Dynamic in Vivo Binding of STAT5 to Growth Hormone-Regulated Genes in Intact Rat Liver. Sex-Specific Binding at Low- But Not High-Affinity STAT5 Sites

Abstract. The effect of corticosterone and dexamethasone on the production of growth hormone and prolactin was studied in rat pituitary tumour cells (GH3-cells) in culture. Corticosterone and dexamethasone caused a dose-dependent stimulation of growth hormone synthesis, and the highest concentration (10−6 mol/l) increased growth hormone levels to 250% of controls. This concentration, however, decreased prolactin synthesis to 25% of the control values. The cytosol fractions from monolayer cultures as well as from tumours of GH3-cells were found to possess receptor molecules for glucocorticoid hormones, having a sedimentation constant close to 8 S in a salt-free buffer and 4 S in the presence of 0.5 mol/l KCL. Isoelectric point of the receptor was 5.8. Scatchard analysis showed one single class of binding sites with high affinity (Kd 2.1 ± 0.4 (sd × 10−9 mol/l). Studies on the steroid specificity revealed that dexamethasone had the highest affinity for the receptor. Corticosterone, cortisol and progesterone had also high affinity, whereas testosterone and oestradiol-17β had no significant affinity for the receptors. After in vivo administration of [3H]dexamethasone to GH3 tumour-bearing rats, radioactivity could be extracted from purified nuclei bound to 4 S macromolecules. The presence of receptors for glucocorticosteroid hormones in the GH3-cells, suggests that these hormones may alter growth hormone and prolactin production at the anterior pituitary level.

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Effects of incubation at physiological temperatures on the concentration-dependence of [2-14C]malonyl-CoA binding to rat liver mitochondria

Biochemical Journal ◽

10.1042/bj2310343 ◽

1985 ◽

Vol 231 (2) ◽

pp. 343-347 ◽

Cited By ~ 3

Author(s):

V A Zammit ◽

C G Corstorphine

Keyword(s):

Rat Liver ◽

Specific Binding ◽

Liver Mitochondria ◽

Rat Liver Mitochondria ◽

Temperature Dependent ◽

Malonyl Coa ◽

Saturation Kinetics ◽

Different Temperatures ◽

Cpt I

Specific binding of [2-14C] malonyl-CoA to rat liver mitochondria was measured at different temperatures and after various periods of time of exposure of the mitochondria to the ligand. Incubation of mitochondria at 37 degrees C in the absence of malonyl-CoA resulted in a decrease in their ability to bind malonyl-CoA at all concentrations tested (up to 55 microM). However, incubation of mitochondria in the presence of malonyl-CoA resulted in the loss of the binding only by a low-affinity component. By contrast, there was an increase in the binding that occurred at low, physiological, concentrations of malonyl-CoA. These differences in the response of the two binding components to incubation conditions were used to obtain quantitative data about their respective saturation kinetics. Evidence was obtained that, whereas the high-affinity component approached saturation hyperbolically with respect to malonyl-CoA concentration, the low-affinity component had sigmoidal characteristics. The concentrations of malonyl-CoA required to half-saturate the two components were 2-3 microM and 30 microM for the high- and low-affinity components respectively. Evidence was also obtained for the involvement of a temperature-dependent transition, that occurred at around 25 degrees C, in the modulation of malonyl-CoA binding to the mitochondria. The possible physiological roles of the two components of malonyl-CoA binding in relation to the regulation of overt carnitine palmitoyltransferase (CPT I) activity in vivo are discussed.

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The galactose-specific receptor system in rat liver during development

Development ◽

10.1242/dev.100.1.13 ◽

1987 ◽

Vol 100 (1) ◽

pp. 13-22

Author(s):

L. Dini ◽

L. Conti-Devirgiliis ◽

S. Russo-Caia

Keyword(s):

Endothelial Cells ◽

Ligand Binding ◽

Rat Liver ◽

Binding Capacity ◽

Specific Binding ◽

Cell Types ◽

Receptor System ◽

In Situ Experiments

The number and distribution of galactose-specific binding sites were investigated in rat liver cells during perinatal development. Ligand binding to hepatocytes, macrophages and endothelial cells was followed with in vitro and in situ experiments by electron microscopy, using lactosylated bovine serum albumin adsorbed onto 5 nm colloidal gold particles as ligand. Binding capacity, starting at a late stage of fetal development, is very low both on the hepatocyte and on the macrophage surface, which show single particles statistically distributed. By contrast, bound particles are absent from fetal endothelial cells, which also lack the typical coated regions. In vivo, experiments at 37 degrees C show that endocytosis occurs to some extent in prenatal life. These results indicate that the expression of galactose-specific receptors' activity on the different liver cell types follows different developmental patterns, which are independently modulated.

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Characterization by saturation studies of the in vivo binding of [3H]flunitrazepam in mouse brain

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y91-025 ◽

1991 ◽

Vol 69 (2) ◽

pp. 176-180 ◽

Cited By ~ 1

Author(s):

Peter T.-H. Wong

Keyword(s):

Mouse Brain ◽

Incubation Temperature ◽

Specific Binding ◽

Protein A ◽

Receptor Occupancy ◽

Acute Tolerance ◽

Alternative Methods ◽

In Vivo Binding

The in vivo binding of [3H]flunitrazepam ([3H]Fln) was characterized in seven regions of the mouse brain. The binding showed saturability and linear Scatchard plots. Hill coefficients were close to unity. Data fitting to a hyperbola by least squares yielded consistent Kd values for all regions studied (0.36–0.6 pmol/mg protein). Bmax values ranged from 0.14 to 0.89 pmol/mg protein, a sixfold regional variation. The order of binding is as follows: cortex > hippocampus > midbrain = thalamus/hypothalamus > striatum ≥ cerebellum > brainstem, consistent with that obtained by in vitro binding. The in vivo receptor density and affinity are apparently lower in comparison with in vitro parameters. This is consistent with the observation that the Kd increases and Bmax decreases in vitro when the incubation temperature is increased from 0 °C. Non-specific binding has been estimated by displacement of in vivo binding by unlabelled ligand in vitro as well as by pretreatment with unlabelled ligand. The two alternative methods were compared and evaluated. It is concluded that the displacement method provides more reliable estimates of the nonspecific binding. Diazepam-sensitive mice did not differ from the control mice in the in vivo [3H]Fln binding. However, mice pretreated with diazepam 1 or 2 days before have binding reduced by 70 or 30%, respectively. The reduced binding may be explained by receptor occupancy by residual oxazepam. However, the low concentration of the residual oxazepam is an unlikely cause of the phenomenon of "acute tolerance" observed in these mice.Key words: benzodiazepines, in vivo binding, characterization, diazepam-sensitive mice, acute tolerance.

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