Pharmacological profile of encounter-induced hyperactivity in isolation-reared mice.

We have recently found that isolation-reared mice show hyperactivity during an encounter with an intruder. However, it is not known whether encounter-induced hyperactivity may model some aspects of psychiatric disorders. The present study examined the pharmacological profile of encounter-induced hyperactivity in isolation-reared mice. Encounter-induced hyperactivity was reduced by acute administration of various antidepressants including the tricyclic antidepressant desipramine (10 mg/kg), the selective serotonin (5-HT) reuptake inhibitors fluvoxamine (10 mg/kg) and paroxetine (10 mg/kg), the 5-HT/noradrenaline reuptake inhibitors venlafaxine (10 mg/kg) and duloxetine (10 mg/kg), the antipsychotic drug risperidone (0.01 mg/kg), the 5-HT2 antagonist ritanserin (1 mg/kg), and the glucocorticoid receptor antagonist RU-43044 (30 mg/kg). The α2 adrenoceptor agonist clonidine (0.03 mg/kg) and the 5-HT4 receptor agonist BIMU8 (30 mg/kg) also reduced encounter-induced hyperactivity. The effect of desipramine was blocked by the α2 adrenoceptor antagonist idazoxan (0.3 mg/kg). The effect of fluvoxamine was blocked by the 5-HT4 receptor antagonist GR125487 (3 mg/kg), but not the 5-HT1A receptor antagonist WAY100635 (1 mg/kg), the 5-HT3 receptor antagonist azasetron (3 mg/kg), or the 5-HT6 receptor antagonist SB399885 (3 mg/kg). The effect of venlafaxine was blocked by the simultaneous administration of idazoxan (0.3 mg/kg) and GR125487 (3 mg/kg), but not by either compound alone. These findings suggest that encounter-induced hyperactivity in isolation-reared mice is a robust model for testing the pharmacological profile of antidepressants, although the range of antidepressants tested is limited and some non-antidepressants are also effective. The present study also shows a key role of α2 and 5-HT4 receptors in the antidepressant effect in this model.

Glucocorticoid analogues: potential therapeutic alternatives for treating inflammatory muscle diseases.

Glucocorticoids (GCs) have been prescribed to treat a variety of diseases, including inflammatory myopathies and Duchenne muscular dystrophy for over 50 years. However, their prescription remains controversial due to the significant side effects associated with the chronic treatment. It is a common belief that the clinical efficacy of GCs is due to their transrepression of pro-inflammatory genes through inhibition of inflammatory transcription factors (i.e. NF-κB, AP-1) whereas the adverse side effects are attributed to the glucocorticoid receptor (GR)-mediated transcription of target genes (transactivation). The past decade has seen an increased interest in the development of GR modulators that maintain the effective anti-inflammatory properties but lack the GR-dependent transcriptional response as a safe alternative to traditional GCs. Many of these analogues or "dissociative" compounds show potential promise in in vitro studies but fail to reach human clinical trials. In this review, we discuss molecular effects of currently prescribed GCs on skeletal muscle and also discuss the current state of development of GC analogues as alternative therapeutics for inflammatory muscle diseases.

Role of prefrontal dopaminergic neurotransmission in glucocorticoid receptor-mediated modulation of methamphetamine-induced hyperactivity.

Glucocorticoids are involved in psychostimulant-induced hyperactivity, but the exact mechanism is not known. This study used the selective glucocorticoid receptor antagonist, RU-43044, to determine whether prefrontal neurotransmission is involved in glucocorticoid-mediated modulation of methamphetamine (METH)-induced hyperactivity in mice. Pretreatment with RU-43044 (10-30 mg/kg) attenuated the increased spontaneous locomotor activity induced by METH (1-2 mg/kg). The psychostimulant effect of METH was also attenuated by adrenalectomy. RU-43044 inhibited METH-induced increases in extracellular dopamine (DA), but not serotonin (5-HT), levels in the prefrontal cortex, but did not affect METH-induced increases in extracellular DA levels in the nucleus accumbens shell, although it inhibited increases in extracellular 5-HT levels. Adrenalectomy also attenuated the METH-induced increases in extracellular DA levels in the prefrontal cortex. RU-43044 did not affect METH-induced increases in plasma corticosterone levels. These findings suggest that glucocorticoid receptors are involved in METH-induced hyperactivity, and that prefrontal dopaminergic neurotransmission plays a role in glucocorticoid-mediated modulation of METH-induced behavioral changes.

Inhibition of iNOS expression and NO production by anti-inflammatory steroids. Reversal by histone deacetylase inhibitors.

In inflammation, nitric oxide (NO) is produced by inducible nitric oxide synthase (iNOS) induced by bacterial products and cytokines, and NO acts as a regulatory and pro-inflammatory mediator. Glucocorticoids are powerful anti-inflammatory agents that inhibit the expression of iNOS and various other inflammatory factors. Histone deacetylation has been recently described as a novel mechanism how glucocorticoids down-regulate transcriptional activation of some inflammatory genes. The aim of the present study was to investigate the effects of inhibitors of histone deacetylation on the suppressive effects of glucocorticoids on NO production and iNOS expression. Dexamethasone and a dissociated glucocorticoid RU24858 inhibited NO production, and iNOS protein and mRNA expression in macrophages exposed to bacterial lipopolysaccharide (LPS). In the presence of a glucocorticoid receptor (GR) antagonist mifepristone, dexamethasone and RU24858 had no effect on NO production. The role of histone deacetylation in the glucocorticoid effect was studied by using three structurally different inhibitors of histone deacetylases (HDACs): trichostatin A, apicidin and MC1293. HDAC inhibitors reversed the effects of dexamethasone and RU24858 on iNOS expression and NO production. Stably transfected A549/8 cells containing luciferase gene under the control of human iNOS promoter were used in promoter-activity studies. iNOS promoter activity induced by IL-1beta was inhibited by dexamethasone and the inhibitory effect was reversed by HDAC inhibitor trichostatin A. The results suggest that glucocorticoids inhibit iNOS expression and NO production by a GR-mediated and GRE-independent manner through histone deacetylation and transcriptional silencing.

Analysis of the dissociated steroid RU24858 does not exclude a role for inducible genes in the anti-inflammatory actions of glucocorticoids.

Although repression of inflammatory gene expression makes glucocorticoids powerful anti-inflammatory agents, side effects limit usage and drive the search for improved glucocorticoid receptor (GR) ligands. In A549 pulmonary cells, dexamethasone and the prototypical dissociated ligand RU24858 (Mol Endocrinol 11:1245-1255, 1997) repress interleukin (IL)-1beta-induced expression of cyclooxygenase (COX)-2 and IL-8. Although RU24858 is a weaker GR ligand, both glucocorticoids showed similar efficacies on transrepression of nuclear factor kappaB (NF-kappaB)-dependent transcription, whereas RU24858 yielded less than 12% of the response to dexamethasone on a classic glucocorticoid response element (GRE) reporter (transactivation). Modest NF-kappaB-dependent transrepression ( approximately 40%), along with analysis of IL-8 transcription rate and the accumulation of unspliced nuclear RNA, indicates that transrepression does not fully account for the repression of genes such as IL-8. This was confirmed by the finding that mRNA degradation is increased by both dexamethasone and RU24858. Analysis of IL-1beta-induced steady-state mRNA levels for IL-8 and COX-2 show that dexamethasone- and RU24858-dependent repression of these genes is attenuated by inhibitors of transcription and protein synthesis. Because similar effects were observed with respect to COX-2 and IL-8 protein expression, we conclude that glucocorticoid-dependent gene expression is necessary for repression by both glucocorticoids. Despite RU24858 being defective at classic GRE-dependent transactivation, both dexamethasone and RU24858 induced the expression of potentially anti-inflammatory genes and metabolic genes, suggesting the importance of nontraditional glucocorticoid-dependent gene expression. Thus, classic transactivation- and transrepressionbased screens for anti-inflammatory "dissociated" GR ligands may be flawed because they may not reflect the effects on real glucocorticoid-inducible genes.

Effects of dissociated glucocorticoids on OPG and RANKL in osteoblastic cells.

Glucocorticoids are effective anti-inflammatory and immunosuppressive agents, but their use is often associated with debilitating side effects such as glucocorticoid-induced osteoporosis. Newly developed glucocorticoid analogues such as the so-called dissociated glucocorticoids are potent immunosuppressants and have the potential for fewer side effects. The effects of these new analogues on osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) in osteoblastic cells have not been studied. OPG and RANKL are osteoblast-derived proteins pivotal to the regulation of bone mass. RANKL stimulates bone resorption by increasing osteoclast differentiation, activation and survival. OPG is the decoy receptor for RANKL and thus inhibits bone resorption. Here, we show that dexamethasone, prednisolone, deflazacort and the dissociated glucocorticoids, RU24858, RU40066, RU24782, AL438-F1 and ZK216348 significantly inhibit OPG production in two human osteoblastic cell lines (MG63 and hFOB). The potency for OPG inhibition was ligand and cell-type specific. In both cell types, dexamethasone and prednisolone were the most potent ligands inhibiting OPG production with IC(50)s of approximately 0.1 nM and 10 nM respectively. In MG63 cells, deflazacort and the RU compounds were the next most potent ligands followed by AL438-F1 and ZK216348. In hFOB cells, however, the RU compounds were the least potent ligands with an IC(50) 74 times higher than in MG63 cells. In contrast, the level of maximum inhibition or effectiveness of OPG inhibition did not vary between cell types but did vary according to the ligand. Dexamethasone, prednisolone, deflazacort and the RU compounds all inhibited OPG production by a maximum of approximately 70-80%, whereas AL438-F1 and ZK 216348 inhibited OPG production by a maximum of only 40-50% at 1 microM. All of the dissociated glucocorticoids and deflazacort were poor stimulators of RANKL gene expression stimulating by only approximately 1-3-fold compared to 7-fold by prednisolone. These data demonstrate that deflazacort and the dissociated glucocorticoids are weak stimulators of the RANKL:OPG ratio compared to prednisolone. Therefore, these compounds have the potential to cause less bone loss than that seen with prednisolone, though this was not investigated here.

The blocking effect of essential controller medications during aspirin challenges in patients with aspirin-exacerbated respiratory disease.

BACKGROUND: The blocking effect of controller medications for asthma could have an effect on the outcome of aspirin challenges in patients suspected of having aspirin-exacerbated respiratory disease (AERD). OBJECTIVE: To evaluate whether there was any blocking effect of long-acting beta2-agonists, systemic corticosteroids, and/or inhaled corticosteroids alone or as co-therapy with leukotriene modifier drugs (LTMDs). METHODS: Between 1981 and 2004, 678 patients with suspected AERD were admitted for aspirin challenge and desensitization. All patients had asthma, chronic sinusitis, nasal polyposis, and at least 1 historical reaction to a nonsteroidal anti-inflammatory drug. Asthma controller medications taken during aspirin challenge were recorded and analyzed with respect to their potential effects on 4 possible outcomes of aspirin challenge, namely, naso-ocular reaction, lower airway reaction, classic upper and lower airway reaction, or a negative challenge result. RESULTS: When compared with AERD patients who received no controller medications, the combined use of LTMDs, inhaled corticosteroids, and long-acting beta2-agonists led to a statistically significant change in aspirin challenge outcomes (P = .009), mainly shifting the reaction from a classic upper and lower respiratory tract reaction to naso-ocular reactions only. LTMDs appeared to have the strongest effect (P < .001) in blocking lower respiratory tract reactions. Systemic corticosteroids did not have the same effects. Blocking of both upper and lower respiratory tract reactions to aspirin as a result of taking controller medications did not occur. CONCLUSION: Controller medications are frequently needed to stabilize airways of patients with AERD. LTMDs alone or in combination with other controllers blocked lower respiratory tract reactions during aspirin challenge in some patients with AERD but did not change the overall rate of positive aspirin challenge results and did not lead to false-negative challenges.

Inhibition of tristetraprolin expression by dexamethasone in activated macrophages.

Tristetraprolin (TTP) is a factor that regulates mRNA stability and the expression of certain inflammatory genes. In the present study, we found that TTP expression was increased in macrophages exposed to bacterial lipopolysaccharide (LPS). Dexamethasone and dissociated steroid RU24858 inhibited LPS-induced TTP protein and mRNA expression and the inhibitory effect was reversed by a glucocorticoid receptor antagonist mifepristone. Histone deacetylase inhibitors trichostatin A (TSA) and apicidin reduced the inhibitory effect of dexamethasone and RU24858 on TTP expression, but the glucocorticoids did not alter TTP mRNA half-life. These results suggest that anti-inflammatory steroids reduce TTP expression in activated macrophages by a glucocorticoid response element (GRE)-independent mechanism, possibly through histone deacetylation and transcriptional silencing.

Progesterone receptor is not required for progesterone action in the rat corpus luteum of pregnancy.

In this study, we investigated whether progesterone exerts a local action regulating the function of the corpus luteum of pregnancy in rats. The luteal activities of the enzymes 3beta-hydroxysteroid dehydrogenase (3beta-HSD), involved in progesterone biosynthesis, and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), that catabolizes progesterone and reduces progesterone secretion by the corpus luteum, were evaluated after intrabursal ovarian administration of progesterone in pregnant rats that had received a luteolytic dose of prostaglandin F2alpha (PGF2alpha). Luteal 3beta-HSD activity decreased and 20alpha-HSD activity increased after PGF2alpha treatment (100 microg x 2 intraperitoneally on Day 19 of pregnancy at 12:00 p.m. and 4:00 p.m.) when compared with controls sacrificed at 8:00 p.m. on Day 20 of pregnancy. This effect of PGF2alpha on the luteal 3beta-HSD and 20alpha-HSD activities was abolished in animals that also received an intraovarian dose of progesterone (3 microg/ovary on Day 19 of pregnancy at 8:00-9:00 a.m.). In a second functional study, luteal cells obtained from 19-day pregnant rats responded to the synthetic progestin promegestone (R5020) in a dose-dependent manner, with an increase in the progesterone output. In addition, the glucocorticoid agent hydrocortisone did not affect progesterone accumulation in the same luteal cell culture. We also examined by immunocytochemistry the expression of progesterone receptors (PR) in the corpora lutea during pregnancy and demonstrated the absence of PR in this endocrine gland in all the days of pregnancy studied. In the same pregnant rats, positive staining for PR was observed in cells within the uteroplacental unit, such as cells of the decidua basalis and trophoblast giant cells of the junctional zone. In addition, positive PR staining was observed in the ovarian granulosa and theca cells of growing follicles, but not in corpora lutea of ovaries obtained from cycling rats at proestrus. In summary, this report provides further evidence of a local action of progesterone regulating luteal function in the rat despite the absence of a classic PR.

A possible mechanism for inhibition of angiogenesis by angiostatic steroids: induction of capillary basement membrane dissolution.

A new class of angiostatic steroids acts independently of previously identified steroid functions to inhibit angiogenesis when administered with heparin. Development of angiostatic steroids as therapeutic modulators of blood vessel growth would be greatly facilitated if their mode of action were thoroughly understood. However, the mechanism by which these steroids produce capillary regression is not known. The distributions of fibronectin and laminin were studied in growing and regressing capillaries by immunofluorescence microscopy to determine whether capillary basement membrane (BM) alterations could be involved in the mechanism of antiangiogenesis. In normal 8-day-old chick chorioallantoic membrane, fibronectin and laminin appeared in continuous linear patterns within BM surrounding growing capillaries. In contrast, chorioallantoic membranes treated with combinations of angiostatic steroid and heparin exhibited capillary BM fragmentation and eventually complete loss of fibronectin and laminin from regions of capillary involution. Capillary BM breakdown correlated with capillary retraction, endothelial cell rounding, and associated capillary regression. BM surrounding large vessels, neighboring epithelium, and nongrowing capillaries were not affected. Capillary BM dissolution is the first biochemical action identified for this new class of antiangiogenic steroids.

Collagen and elastin synthesis in human stroma and breast carcinoma cell lines: modulation by the extracellular matrix.

The desmoplastic response to breast carcinoma is being studied. The stimulation of stromal cell proliferation by a preformed breast tumor matrix was shown. An additional mechanism for stimulating scleroprotein deposition is described here. On a per-cell basis, the synthesis of collagen and elastin was increased by 50% and 70%, respectively, in fibroblasts grown on the preformed breast tumor matrix compared to the same cells grown on plastic or on their own preformed matrix. Breast tumor cells themselves synthesized small amounts of collagen and elastin compared to fibroblasts. These levels were unchanged when breast tumor cells were grown on the preformed matrix of fibroblasts. Addition of steroid hormones to cultured cells grown on plastic or on preformed matrices in various combinations, did not change the levels of either collagen or elastin synthesis. The matrix of human breast tumor cells exerts a dual effect; it is mitogenic for fibroblasts, and also stimulates the level of collagen and elastin synthesis, events that could contribute to the formation of the desmoplastic response to human breast cancer in situ.

Glucocorticoid receptor and in vitro sensitivity to steroid hormones in human lymphoproliferative diseases and myeloid leukemia.

The glucocorticoid receptor (GR) quantitation by a whole-cell assay and/or cytosol technique and the in vitro sensitivity to steroids have been assessed in peripheral blood cells from normal donors and patients with chronic lymphatic leukemia (CLL), acute lymphoblastic leukemia (ALL), lymphosarcoma cell leukemia (LSCL), acute nonlymphatic leukemia (ANLL), and chronic myeloid leukemia (CML). Within the lymphoproliferative diseases, ALL cells exhibited the highest GR concentration (regardless of the method used) and the highest in vitro inhibition of spontaneous [3H]thymidine ([3H]TdR) uptake by glucocorticoids. A significant relationship between GR concentration (whole-cell assay) and in vitro sensitivity to dexamethasone was also found. On the contrary, CLL cells presented the highest sensitivity to glucocorticoids in PHA-stimulated cell cultures. Cells from the only two ALL patients who did not undergo a remission after glucocorticoid-inclusive chemotherapy had both the lowest in vitro sensitivity to dexamethasone and the lowest GR concentration with whole-cell assay. Concerning myeloid leukemia, ANLL patients had GR concentrations slightly higher than those found in the ALL group but exhibited the lowest degree of inhibition of spontaneous [3H]TdR uptake by dexamethasone (stimulatory effects occurred in some cases). CML cells exhibited an inhibition degree by in vitro glucocorticoids significantly higher than that of ANLL cells but not different from that of lymphoproliferative diseases. No clear relationship among GR pattern, in vitro cell sensitivity to glucocorticoids, and clinicohematologic parameters was observed in myeloid leukemia-bearing patients.

Effect of cold exposure on phosphoenolpyruvate carboxykinase (GTP) activity and cyclic amp concentration in livers of starved rats. Role of glucorticoids.

The effect of cold exposure (5 degrees C) on the concentration of cyclic AMP and on the activity of phosphoenolpyruvate carboxykinase (GTP: oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32) was investigated in the liver of intact and adrenalectomized starved rats. Intact starved rats responded to cold exposure with a large increase in both the concentration of hepatic cyclic AMP and the activity of phosphoenolpyruvate carboxykinase above the starvation level. Adrenalectomy did not impair the cold-induced maximum elevation of cyclic AMP but totally prevented the response of the enzyme to cold. Yet, this response was completely restored by hydrocortisone treatment, while the steroid per se had no effect on enzyme activity. In isolated perfused livers of intact starved rats dibutyryl cyclic AMP provoked an immediate dramatic increase in phosphoenolpyruvate carboxykinase activity above the starvation level even if mRNA synthesis was inhibited by cordycepin. However, cyclic AMP was ineffective in increasing enzyme activity in livers of adrenalectomized rats. From these results it is suggested (i) that in starved rats the adaptation to the enhanced glucose demand provoked by cold exposure includes the induction of hepatic phosphoenolpyruvate carboxykinase above the starvation level, (ii) that this induction is due to the cold-induced increase in hepatic cyclic AMP levels, (iii) that cyclic AMP stimulates enzyme synthesis at a post-transcriptional step and (iv) that the cold-induced cyclic AMP-mediated induction of phosphoenolpyruvate carboxykinase above the starvation level requires the "permissive" effect of glucocorticoids.

Hormonal induction of glutamate dehydrogenase in rat liver.

Glutamate dehydrogenase rapidly increases in microsomes and appears in the cytoplasm after administration of cortisone, cAMP, hydrocortisone-acetate. Prolonged administration of ACTH maintains high level of enzyme in the mitochondria and microsomes. Hydrocortisone-acetate, insulin and cortisone decrease drastically enzyme in mitochondria.

Dihydrotestosterone formation in cultured human fibroblasts. Comparison of cells from normal subjects and patients with familial incomplete male pseudohermaphroditism, Type 2.

The conversion of [1,2-3H]testosterone to [3H]dihydrotestosterone has been assessed in fibroblast monolayers grown from skin biopsies of foreskin, scrotum, and various nongential skins from 31 control men who varied in age from newborn to 25 years and three 46,XY subjects with hereditary male pseudohermaphroditism. Under the standardized conditions utilized in this study, the rate of dihydrotestosterone formation was greater in fibroblasts grown from genital skin (foreskin and scrotum) passages exhibit the same differentiation in dihydrotestosterone formation as the skin from which the fibroblasts were grown. Furthermore, 5alpha-reductase, the enzyme that converts testosterone to dihydrotestosterone, exhibits apparent similar substrate specificity in control foreskin fibroblasts and in the foreskin itself. Fibroblasts grown from the foreskin of two patients with familial incomplete male pseudohermaphroditism, type 2, an autosomal recessive disorder of phenotypic sexual differentiation, showed a marked deficiency in the capacity to form dihydrotestosterone. In contrast, fibroblasts grown from the scrotum of one 46,XY male with familial incomplete male pseudohermaphroditism, type 1, an apparent X-linked disorder of phenotypic sexual differentiation, formed dihydrotestosterone at a normal rate.

Effects of ten steroids on acridine orange uptake and beta-N-acetylglucosaminidase levels in rat liver lysosomes.

Ten steroids have been compared for their ability to modify the rate of uptake of acridine orange by rat liver and by rat liver lysosomes in vivo. The short-term effects of the ten steroids on the specific activity of a lysosomal enzyme, beta-N-acetylglucosaminidase, were also compared. Five of the ten steroids were administered as tritium-labelled compounds and the concentration of steroids or metabolites was measured in rat liver and liver lysosomes at 2.5h and 3.75h after administration. Cortisone acetate, etiocholanolone (5-beta-androstan-3-alpha-01-17-one) and testosterone accelerate and increase the uptake of acridine orange by rat liver lysosomes. Deoxycorticosterone, corticosterone, triamcinolone (9-alpha-fluoro-11-beta, 17, 21-trihydroxy-16-alpha-methyl-pregna-1, 4-diene-3, 20-dione), estradiol-17-beta and progesterone appear to inhibit the uptake of acridine orange by rat liver lysosomes at 2.5 hours. Cortisol and dexamethasone (9-alpha-fluoro-11-beta, 17, 21-trihydroxy-16-alpha-methyl-pregna-1, 4-diene-3, 20-dione) had little effect. All steroids with the exception of etiocholanolone and deoxycorticosterone increase with the specific activity of beta-N-acetylglucosaminidase in the lysosomal fraction at 2.5h. None of the effects at 2.5h are due to lowered protein levels. Lysosomal concentrations of radioactivity following the administration of tritiated steroids were greated for the glucocorticoids, corticosterone and cortisol. Estradiol-17-beta, progesterone and testosterone showed much lower concentrations of radioactivity in isolated lysosomes. Most of the lysosomal radioactivity (73-96%) was associated with the soluble fraction of the disrupted lysosomes.

Hypertensive action of 18-hydroxydeoxycorticosterone.

18-Hydroxydeoxycorticosterone is an adrenal steroid hormone causing salt and water retention and is secreted in greatly increased amounts in response to the pituitary hormone adrenocorticotropic hormone. Its production is abnormally high in some forms of hypertension in man and rat. Direct proof that 18-hydroxydeoxycorticosterone is capable of causing hypertension is present. Daily subcutaneous injections of 200 micrograms, a low physiological dose, significantly increase the blood pressure of unilaterally nephrectomized saline-treated rats after 2 weeks. This strengthens the hypothesis that 18-hydroxydeoxycorticosterone contributes to the etiology of hypertension, possibly by a mechanism involving stressinduced release of adrenocorticotropic hormone.