Co-localization of brain corticosteroid receptors in the rat hippocampus.

New developments in corticosteroid receptor research enabled us to perform a highly detailed study on the neuroanatomical topography of MR and GR in the rat hippocampus. Receptor immunocytochemistry was used to map the distribution of GR protein with the help of a monoclonal antibody raised against the purified rat liver GR-hormone complex. Furthermore, in situ hybridization with 35S-labeled RNA probes, which were transcribed from cDNAs complementary to either a fragment of the rat brain MR gene or to the rat liver GR gene, was applied to investigate the localization of MR and GR mRNA in the limbic brain. The pyramidal neurons of cell field Ca1 and CA2 and the granular neurons of the dentate gyrus showed marked GR immunoreactivity (GRir) as well as intense labeling of GR mRNA. The radiolabeled density of GR mRNA in cell fields CA3 and CA4 was considerable less, whereas low-to-almost-undetectable levels of GRir could be observed in these regions. MR mRNA appeared to be evenly distributed over all cell fields of the hippocampus and the dentate gyrus. The topography of GRir, GR mRNA and MR mRNA was found to agree with the cellular distribution of MR and GR binding sites in the hippocampus. Moreover, the microanatomy of MR and GR in the hippocampus appeared to overlap. Our data strongly suggest that MR and GR are co-expressed in the majority of pyramidal and granular neurons of the hippocampal formation. This assumption is based on coherence in the detection of different aspects of the receptor cycle of MR and GR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of benzo(a)pyrene to rat liver nuclear matrix

Binding of benzo(a)pyrene (B(a)P) to nuclei isolated from rat liver was investigated. After incubation with ¹⁴C-B(a)P, the nuclei were subfractionated into an envelope fraction, two chromatin fractions and a matrix fraction. About 50% of the B(a)P that entered the nuclei was associated with the matrix fraction. Covalently bound B(a)P in the matrix fraction also exceeded that in the chromatin fractions. The radioactivity of ¹⁴C-B(a)P attained by the matrix DNA was 3–5 times higher than that attained by the chromatin DNAs. These findings suggest that the nuclear matrix is a major intranuclear binding site of B(a)P.     

Binding of a 23 kD endonuclease to the rat liver nuclear matrix

In a previous paper we have described a 23 kD nuclear endonuclease (p23) that was mostly found to exist in a state of association with the isolated rat hepatocyte nuclear matrix. To investigate the nature of this interaction, the nuclear matrix was prepared using different procedures and examined for the presence/absence of the enzyme by activity gel analysis. Treatment of isolated nuclei with sodium tetrathionate (NaTT), a sulfhydryl-cross-linking agent, led to the complete recovery of p23 in the nuclear matrix, whereas incubation of nuclei with dithiothreitol (DTT), a sulfhydryl-reducing agent, led to its complete solubilization and resulting absence from the nuclear matrix. Exposure of the isolated nuclear matrix to DTT in high-ionic strength buffer, a procedure that promotes the solubilization of the internal nuclear matrix, caused the nearly complete solubilization of p23. It was concluded that disulfide bonds play an essential role in the association of p23 with the nuclear matrix and that p23 is mostly localized in the nuclear matrix interior.     

Binding of triiodothyronine to the nuclear matrix of the rat liver. The effect of thyroid hormones on the phosphorylation of nuclear matrix proteins

The interaction of thyroid hormones with rat liver nuclear matrix proteins was studied. It was shown that the nuclear matrix contains the sites which bind triiodothyronine with a high affinity (Ka = 1.07 X 10(9) M-1) and limited capacity (maximal binding capacity--28.5 fmol triiodothyronine/100 micrograms protein). Electrophoretic analysis of triiodothyronine-binding matrix proteins revealed that the molecular mass of the major triiodothyronine-binding fraction is 50 000-52 000 Da. Injections of triiodothyronine to thyroidectomized animals stimulated the phosphorylation of all protein fractions of the nuclear matrix.     

Binding of the glucocorticoid receptor to the rat liver nuclear matrix. The role of disulfide bond formation

The nuclear matrix is a putative skeletal structure which has been implicated in many nuclear functions. To assess a possible role of the nuclear matrix in glucocorticoid action, purified rat liver nuclei containing glucocorticoid-receptor complexes were treated with DNase I +/- RNase A followed by 1.6 M NaCl, thus yielding salt-extractable and salt-resistant (nuclear matrix) fractions. The subnuclear distribution of hormone-receptor complexes was determined by following the fate of unmetabolized radiolabel after injection of labeled triamcinolone acetonide into adrenalectomized animals and subjecting various subfractions to immunoblotting using a monoclonal antibody which recognizes the glucocorticoid receptor. Both techniques indicated that 50-70% of the total nuclear hormone-receptor complexes were recovered in the nuclear matrix fraction. Previous results (Kaufmann, S. H., and Shaper, J. H. (1984) Exp. Cell Res. 155, 477-495) suggest that a variety of nuclear polypeptides become nuclease- and salt-resistant as a result of the formation of intermolecular disulfide bonds. The following evidence suggests that disulfide bonds mediate the association between the glucocorticoid receptor and the nuclear matrix. When nuclei were isolated in the absence of sulfhydryl-blocking and -cross-linking reagents, sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions revealed that the receptor was present as a high molecular weight disulfide-cross-linked complex. When nuclei were isolated in the presence of the irreversible sulfhydryl-blocking reagent iodoacetamide, the disulfide bonds which cross-linked the receptor into high molecular weight complexes were absent; and 85-100% of the hormone-receptor complexes were salt-extractable. When nuclei (isolated in the absence of iodoacetamide) were treated with the sulfhydryl-cross-linking reagent sodium tetrathionate, greater than 95% of the nuclear hormone-receptor complexes became resistant to extraction with nucleases and 1.6 M NaCl. The implications of these results for other matrix-associated nuclear functions are discussed.     

Effect of corticosteroid hormones on electrical activity in rat hippocampus.

Pyramidal neurons in the rat CA1 hippocampal area contain both mineralocorticoid (MR) and glucocorticoid receptors (GR) which bind the endogenous adrenal steroid corticosterone with differential affinity. With intracellular electrophysiological recording techniques we have investigated how corticosterone affects the membrane properties of these cells. We observed that low doses (1 nM) of corticosterone or aldosterone can, through MR, reduce the spike frequency accommodation and afterhyperpolarization (AHP) evoked by a short depolarizing current in pyramidal neurons. As the accommodation/AHP can be considered as an intrinsic mechanism of CA1 neurons to attenuate transmission of excitatory input, the MR-mediated action might potentially enhance cellular excitability in the CA1 area. Higher doses of corticosterone or selective glucocorticoids were able to reverse the MR-mediated effect on accommodation/AHP, eventually increasing particularly the amplitude of the AHP. GR-mediated events may thus potentially suppress excitability in the hippocampal CA1 area. Not only current- but also transmitter-induced membrane effects were affected by the steroids. Firstly, GR-ligands were able to suppress a temporary noradrenaline-evoked decrease in accommodation/AHP. Secondly, membrane hyperpolarizations induced by serotonin were reduced by MR-agonists. We propose that cellular excitability in the hippocampus is at least partly under control of coordinative, antagonistic MR- and GR-mediated effects on electrical activity.     

Binding of 5-bromouracil-containing S/MAR DNA to the nuclear matrix.

Substitution of thymine with 5-bromouracil in DNA is known to change interaction between DNA and proteins, thereby inducing various biological phenomena. We hypothesize that A/T-rich scaffold/nuclear matrix attachment region (S/MAR) sequences are involved in the effects of 5-bromodeoxyuridine. We examined an interaction between DNA containing an intronic S/MAR sequence of the immunoglobulin heavy chain gene and nuclear halos prepared from HeLa cells. Upon substitution with 5-bromouracil, the S/MAR DNA bound more tightly to the nuclear halos. The multi-functional nuclear matrix protein YY1 was also found to bind more strongly to 5-bromouracil-substituted DNA containing its recognition motif. These results are consistent with the above hypothesis.     

Concepts related to salt resistant estradiol receptors in rat uterine nuclei: nuclear matrix

When ³H-estradiol (0.1 μg) is injected into immature female rats, virtually all of the label that is recovered with uterine nuclei can be solubilized by 0.6 M KCl. Salt resistant uterine nuclear estrogen binding sites do not become labeled within one hour after the injection of ³H-estradiol, but these sites do exist and can be revealed when isolated nuclei are subjected to an $\text{in vitro}$ estradiol exchange assay. These saturable, high affinity salt resistant sites appear to be associated with the uterine nuclear matrix, a residual structure of the nucleus.     

Nuclear receptors. II. Intestinal corticosteroid receptors

Two corticosteroid receptors have been cloned; they are the glucocorticoid receptor and the mineralocorticoid receptor. These receptors are members of the steroid/thyroid/retinoid receptor family of nuclear transactivating factors, which are characterized by two highly conserved zinc fingers in the central DNA binding domain, a COOH-terminal domain that encompasses the ligand binding site, and a variable NH(2)-terminal domain. In addition to these cloned receptors, other corticosteroid receptors have recently been identified in intestine. Steroid binding studies have identified two novel putative corticosteroid receptors in intestinal epithelia, and molecular cloning studies have detected two low-affinity receptors in small intestine that are activated by corticosteroids and induce CYP3A gene expression. This article focuses on the identification of these novel corticosteroid receptors and the potential role they may play in intestinal physiology.     

Binding of glucocorticoid receptors to DNA.

DNA has been implicated as the nuclear acceptor for receptor-glucocorticoid complexes. The present study concerns the interaction of these complexes, isolated from cultured rat hepatoma cells, with purified DNA. This association is rapid, reaching a maximum within a few minutes at 0 degrees, whereas dissociation requires several hours. DNA binds neither free glucocorticoids nor those complexed with transcortin or cytosol proteins different from the receptor. Receptors which are not complexed by steroid have little or no affinity for DNA. "Activation," necessary for the binding of receptor-steroid complexes to isolated nuclei, also enhances DNA binding. The capacity of DNA for binding receptor-steroid complexes is large; saturation was not observed at the complex concentrations studied, using either crude or partially purified receptor preparations. The association of complexes with DNA is inhibited by divalent cations, at increasing ionic strengths, and by mercurial reagents. Complexes bind equally well to bacterial, bacteriophage, or rat DNA; however, there was either no or substantially reduced binding by bacterial 23 S rRNA. The binding of complexes to native DNA is roughly 3-fold greater than to denatured DNA. These characteristics are consistent with the possibility that DNA is the nuclear acceptor for receptor-glucocorticoid complexes; however, the actual composition of the acceptor sites remains unknown.     

Novel adenosine receptors in rat hippocampus. Identification and characterization

2-Chloro[3H]adenosine, a stable analog of adenosine, was used to investigate the presence of adenosine receptors in rat hippocampal membranes that may mediate the depressant effects of adenosine on synaptic transmission in this tissue. Equilibrium binding studies reveal the presence of a previously undescribed class of receptors with a KD of 4.7 microM and a Bmax of 130 pmol/mg of protein. Binding is sensitive to alkylxanthines and to a number of adenosine-related compounds. The pharmacological properties of this binding site are distinct from those of the A1 and A2 adenosine receptors associated with adenylate cyclase. The results suggest that this adenosine binding site is a novel central purinergic receptor through which adenosine may regulate hippocampal excitability.     

Binding of beryllium to nuclear acidic proteins.

In vitro beryllium (Be) binding to rat liver nuclei has been reassessed (KAss = 2.0 X 10(6) M: n = 17 nmol Be/mg protein). Be also binds to rat liver nucleoli (KAss approx. 4 X 10(6) M: n = 10 nmol Be/mg protein). Examination of rat liver chromatin fractionated on a hydroxyapatite column shows that Be does not bind to histone or to the non-histone protein eluted by 0.05 M sodium phosphate. Be is strongly bound to the non-histone proteins eluted by 0.2 M sodium phosphate (KAss = 1.1 X 10(6) M: n = 55 nmol Be/mg protein) and also to the same extent to the fraction containing DNA which is subsequently eluted from the column. Evidence is provided that the latter binding is not due to DNA. The fractions containing the Be-binding proteins also contain the proteins which are phosphorylated to the greater extent.