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Melatonin effects on glucocorticoid receptors in rat brain and pituitary: Significance in adrenocortical regulation
0020-71 IX/91 $3.00 + 0.00 Copyright0 1991 PergamonPressplc
Inr. J. Biochem. Vol. 23, No. 4, pp. 479481, 1991 Printedin Great Britain.All rightsreserved
MELATONIN EFFECTS ON GLUCOCORTICOID RECEPTORS IN RAT BRAIN AND PITUITARY: SIGNIFICANCE IN ADRENOCORTICAL REGULATION C. MARINOVA, S. F%RSENGIEV, R. KONAKCHIEVA, A. ILIEVA and V. PATCHEV* Institute
of Biology and Immunology of Reproduction,
Bulgarian Academy of Sciences, 1113 Sofia,
Bulgaria (Received 4 July 1990)
The effects of chronic melatonin treatment on glucocorticoid binding sites in hippocampus, hypothalamus and pituitary were investigated in rats, subjected to long-term manipulation of circulating corticosterone concentrations. 2. Melatonin treatment decreased the affinity of glucoeorticoid receptors. 3. The effect of melatonin was apparent in the presence of normal or enhanced systemic corticosterone
Abstract-l.
levels, but not in long-term adrenalectomized
animals.
INTRODUCTION
MATERIALS AND METHODS
The secretory patterns of most of the hormones of the hypothalamic-pituitary-adrenal (HPA) axis, and of the pineal gland are characterized by a well-expressed circadian rhythmicity, although a casual relationship cannot be readily implied (Arendt, 1988). A second common feature of these endocrine systems is that they both are activated by stress. However, the physiological significance of stress-related increase in melatonin production and release remains still obscure, although a central tranquilizing action of melatonin has been presumed (Cramer et al., 1974; Vollrath et al., 1981). Numerous recent findings suggest that melatonin might be in fact involved in modulation of neurotransmission, since specific receptors for the pineal hormone have been described in several brain regions [for review see Stankov and Reiter, (1990)]. However, neither acute nor chronic melatonin administration was able to modify glucocorticoid secretion (Wright et al., 1986; Waldhauser et al., 1987). A major neuroendocrine consequence of long-term stress is the sustained elevation of circulating glucocorticoids, owning to the steroid-induced downregulation of central control mechanisms of the HPA axis, and resulting in an inability to terminate the adrenocortical responses to stress (Sapolsky et al., 1986). The neurochemical basis of this phenomenon has been assumed to consist in alterations of hippocampal corticosterone receptors, which are considered as a very sensitive control mechanism of the HPA axis (McEwen et al., 1986). In the present study we investigated the effect of exogenous melatonin on glucocorticoid receptors in two brain regions (hippocampus and hypothalamus) and pituitary by superimposing the melatonin treatment on long-term manipulations of circulating glucocorticoid levels, known to produce distinct changes in receptors for adrenal steroid hormones.
Adult male Wistar rats weighing 160-180g were used. The animals were housed in groups of 6 under controlled illumination (L : D 14 : 10 hr) and had free access to standard lab chow and tap water. A part of the rats were adrenalectomized bilaterally under hexobarbital anaesthesia (30 mg/kg b.w). Adrenalectomized (ADX) animals were provided with 1% saline as drinking solution. Shamoperated rats served as controls. After two weeks of recovery the animals were divided into separate treatment groups as follows:
*To whom all correspondence
should be addressed.
(a) (b) (c) (d) (e) (f)
long-term ADX; long-term ADX with melatonin treatment; chronic corticosterone (CS) overdosage; CS overdosage with melatonin treatment; sham-operated controls; sham-operated controls with melatonin treatment.
Hormonal treatment was provided for 6 days according to the following schedule: corticosterone (Sigma, F.R.G.) was suspended in absolute ethanol and glycerine (v/v l/9) and sonificated to obtain a homogenous stock suspension, containing 6mg corticosterone/ml. Before use it was dissolved in 0.5% saline to a final concentration of 6Oj~g/ml and provided to the animals as drinking fluid. The fluid consumption was checked daily and the averaged corticosterone intake per rat was calculated for each group. All animals which were not subjected to CS supplementation received an equivalent volume of the vehicle, added to the drinking fluid. Melatonin (Sigma, U.S.A.) was dissolved in absolute ethanol to an initial concentration of I .25 mg/ml. Before use it was diluted in physiological saline to a concentration of 25 pg/ml and was applied once daily in a dose of 50 pg/kg b.w. The hormone was injected S.C.4 hr before the onset of the dcrk period. Subjects from the corresponding paired groups received daily saline injections. After 6 days of treatment the animals were killed 1 hr after the onset of the dark period. Pituitary, hypothalamus and hippocampus were rapidly dissected on ice and stored in liquid nitrogen until assay. Cytosol receptors for corticosterone were assayed using [‘Hlcorticosterone (Amersham, U.K.) as described elsewhere (Snochowski ef al., 1980; Reul et al., 1987). Non-specific binding was determined in parallel incubations containing SOO-foldexcess of unlabelled corticosterone and lOO-fold excess of dexamethasone. Cytosolic 479
C. MARINOVA et al.
480
protein concentrations were measured according to Lowry et al. (1951). Data from receptor assays were subjected to Scatchard analysis using a computerized approach (Chamness and McGuire, 1975). The tabulated data for Kd and B,, represent averaged values from two independent experiments in which identical treatment schedules were
used. The efficiency of CS supplementation was assessed by the weight regression of the thymus gland. These data were processed by one-way ANOVA, followed by a c-test. RESULTS
Averaged daily corticosterone intake and changes in thymus weight, resulting from manipulations of circulating steroid levels, are depicted in Table 1. The averaged amounts of ingested corticosterone did not differ in melatonin-treated and non-treated animals. Thymus weights were significantly increased following long-term ADX. Oral CS supplementation resulted in a significant thymolysis, and melatonin treatment was associated with an additional decrease of the thymus weight. The data from glucocorticoid receptor determinations, are presented in the following tables. In all investigated tissues adrenalectomy caused a marked increase of B,, , as compared to the controls. K,, values also increased following long-term ADX. The effect of CS supplementation in rats, which did not receive melatonin, were inconsistent: B,, decreased in the hippocampus, remained unchanged in the hypothalamus, and was elevated in the pituitary; in all tissues Kd returned to the level observed in control rats. Melatonin treatment in control animals increased Kd of glucocorticoid receptors in all structures investigated, and B,, in hypothalamus and pituitary. No major changes were observed after melatonin administration in adrenalectomized rats. Melatonin treatment accompanying oral corticosterone overdosage resulted in marked increase of B,, and Kd in hippocampus; however, no considerable changes could be observed in the remaining tissues, excepted a decrease of receptor number in the pituitary. DISCUSSION
A daily oral dose of 630 pg corticosterone has been shown to prevent ADX-induced thymus enlargement in rats (Akana et al., 1985). the averaged daily CS intake in our experiments exceeded by far that value; thus, the treatment schedule can be expected to provide supraphysiological circulating levels of CS. The thymolysis, observed in CS-supplemented animals, documents that the steroid dose is indeed far above that required for the maintenance of normal glucocorticoid environment. The potentiation of C&induced thymolysis by melatonin cannot be readily explained. Melatonin has been presumed to affect in some aspects the immune system (Maestroni et al., 1988; Arendt, 1990); however, the thymus gland has not been considered as a selective target of melatonin action yet. The changes in hippocampal glucocorticoid receptors following long-term manipulation of endogenous CS levels correspond fairly to those, reported by
Table I
Thymus
Treatment BrO”P
n
ADX + NaCl ADX + MEL CS + NaCl CS + MEL CONT + NaCl CONT + MEL
10 I2 IO 12 14 14
Daily CS intake (mg/rat) -
weight (mg) 751 f 176* 653 f 115. 236 + 38’ 134 +_18’t 406+54 445k61
3.45 * 1.03 3.24 + 1.08 -
Averaged daily corticosterone intake and changes in the weight of the thymus gland (mean + SD; *indicates significant differences as compared to controls; tindicates a significant difference as compared to the paired treatment group; level of significance P < 0.05). Table 2 Treatment PTOUD
ADX + NaCl ADX + MEL CS + NaCl CS + MEL CONT + NaCl CONT + MEL
Kd (nM) 11.5 11.6 3.7 12.5 4.6 10.3
Changes in hippocampal
SD Bmm (nM1 (fmol/mn urotein) 1.4 1.5 1.2 1.8 1.4 3.8
40.3 21.2 10.6 46.7 18.2 14.7
Confidence limits 34.249.6 8.3 - 27.7 7.6 - 21.6 28.&l 05 10.0-53.5 9.638.7
glucocorticoid receptors. Table 3
Treatment EIOUD
ADX + NaCl ADX + MEL CS + NaCl CS + MEL CONT + NaCl CON-r + MEL
Kd (nMj
SD (nM)
(fmollma erotein)
B mm
Confidence limits
24 15.3 13.7 16.5 9.8 26.9
1.8 7.5 4.1 6.7 3.0 I.8
55 53.7 17.6 19.3 15.5 53.1
10.5-93 32-121 12.1 f 34.6 12.163 10.9-30.9 26.5-93
Changes in hypothalamic
glucocorticoid
receptors.
Table 4 WOW
Kd (nM)
SD (nM)
ADX + NaCl ADX + MEL CS + NaCl CS + MEL CONT + NaCl CONT + MEL
7.8 8.5 2.5 4.1 2.2 10.4
1.2 2.6 I.5 1.6 0.5 6.9
Treatment
Changes in pituitary glucocorticoid
B In.1
(fmol/ma protein) 57.9 59.2 16.6 9.3 8.2 20.3
Confidence limits 28.9-95 26124 13-29 7-24 6.8-12 11.3-70
receptors.
McEwen et al. (1986): ADX increased the number of receptors, whereas CS treatment reduced it below the control value. A similar effect of long-term ADX was observed in hypothalamus and pituitary as well; however, decreased number of glucocorticoid binding sites, following CS treatment, was not present in those structures. Since corticosterone-preferring receptors are restricted almost exclusively to limbic structures (hippocampus, septum and amygdala), and their vulnerability to excessive doses of corticosterone is obviously higher than that of glucocorticoid-preferring sites in hypothalamus and pituitary (McEwen et al., 1986), we may infer that the scheme of steroid treatment, used by us, fulfilled its predetermination only concerning hippocampal, but not hypothalamic and pituitary glucocorticoid receptors. Melatonin treatment exerted an uniform and ubiquitous effect on glucocorticoid binding sites: in all structures, in the presence of corticosterone (i.e. in control and CS-treated rats) it caused a more or less pronounced decrease of receptor affinity. This effect
Melatonin and corticosterone
could not be observed in adrenalectomized animals. Since the efficiency of corticosterone treatment appears to be confined mainly in hippocampal receptors, the increase of B,, following melatonin administration in CS-supplemented rats indicates that melatonin probably counteracts the effect of CS also in this respect. The present data suggest that chronic melatonin treatment is probably able to affect glucocorticoid receptors in the brain and the pituitary. Although the strength of melatonin effect is not equal in all structures investigated, two major common features should be noted: (a) melatonin decreased affinity of the glucocorticoid receptors toward their natural ligand; (b) melatonin effect was apparent only in the presence of corticosterone. The physiological significance of these findings remains to be clarified. Our current presumption is that by decreasing the sensitivity of glucocorticoid receptors to their endogenous ligand melatonin “protects” the binding sites from the deleterious influence of enhanced corticosterone concentrations. Additionally, melatonin might prevent hippocampal receptor loss, which usually occurs following chronic stress or long-term exposure to elevated circulating levels of corticosterone. REFERENCES Akana S. F., Cascio C. S., Shinsako J. and Dallman M. F. (1985) Corticosterone: narrow range required for normal body and thymus weight and ACTH. Am. J. Physiol. 249, R527-R532. Arendt J. (1988) Melatonin. Clin. Endocrin. 29, 205-229. Arendt J. (1990) Significance of melatonin to chronobiology: immunological correlations. In Role of Melatonin and Pineal Peptides in Neuroimmunomodulation; Proceed-
receptors
481
ings of the International School of Pharmacology, Workshop, Erice (Italy), 3-10 June 1990.
3rd
Chamness G. C. and McGuire W. I. (1975) Scatchard plots: common errors in correction and interpretation. Steroids 26, 538-544
Cramer H., Rudolph J. and Consbruch V. (1974) Adtrances in Biochemical Psychopharmacology, Vol. 1I, pp. 187-191. Raven Press, New York. Lowry 0. H., Rosenbrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurements with the Folin nhenol reagent. J.‘biol. Chem. 193, 265-275. Maestroni G. J. M., Conti A. and Pierpaoli N. (1988) Role of the pineal gland in immunity: III. Melatonin antagonizes the immunosuppressive effect of acute stress via an opiatergic mechanism. Immunology 63, 465-469. McEwen B. S.. de Kloet E. R. and Rostene W. (1986) Adrenal steroid receptors and actions in the nkrvous system. Physiol. Rev. 66, 1121-l 188.
Reul J. M. H. M., van den Bosch F. R. and de Kloet E. R. (1987) Relative occupation of type-1 and type-11 corticosteroid receptors in rat brain following stress and dexamethasone treatment: functional implications. J. Endocrin. 115, 459467.
Sapolsky R. M., Krey L. C. and McEwen B. S. (1986) The neuroendocrinology of stress and aging: the glucocorticoid cascade hypothesis. Endocrin. Rev. 7, 284-301. Snochowski M., Dahlberg E. and Gustafsson J.-A. (1980) Characterization and quantification of the androgen and glucocorticoid receptors in rat skeletal muscle cytosol. Eur. J. Biochem. 111,603410. Stankov B. and Reiter R. (1990) Melatonin receptors: current status, facts, and hypotheses. Life Sci. 46, 971-982. Vollrath L., Semm P. and Gammel G. (1981) Sleep induction bv intranasal auolication of melatonin. Adv. Biosci. 29, 327-329.
*s
Waldhauser F., Steiger H. and Vorkapic P. (1987) Melatonin secretion in man and the influence of exogenous melatonin on some physiological and behavioural variables. Adv. Pineal Res. 2, 207-223. Wright J., Aldhous M., Franey C., English J. and Arendt J. (1986) The e!Tects of exogenous melatonin on endocrine function in man. Clin. Endocrin. 24, 375-382.
Inr. J. Biochem. Vol. 23, No. 4, pp. 479481, 1991 Printedin Great Britain.All rightsreserved
MELATONIN EFFECTS ON GLUCOCORTICOID RECEPTORS IN RAT BRAIN AND PITUITARY: SIGNIFICANCE IN ADRENOCORTICAL REGULATION C. MARINOVA, S. F%RSENGIEV, R. KONAKCHIEVA, A. ILIEVA and V. PATCHEV* Institute
of Biology and Immunology of Reproduction,
Bulgarian Academy of Sciences, 1113 Sofia,
Bulgaria (Received 4 July 1990)
The effects of chronic melatonin treatment on glucocorticoid binding sites in hippocampus, hypothalamus and pituitary were investigated in rats, subjected to long-term manipulation of circulating corticosterone concentrations. 2. Melatonin treatment decreased the affinity of glucoeorticoid receptors. 3. The effect of melatonin was apparent in the presence of normal or enhanced systemic corticosterone
Abstract-l.
levels, but not in long-term adrenalectomized
animals.
INTRODUCTION
MATERIALS AND METHODS
The secretory patterns of most of the hormones of the hypothalamic-pituitary-adrenal (HPA) axis, and of the pineal gland are characterized by a well-expressed circadian rhythmicity, although a casual relationship cannot be readily implied (Arendt, 1988). A second common feature of these endocrine systems is that they both are activated by stress. However, the physiological significance of stress-related increase in melatonin production and release remains still obscure, although a central tranquilizing action of melatonin has been presumed (Cramer et al., 1974; Vollrath et al., 1981). Numerous recent findings suggest that melatonin might be in fact involved in modulation of neurotransmission, since specific receptors for the pineal hormone have been described in several brain regions [for review see Stankov and Reiter, (1990)]. However, neither acute nor chronic melatonin administration was able to modify glucocorticoid secretion (Wright et al., 1986; Waldhauser et al., 1987). A major neuroendocrine consequence of long-term stress is the sustained elevation of circulating glucocorticoids, owning to the steroid-induced downregulation of central control mechanisms of the HPA axis, and resulting in an inability to terminate the adrenocortical responses to stress (Sapolsky et al., 1986). The neurochemical basis of this phenomenon has been assumed to consist in alterations of hippocampal corticosterone receptors, which are considered as a very sensitive control mechanism of the HPA axis (McEwen et al., 1986). In the present study we investigated the effect of exogenous melatonin on glucocorticoid receptors in two brain regions (hippocampus and hypothalamus) and pituitary by superimposing the melatonin treatment on long-term manipulations of circulating glucocorticoid levels, known to produce distinct changes in receptors for adrenal steroid hormones.
Adult male Wistar rats weighing 160-180g were used. The animals were housed in groups of 6 under controlled illumination (L : D 14 : 10 hr) and had free access to standard lab chow and tap water. A part of the rats were adrenalectomized bilaterally under hexobarbital anaesthesia (30 mg/kg b.w). Adrenalectomized (ADX) animals were provided with 1% saline as drinking solution. Shamoperated rats served as controls. After two weeks of recovery the animals were divided into separate treatment groups as follows:
*To whom all correspondence
should be addressed.
(a) (b) (c) (d) (e) (f)
long-term ADX; long-term ADX with melatonin treatment; chronic corticosterone (CS) overdosage; CS overdosage with melatonin treatment; sham-operated controls; sham-operated controls with melatonin treatment.
Hormonal treatment was provided for 6 days according to the following schedule: corticosterone (Sigma, F.R.G.) was suspended in absolute ethanol and glycerine (v/v l/9) and sonificated to obtain a homogenous stock suspension, containing 6mg corticosterone/ml. Before use it was dissolved in 0.5% saline to a final concentration of 6Oj~g/ml and provided to the animals as drinking fluid. The fluid consumption was checked daily and the averaged corticosterone intake per rat was calculated for each group. All animals which were not subjected to CS supplementation received an equivalent volume of the vehicle, added to the drinking fluid. Melatonin (Sigma, U.S.A.) was dissolved in absolute ethanol to an initial concentration of I .25 mg/ml. Before use it was diluted in physiological saline to a concentration of 25 pg/ml and was applied once daily in a dose of 50 pg/kg b.w. The hormone was injected S.C.4 hr before the onset of the dcrk period. Subjects from the corresponding paired groups received daily saline injections. After 6 days of treatment the animals were killed 1 hr after the onset of the dark period. Pituitary, hypothalamus and hippocampus were rapidly dissected on ice and stored in liquid nitrogen until assay. Cytosol receptors for corticosterone were assayed using [‘Hlcorticosterone (Amersham, U.K.) as described elsewhere (Snochowski ef al., 1980; Reul et al., 1987). Non-specific binding was determined in parallel incubations containing SOO-foldexcess of unlabelled corticosterone and lOO-fold excess of dexamethasone. Cytosolic 479
C. MARINOVA et al.
480
protein concentrations were measured according to Lowry et al. (1951). Data from receptor assays were subjected to Scatchard analysis using a computerized approach (Chamness and McGuire, 1975). The tabulated data for Kd and B,, represent averaged values from two independent experiments in which identical treatment schedules were
used. The efficiency of CS supplementation was assessed by the weight regression of the thymus gland. These data were processed by one-way ANOVA, followed by a c-test. RESULTS
Averaged daily corticosterone intake and changes in thymus weight, resulting from manipulations of circulating steroid levels, are depicted in Table 1. The averaged amounts of ingested corticosterone did not differ in melatonin-treated and non-treated animals. Thymus weights were significantly increased following long-term ADX. Oral CS supplementation resulted in a significant thymolysis, and melatonin treatment was associated with an additional decrease of the thymus weight. The data from glucocorticoid receptor determinations, are presented in the following tables. In all investigated tissues adrenalectomy caused a marked increase of B,, , as compared to the controls. K,, values also increased following long-term ADX. The effect of CS supplementation in rats, which did not receive melatonin, were inconsistent: B,, decreased in the hippocampus, remained unchanged in the hypothalamus, and was elevated in the pituitary; in all tissues Kd returned to the level observed in control rats. Melatonin treatment in control animals increased Kd of glucocorticoid receptors in all structures investigated, and B,, in hypothalamus and pituitary. No major changes were observed after melatonin administration in adrenalectomized rats. Melatonin treatment accompanying oral corticosterone overdosage resulted in marked increase of B,, and Kd in hippocampus; however, no considerable changes could be observed in the remaining tissues, excepted a decrease of receptor number in the pituitary. DISCUSSION
A daily oral dose of 630 pg corticosterone has been shown to prevent ADX-induced thymus enlargement in rats (Akana et al., 1985). the averaged daily CS intake in our experiments exceeded by far that value; thus, the treatment schedule can be expected to provide supraphysiological circulating levels of CS. The thymolysis, observed in CS-supplemented animals, documents that the steroid dose is indeed far above that required for the maintenance of normal glucocorticoid environment. The potentiation of C&induced thymolysis by melatonin cannot be readily explained. Melatonin has been presumed to affect in some aspects the immune system (Maestroni et al., 1988; Arendt, 1990); however, the thymus gland has not been considered as a selective target of melatonin action yet. The changes in hippocampal glucocorticoid receptors following long-term manipulation of endogenous CS levels correspond fairly to those, reported by
Table I
Thymus
Treatment BrO”P
n
ADX + NaCl ADX + MEL CS + NaCl CS + MEL CONT + NaCl CONT + MEL
10 I2 IO 12 14 14
Daily CS intake (mg/rat) -
weight (mg) 751 f 176* 653 f 115. 236 + 38’ 134 +_18’t 406+54 445k61
3.45 * 1.03 3.24 + 1.08 -
Averaged daily corticosterone intake and changes in the weight of the thymus gland (mean + SD; *indicates significant differences as compared to controls; tindicates a significant difference as compared to the paired treatment group; level of significance P < 0.05). Table 2 Treatment PTOUD
ADX + NaCl ADX + MEL CS + NaCl CS + MEL CONT + NaCl CONT + MEL
Kd (nM) 11.5 11.6 3.7 12.5 4.6 10.3
Changes in hippocampal
SD Bmm (nM1 (fmol/mn urotein) 1.4 1.5 1.2 1.8 1.4 3.8
40.3 21.2 10.6 46.7 18.2 14.7
Confidence limits 34.249.6 8.3 - 27.7 7.6 - 21.6 28.&l 05 10.0-53.5 9.638.7
glucocorticoid receptors. Table 3
Treatment EIOUD
ADX + NaCl ADX + MEL CS + NaCl CS + MEL CONT + NaCl CON-r + MEL
Kd (nMj
SD (nM)
(fmollma erotein)
B mm
Confidence limits
24 15.3 13.7 16.5 9.8 26.9
1.8 7.5 4.1 6.7 3.0 I.8
55 53.7 17.6 19.3 15.5 53.1
10.5-93 32-121 12.1 f 34.6 12.163 10.9-30.9 26.5-93
Changes in hypothalamic
glucocorticoid
receptors.
Table 4 WOW
Kd (nM)
SD (nM)
ADX + NaCl ADX + MEL CS + NaCl CS + MEL CONT + NaCl CONT + MEL
7.8 8.5 2.5 4.1 2.2 10.4
1.2 2.6 I.5 1.6 0.5 6.9
Treatment
Changes in pituitary glucocorticoid
B In.1
(fmol/ma protein) 57.9 59.2 16.6 9.3 8.2 20.3
Confidence limits 28.9-95 26124 13-29 7-24 6.8-12 11.3-70
receptors.
McEwen et al. (1986): ADX increased the number of receptors, whereas CS treatment reduced it below the control value. A similar effect of long-term ADX was observed in hypothalamus and pituitary as well; however, decreased number of glucocorticoid binding sites, following CS treatment, was not present in those structures. Since corticosterone-preferring receptors are restricted almost exclusively to limbic structures (hippocampus, septum and amygdala), and their vulnerability to excessive doses of corticosterone is obviously higher than that of glucocorticoid-preferring sites in hypothalamus and pituitary (McEwen et al., 1986), we may infer that the scheme of steroid treatment, used by us, fulfilled its predetermination only concerning hippocampal, but not hypothalamic and pituitary glucocorticoid receptors. Melatonin treatment exerted an uniform and ubiquitous effect on glucocorticoid binding sites: in all structures, in the presence of corticosterone (i.e. in control and CS-treated rats) it caused a more or less pronounced decrease of receptor affinity. This effect
Melatonin and corticosterone
could not be observed in adrenalectomized animals. Since the efficiency of corticosterone treatment appears to be confined mainly in hippocampal receptors, the increase of B,, following melatonin administration in CS-supplemented rats indicates that melatonin probably counteracts the effect of CS also in this respect. The present data suggest that chronic melatonin treatment is probably able to affect glucocorticoid receptors in the brain and the pituitary. Although the strength of melatonin effect is not equal in all structures investigated, two major common features should be noted: (a) melatonin decreased affinity of the glucocorticoid receptors toward their natural ligand; (b) melatonin effect was apparent only in the presence of corticosterone. The physiological significance of these findings remains to be clarified. Our current presumption is that by decreasing the sensitivity of glucocorticoid receptors to their endogenous ligand melatonin “protects” the binding sites from the deleterious influence of enhanced corticosterone concentrations. Additionally, melatonin might prevent hippocampal receptor loss, which usually occurs following chronic stress or long-term exposure to elevated circulating levels of corticosterone. REFERENCES Akana S. F., Cascio C. S., Shinsako J. and Dallman M. F. (1985) Corticosterone: narrow range required for normal body and thymus weight and ACTH. Am. J. Physiol. 249, R527-R532. Arendt J. (1988) Melatonin. Clin. Endocrin. 29, 205-229. Arendt J. (1990) Significance of melatonin to chronobiology: immunological correlations. In Role of Melatonin and Pineal Peptides in Neuroimmunomodulation; Proceed-
receptors
481
ings of the International School of Pharmacology, Workshop, Erice (Italy), 3-10 June 1990.
3rd
Chamness G. C. and McGuire W. I. (1975) Scatchard plots: common errors in correction and interpretation. Steroids 26, 538-544
Cramer H., Rudolph J. and Consbruch V. (1974) Adtrances in Biochemical Psychopharmacology, Vol. 1I, pp. 187-191. Raven Press, New York. Lowry 0. H., Rosenbrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurements with the Folin nhenol reagent. J.‘biol. Chem. 193, 265-275. Maestroni G. J. M., Conti A. and Pierpaoli N. (1988) Role of the pineal gland in immunity: III. Melatonin antagonizes the immunosuppressive effect of acute stress via an opiatergic mechanism. Immunology 63, 465-469. McEwen B. S.. de Kloet E. R. and Rostene W. (1986) Adrenal steroid receptors and actions in the nkrvous system. Physiol. Rev. 66, 1121-l 188.
Reul J. M. H. M., van den Bosch F. R. and de Kloet E. R. (1987) Relative occupation of type-1 and type-11 corticosteroid receptors in rat brain following stress and dexamethasone treatment: functional implications. J. Endocrin. 115, 459467.
Sapolsky R. M., Krey L. C. and McEwen B. S. (1986) The neuroendocrinology of stress and aging: the glucocorticoid cascade hypothesis. Endocrin. Rev. 7, 284-301. Snochowski M., Dahlberg E. and Gustafsson J.-A. (1980) Characterization and quantification of the androgen and glucocorticoid receptors in rat skeletal muscle cytosol. Eur. J. Biochem. 111,603410. Stankov B. and Reiter R. (1990) Melatonin receptors: current status, facts, and hypotheses. Life Sci. 46, 971-982. Vollrath L., Semm P. and Gammel G. (1981) Sleep induction bv intranasal auolication of melatonin. Adv. Biosci. 29, 327-329.
*s
Waldhauser F., Steiger H. and Vorkapic P. (1987) Melatonin secretion in man and the influence of exogenous melatonin on some physiological and behavioural variables. Adv. Pineal Res. 2, 207-223. Wright J., Aldhous M., Franey C., English J. and Arendt J. (1986) The e!Tects of exogenous melatonin on endocrine function in man. Clin. Endocrin. 24, 375-382.