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Tetrabenazine-induced depletion of brain monoamines: Mechanism by which desmethylimipramine protects cortical norepinephrine
European Journal of Pharmacology, 102 (1984) 431-436
431
Elsevier
T E T R A B E N A Z I N E - I N D U C E D D E P L E T I O N OF BRAIN M O N O A M I N E S : M E C H A N I S M BY W H I C H D E S M E T H Y L I M I P R A M I N E P R O T E C T S CORTICAL N O R E P I N E P H R I N E D O U G L A S J. PETTIBONE *, A. B A R B A R A P F L U E G E R and JAMES A. T O T A R O
Department of Pharmacology, Merck Institute for Therapeutic Research, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486, U.S.A. Received 6 December 1983, revised MS received 28 February 1984, accepted 3 April 1984
D.J. PETTIBONE, A.B. PFLUEGER and J.A. TOTARO, Tetrabenazine-induced depletion of brain monoamines: mechanism by which desmethylimipramine protects cortical norepinephrine, European J. Pharmacol. 102 (1984) 431-436. Pretreatment of rats with desmethylimipramine (DMI) (5 mg/kg) significantly antagonized the tetrabenazine (TBZ)-induced (3 mg/kg) depletion of cortical norepinephrine (NE) in rats. This protective effect of DMI was fully blocked by the specific a2-adrenergic antagonist, RX 781094. The doses of DMI which antagonized the depleting effects of TBZ caused rapid, maximal reductions in cortical dihydroxyphenylalanine (DOPA) accumulation, effects which were also blocked by RX 781094. Like DMI, the a2-adrenergic agonist, clonidine (50 gg/kg) decreased cortical DOPA accumulation and rapidly prevented the TBZ-evoked loss of NE in this brain region. On the other hand, doses of RX 781094 (0.5-2.0 mg/kg) which accelerated cortical DOPA synthesis, enhanced significantly the depleting effects of a threshold dose of TBZ (0.6 mg/kg). These findings suggest that DMI reduces the rate of TBZ-induced depletion of cortical NE by lowering NE turnover through the indirect stimulation of a2-adrenoceptors. Tetrabenazine
DMI
NE turnover
a2-Adrenergic receptors
1. Introduction
It is generally believed that the potent and selective norepinephrine (NE) uptake-inhibiting property of the antidepressant desmethylimipramine (DMI) is responsible for its behavioral antitetrabenazine (TBZ) activity in rodents. However, there are very few reports which provide information regarding the neurochemical interactions of TBZ and anti-TBZ agents, such as DMI. In an effort to gain a better understanding of the neurochemical events accompanying a n d / o r underlying TBZ antagonism, we examined the interaction effects of TBZ and DMI on brain regional monoamines and determined that DMI reduced the rate of N E depletion by TBZ but not that of dopamine (DA) or serotonin (5-HT) (Pettibone et al., 1984). The findings presented in this report suggest that the protective effects of DMI are due to decreased * To w h o m all correspondence should be addressed. 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
cortical N E turnover, most likely through the stimulation of an a2-adrenergic mechanism.
2. Materials and methods 2.1. Animals
Adult, male, Sprague-Dawley rats (150-200 g) having free access to standard laboratory rat chow and tap water were used in all experiments. All drugs were administered intraperitoneally except for TBZ which was injected subcutaneously. Animals were sacrificed by decapitation and fronto-parietal cortex was rapidly dissected and frozen on dry ice. 2.2. Assays
N E and DOPA were assayed by HPLC/electrochemical detection by the method of Hefti et al. (1980).
432
2.3. Drugs
TABLE 2 Effects of DMI on cortical NE synthesis a.
DMI HC1 (USV Pharmaceutical Corp., Tuckahoe, NY), TBZ methane sulfonate (Hoffm a n n - L a R o c h e , Inc., N u t l e y , N J), a n d c l o n i d i n e HC1 ( B o e h r i n g e r - I n g e l h e i m Ltd., R i d g e f i e l d , C T ) w e r e g r a t e f u l l y r e c e i v e d as gifts. N S D 1015 ( m - h y d r o x y b e n z y l h y d r a z i n e ) was p u r c h a s e d f r o m A l d r i c h C h e m i c a l C o . ( M i l w a u k e e , W I ) . R X 781094 (2-(2,3-dihydro-l,4-benzodioxin-2-yl)-4,5-dihydro1 H - i m i d a z o l e H C I ) was s y n t h e s i z e d in t h e M e d i c i n a l C h e m i s t r y D e p a r t m e n t at t h e M e r c k S h a r p & D o h m e R e s e a r c h L a b o r a t o r i e s , W e s t P o i n t , PA.
2.4. Statistics D i f f e r e n c e s b e t w e e n g r o u p s ( P < 0.05) w e r e determined by ANOVA and Duncan's Multiple R a n g e test (2-tailed)i
3. Results
3.1. Blockade of the protective effects of DMI by R X 781094 T h e a d m i n i s t r a t i o n of D M I (5 m g / k g ) signific a n t l y a n t a g o n i z e d (by 72%) the d e p l e t i o n o f c o r t i cal N E i n d u c e d b y T B Z (3 m g / k g ; t a b l e 1). Pretreatment with the selective a2-adrenergic
TABLE 1 Effect of RX 781094 on DMl-induced protection of cortical NE depletion by TBZ a. Treatment
%change
(mg/kg)
Cortical NE (pmol/mg tissue)
Control DMI (5) TBZ (3) RX 781094 (2.5) DMI + TBZ RX 781094 + TBZ RX 781094+ DMI +TBZ
0.87 + 0.05 0.94 + 0.i0 0.48 + 0.04 b.¢ 0.90 + 0.05 0.76+0.05 0.41 + 0.06 b.¢ 0.41+0.06 b.¢
+8 -- 45 +3 - 13 -- 53 -53
a Rats received RX 781094 (i.p.) or its vehicle (H20) 30 rain before DMI (i.p.) or its vehicle (H20). Thirty rain later they received TBZ (s.c.) or its vehicle (H20) and were sacrificed 15 rain thereafter. Values are group means __S.E.M. (n ~ 4-5). b p < 0.05 vs. control group, c p < 0.05 vs. DMI + TBZ group.
Treatment (mg/kg i.p.)
Cortical DOPA (pmol/mg tissue)
~change
Control DMI (2.5) DMI (5.0) DMI (10.0) DMI (15.0)
0.94 + 0.06 0.63 + 0.03 b 0.58 + 0.04 b 0.58 + 0.03 b 0.61 + 0.04 b
-- 33 -- 38 -- 38 --35
a Rats were injected with DMI (i.p,) or its vehicle (H20) 30 min before NSD 1015 (125 mg/kg i.p.) and sacrificed 30 min thereafter. Values are group means + S.E.M. (n = 5). b p < 0.05 vs. control group (NSD 1015 alone).
a n t a g o n i s t R X 781094 (2.5 m g / k g ; D o x e y et al., 1983; D e t t m a r et al., 1983), h o w e v e r , fully p r e v e n t e d the p r o t e c t i v e effects o f D M I ( t a b l e 1).
3.2. Effects of DMI on cortical NE turnover T h e in v i v o r a t e o f c o r t i c a l N E s y n t h e s i s was estimated by measuring the rate of DOPA format i o n .after a r o m a t i c a m i n o a c i d d e c a r b o x y l a s e inh i b i t i o n ( A A A D ) b y N S D 1015 ( C a r l s s o n et al., 1972; R e i n h a r d a n d R o t h , 1982). D M I at 5 m g / k g m a x i m a l l y r e d u c e d ( b y 38%) t h e r a t e o f c o r t i c a l D O P A a c c u m u l a t i o n ( t a b l e 2). T h i s r e d u c t i o n in N E s y n t h e s i s was e v i d e n t w h e n D M I was a d m i n i s t e r e d as e a r l y as 15 r a i n b e f o r e N S D 1015 a n d l a s t e d at least a n o t h e r 90 m i n ( t a b l e 3). P r e t r e a t m e n t w i t h R X 781094 s i g n i f i c a n t l y a t t e n u a t e d the r e d u c t i o n in c o r t i c a l D O P A a c c u m u l a t i o n o b -
TABLE 3 Time-related effects of DMI on cortical NE synthesis a. Treatment (time before NSD 1015, rain)
Cortical DOPA (pmol/mg tissue)
%change
Vehicle (5) DMI (5) DMI (15) DMI (30) DMI (60) DMI (90)
0.52 + 0.05 0.45 _+0.03 0.38+0.03 b 0.36 + 0.01 b 0.39 + 0.03 b 0.35 +_0.01 b
- 13 --27 -- 31 -- 25 -- 33
a Rats were administered DMI (5 mg/kg i.p.) or its vehicle (H20) at the indicated times before NSD 1015 (125 mg/kg i.p.) and sacrificed 30 min thereafter. Values are group means + S.E.M. (n = 5). b p < 0.05 vs. vehicle-treated group.
433 TABLE 4
TABLE 5
Effects of R X 781094 o n D M I - i n d u c e d r e d u c t i o n in cortical N E synthesis a
Dose-related p r o t e c t i o n of T B Z - i n d u c e d d e p l e t i o n of cortical N E b y c l o n i d i n e a.
Treatment
Cortical DOPA ( p m o l / m g tissue)
%change
Treatment (mg/kg)
Control DMI R X 781094 R X 781094 + D M I
0.40 + 0.02 0.25 + 0.01 b 0.37+0.01 0.33 + 0.02 b,¢
-- 47 - 7 -- 17
Control Clonidine T B Z (3) Clonidine Clonidine Clonidine Clonidine
a R a t s were treated w i t h R X 781094 (250 # g / k g i.p.) or its vehicle ( H 2 0 ) 20 rain before D M I (3 m g / k g i.p.) or its vehicle ( H 2 0 ) . T h i r t y rain l a t e r they received N S D 1015 (125 m g / k g i.p.) a n d sacrificed 30 rain thereafter. Values are g r o u p m e a n s + S.E.M. (n = 5). b p < 0.05 vs. control g r o u p ( N S D 1015 alone), c p < 0.05 vs. D M I - t r e a t e d group.
served after DMI (table 4). This small dose of RX 781094 (250 #g/kg) did not influence NE synthesis by itself.
3.3. Blockade of TBZ-induced depletion of cortical NE by clonidine Similar to the effects of DMI, the administration of clonidine (250 #g/kg) as early as 20 min before TBZ inhibited the depletion of NE (fig. 1), an effect which lasted for at least 2 h. This protective effect of clonidine was observed with doses as
1.O E O
E Q.
/ 0.5
.......
"'
-
(0.025)+TBZ (0.05) + T B Z (0.10)+TBZ (0.25) + T B Z
%change
1.17 + 0.09 1.41 + 0.04 0.62 + 0.04 0.96 + 0 . 0 6 1.06 + 0.02 1.07+0.03 1.07 + 0.04
_ + 21 -- 47 -- 18 - 9 -9 - 9
c b,c b b,c c c c
a R a t s were injected w i t h c l o n i d i n e (i.p.) or its vehicle ( H 2 0 ) 40 rain before T B Z (s.c.) or its vehicle ( H 2 0 ) a n d sacrificed 15 rain thereafter. Values are group m e a n s + S.E.M. (n = 5). b p < 0.05 vs. control group, c p < 0.05 vs. T B Z a l o n e - t r e a t e d group.
low as 25 #g/kg i.p. (table 5). The doses of clonidine which fully blocked the TBZ-induced loss of NE, maximally reduced cortical DOPA accumulation by about 50% (table 6).
3.4. Enhancement of TBZ potency by R X 781094 If reducing NE turnover through a2-adrenergic stimulation reduces the effectiveness of TBZ to deplete cortical NE, then the administration of a2-adrenergic antagonists, which are known to augment NE turnover, might be expected to enhance the potency of TBZ. Pretreatment with RX 781094 significantly enhanced the rate of cortical
T TABLE 6
#
~.
•
UJ Z
•
0---0
Effects of c l o n l d i n e or R X 781094 o n cortical N E synthesis a
Control
TBZ-treated
6 t. o
I=t,. Veh 20
(0.25)
Cortical N E ( p m o l / m g tissue)
i
I 60
=
I 120
Clonidine Pretreatment (rain) Fig. 1. I n h i b i t i o n of T B Z - i n d u c e d d e p l e t i o n of cortical N E b y clonidine. R a t s were a d m i n i s t e r e d c l o n i d i n e (250 # g / k g i.p.) or its vehicle ( H 2 0 ) at the i n d i c a t e d times before T B Z (3 m g / k g s.c.) or its vehicle ( H 2 0 ) a n d sacrificed 15 rain later. Values are g r o u p m e a n s 5: S.E.M. (n = 5). * P < 0.05 vs. control, t p < 0.05 vs. T B Z alone.
Treatment (mg/kg)
Cortical D O P A ( p m o l / m g tissue)
C o n t r o l ( N S D 1015 alone) R X 781094 (0.5) R X 781094 (1.0) R X 781094 (2.0) C l o n i d i n e (0.05) C l o n i d i n e (0.10) C l o n i d i n e (0.25)
0.34 0.55 0.61 0.62 0.15 0.19 0.16
+ 0.03 + 0.04 + 0.04 + 0.04 + 0.01 -t- 0.01 + 0.02
b b b b b b
%change
+ 62 + 79 + 82 -- 56 - - 43 -- 53
a R a t s were a d m i n i s t e r e d R X 781094 (i.p.), c l o n i d i n e (i.p.) or H 2 0 (i.p.) 30 rain b e f o r e N S D 1015 (125 m g / k g i.p.) a n d sacrificed 30 m i n thereafter. Values are g r o u p m e a n s + S.E.M. (n = 5). b p < 0.05 vs. control group.
434 TABLE 7 E n h a n c e m e n t of the TBZ-induced depletion of cortical N E by the a2-adrenergic antagonist, RX 781094 a. Treatment (mg/kg) Control R X 781094 RX 781094 R X 781094 TBZ (0.6) R X 781094 R X 781094 RX 781094
(0.5) (1.0) (2.0) (0.5) + TBZ (1.0)+ TBZ (2.0)+TBZ
Cortical N E ( p m o l / m g tissue)
%change
0.88 4- 0.07 0.96 4- 0.09 0.96 4- 0.05 0.92 4- 0.07 0.75 4-0.08 0.69 4- 0.07 0.68 4- 0.03 b 0.504-0.04 b.c
+9 +9 +5 - 15 - 22 -- 23 --43
a Rats were treated with RX 781094 (i.p.) or its vehicle ( H 2 0 ) 45 min before TBZ (s.c.) or its vehicle ( H 2 0 ) and were sacrificed 15 rain thereafter. Values represent group means + S.E.M. (n = 5). b p < 0.05 vs. control group, c p < 0.05 vs. TBZ alone-treated group.
DOPA accumulation after NSD 1015 (table 6) and at doses (0.5-2.0 ng/kg) which did not influence cortical NE by themselves, dose-dependently potentiated the depletion of NE by a threshold dose of TBZ (0.6 mg/kg; table 7).
4. Discussion
In the previous report (Pettibone et al., 1984) we observed that the antidepressant, clorgyline, equally prevented the TBZ-induced depletion of brain NE, DA and 5-HT. Since these monoamines are known to be substrates of the form of monoamine oxidase (MAO) inhibited by clorgyline (Johnston, 1968; Neff et al., 1974; Yang and Neff, 1974) it is likely that the protective effects of clorgyline resulted from reduced deamination of these neurotransmitters after their release from granular storage by TBZ. The inhibition by DMI of NE but not DA or 5-HT depletion, however indicates a different mechanism accounting for the interference with the actions of TBZ, perhaps being related to the selective inhibition of NE uptake by DMI. The acute peripheral administration of DMI presumably increases the synaptic levels of NE (Racagni et al., 1982) which leads to decreased firing of brain noradrenergic neurons (Nyback et al., 1975; Cedarbaum and Aghajanian, 1977; Quinaux et al.,
1982) and NE turnover (Schubert et al., 1970; Sugrue, 1980) at least in part through the stimulation of ~x2-adrenergic receptors (Cedarbaum and Aghajanian, 1977; Sugrue, 1980). In the present studies the reduction in NE turnover by acute DMI was confirmed (table 2) as indicated by decreased cortical DOPA accumulation after AAAD inhibition. This effect of DMI appears to be mediated by ct2-adrenergic receptors since it was blocked by low doses of the specific a Eantagonist RX 781094 (table 4). The specificity of RX 781094 on the noradrenergic system is substantiated by our unpublished findings that at doses as high as 20 mg/kg, RX 781094 did not influence brain 5-HT or DA synthesis. At lower doses (2.0 mg/kg), however, significant increases in cortical NE synthesis (table 6) occurred, an effect commonly associated with a2-adrenergic antagonism (Sugrue, 1980; Fludder and Leonard, 1978). The DMI-induced reduction in NE turnover appears to mediate the protective effects of DMI against the depletion of cortical NE by TBZ; the reduction in cortical DOPA accumulation and inhibition of the loss of cortical NE induced by TBZ are concurrent and are observed within the same dose range of DMI. Furthermore, because both effects of DMI are antagonized by RX 781094, an aE-adrenergic mechanism may be involved. This possibility is reinforced by the findings that low doses of the a2-agonist, clonidine, like DMI, reduces cortical NE turnover and also protects cortical NE from depletion by TBZ. In terms of the protective effects of these compounds on cortical NE, clonidine is more potent and more active than DMI, fully inhibiting the TBZ-induced depletion of this monoamine. This probably results from the direct-acting agonist properties of clonidine while the agonist activity of DMI is most likely indirect, mediated through increased levels of synaptic NE. The higher efficacy of clonidine is also evident when comparing the effects of both compounds on cortical DOPA accumulation (see tables 2,6); the maximal reduction in DOPA formation ( - 50~) is larger than that for DMI ( - 35%) and occurs at a much lower dose. This difference in potencies may explain why clonidine has a longer duration of action in protecting cortical NE (fig. 1) compared
435
to DMI ( < 60 min; Pettibone et al., 1984). It may be that the 30% reduction in cortical DOPA formation by DMI, lasting for at least 90 min (table 3) is not of a sufficient magnitude for sustained protection against TBZ. The importance of the rate of NE turnover in the activity of TBZ is underscored by our experiments with RX 781094; pretreatment with this a2-adrenergic antagonist at doses which stimulate cortical DOPA accumulation causes an ineffective dose of TBZ (0.6 mg/kg) to significantly reduce cortical NE (table 7). This effect of RX 781094 is, however, not as extensive when higher doses of TBZ are used (cf. table 1). It is not known to what extent the protection by DMI of brain NE levels from depletion by TBZ underlies the behavioral TBZ antagonistic properties of this antidepressant. Our finding that reasonable doses (3-5 mg/kg) of DMI antagonizes the effect of TBZ on cortical NE even with the high doses of TBZ used in the behavioral test (Pettibone et al., 1984; Sulser and Soroko, 1965; Howard et al., 1981) is consistent, however, with this possibility. NE uptake inhibition by DMI in itself may not be sufficient for behavioral TBZ antagonism especially when one considers the very rapid and extensive depletion of cortical NE (Pettibone et al., 1984). The reduced rate of brain NE depletion coupled with the inhibition of NE uptake by DMI, however, could provide a sufficient condition for behavioral TBZ antagonism although this would not necessarily mean that all TBZ antagonists act by this mechanism. Clorgyline, for instance, probably exerts its behavioral TBZ antagonism simply by the extensive inhibition of MAO (Christmas et al., 1972). If reducing the rate of TBZ-induced NE depletion is the only factor involved in the behavioral effects of DMI, then one would expect clonidine to be an effective behavioral TBZ antagonist. We are not aware of any reports which have tested this possibility with low, non-sedating doses of clonidine. Appropriate behavioral studies (i.e., with the use of specific a E - a d r e n e r g i c a n t a g o n i s t s ) are r e q u i r e d to d e t e r m i n e the relative importance of the effects of
DMI on NE uptake and/or NE turnover in the behavioral TBZ antagonism by this antidepressant.
Acknowledgements We are grateful to Dr. B.V. Clineschmidt for helpful discussion and Ms. Jan Heebner for the preparation of the manuscript.
References Carlsson, A., W. Kehr, M. Lindqvist, T. Magnusson and C.V. Atack, 1972, Regulation of monoamine metabolism in the central nervous system, Pharmacol. Rev. 24, 371. Cedarbaum, J.M. and G.K. Aghajanian, 1977, Catecholamine receptors on locus coeruleus neurons: pharmacological characterization, European J. Pharmacol. 44, 375. 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. Dettmar, P.W., A.G. Lynn and I.F. Tulloch, 1983, Neuropharmacological studies in rodents on the action of RX 781094, a new selective a2-adrenoceptor antagonist, Neuropharmacol. 22, 729. Doxey, J.C., A.G. Roach and C.F.C. Smith, 1983, Studies on RX 781094: a selective potent and specific antagonist of a2-adrenoceptors, Br. J. Phannacol. 78, 489. Fludder, J.M. and B.E. Leonard, 1978, The action o f the antidepressant drug mianserin on presynaptic a-adrenergic receptors in the rat brain in vivo, Neuropharmacol. 17, 1058. Hefti, F., E. Melamed, R.J. Wurtman, 1980, Partial lesions of the dopaminergic nigrostriatal system in rat brain: biochemical characterization, Brain Res. 195, 123. Howard, J.L., F.E. Soroko and B.R. Cooper, 1981, Empirical behavioral models of depression with emphasis on tetrabenazine antagonism, in: Antidepressants: neurochemical, behavioral and clinical perspectives, S.J. Enna et al., eds., Raven Press, New York, p. 107. Johnston, J.P., 1968, Some observations upon a new inhibitor of monoamine oxidase in brain tissue, Biochem. Pharmacol. 17, 1285. Neff, N.H., H.-Y.T. Yang and J.A. Fuentes, 1974, The use of selective monoamine oxidase inhibitor drugs to modify amine metabolism in brain, Adv. Biochem. Pharmacol. 12, 49. Nyback, H.V., J.R. Walters, G.K. Aghajanian and R.H. Roth, 1975, Tricyclic antidepressants: effects on the firing rate of brain noradrenergie neurons, European J. Pharmacol. 32, 302. Pettibone, D.J., J.A. Totaro and A.B. Pflueger, 1984, Tetrabenazine-induced depletion of brain monoamines. Characterization and interaction with selected antidepressants, European J. Pharmacol. 102, 425. Quinaux, N., J. Scuvee-Moreau and A. Dresse, 1982, Inhibition of in vitro and ex vivo uptake of noradrenaline and 5-hydroxytryptamine by five antidepressants; correlation with the reduction of spontaneous firing rate of central mono-
436 aminergic neurons, Naunyn-Schmiedeb. Arch. Pharmacol. 319, 66. Racagni, G., I. Mocchetti, G. Renna and V. Cuomo, 1982, In vivo studies on central noradrenergic synaptic mechanisms after acute and chronic antidepressant drug treatment: biochemical and behavioral comparison, J. Pharmacol. Exp. Ther. 223, 227. Reinhard, J.F., Jr. and R.H. Roth, 1982, Noradrenergic modulation of serotonin synthesis and metabolism. 1. Inhibition by clonidine in vivo, J. Pharmacol. Exp. Ther. 221,541. Schubert, J., H. Nyback and G. Sedvall, 1970, Effect of antidepressant drugs on accumulation and disappearance of
monoamines formed in vivo from labeled precursors in mouse brain, J. Pharm. Pharmacol. 22, 136. Sugrue, M.F., 1980, Effects of acutely and chronically administered antidepressants on the clonidine-induced decrease in rat brain 3-methoxy-4-hydroxyphenylethylene glycol sulphate content, Life Sci. 28, 377. Sulser, F. and F. Soroko, 1965, On the role of the rate of brain norepinephrine release in the antibenzoquinolizine action of desipramine, Psychopharmacologia (Berlin) 8, 191. Yang, H.-Y.T. and N.F. Neff, 1974, The monoamine oxidases in brain: selective inhibition with drugs and the consequences for the metabolism of biogenic amines, J. Pharmacol. Exp. Ther. 189, 733.
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Elsevier
T E T R A B E N A Z I N E - I N D U C E D D E P L E T I O N OF BRAIN M O N O A M I N E S : M E C H A N I S M BY W H I C H D E S M E T H Y L I M I P R A M I N E P R O T E C T S CORTICAL N O R E P I N E P H R I N E D O U G L A S J. PETTIBONE *, A. B A R B A R A P F L U E G E R and JAMES A. T O T A R O
Department of Pharmacology, Merck Institute for Therapeutic Research, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486, U.S.A. Received 6 December 1983, revised MS received 28 February 1984, accepted 3 April 1984
D.J. PETTIBONE, A.B. PFLUEGER and J.A. TOTARO, Tetrabenazine-induced depletion of brain monoamines: mechanism by which desmethylimipramine protects cortical norepinephrine, European J. Pharmacol. 102 (1984) 431-436. Pretreatment of rats with desmethylimipramine (DMI) (5 mg/kg) significantly antagonized the tetrabenazine (TBZ)-induced (3 mg/kg) depletion of cortical norepinephrine (NE) in rats. This protective effect of DMI was fully blocked by the specific a2-adrenergic antagonist, RX 781094. The doses of DMI which antagonized the depleting effects of TBZ caused rapid, maximal reductions in cortical dihydroxyphenylalanine (DOPA) accumulation, effects which were also blocked by RX 781094. Like DMI, the a2-adrenergic agonist, clonidine (50 gg/kg) decreased cortical DOPA accumulation and rapidly prevented the TBZ-evoked loss of NE in this brain region. On the other hand, doses of RX 781094 (0.5-2.0 mg/kg) which accelerated cortical DOPA synthesis, enhanced significantly the depleting effects of a threshold dose of TBZ (0.6 mg/kg). These findings suggest that DMI reduces the rate of TBZ-induced depletion of cortical NE by lowering NE turnover through the indirect stimulation of a2-adrenoceptors. Tetrabenazine
DMI
NE turnover
a2-Adrenergic receptors
1. Introduction
It is generally believed that the potent and selective norepinephrine (NE) uptake-inhibiting property of the antidepressant desmethylimipramine (DMI) is responsible for its behavioral antitetrabenazine (TBZ) activity in rodents. However, there are very few reports which provide information regarding the neurochemical interactions of TBZ and anti-TBZ agents, such as DMI. In an effort to gain a better understanding of the neurochemical events accompanying a n d / o r underlying TBZ antagonism, we examined the interaction effects of TBZ and DMI on brain regional monoamines and determined that DMI reduced the rate of N E depletion by TBZ but not that of dopamine (DA) or serotonin (5-HT) (Pettibone et al., 1984). The findings presented in this report suggest that the protective effects of DMI are due to decreased * To w h o m all correspondence should be addressed. 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
cortical N E turnover, most likely through the stimulation of an a2-adrenergic mechanism.
2. Materials and methods 2.1. Animals
Adult, male, Sprague-Dawley rats (150-200 g) having free access to standard laboratory rat chow and tap water were used in all experiments. All drugs were administered intraperitoneally except for TBZ which was injected subcutaneously. Animals were sacrificed by decapitation and fronto-parietal cortex was rapidly dissected and frozen on dry ice. 2.2. Assays
N E and DOPA were assayed by HPLC/electrochemical detection by the method of Hefti et al. (1980).
432
2.3. Drugs
TABLE 2 Effects of DMI on cortical NE synthesis a.
DMI HC1 (USV Pharmaceutical Corp., Tuckahoe, NY), TBZ methane sulfonate (Hoffm a n n - L a R o c h e , Inc., N u t l e y , N J), a n d c l o n i d i n e HC1 ( B o e h r i n g e r - I n g e l h e i m Ltd., R i d g e f i e l d , C T ) w e r e g r a t e f u l l y r e c e i v e d as gifts. N S D 1015 ( m - h y d r o x y b e n z y l h y d r a z i n e ) was p u r c h a s e d f r o m A l d r i c h C h e m i c a l C o . ( M i l w a u k e e , W I ) . R X 781094 (2-(2,3-dihydro-l,4-benzodioxin-2-yl)-4,5-dihydro1 H - i m i d a z o l e H C I ) was s y n t h e s i z e d in t h e M e d i c i n a l C h e m i s t r y D e p a r t m e n t at t h e M e r c k S h a r p & D o h m e R e s e a r c h L a b o r a t o r i e s , W e s t P o i n t , PA.
2.4. Statistics D i f f e r e n c e s b e t w e e n g r o u p s ( P < 0.05) w e r e determined by ANOVA and Duncan's Multiple R a n g e test (2-tailed)i
3. Results
3.1. Blockade of the protective effects of DMI by R X 781094 T h e a d m i n i s t r a t i o n of D M I (5 m g / k g ) signific a n t l y a n t a g o n i z e d (by 72%) the d e p l e t i o n o f c o r t i cal N E i n d u c e d b y T B Z (3 m g / k g ; t a b l e 1). Pretreatment with the selective a2-adrenergic
TABLE 1 Effect of RX 781094 on DMl-induced protection of cortical NE depletion by TBZ a. Treatment
%change
(mg/kg)
Cortical NE (pmol/mg tissue)
Control DMI (5) TBZ (3) RX 781094 (2.5) DMI + TBZ RX 781094 + TBZ RX 781094+ DMI +TBZ
0.87 + 0.05 0.94 + 0.i0 0.48 + 0.04 b.¢ 0.90 + 0.05 0.76+0.05 0.41 + 0.06 b.¢ 0.41+0.06 b.¢
+8 -- 45 +3 - 13 -- 53 -53
a Rats received RX 781094 (i.p.) or its vehicle (H20) 30 rain before DMI (i.p.) or its vehicle (H20). Thirty rain later they received TBZ (s.c.) or its vehicle (H20) and were sacrificed 15 rain thereafter. Values are group means __S.E.M. (n ~ 4-5). b p < 0.05 vs. control group, c p < 0.05 vs. DMI + TBZ group.
Treatment (mg/kg i.p.)
Cortical DOPA (pmol/mg tissue)
~change
Control DMI (2.5) DMI (5.0) DMI (10.0) DMI (15.0)
0.94 + 0.06 0.63 + 0.03 b 0.58 + 0.04 b 0.58 + 0.03 b 0.61 + 0.04 b
-- 33 -- 38 -- 38 --35
a Rats were injected with DMI (i.p,) or its vehicle (H20) 30 min before NSD 1015 (125 mg/kg i.p.) and sacrificed 30 min thereafter. Values are group means + S.E.M. (n = 5). b p < 0.05 vs. control group (NSD 1015 alone).
a n t a g o n i s t R X 781094 (2.5 m g / k g ; D o x e y et al., 1983; D e t t m a r et al., 1983), h o w e v e r , fully p r e v e n t e d the p r o t e c t i v e effects o f D M I ( t a b l e 1).
3.2. Effects of DMI on cortical NE turnover T h e in v i v o r a t e o f c o r t i c a l N E s y n t h e s i s was estimated by measuring the rate of DOPA format i o n .after a r o m a t i c a m i n o a c i d d e c a r b o x y l a s e inh i b i t i o n ( A A A D ) b y N S D 1015 ( C a r l s s o n et al., 1972; R e i n h a r d a n d R o t h , 1982). D M I at 5 m g / k g m a x i m a l l y r e d u c e d ( b y 38%) t h e r a t e o f c o r t i c a l D O P A a c c u m u l a t i o n ( t a b l e 2). T h i s r e d u c t i o n in N E s y n t h e s i s was e v i d e n t w h e n D M I was a d m i n i s t e r e d as e a r l y as 15 r a i n b e f o r e N S D 1015 a n d l a s t e d at least a n o t h e r 90 m i n ( t a b l e 3). P r e t r e a t m e n t w i t h R X 781094 s i g n i f i c a n t l y a t t e n u a t e d the r e d u c t i o n in c o r t i c a l D O P A a c c u m u l a t i o n o b -
TABLE 3 Time-related effects of DMI on cortical NE synthesis a. Treatment (time before NSD 1015, rain)
Cortical DOPA (pmol/mg tissue)
%change
Vehicle (5) DMI (5) DMI (15) DMI (30) DMI (60) DMI (90)
0.52 + 0.05 0.45 _+0.03 0.38+0.03 b 0.36 + 0.01 b 0.39 + 0.03 b 0.35 +_0.01 b
- 13 --27 -- 31 -- 25 -- 33
a Rats were administered DMI (5 mg/kg i.p.) or its vehicle (H20) at the indicated times before NSD 1015 (125 mg/kg i.p.) and sacrificed 30 min thereafter. Values are group means + S.E.M. (n = 5). b p < 0.05 vs. vehicle-treated group.
433 TABLE 4
TABLE 5
Effects of R X 781094 o n D M I - i n d u c e d r e d u c t i o n in cortical N E synthesis a
Dose-related p r o t e c t i o n of T B Z - i n d u c e d d e p l e t i o n of cortical N E b y c l o n i d i n e a.
Treatment
Cortical DOPA ( p m o l / m g tissue)
%change
Treatment (mg/kg)
Control DMI R X 781094 R X 781094 + D M I
0.40 + 0.02 0.25 + 0.01 b 0.37+0.01 0.33 + 0.02 b,¢
-- 47 - 7 -- 17
Control Clonidine T B Z (3) Clonidine Clonidine Clonidine Clonidine
a R a t s were treated w i t h R X 781094 (250 # g / k g i.p.) or its vehicle ( H 2 0 ) 20 rain before D M I (3 m g / k g i.p.) or its vehicle ( H 2 0 ) . T h i r t y rain l a t e r they received N S D 1015 (125 m g / k g i.p.) a n d sacrificed 30 rain thereafter. Values are g r o u p m e a n s + S.E.M. (n = 5). b p < 0.05 vs. control g r o u p ( N S D 1015 alone), c p < 0.05 vs. D M I - t r e a t e d group.
served after DMI (table 4). This small dose of RX 781094 (250 #g/kg) did not influence NE synthesis by itself.
3.3. Blockade of TBZ-induced depletion of cortical NE by clonidine Similar to the effects of DMI, the administration of clonidine (250 #g/kg) as early as 20 min before TBZ inhibited the depletion of NE (fig. 1), an effect which lasted for at least 2 h. This protective effect of clonidine was observed with doses as
1.O E O
E Q.
/ 0.5
.......
"'
-
(0.025)+TBZ (0.05) + T B Z (0.10)+TBZ (0.25) + T B Z
%change
1.17 + 0.09 1.41 + 0.04 0.62 + 0.04 0.96 + 0 . 0 6 1.06 + 0.02 1.07+0.03 1.07 + 0.04
_ + 21 -- 47 -- 18 - 9 -9 - 9
c b,c b b,c c c c
a R a t s were injected w i t h c l o n i d i n e (i.p.) or its vehicle ( H 2 0 ) 40 rain before T B Z (s.c.) or its vehicle ( H 2 0 ) a n d sacrificed 15 rain thereafter. Values are group m e a n s + S.E.M. (n = 5). b p < 0.05 vs. control group, c p < 0.05 vs. T B Z a l o n e - t r e a t e d group.
low as 25 #g/kg i.p. (table 5). The doses of clonidine which fully blocked the TBZ-induced loss of NE, maximally reduced cortical DOPA accumulation by about 50% (table 6).
3.4. Enhancement of TBZ potency by R X 781094 If reducing NE turnover through a2-adrenergic stimulation reduces the effectiveness of TBZ to deplete cortical NE, then the administration of a2-adrenergic antagonists, which are known to augment NE turnover, might be expected to enhance the potency of TBZ. Pretreatment with RX 781094 significantly enhanced the rate of cortical
T TABLE 6
#
~.
•
UJ Z
•
0---0
Effects of c l o n l d i n e or R X 781094 o n cortical N E synthesis a
Control
TBZ-treated
6 t. o
I=t,. Veh 20
(0.25)
Cortical N E ( p m o l / m g tissue)
i
I 60
=
I 120
Clonidine Pretreatment (rain) Fig. 1. I n h i b i t i o n of T B Z - i n d u c e d d e p l e t i o n of cortical N E b y clonidine. R a t s were a d m i n i s t e r e d c l o n i d i n e (250 # g / k g i.p.) or its vehicle ( H 2 0 ) at the i n d i c a t e d times before T B Z (3 m g / k g s.c.) or its vehicle ( H 2 0 ) a n d sacrificed 15 rain later. Values are g r o u p m e a n s 5: S.E.M. (n = 5). * P < 0.05 vs. control, t p < 0.05 vs. T B Z alone.
Treatment (mg/kg)
Cortical D O P A ( p m o l / m g tissue)
C o n t r o l ( N S D 1015 alone) R X 781094 (0.5) R X 781094 (1.0) R X 781094 (2.0) C l o n i d i n e (0.05) C l o n i d i n e (0.10) C l o n i d i n e (0.25)
0.34 0.55 0.61 0.62 0.15 0.19 0.16
+ 0.03 + 0.04 + 0.04 + 0.04 + 0.01 -t- 0.01 + 0.02
b b b b b b
%change
+ 62 + 79 + 82 -- 56 - - 43 -- 53
a R a t s were a d m i n i s t e r e d R X 781094 (i.p.), c l o n i d i n e (i.p.) or H 2 0 (i.p.) 30 rain b e f o r e N S D 1015 (125 m g / k g i.p.) a n d sacrificed 30 m i n thereafter. Values are g r o u p m e a n s + S.E.M. (n = 5). b p < 0.05 vs. control group.
434 TABLE 7 E n h a n c e m e n t of the TBZ-induced depletion of cortical N E by the a2-adrenergic antagonist, RX 781094 a. Treatment (mg/kg) Control R X 781094 RX 781094 R X 781094 TBZ (0.6) R X 781094 R X 781094 RX 781094
(0.5) (1.0) (2.0) (0.5) + TBZ (1.0)+ TBZ (2.0)+TBZ
Cortical N E ( p m o l / m g tissue)
%change
0.88 4- 0.07 0.96 4- 0.09 0.96 4- 0.05 0.92 4- 0.07 0.75 4-0.08 0.69 4- 0.07 0.68 4- 0.03 b 0.504-0.04 b.c
+9 +9 +5 - 15 - 22 -- 23 --43
a Rats were treated with RX 781094 (i.p.) or its vehicle ( H 2 0 ) 45 min before TBZ (s.c.) or its vehicle ( H 2 0 ) and were sacrificed 15 rain thereafter. Values represent group means + S.E.M. (n = 5). b p < 0.05 vs. control group, c p < 0.05 vs. TBZ alone-treated group.
DOPA accumulation after NSD 1015 (table 6) and at doses (0.5-2.0 ng/kg) which did not influence cortical NE by themselves, dose-dependently potentiated the depletion of NE by a threshold dose of TBZ (0.6 mg/kg; table 7).
4. Discussion
In the previous report (Pettibone et al., 1984) we observed that the antidepressant, clorgyline, equally prevented the TBZ-induced depletion of brain NE, DA and 5-HT. Since these monoamines are known to be substrates of the form of monoamine oxidase (MAO) inhibited by clorgyline (Johnston, 1968; Neff et al., 1974; Yang and Neff, 1974) it is likely that the protective effects of clorgyline resulted from reduced deamination of these neurotransmitters after their release from granular storage by TBZ. The inhibition by DMI of NE but not DA or 5-HT depletion, however indicates a different mechanism accounting for the interference with the actions of TBZ, perhaps being related to the selective inhibition of NE uptake by DMI. The acute peripheral administration of DMI presumably increases the synaptic levels of NE (Racagni et al., 1982) which leads to decreased firing of brain noradrenergic neurons (Nyback et al., 1975; Cedarbaum and Aghajanian, 1977; Quinaux et al.,
1982) and NE turnover (Schubert et al., 1970; Sugrue, 1980) at least in part through the stimulation of ~x2-adrenergic receptors (Cedarbaum and Aghajanian, 1977; Sugrue, 1980). In the present studies the reduction in NE turnover by acute DMI was confirmed (table 2) as indicated by decreased cortical DOPA accumulation after AAAD inhibition. This effect of DMI appears to be mediated by ct2-adrenergic receptors since it was blocked by low doses of the specific a Eantagonist RX 781094 (table 4). The specificity of RX 781094 on the noradrenergic system is substantiated by our unpublished findings that at doses as high as 20 mg/kg, RX 781094 did not influence brain 5-HT or DA synthesis. At lower doses (2.0 mg/kg), however, significant increases in cortical NE synthesis (table 6) occurred, an effect commonly associated with a2-adrenergic antagonism (Sugrue, 1980; Fludder and Leonard, 1978). The DMI-induced reduction in NE turnover appears to mediate the protective effects of DMI against the depletion of cortical NE by TBZ; the reduction in cortical DOPA accumulation and inhibition of the loss of cortical NE induced by TBZ are concurrent and are observed within the same dose range of DMI. Furthermore, because both effects of DMI are antagonized by RX 781094, an aE-adrenergic mechanism may be involved. This possibility is reinforced by the findings that low doses of the a2-agonist, clonidine, like DMI, reduces cortical NE turnover and also protects cortical NE from depletion by TBZ. In terms of the protective effects of these compounds on cortical NE, clonidine is more potent and more active than DMI, fully inhibiting the TBZ-induced depletion of this monoamine. This probably results from the direct-acting agonist properties of clonidine while the agonist activity of DMI is most likely indirect, mediated through increased levels of synaptic NE. The higher efficacy of clonidine is also evident when comparing the effects of both compounds on cortical DOPA accumulation (see tables 2,6); the maximal reduction in DOPA formation ( - 50~) is larger than that for DMI ( - 35%) and occurs at a much lower dose. This difference in potencies may explain why clonidine has a longer duration of action in protecting cortical NE (fig. 1) compared
435
to DMI ( < 60 min; Pettibone et al., 1984). It may be that the 30% reduction in cortical DOPA formation by DMI, lasting for at least 90 min (table 3) is not of a sufficient magnitude for sustained protection against TBZ. The importance of the rate of NE turnover in the activity of TBZ is underscored by our experiments with RX 781094; pretreatment with this a2-adrenergic antagonist at doses which stimulate cortical DOPA accumulation causes an ineffective dose of TBZ (0.6 mg/kg) to significantly reduce cortical NE (table 7). This effect of RX 781094 is, however, not as extensive when higher doses of TBZ are used (cf. table 1). It is not known to what extent the protection by DMI of brain NE levels from depletion by TBZ underlies the behavioral TBZ antagonistic properties of this antidepressant. Our finding that reasonable doses (3-5 mg/kg) of DMI antagonizes the effect of TBZ on cortical NE even with the high doses of TBZ used in the behavioral test (Pettibone et al., 1984; Sulser and Soroko, 1965; Howard et al., 1981) is consistent, however, with this possibility. NE uptake inhibition by DMI in itself may not be sufficient for behavioral TBZ antagonism especially when one considers the very rapid and extensive depletion of cortical NE (Pettibone et al., 1984). The reduced rate of brain NE depletion coupled with the inhibition of NE uptake by DMI, however, could provide a sufficient condition for behavioral TBZ antagonism although this would not necessarily mean that all TBZ antagonists act by this mechanism. Clorgyline, for instance, probably exerts its behavioral TBZ antagonism simply by the extensive inhibition of MAO (Christmas et al., 1972). If reducing the rate of TBZ-induced NE depletion is the only factor involved in the behavioral effects of DMI, then one would expect clonidine to be an effective behavioral TBZ antagonist. We are not aware of any reports which have tested this possibility with low, non-sedating doses of clonidine. Appropriate behavioral studies (i.e., with the use of specific a E - a d r e n e r g i c a n t a g o n i s t s ) are r e q u i r e d to d e t e r m i n e the relative importance of the effects of
DMI on NE uptake and/or NE turnover in the behavioral TBZ antagonism by this antidepressant.
Acknowledgements We are grateful to Dr. B.V. Clineschmidt for helpful discussion and Ms. Jan Heebner for the preparation of the manuscript.
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