Characterization of specific binding sites for corticosterone in mouse liver plasma membrane

The specific binding of [3H]corticosterone to mouse liver purified plasma membrane fractions is a saturable, reversible, and temperature-dependent process. Only one type of independent and equivalent binding sites has been determined in plasma membrane (Kd = 4.1 nM and Bmax = 3368 fmol/mg). As can be deduced from displacement data obtained in plasma membrane, the high-affinity binding site is different from nuclear glucocorticoid, nuclear progesterone, and Na+, K(+)-ATPase digitalis receptors. Probably this corticosterone binding site or receptor is the same one determined previously for [3H]cortisol in mouse liver plasma membrane. Such beta- and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [3H]corticosterone binding to plasma membranes; therefore, this binding site is independent of these receptors. The binding sites in plasma membranes are not exclusive for corticosterone, but other steroids are also bound with very different affinities.     

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.     

Cortisol 17 beta acid, transcortin, and the heterogeneity of rat brain glucocorticoid receptors

Binding of tracer or competing steroids to transcortin can compromise specificity studies on receptors for adrenal steroids. Recently Alexis et al. have used cortisol 17 beta acid at high concentrations to prevent steroid binding to any transcortin possibly contaminating rat brain cytosol preparations. On the basis of limited specificity studies of [3H]dexamethasone and [3H]corticosterone binding under such conditions, it was claimed that binding sites for the two steroids are indistinguishable, and it is thus unnecessary to invoke distinct binding sites for each glucocorticoid. We have extended these competition studies in the presence of cortisol 17 beta acid, and shown that in rat hippocampus Type I, corticosterone-preferring glucocorticoid receptors can be clearly distinguished both from transcortin and from Type II, dexamethasone-binding glucocorticoid receptors.     

Antagonistic effects of aldosterone on corticosterone-mediated changes in exploratory behavior of adrenalectomized rats

The effect of aldosterone administration on exploratory activity of chronic adrenalectomized (10 days) male rats was investigated. Aldosterone (30 μg/100 g body wt sc) administered 1 hr or 30 min prior to the behavioral test failed to normalize disturbed exploratory activity of adrenalectomized rats, in contrast to the restoration observed after corticosterone, the naturally occurring glucocorticoid of the rat. Administration of the mineralocorticoid 30 min prior to corticosterone prevented the normalization of the behavioral response by the latter steroid. Administration of the same dose of aldosterone 30 min prior to a tracer amount of [³H]corticosterone effectively blocked cell nuclear uptake of radioactive-labeled hormone in the hippocampus. The specific action of corticosterone on exploratory behavior corresponds with the stringent specificity of the neuronal hippocampal corticosterone receptor system. Mineralocorticoid receptors do not seem to be involved in effects on this behavior. The antagonistic action of aldosterone is probably exerted by competitive binding to the corticosterone receptor.     

Photoaffinity labeling with a neuroactive steroid analogue. 6-azi-pregnanolone labels voltage-dependent anion channel-1 in rat brain

Neuroactive steroids modulate the function of gamma-aminobutyric acid, type A (GABA(A)) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3 alpha,5 beta-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABA(A) receptors as evidenced by its ability to inhibit binding of [(35)S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents in Xenopus oocytes and human endothelial kidney 293 cells expressing GABA(A) receptor subunits (alpha(1)beta(2)gamma(2)). [(3)H]6-AziP produced time- and concentration-dependent photolabeling of protein bands of approximately 35 and 60 kDa in rat brain membranes. The 35-kDa band was half-maximally labeled at a [(3)H]6-AziP concentration of 1.9 microM, whereas the 60-kDa band was labeled at higher concentrations. The photolabeled 35-kDa protein was isolated from rat brain by two-dimensional PAGE and identified as voltage-dependent anion channel-1 (VDAC-1) by both matrix-assisted laser desorption ionization time-of-flight and ESI-tandem mass spectrometry. Monoclonal antibody directed against the N terminus of VDAC-1 immunoprecipitated labeled 35-kDa protein from a lysate of rat brain membranes, confirming that VDAC-1 is the species labeled by [(3)H]6-AziP. The beta(2) and beta(3) subunits of the GABA(A) receptor were co-immunoprecipitated by the VDAC-1 antibody suggesting a physical association between VDAC-1 and GABA(A) receptors in rat brain membranes. These data suggest that neuroactive steroid effects on the GABA(A) receptor may be mediated by binding to an accessory protein, VDAC-1.     

Characterization of an imidazoline/guanidinium receptive site distinct from the alpha 2-adrenergic receptor

alpha 2-Adrenergic receptors recognize a number of molecules with diverse chemical structures, including the yohimban diastereoisomers yohimbine and rauwolscine, catecholamines, guanidinium analogs, and imidazolines, such as clonidine. The affinity of the receptor protein for some of these ligands can vary by 10-100-fold among various tissues and species, suggesting a heterogeneous class of binding sites. Certain cellular effects elicited by the compounds possessing an imidazoline or guanidinium moiety may actually be mediated by a membrane receptor distinct from the alpha 2-adrenergic receptor. To determine whether this imidazoline/guanidinium receptive site (IGRS) and the alpha 2-adrenergic receptor represent distinct proteins, we solubilized and partially characterized the two binding sites in rabbit kidney. This tissue expresses both alpha 2-adrenergic receptors and high affinity imidazoline/guanidinium binding sites, the latter which are rauwolscine-insensitive but can be identified with the benzodioxan [3H]idazoxan. The IGRS and alpha 2-adrenergic receptor in rabbit kidney exhibit distinct ligand recognition properties, which are maintained after solubilization and partial purification. In addition, the two receptors can be physically separated by heparin-agarose or lectin affinity chromatography indicating that the two binding sites are distinct entities. [3H]Idazoxan binding is trypsin-sensitive, indicating that the IGRS is a protein rather than a lipid component of the plasma membrane. [3H]Idazoxan binding is not inhibited by endogenous agonists for known neurotransmitter receptors. However, the IGRS does recognize clonidine-displacing substance, a small non-catechol compound isolated from calf brain, suggesting the existence of a previously uncharacterized hormonal/neurotransmitter receptor system.     

Binding of glucocorticoid receptors to mammary chromatin acceptor sites

We have recently characterized the interaction of mouse mammary estrogen receptors (ER) with mammary chromatin acceptor sites and demonstrated that ER from estrogen resistant lactating mammary glands do not bind to chromatin. In this study we have characterized the chromatin binding of the glucocorticoid receptor from mouse mammary glands isolated from nulliparous and lactating mice in order to better understand the relationship between receptor binding to chromatin and steroidogenic sensitivity of the tissue. Mammary chromatin was linked covalently to cellulose and deproteinized sequentially by 0-8 M Gdn-HCl. Binding to intact chromatin as well as to chromatin deproteinized by Gdn-HCl was determined using partially purified [3H]dexamethasone labelled glucocorticoid-receptor complexes (GR) obtained by fractionation on DEAE-cellulose columns. The binding of [3H]GR from mammary glands of nulliparous mice to chromatin fractions from the same tissue revealed maximal binding activity (acceptor sites) on chromatin previously extracted with 5-6 M Gdn-HCl. Binding of [3H]GR was of high affinity (Kd = 0.2 nM) and saturable. A simultaneous comparison of the chromatin binding patterns for [3H]ER and [3H]GR isolated from mammary glands of nulliparous mice revealed that the chromatin subfractions obtained with 4-6 M Gdn-HCl extraction contained acceptor sites for both [3H]ER and [3H]GR; however, while the [3H]ER bound to a 4.5 M and a 5.5 M site, the [3]GR bound a 5 M and a 6 M site. Competition experiments supported the steroid receptor specificity of the chromatin acceptor sites. Thus, the 4-6 M chromatin fractions contain distinct acceptor sites for the glucocorticoid receptor and for the estrogen receptor. In addition our studies reveal that the binding patterns of [3H]GR isolated from mammary glands of nulliparous and lactating mice to their homologous chromatin is essentially similar. Thus, in contrast to estrogen receptors, glucocorticoid receptors from lactating mammary glands are able to effectively bind to chromatin acceptor sites which supports our previous suggestion that the estrogenic insensitivity of lactating mouse mammary glands may at least be in part due to the impeded interaction of ER with chromatin acceptor sites.     

[3H]Fluphenazine Binding to Brain Membranes: Simultaneous Measurement of D-1 and D-2 Receptor Sites

[3H]Fluphenazine was used to label both D-1 and D-2 dopamine receptors in mouse striatal membranes. The D-1 and D-2 specific binding of [3H]fluphenazine was discriminated by the dopamine antagonists SCH-23390 (D-1 selective) and spiperone (D-2 selective). Saturation analyses of these two sites yielded a D-1 receptor density in mouse striatum of 1,400 fmol/mg of protein and a D-2 receptor density of 700 fmol/mg of protein. The affinity of [3H]fluphenazine for the D-2 site was slightly greater than for the D-1 site; the equilibrium dissociation constant (KD) was 0.7 versus 3.2 nM, respectively. Assay conditions are described that reduce nonspecific binding of [3H]fluphenazine to acceptable levels (35% of total binding at 1 nM [3H]fluphenazine). By comparison of displacement curves from a series of dopaminergic and nondopaminergic ligands, the pharmacological specificity of [3H]fluphenazine binding in mouse striatum was demonstrated to be dopaminergic. Only small amounts of dopamine-specific (apomorphine-sensitive) [3H]fluphenazine binding were found in other brain regions. However, chlorpromazine displaced considerable [3H]fluphenazine from all brain regions, including cerebellum, suggesting the presence of a [3H]fluphenazine binding site with a phenothiazine specificity.     

Interaction of rat liver glucocorticoid receptor with a newly synthesized antisteroid ZK98299

Steroid receptor antagonists are important biochemical probes for understanding the mode of steroid hormone action. We have studied the interaction between rat liver glucocorticoid receptor and a newly synthesized antisteroid ZK98299 (13-antigestagen; [11-beta-(4-dimethylaminophenyl)-17a-hydroxy-17 beta-(3- hydroxypropyl)-13 alpha-methyl-4,9-gonadien-3-one]). Glucocorticoid receptor from freshly prepared hepatic cytosol bound [3H]ZK98299 with affinity approximately equal to that of [3H]triamcinolone acetonide. The binding of both steroids reached a maximum at 4 h at 0 degrees C. Both ligands were able to compete for the steroid binding site but progesterone, estradiol and dihydrotestosterone (DHT) failed to compete for the [3H]ZK98299 and [3H]triamcinolone acetonide binding. While [3H]ZK98299 binding to glucocorticoid receptor could occur in the presence of iodoacetamide and N-ethylmaleimide (NEM), [3H]triamcinolone acetonide binding capacity was completely abolished following such treatments. The [3H]ZK98299-receptor complexes sedimented as 9 S and 4 S molecules under control (4 degrees C) and receptor transforming (23 degrees C) conditions, and exhibited a faster rate of dissociation at 23 degrees C when compared with [3H]triamcinolone acetonide-receptor complexes. These results indicate that ZK98299 interacts with hepatic glucocorticoid receptor. The differential effects of iodoacetamide and NEM on the interaction of glucocorticoid receptor with ZK98299 and triamcinolone acetonide, and the faster rate of dissociation of [3H]ZK98299-receptor complexes suggest that treatment with these agents (NEM and iodoacetamide) results in distinct conformational changes in glucocorticoid receptor structure with respect to triamcinolone acetonide and ZK98299 binding. Alternatively, ZK98299 may be interacting with a site which is distinct from one which accepts triamcinolone acetonide.     

Characterization of 5-HT transporter and receptor system in HeLaS3 cells by [(3)H]8-OH-DPAT and other serotonergic ligands

[(3)H]8-OH-DPAT is a selective ligand for labeling 5-HT(1A) receptor sites. In competition binding experiments, we found that classic biogenic amine transporter inhibitors displaced [(3)H]8-OH-DPAT binding at its high-affinity binding sites in HeLaS3 cells. [(125)I]RTI-55 and [(3)H]paroxetine are known to specifically label amine transporter sites, and this was observed in our cells. Displacement studies showed that 8-OH-DPAT displayed affinity in a dose-dependent manner for the labeled amine transporter sites. These data suggest that [(3)H]8-OH-DPAT binds to amine uptake sites in HeLaS3 cells. A variety of drugs targeting different classes of receptors did not significantly affect [(3)H]8-OH-DPAT binding. Moreover, we determined the specific binding effects of various serotonergic ligands (i.e. [(125)I]cyanopindolol, [(3)H]ketanserin/[(3)H]mesulergine, [(3)H]GR-65630, [(3)H]GR-113808 and [(3)H]LSD) that specifically labeled 5-HT(1), 5-HT(2), 5-HT(3), 5-HT(4) and 5-HT(5-7) receptors, respectively. It is suggested that HeLaS3 cells contain distinct types of the related to 5-HT receptor recognition binding sites. These observations could help elucidate the relevant characteristics of different types of 5-HT receptors and 5-HT membrane transporters in tumor cells and their role in tumorigenesis.     

Muscarinic Receptor Subclasses in the Chick Embryo Retina: Influence of Corticosterone Treatment

The present study was performed on retinas of chick embryos receiving at day 8 of incubation an intracerebral injection of 0.02 microgram of corticosterone. We had previously shown with the use of [3H]quinuclidinylbenzilate [( 3H]QNB) that such treatment induced the appearance of two muscarinic binding sites in the treated retinas, whereas only one was detectable in the controls. In the present study we investigated muscarinic cholinergic receptor subclasses with agonist and antagonist binding. Agonist binding was studied by varying the concentrations of carbachol and acetylcholine (10(-9) M-10(-5) M) in the presence of a constant concentration (0.2 nM) of [3H]QNB. Two subpopulations of receptors were revealed, a high- and a low-affinity receptor, in both treated and control retinas. However, in the hormone-treated retinas, the two subpopulations significantly differed from the controls in their affinity and in their relative percentage among the total receptor population. Moreover, using pirenzepine, an antagonist known to have the capacity to distinguish between muscarinic cholinergic subclasses, two receptor subpopulations were found to be present in the hormone-treated retinas but a single one in the controls. It is suggested that hormone treatment can either induce the appearance of a new subclass of muscarinic cholinergic receptors or favor the maturation of a population of retinal cells having these receptors. Pirenzepine binding in retinas from intact embryos of 7, 9, and 11 days of incubation revealed one receptor subpopulation. Thus, these findings are more consistent with the hypothesis that corticosterone effects the target cells, either inducing changes in muscarinic receptor and/or modifying the receptor environment.     

Effect of cellular differentiation on 11beta-hydroxysteroid dehydrogenase activity in the intestine.

11beta-Hydroxysteroid dehydrogenase (11betaHSD) converts endogenous glucocorticoids to their biologically inactive 11-dehydro derivatives and is therefore able to determine, at least in part, the biological action of glucocorticoids. Type 1 11betaHSD has both oxidase and reductase activities interconverting corticosterone and 11-dehydrocorticosterone, whereas type 2 11betaHSD has only oxidase activity converting corticosterone to 11-dehydrocorticosterone. Since 11betaHSD expression is regulated during development and by hormones in a tissue-specific manner and since glucocorticoids play an important role in postnatal intestinal maturation, we investigated the role of corticosteroids and cytodifferentiation in the regulation of intestinal 11betaHSD. Using rat intestinal organ cultures and epithelial cell lines derived from rat small intestine (IEC-6, IEC-18) and from human colon adenocarcinoma (Caco-2, HT-29), we analyzed the effect of corticosteroids and cytodifferentiation on 11betaHSD. Screening of the clonal cell lines showed that Caco-2 cells expressed by far the greatest 11betaHSD2 oxidase activity, lower activity was observed in HT-29 cells, and lowest activity was seen in IEC cells. Treatment with dexamethasone (50 nM) increased the activity of 11betaHSD2 in IEC-6 cells (+59%) and HT-29 cells (+31%), whereas aldosterone (50 nM) stimulated 11betaHSD2 in IEC-6 cells only (+31%). Caco-2 cells and IEC-18 cells did not respond to corticosteroids. Growth of IEC-6 cells on Matrigel, treatment of HT-29 cells with butyrate, and postconfluency of Caco-2 cells increased not only the markers of cytodifferentiation, such as alkaline phosphatase and sucrase, but also the activity of 11betaHSD2 in all of these cell lines (IEC-6, +96%; HT-29, +139%; Caco-2, +95%). Addition of corticosteroids to these more differentiated cell cultures did not enhance 11betaHSD2 activity. In intestinal organ cultures of suckling rat small intestine, dexamethasone and aldosterone stimulated 11betaHSD by more than 300%. We conclude that corticosteroids markedly and differentially regulate intestinal 11betaHSD2 and that cytodifferentiation of intestinal epithelial cells is associated with upregulation of 11betaHSD2 activity that is independent of corticosteroids.     

Selective covalent labeling of cysteines in bovine serum albumin and in hepatoma tissue culture cell glucocorticoid receptors by dexamethasone 21-mesylate

The specificity of protein labeling by an affinity label of glucocorticoid receptors, dexamethasone 21-mesylate (Dex-Mes), was investigated using bovine serum albumin (BSA) as a model. During the early stages of [3H]Dex-Mes labeling at pH 8.8, approximately 90% of the covalent bond formation occurred at the one non-oxidized cysteine (Cys-34) of BSA. The nonspecific labeling was equally distributed over the rest of the BSA molecule. [3H]Dex-Mes labeling of Cys-34 was totally, and specifically inhibited by nearly stoichiometric amounts of the thiol-specific reagent methyl methanethiolsulfonate (MMTS). Thus both Dex-Mes and MMTS appear to react very selectively with thiols under our conditions. In reactions with hepatoma tissue culture (HTC) cell glucocorticoid receptors, MMTS was equally efficient in preventing [3H]dexamethasone binding to receptors and [3H]Dex-Mes labeling of the 98-kDa receptor protein. These results indicate that Dex-Mes labeling of the glucocorticoid receptor involves covalent reaction with at least one cysteine in the steroid binding site of the receptor. Small (approximately 1600-dalton) fragments of the [3H]Dex-Mes-labeled 98-kDa receptor were generated by limit proteolysis with trypsin, chymotrypsin, and Staphylococcus aureus V8 protease under denaturing conditions. Data from these fragments on 15% sodium dodecyl sulfate-polyacrylamide gels were consistent with all of the covalent [3H] Dex-Mes being located on one or a few cysteines in one approximately 15-residue stretch of the receptor. Further studies revealed no differences in the limit protease digestion patterns of activated and unactivated [3H]Dex-Mes-labeled receptors with trypsin, chymotrypsin, or V8 protease under denaturing conditions. These data suggest that activation does not cause any major covalent modifications of the amino acids immediately surrounding the affinity-labeled cysteine(s) of the steroid binding site.     

Identification of specific [3H]adenine-binding sites in rat brain membranes

We recently reported that adenine acts as a neurotrophic factor independent of adenosine or P2 receptors in cultured Purkinje cells [Watanabe S. et al. (2003) J. Neurosci. Res. 74, 754-759], suggesting the presence of specific receptors for adenine in the brain. In this study, the characterization of adenine-binding activity in the rat brain was performed to further characterize the receptor-like adenine-binding sites. Specific binding sites for [(3)H]adenine were detected in membrane fractions prepared from rat brains. The kinetics of [(3)H]adenine binding to membranes was described by the association and dissociation rate constants, 8.6 x 10(5) M(-1) min(-1) and 0.118 +/- 0.045 min(-1), respectively. A single binding site for [(3)H]adenine with a K (D) of 157.1 +/- 20.8 nM and a B (max) of 16.3 +/- 1.1 pmol/mg protein (n = 6) was demonstrated in saturation experiments. A displacement study involving various related compounds showed that the [(3)H]adenine binding was highly specific for adenine. It was also found that [(3)H]adenine-binding activity was inhibited by adenosine, although other adenosine receptor ligands were ineffective as to [(3)H]adenine binding. The brain, especially the cerebellum and spinal cord, showed the highest [(3)H]adenine-binding activity of the tissues examined. These results are consistent with the presence of a novel adenine receptor in rat brain membranes.     

In vivo competitive autoradiographic study of [3H]corticosterone and [3H]aldosterone binding sites within mouse brain hippocampus

The binding sites for [3H]corticosterone (3HB) and [3H]aldosterone (3HA) within the hippocampal area of the mouse brain have been studied by autoradiography in competition experiments. Excess unlabelled aldosterone (A) or corticosterone (B) both abolished the nuclear accumulation of radioactivity within neurons observed after injection of either 3HA or 3HB. Experiments where a subcutaneous injection of a "pure glucocorticoid' RU26988 was given before injection of 3HA alone showed a marked accumulation of radioactivity within neuronal nuclei of the hippocampus suggesting the presence of 3HA binding sites distinct from classical type II glucocorticoid receptors. In addition, when RU26988 was given before the injection of 3HA associated with a 30- or 100-fold excess of either A or B, the cell nuclear accumulation of radioactivity was no longer observed. These results showed that in our in vivo experimental conditions, B displayed the same ability as A to occupy 3HA binding sites, supporting the view that in mouse hippocampal neuronal nuclei, the aldosterone-binding and corticosterone-preferring sites represent the same molecular entity.     

Binding characteristics of [3H]delta(5)-androstene-3beta,17beta-diol to a nuclear protein in the rabbit vagina

In this study, we investigated the binding characteristics of [3H]Delta(5)-androstene-3beta,17beta-diol to rabbit vaginal cytosolic and nuclear extracts and in freshly excised intact tissue strips. [3H]delta(5)-Androstene-3beta,17beta-diol bound to a protein(s) in the vaginal nuclear extract with high affinity (K(d)=3-5 nM) and limited capacity (50-100 fmol/mg protein). No specific binding was detected in the cytoplasmic extracts. Competitive binding studies showed that binding of [3H]delta(5)-androstene-3beta,17beta-diol was effectively displaced with unlabeled delta(5)-androstene-3beta,17beta-diol but not with dehydroepiandrosterone, testosterone, dihydrotestosterone, triamcinolone acetonide, or progesterone. However, estradiol at high concentrations partially displaced bound [3H]delta(5)-androstene-3beta,17beta-diol. Incubation of freshly excised vaginal tissue strips with [3H]delta(5)-androstene-3beta,17beta-diol in the absence or presence of excess unlabeled delta(5)-androstene-3beta,17beta-diol for 1h at 37 degrees C resulted in specific binding to a soluble macromolecule in the nuclear KCl extracts. In addition, quantitative measurement of estrogen receptor, androgen receptor and delta(5)-androstene-3beta,17beta-diol binding protein was performed by equilibrium ligand binding assays using extracts of distal vaginal tissue from intact animals or ovariectomized animals treated for 2 weeks with vehicle, estradiol, testosterone, or estradiol plus testosterone. These changes in steroid hormone levels resulted in opposing trends between the estrogen receptor and delta(5)-androstene-3beta,17beta-diol binding protein, suggesting that delta(5)-androstene-3beta,17beta-diol binding protein is regulated differently by the hormonal milieu than the estrogen receptor. These data suggest that rabbit vaginal tissue expresses a novel binding protein which specifically binds delta(5)-androstene-3beta,17beta-diol and is distinct from the androgen and estrogen receptors.     

Binding of dexamethasone to rat liver nuclei in vivo and in vitro: evidence for two distinct binding sites

The binding of [3H]dexamethasone (DEX) to rat liver nuclei in vitro and in vivo have been compared. In vitro, purified nuclei displayed a single class of specific glucocorticoid binding sites with a dissociation constant (Kd) of approximately 10(-7) M for [3H]DEX at 4 degrees C. The glucocorticoid agonists prednisolone, cortisol, and corticosterone and the antagonists progesterone and cortexolone competed avidly for this site, but the potent glucocorticoid triamcinolone acetonide (TA) competed poorly in vitro. Nuclei isolated from the livers of intact rats contained 1-2 X 10(4) [3H]DEX binding sites/nucleus. Up to 85% of the binding sites were recovered in the nuclear envelope (NE) fraction when NE were prepared either before or after labeling with [3H]DEX in vitro. After adrenalectomy, the specific [3H]DEX binding capacity of both nuclei and NE decreased to 15-20% of control values, indicating sensitivity of the binding sites to hormonal status of the animals. Efforts to restore the binding capacity by administration of exogenous glucocorticoids, however, were unsuccessful. After labeling of rat liver nuclei in vivo by intraperitoneal injection of [3H]DEX or [3H]TA into living animals, the steroid specificity and subnuclear localization of radiolabel were different. Both [3H]TA (which did not bind in vitro) and [3H]DEX became localized to nuclei in a saturable fashion in vivo. With either of these ligands, approximately 20% of the total nuclear radiolabel was recovered in the NE fraction. These results suggest the presence of two separate and distinct binding sites in rat liver nuclei, one which is localized to the NE and binds [3H]DEX (but not [3H]TA) in vitro, and another which is not localized to the NE but binds [3H]DEX and [3H]TA in vivo.     

A comparison of the high-affinity peripheral benzodiazepine receptor ligands DAA1106 and (R)-PK11195 in rat models of neuroinflammation: implications for PET imaging of microglial activation

Activated microglia are an important feature of many neurological diseases and can be imaged in vivo using 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a ligand that binds the peripheral benzodiazepine receptor (PBR). N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl) acetamide (DAA1106) is a new PBR-specific ligand that has been reported to bind to PBR with higher affinity compared with PK11195. We hypothesized that this high-affinity binding of DAA1106 to PBR will enable better delineation of microglia in vivo using positron emission tomography. [(3)H]DAA1106 showed higher binding affinity compared with [(3)H](R)-PK11195 in brain tissue derived from normal rats and the rats injected intrastriatally with 6-hydroxydopamine or lipopolysaccharide at the site of the lesion. Immunohistochemistry combined with autoradiography in brain tissues as well as correlation analyses showed that increased [(3)H]DAA1106 binding corresponded mainly to activated microglia. Finally, ex vivo autoradiography and positron emission tomography imaging in vivo showed greater retention of [(11)C]DAA1106 compared with [(11)C](R)-PK11195 in animals injected with either lipopolysaccaride or 6-hydroxydopamine at the site of lesion. These results indicate that DAA1106 binds with higher affinity to microglia in rat models of neuroinflammation when compared with PK11195, suggesting that [(11)C]DAA1106 may represent a significant improvement over [(11)C](R)-PK11195 for in vivo imaging of activated microglia in human neuroinflammatory disorders.