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Effects of clorgyline and deprenil on corticosterone levels in rats
European Journal of Pharmacology, 81 (1982) 349- 355
349
Elsevier Biomedical Press
E F F E C T S O F CLORGYLINE AND D E P R E N I L ON C O R T I C O S T E R O N E LEVELS IN RATS * M. ANGELES VENTURA **
Unitb de Neuropharmacologie, B~t 440, Universitb Paris XI, 91405 Orsay Cedex, France Received 25 September 1981, revised MS received 12 February 1982, accepted 19 April 1982
M.A. VENTURA, Effects of clorgyline and deprenil on corticosterone levels in rats, European J. Pharmacol. 81 (1982) 349-355. Adrenal and plasma corticosterone levels were measured in adult female rats after i.p. administration of specific MAO inhibitors: clorgyline (IMAO-A) and deprenil (IMAO-B). Brain and adrenal MAO activity was also determined. The IMAO effect was found to depend on 3 interrelated factors: (a) dose of drug, (b) time after injection, (c) specificity in MAO inhibition. One hour after injection, clorgyline (1 and 2.5 mg/kg ) but not deprenil (20 mg/kg), appeared to inhibit the adrenocortical stress response to i.p. injection. At higher doses, MAO-B inhibition by deprenil (40 mg/kg) induced a moderate but sustained increase in corticosterone levels, while MAO-A inhibition by clorgyline (5, 10, 20 mg/kg) resulted in a large and sharp rise. This effect of clorgyline was potentiated with a-methyl-p-tyrosine and blocked with p-chlorophenylalanine. On this basis, the involvement of serotonergic mechanisms could be considered to explain the stimulatory effect of clorgyline on adrenal cortical function. Rat corticosterone levels
Clorgyline
Deprenil
p-Chlorophenylalanine
1. Introduction
The pharmacological approach has been widely used to study the interactions between central nervous system and endocrine glands (De Wied and De Jong, 1974; Buckingham, 1980). As far as the hypothalamo-hypophyseal-adrenal axis is concerned, the results obtained with monoamine oxidase inhibitors (IMAO) are controversial (De Wied and De Jong, 1974; Keim and Sigg, 1977). Differences in drugs and animal species used could account for some of the discrepancies, as could the fact that several aspects of the adrenal function can be affected: i.e. direct stimulation, response to stress, circadian rhythm. On the other hand, the role of the different * Part of this investigation was presented to the Second International Symposium on Catecholamines and Stress, Smolenice Castle, Czechoslovakia, 1979 (Ventura et al., 1980). ** Present address: Pharmacologic Biochimique, CHU Cochin-Port Royal, 24 rue du Fg St Jacques, F-75014 Paris, France. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
a-Methyl-p-tyrosine
neurotransmitters in the control of adrenocortical function is not clearly established (See Weiner and Ganong, 1978; Meites and Sonntag, 1981, for review). There is some agreement about the inhibitory action of norepinephrine (NE), whereas epinephrine (E) and dopamine (DA) do not seem involved. Evidence has been obtained for both an inhibitory and excitatory effect of serotonin (5HT), but pharmacological studies particularly support the latter hypothesis (Fuller, 1981). Histamine seems to exert some stimulatory activity. The IMAO used up to now in such studies (nialamine, iproniazid, pargyline, etc...) do not act with the same degree of efficiency upon the two forms of MAO and consequently, the levels of the different neurotransmitters are not affected to the same extent (Christmas et al., 1972): this could explain some of the discrepancies observed. In this context, we studied the response of the adrenal cortex to the specific inhibitors, clorgyline (CI: MAO-A) (Johnston, 1968) and deprenil (D: MAO-B) (Knoll et al., 1965) by measuring corticosterone levels in plasma and adrenals. The re-
350 suits showed that clorgyline drastically increased both parameters. Subsequently, we studied the effect of a-methyl-p-tyrosine (a-MT) and p-chlorophenylalanine (PCPA), well known to block the synthesis of catecholamines and serotonin respectively, on the adrenocortical stimulation induced by clorgyline.
2. Materials and methods
2.1. Drugs The following drugs were used: clorgyline and (RS)-deprenil hydrochloride (May and Baker), D,L-a-methyl-p-tyrosine methyl ester hydrochloride (Regis and Co), D,L-p-chlorophenylalanine methyl ester hydrochloride (Labkemi AB). The drugs were dissolved in cold saline immediately before each experiment and administered by intraperitoneal (i.p.) injection (1 ml/kg).
2.3. Experimental schedule In the first part of the study, three series of experiments were carried out: (a) a single injection of 1, 5, 10, 20 m g / k g of clorgyline, or 20, 40 m g / k g of deprenil, 1 or 8 h before sacrifice; (b) in order to test the effects of chronic treatment, some animals were given 5 or 10 m g / k g of clorgyline, or 20 m g / k g of deprenil, daily for 1 week, and killed 8 h after the last injection; (c) the additive effect of clorgyline (2.5 mg/kg) and deprenil (20 m g / k g ) was tested 1 h after injection. In the second part of the study, a-MT (250 mg/kg, 9 h before sacrifice) and PCPA (300 mg/kg, 24 h before sacrifice) were used, alone or associated with two doses of clorgyline (2.5 or 5 mg/kg, 1 h before sacrifice). In each series, a control group (s) was injected with saline (1 ml/kg) under the same conditions as drug-injected groups. These controls were used for statistical comparisons. In addition, basal levels in intact animals (i) are given for reference.
2.2. Animals 2.4. Assay procedures Adult Sherman female rats bred in our laboratory (thus easily available) weighing 250-300g were used. The phase of the estrous cycle was not verified. According to Audrian et al. (1978), intact Sherman rats killed at random between 8 and 9 h 30 a.m. are distributed as follows: proestrus (P), 12%; estrus (E), 35%; meta + diestrus (M + D), 53%. The corresponding corticosterone levels reach 1 8 ± 2 (P), 1 0 ± 0 . 4 (E) and 6 ± 0 . 2 ( M + D ) /zg/100 ml, respectively. In our intact groups, the mean was 8.6 ± 1.2, intermediate among E and M + D which account for 88% of the cases. On this basis, we found it not essential to verify the estrous cycle phase since this would have been (a) stressful if done in vivo, (b) time- and drug-consuming if done 'post-mortem'. The experiments were carried out during spring and summer. The animals were kept under natural d a y / n i g h t cycle, at a constant temperature (21 ± I°C) with tap water and commercial food ad libitum. The animals were weighed and put in individual cages at 5 p.m. the day before sacrifice. They were killed by cervical dislocation between 9 and 11 a.m., in all the experiments.
After sacrifice 2 ml of blood were collected by aortic puncture. The adrenals and brain were removed quickly and kept in ice-cold 0.9% KC1 or 4% TCA (trichloroacetic acid), for enzyme assay (brain and adrenal) and for corticosterone (adrenal) measurement respectively, until processing of the samples the same day. Corticosterone in adrenals and plasma was measured fluorometrically according to De Moor et al. (1962). Total MAO activity was determined using tryptamine [14C]bisuccinate (side chain label) as substrate (Wurtman and Axelrod, 1963; Parvez and Parvez, 1973) while kynuramine was used to estimate form B activity (Kraml, 1965). Tissue protein content was determined according to Lowry et al. (1951).
2.5. Statistical analysis The statistical analysis of the data, according to procedures described in Sokal and Rohlf (1969), included: (a) basic computations (~, S.E.M., s 2, etc.); (b) F-max test for homogeneity of variances; (c) Log transformation, if required; (d) analysis of
351 variance ( A N O V A ) , one or two-way, model 1, unequal sample size; (e) when the overall A N O V A was significant, two types of comparisons a m o n g means were performed:
2.5.1. A posteriori Student-Newman-Keuls (SNK) test for the first part of the study (i.e. experiments using different concentrations of the same drug).
2.5.2. A priori Comparisons by the 'contrasts' method for the second part (i.e. experiment including several drugs). The S N K test was performed on the basis of a level of significance ( a ) -- 0.05. It provides the 'upper' limit of significance, but not 'true' a values. On the other hand, the 'contrasts' a priori m e t h o d provides 'true' a values. 'Basal' (i) and 'control' (s) levels were c o m p a r e d using Student's t-test for unequal sample size.
3. Results The experiments were carried out in two different environments due to the transfer of the laboratory to another building. During the first part of the study, the animals were reared in the general animal room, whereas in the second part, they were kept in a special room, and thus subjected to less disturbance. In the first set of experiments, but not in the second, plasma corticosterone levels were found higher in saline-injected than in intact animals, 1 h after injection. This fact was p r o b a b l y related to the differences in experimental conditions. The sensitivity of the adrenal cortex to all kind of stimuli, particularly in the female (Le Mevel et al., 1979) is well established, as well as the role of previous history of exposure to stimulation (Ader, 1970).
pg / 100 ml SINGLE
I
10080-
60-
TREATMENT
TI 40-
20-
16191al7171 i
s 1 51020
S l 51o2o
lh
S2o*O'
8h
lh
8h
i
clorgyline
S2o*O t
deprenil
Fig. I. Plasma corticosterone levels (mean--+S.E.M.) in rats, 1 or 8 h after a single treatment with clorgyline or deprenil (hatched or lined columns respectively), i = intact; s--saline; l, 5, 10, 20, 40: doses used (mg/kg). Inside columns: number of animals per group. Statistics: ANOVA (clorgyline): overall, time and dose factors and interaction, P<0.001. ANOVA (deprenil): overall, time and dose factors, P<0.001; interaction, n.s. SNK test: * means groups significantly different from their controls (open columns), at least for a=0.05.
352
3.1. Effect of single doses of clorgyline and deprenil (fig. 1)
3.2. Effect of chronic treatment with clorgyline and deprenil (fig. 2)
Saline-injected a n i m a l s (s) p r e s e n t e d p l a s m a c o r t i c o s t e r o n e levels significantly higher than d i d i n t a c t (i) a n i m a l s 1 h ( b u t n o t 8 h) after injection. N o difference was f o u n d in a d r e n a l levels (not shown). Plasma and adrenal corticosterone concentrations were lower than the c o n t r o l s 1 h after 1 m g / m l of clorgyline. Conversely, the o t h e r doses o f the d r u g (5, 10, 20 m g / k g ) i n d u c e d strong increases in c o r t i c o s t e r o n e levels, within the s a m e range. Eight hours after injection, the values were n o t significantly different from the controls, exc e p t in p l a s m a for the 10 a n d 20 m g / k g doses. T h e lower d o s e (20 m g / k g ) of d e p r e n i l h a d no effect, whereas the higher (40 m g / k g ) i n d u c e d a m o d e r a t e b u t s u s t a i n e d increase in p l a s m a a n d a d r e n a l ( n o t shown) c o r t i c o s t e r o n e levels, for at least 8 h.
D a i l y injection for 1 week with 5 or 10 m g / k g of clorgyline or 20 m g / k g of deprenil, resulted in a m o d e r a t e increase in p l a s m a a n d a d r e n a l (not shown) corticosterone.
CHRONIC TREATMENT
3.3. Additive effect of clorgyline and deprenil (fig. 2) Doses of clorgyline (2.5 m g / k g ) a n d d e p r e n i l (20 m g / k g ) ineffective b y themselves i n d u c e d a m a r k e d increase in c o r t i c o s t e r o n e levels in b o t h p l a s m a a n d a d r e n a l ( n o t shown) when a d m i n i s tered together.
3. 4. Interaction of ot-MT and PCPA with clorgyline (table 1) T h e a d m i n i s t r a t i o n of a - M T evoked a rise in c o r t i c o s t e r o n e levels, m o d e r a t e in the a d r e n a l a n d
ADDITIVE EFFECT pg/lOO ml
IJg/ 100 ml
• o.
IJ.
,oo.
i
.
1
0
~
20
i
s
cl s C11o d2o
i
S
d2 o
Cho
cl~
i
s
i
cl2. 5
cl2.5 d2o cl.d dzo
s
cl.d
Fig. 2. Plasma corticosterone levels (mean±S.E.M.) in rats. Left: following daily treatment for one week with clorgyline (cl) or deprenil (d), and sampling 8 h after the last injection; right: 1 h after a single injection of clorgyline and/or deprenil, i--intact; s--saline; 2.5, 5, 10, 20: doses used (mg/kg). Inside columns: number of animals per group. Statistics: ANOVA: P<0.001. SNK test: the results are given below the diagrams. Under the lines, the treatment groups are arrayed in order of magnitude of their means, from smaller (left) to higher (right). Each line represents a non-significant subset of means (a=0.05). In other words, two means are significantly different when they are not placed below the same line.
353 TABLE I Interaction of a-MT (300 mg/kg, 9 h before sacrifice) and PCPA (300 mg/kg, 24 h before sacrifice) with the differential effects of two doses of clorgyline (CL, 2.5 and 5 mg/kg, 1 h before sacrifice) on morning corticosterone levels. Data are means-+S.E.M. ( # g / m g adrenal protein × l02, or #g/100 ml of plasma), followed by the number of animals per group, in parentheses. Statistical analysis: one-way ANOVA followed by the identification of the different sources of variation, using the 'contrasts' a priori procedure. Control
a-MT
PCPA
4.4--+0.32 (7) 5.8-+0.96 (9) 34.2-+ 1.96 (7)
13.7-+ 2.12 (10) 10.6-+ 0.92 (7) 44.4-+ 5.23 (7)
16.3-+2.97 (10) 21.9--+2.64 (10) 23.7-+2.63 (10)
8.0-+ 1.53 (7) 15.3 -+ 3.59 (9) 69.4--+3.75 (7)
52.2-+ 10.1 (10) 58.4-+ 5.30 (7) 94.9-+ 10.1 (7)
31.2-+5.52 (10) 40.2-+ 5.58 (10) 39.8-+4.31 (10)
Adrenal
Saline Clorgyline 2.5 m g / k g Clorgyline 5 m g / k g Plasma
Saline Clorgyline 2.5 m g / k g Clorgyline 5 m g / k g
Source of variation
Plasma
Adrenal
Among groups Control vs. a-MT and PCPA Among a-MT and PCPA Saline vs. clorgyline Control CL2. 5 vs. CL 5 a-MT alone vs. a - M T + C L a-MT [ a-MT CL2. 5 vs. a - M T + C L 5 PCPA alone vs. P C P A + C L PCPA [ PCPA+CL2. 5 vs. P C P A + C L s
P<0.001 P<0.001 P<0.002 P<0.001 P<0.001 P<0.01 n.s. n.s. n.s.
P<0.001 P<0.001 n.s. P<0.001 P<0.001 P<0.02 P<0.001 P<0.05 n.s.
more marked in plasma. Clorgyline 2.5 m g / k g did not modify this response, but the 5 m g / k g dose induced a very large increase, in both plasma and adrenal corticosterone. PCPA injection resulted in some enhancement of the steroid levels. The simultaneous administration of clorgyline (2.5 or 5 m g / k g ) did not change the PCPA effect at any of the two doses used.
3.5. Control of drug efficiency: MA 0 inhibition levels After clorgyline treatment, MAO inhibition was more pronounced in the adrenal than in brain. In the adrenal, about 90% inhibition was obtained with 2.5 or 5 mgykg doses, 1 or 8 h after injection, with tryptamine or kynuramine as substrates (common or MAO-B specific, respectively). On the other hand, the expected specificity was observed in the brain: 70-76% inhibition with tryptamine, and 32-52% with kynuramine, 1 or 8 h after 5
m g / k g injection. Only a 10% difference was observed between the 2.5 and 5 m g / k g doses. The inhibitory effect of deprenil increased markedly with dose and time after injection. In the adrenal, 1 h after 20 mg/kg, 40 and 15% inhibition was observed with common or MAO-B substrates, respectively. Using 40 mg/kg, the same degree of inhibition was obtained with both substrates: 57 and 90%, 1 and 8 h after injection respectively. In brain, I h after injection of 20 mg/kg, 8 and 77% inhibition was found using tryptamine and kynuramine as substrates. With 40 mg/kg, 45-70% (common) and 83-88% (MAO-B) inhibition was noticed, 1 or 8 h respectively, after treatment. The simultaneous injection of clorgyline (2.5 mg/kg) and deprenil (20 mg/kg) induced a nearly maximal inhibition of MAO activity with both substrates in adrenal as well as in brain, a-MT treatment did not modify substantially MAO activity in either control or clorgyline-injected
354 animals. No MAO assays were performed in chronic IMAO or PCPA experiments.
4. Discussion
In the first set of experiments, higher plasma corticosterone levels were found in saline-injected than in intact animals 1 h after injection. This allowed us to elicit two different effects of clorgyline, depending on the dose used: (a) small doses (1 and 2.5 mg/kg) appeared to inhibit the response of the adrenal cortex to the mild stress of i.p. injection; (b) higher doses (5, 10, 20 mg/kg) elicited a strong increase in adrenal as well as plasma corticosterone. In spite of the large difference in the adrenal response to 2.5 and 5 m g / k g of clorgyline, as little as 10% change in total MAO activity was observed in brain. Since clorgyline is a specific inhibitor of MAO-A, this difference might be related to changes in MAO-A activity. Both effects of clorgyline will be now discussed separately. The puzzling results reviewed by De Wied and De Jong (1974), concerning the stress-blocking ability of some classical IMAO, could be explained in part by our results: clorgyline (IMAOA), but not deprenil (IMAO-B), revealed itself active in this regard. This might imply that the stress response depends on MAO-A substrates, (namely, N E and 5-HT, according to Youdim and Holzbauer (1976)). Ganong (1980) has proposed a central a-adrenergic inhibitory control for the adrenocortical response to stress. The bulk of the data published have shown no stimulatory effect of the IMAO on the adrenal cortex (De Wied and De Jong, 1974). According to the present results, the response appears to depend on three interrelated factors: (a) dose of drug, (b) interval after administration, (c) specificity in MAO inhibition. One hour after their injection, the doses of clorgyline able to raise corticosterone values were relatively high, and thus involved deep inhibition of MAO-A. Moreover, clorgyline appeared more effective than deprenil, in spite of the fact that Yang and Neff (1974) found similar dose-response curves for MAO-A and MAO-B inhibition, respec-
tively. Conversely, 8h after injection, deprenil maintained the increased corticosterone levels more efficiently than clorgyline, while it strongly inhibited MAO-B throughout the period studied. In fact, dose-dependent increases in corticosterone levels were observed at that time with the higher doses of clorgyline. Since the MAO-A was, in all likelihood, already inhibited only MAO-B inhibition could be expected to increase further at these doses. On this basis, two components can be described for the response of the adrenal cortex to specific IMAO: (a) a large and sharp increase in corticosterone levels, related to MAO-A inhibition, and (b) a moderate and sustained effect, related to MAO-B inhibition. In rat brain, N E and 5-HT are preferred sustrates for MAO-A and phenylethylamine for MAO-B, whilst DA is a common substrate (Youdim and Holzbauer, 1976; Fowler et al. 1981). Therefore, clorgyline increases brain N E and 5-HT in a selective manner (Christmas et al. 1972; Yang and Neff, 1974). In the second part of the study, an attempt was made to identify the role of these neurotransmitters in the stimulatory effect of clorgyline on the adrenal cortex. Moderate increases in corticosterone levels were found after inhibition of N E synthesis by c~-MT, or of 5-HT s3Jnthesis by PCPA, as already described (Buckingham, 1980; Fuller, 1981). In order to discuss the interaction of clorgyline with ~-MT and PCPA, the differential effect of low and high doses of the drug on corticosterone levels should be kept in mind. When the levels of N E are lowered by a-MT, the differential effect of clorgyline is maintained, particularly in the adrenal. The stimulatory effect of the higher dose is even potentiated, to a certain extent, since corticosterone reaches the maximal levels found in this study. On the contrary, the difference between both doses of clorgyline is abolished when 5-HT levels are decreased by PCPA. Consequently, the stimulatory effect of high doses of clorgyline on the adrenal cortex most likely involves serotonergic mechanisms. Moreover, after clorgyline treatment, brain 5-HT levels increase to a higher extent than do NE levels (Yang and Neff, 1974). Finally recent pharmacological studies point to a stimulatory role
355 of 5-HT in the control of adrenocortical function ( F u l l e r , 1981). As an alternative explanation, a direct effect of c l o r g y l i n e o r d e p r e n i l , e.g. o n s t e r o i d o g e n e s i s , as shown for tranylcypromine (Lorenzen and G a n o n g , 1967), c a n n o t b e r u l e d o u t i n t h e a b s e n c e of in vitro studies. Nevertheless, the results obtained with PCPA plus clorgyline, or deprenil plus clorgyline, are evidence against this possibility. The present work was aimed at providing an insight into the pharmacological effects of IMAO on adrenal function, using the specific inhibitors, clorgyline and deprenil. Thus, a detailed discussion on adrenocortical controlling mechanisms was e x c l u d e d (see W e i n e r a n d G a n o n g , 1978; M e i t e s a n d S o n n t a g , 1981).
Acknowledgements The author is greatly indebted to Dr. Kordon for his valuable comments as well as to Mr. Caudron (Fortran Program), Dr. Gardey (preparation of the manuscript) and Miss Thiroux (expert secretarial assistance). M.A. Ventura was a post-doctoral fellow of the French Health Ministry and the Spanish Education and Science Ministry.
References Ader, R., 1970, The effects of early experience on the adrenocortical response to different magnitudes of stimulation, Physiol. Behav. 5, 837. Aud'rian, M., G. Beraud, G. Lescoat, J. Eliot and J. Maniey, 1978, Influence du cycle oestrien, de l'ovariectomie et de la lactation sur les variations de la corticost&on6mie basale ou aprb,s agression chez la ratte, C.R. Soc. Biol. 172, 33. Buckingham, J.C., 1980, Corticotrophin releasing factor, Pharmacol. Rev. 31,253. Christmas, A.J., C.J. Coulson, D.R. Maxwell and D. Riddell, 1972, A comparison of the pharmacological and biochemical properties of substrate selective monoamine oxidase inhibitors, Br. J. Pharmacol. 45, 490. De Moor, P., O. Steeno, M. Raskin and A. Hendrikx, 1960, Fluorometric determination of free plasma l l-hydroxycorticosteroids in man, Acta Endocrinol. 33, 297. De Wied, D. and W, De Jong, 1974, Drug effects and hypothalamic-anterior pituitary function, Ann. Rev. Pharmacol. 14, 389. Fowler, C.J., L. Oreland and B.A. Callingham, 1981, The acetylenic monoamine oxidase inhibitors clorgyline, depre-
nil, pargyline and J-508: their properties and applications, J. Pharm. Pharmacol. 33, 341. Fuller, R.W., 1981, Serotonergic stimulation of pituitary adrenocortical function in rats, Neuroendocrinol. 32, 118. Ganong, W.F., 1980, Participation of brain monoamines in the regulation of neuroendocrine activity under stress, in: Catecholamines and stress: Recent Advances, eds. E. Usdin, R. Kvetnansky and I.J. Kopin (Elsevier North Holland, New York) p. 115. Johnston, J.P., 1968, Some observations on a new inhibitor of MAO in brain tissue, Biochem. Pharmacol. 17, 1285. Keim, K.L., and E.B. Sigg, 1977, Plasma corticosterone and brain catecholamines in stress: effect of psychotropic drugs, Pharmacol. Biochem. Behav. 6, 79. Knoll, J., Z. Ecseri, K. Kelemen, J. Nievel and B. Knoll, 1965, Phenylisopropylmethylpropinylamine (E-250), a new spectrum psychic energizer, Arch. Int. Pharmacodyn. 155, 154. Kraml, M., 1965, A rapid microfluorometric determination of monoamine oxidase, Biochem. Pharmacol. 14, 1683. Le Mevel, J.C., S. Abitbol, G. Beraud and J. Maniey, 1979, Temporal changes in plasma adrenocorticotropin concentration after repeated neurotropic stress in male and female rats, Endocrinology 105, 812. Lorenzen, L.C. and W.F. Ganong, 1967, Effect of drugs related to a-ethyl-tryptamine on stress-induced ACTH secretion in the dog, Endocrinology 80, 889. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Meites, J. and W.E. Sonntag, 1981, Hypothalamic hypophysiotropic hormones and neurotransmitter regulation: Current views, Ann. Rev. Pharmacol. Toxicol. 21,295. Parvez, H. and S. Parvez, 1973, Micro radioisotopic determination of enzymes catechol-O-methyl-transferase, phenylethanolamine-N-methyl transferase and monoamine oxidase in a single concentration of tissue homogenate, Clin.. Chim. Acta 46, 85. Sokal, R.R. and F.J. Rohlf, i969, Biometry, San Francisco: Freeman W.H. and Co. Ventura, M.A., H. Parvez, S. Parvez and M.B.H. Youdim, 1980, Mechanism of adrenocortical stimulation by specific MAO-A inhibitors, in: Catecholamines and Stress: Recent advances, eds. E. Usdin, R. Kvetnansky and I.J. Kopin (Elsevier North Holland, New York) p. 359. Weiner, R.I. and W.F. Ganong, 1978, Role of brain monoamines and histamine in regulation of anterior pituitary secretion, Physiol. Rev. 58, 905. Wurtman, R.J. and J. Axelrod, 1963, A sensitive and specific assay for the estimation of monoamine oxidase, Biochem. Pharmacol. 12, 1439. Yang, H.Y.T. and N.H. Neff, 1974, The monoamine oxidase of brain: selective inhibition with drugs and the consequences for the metabolism of the biogenic amines, J. Pharmacol. Exp. Ther. 189, 733. Youdim, M.B.H. and M. Holzbauer, 1976, Physiological and pathological changes in tissue monoamine oxidase activity, J. Neural Transm. 38, 193.
349
Elsevier Biomedical Press
E F F E C T S O F CLORGYLINE AND D E P R E N I L ON C O R T I C O S T E R O N E LEVELS IN RATS * M. ANGELES VENTURA **
Unitb de Neuropharmacologie, B~t 440, Universitb Paris XI, 91405 Orsay Cedex, France Received 25 September 1981, revised MS received 12 February 1982, accepted 19 April 1982
M.A. VENTURA, Effects of clorgyline and deprenil on corticosterone levels in rats, European J. Pharmacol. 81 (1982) 349-355. Adrenal and plasma corticosterone levels were measured in adult female rats after i.p. administration of specific MAO inhibitors: clorgyline (IMAO-A) and deprenil (IMAO-B). Brain and adrenal MAO activity was also determined. The IMAO effect was found to depend on 3 interrelated factors: (a) dose of drug, (b) time after injection, (c) specificity in MAO inhibition. One hour after injection, clorgyline (1 and 2.5 mg/kg ) but not deprenil (20 mg/kg), appeared to inhibit the adrenocortical stress response to i.p. injection. At higher doses, MAO-B inhibition by deprenil (40 mg/kg) induced a moderate but sustained increase in corticosterone levels, while MAO-A inhibition by clorgyline (5, 10, 20 mg/kg) resulted in a large and sharp rise. This effect of clorgyline was potentiated with a-methyl-p-tyrosine and blocked with p-chlorophenylalanine. On this basis, the involvement of serotonergic mechanisms could be considered to explain the stimulatory effect of clorgyline on adrenal cortical function. Rat corticosterone levels
Clorgyline
Deprenil
p-Chlorophenylalanine
1. Introduction
The pharmacological approach has been widely used to study the interactions between central nervous system and endocrine glands (De Wied and De Jong, 1974; Buckingham, 1980). As far as the hypothalamo-hypophyseal-adrenal axis is concerned, the results obtained with monoamine oxidase inhibitors (IMAO) are controversial (De Wied and De Jong, 1974; Keim and Sigg, 1977). Differences in drugs and animal species used could account for some of the discrepancies, as could the fact that several aspects of the adrenal function can be affected: i.e. direct stimulation, response to stress, circadian rhythm. On the other hand, the role of the different * Part of this investigation was presented to the Second International Symposium on Catecholamines and Stress, Smolenice Castle, Czechoslovakia, 1979 (Ventura et al., 1980). ** Present address: Pharmacologic Biochimique, CHU Cochin-Port Royal, 24 rue du Fg St Jacques, F-75014 Paris, France. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
a-Methyl-p-tyrosine
neurotransmitters in the control of adrenocortical function is not clearly established (See Weiner and Ganong, 1978; Meites and Sonntag, 1981, for review). There is some agreement about the inhibitory action of norepinephrine (NE), whereas epinephrine (E) and dopamine (DA) do not seem involved. Evidence has been obtained for both an inhibitory and excitatory effect of serotonin (5HT), but pharmacological studies particularly support the latter hypothesis (Fuller, 1981). Histamine seems to exert some stimulatory activity. The IMAO used up to now in such studies (nialamine, iproniazid, pargyline, etc...) do not act with the same degree of efficiency upon the two forms of MAO and consequently, the levels of the different neurotransmitters are not affected to the same extent (Christmas et al., 1972): this could explain some of the discrepancies observed. In this context, we studied the response of the adrenal cortex to the specific inhibitors, clorgyline (CI: MAO-A) (Johnston, 1968) and deprenil (D: MAO-B) (Knoll et al., 1965) by measuring corticosterone levels in plasma and adrenals. The re-
350 suits showed that clorgyline drastically increased both parameters. Subsequently, we studied the effect of a-methyl-p-tyrosine (a-MT) and p-chlorophenylalanine (PCPA), well known to block the synthesis of catecholamines and serotonin respectively, on the adrenocortical stimulation induced by clorgyline.
2. Materials and methods
2.1. Drugs The following drugs were used: clorgyline and (RS)-deprenil hydrochloride (May and Baker), D,L-a-methyl-p-tyrosine methyl ester hydrochloride (Regis and Co), D,L-p-chlorophenylalanine methyl ester hydrochloride (Labkemi AB). The drugs were dissolved in cold saline immediately before each experiment and administered by intraperitoneal (i.p.) injection (1 ml/kg).
2.3. Experimental schedule In the first part of the study, three series of experiments were carried out: (a) a single injection of 1, 5, 10, 20 m g / k g of clorgyline, or 20, 40 m g / k g of deprenil, 1 or 8 h before sacrifice; (b) in order to test the effects of chronic treatment, some animals were given 5 or 10 m g / k g of clorgyline, or 20 m g / k g of deprenil, daily for 1 week, and killed 8 h after the last injection; (c) the additive effect of clorgyline (2.5 mg/kg) and deprenil (20 m g / k g ) was tested 1 h after injection. In the second part of the study, a-MT (250 mg/kg, 9 h before sacrifice) and PCPA (300 mg/kg, 24 h before sacrifice) were used, alone or associated with two doses of clorgyline (2.5 or 5 mg/kg, 1 h before sacrifice). In each series, a control group (s) was injected with saline (1 ml/kg) under the same conditions as drug-injected groups. These controls were used for statistical comparisons. In addition, basal levels in intact animals (i) are given for reference.
2.2. Animals 2.4. Assay procedures Adult Sherman female rats bred in our laboratory (thus easily available) weighing 250-300g were used. The phase of the estrous cycle was not verified. According to Audrian et al. (1978), intact Sherman rats killed at random between 8 and 9 h 30 a.m. are distributed as follows: proestrus (P), 12%; estrus (E), 35%; meta + diestrus (M + D), 53%. The corresponding corticosterone levels reach 1 8 ± 2 (P), 1 0 ± 0 . 4 (E) and 6 ± 0 . 2 ( M + D ) /zg/100 ml, respectively. In our intact groups, the mean was 8.6 ± 1.2, intermediate among E and M + D which account for 88% of the cases. On this basis, we found it not essential to verify the estrous cycle phase since this would have been (a) stressful if done in vivo, (b) time- and drug-consuming if done 'post-mortem'. The experiments were carried out during spring and summer. The animals were kept under natural d a y / n i g h t cycle, at a constant temperature (21 ± I°C) with tap water and commercial food ad libitum. The animals were weighed and put in individual cages at 5 p.m. the day before sacrifice. They were killed by cervical dislocation between 9 and 11 a.m., in all the experiments.
After sacrifice 2 ml of blood were collected by aortic puncture. The adrenals and brain were removed quickly and kept in ice-cold 0.9% KC1 or 4% TCA (trichloroacetic acid), for enzyme assay (brain and adrenal) and for corticosterone (adrenal) measurement respectively, until processing of the samples the same day. Corticosterone in adrenals and plasma was measured fluorometrically according to De Moor et al. (1962). Total MAO activity was determined using tryptamine [14C]bisuccinate (side chain label) as substrate (Wurtman and Axelrod, 1963; Parvez and Parvez, 1973) while kynuramine was used to estimate form B activity (Kraml, 1965). Tissue protein content was determined according to Lowry et al. (1951).
2.5. Statistical analysis The statistical analysis of the data, according to procedures described in Sokal and Rohlf (1969), included: (a) basic computations (~, S.E.M., s 2, etc.); (b) F-max test for homogeneity of variances; (c) Log transformation, if required; (d) analysis of
351 variance ( A N O V A ) , one or two-way, model 1, unequal sample size; (e) when the overall A N O V A was significant, two types of comparisons a m o n g means were performed:
2.5.1. A posteriori Student-Newman-Keuls (SNK) test for the first part of the study (i.e. experiments using different concentrations of the same drug).
2.5.2. A priori Comparisons by the 'contrasts' method for the second part (i.e. experiment including several drugs). The S N K test was performed on the basis of a level of significance ( a ) -- 0.05. It provides the 'upper' limit of significance, but not 'true' a values. On the other hand, the 'contrasts' a priori m e t h o d provides 'true' a values. 'Basal' (i) and 'control' (s) levels were c o m p a r e d using Student's t-test for unequal sample size.
3. Results The experiments were carried out in two different environments due to the transfer of the laboratory to another building. During the first part of the study, the animals were reared in the general animal room, whereas in the second part, they were kept in a special room, and thus subjected to less disturbance. In the first set of experiments, but not in the second, plasma corticosterone levels were found higher in saline-injected than in intact animals, 1 h after injection. This fact was p r o b a b l y related to the differences in experimental conditions. The sensitivity of the adrenal cortex to all kind of stimuli, particularly in the female (Le Mevel et al., 1979) is well established, as well as the role of previous history of exposure to stimulation (Ader, 1970).
pg / 100 ml SINGLE
I
10080-
60-
TREATMENT
TI 40-
20-
16191al7171 i
s 1 51020
S l 51o2o
lh
S2o*O'
8h
lh
8h
i
clorgyline
S2o*O t
deprenil
Fig. I. Plasma corticosterone levels (mean--+S.E.M.) in rats, 1 or 8 h after a single treatment with clorgyline or deprenil (hatched or lined columns respectively), i = intact; s--saline; l, 5, 10, 20, 40: doses used (mg/kg). Inside columns: number of animals per group. Statistics: ANOVA (clorgyline): overall, time and dose factors and interaction, P<0.001. ANOVA (deprenil): overall, time and dose factors, P<0.001; interaction, n.s. SNK test: * means groups significantly different from their controls (open columns), at least for a=0.05.
352
3.1. Effect of single doses of clorgyline and deprenil (fig. 1)
3.2. Effect of chronic treatment with clorgyline and deprenil (fig. 2)
Saline-injected a n i m a l s (s) p r e s e n t e d p l a s m a c o r t i c o s t e r o n e levels significantly higher than d i d i n t a c t (i) a n i m a l s 1 h ( b u t n o t 8 h) after injection. N o difference was f o u n d in a d r e n a l levels (not shown). Plasma and adrenal corticosterone concentrations were lower than the c o n t r o l s 1 h after 1 m g / m l of clorgyline. Conversely, the o t h e r doses o f the d r u g (5, 10, 20 m g / k g ) i n d u c e d strong increases in c o r t i c o s t e r o n e levels, within the s a m e range. Eight hours after injection, the values were n o t significantly different from the controls, exc e p t in p l a s m a for the 10 a n d 20 m g / k g doses. T h e lower d o s e (20 m g / k g ) of d e p r e n i l h a d no effect, whereas the higher (40 m g / k g ) i n d u c e d a m o d e r a t e b u t s u s t a i n e d increase in p l a s m a a n d a d r e n a l ( n o t shown) c o r t i c o s t e r o n e levels, for at least 8 h.
D a i l y injection for 1 week with 5 or 10 m g / k g of clorgyline or 20 m g / k g of deprenil, resulted in a m o d e r a t e increase in p l a s m a a n d a d r e n a l (not shown) corticosterone.
CHRONIC TREATMENT
3.3. Additive effect of clorgyline and deprenil (fig. 2) Doses of clorgyline (2.5 m g / k g ) a n d d e p r e n i l (20 m g / k g ) ineffective b y themselves i n d u c e d a m a r k e d increase in c o r t i c o s t e r o n e levels in b o t h p l a s m a a n d a d r e n a l ( n o t shown) when a d m i n i s tered together.
3. 4. Interaction of ot-MT and PCPA with clorgyline (table 1) T h e a d m i n i s t r a t i o n of a - M T evoked a rise in c o r t i c o s t e r o n e levels, m o d e r a t e in the a d r e n a l a n d
ADDITIVE EFFECT pg/lOO ml
IJg/ 100 ml
• o.
IJ.
,oo.
i
.
1
0
~
20
i
s
cl s C11o d2o
i
S
d2 o
Cho
cl~
i
s
i
cl2. 5
cl2.5 d2o cl.d dzo
s
cl.d
Fig. 2. Plasma corticosterone levels (mean±S.E.M.) in rats. Left: following daily treatment for one week with clorgyline (cl) or deprenil (d), and sampling 8 h after the last injection; right: 1 h after a single injection of clorgyline and/or deprenil, i--intact; s--saline; 2.5, 5, 10, 20: doses used (mg/kg). Inside columns: number of animals per group. Statistics: ANOVA: P<0.001. SNK test: the results are given below the diagrams. Under the lines, the treatment groups are arrayed in order of magnitude of their means, from smaller (left) to higher (right). Each line represents a non-significant subset of means (a=0.05). In other words, two means are significantly different when they are not placed below the same line.
353 TABLE I Interaction of a-MT (300 mg/kg, 9 h before sacrifice) and PCPA (300 mg/kg, 24 h before sacrifice) with the differential effects of two doses of clorgyline (CL, 2.5 and 5 mg/kg, 1 h before sacrifice) on morning corticosterone levels. Data are means-+S.E.M. ( # g / m g adrenal protein × l02, or #g/100 ml of plasma), followed by the number of animals per group, in parentheses. Statistical analysis: one-way ANOVA followed by the identification of the different sources of variation, using the 'contrasts' a priori procedure. Control
a-MT
PCPA
4.4--+0.32 (7) 5.8-+0.96 (9) 34.2-+ 1.96 (7)
13.7-+ 2.12 (10) 10.6-+ 0.92 (7) 44.4-+ 5.23 (7)
16.3-+2.97 (10) 21.9--+2.64 (10) 23.7-+2.63 (10)
8.0-+ 1.53 (7) 15.3 -+ 3.59 (9) 69.4--+3.75 (7)
52.2-+ 10.1 (10) 58.4-+ 5.30 (7) 94.9-+ 10.1 (7)
31.2-+5.52 (10) 40.2-+ 5.58 (10) 39.8-+4.31 (10)
Adrenal
Saline Clorgyline 2.5 m g / k g Clorgyline 5 m g / k g Plasma
Saline Clorgyline 2.5 m g / k g Clorgyline 5 m g / k g
Source of variation
Plasma
Adrenal
Among groups Control vs. a-MT and PCPA Among a-MT and PCPA Saline vs. clorgyline Control CL2. 5 vs. CL 5 a-MT alone vs. a - M T + C L a-MT [ a-MT CL2. 5 vs. a - M T + C L 5 PCPA alone vs. P C P A + C L PCPA [ PCPA+CL2. 5 vs. P C P A + C L s
P<0.001 P<0.001 P<0.002 P<0.001 P<0.001 P<0.01 n.s. n.s. n.s.
P<0.001 P<0.001 n.s. P<0.001 P<0.001 P<0.02 P<0.001 P<0.05 n.s.
more marked in plasma. Clorgyline 2.5 m g / k g did not modify this response, but the 5 m g / k g dose induced a very large increase, in both plasma and adrenal corticosterone. PCPA injection resulted in some enhancement of the steroid levels. The simultaneous administration of clorgyline (2.5 or 5 m g / k g ) did not change the PCPA effect at any of the two doses used.
3.5. Control of drug efficiency: MA 0 inhibition levels After clorgyline treatment, MAO inhibition was more pronounced in the adrenal than in brain. In the adrenal, about 90% inhibition was obtained with 2.5 or 5 mgykg doses, 1 or 8 h after injection, with tryptamine or kynuramine as substrates (common or MAO-B specific, respectively). On the other hand, the expected specificity was observed in the brain: 70-76% inhibition with tryptamine, and 32-52% with kynuramine, 1 or 8 h after 5
m g / k g injection. Only a 10% difference was observed between the 2.5 and 5 m g / k g doses. The inhibitory effect of deprenil increased markedly with dose and time after injection. In the adrenal, 1 h after 20 mg/kg, 40 and 15% inhibition was observed with common or MAO-B substrates, respectively. Using 40 mg/kg, the same degree of inhibition was obtained with both substrates: 57 and 90%, 1 and 8 h after injection respectively. In brain, I h after injection of 20 mg/kg, 8 and 77% inhibition was found using tryptamine and kynuramine as substrates. With 40 mg/kg, 45-70% (common) and 83-88% (MAO-B) inhibition was noticed, 1 or 8 h respectively, after treatment. The simultaneous injection of clorgyline (2.5 mg/kg) and deprenil (20 mg/kg) induced a nearly maximal inhibition of MAO activity with both substrates in adrenal as well as in brain, a-MT treatment did not modify substantially MAO activity in either control or clorgyline-injected
354 animals. No MAO assays were performed in chronic IMAO or PCPA experiments.
4. Discussion
In the first set of experiments, higher plasma corticosterone levels were found in saline-injected than in intact animals 1 h after injection. This allowed us to elicit two different effects of clorgyline, depending on the dose used: (a) small doses (1 and 2.5 mg/kg) appeared to inhibit the response of the adrenal cortex to the mild stress of i.p. injection; (b) higher doses (5, 10, 20 mg/kg) elicited a strong increase in adrenal as well as plasma corticosterone. In spite of the large difference in the adrenal response to 2.5 and 5 m g / k g of clorgyline, as little as 10% change in total MAO activity was observed in brain. Since clorgyline is a specific inhibitor of MAO-A, this difference might be related to changes in MAO-A activity. Both effects of clorgyline will be now discussed separately. The puzzling results reviewed by De Wied and De Jong (1974), concerning the stress-blocking ability of some classical IMAO, could be explained in part by our results: clorgyline (IMAOA), but not deprenil (IMAO-B), revealed itself active in this regard. This might imply that the stress response depends on MAO-A substrates, (namely, N E and 5-HT, according to Youdim and Holzbauer (1976)). Ganong (1980) has proposed a central a-adrenergic inhibitory control for the adrenocortical response to stress. The bulk of the data published have shown no stimulatory effect of the IMAO on the adrenal cortex (De Wied and De Jong, 1974). According to the present results, the response appears to depend on three interrelated factors: (a) dose of drug, (b) interval after administration, (c) specificity in MAO inhibition. One hour after their injection, the doses of clorgyline able to raise corticosterone values were relatively high, and thus involved deep inhibition of MAO-A. Moreover, clorgyline appeared more effective than deprenil, in spite of the fact that Yang and Neff (1974) found similar dose-response curves for MAO-A and MAO-B inhibition, respec-
tively. Conversely, 8h after injection, deprenil maintained the increased corticosterone levels more efficiently than clorgyline, while it strongly inhibited MAO-B throughout the period studied. In fact, dose-dependent increases in corticosterone levels were observed at that time with the higher doses of clorgyline. Since the MAO-A was, in all likelihood, already inhibited only MAO-B inhibition could be expected to increase further at these doses. On this basis, two components can be described for the response of the adrenal cortex to specific IMAO: (a) a large and sharp increase in corticosterone levels, related to MAO-A inhibition, and (b) a moderate and sustained effect, related to MAO-B inhibition. In rat brain, N E and 5-HT are preferred sustrates for MAO-A and phenylethylamine for MAO-B, whilst DA is a common substrate (Youdim and Holzbauer, 1976; Fowler et al. 1981). Therefore, clorgyline increases brain N E and 5-HT in a selective manner (Christmas et al. 1972; Yang and Neff, 1974). In the second part of the study, an attempt was made to identify the role of these neurotransmitters in the stimulatory effect of clorgyline on the adrenal cortex. Moderate increases in corticosterone levels were found after inhibition of N E synthesis by c~-MT, or of 5-HT s3Jnthesis by PCPA, as already described (Buckingham, 1980; Fuller, 1981). In order to discuss the interaction of clorgyline with ~-MT and PCPA, the differential effect of low and high doses of the drug on corticosterone levels should be kept in mind. When the levels of N E are lowered by a-MT, the differential effect of clorgyline is maintained, particularly in the adrenal. The stimulatory effect of the higher dose is even potentiated, to a certain extent, since corticosterone reaches the maximal levels found in this study. On the contrary, the difference between both doses of clorgyline is abolished when 5-HT levels are decreased by PCPA. Consequently, the stimulatory effect of high doses of clorgyline on the adrenal cortex most likely involves serotonergic mechanisms. Moreover, after clorgyline treatment, brain 5-HT levels increase to a higher extent than do NE levels (Yang and Neff, 1974). Finally recent pharmacological studies point to a stimulatory role
355 of 5-HT in the control of adrenocortical function ( F u l l e r , 1981). As an alternative explanation, a direct effect of c l o r g y l i n e o r d e p r e n i l , e.g. o n s t e r o i d o g e n e s i s , as shown for tranylcypromine (Lorenzen and G a n o n g , 1967), c a n n o t b e r u l e d o u t i n t h e a b s e n c e of in vitro studies. Nevertheless, the results obtained with PCPA plus clorgyline, or deprenil plus clorgyline, are evidence against this possibility. The present work was aimed at providing an insight into the pharmacological effects of IMAO on adrenal function, using the specific inhibitors, clorgyline and deprenil. Thus, a detailed discussion on adrenocortical controlling mechanisms was e x c l u d e d (see W e i n e r a n d G a n o n g , 1978; M e i t e s a n d S o n n t a g , 1981).
Acknowledgements The author is greatly indebted to Dr. Kordon for his valuable comments as well as to Mr. Caudron (Fortran Program), Dr. Gardey (preparation of the manuscript) and Miss Thiroux (expert secretarial assistance). M.A. Ventura was a post-doctoral fellow of the French Health Ministry and the Spanish Education and Science Ministry.
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