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Corticosterone prevents the increase in noradrenaline-stimulated adenyl cyclase activity in rat hippocampus following adrenalectomy or metopirone
European Journal of Pharmaeologv, 103 (1984) 235-240 Elsevier
235
C O R T I C O S T E R O N E P R E V E N T S T H E INCREASE IN N O R A D R E N A L I N E - S T I M U L A T E D ADENYL CYCLASE ACTIVITY IN RAT H I P P O C A M P U S F O L L O W I N G A D R E N A L E C T O M Y OR METOPIRONE V.J. ROBERTS *, R.L. SINGHAL and D.C.S. ROBERTS **
Department of Pharmacolo,~v, FaculO, of Health Sciences, Universi(v of Ottawa, K i l l 8M5. and * * Department ~f P.s~vchologv, Carleton Universi(v. Ottawa, Ontario KIS 5B6, Canada
Received 18 April 1984, accepted 17 May 1984
V.J. R O B E R T S , R.L. S I N G H A L a n d D.C.S. R O B E R T S , Corticosterone prevents the increase in noradrenaline-stimulated adenvl o'clase activity in rat htppocampus following adrenalectomv or Metopirone, European J. Pharmacol. 103 (1984) 235-240. Corticosterone modulation of the noradrenaline-responsive cyclic A M P generating system was examined in rat hippocampus. A d r e n a l e c t o m y was found to produce a small but significant elevation in the rate of cyclic A M P formation in response to noradrenaline. I m p l a n t a t i o n of corticosterone pellets 5 days prior to sacrifice prevented this a d r e n a l e c t o m y - i n d u c e d increase. Metopirone, an inhibitor of corticosterone synthesis, was also observed to increase cyclic A M P formation. This elevation was seen 2 h following a 50 m g / k g i.p. injection and was completely prevented by corticosterone pellet implantation. Metopirone had no significant effect on cyclic A M P production after 1 h, while a slight but statistically non-significant elevation remained at 4 h. These observations parallel the inhibitory effect of M e t o p i r o n e on corticosterone synthesis as determined by serum corticosterone levels.
Corticosterone
Noradrenaline
Cyclic AMP
Hippocampus
1. Introduction
There is growing evidence that adrenal steroids modulate noradrenaline-stimulated cyclic AMP levels in the brain. Adrenalectomy has been reported by Mobley and Sulser (1980) to increase noradrenaline-induced cyclic A M P accumulation in slices from rat frontal cortex. These effects were reversed by daily corticosterone treatment. Interestingly, the observed elevation in cyclic A M P formation does not appear to depend upon an increase in/~-adrenergic receptor binding. Following adrenalectomy, no significant alteration in B...... or K D values was noted for [3H]DHA binding in either rat frontal cortex (Mobley and Sulser, 1980) or hippocampus (Roberts and Bloom, 1981), despite decreases in rat brain noradrenaline (NA) concentrations (Rastogi and Singhal, 1978). * To whom all correspondence should be addressed: 451 Smytb Road, Ottawa, Ontario, K1H 8M5, Canada. 0014-2999/84/$03.00 ~ 1984 Elsevier Science Publishers B.V.
One problem with adrenalectomy, however, is that because many hormonal responses are affected, the observed effects on central NA mechanisms may be due to some of these secondary changes. We therefore sought convergent evidence for the role of corticosterone on NA receptor mechanisms by using an acute pharmacological manipulation of corticosterone levels. We now report that Metopirone, which inhibits the synthesis of corticosterone, also causes a significant up regulation of the NA-stimulated cyclic AMP generating system.
2. Materials and methods
2.1. Animal preparation Male Wister rats (175-225 g) were maintained on a constant 12 h light, 12 h dark cycle at 23°C.
236
Animals were given free access to food and water. In the case of adrenalectomized (ADX) rats, a 0.9% NaCI solution was substituted for water. Bilateral adrenalectomies were performed 9 days prior to sacrifice. In sham-operated rats, adrenals were located but not removed. Animals receiving corticosterone pellets were anesthetized with ether and pellets were implanted subcutaneously in the back of the neck 5 days prior to sacrifice. Shamoperated rats received an incision but no pellet. Rats treated with Metopirone were given a 50 m g / k g i.p. injection approximately 3 h into the light cycle and sacrificed 1, 2, or 4 h later. All control animals received an equal volume of the vehicle (40% propylene glycol in water).
2.2. Tissue preparation Tissue was prepared according to the method of Harden et al, (1977) with some modifications. Rats were sacrificed by decapitation, the brain rapidly removed and placed on an ice-cold glass plate. Hippocampi were removed freehand, pooled 4-6 per group and sliced in two directions at right angles to each other (0.26 × 0.26 mm) with a McIlwain tissue chopper. The slices were weighed and suspended (50 m g / m l ) in oxygenated (95% O2: 5% CO2) Krebs-Henseleit solution (NaCI 115.3, KC1 4.6, CaC12 2.3, MgSO 4 1.1, N a H C O 3 22.1, KH2PO 4 1.1, glucose 7.8 and disodium EDTA 0.03 mM and 1 t~M Metopirone when indicated). The suspension was incubated in an atmosphere of 95% 02 and 5% CO 2 for 20 min at 3 7 ° C in a shaking Dubnoff water bath. Following incubation, the samples were centrifuged at 600 × g for 20 s. The pellet was re-suspended (50 m g / m l ) in fresh Krebs-Henseleit buffer containing 2.5 ~tCi/ml of [8 -3H]adenine and incubated for an additional 40 min period. Excess radioactivity was removed by washing 3 times by centrifugation (600 × g, 20 s) with pre-warmed and gassed Krebs-Henseleit buffer. Washed pellets were resuspended in Krebs-Henseleit buffer containing 1 mM Na ascorbate, I /zM pargyline, and 1 mM 3-isobutyl1-methylxanthine. Samples were divided into 4 aliquots and incubated in the presence of 0, 1, 10 and 100 t~M NA (37°C; 95% 02: 5% CO2) for 10 min. The reaction was stopped by the addition of
50% trichloroacetic acid (107/xl/ml) and homogenized immediately. A small portion (100 /,1) was removed for protein estimation and the remaining portion centrifuged at 18000 × g for 10 rain. The pellet was discarded and the supernatant saved in glass tubes. Trichloroacetic acid was removed from the supernatant by extracting 3 times with 4 vol. water-saturated ether, then placed in a 60 °C water bath for 10 min to boil off any residual ether.
2.3. Purification and estimation of cyclic A M P Sequential Dowex and alumina chromatography were used to separate radioactive cyclic AMP from other labelled compounds (Salomon et al., 1974). Dowex (50 AG WX4, 200-400 mesh) was prepared by washing with 2 vol. 1 N HCI followed by 10 vol. water. 100 /xl 1 N HC1 was added to 900 /,1 TCA extract (final concentration of 0.1 N HCI) and the mixture was decanted into columns (0.4 × 10 cm) containing 1 ml of prepared Dowex resin. Columns were first washed with 3.5 ml of water and a portion of this flow-through was reserved for counting (3 × 200 ~LI in 10 ml PCS scintillation cocktail). Columns were then washed with an additional 4.5 ml water and a portion (3 × 200 /,1) of elute was saved for counting. To the remaining portion (3.8 ml), 0.253 ml 1.5 M imidazole-HCl, pH 7.2, was added. The mixture was decanted into columns (0.4 × 10 cm) containing 0.6 g neutral alumina which had been washed with 8 ml 0.1 M imidazole-HCl, pH 7.5. The columns were allowed to drain completely of sample and then washed with 1 ml 0.1 M imidazoleHC1, pH 7.5. Three 200 /,1 portions of the elute were saved for counting. The final elute contained the purified [3H]cyclic AMP while the flowthrough from the Dowex column was mainly [~H]ATP. Results are expressed as percent conversion of [~H]ATP to [3H]cyclic AMP and analyzed using the 2-tailed Student's t-test. Brain protein concentrations were measured using a modification of the method of Lowry et al. (1951).
2.3. Purification and estimation of corticosterone Serum corticosterone levels were determined using a modification of the method of Solem and
237 B r i n c k - J o h n s e n (1965). Serum (0.5 ml) was a d d e d to 6 ml m e t h y l e n e chloride. A f t e r vortexing and centrifugation, the serum layer was r e m o v e d and d i s c a r d e d . To remove impurities and metabolites, 1 ml 0.1 N N a O H was a d d e d to methylene chloride, vortexed, centrifuged, a n d removed, A n E T O H / H 2 S O 4 (7 : 3) solution was then mixed with the r e m a i n i n g m e t h y l e n e chloride which was also d i s c a r d e d following centrifugation. The r e m a i n i n g E T O H / H 2 S O 4 mixture was read on a s p e c t r o p h o t o f l u o r o m e t e r at 530 emission, 470 excitation.
2.5. Materials
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S o d i u m a s c o r b a t e (1-ascorbic acid s o d i u m salt), p a r g y l i n e and n o r a d r e n a l i n e were from Sigma C h e m i c a l C o m p a n y (St. Louis, M O ) whereas 3-isob u t y l - l - m e t h y l - x a n t h i n e was o b t a i n e d from Aldrich Chemical C o m p a n y Inc.+ Milwaukee, WI. Imidazole-HC1 was from E a s t m a n K o d a k Co., Rochester, NY. D o w e x (50 A G WX4+ 200-400 mesh) was o b t a i n e d from BioRad. Laboratories, R i c h m o n d , CA, a n d neutral a l u m i n a was from F i s h e r Scientific C o m p a n y , Fairlawn, NJ [8) H ] A d e n i n e a n d PCS scintillation cocktail were o b t a i n e d from A m e r s h a m , Oakville, Ontario. [~H]Cyclic A M P was p u r c h a s e d from New Engl a n d N u c l e a r ( C a n a d a ) Ltd., Lachine, Quebec, whereas M e t o p i r o n e was a generous gift from Ciba-Geigy (Canada).
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NORADRENAL1NE [~M]
Fig. 1. Effect of adrenalectomy on cyclic AMP formation. Adrenalectomies were performed 9 days prior to sacrifice. Corticosterone-treated animals received subcutaneous pellet implantations 5 days prior to sacrifice. Hippocampal slices were prelabelled with [3H]adenine and then incubated in the presence of 0, 1, 10 or 100/~M NA. Results are expressed as % conversion of [3H]ATP to [3H]cyclic AMP. Each value represents the mean+S.E.M, of 4-5 separate determinations obtained from 10-12 animals. *P < 0.05 as compared with control animals.
3. R e s u l t s
A possible i n t e r a c t i o n between a d r e n a l corticoids and the N A - c o u p l e d cyclic A M P generating system was examined, A T P stores in slices of rat h i p p o c a m p i were p r e l a b e l l e d with [~H]adenine and i n c u b a t e d in the presence of various concentrations of N A (0, 1, 10, 100/~M), As can be seen in fig. 1, bilateral a d r e n a l e c t o m y was found to p r o d u c e a slight increase in the rate of cyclic A M P f o r m a t i o n which was statistically significant at a N A c o n c e n t r a t i o n of 10 ~ M . Imp l a n t a t i o n of c o r t i c o s t e r o n e pellets into A D X rats 5 d a y s p r i o r to sacrifice p r e v e n t e d this increase. Metopirone, which inhibits corticosterone synthesis, was also f o u n d to increase cyclic A M P
formation. T r e a t m e n t with M e t o p i r o n e (50 m g / k g ) 2 h p r i o r to sacrifice resulted in an elevation of cyclic A M P levels which was statistically signific a n t at c o n c e n t r a t i o n s of 10 a n d 100 /~M N A . I m p l a n t a t i o n of c o r t i c o s t e r o n e pellets p r e v e n t e d this increase (fig. 2). Steroid pellet i m p l a n t a t i o n h a d no significant effect on the cyclic A M P generating system in control animals. As can be seen in table 1, the corticosterone synthesis i n h i b i t o r had no significant effect on N A - s t i m u l a t e d cyclic A M P f o r m a t i o n 1 h after t r e a t m e n t at any c o n c e n t r a t i o n of N A used. A statistically significant increase in cyclic A M P form a t i o n was o b s e r v e d 2 h following t r e a t m e n t at 10 a n d 100 /~M N A . A slight but nonsignificant
238
"FABLE 2
I g5l/~//~
~METOPIRONE
CORT
1h 2h 4 h
i 0
lO0
~ NORADRENALINE
Serum corticosterone levels from a n i m a l s sacrificed 1, 2 or 4 h after t r e a t m e n t with m e t o p i r o n e or vehicle. Rats were treated a p p r o x i m a t e l y 3 h into the light cycle with a 50 m g / k g i.p. injection of M e t o p i r o n e or an equal volume of the vehicle (409,: p r o p y l e n e glycol in water). A n i m a l s were sacrificed 1, 2 or 4 h later at which time trunk blood was collected for d e t e r m i n a t i o n of serum corticosterone levels. Corticosterone was purified using methylene chloride, N a O H and E T O H / H 2 S O 4 extractions, and measured using a spectrophotofluorometer, Results are expressed as /Lg c o r t i c o s t e r o n e / 1 0 0 ml serum. Each value represents the m e a n + S.E.M. of 4-6 separate d e t e r m i n a t i o n s o b t a i n e d from 10-15 animals. * Statistically significant difference when c o m p a r e d to the control values (P < 0.01). Vehicle (control)
Metopirone (treated)
29.8 ± 5.2 29.2+2.8 18.6+ 3.2
20.0+2.2 18.0+1.2" 16.2+ 3.2
[ ,uM]
Fig. 2. Effect of M e t o p i r o n e on cyclic A M P synthesis. A n i m a l s received a 50 m g / k g i.p. injection 2 h prior to sacrifice. C o n t r o l a n i m a l s received an equal v o l u m e of the vehicle (40% p r o p y l e n e glycol in water). C o r t i c o s t e r o n e - t r e a t e d a n i m a l s were i m p l a n t e d s u b c u t a n e o u s l y in the back of the neck with corticosterone pellets 5 days prior to sacrifice. H i p p o c a m p a l slices were prelabelled with [3H]adenine and then i n c u b a t e d in the presence of 0, 1, 10 or 100 ~ M NA. Results are expressed as c o n v e r s i o n of [ 3 H ] A T P to [3H]cyclic A M P . Each value represents the m e a n i S . E . M , of 4-6 separate d e t e r m i n a t i o n s obtained from 10-15 animals. * P < 0.01 as c o m p a r e d with control animals.
elevation in NA-stimulated cyclic AMP levels retnained at 4 h. These data parallel the corticosterone synthesis inhibiting effect of Metopirone (table 2). A slight but non-significant drop was observed in serum corticosterone levels 1 h after treatment. This decrease reached statistical significance at 2 h when compared to control values. Metopirone-treated corticosterone levels remained relatively low at 4 h, but were not significantly different from the low control values at 4 h. The difference in the corticosterone levels in the 4 h animals could be due to the time of sacrifice (i.e. later in the day and longer since the i.p. injection).
TABLE 1 N o r a d r e n a l i n e - s t i m u l a t e d cyclic A M P p r o d u c t i o n in a n i m a l s sacrificed 1, 2 or 4 h after M e t o p i r o n e treatment. M e t o p i r o n e - t r e a t e d rats received a 50 m g / k g i.p, injection 1, 2 or 4 h prior to sacrifice. C o n t r o l a n i m a l s received an equal volume of the vehicle (40% p r o p y l e n e glycol in water). H i p p o c a m p a l slices were prelabelled with [3H]adenine and then i n c u b a t e d in the presence of 0. 1, 10 or 100 ~ M NA. Results are expressed as % conversion of [3 H ] A T P to [3 H]cyclic A M P . Each value represents the mean ± S,E.M. of 4-6 separate d e t e r m i n a t i o n s o b t a i n e d from 10-15 animals. Cyclic A M P f o r m a t i o n in the vehicle-treated a n i m a l s from the 1, 2 and 4 h g r o u p s was not significantly different. D a t a from these groups were pooled and represented as the control group (16 separate d e t e r m i n a t i o n s , from 48 animals). * Statistically significant difference when c o m p a r e d to the control values (P < 0.01). Time
N o r a d r e n a l i n e (p,M) 0
Vehicle (control) 1 h Metopirone 2 h Metopirone 4 h Metopirone
0.260 0.287 0.274 0.278
1 ± ± ± ±
0.003 0.025 0.011 0.006
0.849 0.762 0.950 1.026
10 ± ± ± ±
0.042 0.043 0.061 0.111
1.399 1.345 1.605 1.530
100 +_0.046 + 0.040 _+0.079 * ± 0.126
1.659 1.670 2.035 1.787
± ± ± ±
0.059 0.043 0.128 * 0.173
239 The effect of 1 /~M Metopirone was also tested in vitro and exerted no significant effect on the cyclic AMP generating system in hippocampal slices obtained from control animals. As the proportion of Metopirone available to brain tissue is not known, further concentrations of Metopirone should be tested to confirm this result. Expression of the data as a percent of [3H]cyclic A M P formed from [3H]ATP stores reduces complications due to unequal tissue sizes, variations in protein levels, or to unmeasurable loss of sample. Nevertheless, protein levels were determined to ensure a relatively uniform sample preparation and were consistently between 1.7 and 2.4 mg per ml of the homogenate (data not shown).
4. D i s c u s s i o n
Mobley and Sulser (1980) reported an increase in NA-stimulated cyclic A M P synthesis in rat frontal cortex 2 weeks following bilateral adrenalectomy. In the present study, we report a less pronounced but significant increase in NA-stimulated cyclic A M P synthesis in rat hippocampus 9 days following bilateral adrenalectomy. This smaller increase could perhaps be due to the different brain region examined, the shorter period following adrenalectomy, or to difference(s) in assay procedure. Both increases were prevented by corticosterone replacement. Data obtained from A D X animals must be interpreted with caution because adrenalectomy causes many changes such as in electrolyte balance, carbohydrate metabolism (Exton et al., 1972) as well as in A C T H and endorphin secretion (Guillemin et al., 1977). Changes observed a week following such a drastic insult might be secondary to these effects, or due to some compensatory response. We therefore sought to obtain convergent evidence for the role of corticosterone in NA-stimulated adenyl cyclase activity using a pharmacological manipulator, Metopirone. Metopirone has been shown to inhibit the ll-/~-hydroxylating enzyme responsible for the synthesis of corticosterone (Liddle et al., 1958). In the present study, an injection of 50 m g / k g Metopirone reduced serum corticosterone levels to 62% of c o n -
trol values within 2 h. In addition, this treatment produced a significant increase in NA-stimulated cyclic AMP production which was prevented by implantation of corticosterone pellets. These pellets have been found to effectively maintain plasma corticosterone levels within the normal range (Bialik et al., 1982). This would presumably inhibit de novo synthesis of corticosterone in adrenal glands and thus render the Metopirone inhibition of corticosterone synthesis inconsequential. Metopirone had no significant effect in vitro which suggests that it has an indirect action on hippocampal NA-stimulated cyclic A M P formation which we presume to be mediated by its inhibitory effect on corticosterone synthesis. The present study suggests that corticosterone regulates cyclic A M P formation in the brain, and that this modulatory process can occur in as little as 2 h. At present, we can only speculate as to the nature of this interaction. The biochemical sequelae of NA receptor stimulation in brain are not yet clearly understood. However, there are growing parallels between well-studied peripheral reactions and those found in the central nervous system. In liver, adrenergic stimulation is known to produce glycogen breakdown through an adenyl cyclasemediated phosphorylation of glycogen phosphorylase. This reaction requires the "permissive" effect of corticosterone (Extort et al., 1972). Adrenalectomy causes blockade of glycogen hydrolysis and, presumably as a compensatory response, also causes an up regulation of NA-stimulated adenyl cyclase (Wolfe et al., 1976). It is possible that a similar mechanism is operative in brain as well. The enzymes responsible for glycogen metabolism, glycogen phosphorylase and phosphorylase kinase are present in brain and the activation of these enzymes has been shown to be dependent upon cyclic A M P (Wilkening and Makman, 1977). NA-stimulated glycogenolysis has been reported in brain cortical slices, and this effect is mediated via ~-adrenergic receptors and cyclic A M P formation (Quach et al., 1982; Magistretti et al., 1981). If the parallels between central and peripheral mechanisms hold this would not only explain why adrenolectomy up regulates NAstimulated adenyl cyclase, but would also suggest that glycogenolysis in brain is dependent on
240
adrenal glucocorticoids as well. The biogenic amine hypothesis of effective disorders states that depression is caused by a functional deficiency of serotonin (Coppen, 1967) a n d / o r N A (Schildkraut, 1965). There is much clinical evidence suggesting that adrenal corticoids may also have a role in effective disorders (Carroll, 1978; Sachar, 1980). Steroid imbalances and steriod therapy are often accompanied by depression or euphoria (Carpenter and Gruen, 1982). If biogenic amines and steroids are critically linked to brain energy balance, then we suggest that mood disorders are a consequence of an imbalance in brain oxidative metabolism. Just as low blood sugar levels can induce fatigue and depression, endogenous depression may also be due to an abnormal glucose metabolism in the brain. This idea, though speculative, has heuristic value.
Acknowledgements This work was supported by grants from the Ontario Menta[ Health Foundation (to R.L.S.) and the Medical Research Council of Canada (to R.L,S. and D.C.S.R.).
References Bialik, B., B.A. Pappas and D.C,S. Roberts, 1982. Adrenalectomy and Metopirone disrupt acquisition of active avoidance behavior, Soc, Neurosci. Abst. 19.1. Carpenter, W.J., Jr. and P.H. Gruen, 1982, Cortisol's effect on human functioning, J. Clin. Psychopharm. 2, 91. Carroll, B.J., 1978, Neuroendocrine function in psychiatric disorders, in: Psychopharmacology: a Generation of Progress, eds. M.A. Lipton, A. DiMascio and K.F. Killam (Raven Press, New York). Coppen, A., 1967, The biochemistry of affective disorders, Br. J. Psychiat. 113, 1237. Exton, J.A., N. Freidman, E.H.-A. Wong, J.P. Brineaux, J.D. Corbin and C.R. Park, 1972, Interaction of glucocorticoids with glucagon and epinephrine in the control of gluconeogenesis and glycogenolysis in liver and of lipolysis in adipose tissue, J. Biol. Chem. 247, 3579. Guillemin, R., T. Vargo, J. Rossier, S. Minick, N. Ling, C. Rivier, W. Vale and F. Bloom, 1977, Beta-endorphin and adrenocorticotropin are secreted concomitantly by pituitary gland, Science 197, 1367.
Harden, T.K., B.B. Wolfe, J.R. Sporn, B.K. Poulos and P.B. Molinoff, 1977, Effects of 6-hydroxydopamine on the development of the beta adrenergic receptor/adenylate cyclasc system in rat cerebral cortex, J. Pharmacol. Exp. Ther. 203. 132. Liddle, G.W., D. Island. E.M. Lance and A.P. Harris, 1958, Alterations of adrenal steroid patterns in man resulting from treatment with a chemical inhibitor of 11-beta-hydroxylase, J. Clin. Endocrinol. 18, 906. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with a folin phenol reagent, J. Biol. Chem. 193, 265. Magistretti, P.J., J.H. Morrison, W.J, Shoemaker, V. Sapin and F.E. Bloom, 1981. VIP induces glycogenolysis in mouse cortical slices: a possible regulatory mechanism for the local control of energy metabolism, Proc. Natl. Acad. Sci. U.S.A. 78, 6535. Mobley, P.L. and F. Sulser, 1980, Adrenal corticoids regulate sensitivity of noradrenaline receptor-coupled adenylate cyclase in brain, Nature 286, 608. Quach, T.T., C. Rose, A.M. Duchemin and J.C. Schwartz, 1982, Glycogenolysis reduced by serotonin in brain: identification of a new class of receptor, Nature 298, 373. Rastogi, R.B. and R.L. Singhal, 1978, Evidence for the role of adrenocortical hormones in the regulation of noradrenaline and dopamine metabolism in certain brain areas, Br. J. Pharmaco[. 62, 131. Roberts. D.C.S. and F.E. Bloom, 1981, Adrenal steroid-induced changes in beta-adrenergic receptor binding in rat hippocampus, European J. Pharmacol. 74, 37. Sachar, E.J., G. Asnis, U. Halbreich. R.S. Nathan and F. Halpern, 1980, Recent studies in the neuroendocrinology of major depressive disorders, Psychiat. Clin. N. Am. 3, 313. Salomon, Y.. C. Londos and M. Rodbell, 1974, A highly sensitive adenylate cyelase assay, Anal. Bioehem. 58, 541. Schildkraut, J.J., 1965, The catecholamine hypothesis of affeclive disorders. A review of supporting evidence, Am. J. Psychiat. 122, 509. Solem, J.H. and T. Brinck-Johnsen, 1965, An evaluation of a method for determination of the corticosteroids in minute quantities of mouse plasma. Stand. J. Clin. Lab. Invest. 17. Suppl. 80, 1. Wilkening, D, and M.H. Makman, 1977, Activation of glycogen phosphorylase inrat caudate nucleus slices by L-isopropylnorepinephrine and dibutyryl cyclic AMP, J. Neurochem. 28, 1001. Wolfe, B.B., T.K. Harden and P.B. Molinoff, 1976, Beta-adrenergic receptors in rat liver: effects of adrenalectomy, Proc. Natl. Acad. Sci. U.S.A. 73, 1343.
235
C O R T I C O S T E R O N E P R E V E N T S T H E INCREASE IN N O R A D R E N A L I N E - S T I M U L A T E D ADENYL CYCLASE ACTIVITY IN RAT H I P P O C A M P U S F O L L O W I N G A D R E N A L E C T O M Y OR METOPIRONE V.J. ROBERTS *, R.L. SINGHAL and D.C.S. ROBERTS **
Department of Pharmacolo,~v, FaculO, of Health Sciences, Universi(v of Ottawa, K i l l 8M5. and * * Department ~f P.s~vchologv, Carleton Universi(v. Ottawa, Ontario KIS 5B6, Canada
Received 18 April 1984, accepted 17 May 1984
V.J. R O B E R T S , R.L. S I N G H A L a n d D.C.S. R O B E R T S , Corticosterone prevents the increase in noradrenaline-stimulated adenvl o'clase activity in rat htppocampus following adrenalectomv or Metopirone, European J. Pharmacol. 103 (1984) 235-240. Corticosterone modulation of the noradrenaline-responsive cyclic A M P generating system was examined in rat hippocampus. A d r e n a l e c t o m y was found to produce a small but significant elevation in the rate of cyclic A M P formation in response to noradrenaline. I m p l a n t a t i o n of corticosterone pellets 5 days prior to sacrifice prevented this a d r e n a l e c t o m y - i n d u c e d increase. Metopirone, an inhibitor of corticosterone synthesis, was also observed to increase cyclic A M P formation. This elevation was seen 2 h following a 50 m g / k g i.p. injection and was completely prevented by corticosterone pellet implantation. Metopirone had no significant effect on cyclic A M P production after 1 h, while a slight but statistically non-significant elevation remained at 4 h. These observations parallel the inhibitory effect of M e t o p i r o n e on corticosterone synthesis as determined by serum corticosterone levels.
Corticosterone
Noradrenaline
Cyclic AMP
Hippocampus
1. Introduction
There is growing evidence that adrenal steroids modulate noradrenaline-stimulated cyclic AMP levels in the brain. Adrenalectomy has been reported by Mobley and Sulser (1980) to increase noradrenaline-induced cyclic A M P accumulation in slices from rat frontal cortex. These effects were reversed by daily corticosterone treatment. Interestingly, the observed elevation in cyclic A M P formation does not appear to depend upon an increase in/~-adrenergic receptor binding. Following adrenalectomy, no significant alteration in B...... or K D values was noted for [3H]DHA binding in either rat frontal cortex (Mobley and Sulser, 1980) or hippocampus (Roberts and Bloom, 1981), despite decreases in rat brain noradrenaline (NA) concentrations (Rastogi and Singhal, 1978). * To whom all correspondence should be addressed: 451 Smytb Road, Ottawa, Ontario, K1H 8M5, Canada. 0014-2999/84/$03.00 ~ 1984 Elsevier Science Publishers B.V.
One problem with adrenalectomy, however, is that because many hormonal responses are affected, the observed effects on central NA mechanisms may be due to some of these secondary changes. We therefore sought convergent evidence for the role of corticosterone on NA receptor mechanisms by using an acute pharmacological manipulation of corticosterone levels. We now report that Metopirone, which inhibits the synthesis of corticosterone, also causes a significant up regulation of the NA-stimulated cyclic AMP generating system.
2. Materials and methods
2.1. Animal preparation Male Wister rats (175-225 g) were maintained on a constant 12 h light, 12 h dark cycle at 23°C.
236
Animals were given free access to food and water. In the case of adrenalectomized (ADX) rats, a 0.9% NaCI solution was substituted for water. Bilateral adrenalectomies were performed 9 days prior to sacrifice. In sham-operated rats, adrenals were located but not removed. Animals receiving corticosterone pellets were anesthetized with ether and pellets were implanted subcutaneously in the back of the neck 5 days prior to sacrifice. Shamoperated rats received an incision but no pellet. Rats treated with Metopirone were given a 50 m g / k g i.p. injection approximately 3 h into the light cycle and sacrificed 1, 2, or 4 h later. All control animals received an equal volume of the vehicle (40% propylene glycol in water).
2.2. Tissue preparation Tissue was prepared according to the method of Harden et al, (1977) with some modifications. Rats were sacrificed by decapitation, the brain rapidly removed and placed on an ice-cold glass plate. Hippocampi were removed freehand, pooled 4-6 per group and sliced in two directions at right angles to each other (0.26 × 0.26 mm) with a McIlwain tissue chopper. The slices were weighed and suspended (50 m g / m l ) in oxygenated (95% O2: 5% CO2) Krebs-Henseleit solution (NaCI 115.3, KC1 4.6, CaC12 2.3, MgSO 4 1.1, N a H C O 3 22.1, KH2PO 4 1.1, glucose 7.8 and disodium EDTA 0.03 mM and 1 t~M Metopirone when indicated). The suspension was incubated in an atmosphere of 95% 02 and 5% CO 2 for 20 min at 3 7 ° C in a shaking Dubnoff water bath. Following incubation, the samples were centrifuged at 600 × g for 20 s. The pellet was re-suspended (50 m g / m l ) in fresh Krebs-Henseleit buffer containing 2.5 ~tCi/ml of [8 -3H]adenine and incubated for an additional 40 min period. Excess radioactivity was removed by washing 3 times by centrifugation (600 × g, 20 s) with pre-warmed and gassed Krebs-Henseleit buffer. Washed pellets were resuspended in Krebs-Henseleit buffer containing 1 mM Na ascorbate, I /zM pargyline, and 1 mM 3-isobutyl1-methylxanthine. Samples were divided into 4 aliquots and incubated in the presence of 0, 1, 10 and 100 t~M NA (37°C; 95% 02: 5% CO2) for 10 min. The reaction was stopped by the addition of
50% trichloroacetic acid (107/xl/ml) and homogenized immediately. A small portion (100 /,1) was removed for protein estimation and the remaining portion centrifuged at 18000 × g for 10 rain. The pellet was discarded and the supernatant saved in glass tubes. Trichloroacetic acid was removed from the supernatant by extracting 3 times with 4 vol. water-saturated ether, then placed in a 60 °C water bath for 10 min to boil off any residual ether.
2.3. Purification and estimation of cyclic A M P Sequential Dowex and alumina chromatography were used to separate radioactive cyclic AMP from other labelled compounds (Salomon et al., 1974). Dowex (50 AG WX4, 200-400 mesh) was prepared by washing with 2 vol. 1 N HCI followed by 10 vol. water. 100 /xl 1 N HC1 was added to 900 /,1 TCA extract (final concentration of 0.1 N HCI) and the mixture was decanted into columns (0.4 × 10 cm) containing 1 ml of prepared Dowex resin. Columns were first washed with 3.5 ml of water and a portion of this flow-through was reserved for counting (3 × 200 ~LI in 10 ml PCS scintillation cocktail). Columns were then washed with an additional 4.5 ml water and a portion (3 × 200 /,1) of elute was saved for counting. To the remaining portion (3.8 ml), 0.253 ml 1.5 M imidazole-HCl, pH 7.2, was added. The mixture was decanted into columns (0.4 × 10 cm) containing 0.6 g neutral alumina which had been washed with 8 ml 0.1 M imidazole-HCl, pH 7.5. The columns were allowed to drain completely of sample and then washed with 1 ml 0.1 M imidazoleHC1, pH 7.5. Three 200 /,1 portions of the elute were saved for counting. The final elute contained the purified [3H]cyclic AMP while the flowthrough from the Dowex column was mainly [~H]ATP. Results are expressed as percent conversion of [~H]ATP to [3H]cyclic AMP and analyzed using the 2-tailed Student's t-test. Brain protein concentrations were measured using a modification of the method of Lowry et al. (1951).
2.3. Purification and estimation of corticosterone Serum corticosterone levels were determined using a modification of the method of Solem and
237 B r i n c k - J o h n s e n (1965). Serum (0.5 ml) was a d d e d to 6 ml m e t h y l e n e chloride. A f t e r vortexing and centrifugation, the serum layer was r e m o v e d and d i s c a r d e d . To remove impurities and metabolites, 1 ml 0.1 N N a O H was a d d e d to methylene chloride, vortexed, centrifuged, a n d removed, A n E T O H / H 2 S O 4 (7 : 3) solution was then mixed with the r e m a i n i n g m e t h y l e n e chloride which was also d i s c a r d e d following centrifugation. The r e m a i n i n g E T O H / H 2 S O 4 mixture was read on a s p e c t r o p h o t o f l u o r o m e t e r at 530 emission, 470 excitation.
2.5. Materials
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S o d i u m a s c o r b a t e (1-ascorbic acid s o d i u m salt), p a r g y l i n e and n o r a d r e n a l i n e were from Sigma C h e m i c a l C o m p a n y (St. Louis, M O ) whereas 3-isob u t y l - l - m e t h y l - x a n t h i n e was o b t a i n e d from Aldrich Chemical C o m p a n y Inc.+ Milwaukee, WI. Imidazole-HC1 was from E a s t m a n K o d a k Co., Rochester, NY. D o w e x (50 A G WX4+ 200-400 mesh) was o b t a i n e d from BioRad. Laboratories, R i c h m o n d , CA, a n d neutral a l u m i n a was from F i s h e r Scientific C o m p a n y , Fairlawn, NJ [8) H ] A d e n i n e a n d PCS scintillation cocktail were o b t a i n e d from A m e r s h a m , Oakville, Ontario. [~H]Cyclic A M P was p u r c h a s e d from New Engl a n d N u c l e a r ( C a n a d a ) Ltd., Lachine, Quebec, whereas M e t o p i r o n e was a generous gift from Ciba-Geigy (Canada).
,
:ADX. CORT
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0
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,
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10
100
NORADRENAL1NE [~M]
Fig. 1. Effect of adrenalectomy on cyclic AMP formation. Adrenalectomies were performed 9 days prior to sacrifice. Corticosterone-treated animals received subcutaneous pellet implantations 5 days prior to sacrifice. Hippocampal slices were prelabelled with [3H]adenine and then incubated in the presence of 0, 1, 10 or 100/~M NA. Results are expressed as % conversion of [3H]ATP to [3H]cyclic AMP. Each value represents the mean+S.E.M, of 4-5 separate determinations obtained from 10-12 animals. *P < 0.05 as compared with control animals.
3. R e s u l t s
A possible i n t e r a c t i o n between a d r e n a l corticoids and the N A - c o u p l e d cyclic A M P generating system was examined, A T P stores in slices of rat h i p p o c a m p i were p r e l a b e l l e d with [~H]adenine and i n c u b a t e d in the presence of various concentrations of N A (0, 1, 10, 100/~M), As can be seen in fig. 1, bilateral a d r e n a l e c t o m y was found to p r o d u c e a slight increase in the rate of cyclic A M P f o r m a t i o n which was statistically significant at a N A c o n c e n t r a t i o n of 10 ~ M . Imp l a n t a t i o n of c o r t i c o s t e r o n e pellets into A D X rats 5 d a y s p r i o r to sacrifice p r e v e n t e d this increase. Metopirone, which inhibits corticosterone synthesis, was also f o u n d to increase cyclic A M P
formation. T r e a t m e n t with M e t o p i r o n e (50 m g / k g ) 2 h p r i o r to sacrifice resulted in an elevation of cyclic A M P levels which was statistically signific a n t at c o n c e n t r a t i o n s of 10 a n d 100 /~M N A . I m p l a n t a t i o n of c o r t i c o s t e r o n e pellets p r e v e n t e d this increase (fig. 2). Steroid pellet i m p l a n t a t i o n h a d no significant effect on the cyclic A M P generating system in control animals. As can be seen in table 1, the corticosterone synthesis i n h i b i t o r had no significant effect on N A - s t i m u l a t e d cyclic A M P f o r m a t i o n 1 h after t r e a t m e n t at any c o n c e n t r a t i o n of N A used. A statistically significant increase in cyclic A M P form a t i o n was o b s e r v e d 2 h following t r e a t m e n t at 10 a n d 100 /~M N A . A slight but nonsignificant
238
"FABLE 2
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~METOPIRONE
CORT
1h 2h 4 h
i 0
lO0
~ NORADRENALINE
Serum corticosterone levels from a n i m a l s sacrificed 1, 2 or 4 h after t r e a t m e n t with m e t o p i r o n e or vehicle. Rats were treated a p p r o x i m a t e l y 3 h into the light cycle with a 50 m g / k g i.p. injection of M e t o p i r o n e or an equal volume of the vehicle (409,: p r o p y l e n e glycol in water). A n i m a l s were sacrificed 1, 2 or 4 h later at which time trunk blood was collected for d e t e r m i n a t i o n of serum corticosterone levels. Corticosterone was purified using methylene chloride, N a O H and E T O H / H 2 S O 4 extractions, and measured using a spectrophotofluorometer, Results are expressed as /Lg c o r t i c o s t e r o n e / 1 0 0 ml serum. Each value represents the m e a n + S.E.M. of 4-6 separate d e t e r m i n a t i o n s o b t a i n e d from 10-15 animals. * Statistically significant difference when c o m p a r e d to the control values (P < 0.01). Vehicle (control)
Metopirone (treated)
29.8 ± 5.2 29.2+2.8 18.6+ 3.2
20.0+2.2 18.0+1.2" 16.2+ 3.2
[ ,uM]
Fig. 2. Effect of M e t o p i r o n e on cyclic A M P synthesis. A n i m a l s received a 50 m g / k g i.p. injection 2 h prior to sacrifice. C o n t r o l a n i m a l s received an equal v o l u m e of the vehicle (40% p r o p y l e n e glycol in water). C o r t i c o s t e r o n e - t r e a t e d a n i m a l s were i m p l a n t e d s u b c u t a n e o u s l y in the back of the neck with corticosterone pellets 5 days prior to sacrifice. H i p p o c a m p a l slices were prelabelled with [3H]adenine and then i n c u b a t e d in the presence of 0, 1, 10 or 100 ~ M NA. Results are expressed as c o n v e r s i o n of [ 3 H ] A T P to [3H]cyclic A M P . Each value represents the m e a n i S . E . M , of 4-6 separate d e t e r m i n a t i o n s obtained from 10-15 animals. * P < 0.01 as c o m p a r e d with control animals.
elevation in NA-stimulated cyclic AMP levels retnained at 4 h. These data parallel the corticosterone synthesis inhibiting effect of Metopirone (table 2). A slight but non-significant drop was observed in serum corticosterone levels 1 h after treatment. This decrease reached statistical significance at 2 h when compared to control values. Metopirone-treated corticosterone levels remained relatively low at 4 h, but were not significantly different from the low control values at 4 h. The difference in the corticosterone levels in the 4 h animals could be due to the time of sacrifice (i.e. later in the day and longer since the i.p. injection).
TABLE 1 N o r a d r e n a l i n e - s t i m u l a t e d cyclic A M P p r o d u c t i o n in a n i m a l s sacrificed 1, 2 or 4 h after M e t o p i r o n e treatment. M e t o p i r o n e - t r e a t e d rats received a 50 m g / k g i.p, injection 1, 2 or 4 h prior to sacrifice. C o n t r o l a n i m a l s received an equal volume of the vehicle (40% p r o p y l e n e glycol in water). H i p p o c a m p a l slices were prelabelled with [3H]adenine and then i n c u b a t e d in the presence of 0. 1, 10 or 100 ~ M NA. Results are expressed as % conversion of [3 H ] A T P to [3 H]cyclic A M P . Each value represents the mean ± S,E.M. of 4-6 separate d e t e r m i n a t i o n s o b t a i n e d from 10-15 animals. Cyclic A M P f o r m a t i o n in the vehicle-treated a n i m a l s from the 1, 2 and 4 h g r o u p s was not significantly different. D a t a from these groups were pooled and represented as the control group (16 separate d e t e r m i n a t i o n s , from 48 animals). * Statistically significant difference when c o m p a r e d to the control values (P < 0.01). Time
N o r a d r e n a l i n e (p,M) 0
Vehicle (control) 1 h Metopirone 2 h Metopirone 4 h Metopirone
0.260 0.287 0.274 0.278
1 ± ± ± ±
0.003 0.025 0.011 0.006
0.849 0.762 0.950 1.026
10 ± ± ± ±
0.042 0.043 0.061 0.111
1.399 1.345 1.605 1.530
100 +_0.046 + 0.040 _+0.079 * ± 0.126
1.659 1.670 2.035 1.787
± ± ± ±
0.059 0.043 0.128 * 0.173
239 The effect of 1 /~M Metopirone was also tested in vitro and exerted no significant effect on the cyclic AMP generating system in hippocampal slices obtained from control animals. As the proportion of Metopirone available to brain tissue is not known, further concentrations of Metopirone should be tested to confirm this result. Expression of the data as a percent of [3H]cyclic A M P formed from [3H]ATP stores reduces complications due to unequal tissue sizes, variations in protein levels, or to unmeasurable loss of sample. Nevertheless, protein levels were determined to ensure a relatively uniform sample preparation and were consistently between 1.7 and 2.4 mg per ml of the homogenate (data not shown).
4. D i s c u s s i o n
Mobley and Sulser (1980) reported an increase in NA-stimulated cyclic A M P synthesis in rat frontal cortex 2 weeks following bilateral adrenalectomy. In the present study, we report a less pronounced but significant increase in NA-stimulated cyclic A M P synthesis in rat hippocampus 9 days following bilateral adrenalectomy. This smaller increase could perhaps be due to the different brain region examined, the shorter period following adrenalectomy, or to difference(s) in assay procedure. Both increases were prevented by corticosterone replacement. Data obtained from A D X animals must be interpreted with caution because adrenalectomy causes many changes such as in electrolyte balance, carbohydrate metabolism (Exton et al., 1972) as well as in A C T H and endorphin secretion (Guillemin et al., 1977). Changes observed a week following such a drastic insult might be secondary to these effects, or due to some compensatory response. We therefore sought to obtain convergent evidence for the role of corticosterone in NA-stimulated adenyl cyclase activity using a pharmacological manipulator, Metopirone. Metopirone has been shown to inhibit the ll-/~-hydroxylating enzyme responsible for the synthesis of corticosterone (Liddle et al., 1958). In the present study, an injection of 50 m g / k g Metopirone reduced serum corticosterone levels to 62% of c o n -
trol values within 2 h. In addition, this treatment produced a significant increase in NA-stimulated cyclic AMP production which was prevented by implantation of corticosterone pellets. These pellets have been found to effectively maintain plasma corticosterone levels within the normal range (Bialik et al., 1982). This would presumably inhibit de novo synthesis of corticosterone in adrenal glands and thus render the Metopirone inhibition of corticosterone synthesis inconsequential. Metopirone had no significant effect in vitro which suggests that it has an indirect action on hippocampal NA-stimulated cyclic A M P formation which we presume to be mediated by its inhibitory effect on corticosterone synthesis. The present study suggests that corticosterone regulates cyclic A M P formation in the brain, and that this modulatory process can occur in as little as 2 h. At present, we can only speculate as to the nature of this interaction. The biochemical sequelae of NA receptor stimulation in brain are not yet clearly understood. However, there are growing parallels between well-studied peripheral reactions and those found in the central nervous system. In liver, adrenergic stimulation is known to produce glycogen breakdown through an adenyl cyclasemediated phosphorylation of glycogen phosphorylase. This reaction requires the "permissive" effect of corticosterone (Extort et al., 1972). Adrenalectomy causes blockade of glycogen hydrolysis and, presumably as a compensatory response, also causes an up regulation of NA-stimulated adenyl cyclase (Wolfe et al., 1976). It is possible that a similar mechanism is operative in brain as well. The enzymes responsible for glycogen metabolism, glycogen phosphorylase and phosphorylase kinase are present in brain and the activation of these enzymes has been shown to be dependent upon cyclic A M P (Wilkening and Makman, 1977). NA-stimulated glycogenolysis has been reported in brain cortical slices, and this effect is mediated via ~-adrenergic receptors and cyclic A M P formation (Quach et al., 1982; Magistretti et al., 1981). If the parallels between central and peripheral mechanisms hold this would not only explain why adrenolectomy up regulates NAstimulated adenyl cyclase, but would also suggest that glycogenolysis in brain is dependent on
240
adrenal glucocorticoids as well. The biogenic amine hypothesis of effective disorders states that depression is caused by a functional deficiency of serotonin (Coppen, 1967) a n d / o r N A (Schildkraut, 1965). There is much clinical evidence suggesting that adrenal corticoids may also have a role in effective disorders (Carroll, 1978; Sachar, 1980). Steroid imbalances and steriod therapy are often accompanied by depression or euphoria (Carpenter and Gruen, 1982). If biogenic amines and steroids are critically linked to brain energy balance, then we suggest that mood disorders are a consequence of an imbalance in brain oxidative metabolism. Just as low blood sugar levels can induce fatigue and depression, endogenous depression may also be due to an abnormal glucose metabolism in the brain. This idea, though speculative, has heuristic value.
Acknowledgements This work was supported by grants from the Ontario Menta[ Health Foundation (to R.L.S.) and the Medical Research Council of Canada (to R.L,S. and D.C.S.R.).
References Bialik, B., B.A. Pappas and D.C,S. Roberts, 1982. Adrenalectomy and Metopirone disrupt acquisition of active avoidance behavior, Soc, Neurosci. Abst. 19.1. Carpenter, W.J., Jr. and P.H. Gruen, 1982, Cortisol's effect on human functioning, J. Clin. Psychopharm. 2, 91. Carroll, B.J., 1978, Neuroendocrine function in psychiatric disorders, in: Psychopharmacology: a Generation of Progress, eds. M.A. Lipton, A. DiMascio and K.F. Killam (Raven Press, New York). Coppen, A., 1967, The biochemistry of affective disorders, Br. J. Psychiat. 113, 1237. Exton, J.A., N. Freidman, E.H.-A. Wong, J.P. Brineaux, J.D. Corbin and C.R. Park, 1972, Interaction of glucocorticoids with glucagon and epinephrine in the control of gluconeogenesis and glycogenolysis in liver and of lipolysis in adipose tissue, J. Biol. Chem. 247, 3579. Guillemin, R., T. Vargo, J. Rossier, S. Minick, N. Ling, C. Rivier, W. Vale and F. Bloom, 1977, Beta-endorphin and adrenocorticotropin are secreted concomitantly by pituitary gland, Science 197, 1367.
Harden, T.K., B.B. Wolfe, J.R. Sporn, B.K. Poulos and P.B. Molinoff, 1977, Effects of 6-hydroxydopamine on the development of the beta adrenergic receptor/adenylate cyclasc system in rat cerebral cortex, J. Pharmacol. Exp. Ther. 203. 132. Liddle, G.W., D. Island. E.M. Lance and A.P. Harris, 1958, Alterations of adrenal steroid patterns in man resulting from treatment with a chemical inhibitor of 11-beta-hydroxylase, J. Clin. Endocrinol. 18, 906. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with a folin phenol reagent, J. Biol. Chem. 193, 265. Magistretti, P.J., J.H. Morrison, W.J, Shoemaker, V. Sapin and F.E. Bloom, 1981. VIP induces glycogenolysis in mouse cortical slices: a possible regulatory mechanism for the local control of energy metabolism, Proc. Natl. Acad. Sci. U.S.A. 78, 6535. Mobley, P.L. and F. Sulser, 1980, Adrenal corticoids regulate sensitivity of noradrenaline receptor-coupled adenylate cyclase in brain, Nature 286, 608. Quach, T.T., C. Rose, A.M. Duchemin and J.C. Schwartz, 1982, Glycogenolysis reduced by serotonin in brain: identification of a new class of receptor, Nature 298, 373. Rastogi, R.B. and R.L. Singhal, 1978, Evidence for the role of adrenocortical hormones in the regulation of noradrenaline and dopamine metabolism in certain brain areas, Br. J. Pharmaco[. 62, 131. Roberts. D.C.S. and F.E. Bloom, 1981, Adrenal steroid-induced changes in beta-adrenergic receptor binding in rat hippocampus, European J. Pharmacol. 74, 37. Sachar, E.J., G. Asnis, U. Halbreich. R.S. Nathan and F. Halpern, 1980, Recent studies in the neuroendocrinology of major depressive disorders, Psychiat. Clin. N. Am. 3, 313. Salomon, Y.. C. Londos and M. Rodbell, 1974, A highly sensitive adenylate cyelase assay, Anal. Bioehem. 58, 541. Schildkraut, J.J., 1965, The catecholamine hypothesis of affeclive disorders. A review of supporting evidence, Am. J. Psychiat. 122, 509. Solem, J.H. and T. Brinck-Johnsen, 1965, An evaluation of a method for determination of the corticosteroids in minute quantities of mouse plasma. Stand. J. Clin. Lab. Invest. 17. Suppl. 80, 1. Wilkening, D, and M.H. Makman, 1977, Activation of glycogen phosphorylase inrat caudate nucleus slices by L-isopropylnorepinephrine and dibutyryl cyclic AMP, J. Neurochem. 28, 1001. Wolfe, B.B., T.K. Harden and P.B. Molinoff, 1976, Beta-adrenergic receptors in rat liver: effects of adrenalectomy, Proc. Natl. Acad. Sci. U.S.A. 73, 1343.