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Comparative effects of clozapine and α-adrenoceptor blocking drugs on regional noradrenaline metabolism in rat brain
225
European Journal of Pharmacology, 52 (1978) 225--230 © Elsevier/North-Holland Biomedical Press
COMPARATIVE EFFECTS OF CLOZAPINE AND a-ADRENOCEPTOR BLOCKING DRUGS ON REGIONAL NORADRENALINE METABOLISM IN RAT BRAIN * BRIAN A. McMILLEN and PARKHURST A. SHORE Department of Pharmacology, University of Texas Health Science Center, Dallas, Texas 75235, U.S.A.
Received 4 April 1978, revised MS received 7 July 1978, accepted 3 August 1978
B.A. McMILLEN and P.A. SHORE, Comparative effects of clozapine and o~-adrenoceptor blocking drugs on regional noradrenaline metabolism in rat brain, European J. Pharmacol. 52 (1978) 225--230. Clozapine increased brain noradrenaline (NA) metabolism, as indicated by changes in 3-methoxy-4-hydroxyphenylglycol sulfate content, in brain regions corresponding to the predominance of a- over ~-receptors, i.e., hypothalamus, medulla, midbrain and cortex, but not corpus striatum or cerebellum. Phenoxybenzamine had a stronger effect in the hypothalamus than did clozapine, but did not change cortical NA metabolism within a 60 min treatment time; however, cortical NA metabolism was increased 150 min after phenoxybenzamine. The delayed effect of phenoxybenzamine may be due to either a poor affinity for some central receptors or a slow rate of entry into certain brain regions. Thioridazine and the benzodioxane, dibozane, had regional effects similar to clozapine. The similarity between clozapine and dibozane in their effects on regional brain NA metabolism may reflect a preference for presynaptic a-receptors. It is unlikely that the antipsychotic activity of clozapine is related to a specific adrenolytic effect, but may reflect the combined activity of this drug on several transmitter systems. Clozapine
Phenoxybenzamine
Noradrenaline
1. I n t r o d u c t i o n T h e a n t i p s y c h o t i c drug, c l o z a p i n e , has b e e n the s u b j e c t o f c o n s i d e r a b l e research i n t e r e s t as it causes little or n o e x t r a p y r a m i d a l d y s f u n c tion (Stille et al., 1 9 7 1 ; Hippius, 1975), a l t h o u g h the d r u g e n h a n c e s d o p a m i n e (DA) t u r n o v e r in the n e o s t r i a t a l a n d m e s o l i m b i c dopaminergic systems (And6n and Stock, 1 9 7 3 ; Bartholini et al., 1 9 7 2 ; Zivkovic e t al., 1975). H o w e v e r , c l o z a p i n e has e f f e c t s o n central t r a n s m i t t e r s y s t e m s o t h e r t h a n DA. C o s t a ( 1 9 7 7 ) has r e p o r t e d t h a t 7 - a m i n o b u t y r i c acid ( G A B A ) t u r n o v e r t i m e is d e c r e a s e d b y clozapine. Whole brain n o r a d r e n a l i n e (NA) m e t a b * Supported by Public Health Service Grant MH05831.
Antipsychotic
olism in rats is increased b y doses o f clozap i n e within t h e clinical dose range (Bartholini et al., 1 9 7 3 ; Keller e t al., 1 9 7 3 ) a n d c l o z a p i n e has c o n s i d e r a b l e a n t i m u s c a r i n i c e f f e c t s in several in vitro t e s t s y s t e m s (Miller a n d Hiley, 1 9 7 4 ; S n y d e r et al., 1974). T h e s e p u t a t i v e t r a n s m i t t e r s as well as D A are t h o u g h t t o be involved in e x t r a p y r a m i d a l a n d limbic functions (see Costa, 1977). It is i n t e r e s t i n g t h a t thioridazine, another atypical antipsychotic drug, increases brain N A m e t a b o l i s m t o t h e s a m e e x t e n t as c l o z a p i n e w h e n a d m i n i s t e r e d in a 50% g r e a t e r dose (Keller et al., 1 9 7 3 ) , a p o t e n c y d i f f e r e n c e w h i c h c o r r e s p o n d s to the d i f f e r e n c e in clinical doses. I t is c o n c e i v a b l e t h a t c l o z a p i n e e x e r t s its a n t i p s y c h o t i c actions, in p a r t at least, t h r o u g h a n o r a d r e n e r g i c m e c h a n i s m . In an a t t e m p t t o
226 determine whether clozapine's central effects on NA metabolism represent a novel action or are simply those shared by known blockers of a-adrenoceptors, the drug was compared with phenoxybenzamine and other u-receptor antagonists for their effects on NA metabolism in specific brain regions. Changes in NA metabolism were determined by the effects of these drugs on the regional concentration of the NA metabolite, 3-methoxy-4-hydroxyphenylglycol sulfate (MOPEG-SO4). 2. Materials and methods 2.1. Animals and drugs Female Sprague-Dawley rats, 2 0 0 - - 2 3 0 g (Holtzman), maintained on a 12 h light/dark cycle, were treated with drugs i.p. and killed at the times noted in the tables. Phenoxybenzamine (Smith, Kline and French Laboratories) was dissolved (40 mg/ml) in propyleneglycol and diluted to 10 mg/ml with water. Clozapine (Sandoz Pharmaceuticals) was dissolved (10 mg/ml) in 0.1 N HC1 and diluted to 3.0 mg/ml with water. Dibozane (McNeil Laboratories) was dissolved (10 mg/ml) in 0.1 N HC1. Thioridazine concentrate (Sandoz Pharmaceuticals) was diluted with water to 10 mg/ml. All doses refer to the free base. 2.2. MOPEG-S04 assay Animals were killed by chloroform asphyxiation and the brains were rapidly removed and chilled in ice-cold saline. The brains were dissected into 6 regions (Glowinski and Iversen, 1966): cerebellum, medulla-pons, hypothalamus, corpus striatum, cortex-hippocampus and midbrain. The brain regions from 2--4 animals were pooled and frozen until assayed the next day. The procedure of Meek and Neff (1972) for the fluorometric assay of MOPEC-SO4 was used with minor modifications. Briefly, brains were homogenized in 0.2 M ZnSO4, mixed with 0.2 M Ba(OH)2 and centrifuged at 3 0 , 0 0 0 × g for 10 min as described by Meek and Neff (1972). The supernatants were adjusted to pH 7.5--7.7
B.A. McMILLEN, P.A. SHORE with 0.2 M ZnSO4 and centrifuged again (T.K. Keeton, personal communication). The resulting supernatant fractions were deeantcd-e~t~ 5.0 mm X 5.0 cm DEAE Sephadex A-25 anion exchange columns (Pharmacia). After the supernatants passed through, the columns were washed with 4.0 ml of 0.06 N HC1. MOPEG-SO4 was eluted with 0.15 N HC1. We found that increasing the amounts of tissue increased the rate of MOPEG-SO4 elution. Therefore, with cortical samples (pooled from two animals), the first 2.0 ml of eluate were discarded while with the smaller brain regions (pooled from 4 animals) or with MOPEG-SO4 standards (Ro 4-6028, Hoffman-LaRoche), substituting water for tissue, the first 3.0 ml were discarded. In each case the subsequent 4.0 ml of eluate contained all of the MOPEGSO4. To the 4.0 ml of eluate were added 0.2 ml of 1.0% cysteine and 0.2 ml 60% HC104. After mixing, 2.0 ml were taken for fluorphor development. Samples were heated in a 100°C water bath for 12 min and cooled in tap water. Freshly redistilled ethylenediamine (0.3 ml) was added, mixed thoroughly, and the samples heated in a boiling water bath for 5 min. After the samples cooled to room temperature, fluorescence was determined in an Aminco-Bowman spectrofluorometer at 325 n m / 4 7 0 nm (excitation/emission, uncorrected instrument values). Relative fluorescence was compared to a standard curve of MOPEG-SO4 dissolved in 0.15 N HC1 which had been previously passed through a DEAE Sephadex A-25 column. This treatment of the acid enhanced the fluorescence of MOPEG-SO4 standards, suggesting that the columns had removed an interfering impurity. All values were corrected for recovery of MOPEG-SO4 which averaged 88 + 9%. 3. Results
3.1. Effects o f clozapine and phenoxybenzamine on regional brain NA metabolism Clozapine was used in doses of 3 and 6 mg/ kg, a range comparable to the daily clinical
ANTIPSYCHOTIC AND ADRENOLYTIC DRUGS
227
dose. The dose of 6 mg/kg has been reported to increase whole brain MOPEG-SO4 levels by 25% in 2 h (Keller et al., 1973). In the present study, a shorter (60 min) treatment was used to help elucidate regional effects before a maximal accumulation of MOPEGSO4 could occur. Phenoxybenzamine was used in a dose of 40 mg/kg after preliminary experiments indicated that lower doses do n o t saturate a-receptors. For example 17 h after 25 mg/kg of phenoxybenzamine, whole brain MOPEG-SO4 concentrations increased significantly from 0.157 /zg/g to 0.200 /zg/g. If an additional 40 mg/kg was administered 2.5 h before killing the animals (14.5 h after the first dose), MOPEG-SO4 levels increased to 0.403 #g/g. Thus the 40 mg/kg dose of phenoxybenzamine was used in the current study. This dose of phenoxybenzamine was previously reported to cause a 47% increase of MOPEG-SO4 concentrations in rat whole brains (McMillen and Shore, 1977). The data in table 1 show that 6.0 mg/kg clozapine increased MOPEG-SO4 concentrations in the hypothalamus, medulla, midbrain and cortex. This result is in harmony with the
whole brain dose--response curve reported by Keller et al. (1973). Phenoxybenzamine produced similar regional effects except for the cortex where MOPEG-SO4 levels were unchanged. Maintaining the clozapine-treated animals within 1.0°C of normal b o d y temperature (ambient temperature 32°C), instead of allowing the 2.0--2.5 ° decrease seen at room temperature, slightly enhanced the effects of clozapine on NA metabolism. The most striking differences between the two drugs 60 min after administration were in the hypothalamus, where phenoxybenzamine had a greater effect than did clozapine, and in the cortex, where clozapine, b u t n o t phenoxybenzamine, significantly increased NA metabolism. 3.2. Effects o f various drugs on MOPEG-S04 concentrations in hypothalamus and cortex To examine further the apparent differences between phenoxybenzamine and clozapine in their relative effects on hypothalamus and cortex, the effects of other drugs on these two areas were studied. The effects of thi-
TABLE 1 T h e effects of c l o z a p i n e a n d p h e n o x y b e n z a m i n e o n regional b r a i n n o r e p i n e p h r i n e m e t a b o l i s m . N u m b e r s in p a r e n t h e s e s r e p r e s e n t t h e n u m b e r o f e x p e r i m e n t s . 4 brains were dissected a n d t h e regions p o o l e d for e a c h e x p e r i m e n t . All drugs were i n j e c t e d 60 m i n b e f o r e sacrifice. C o r t e x includes h i p p o c a m p u s . MOPEG-SO4 pg/g _+S.E.M.
C o n t r o l (9) 3.0 m g / k g c l o z a p i n e (5) 6.0 m g / k g c l o z a p i n e (6) 6.0 m g / k g c l o z a p i n e + 32°C (6) 40.0 mg/kg phenoxybenzamine (8)
Hypothalamus
Striatum
Midbrain
Medulla
Cerebellum
Cortex
0.224 -+0.034 0.289 _+0.023 0 . 3 4 7 1~3 -+0,016 0,408 2 _+0,037 0.478 2 -+0.039
0.114 -+0,022 0.139 +0.009 0.125 +0.022 0.150 -+0.017 0.147 _+0.034
0.176 -+0.008 0.208 -+0.023 0.223 -+0.030 0.330 2 -+0.032 0.268 2 -+0.021
0.150 +0.015 0.190 -+0.021 0.192 -+0.017 0.261 2 -+0.045 0.204 4 -+0.009
0,028 +0.006 0.039 -+0.004 0.033 _+0.004 0,031 _+0.005 0,032 -+0.006
0.123 _+0.006 0.144 -+0.017 0.177 1 _+0.024 0 . 1 9 2 2,3 -+0.020 0.133 _+0.013
1 p < 0 . 0 5 ; 2 p < 0.01 c o m p a r e d to c o n t r o l ( D u n n e t t ' s t-test). 3 Differs f r o m p h e n o x y b e n z a m i n e P < 0,05 ( S t u d e n t ' s t-test); 4 differs f r o m c o n t r o l P < 0.01 ( S t u d e n t ' s t-test).
228
B.A. M c M I L L E N , P.A. S H O R E
oridazine, another atypical antipsychotic, and dibozane, a benzodioxane a-receptor blocker, were compared with clozapine and phenoxybenzamine (table 2). The dose of thioridazine was chosen because of an earlier report that a 50% greater molar dose of thiorodazine is needed to increase whole brain MOPEG-SO4 levels to those seen with clozapine (Keller et al., 1973). The dose of dibozane was chosen after a preliminary dose--response curve for whole brain MOPEG-SO4 levels indicated that 20 mg/kg of dibozane was a b o u t equivalent to 40 mg/kg of phenoxybenzamine (McMillen and Shore, 1977). Table 2 shows that both thioridazine and dibozane had effects similar to clozapine in that both hypothalamic and cortical NA metabolism were increased. In control animals and those treated with clozapine, thioridazine or dibozane, the ratios for MOPEG-SO4 concentrations in hypothalamus and cortex ranged from 1.96 to 2.50. Phenoxybenzamine did n o t increase MOPEG-SO4 levels in the cortex within this 60 min treatment time, the hypothalamus to cortex ratio for MOPEG-SO4 content being 3.34. However, longer (150 min) treatment with phenoxybenzamine caused a significant increase
in cortical NA metabolism such that the hypothalamus to cortex ratio changed to 2.16. 4. Discussion
The further effect of a second dose of phenoxybenzamine on MOPEG-SO4 accumulation suggests that central a-receptors are not completely blocked by a 25 mg/kg dose of phenoxybenzamine and we have no evidence to suggest that even 40 mg/kg of phenoxybenzamine has saturated a-receptors. This may be due to either a poor affinity of phenoxybenzamine for some central receptors or to poor accessibility of some brain regions to the drug. The difficulty in achieving saturation of central a-receptors with a blocking agent thought to exert an irreversible blockade and the delayed increase of MOPEG-SO4 concentration in the cortex (table 2) suggest that observations made while using phenoxybenzamine as a central a-blocker should be interpreted with care because of the apparent difficulty in blocking all central a-receptors with this drug. Low doses of clozapine and a shorter time course for the accumulation of MOPEG-SO4
TABLE 2 T h e effects o f a - a d r e n o c e p t o r a n t a g o n i s t s o n MOPEG-SO4 c o n t e n t o f rat h y p o t h a l a m u s a n d cortex. R a t s were i n j e c t e d w i t h c l o z a p i n e (6.0 m g / k g i.p.), t h i o r i d a z i n e (10 m g / k g i.p.), d i b o z a n e (20 m g / k g i.p.) or p h e n o x y b e n z a m i n e (40 m g / k g i.p.) a n d killed 60 rain later. N u m b e r s in p a r e n t h e s e s r e p r e s e n t t h e n u m b e r of exp e r i m e n t s . C o r t e x includes t h e h i p p o c a m p u s . T h e r a t i o o f t h e MOPEG-SO4 c o n t e n t s in t h e t w o regions is s h o w n in t h e last c o l u m n . MOPEG-SO4 pg/g + S.E.M.
Control Clozapine Thioridazine Dibozane Phenoxybenzamine 60 m i n 150min
Hypothalamus
Cortex
0.252 0.347 0.380 0.397
0.120 0.177 0.152 0.171
-+ 0 . 0 2 8 (13) -+ 0 . 0 1 6 (6) 3 + 0 . 0 1 4 (6) I + 0 . 0 3 7 (7) 2
0 . 4 6 2 + 0 . 0 2 7 (13) 2 0.431+0.044 (9) 2
I Differs f r o m c o n t r o l , P < 0.05 ( D u n n e t t ' s t-test). 2 Differs f r o m c o n t r o l , P < 0.01 ( D u n n e t t ' s t-test). 3 Differs f r o m c o n t r o l , P < 0.01 ( S t u d e n t ' s t-test).
H/C -+ 0 . 0 0 5 (13) + 0 . 0 2 4 (6) 2 -+ 0 . 0 0 9 (6) 3 + 0 . 0 1 6 (8) 2
2.10 1.96 2.50 2.32
0 . 1 3 8 + 0 . 0 1 0 (12) 0 . 2 0 0 + 0 . 0 1 5 (7) 2
3.34 2.16
ANTIPSYCHOTIC AND ADRENOLYTIC DRUGS were chosen in order to determine whether this drug may have regional effects which might otherwise be obscured by the large increases of whole brain MOPEG-SO4 concentrations elicited by higher doses of clozapine (Keller et al., 1973). The data in table 1 show that the regional clozapine-induced increase of NA metabolism parallels the known distribution of a-receptors (U'Prichard et al., 1977). No significant changes occur in the striatum and cerebellum where ~-receptors predominate (Bylund and Snyder, 1976). Both clozapine and thioridazine resemble dibozane in their effects on hypothalamic and cortical NA metabolism. This may reflect a relative preference by clozapine for presynaptic a-receptors, as the benzodioxanes, piperoxane and dibozane, are considered to be preferential presynaptic antagonists, while phenoxybenzamine may have a relative preference for postsynaptic receptors (Cubeddu et al., 1974; And~n et al., 1976; Franklin and Herberg, 1977). Clozapine lowers NA and metaraminol in the rat heart, an effect which is inhibited by ganglionic blockade (Dorris and Shore, 1976). Such an effect may be interpreted as suggestive of presynaptic a-inhibition (see Langer, 1977). Clozapine is an inhibitor of NA-stimulated adenylate cyclase in rat limbic forebrain at concentrations much lower than obtained from clinical doses of clozapine (Blumberg et al., 1976), there being no correlation between antipsychotic efficacy and inhibition of NA-stimulated c-AMP formation. In h a r m o n y with the report by Blumberg and coworkers for a central a-receptor effect of clozapine is the observation that clonidine inhibits the brain NA lowering effect of clozapine (Bartholini et al., 1973) as well as phenoxybenzamine (Braestrup and Nielsen, 1976). From the findings presented here it may be seen that the atypical antipsychotic drugs, clozapine and thioridazine, do n o t appear to exert effects on brain NA metabolism that differ significantly from known a-blockers which have n o t been reported as having antipsychotic activity.
229 Although clozapine is an effective antipsychotic drug and elevates striatal and mesolimbic DA metabolism, the extrapyramidal dysfunctions in man and animals observed with commonly employed neuroleptics do not occur with clozapine (Stille et al., 1971; Hippius, 1975). In addition, clozapine is a poor inhibitor of the behavioral effects of amphetamine and apomorphine (Stille et al., 1971). From all that is now known of clozapine's effects on various transmitters, it is clear that the effects of clozapine on central nervous system functions are complex. If schizophrenia is considered the result of a functional imbalance between several neuronal systems, including the dopaminergic system, then it is possible that classical neuroleptics restore the balance by a strong inhibition of DA receptors. Clozapine and other atypical antipsychotic drugs may help restore normal behavior by the sum of their effects on multiple neuronal systems including DA, NA, GABA and acetylcholine-containing neurons.
Acknowledgement The authors thank Ms. Karen Johnson for her skilled technical assistance.
References
And4n, N.E., M. Grabowska and U. StrSm, 1976, Different a-adrenoceptors in the central nervous system mediating biochemical and functional effects of clonidine and receptor blocking agents, Naunyn-Schmiedeb. Arch. Pharmacol. 292, 43. And~n, N.E. and G. Stock, 1973, Effects of clozapine on the turnover of dopamine in the corpus striatum and in the limbic system, J. Pharm. Pharmacol. 25,346. Bartholini, G., W. Haefely, M. Jalfre, H.H. Keller and A. Pletscher, 1972, Effects of clozapine on cerebral catecholaminergic neurone systems, Brit. J. Pharmacol. 46,736. Bartholini, G., H.H. Keller and A. Pletscher, 1973, Effect of neuroleptics on endogenous norepinephrine in rat brain, Neuropharmacol. 12,751. Blumberg, J.B., J. Vetulani, R.J. Stawarz and F.
230 Sulser, 1976, The noradrenergic cyclic AMP generating system in the limbic forebrain: pharmacological characterization in vitro and possible role of limbic noradrenergic mechanisms in the mode of action of antipsychotics, European J. Pharmacol. 3 7 , 3 5 7 . Braestrup, C. and M. Nielsen, 1976, Regulation in the central norepinephrine neurotransmission induced in vivo by ~-adrenoceptor active drugs. J. Pharmacol. Exptl. Therap. 198, 596. Bylund, D.B. and S.H. Snyder, 1976, ~-Adrenergic receptor binding in membrane preparations from mammalian brain, Mol. Pharmacol. 12, 568. Costa, E., 1977, Introduction: morphine, amphetamine and noncataleptogenic neuroleptics, in: Nonstriatal Dopaminergic Neurons, eds. E. Costa and G.L. Gessa (Raven Press, New York) p. 577. Cubeddu, L., E.M. Barnes, S.Z. Langer and N. Weiner, 1974, Release of norepinephrine and dopamine~-hydroxylase by nerve stimulation. I. Role of neuronal and extraneuronal uptake and of presynaptic c~-receptors, J. Pharmacol. Exptl. Therap. 190,431. Dorris, R.L. and P.A. Shore, 1976, On the mechanism of action of clozapine on the adrenergic neurone, Brit. J. Pharmacol. 56,279. Franklin, K.B.J. and L.J. Herberg, 1977, Presynaptic ~-adrenoceptors: the depression of self-stimulation by clonidine and its restoration by piperoxane but not by phentolamine or phenoxybenzamine, European J. Pharmacol. 43, 33. Glowinski, J. and L.L. Iversen, 1966, Regional studies of catecholamines in the rat brain, J. Neurochem. 13,655. Hippius, H., 1975, On the relations between antipsychotic and extrapyramidal effects of psychoactive drugs, in: Antipsychotic Drugs, Pharmacodynamics and Pharmacokinetics, eds. G. Sedvall,
B.A. McMILLEN, P.A. SHORE B. Uvnas and Y. Zotterman (Pergamon Press, Oxford and New York) p. 437. Keller, H.H., G. Bartholini and A. Pletscher, 1973, Increase of 3-methoxy-4-hydroxyphenylethylene glycol in rat brain by neuroleptic drugs, European J. Pharmacol. 23, 183. Langer, S.Z., 1977, Presynaptic receptors and their role in the regulation of transmitter release, Brit. J. Pharmacol. 60, 481. McMillen, B.A. and P.A. Shore, 1977, The relative functional availability of brain noradrenaline and dopamine storage pools, J. Pharm. Pharmacol. 29, 780. Meek, J.L. and N.H. Neff, 1972, Fluorometric estimation of 4-hydroxy-3-methoxyphenylethyleneglycol sulphate in brain, Brit. J. Pharmacol. 45, 435. Miller, R.J. and C.R. Hiley, 1974, Antimuscarinic properties of neuroleptics and drug-induced Parkinsonism, Nature 248,596. Snyder, S.H., D. Greenberg and H.I. Yamamura, 1974, Antischizophrenic drugs and brain cholinergic receptors, Arch. Gen. Psychiat. 31, 58. Stille, G., H. Lauener and E. Eichenberger, 1971, The pharmacology of 8-chloro-ll-(4-methyl-l-piperazinyl)-5h-dibenzo[b,e ] [ 1,4 ] diazepine (clozapine), I1 Farmaco 26,603. U'Prichard, D.C., D.A. Greenberg and S.H. Snyder, 1977, Binding characteristics of a radiolabeled agonist and antagonist at central nervous system noradrenergic s-receptors, Mol. Pharmacol. 13, 454. Zivkovic, B., A. Guidotti, A. Revuelta and E. Costa, 1975, Effect of thioridazine, clozapine and other antipsychotics on the kinetic state of tyrosine hydroxylase and on the turnover rate of dopamine in striatum and nucleus accumbens, J. Pharmacol. Exptl. Therap. 194, 37.
European Journal of Pharmacology, 52 (1978) 225--230 © Elsevier/North-Holland Biomedical Press
COMPARATIVE EFFECTS OF CLOZAPINE AND a-ADRENOCEPTOR BLOCKING DRUGS ON REGIONAL NORADRENALINE METABOLISM IN RAT BRAIN * BRIAN A. McMILLEN and PARKHURST A. SHORE Department of Pharmacology, University of Texas Health Science Center, Dallas, Texas 75235, U.S.A.
Received 4 April 1978, revised MS received 7 July 1978, accepted 3 August 1978
B.A. McMILLEN and P.A. SHORE, Comparative effects of clozapine and o~-adrenoceptor blocking drugs on regional noradrenaline metabolism in rat brain, European J. Pharmacol. 52 (1978) 225--230. Clozapine increased brain noradrenaline (NA) metabolism, as indicated by changes in 3-methoxy-4-hydroxyphenylglycol sulfate content, in brain regions corresponding to the predominance of a- over ~-receptors, i.e., hypothalamus, medulla, midbrain and cortex, but not corpus striatum or cerebellum. Phenoxybenzamine had a stronger effect in the hypothalamus than did clozapine, but did not change cortical NA metabolism within a 60 min treatment time; however, cortical NA metabolism was increased 150 min after phenoxybenzamine. The delayed effect of phenoxybenzamine may be due to either a poor affinity for some central receptors or a slow rate of entry into certain brain regions. Thioridazine and the benzodioxane, dibozane, had regional effects similar to clozapine. The similarity between clozapine and dibozane in their effects on regional brain NA metabolism may reflect a preference for presynaptic a-receptors. It is unlikely that the antipsychotic activity of clozapine is related to a specific adrenolytic effect, but may reflect the combined activity of this drug on several transmitter systems. Clozapine
Phenoxybenzamine
Noradrenaline
1. I n t r o d u c t i o n T h e a n t i p s y c h o t i c drug, c l o z a p i n e , has b e e n the s u b j e c t o f c o n s i d e r a b l e research i n t e r e s t as it causes little or n o e x t r a p y r a m i d a l d y s f u n c tion (Stille et al., 1 9 7 1 ; Hippius, 1975), a l t h o u g h the d r u g e n h a n c e s d o p a m i n e (DA) t u r n o v e r in the n e o s t r i a t a l a n d m e s o l i m b i c dopaminergic systems (And6n and Stock, 1 9 7 3 ; Bartholini et al., 1 9 7 2 ; Zivkovic e t al., 1975). H o w e v e r , c l o z a p i n e has e f f e c t s o n central t r a n s m i t t e r s y s t e m s o t h e r t h a n DA. C o s t a ( 1 9 7 7 ) has r e p o r t e d t h a t 7 - a m i n o b u t y r i c acid ( G A B A ) t u r n o v e r t i m e is d e c r e a s e d b y clozapine. Whole brain n o r a d r e n a l i n e (NA) m e t a b * Supported by Public Health Service Grant MH05831.
Antipsychotic
olism in rats is increased b y doses o f clozap i n e within t h e clinical dose range (Bartholini et al., 1 9 7 3 ; Keller e t al., 1 9 7 3 ) a n d c l o z a p i n e has c o n s i d e r a b l e a n t i m u s c a r i n i c e f f e c t s in several in vitro t e s t s y s t e m s (Miller a n d Hiley, 1 9 7 4 ; S n y d e r et al., 1974). T h e s e p u t a t i v e t r a n s m i t t e r s as well as D A are t h o u g h t t o be involved in e x t r a p y r a m i d a l a n d limbic functions (see Costa, 1977). It is i n t e r e s t i n g t h a t thioridazine, another atypical antipsychotic drug, increases brain N A m e t a b o l i s m t o t h e s a m e e x t e n t as c l o z a p i n e w h e n a d m i n i s t e r e d in a 50% g r e a t e r dose (Keller et al., 1 9 7 3 ) , a p o t e n c y d i f f e r e n c e w h i c h c o r r e s p o n d s to the d i f f e r e n c e in clinical doses. I t is c o n c e i v a b l e t h a t c l o z a p i n e e x e r t s its a n t i p s y c h o t i c actions, in p a r t at least, t h r o u g h a n o r a d r e n e r g i c m e c h a n i s m . In an a t t e m p t t o
226 determine whether clozapine's central effects on NA metabolism represent a novel action or are simply those shared by known blockers of a-adrenoceptors, the drug was compared with phenoxybenzamine and other u-receptor antagonists for their effects on NA metabolism in specific brain regions. Changes in NA metabolism were determined by the effects of these drugs on the regional concentration of the NA metabolite, 3-methoxy-4-hydroxyphenylglycol sulfate (MOPEG-SO4). 2. Materials and methods 2.1. Animals and drugs Female Sprague-Dawley rats, 2 0 0 - - 2 3 0 g (Holtzman), maintained on a 12 h light/dark cycle, were treated with drugs i.p. and killed at the times noted in the tables. Phenoxybenzamine (Smith, Kline and French Laboratories) was dissolved (40 mg/ml) in propyleneglycol and diluted to 10 mg/ml with water. Clozapine (Sandoz Pharmaceuticals) was dissolved (10 mg/ml) in 0.1 N HC1 and diluted to 3.0 mg/ml with water. Dibozane (McNeil Laboratories) was dissolved (10 mg/ml) in 0.1 N HC1. Thioridazine concentrate (Sandoz Pharmaceuticals) was diluted with water to 10 mg/ml. All doses refer to the free base. 2.2. MOPEG-S04 assay Animals were killed by chloroform asphyxiation and the brains were rapidly removed and chilled in ice-cold saline. The brains were dissected into 6 regions (Glowinski and Iversen, 1966): cerebellum, medulla-pons, hypothalamus, corpus striatum, cortex-hippocampus and midbrain. The brain regions from 2--4 animals were pooled and frozen until assayed the next day. The procedure of Meek and Neff (1972) for the fluorometric assay of MOPEC-SO4 was used with minor modifications. Briefly, brains were homogenized in 0.2 M ZnSO4, mixed with 0.2 M Ba(OH)2 and centrifuged at 3 0 , 0 0 0 × g for 10 min as described by Meek and Neff (1972). The supernatants were adjusted to pH 7.5--7.7
B.A. McMILLEN, P.A. SHORE with 0.2 M ZnSO4 and centrifuged again (T.K. Keeton, personal communication). The resulting supernatant fractions were deeantcd-e~t~ 5.0 mm X 5.0 cm DEAE Sephadex A-25 anion exchange columns (Pharmacia). After the supernatants passed through, the columns were washed with 4.0 ml of 0.06 N HC1. MOPEG-SO4 was eluted with 0.15 N HC1. We found that increasing the amounts of tissue increased the rate of MOPEG-SO4 elution. Therefore, with cortical samples (pooled from two animals), the first 2.0 ml of eluate were discarded while with the smaller brain regions (pooled from 4 animals) or with MOPEG-SO4 standards (Ro 4-6028, Hoffman-LaRoche), substituting water for tissue, the first 3.0 ml were discarded. In each case the subsequent 4.0 ml of eluate contained all of the MOPEGSO4. To the 4.0 ml of eluate were added 0.2 ml of 1.0% cysteine and 0.2 ml 60% HC104. After mixing, 2.0 ml were taken for fluorphor development. Samples were heated in a 100°C water bath for 12 min and cooled in tap water. Freshly redistilled ethylenediamine (0.3 ml) was added, mixed thoroughly, and the samples heated in a boiling water bath for 5 min. After the samples cooled to room temperature, fluorescence was determined in an Aminco-Bowman spectrofluorometer at 325 n m / 4 7 0 nm (excitation/emission, uncorrected instrument values). Relative fluorescence was compared to a standard curve of MOPEG-SO4 dissolved in 0.15 N HC1 which had been previously passed through a DEAE Sephadex A-25 column. This treatment of the acid enhanced the fluorescence of MOPEG-SO4 standards, suggesting that the columns had removed an interfering impurity. All values were corrected for recovery of MOPEG-SO4 which averaged 88 + 9%. 3. Results
3.1. Effects o f clozapine and phenoxybenzamine on regional brain NA metabolism Clozapine was used in doses of 3 and 6 mg/ kg, a range comparable to the daily clinical
ANTIPSYCHOTIC AND ADRENOLYTIC DRUGS
227
dose. The dose of 6 mg/kg has been reported to increase whole brain MOPEG-SO4 levels by 25% in 2 h (Keller et al., 1973). In the present study, a shorter (60 min) treatment was used to help elucidate regional effects before a maximal accumulation of MOPEGSO4 could occur. Phenoxybenzamine was used in a dose of 40 mg/kg after preliminary experiments indicated that lower doses do n o t saturate a-receptors. For example 17 h after 25 mg/kg of phenoxybenzamine, whole brain MOPEG-SO4 concentrations increased significantly from 0.157 /zg/g to 0.200 /zg/g. If an additional 40 mg/kg was administered 2.5 h before killing the animals (14.5 h after the first dose), MOPEG-SO4 levels increased to 0.403 #g/g. Thus the 40 mg/kg dose of phenoxybenzamine was used in the current study. This dose of phenoxybenzamine was previously reported to cause a 47% increase of MOPEG-SO4 concentrations in rat whole brains (McMillen and Shore, 1977). The data in table 1 show that 6.0 mg/kg clozapine increased MOPEG-SO4 concentrations in the hypothalamus, medulla, midbrain and cortex. This result is in harmony with the
whole brain dose--response curve reported by Keller et al. (1973). Phenoxybenzamine produced similar regional effects except for the cortex where MOPEG-SO4 levels were unchanged. Maintaining the clozapine-treated animals within 1.0°C of normal b o d y temperature (ambient temperature 32°C), instead of allowing the 2.0--2.5 ° decrease seen at room temperature, slightly enhanced the effects of clozapine on NA metabolism. The most striking differences between the two drugs 60 min after administration were in the hypothalamus, where phenoxybenzamine had a greater effect than did clozapine, and in the cortex, where clozapine, b u t n o t phenoxybenzamine, significantly increased NA metabolism. 3.2. Effects o f various drugs on MOPEG-S04 concentrations in hypothalamus and cortex To examine further the apparent differences between phenoxybenzamine and clozapine in their relative effects on hypothalamus and cortex, the effects of other drugs on these two areas were studied. The effects of thi-
TABLE 1 T h e effects of c l o z a p i n e a n d p h e n o x y b e n z a m i n e o n regional b r a i n n o r e p i n e p h r i n e m e t a b o l i s m . N u m b e r s in p a r e n t h e s e s r e p r e s e n t t h e n u m b e r o f e x p e r i m e n t s . 4 brains were dissected a n d t h e regions p o o l e d for e a c h e x p e r i m e n t . All drugs were i n j e c t e d 60 m i n b e f o r e sacrifice. C o r t e x includes h i p p o c a m p u s . MOPEG-SO4 pg/g _+S.E.M.
C o n t r o l (9) 3.0 m g / k g c l o z a p i n e (5) 6.0 m g / k g c l o z a p i n e (6) 6.0 m g / k g c l o z a p i n e + 32°C (6) 40.0 mg/kg phenoxybenzamine (8)
Hypothalamus
Striatum
Midbrain
Medulla
Cerebellum
Cortex
0.224 -+0.034 0.289 _+0.023 0 . 3 4 7 1~3 -+0,016 0,408 2 _+0,037 0.478 2 -+0.039
0.114 -+0,022 0.139 +0.009 0.125 +0.022 0.150 -+0.017 0.147 _+0.034
0.176 -+0.008 0.208 -+0.023 0.223 -+0.030 0.330 2 -+0.032 0.268 2 -+0.021
0.150 +0.015 0.190 -+0.021 0.192 -+0.017 0.261 2 -+0.045 0.204 4 -+0.009
0,028 +0.006 0.039 -+0.004 0.033 _+0.004 0,031 _+0.005 0,032 -+0.006
0.123 _+0.006 0.144 -+0.017 0.177 1 _+0.024 0 . 1 9 2 2,3 -+0.020 0.133 _+0.013
1 p < 0 . 0 5 ; 2 p < 0.01 c o m p a r e d to c o n t r o l ( D u n n e t t ' s t-test). 3 Differs f r o m p h e n o x y b e n z a m i n e P < 0,05 ( S t u d e n t ' s t-test); 4 differs f r o m c o n t r o l P < 0.01 ( S t u d e n t ' s t-test).
228
B.A. M c M I L L E N , P.A. S H O R E
oridazine, another atypical antipsychotic, and dibozane, a benzodioxane a-receptor blocker, were compared with clozapine and phenoxybenzamine (table 2). The dose of thioridazine was chosen because of an earlier report that a 50% greater molar dose of thiorodazine is needed to increase whole brain MOPEG-SO4 levels to those seen with clozapine (Keller et al., 1973). The dose of dibozane was chosen after a preliminary dose--response curve for whole brain MOPEG-SO4 levels indicated that 20 mg/kg of dibozane was a b o u t equivalent to 40 mg/kg of phenoxybenzamine (McMillen and Shore, 1977). Table 2 shows that both thioridazine and dibozane had effects similar to clozapine in that both hypothalamic and cortical NA metabolism were increased. In control animals and those treated with clozapine, thioridazine or dibozane, the ratios for MOPEG-SO4 concentrations in hypothalamus and cortex ranged from 1.96 to 2.50. Phenoxybenzamine did n o t increase MOPEG-SO4 levels in the cortex within this 60 min treatment time, the hypothalamus to cortex ratio for MOPEG-SO4 content being 3.34. However, longer (150 min) treatment with phenoxybenzamine caused a significant increase
in cortical NA metabolism such that the hypothalamus to cortex ratio changed to 2.16. 4. Discussion
The further effect of a second dose of phenoxybenzamine on MOPEG-SO4 accumulation suggests that central a-receptors are not completely blocked by a 25 mg/kg dose of phenoxybenzamine and we have no evidence to suggest that even 40 mg/kg of phenoxybenzamine has saturated a-receptors. This may be due to either a poor affinity of phenoxybenzamine for some central receptors or to poor accessibility of some brain regions to the drug. The difficulty in achieving saturation of central a-receptors with a blocking agent thought to exert an irreversible blockade and the delayed increase of MOPEG-SO4 concentration in the cortex (table 2) suggest that observations made while using phenoxybenzamine as a central a-blocker should be interpreted with care because of the apparent difficulty in blocking all central a-receptors with this drug. Low doses of clozapine and a shorter time course for the accumulation of MOPEG-SO4
TABLE 2 T h e effects o f a - a d r e n o c e p t o r a n t a g o n i s t s o n MOPEG-SO4 c o n t e n t o f rat h y p o t h a l a m u s a n d cortex. R a t s were i n j e c t e d w i t h c l o z a p i n e (6.0 m g / k g i.p.), t h i o r i d a z i n e (10 m g / k g i.p.), d i b o z a n e (20 m g / k g i.p.) or p h e n o x y b e n z a m i n e (40 m g / k g i.p.) a n d killed 60 rain later. N u m b e r s in p a r e n t h e s e s r e p r e s e n t t h e n u m b e r of exp e r i m e n t s . C o r t e x includes t h e h i p p o c a m p u s . T h e r a t i o o f t h e MOPEG-SO4 c o n t e n t s in t h e t w o regions is s h o w n in t h e last c o l u m n . MOPEG-SO4 pg/g + S.E.M.
Control Clozapine Thioridazine Dibozane Phenoxybenzamine 60 m i n 150min
Hypothalamus
Cortex
0.252 0.347 0.380 0.397
0.120 0.177 0.152 0.171
-+ 0 . 0 2 8 (13) -+ 0 . 0 1 6 (6) 3 + 0 . 0 1 4 (6) I + 0 . 0 3 7 (7) 2
0 . 4 6 2 + 0 . 0 2 7 (13) 2 0.431+0.044 (9) 2
I Differs f r o m c o n t r o l , P < 0.05 ( D u n n e t t ' s t-test). 2 Differs f r o m c o n t r o l , P < 0.01 ( D u n n e t t ' s t-test). 3 Differs f r o m c o n t r o l , P < 0.01 ( S t u d e n t ' s t-test).
H/C -+ 0 . 0 0 5 (13) + 0 . 0 2 4 (6) 2 -+ 0 . 0 0 9 (6) 3 + 0 . 0 1 6 (8) 2
2.10 1.96 2.50 2.32
0 . 1 3 8 + 0 . 0 1 0 (12) 0 . 2 0 0 + 0 . 0 1 5 (7) 2
3.34 2.16
ANTIPSYCHOTIC AND ADRENOLYTIC DRUGS were chosen in order to determine whether this drug may have regional effects which might otherwise be obscured by the large increases of whole brain MOPEG-SO4 concentrations elicited by higher doses of clozapine (Keller et al., 1973). The data in table 1 show that the regional clozapine-induced increase of NA metabolism parallels the known distribution of a-receptors (U'Prichard et al., 1977). No significant changes occur in the striatum and cerebellum where ~-receptors predominate (Bylund and Snyder, 1976). Both clozapine and thioridazine resemble dibozane in their effects on hypothalamic and cortical NA metabolism. This may reflect a relative preference by clozapine for presynaptic a-receptors, as the benzodioxanes, piperoxane and dibozane, are considered to be preferential presynaptic antagonists, while phenoxybenzamine may have a relative preference for postsynaptic receptors (Cubeddu et al., 1974; And~n et al., 1976; Franklin and Herberg, 1977). Clozapine lowers NA and metaraminol in the rat heart, an effect which is inhibited by ganglionic blockade (Dorris and Shore, 1976). Such an effect may be interpreted as suggestive of presynaptic a-inhibition (see Langer, 1977). Clozapine is an inhibitor of NA-stimulated adenylate cyclase in rat limbic forebrain at concentrations much lower than obtained from clinical doses of clozapine (Blumberg et al., 1976), there being no correlation between antipsychotic efficacy and inhibition of NA-stimulated c-AMP formation. In h a r m o n y with the report by Blumberg and coworkers for a central a-receptor effect of clozapine is the observation that clonidine inhibits the brain NA lowering effect of clozapine (Bartholini et al., 1973) as well as phenoxybenzamine (Braestrup and Nielsen, 1976). From the findings presented here it may be seen that the atypical antipsychotic drugs, clozapine and thioridazine, do n o t appear to exert effects on brain NA metabolism that differ significantly from known a-blockers which have n o t been reported as having antipsychotic activity.
229 Although clozapine is an effective antipsychotic drug and elevates striatal and mesolimbic DA metabolism, the extrapyramidal dysfunctions in man and animals observed with commonly employed neuroleptics do not occur with clozapine (Stille et al., 1971; Hippius, 1975). In addition, clozapine is a poor inhibitor of the behavioral effects of amphetamine and apomorphine (Stille et al., 1971). From all that is now known of clozapine's effects on various transmitters, it is clear that the effects of clozapine on central nervous system functions are complex. If schizophrenia is considered the result of a functional imbalance between several neuronal systems, including the dopaminergic system, then it is possible that classical neuroleptics restore the balance by a strong inhibition of DA receptors. Clozapine and other atypical antipsychotic drugs may help restore normal behavior by the sum of their effects on multiple neuronal systems including DA, NA, GABA and acetylcholine-containing neurons.
Acknowledgement The authors thank Ms. Karen Johnson for her skilled technical assistance.
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