 nM (P less than 0.02) and 2.8 nM (P less than 0.01) added [3H]paf with an increased number of paf binding sites in the Scatchard plot analysis of the data. Specific paf binding was functionally active since paf mediated a cellular [Ca2+]i rise. The protein kinase C (PKC) activator PMA (1 nM, 37 degrees C) expressed specific [3H]paf binding already after a 15-min incubation period, indicating a PKC activation as the decisive step of paf receptor expression. LDL also stimulated the paf degrading cellular acetylhydrolase significantly by increasing both Km (9.4 +/- 1.9 vs. 2.0 +/- 0.5 microM, P less than 0.02) and vmax (0.5 +/- 0.2 vs. 0.2 +/- 0.0 nmol/min per mg cell protein, P less than 0.02). The data demonstrate that LDL increases the number of paf receptors on monocyte/macrophage-like U 937 cells and interferes with the dynamics and/or synthesis of the cellular acetyl hydrolase. These effects could be of importance in the pathogenesis of atherosclerosis.     

Cytosol glucocorticoid receptor in the neoplastic epithelial duct cell line from human salivary gland (HSG)

Neoplastic epithelial duct cell line from human salivary gland (HSG cell) contained cytosol glucocorticoid receptor. Scatchard analysis of cytosol indicated that the dissociation constant (Kd) was 5.6-6.5 nmol/l and the number of binding sites was 83-92 fmol/mg protein. A competitive assay showed that the binding sites for [3H]triamcinolone acetonide were specific to glucocorticoid. Glycerol density gradient centrifugation displayed that the [3H]triamcinolone acetonide receptor complexes sedimented in the 8.5 S region under low salt conditions and in the 4.2 S region under high salt condition (0.4 M KCl). The same high salt conditions induced an increased binding of [3H]triamcinolone acetonide complexes for DNA-cellulose.