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Adrenal Steroid Receptor Activation in Rat Brain and Pituitary Following Dexamethasone: Implications for the Dexamethasone Suppression Test Andrew H. Miller, Robert L. Spencer, Mark Pulera, Susan Kang, Bruce S. McEwen, and Marvin Stein
The dexamethasone suppression test (DST) has been used extensively to evaluate feedback inhibition of the hypothalamic-pituitary--adrenal (HPA ) axis by adrenal steroids. Nevertheless, it remains unclear at what level of the HPA axis and through which adrenal steroid receptor subtype dexamethasone exerts its inhibitory effect. Because adrenal steroid receptor activation is an important prerequisite for dexamethasone to affect cellular function, HPA axis tissues that exhibit evidence of receptor activation following dexamethasone administration are likely site(s) of action for this synthetic hormone to inhibit HPA axis activity. Therefore, type-! and type.ll adrenal steroid receptor activation was assessed in the pituitary, hypothalamus, and hippocampus of intact and adrenalectomized rats after overnight exposure to various oral doses of dexamethasone. Results with dexamethasone were compared to similar studies using corticosterone, the endogenous glucocorticoid of the rat. All dexamethasone doses led to significant type-I! receptor activation in the pituitary, whereas only an exceedingly high dexamethasone dose activated type.ll receptors in the hippocampus and hypothalamus. Dexamethasone had little effect on type ! receptors in any tissue at any dose. In contrast, corticosterone significantly activated type4 receptors in all tissues, whereas it activated type.ll receptors in the brain and not the pituitary at physiological concentrations. Because dexamethasone activated pituitary type-ll receptors at blood concentrations that did not activate type-II receptors in the brain, these results suggest that the DST in humans may primarily be a measure of type-ll adrenal steroid receptor feedback inhibition at the level of the pituitary.
Introduction Dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis in patients with major depression is one of the most consistent findings in all of biological psychiatry. Abnormalities include hypersecretion of the adrenal steroid, cortisol (Sachar 1975; Carroll et al 1976); nonsuppression of cortisol and corticotropin (ACTH) following administration of the synthetic glucocorticoid, dexamethasone (Carroll et al 1981; Kalin et al 1982; From the Department of Psychiatry (AHM, MP, SK, MS), Mount Sinai School of Medicine; and the Laboratory of Neuroendocrinology (PLS, BSM), Rockefeller University, New York, NY Address reprint requests to Andrew H. Miller, M.D., Box 1229, Annenbe~g 22-66, Department of Psychiatry, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. R~eived April 23, 1992; revised August 6, 1992. © 1992 Society of Biological Psychiatry
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Holsboer 1983; Arana et al 1985); a blunted ACTH response to corticotropin-releasing hormone (CRH) (Gold et al 1984, 1986; Holsboer et al 1984, 1986; Risch et al 1988) and an increased cortisol response to ACTH (Amsterdam et al 1983; Kalin et al 1987; Jaeckle et al 1987; Krishnan et al 1990). These abnormalities are believed to be related in part to an overactivation of limbic structures, including the hypothalamus and hippocampus, resulting in hypersecretion of CRH (Nemeroff et al 1984; Gold et al 1984). The mechanism of this limbic overactivation is unknown, although resistance of adrenal steroid receptors to feedback inhibition at or above the level of the hypothalamus is believed to be involved (Sapolsky et al 1984b; Sapolsky et al 1986; Gold et al 1988). There are two types of adrenal steroid receptors; type-I adrenal steroid receptors, which are commonly referred to as mineralocorticoid receptors, and type-If adrenal steroid receptors, which are also known as glucocorticoid receptors (Reul and de Kloet 1985). Type-I receptors have a high affinity for endogenous adrenal steroid hormones and are believed to play a role in the regulation of circadian fluctuations in corticosteroids (Beaumont and Fanestil 1983; de KIoet and Reui 1987; Sutanto and de Kloet 1987). Type-ll receptors, on the other hand, have a lower affinity for endogenous adrenal steroids than type-I receptors and therefore are believed to be more important in termination of the corticosteroid response to stress when endogenous levels of glucocorticoids are high (Reui and de Kloet 1985; de KIoet and Reul 1987). In light of these characteristics, dysregulation of either or both receptor subtypes may be involved in altered HPA axis function in major depression. The dexamethasone suppression test (DST) has been used extensively to evaluate dysregulated adrenal steroid feedback inhibition in the depressive disorders. However, it remains unclear at what level of the HPA axis dexamethasone exerts its inhibitory effect. Moreover, it has not been established through which adrenal steroid receptor subtype dcxamethasone acts. In order for dexamethasone to inhibit HPA axis function, an important prerequisite is the activation of type-I and/or type-ll adrenal steroid receptors in relevant HPA tissues. Previous studies suggest that depending on the mode of administration dexamethasone may be more effective in activating type-II receptors in the pituitary (and peripheral tissues in general) than in the brain (de Kioet et al 1974, 1975; Miller et al 1990; Spencer et al 1990). The DST may therefore provide information on type-If adrenal steroid receptor feedback inhibition, primarily at the level of the pituitary with consequent implications for the interpretation of abnormal DST results. To further investigate at what level of the HPA axis and through which receptor subtype dexamethasone acts, the ability of various oral doses of dexamethasone to activate typeI and type-II adrenal steroid receptors in the pituitary, hypothalamus, and hippocampus was assessed in intact and adrenalectomized Sprague-Dawley rats. To compare binding parameters in the pituitary and brain to other peripheral tissues, receptor binding was also measured in the spleen, which has been shown to express both type 1 and type-If adrenal steroid receptors (Miller et al 1990). Types I and II receptor activation was estimated by comparing adrenal steroid receptor binding in the various treatment groups (Meaney et al 1988; Spencer et al 1990; Miller et al 1990, 1991a). Prevailing models of adrenal steroid action propose that the receptor, when bound by steroid, undergoes a conformational change referred to as activation or transformation (Munck and Foley 1980; Waiters 1985). The activated form of the receptor has a high affinity for deoxyribose nucleic acid (DNA) and is found exclusively in the nucleus. Adrenal steroid receptors appear to be unique from other steroid receptors in that only the unactivated form of the receptor can rebind steroid and participate in an in
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vitro exchange assay (Chou and Luttge 1988; Spencer et al 1990; Meaney et al 1988). Therefore. adrenal steroid receptors activated in vivo will not rebind radiolabeled steroid in vitro and thu~ cannot be measured in an in vitro cytosolic exchange assay. Taking advantage of this unique property, the amount of adrenal steroid receptor activation that occurs in vivo can be estimated by comparing the number of binding sites measured by an exchange assay in the presence of varying concentrations of circulating steroid hormone; the greater the receptor activation, the lower the number of measurable binding sites, and vice versa (Meaney et al 1988; Spencer et al 1990; Miller et al 1990, 1991a). Of note is that this estimate of receptor activation (i.e., decreased receptor binding following an acute hormone exposure) has been shown to highly correlate with changes in cellular function that are mediated by adrenal steroid receptors (Miller et al 1991a). A similar paradigm was used to estimate receptor activation in the above-noted tissues after exposure to corticosterone, the endogenous glucocorticoid of the rat. Several studies have used naturally occurring glucocorticoids to investigate feedback inhibition in human depressives (Young et al 1991), however as with dexamethasone, it remains unclear through which receptor subtype and in what HPA axis tissue(s) these hormones act.
Methods Subjects Young, adult male Sprague-Dawley rats (300-350g) were used in all treatment groups. Animals were housed 2 to 3 per cage and maintained on a 12-hr light--dark cycle (lights on at 7 AM) in an animal room separate from the laboratory. Rat chow and tap water were provided ad iibitum except in adrenalectomy experiments, in which case 0.9% saline was substituted for tap water. Where indicated, bilateral adrenalectomies were performed using standard aseptic surgical techniques on animals fully anesthetized with the inhalant, methoxyflurane (Metofane; Pitman-moore, Washington Crossing, NJ).
Design Dexamethasone Studies Two separate experiments were conducted using dexamethasone treatment; one employing intact animals and the other employing adrcnalectomized ~imals. In the dexamethasone experiment on intact animals, the experiment was conducted on two separate days, each using four treatment groups (n - 4 per group per day) based on the concentration of dexamethasone in the drinking water: 0, 0.3 ~,g/ml, 0.8 gg/ml, and 10 p~g/ml dexamethasone. The low and medium doses of dexamethasone were designed to reproduce dexamethasone blood concentrations consistent with those found in humans during a l-mg DST. The highest dose was used to achieve saturation of receptors in all tissues. Appropriate amounts of stock dexamethasone [10 mg/ml in 100% ethanol (EtOH)] or EtOH alone were added to the drinking water at 6 pM, the night before the experiment. The final concentration of EtOH in the drinking water of all rats was stano dardized at 0.078% wt/vol. No other source of fluids was provided in any of the experiments. At 9 AM the following day, all animals were killed by rapid decapitation. Trunk blood was obtained for determination of the plasma dexamethasone concentration, and the indicated brain regions and peripheral tissues were surgically removed for binding studies. Tissue for binding assays was rapidly frozen on dry ice and stored at - 8 0 ° C.
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In the experiment on adrenalectomized animals, five treatment groups were used (n = 4 per group): intact, acLrena!ecto~ed~ adrenalectomized + 0.3 ttg/ml dexamethasone in the drinking water, adrenalectomized + 0.8 ttg/ml dexamethasone, and adrenalectomized + 10 ~tg/ml dexamethasone. Adrenalectomies were performed between 20 and 24 hr before sacrifice, a time period that allows adequate time for clearance of endogenous corticosterone (Spencer et al 1990). The drinking water containing dexamethasone was prepared in the same manner as the experiment on intact animals, and animals were sacrificed and tissues removed as previously indicated.
Corticosterone Studies As with dexamethasone, two separate experiments were conducted using corticosterone treatment, one using intact animals and the other using adrenalectomized animals. In the experiment on intact animals, four treatment groups (n -- 5 per group) were used based on the concentration of corticosterone in the drinking water: 0, 200 ttg/ml, 400 ttgtml, and 600 ttg/ml corticosterone. Appropriate amounts of stock corticosterone (40 mg/ml in 100% ethanol) were added to the drinking water at 6 PM the night before the experiment. The final concentration of EtOH in the drinking water of all rats was standardized at 1.17% wt/vol. The higher EtOH concentrations in corticosterone-treated animals was necessitated by the relative low solubility of corticosterone in alcohol and H20. At 9 AM, following overnight corticosterone exposure, animals were killed by rapid decapitation, and blood and tissue samples were removed and processed as indicated in the dexamethasone studies. In the experiment on adrenalectomized animals, five treatment groups were used (n = 4-5 per group); intact, adrenalectomized, adrenalectomized + 200 ttg/ml corticosterone in the drinking water, adrenalectomized + 400 ttg/ml corticosterone, and adrenalectomized + 600 ttg/ml corticosterone. Adrenalectomies were performed as in the dexamethasone experiments. Corticosterone was prepared as indicated, and animals were sacrificed and tissues removed as noted earlier.
Bindin8 Assay Adrenal steroid receptor binding was determined using a previously described in vitro cytosolic exchange assay (Miller et al 1990; Spencer et al 1990). Tissue was homogenized and centrifuged at 105,000 g for 60 rain at 4°C. The supernatant cytosol was then added to incubation solutions containing radiolabeled steroids with or without unlabeled competitors. The cytosol-steroid mixture was incubated at 4 ° C with steroids for 18--22 hr. Shorter time periods have been shown to be insufficient in allowing full exchange of radiolabeled steroid with adrenal steroid receptors when competing steroid is present (HoHim et al 1983; Meaney et al 1988). Bound radiolabeled steroid was separated from unbound steroid by filtration through mini columns containing 1.25 ml of LH-20 Sephadex (Pharmacia, Piscataway, NJ). Scintillation flour (Ready Safe, Beckman, Fullerton, CA) was added to the eluate containing the bound fraction of steroid, and tritium (3H) radioactivity was determined in a liquid scintillation couter (LKB Wallac Beta 1214; LKB, Uppsala, Sweden, 33% efficiency). The homogenization and incubation buffer was comprised of I0 mM tris(hydroxymethyl)aminomethane, 1 mM EDTA, 20 mM molybdic acid, 5 mmol/L dithiothreitol and 10% glycerin in double-distilled water (pH -7.4). Tissue was homogenized in a volume of I-3.5 ml of buffer, yielding a final protein concentration of 0.5--1.5 mg
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protein/ml cytosol. For single-point assays, type-I receptor binding was determined from the binding of [3H]dexamethasone (10 nmol/L) in the presence of the type-II competitor, RU28362 (0.5 p~mol/L) (Coirini et al 1985). [3H]Dexamethasone has been found to be an effective radioligand for measuring type-I receptor binding sites in vitro and gives type-I receptor binding levels similar to [3H]aldosterone (Luttge et al 1989a; Spencer et al 1990). Type-ll binding was defined as the amount of total [3H]dexamethasone binding displaced by RU28362 (0.5 Ixmol/L). Binding in the presence of an excess of corticosterone (2.5 i~mol/L) was used for determining nonspecific binding. Nonspecific binding was reliably < 5% of total binding. Specific binding was expressed as femtomoles per mg of cytosol protein. For saturation binding studies, a range of five concentrations of [3H]dexamethasone (0.3-10 nmol/L) was used. Protein content was determined by the method of Bradford (1976), with use of bovine serum albumin as a standard.
Steroids [6,7-3H(N)]-Dexamethasone (49.9 Ci/nmol/L) was obtained from New England Nuclei,," (Boston, MA). Unlabeled corticosterone was obtained from Steraloids (Wilton, NH) and dexamethasone was obtained from Sigma (St. Louis, MO.). RU28362, the type-ll receptor agonist, was a gift from Koussel-Uclaf (Romainville, France).
Plasma Dexamethasone and Corticosterone Plasma dexamethasone levels were determined using a direct radioimmunoassay procedure as described by Lo et al (1989). Assay sensitivity is 1 pg of dexamethasone, and coefficients of variation within and between assays range from 1%-3% and 3%-8%, respectively. Total serum corticosterone was measured in trunk blood by radioimmunoassay using rabbit antiserum raised against corticosterone-21-hemisuccinate BSA (B21-42; Endocrine Sciences, Tarzana, CA). Assay sensitivity is 10 pg of corticosterone, and coefficients of variation within and between assays range from 2%-5% and 6%-10%, respectively.
Statistical Analysis A one-way analysis of variance (ANOVA) was used to evaluate the effects of steroid treatment on type-I and type-ll receptor binding, except in the case of the dexamethasone studies on intact rats, in which a two-way ANOVA was used to evaluate dexamethasone effects, day effects, and dexamethasone*day interactions. Contrasts between treatment groups were made using t-tests (two-tailed); Bonferonni correction was applied for multiple comparisons except where indicated (Cohen and Cohen 1983). For saturation binding, the dissociation constant (Kd) (in nmol/L) was derived from Scatchard analysis (Limbird 1986), and the binding maximum (in fmoles/mg protein) was determined at the 10 nmol/L [3H]dexamethasone concentration. Results
Dexamethasone Studies Experiment 1. In the first experiment, the effect of various oral doses of dexamethasone on type-II adrenal steroid receptor binding was evaluated in intact rats. The dexamethasone blood levels for each group are shown in Table 1 (top). It should be noted that the mean
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Table 1. Mean ("4-SEM) Dexamethasone Blood Concentrations in Intact (Top) and Adrenalectomized (Bottom) Rats
Ikxamethasone treatment group
Dexamethasone(.g/ml)
Intact 0.3 ItWmi 0.8 Itg/ml 10 ttg/ml ADX Intact ADX ~- 0.3 Itg/ml ADX + 0.8 Itg/ml ADX + 10 Itg/ml
60 ~g/dl) of corticosterone were achieved. Therefore, factors such as CBG, which may serve to buffer peripheral tissues from corticosterone, appear only to be relevant under physiological conditions. Because dexamethasone activated pituitary type-If receptors at blood concentrations that did not activate brain type-II receptors in the rat, it seems unlikely that any potential effects of dexamethasone on the brain to inhibit HPA axis function can be separated from its effect on the pituitary. This latter point may have implications for the interpretation of the DST in humans. Our data from rats suggests that the DST in humans may prima~ily be a measure of type-II adrenal steroid receptor feedback inhibition at the level of the pituitary. This conclusion is based on a comparison of the dexamethasone blood levels required to achieve significant activation of type-II receptors in the rat brain and pituitary with the dexamethasone blood levels obtained in humans during a l-mg DST. For example, maximum mean dexamethasone blood levels in humans 30 min-2 hr following ingestion of 1-1.5 mg of dexamethasone are in the range of 10-28 ng/ml (Duggan et al 1975; Wiedemann and Holsboer 1987a,b). These dexamethasone blood concentrations are approximately tenfold lower than those levels that were found to lead to significant activation of brain type-If receptors in the rat. Moreover, these peak levels in humans overlap with the dexamethasone blood concentrations in rats of the low- and mediumdose groups in the dexamethasone experiment on adrenalextomized animals and the medium-dose group of the dexamethasone experiment on intact animals, both instances in which there was no evidence of significant activation of brain type-If receptors. Of note is that the dexamethasone blood levels in the rats were obte'ned 2-hr into their light period (9 AM), and rats drink primarily in the first-half of their dark period (7 PM-I AM). Therefore, it is likely that the peak dexamethasone blood levels in these animals were much higher than those observed at the time of sacrifice. Dexamethasone blood levels in humans at 8 AM following 11 PM ingestion of 1 mg dexamethasone are in the 0.5-4 ng/ml range, which overlap with our low-dose intact/dexamethasone treatment group (Asnis et al 1989; Klein and Berger 1987). It should be pointed out that the phmmacokinetics of dexamethasone may differ in the human as compared to the rat. Furthermore, although studies in humans and rats indicate that the type.ll receptor in these two species is similar in (l) affinity for dexamethasone (Sarrieau et al 1988), (2) steroid specificity (Yu et al 1981), (3) protein structure (Yu et al 1981), and (4) magnitude of binding sites in the pituitary, hippocampus, and hypothalamus (Tsuboi et al 1979), caution should be taken against drawing direct comparisons of receptor activation by steroid hormones between these species. Nevertheless, the results of these studies may offer important insights into human adrenal steroid receptor physiology following dexamethasone administration. Although dexamethasone bioavailability significantly confounds attempts to interpret DST results, several studies that have controlled for dexamethasone blood level have demonstrated DST abnormalities in depressed patients OViedemann and Holsboer 1987a,b). Taken together with these results, our findings raise several important considerations for
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examining feedback inhibition in the depressive disorders. First, as noted previously, an abnormal DST may primarily represent altered type-ll receptor function in the pituitary of depressed patients. In keeping with this notion is evidence of adrenal steroid receptor dysfunction in other peripheral tissues of patients with major depression. For example, some studies have shown that depressed patients exhibit decreased adrenal steroid receptors in peripheral blood mononuclear cells, which may be related to an impaired nutoregulation of receptor expression (Gormely et al 1985; Whalley et al 1986; Rupprecht et al 1991). In addition, the sensitivity of immune cells to the inhibitory effects of dexamethasone or cortisol in vitro and in vivo has been found to be reduced in depressed patients (Miller et al 1987; Lowy et al 1988; Miller et al 1991b). Furthermore, unlike controls, depressives pretreated with dexamethasone exhibit a significant rise in ACTH and cortisol following CRI-I, providing further evidence of impaired feedback inhibition at the level of the pituitary (Holsboer et al 1987; Bardeleben and Holsboer 1991). However, intact feedback inhibition is supported by findings which indicate that blunted ACTH responses to CRH in depressives are eliminated by reducing circulating endogenous glucocorticoids with metyrapone (Bardeleben et al 1988). These apparent inconsistencies in HPA axis alterations may represent more than one underlying pathology of the HPA axis in depressed patients and/or different time points along a continuum of evolving HPA axis pathology. A second implication of our study is that the DST does not appear to provide information on adrenal steroid receptor feedback inhibition in the hippocampus and hypothalamus. Evidence indicates that there is pathology in these brain regions that may have relevance to HPA axis disturbances in depressed patients. For example, elevated CRH levels in the cerebrospinal fluid of depressed patients along with downregulation of CRH receptors in the frontal cortex of suicide victims suggest that altered HPA axis activity is in part dw. to hypersecretion of CRH secondary to pathology at or above the level of the hypothalamus (Nemeroff et al 1984, 1988, 1991). Moreover, taken together with the role of the hippocampus in HPA axis feedback inhibition (Sapolsky et al 1984a,b, Herman et al 1989), the increased sensitivity of hippocampal neurons to the effects of chronic stress and/or elevated glucocorticoids further support the notion that the roots of HPA axis pathology in depression may reside in the brain (Sapolsky et al 1984a, Sapolsky et al 1986). Therefore, alternative strategies need to be developed to examine feedback inhibition in relevant brain regions. As seen in our studies, infusions of naturally occurring glucocorticoids may allow preferential access of adrenal steroids to the hippocampus and hypothalamus and thereby provide a reasonable probe of feedback inhibition in these brain regions. However, high concentrations of these hormones are likely to activate pituitary type-ll receptors in addition to brain type-ll receptors making interpretation difficult. Furthermore, naturally occuring glucocorticoids activate type-I receptors in all HPA axis tissues at doses that might otherwise selectively activate type-ll receptors in the brain. Future studies examining adrenal steroid feedback inhibition in the brain may require infusions of low-dose cortisol in the presence of type-I receptor antagonists, such as RU28318 (Ratka et ai 1989). Finally, our studies suggest that the role of type-i adrenal swroid receptors in HPA axis disturbances has not been adequately addressed by studies using dexamethasone or cortisol. Because type-I receptors may regulate basal corticosteroid secretion, hypercortisolism in the absence of DST nonsuppression may represent a type-I receptor defect. Furthermore, since chronic activation of type-I receptors can lead to downregulation of type-ll receptors (Luttge et al 1989b), type-I receptors may also play a role in altered
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type-ll receptor expression and/or responsiveness. Low-dose infusions with the type-I agonist, aldosterone, followed by measurement of ACTH in patients pretreated with metyrapone may shed light on this possibility. In conclusion, animal studies examining adrenal steroid receptor activation in HPA axis tissues following various hormone exposures may provide important insights into clinical research protocols geared to evaluate adrenal steroid feedback inhibition in human subjects. Animal studies can indicate wh;ch receptor subtypes are activated in which HPA axis tissues in any given protocol. Such data is relevant for the interpretation of results as well as the development of new approaches to the study of H ° A axis pathology in psychiatric disorders. This research was supported in part by a National Institute of Mental Health Research Scientist Development Award (MH00680)to AHM and research grant (MH47674)to AHM and RLS. The authors would like to thank Abrar Husain, HeatherModay, and Rich Rhee for technical assistance and manuscriptpreparation. In addition, we would like to thank Tom Cooper for determination of plasma dexamethasone concentrations.
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Luttge WG, Rupp ME, Davda MM (1989b): Aldosterone-stimulated down-regulation of both type I and type II adrenocorticosteroid receptors in mouse brain is mediated via type I receptors. Endocrinology 125:817-824. Meaney MJ, Viau V, Aitken DH, Bhatnagar S (1988): Stress-induced occupancy and transiocation of hippocampal glucocorticoid receptors. Brain Res 445:198-203. Miller AH, Asnis GM, Lackner G, Norin AJ (1987): The in vitro effect of cortisol on natural killer cell activity in patients with major depressive disorder. Psychopharmacol Bull 23(3):502-504. Miller AH, Spencer RL, Stein M, McEwen BS (1990): Adrenal steroid receptor binding in spleen and thymus after stress or dexamethasone. Am J Physiol 259:E405-E412. Miller AH, Spencer RL, Trestman RL, Kim C, McEwen BS, Stein M (1991a): Adrenal steroid receptor activation in vivo and immune function. Am J Physiol 261:EI26--EI31. Miller AH, Asnis GM, Lackner C, Halbreich U, Norin AJ (1991b): Depression, natural killer cell activity, and cortisol secretion. Biol Psychiatry 29:878-886. Munck A, Foley R (1980): Activated and non-activated glucocortieoid-receptorcomplexes in rat thymus cells: Kinetics of formation and relation to steroid structure. J Steroid Biochem 12:225230, Nemeroff CB, Widerlov E, Bissette G, et al (1984): Elevated concentrations of CSF corticotropinreleasing factor-like immunoreactivity in depressed patients. Science 226:1342-1344. Nemeroff CB, Owens MJ, Bissette G, Andorn AC, Stanely M (1988): Reduced corticotropin releasing factor binding sites in the frontal cortex of suicide victims. Arch Gen Psychiatry 45:577-579. Nemeroff CB, Bissette G, Akil H, Fink M (1991): Neuropeptide concentrations in the cerebrospinal fluid of depressed patients treated with electroconvulsivetherapy: Corticotropin-releasingfactor, B-endorphin and somatostatin. Br J Psychiatry 158:59-63. R,',tka A, Sutanto W, Bloemers M, de Kloet ER (1989): On the role of brain mineralocorticoid (type I) and glucocorticoid (type 11)receptors in neuroendocrine regulation. Neuroendocrinology 50:117-123. Reul JM, de Kloet ER (1985): Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Endocrinology 117:2505-251 I. Reul JM, van den Bosch FR, deKIoet ER (1987): Relative occupation of type ! and type 11 corticosteroid receptors in rat brain following stress and dexamethasone treatment: Functional implications. J Endocrinol 115:459-467, Risch SC, Shahkrokh G, Rapaport MH, et al (1988): Neuroendocrine effects of intravenous ovine corticotropin-releasing factor in affective disorder patients and normal controls. Biol Psychiatry 23:755-758. Rupprecht K, KornhuberJ, Wodarz N, et ai (1991): Disturbed glucocorticoidreceptor autoregulation and corticotropin response to dexamethasone in depressives pretreated with metyrapone. Biol Psychiatry 29:1099-1109. Sachar El (1975): Neuroendocrine abnormalities in depressive illness. In Sachar El (ed.), Topics in Psychoendocrinology. New York: Gmne and Stratton, pp 135-156. Sapolsky RM, McEwen BS (1985): Down-regulation of neural corticosterone receptors by corticosterone and dexamethasone. Brain Res 339:161-165. Sapolsky RM, Krey LC, McEwen BS (1984a): Prolonged glucocorticoid exposure reduces hippocampal neuron number, lmplicatiol~s for aging. J Neurosci 5:1221-1226. Sapolsky RM, Krey LC, McEwen BS (1984b): Glucocorticoid sensitive hippocampal neurons are involved in terminating the adrenocortical stress response, Proc Natl Acad Sci USA 81:61746177. Sapolsky RM, Krey LC, McEwen BS (1986): The neuroendocrinology of stress and aging: The glucocorticoid cascade hypothesis. Endocrinol Res 7:284. Sarrieau A, Dussaillant M, Sapolsky RM, et al (1988): Glucocorticoid binding sites in human temporal cortex. Brain Res 442:157-160.
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Spencer RL, Young EA, Choo PH, McEwen BS (1990): Adrenal steroid type I and II receptor binding: Estimates of in vivo receptor number, occupancy, and activation with vanying level of steroid. Brain Res 514:37-48. Spencer RL, Miller AH, Stein M, McEwen BS (1991): Corticostemne regulation of type I and type I1 adrenal steroid receptors in brain, pituitary, and immune tissue. Brain Res 549:236246. Sutanto W, de Kloet ER (1987): Species-specificity of corticosteroid receptors in hamster and rat brains. Endocrinology 121:1405-1411. Tomello S, Orti E, de Nicola AF, Rainbow TC, McEwen BS (1982): Regulation of glucocorticoid receptors in brain by corticosterone treatment of adrenalectomized rats. Neuroendocrinology 35:411-417. Tsuboi S, Kawashima R, Tomioka O, Nakata M, Sakamoto N, Fujita T (1979): Glucocorticoid binding proteins of human brain cytosol. Brain Res 179:181-185. Waiters MR (1985): Steroid hormone receptors and the nucleus. Endocr Rev 6:512-543. Whalley LJ, Borthwick N, Copolov D, Dick H, Christie JE, Fink G (1986): Glucocorticoid receptors and depression. Br Med J 292:859-861. Wiedemann K, Holsboer F (1987a): Plasma dexamethasone levels in depression: Oral vs. I.V. administration. Psychiatry Res 20:83-85. Wiedemann K, Holsboer F (1987b): Plasma dexamethasone kinetics during the DST after oral and intravenous administration of the test drag. Biol Psychiatry 22:1340-1348. Young EA, Haskett RF, Murphy-Weinberg V, Watson SJ, Akil H (1991): Loss of glucocorticoid fast feedback in depression. Arch Gen Psychiatry 48:693-699. Yu Z, Wrange O, Boethius J, Gustafsson J, Granholm L (1981): A qualitative comparison of the glucocorticoid receptor in cytosol from human brain and rat brain. Brain Res 223:325-333.
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Adrenal Steroid Receptor Activation in Rat Brain and Pituitary Following Dexamethasone: Implications for the Dexamethasone Suppression Test Andrew H. Miller, Robert L. Spencer, Mark Pulera, Susan Kang, Bruce S. McEwen, and Marvin Stein
The dexamethasone suppression test (DST) has been used extensively to evaluate feedback inhibition of the hypothalamic-pituitary--adrenal (HPA ) axis by adrenal steroids. Nevertheless, it remains unclear at what level of the HPA axis and through which adrenal steroid receptor subtype dexamethasone exerts its inhibitory effect. Because adrenal steroid receptor activation is an important prerequisite for dexamethasone to affect cellular function, HPA axis tissues that exhibit evidence of receptor activation following dexamethasone administration are likely site(s) of action for this synthetic hormone to inhibit HPA axis activity. Therefore, type-! and type.ll adrenal steroid receptor activation was assessed in the pituitary, hypothalamus, and hippocampus of intact and adrenalectomized rats after overnight exposure to various oral doses of dexamethasone. Results with dexamethasone were compared to similar studies using corticosterone, the endogenous glucocorticoid of the rat. All dexamethasone doses led to significant type-I! receptor activation in the pituitary, whereas only an exceedingly high dexamethasone dose activated type.ll receptors in the hippocampus and hypothalamus. Dexamethasone had little effect on type ! receptors in any tissue at any dose. In contrast, corticosterone significantly activated type4 receptors in all tissues, whereas it activated type.ll receptors in the brain and not the pituitary at physiological concentrations. Because dexamethasone activated pituitary type-ll receptors at blood concentrations that did not activate type-II receptors in the brain, these results suggest that the DST in humans may primarily be a measure of type-ll adrenal steroid receptor feedback inhibition at the level of the pituitary.
Introduction Dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis in patients with major depression is one of the most consistent findings in all of biological psychiatry. Abnormalities include hypersecretion of the adrenal steroid, cortisol (Sachar 1975; Carroll et al 1976); nonsuppression of cortisol and corticotropin (ACTH) following administration of the synthetic glucocorticoid, dexamethasone (Carroll et al 1981; Kalin et al 1982; From the Department of Psychiatry (AHM, MP, SK, MS), Mount Sinai School of Medicine; and the Laboratory of Neuroendocrinology (PLS, BSM), Rockefeller University, New York, NY Address reprint requests to Andrew H. Miller, M.D., Box 1229, Annenbe~g 22-66, Department of Psychiatry, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. R~eived April 23, 1992; revised August 6, 1992. © 1992 Society of Biological Psychiatry
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Holsboer 1983; Arana et al 1985); a blunted ACTH response to corticotropin-releasing hormone (CRH) (Gold et al 1984, 1986; Holsboer et al 1984, 1986; Risch et al 1988) and an increased cortisol response to ACTH (Amsterdam et al 1983; Kalin et al 1987; Jaeckle et al 1987; Krishnan et al 1990). These abnormalities are believed to be related in part to an overactivation of limbic structures, including the hypothalamus and hippocampus, resulting in hypersecretion of CRH (Nemeroff et al 1984; Gold et al 1984). The mechanism of this limbic overactivation is unknown, although resistance of adrenal steroid receptors to feedback inhibition at or above the level of the hypothalamus is believed to be involved (Sapolsky et al 1984b; Sapolsky et al 1986; Gold et al 1988). There are two types of adrenal steroid receptors; type-I adrenal steroid receptors, which are commonly referred to as mineralocorticoid receptors, and type-If adrenal steroid receptors, which are also known as glucocorticoid receptors (Reul and de Kloet 1985). Type-I receptors have a high affinity for endogenous adrenal steroid hormones and are believed to play a role in the regulation of circadian fluctuations in corticosteroids (Beaumont and Fanestil 1983; de KIoet and Reui 1987; Sutanto and de Kloet 1987). Type-ll receptors, on the other hand, have a lower affinity for endogenous adrenal steroids than type-I receptors and therefore are believed to be more important in termination of the corticosteroid response to stress when endogenous levels of glucocorticoids are high (Reui and de Kloet 1985; de KIoet and Reul 1987). In light of these characteristics, dysregulation of either or both receptor subtypes may be involved in altered HPA axis function in major depression. The dexamethasone suppression test (DST) has been used extensively to evaluate dysregulated adrenal steroid feedback inhibition in the depressive disorders. However, it remains unclear at what level of the HPA axis dexamethasone exerts its inhibitory effect. Moreover, it has not been established through which adrenal steroid receptor subtype dcxamethasone acts. In order for dexamethasone to inhibit HPA axis function, an important prerequisite is the activation of type-I and/or type-ll adrenal steroid receptors in relevant HPA tissues. Previous studies suggest that depending on the mode of administration dexamethasone may be more effective in activating type-II receptors in the pituitary (and peripheral tissues in general) than in the brain (de Kioet et al 1974, 1975; Miller et al 1990; Spencer et al 1990). The DST may therefore provide information on type-If adrenal steroid receptor feedback inhibition, primarily at the level of the pituitary with consequent implications for the interpretation of abnormal DST results. To further investigate at what level of the HPA axis and through which receptor subtype dexamethasone acts, the ability of various oral doses of dexamethasone to activate typeI and type-II adrenal steroid receptors in the pituitary, hypothalamus, and hippocampus was assessed in intact and adrenalectomized Sprague-Dawley rats. To compare binding parameters in the pituitary and brain to other peripheral tissues, receptor binding was also measured in the spleen, which has been shown to express both type 1 and type-If adrenal steroid receptors (Miller et al 1990). Types I and II receptor activation was estimated by comparing adrenal steroid receptor binding in the various treatment groups (Meaney et al 1988; Spencer et al 1990; Miller et al 1990, 1991a). Prevailing models of adrenal steroid action propose that the receptor, when bound by steroid, undergoes a conformational change referred to as activation or transformation (Munck and Foley 1980; Waiters 1985). The activated form of the receptor has a high affinity for deoxyribose nucleic acid (DNA) and is found exclusively in the nucleus. Adrenal steroid receptors appear to be unique from other steroid receptors in that only the unactivated form of the receptor can rebind steroid and participate in an in
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vitro exchange assay (Chou and Luttge 1988; Spencer et al 1990; Meaney et al 1988). Therefore. adrenal steroid receptors activated in vivo will not rebind radiolabeled steroid in vitro and thu~ cannot be measured in an in vitro cytosolic exchange assay. Taking advantage of this unique property, the amount of adrenal steroid receptor activation that occurs in vivo can be estimated by comparing the number of binding sites measured by an exchange assay in the presence of varying concentrations of circulating steroid hormone; the greater the receptor activation, the lower the number of measurable binding sites, and vice versa (Meaney et al 1988; Spencer et al 1990; Miller et al 1990, 1991a). Of note is that this estimate of receptor activation (i.e., decreased receptor binding following an acute hormone exposure) has been shown to highly correlate with changes in cellular function that are mediated by adrenal steroid receptors (Miller et al 1991a). A similar paradigm was used to estimate receptor activation in the above-noted tissues after exposure to corticosterone, the endogenous glucocorticoid of the rat. Several studies have used naturally occurring glucocorticoids to investigate feedback inhibition in human depressives (Young et al 1991), however as with dexamethasone, it remains unclear through which receptor subtype and in what HPA axis tissue(s) these hormones act.
Methods Subjects Young, adult male Sprague-Dawley rats (300-350g) were used in all treatment groups. Animals were housed 2 to 3 per cage and maintained on a 12-hr light--dark cycle (lights on at 7 AM) in an animal room separate from the laboratory. Rat chow and tap water were provided ad iibitum except in adrenalectomy experiments, in which case 0.9% saline was substituted for tap water. Where indicated, bilateral adrenalectomies were performed using standard aseptic surgical techniques on animals fully anesthetized with the inhalant, methoxyflurane (Metofane; Pitman-moore, Washington Crossing, NJ).
Design Dexamethasone Studies Two separate experiments were conducted using dexamethasone treatment; one employing intact animals and the other employing adrcnalectomized ~imals. In the dexamethasone experiment on intact animals, the experiment was conducted on two separate days, each using four treatment groups (n - 4 per group per day) based on the concentration of dexamethasone in the drinking water: 0, 0.3 ~,g/ml, 0.8 gg/ml, and 10 p~g/ml dexamethasone. The low and medium doses of dexamethasone were designed to reproduce dexamethasone blood concentrations consistent with those found in humans during a l-mg DST. The highest dose was used to achieve saturation of receptors in all tissues. Appropriate amounts of stock dexamethasone [10 mg/ml in 100% ethanol (EtOH)] or EtOH alone were added to the drinking water at 6 pM, the night before the experiment. The final concentration of EtOH in the drinking water of all rats was stano dardized at 0.078% wt/vol. No other source of fluids was provided in any of the experiments. At 9 AM the following day, all animals were killed by rapid decapitation. Trunk blood was obtained for determination of the plasma dexamethasone concentration, and the indicated brain regions and peripheral tissues were surgically removed for binding studies. Tissue for binding assays was rapidly frozen on dry ice and stored at - 8 0 ° C.
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In the experiment on adrenalectomized animals, five treatment groups were used (n = 4 per group): intact, acLrena!ecto~ed~ adrenalectomized + 0.3 ttg/ml dexamethasone in the drinking water, adrenalectomized + 0.8 ttg/ml dexamethasone, and adrenalectomized + 10 ~tg/ml dexamethasone. Adrenalectomies were performed between 20 and 24 hr before sacrifice, a time period that allows adequate time for clearance of endogenous corticosterone (Spencer et al 1990). The drinking water containing dexamethasone was prepared in the same manner as the experiment on intact animals, and animals were sacrificed and tissues removed as previously indicated.
Corticosterone Studies As with dexamethasone, two separate experiments were conducted using corticosterone treatment, one using intact animals and the other using adrenalectomized animals. In the experiment on intact animals, four treatment groups (n -- 5 per group) were used based on the concentration of corticosterone in the drinking water: 0, 200 ttg/ml, 400 ttgtml, and 600 ttg/ml corticosterone. Appropriate amounts of stock corticosterone (40 mg/ml in 100% ethanol) were added to the drinking water at 6 PM the night before the experiment. The final concentration of EtOH in the drinking water of all rats was standardized at 1.17% wt/vol. The higher EtOH concentrations in corticosterone-treated animals was necessitated by the relative low solubility of corticosterone in alcohol and H20. At 9 AM, following overnight corticosterone exposure, animals were killed by rapid decapitation, and blood and tissue samples were removed and processed as indicated in the dexamethasone studies. In the experiment on adrenalectomized animals, five treatment groups were used (n = 4-5 per group); intact, adrenalectomized, adrenalectomized + 200 ttg/ml corticosterone in the drinking water, adrenalectomized + 400 ttg/ml corticosterone, and adrenalectomized + 600 ttg/ml corticosterone. Adrenalectomies were performed as in the dexamethasone experiments. Corticosterone was prepared as indicated, and animals were sacrificed and tissues removed as noted earlier.
Bindin8 Assay Adrenal steroid receptor binding was determined using a previously described in vitro cytosolic exchange assay (Miller et al 1990; Spencer et al 1990). Tissue was homogenized and centrifuged at 105,000 g for 60 rain at 4°C. The supernatant cytosol was then added to incubation solutions containing radiolabeled steroids with or without unlabeled competitors. The cytosol-steroid mixture was incubated at 4 ° C with steroids for 18--22 hr. Shorter time periods have been shown to be insufficient in allowing full exchange of radiolabeled steroid with adrenal steroid receptors when competing steroid is present (HoHim et al 1983; Meaney et al 1988). Bound radiolabeled steroid was separated from unbound steroid by filtration through mini columns containing 1.25 ml of LH-20 Sephadex (Pharmacia, Piscataway, NJ). Scintillation flour (Ready Safe, Beckman, Fullerton, CA) was added to the eluate containing the bound fraction of steroid, and tritium (3H) radioactivity was determined in a liquid scintillation couter (LKB Wallac Beta 1214; LKB, Uppsala, Sweden, 33% efficiency). The homogenization and incubation buffer was comprised of I0 mM tris(hydroxymethyl)aminomethane, 1 mM EDTA, 20 mM molybdic acid, 5 mmol/L dithiothreitol and 10% glycerin in double-distilled water (pH -7.4). Tissue was homogenized in a volume of I-3.5 ml of buffer, yielding a final protein concentration of 0.5--1.5 mg
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protein/ml cytosol. For single-point assays, type-I receptor binding was determined from the binding of [3H]dexamethasone (10 nmol/L) in the presence of the type-II competitor, RU28362 (0.5 p~mol/L) (Coirini et al 1985). [3H]Dexamethasone has been found to be an effective radioligand for measuring type-I receptor binding sites in vitro and gives type-I receptor binding levels similar to [3H]aldosterone (Luttge et al 1989a; Spencer et al 1990). Type-ll binding was defined as the amount of total [3H]dexamethasone binding displaced by RU28362 (0.5 Ixmol/L). Binding in the presence of an excess of corticosterone (2.5 i~mol/L) was used for determining nonspecific binding. Nonspecific binding was reliably < 5% of total binding. Specific binding was expressed as femtomoles per mg of cytosol protein. For saturation binding studies, a range of five concentrations of [3H]dexamethasone (0.3-10 nmol/L) was used. Protein content was determined by the method of Bradford (1976), with use of bovine serum albumin as a standard.
Steroids [6,7-3H(N)]-Dexamethasone (49.9 Ci/nmol/L) was obtained from New England Nuclei,," (Boston, MA). Unlabeled corticosterone was obtained from Steraloids (Wilton, NH) and dexamethasone was obtained from Sigma (St. Louis, MO.). RU28362, the type-ll receptor agonist, was a gift from Koussel-Uclaf (Romainville, France).
Plasma Dexamethasone and Corticosterone Plasma dexamethasone levels were determined using a direct radioimmunoassay procedure as described by Lo et al (1989). Assay sensitivity is 1 pg of dexamethasone, and coefficients of variation within and between assays range from 1%-3% and 3%-8%, respectively. Total serum corticosterone was measured in trunk blood by radioimmunoassay using rabbit antiserum raised against corticosterone-21-hemisuccinate BSA (B21-42; Endocrine Sciences, Tarzana, CA). Assay sensitivity is 10 pg of corticosterone, and coefficients of variation within and between assays range from 2%-5% and 6%-10%, respectively.
Statistical Analysis A one-way analysis of variance (ANOVA) was used to evaluate the effects of steroid treatment on type-I and type-ll receptor binding, except in the case of the dexamethasone studies on intact rats, in which a two-way ANOVA was used to evaluate dexamethasone effects, day effects, and dexamethasone*day interactions. Contrasts between treatment groups were made using t-tests (two-tailed); Bonferonni correction was applied for multiple comparisons except where indicated (Cohen and Cohen 1983). For saturation binding, the dissociation constant (Kd) (in nmol/L) was derived from Scatchard analysis (Limbird 1986), and the binding maximum (in fmoles/mg protein) was determined at the 10 nmol/L [3H]dexamethasone concentration. Results
Dexamethasone Studies Experiment 1. In the first experiment, the effect of various oral doses of dexamethasone on type-II adrenal steroid receptor binding was evaluated in intact rats. The dexamethasone blood levels for each group are shown in Table 1 (top). It should be noted that the mean
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Table 1. Mean ("4-SEM) Dexamethasone Blood Concentrations in Intact (Top) and Adrenalectomized (Bottom) Rats
Ikxamethasone treatment group
Dexamethasone(.g/ml)
Intact 0.3 ItWmi 0.8 Itg/ml 10 ttg/ml ADX Intact ADX ~- 0.3 Itg/ml ADX + 0.8 Itg/ml ADX + 10 Itg/ml
60 ~g/dl) of corticosterone were achieved. Therefore, factors such as CBG, which may serve to buffer peripheral tissues from corticosterone, appear only to be relevant under physiological conditions. Because dexamethasone activated pituitary type-If receptors at blood concentrations that did not activate brain type-II receptors in the rat, it seems unlikely that any potential effects of dexamethasone on the brain to inhibit HPA axis function can be separated from its effect on the pituitary. This latter point may have implications for the interpretation of the DST in humans. Our data from rats suggests that the DST in humans may prima~ily be a measure of type-II adrenal steroid receptor feedback inhibition at the level of the pituitary. This conclusion is based on a comparison of the dexamethasone blood levels required to achieve significant activation of type-II receptors in the rat brain and pituitary with the dexamethasone blood levels obtained in humans during a l-mg DST. For example, maximum mean dexamethasone blood levels in humans 30 min-2 hr following ingestion of 1-1.5 mg of dexamethasone are in the range of 10-28 ng/ml (Duggan et al 1975; Wiedemann and Holsboer 1987a,b). These dexamethasone blood concentrations are approximately tenfold lower than those levels that were found to lead to significant activation of brain type-If receptors in the rat. Moreover, these peak levels in humans overlap with the dexamethasone blood concentrations in rats of the low- and mediumdose groups in the dexamethasone experiment on adrenalextomized animals and the medium-dose group of the dexamethasone experiment on intact animals, both instances in which there was no evidence of significant activation of brain type-If receptors. Of note is that the dexamethasone blood levels in the rats were obte'ned 2-hr into their light period (9 AM), and rats drink primarily in the first-half of their dark period (7 PM-I AM). Therefore, it is likely that the peak dexamethasone blood levels in these animals were much higher than those observed at the time of sacrifice. Dexamethasone blood levels in humans at 8 AM following 11 PM ingestion of 1 mg dexamethasone are in the 0.5-4 ng/ml range, which overlap with our low-dose intact/dexamethasone treatment group (Asnis et al 1989; Klein and Berger 1987). It should be pointed out that the phmmacokinetics of dexamethasone may differ in the human as compared to the rat. Furthermore, although studies in humans and rats indicate that the type.ll receptor in these two species is similar in (l) affinity for dexamethasone (Sarrieau et al 1988), (2) steroid specificity (Yu et al 1981), (3) protein structure (Yu et al 1981), and (4) magnitude of binding sites in the pituitary, hippocampus, and hypothalamus (Tsuboi et al 1979), caution should be taken against drawing direct comparisons of receptor activation by steroid hormones between these species. Nevertheless, the results of these studies may offer important insights into human adrenal steroid receptor physiology following dexamethasone administration. Although dexamethasone bioavailability significantly confounds attempts to interpret DST results, several studies that have controlled for dexamethasone blood level have demonstrated DST abnormalities in depressed patients OViedemann and Holsboer 1987a,b). Taken together with these results, our findings raise several important considerations for
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examining feedback inhibition in the depressive disorders. First, as noted previously, an abnormal DST may primarily represent altered type-ll receptor function in the pituitary of depressed patients. In keeping with this notion is evidence of adrenal steroid receptor dysfunction in other peripheral tissues of patients with major depression. For example, some studies have shown that depressed patients exhibit decreased adrenal steroid receptors in peripheral blood mononuclear cells, which may be related to an impaired nutoregulation of receptor expression (Gormely et al 1985; Whalley et al 1986; Rupprecht et al 1991). In addition, the sensitivity of immune cells to the inhibitory effects of dexamethasone or cortisol in vitro and in vivo has been found to be reduced in depressed patients (Miller et al 1987; Lowy et al 1988; Miller et al 1991b). Furthermore, unlike controls, depressives pretreated with dexamethasone exhibit a significant rise in ACTH and cortisol following CRI-I, providing further evidence of impaired feedback inhibition at the level of the pituitary (Holsboer et al 1987; Bardeleben and Holsboer 1991). However, intact feedback inhibition is supported by findings which indicate that blunted ACTH responses to CRH in depressives are eliminated by reducing circulating endogenous glucocorticoids with metyrapone (Bardeleben et al 1988). These apparent inconsistencies in HPA axis alterations may represent more than one underlying pathology of the HPA axis in depressed patients and/or different time points along a continuum of evolving HPA axis pathology. A second implication of our study is that the DST does not appear to provide information on adrenal steroid receptor feedback inhibition in the hippocampus and hypothalamus. Evidence indicates that there is pathology in these brain regions that may have relevance to HPA axis disturbances in depressed patients. For example, elevated CRH levels in the cerebrospinal fluid of depressed patients along with downregulation of CRH receptors in the frontal cortex of suicide victims suggest that altered HPA axis activity is in part dw. to hypersecretion of CRH secondary to pathology at or above the level of the hypothalamus (Nemeroff et al 1984, 1988, 1991). Moreover, taken together with the role of the hippocampus in HPA axis feedback inhibition (Sapolsky et al 1984a,b, Herman et al 1989), the increased sensitivity of hippocampal neurons to the effects of chronic stress and/or elevated glucocorticoids further support the notion that the roots of HPA axis pathology in depression may reside in the brain (Sapolsky et al 1984a, Sapolsky et al 1986). Therefore, alternative strategies need to be developed to examine feedback inhibition in relevant brain regions. As seen in our studies, infusions of naturally occurring glucocorticoids may allow preferential access of adrenal steroids to the hippocampus and hypothalamus and thereby provide a reasonable probe of feedback inhibition in these brain regions. However, high concentrations of these hormones are likely to activate pituitary type-ll receptors in addition to brain type-ll receptors making interpretation difficult. Furthermore, naturally occuring glucocorticoids activate type-I receptors in all HPA axis tissues at doses that might otherwise selectively activate type-ll receptors in the brain. Future studies examining adrenal steroid feedback inhibition in the brain may require infusions of low-dose cortisol in the presence of type-I receptor antagonists, such as RU28318 (Ratka et ai 1989). Finally, our studies suggest that the role of type-i adrenal swroid receptors in HPA axis disturbances has not been adequately addressed by studies using dexamethasone or cortisol. Because type-I receptors may regulate basal corticosteroid secretion, hypercortisolism in the absence of DST nonsuppression may represent a type-I receptor defect. Furthermore, since chronic activation of type-I receptors can lead to downregulation of type-ll receptors (Luttge et al 1989b), type-I receptors may also play a role in altered
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type-ll receptor expression and/or responsiveness. Low-dose infusions with the type-I agonist, aldosterone, followed by measurement of ACTH in patients pretreated with metyrapone may shed light on this possibility. In conclusion, animal studies examining adrenal steroid receptor activation in HPA axis tissues following various hormone exposures may provide important insights into clinical research protocols geared to evaluate adrenal steroid feedback inhibition in human subjects. Animal studies can indicate wh;ch receptor subtypes are activated in which HPA axis tissues in any given protocol. Such data is relevant for the interpretation of results as well as the development of new approaches to the study of H ° A axis pathology in psychiatric disorders. This research was supported in part by a National Institute of Mental Health Research Scientist Development Award (MH00680)to AHM and research grant (MH47674)to AHM and RLS. The authors would like to thank Abrar Husain, HeatherModay, and Rich Rhee for technical assistance and manuscriptpreparation. In addition, we would like to thank Tom Cooper for determination of plasma dexamethasone concentrations.
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