Reduced cell sensitivity to glucocorticoid hormones in hypercholesterolemia

The number of 3H-dexamethasone binding sites in lymphocytes of subjects with hypercholesterolemia (HCS) was found to be decreased as compared to the receptor level in normolipidemic patients (N). In HCS-lymphocytes, the dexamethasone-induced inhibition of 3H-thymidine and 14C-acetate incorporation was less pronounced (by 20% and 22%, respectively) than in control cells, which is suggestive of the decreased sensitivity of HCS-lymphocytes to the hormone. An addition of 5-25% HCS blood sera to human skin fibroblast cultures caused a 10-50% decrease in the number of 3H-dexamethasone binding sites and diminished the Kd values 2-3 times. Lipid-depleted HCS-sera had no effect on the glucocorticoid reception in fibroblasts, whereas very low (VLDL) and low (LDL) density lipoproteins inhibited the 3H-dexamethasone binding to the cells. The most potent inhibiting effect was exerted by VLDL (both N-VLDL and HCS-VLDL). HCS-VLDL were more effective than N-LDL. HCS-HDL and N-HDL did not effect the 3H-dexamethasone binding to fibroblasts. In cells preincubated with VLDL dexamethasone inhibited the incorporation of 3H-thymidine and 14C-acetate less intensively (by 27% and 20%, respectively) than in control fibroblasts. The experimental results are suggestive of a decreased sensitivity of peripheral HCS-cells to glucocorticoids, which may shed some light on the mechanism of hypercholesterolemia realization into coronary heart disease and atherosclerosis.     

The principal glucocorticoid binding macromolecule in hepatoma cells in culture is similar to corticosteroid Binder II of rat liver cytosol

Reuber H-35 hepatoma (H4-II-E-C3) and HTC cells are known to retain differentiated corticosteroid induced functions in cell culture and to bind corticosteroids to macromolecules in cytosol which subsequently enter the cell nuclear fraction. Using both cell types we have demonstrated the major macromolecular fraction in cytosol to have properties (elution position from DEAE columns, pI, ³H-dexamethasone binding), very similar to those of rat liver corticosteroid Binder II which may be the hormone receptor.     

Rat ovary glucocorticoid receptor: Identification and characterization

A soluble, thermolabile protein with characteristics typical of glucocorticoid receptors has been identified in the ovaries of estrogenstimulated hypophysectomized immature rats. After the incubation of ³H-dexamethasone with ovarian cytosol, fractionation on a Sephadex G-200 column reveals a peak of radioactivity which elutes at the void volume. This peak, which represents saturable ³H-dexamethasone binding, disappears following heating (4 ° C × 15 min) or treatment of the cytosol with pronase. Scatchard analysis of the ³H-dexamethasone binding to cytosol shows it to be high affinity (Kd=5.1 nM) and saturable, with 327 fmol binding sites/mg cytosol protein. Binding site number rises linearly with increasing cytosol protein concentrations. The relative abilities of various steroids to inhibit ³H-dexamethasone binding are: triamcinolone acetonide ≥ dexamethasone > cortisol = progesterone > dihydrotestosterone > estradiol. This binding protein sediments at 9 S on a sucrose gradient, has a mean Stokes radius of 105 Å on gel exclusion chromatography, and has a calculated molecular weight of 388, 000 daltons and a frictional ratio of 2.1. ³H-Dexamethasone is not metabolized and does not bind specifically to serum. We have identified a protein in the rat ovary with characteristics of a glucocorticoid receptor and propose that this protein may be responsible for mediating direct effects of glucocorticoids on the ovary.     

Endotoxin inhibition of glucocorticoid enzyme induction and in vivo 3H-dexamethasone labelling of rat liver nuclei

1. The effect of endotoxin on glucocorticoid (GC) induction of liver TO and TAT was investigated. 2. It was found that endotoxin inhibited not only TO GC induction, but also that of TAT, though to a lesser extent (17.41%). 3. Endotoxin did not influence the binding capacity of liver cytosol for 3H-dexamethasone at the second hour after the toxin administration. 4. In in vivo experiments endotoxin inhibited with 57.2% the binding of 3H-dexamethasone to hepatic nuclei. 5. It is suggested that the lower extent of endotoxin inhibition of GC induction of TAT may be due to the counteracting action of some inductor(s) for TAT only.     

Glucocorticoid hormone receptor mediated induction of metallothionein synthesis in HeLa cells

HeLa cells grown in chemically defined medium lacking glucocorticoids synthesize metallothioneins, low molecular-weight heavy-metal binding proteins. Dexamethasone and hydrocortisone increase the rate of metal-lothionein synthesis five- to ten-fold. Maximal induction is achieved with 10^–8M dexamethasone and 10^–7M hydrocortisone. Half-maximal induction is achieved at 5 ± 10^–9M dexamethasone and 5 ± 10^–8M hydrocortisone. Although carried for many generations in the absence of any glucocorticoids, HeLa cells (clone S) contain 25,000 specific ³H-dexamethasone receptors that translocate into the nucleus after one hour of incubation. ³H-dexamethasone binds to a single class of receptors with an apparent Kd = 18.8 nM. A variety of steroids can be classified into three classes, based on their effect on metallothionein synthesis: (a) full agonists (optimal inducers), (b) intermediate effectors which have either partial agonist or antagonist activities, and (c) inactive steroids. There is a correlation between the effects on metallothionein synthesis of different steroids and their ability to compete with ³H-dexamethasone binding. We conclude that metallothionein is induced in HeLa cells by a glucocorticoid receptor mediated mechanism.     

Evidence that type II insulin-like growth factor receptor is coupled to calcium gating system

In competent Balb/c 3T3 cells primed with epidermal growth factor (primed competent cells), insulin-like growth factor-II (IGF-II) stimulated calcium influx in a concentration dependent manner with the ED50 of 450 pM. When receptor-bound [125I]IGF-II was cross-linked by use of disuccinimidyl suberate, a 240 K-Da protein was radiolabeled. Excess amount of unlabeled IGF-II inhibited the affinity-labeling of the 240 K-Da protein. To further examine whether IGF-II stimulates calcium influx by acting on the type II IGF receptor, we employed polyclonal antibody raised against rat type II IGF receptor, R-II-PABl. This antibody immunoprecipitated the type II IGF receptor and inhibited IGF-II binding in Balb/c 3T3 cell membrane without affecting IGF-I binding. In primed competent cells, R-II-PABl elicited an agonistic action in stimulating [3H]thymidine incorporation. Under the same condition, R-II-PABl elicited a marked stimulation of calcium influx. These results suggest that, in Balb/c 3T3 cells, 1) relatively low concentrations of IGF-II act mainly on the type II IGF receptor; 2) the type II IGF receptor is coupled to a calcium gating system; and 3) binding of a ligand to the type II IGF receptor leads to the stimulation of DNA synthesis.     

Binding of 3H-aldosterone and 3H-dexamethasone in primary monolayer cultures of kidney cortical collecting tubule (CCT) cells

Specific binding of corticosteroids in cultured CCT cells was measured as a function of time, of temperature, of pH, and of concentration. Scatchard analysis revealed the existence of two species of binding sites for both, 3H-aldosterone type I A: KD = 2.3.10(-9) M, N = 33.10(-17) mol/10(4) cells; type I B: KD = 51.10(-9) M, N = 55.10(-17) mol/10(4) cells) and dexamethasone (type II A: KD = 4.7.10(-9) M, N = 2.3.10(17) mol/10(4) cells; type II B: KD = 22.10(-9) M, N = 6.5.10(-17) mol/10(4) cells). The data demonstrate that CCT cells in primary monolayer culture express corticosteroid binding sites similar to cells of the CCT in vivo.     

Effect of colchicine on the depletion and replenishment of cytoplasmic glucocorticoid receptor in rat liver after administration of glucocorticoid

Pretreatment of rats with colchicine (3 mg/kg body weight) modified the time course of depletion of the cytoplasmic binding sites for 3H-dexamethasone after administration of prednisolone (0.5 or 1.5 mg/kg body weight). Colchicine also decreased the rate of the cytoplasmic receptor replenishment which was confirmed by application of this drug after completion of the cytoplasmic receptor translocation to nuclei (30 min after prednisolone injection). Addition of colchicine to the incubation mixture for in vitro binding of 3H-dexamethasone-labelled liver cytosol to isolated liver nuclei suspended in TKMS buffer (50 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM MgCl2 and 250 mM sucrose) evoked no measurable changes in the rate of the nuclear binding.     

Studies on the nuclear binding of steroid hormone-receptor complex; binding of liver and thymus dexamethasone-receptor complex and prostate dihydrotestosterone-receptor complex to nuclei from various tissues.

To examine the binding specificity of steroid hormone-cytoplasmic receptor complexes to nuclei, binding of 3H-dexamethasone (Dex)-liver, 3H-Dex-thymus and 3H-dihydrotestosterone (DHT)-prostate receptor complexes to nuclei from liver, prostate, thymus, spleen and kidney was studied. It was observed that a significant amount of steroid-receptor complexes was bound to any nuclei used in the present study and the extent of the binding of receptor complexes to nuclei from homologous tissues was not always greater than that to nuclei from heterogenous tissues. However, a significant portion of the 3H-Dex-liver and 3H-DHT-prostate receptor complexes was not absorbed by nuclei from kidney, spleem, and thymus, and the unabsorbed complexes were efficiently bound to liver and prostate nuclei. The results obtained indicate that two types of receptor complex with regard to nuclear binding were present in cytosols of liver and prostate; one binds to nuclei from kidney, spleen, thymus, liver and prostate and the other does not bind to nuclei from kidney, spleen and thymus but does bind to nuclei of liver and prostate. The latter type of receptor complex was not observed in the cytosol from the thymus.     

Evidence of type II estrogen receptor in human osteoblast-like cells

Osteoblast-like cells isolated from human bone bioptic specimens were characterized and analysed for the presence of type II estrogen receptor (type II EBS). The amount of type II EBS was measured by a whole-cell assay at 4 degrees C for 2.5 h using [(3)H]-estradiol as tracer. Saturation analysis, used to investigate the binding characteristic of type II EBS, resulted in a sigmoid curve. Scatchard analysis showed the binding affinity of the estrogen receptor, yielding a concave plot. The dissociation constant (K(d)), determined from the [(3)H]-estradiol concentration required for half saturation was about 12+/-2 nM (SD). The number of type II EBS, estimated at maximum binding, was 197,000+/-8800 sites per cell. If the regulation of the receptor by flavonoids would be confirmed, the evidence of type II EBS in osteoblast-like cells could suggest a direct action of ipriflavone and others flavonoids on bone density in postmenopausal osteoporosis.     

Placental transfer of dexamethasone in near-term sheep

The placental transfer of 3H-dexamethasone was studied in six near-term sheep. The placental clearance of 3H-dexamethasone was 18.8 +/- 3.5 ml/min per kg of fetal weight. The clearance of dexamethasone by the fetal tissues was 9.3 +/- w.5 ml/min per kg. The maximum placental clearance was 285 +/- 24 ml/min and the degree of diffusion limitation to the placental transfer of dexamethasone was 78 +/- 4%. The placental transfer of dexamethasone is therefore limited primarily by the nature of the placental barrier.     

A two-receptor model for salmon gonadotropins (GTH I and GTH II).

The possible existence of distinct receptors for salmon gonadotropins (GTH I and GTH II) and the distribution of the receptor(s) were studied through examination of the binding of coho salmon (Oncorhynchus kistuch) GTH I and GTH II to membranes from thecal layers and granulosa cells of salmon ovaries. Purified coho salmon gonadotropins were iodinated by the lactoperoxidase method. Crude membrane preparations were obtained from thecal layers, granulosa cells, and whole ovaries of coho salmon in the postvitellogenic/preovulatory phase. Binding of 125I-GTH I to membranes from thecal layers, granulosa cells, and whole ovaries, and binding of 125I-GTH II to thecal layer cell membranes could be inhibited by both GTHs, but GTH I was more potent than GTH II. In contrast, GTH II was more potent than GTH I in inhibiting 125I-GTH II binding to membranes from granulosa cells and whole ovaries, but the inhibition curves were not parallel. Scatchard plot analysis suggested that there was a single type of receptor in the thecal layers for both GTHs, whereas in the granulosa cells there was more than one type of receptor for both GTHs. Based on these results, a two-receptor model for the postvitellogenic/preovulatory salmon ovary is proposed with the following features: 1) there are two types of gonadotropin receptors in the salmon ovary, type I and type II; 2) the type I receptor binds both GTHs, but with higher affinity for GTH I, whereas the type II receptor is highly specific for GTH II and may have only limited interaction with GTH I; and 3) the type I receptor is present in both thecal cells and granulosa cells, whereas the type II receptor is present in granulosa cells.     

Human T lymphocyte cAMP-dependent protein kinase: subcellular distributions and activity ranges of type I and type II isozymes.

The role of the type I and type II protein kinase A isozymes in the regulation of human T lymphocyte immune effector functions has not been ascertained. To approach this question, we first characterized the distribution and enzyme activities of the type I and type II protein kinase A (PKA) isozymes in normal, human T lymphocytes. T cells possess both type I and type II isozymes with an activity ratio of 5.0:1 +/- 0.71 (mean +/- SD). The type I isozyme associates predominately with the plasma membrane whereas the type II isozyme localizes primarily to the cytosol. Analyses of isozyme activities demonstrated that T cells from approximately one-third of 16 healthy donors exhibited significantly higher type II isozyme activities (higher type II, type IIH) than the remaining donors (lower type II, type IIL) (mean = 605 +/- 75 pmol.min-1.mg protein-1, P less than 0.001). Scatchard analyses of [3H]cAMP binding in the cytosolic fraction demonstrated similar Kd values (type IIH, 1.1 x 10(-7) M; type IIL, 9.0 x 10(-8) M); however, the Bmax (maximal binding) of the type IIH was 400 fmol/mg protein compared to the Bmax of the type IIL of 126 fmol/mg protein. Scatchard analysis of [3H]cAMP binding to the type I isozyme associated with membrane fragments had a Kd of 5.6 x 10(-8) M and a Bmax of 283 fmol/mg protein. Eadie-Hofstee plots of type IIH and type IIL gave a Km and Vmax of 2.3 mg/ml and 1.5 nmol.mg-1.min-1, and 2.1 mg/ml and 1.6 nmol.mg-1.min-1, respectively. The 3.2-fold higher maximal binding of the type II isozyme in one-third of healthy donors may reflect a greater amount of isozyme protein. The compartmentalization of type I PKA isozyme to the plasma membrane and type II PKA isozyme to the cytosol may serve to localize the isozymes to their respective substrates in T lymphocytes.     

HepG2 cells predominantly express the type II interleukin 1 receptor (biochemical and molecular characterization of the IL-1 receptor).

In this study we have characterized the cell surface interleukin 1 (IL-1) receptor in HepG2 hepatoma cells. We found that HepG2 cells bind both IL-1 alpha and beta with high affinity, KDs of 136 and 180 pM and receptor densities of 16,000 and 8500 binding sites/cell respectively. The binding sites appeared to be predominantly type II since phorbol ester treatment of the cells, which selectively downregulates type II IL-1 receptors, reduced binding by 68% while treatment of the cells with an inhibitory monoclonal antibody specific for the type I receptor had no significant effect on IL-1 binding. Competition studies with a modified IL-1 beta analog (Glu4) also revealed binding kinetics more consistent with binding to type II receptors than to type I. Crosslinking and ligand blotting with human 125I-IL-1 demonstrated the presence of two bands, a 78 kDa band typical of crosslinking to type II (p60) receptor, and a 98 kDa band, typical of crosslinking to the type I (p80) receptor. Low level expression of the type I receptor was consistent with molecular biological studies employing polymerase chain reaction (PCR) amplification which indicated that mRNA for the type I receptor was produced by the HepG2 cells. Functional receptors were demonstrated by the induction of IL-8 by IL-1 stimulated cells.     

Mitogenic response of human SH-SY5Y neuroblastoma cells to insulin-like growth factor I and II is dependent on the stage of differentiation

Human insulin-like growth factor I and II (IGF-I and IGF-II) in concentrations of 1-30 ng/ml, were shown to stimulate ornithine decarboxylase activity and [3H]thymidine incorporation in human SH-SY5Y neuroblastoma cells. Proliferation of these cells was also stimulated by IGF-I and II when added to RPMI 1640 medium, fortified with selenium, hydrocortisone, transferrin, and beta-estradiol. Labeled IGF-I and II bound to SH-SY5Y cells. The cross-reaction pattern of IGF-I, IGF-II, and insulin in competing with the binding of labeled IGF-I and IGF-II, respectively, indicated that SH-SY5Y cells express both type I and type II IGF receptors. Treatment of SH-SY5Y cells for 4 d with 12-O-tetradecanoylphorbol-13-acetate (TPA), which resulted in morphological and functional differentiation and growth inhibition, abolished the mitogenic response to both IGF-I and II. Concomitantly, the binding of IGF-II disappeared almost totally, which offers a possible explanation for the reduced biological response to IGF-II after TPA treatment. In contrast, the IGF-I binding in TPA-treated cells was only reduced to approximately 70% of the binding to control cells. It is therefore not excluded that the IGF-I receptor could be uncoupled by TPA, with persistent binding capacity for IGF-I.     

A study of cAMP binding proteins on intact and disrupted sperm cells using 8-azidoadenosine 3',5'-cyclic monophosphate

The photoaffinity probe (32P) 8-N3 cAMP was used to label the cAMP binding proteins in washed ejaculated human sperm. Three saturable binding proteins were photolabeled in both intact and disrupted cells with apparent molecular weights of 55,000, 49,000 and 40,000 daltons corresponding to the regulatory subunits of type II and type I cAMP-dependent protein kinase (cAMP-PK) and to an endogenous proteolytic product of the regulatory subunits, respectively. Photoincorporation in the three proteins could be totally blocked by preincubating the cells with cAMP. Cell-free seminal plasma was found to be free of detectable (32P) 8-N3 cAMP-binding proteins. The 8-N, cAMP was also effective in stimulating endogenous cAMP-PK activity in intact and disrupted sperm. A substantial amount of (32P) 8-N3 cAMP binding to types I and II regulatory subunits and cAMP-PK activity was detected on washed intact cells. Intact cells bound 1.80 pmol of (32P) 8-N3 cAMP/mg protein and had cAMP-PK activity of 824 units/10(8) cells. Disrupted cells bound 3.95 pmol (32P) 8-N3 cAMP/mg protein and had a cAMP-PK activity of 2,206 units/10(8) cells. The data presented support the concept of two classes of cAMP receptors being differentially available to externally added (32P) 8-N3 cAMP and proteases. Cellular membrane integrity and membrane sidedness are discussed as possible explanations for the observation reported.     

Isolation and characterization of differentiated alveolar type II cells from fetal human lung

A method has been developed for isolating differentiated type II cells from human lung of 18-24-week gestation. The procedure involves an initial 4-day culture of lung explants in the presence of dexamethasone (10 nM) and triiodothyronine (2 nM). Type II cells (and fibroblasts) are isolated by trypsin digestion of the explants, two differential adherence steps and incubation overnight in primary culture. This method provides a high yield of type II cells ((50 +/- 15) X 10(6) cells/g wet weight of explant) with a purity of 85 +/- 5% in 16 experiments. The type II cells contain numerous perinuclear granules which stain darkly with toluidine blue and Papanicolaou stain; electron microscopy showed these inclusions to be lamellar bodies with tightly stacked, well defined lamellae. Type II cells, but not fibroblasts, were positive by immunofluorescence histology for surfactant apoprotein and binding of Maclura pomifera lectin which binds to the surface of type II but not type I cells in vivo. The rate of both [3H]acetate and [3H]choline incorporation into phosphatidylcholine (PC) was several-fold greater in type II cells than fibroblasts; the saturation of PC was 36.2 and 25.9%, respectively. Release of saturated PC was stimulated by terbutaline, the ionophore A23187, and tetradecanoyl phorbol acetate in type II cells but not fibroblasts. We conclude that differentiated type II cells can be isolated in relatively high yield and purity from hormone-treated explants of fetal human lung.     

Nuclear type II [3H]estradiol binding site ligands: inhibition of ER-positive and ER-negative cell proliferation and c-Myc and cyclin D1 gene expression

These studies assessed the effects of 3,4-dihydroxybenzalacetone (ZN-1) and 1-(3,4-dihydroxyphenyl)-2-propanol (ZN-2) on MCF-7 cell proliferation. The compounds blocked [3H]estradiol binding to nuclear type II sites, but did not compete for [3H]estradiol binding to recombinant ERalpha or ERbeta. ZN-1 and ZN-2 inhibited the proliferation of ERalpha and ERbeta positive (MCF-7) and negative (MCF-10A) breast cells, further ruling out direct binding to ER in the mechanism of action of these compounds. Pre-loading type II sites with ZN-1 or ZN-2 reduced [3H]estradiol exchange, strongly suggesting the drugs were binding covalently. ZN-1 treatment resulted in complete occupancy of type II sites and sustained (9 days) inhibition of MCF-7 cell proliferation following its removal from the tissue culture medium. This cell growth inhibition was not due to non-specific toxicity, as the numbers of viable, attached cells per dish (determined by trypan blue dye exclusion) remained constant throughout this 9-day period and eventually reversed by day 19. ZN-2 effects on cell proliferation reversed more rapidly following discontinuation of treatment, a response consistent with the inability of the compound to totally block type II binding. Both ZN-1 and ZN-2 blocked estradiol stimulation of c-Myc and cyclin D1 gene expression in MCF-7 cells, two events that are clearly coupled to cell cycle progression. We suspect this may occur through ZN-1 or ZN-2 modification of nucleosome function and/or chromatin remodeling since nuclear type II sites are localized to a complex of histones H3 and H4 (Shoulars et. al, J Steroid Biochem. Mol. Biol. 96: 19-30, 2005).     

Modulation of Pseudomonas aeruginosa adherence to collagen type I and type II by carbohydrates

Abstract This study was undertaken to examine if receptor recognizing saccharides may be involved in the adherence of Pseudomonas aeruginosa to collagen type I and type II. We performed an adherence inhibition assay: cells of individual P. aeruginosa isolates attached to immobilized collagen type I or type II in the presence of monosaccharides, which could serve as blockers of bacterial receptors. Bacterial binding to collagen type I molecules was inhibited to the highest degree by sugar composition d -galactose/ d -mannose/ N -acetylneuraminic acid (5:5:1), whereas attachment of P. aeruginosa to collagen type II was inhibited by composition d -glucose/ d -galactose (1:1). The same strains which were sensitive to inhibition of binding to collagen type II by both collagen types, were also sensitive to blocking by composition d -glucose/ d -galactose. It suggests that saccharides play a role in adherence of P. aeruginosa to collagen type I and type II, and a common receptor for both types of collagen may be available on the surface of P. aeruginosa cells.     

Incorporation of dexamethasone by Candida albicans

The incorporation of 3H-dexamethasone into Candida albicans has been studied. The results indicate that the steroid is incorporated unchanged and primarily into the cell wall and membrane of the organism. The incorporation appears to be of a noncovalent type.