Biopotency and nuclear binding of glucocorticoids

Nuclear binding abilities of 3 glucocorticoids, dexamethasone (Dex), prednisolone (Pred) and corticosterone (Cort), which exhibited different biopotencies were compared in vitro. cytosols labelled with 3H-Dex, 3H-Pred and 3H-Cort from the rat liver prepared by incubation at 0 degrees C for 16 hr were bound to isolated liver nuclei in rates of approximately 25%, 9% and 1% of added radioactivity, respectively. Nuclear binding rates observed were correlated with biopotencies of these steroids. Time course studies of the cytosol binding revealed that the difference in the nuclear binding ability of these ligands was attributable, at least in part, to the metabolic transformation of ligands during the incubation period. A significant portion of 3H-Pred and 3H-Cort was transformed to polar metabolite(s) even under the incubation conditions at 0 degrees C. Kd's of the cytosol binding to 3H-Dex which was metabolically stable were decreased with the length of incubation time, significantly lower Kd being observed in the cytosol incubated for 16 hr than in those incubated for 2 and 6 hr. Kd's and the number of maximum binding sites were erratic when the ligands received biotransformation during the course of incubation. Transformed 3H-Pred and 3H-Cort during the incubation still exhibited features of the protein bound state. Besides biotransformation of ligands, structure related difference in the nuclear binding ability of these glucocorticoids was also observed. These observations suggest that metabolic susceptibility as well as structure related ability of the nuclear binding may contribute to the biopotency of glucocorticoids.     

The Mg,Ca, EDTA and ATP dependence of Na binding by rat liver microsomes

The role of ionic interactions in the adenosinetriphosphate (ATP) dependent Na binding by rat liver microsomes was investigated. In the concentration range of 0 to 20 mM, Mg and Ca are demonstrated to compete strongly against Na for microsome binding sites. In the presence of Ca, the nonbiological complexing agent ethylenediaminetetraacetate (EDTA) produced a marked increase in Na binding accompanied by a concomitant decrease in Ca binding. Under similar conditions ATP, which is a weaker complexing agent than EDTA, produced quantitatively smaller but qualitatively similar changes in binding. The data show that the effect of ATP on Na binding is not dependent upon the formation of a hypothetical Na binding intermediate in the hydrolysis of ATP as other investigators have postulated. Rather, the effect of ATP is demonstrated to depend upon the presence of unhydrolyzed ATP and its ability to complex divalent cations, and thereby to reduce divalent cation competition against monovalent cations for membrane binding sites.     

The mechanism of inhibition of Ran-dependent nuclear transport by cellular ATP depletion

Rran-dependent nuclear transport requires a nuclear pool of RanGTP both for the assembly of export complexes and the disassembly of import complexes. Accordingly, in order for these processes to proceed, Ran-dependent nuclear import and export assays in vitro require the addition of GTP to produce RanGTP. Notably, no ATP requirement can be detected for these transport processes in vitro. But in vivo, when cells are depleted of ATP by the addition of sodium azide and 2-deoxyglucose to block ATP production by oxidative phosphorylation and glycolysis, respectively, Ran-dependent nuclear import and export are rapidly inhibited. This raised the question of whether there is an ATP requirement for these nuclear transport pathways in an intact cell that has remained undetected in vitro. Here we report that the free (but not total) GTP concentration rapidly drops to an undetectable level upon ATP depletion as does the availability of RanGTP. Our conclusion is that the inhibition of Ran-dependent nuclear transport observed upon ATP depletion in vivo results from a shortage of RanGTP rather than the inhibition of some ATP-dependent process.     

Effects of ATP and pyrophosphate on properties of glucocorticoid-receptor complexes from rat thymus cells

Cytosols from rat thymus cells incubated with glucocorticoid contain nonactivated and activated receptors and mero-receptor complexes, in relative amounts that depend on the incubation conditions. These forms can be separated by a rapid minicolumn chromatographic technique based on their differential affinities for DNA, DEAE, and hydroxylapatite. We have used this method to examine the effects of ATP, pyrophosphate (PPi), and related compounds on cytosolic complexes. In addition to ATP, already known to promote activation at 0 degrees C, PPi, ADP, and other triphosphates at millimolar concentrations promoted activation of nonactivated complexes. AMP and Pi had little effect. ATP and PPi at millimolar concentrations also reduced binding of activated complexes to DNA. Characterization of the ATP- and PPi-activated complexes by gel filtration and ion exchange chromatography revealed two DNA-binding forms. One was essentially identical (Stokes radius of approximately 5.4 nm, elution from DEAE at approximately 50 mM KCl) to the normal activated complex obtained directly from cells incubated at 37 degrees C. The other had a Stokes radius of approximately 3.1 nm and had no affinity for DEAE. Analysis by minicolumns and gel filtration showed that ATP and PPi prevented formation of mero-receptor complexes, a process which occurs relatively rapidly in untreated thymus cytosols. These compounds did not alter properties of preformed mero-receptor. The accumulation of 3.1-nm complexes in thymus cytosols in which formation of mero-receptor is prevented suggests that this form is an intermediate, normally short-lived, in the conversion of 5.4 nm complexes to mero-receptor.     

Phosphate transport in Halobacterium halobium depends on cellular ATP levels.

Freshly harvested Halobacterium halobium cells grown in the presence of 0.5 mM Pi took up phosphate with a low apparent Km. Import depended on intracellular ATP levels; sodium and proton (electro)chemical gradients alone were not competent to drive Pi uptake. Although most of the phosphate accumulated as Pi in the cells, efflux of Pi was difficult to achieve.     

Constitutive HSP70: oligomerization and its dependence on ATP binding.

The constitutive HSP70 purified from CHO cells, which indicated a single band in SDS-polyacrylamide gel electrophoresis, showed multiple bands in native-polyacrylamide gel electrophoresis. These results indicate that the protein may exist in oligomeric forms. After crosslinking the oligomers with glutaraldehyde, SDS-polyacrylamide gel electrophoresis showed three protein bands of molecular weight 70 kDa, 153 kDa, and 200 kDa corresponded to monomer, dimer, and trimer, respectively. The relative amount of oligomeric forms was dependent upon ATP concentrations: it increased upon hydrolysis of ATP or decreased upon incubation with high concentrations of ATP (1-10 mM). Autoradiographic analysis of the native polyacrylamide gel electrophoresis of HSP70 following incubation with [gamma-32P]ATP revealed that ATP bound to only monomer. These results suggest that the equilibrium between oligomeric forms is dependent on ATP concentrations. Nonetheless, during heat shock, both monomer and oligomer might be indistinguishably associated with some proteins, probably denatured proteins.     

Age dependence of somatostatin levels and somatostatin binding in the rat brain

1. Somatostatin-like immunoreactivity (SLI) and 125I-Tyrl-somatostatin binding were measured from the brains of rats aged 1, 8 and 18 months. 2. Somatostatin binding was reduced in the striatum, frontal cortex, hypothalamus and hippocampus of the 8-month-old rats compared to the 1-month-old group. 3. Somatostatin binding was reduced in the striatum, frontal cortex and hippocampus of the 18-month-old rats compared to the 1-month-old group. 4. The reduction (40%) was most striking in the frontal cortex. 5. In no area of the brain did changes in SLI differ significantly between the different age groups.     

Cytosolic and nuclear binding proteins for 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat thymus

The existence of a high-affinity, low-capacity 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-binding species was demonstrated in cytosol from rat thymus. It was sensitive to heat and to pronase, trypsin or chymotrypsin but not to DNAase or RNAase, indicating that it was a protein. An excess of unlabelled 2,3,7,8-tetrachlorodibenzofuran or β-naphthoflavone displaced [³H]TCDD from the binder whereas phenobarbital, pregnenolone-16-α-carbonitrile or dexamethasone did not compete. Using a dextra-coated charcoal assay, the apparent dissociation constant (K_d) of the [³H]TCDD-binder complex was determined to 0.36 nM and the apparent maximum amount of binding sites (B_max) to 68 fmol/mg of cytosolic protein. When analyzed by sucrose density-gradient centrifugation at high ionic strength, the [³H]TCDD-binder complex sedimented at 4?5 S; at low ionic strength the complex sedimented more rapidly, probably due to aggregation. All these data support the interpretation that the demonstrated cytosolic TCDD-binder represents the receptor protein for TCDD, as previously described for rat and mouse liver. Following intravenous administration of [^3H]TCDD, a low-capacity [³H]TCDD-macromolecule complex was extractable from thymic cell neuclei; this complex behaved identically to the cytosolic [³H]TCDD-receptor complex when exposed to heat or to hydrolytic enzymes and was therefore alos identified as a protein. The nuclear [³H]TCDD-protein complex sedimented at 4–5 S at high ionic strength. Furthermore, a maximum uptake of [³H]TCDD in thymic nuclei was observed simultaneously with a decline in cytosolic radioactivity (at 3 h post-injection). These findings suggest that the nuclear [³H]TCDD-protein complex represented [³H]TCDD-receptor complex translocated from the cytoplasm. In conclusion, the rat thymus contains a cytosolic TCDD receptor at a concentration similar to that of the rat hepatic receptor. However, in vivo experiments showed that the nuclear uptake of [³H]TCDD (expressed as dpm/mg GNA) in the thymus was only about 6% of that in liver. Further studies are needed for an understanding of the mechanism behind this discrepancy.     

Increased nuclear size in BHK21 cells after treatment with non-toxic levels of calf thymus histones.

The effect of treatment with either whole calf thymus histones, or individually isolated histones, or polyarginine, or polylysine, on the nuclear size of BHK21 cells has been investigated. Incubation of the cells with mixed histones (12.5--44 microgram/ml) for 1 h considerably increased nuclear size. Increasing the histone concentration and/or the incubation time resulted in a decrease in the effect and could result in no change in nuclear size. Treatment of the cells with polyarginine or polylysine did not affect nuclear size. Experiments with individually isolated histones showed that the nuclear size effect was almost exclusively due to the histone H4. It is argued that the changes observed most likely resulted from interaction of H4 with the nucleus, and could reflect the properties of this particular histone molecule.     

The ATP4- receptor of rat mast cells.

The concentration-dependence on exogenous ATP of activation and inhibition of mast-cell histamine secretion, phosphatidylinositol labelling and leakage of metabolites shows that all these functions are regulated by the free acid ATP4-. Maximal histamine secretion and phosphatidylinositol labelling occur with ATP4- at approx. 2 microM, but higher concentrations, which cause inhibition of secretion and phosphatidylinositol labelling, are required to maximize leakage of 32P-labelled metabolites. Both enhancement and inhibition of phosphatidylinositol labelling (due to low and high concentrations of ATP4- respectively) are rapid in onset; histamine secretion is characterized by a delay, especially at low concentrations of ATP4- (approx. 1 microM). Phosphatidylinositol labelling and histamine secretion are dependent on extracellular Ca2+. Metabolite leakage due to the presence of exogenous ATP4- is slow and does not require Ca2+. Of 18 analogues of ATP that were tested, only four were agonists for secretion, and only these four permitted leakage of 32P-labelled metabolites. It is argued that activation and inhibition of histamine secretion, phosphatidylinositol labelling and metabolite leakage are all initiated by ATP4- acting at the same receptor. For mast cells stimulated with ATP4- enhancement of phosphatidylinositol metabolism is not sufficient by itself to cause Ca2+-dependent secretion.