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In vivo identification of muscarinic cholinergic receptor binding in rat brain
170
Brain Research, 80 (19;4i i )i) 17(', Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
In vivo identification of muscarinic cholinergic receptor binding in rat brain
H E N R Y I. Y A M A M U R A ,
M I C H A E L J. K U H A R AND S O L O M O N H. S N Y D E R
Departments of Pharmacology and Experimental Therapeutics, and Psychiatry and the Behavioral Sciences, Baltimore, Md. 21205 (U.S.A.) (Accepted July 23rd, 1974)
Muscarinic cholinergic receptor binding has been identified biochemically in vitro by the binding of cholinergic agonists 7,'5 and antagonists 4-9.14.t~.19 ~. 3-Quinuclidinyl benzilate ( Q N B ) i s a potent central muscarinic antagonist1, ':~. Using [SH]QNB. we have identified its binding selectively to muscarinic cholinergic receptor sites in mammalian brain 19,2° and guinea pig ileum '~1. Specific [aH]QNB binding in the brain and guinea pig ileum in ~,itro is displaced by muscarinic cholinergic agonists and antagonists m proportion to their pharmacological potency but is not affected by nicotinic cholinergic and other classes of drugs '~0,21. In the monkey brain. [;~H]QN B binding is highest in the extrapyramidal areas tputamen and caudate nucleus ~. which contain the highest acetylcholine levels in the brain, is intermediate in the hippocampus and lowest in the cerebellum and white matter areas in which only negligible binding of [3H]QNB associated specifically with muscarlnic receptor can be identifiedl.~ We sought to identify muscarinic cholinergic receptor binding in vivo in order to evaluate the feasibility of light autoradiographic localization of [3H]QNB and to evaluate alterations in receptor binding after pharmacological and physiological manipulations. Several workers have measured the accumulation of cholinergic drugs in mammalian brain, but most of the accumulation observed was rather evenly distributed throughout the brain 2,3,IT,Is. We here report the accumulation of [3H]QNB by various regions of rat brain in a fashion which indicates that QNB selectively labels the muscarinic cholinergic receptor in vivo. Male Sprague-Dawley rats. 150-200 g, were anesthetized with ether and each received a rapid injection, via the tail vein. of [aH]QNB (4 Ci/mmole) in saline. Rats received various concentrations of [3H]QNB in 0.3 ml saline and were sacrificed at different time intervals. Rats were decapitated, their brains and peripheral tissues rapidly removed and placed in vials containing 1 ml of NEN solubilizer tProtosol) and incubated overnight at 37 ~C. Each vial contained 15 ml Hvdromix I Yorktown Research, New York) fluor and was counted in a Packard Tri-Carb Model No. 3375 by liquid scintillation spectroscopy with an efficiency of 20 ~,. In vivo metabolism of [3H]QNB was determined in samples in which rats received 28 nmoles of [aH]QNB and were sacrificed 2 h after injection. A small portion of the brain was dissected and homogenized in 3 ml of 0.2 °/o,acetic acid in 96 "/~,,ethanol, left
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Fig. I. Time course of [aH]QNB accumulation into various regions of rat brain. Rats were given 60/;Ci of [:3HIQNB (4 Ci/mmole) intravenously via the tail vein in 0.3 ml of saline and sacrificed after various time intervals. Brain regions were placed in scintillation vials containing 1 ml of NEN solubilizer (Protosol) and incubated at 37 'C until solubilized. Each point represents the mean of at least 3 animals which varied by no more than 10-15~o. at a m b i e n t temperature for 30 min and centrifuged at 20,000 × g For 20 rain. A n aliquot of the s u p e r n a t a n t was c h r o m a t o g r a p h e d on Silica-gel with fluorescent indicator with authentic [aH]QNB in two solvent systems: heptane t o l u e n e - d i e t h y l a m i n e (2:1:1 and 4:1:1) and n - b u t a n o l - a c e t i c acid-water (4:1:1). Radiolabeled Q N B extracted from the brain homogenate migrated as a single peak with authentic [3H]QN B and unlabeled Q N B in both solvent systems. [3H]QNB is accumulated rapidly by the regions of the rat brain examined with peak levels in all regions at 2.5 rain, the first time p o i n t that we measured. In the cerebellum, [3H]QNB levels then fall so rapidly that by 10 rain they are only a b o u t half of peak values. Thereafter cerebellar Q N B levels decline to values a b o u t one-fifth of peak levels after 60 min a n d then remain c o n s t a n t for up to 24 h (Fig. 1). By contrast,
TABLE 1 REGIONAL
ACCUMULATION
OF
[3H]QNB I N
SALINE
AND
ATROPINE
PRETREATED
RAT
BRAIN
Rats were pretreated 30 rain either with saline or atropine (50 mg/kg, i,m.) and then injected with 28 nmoles of [3H]QNB (4 Ci/mmole) in a 0.3 ml volume via the tail vein and sacrificed 1 h after [3H]QNB injection. Each tissue was placed in a vial containing I ml of Protosol and incubated for 48 h at 37 ~C. Fifteen ml of Hydromix fluor was added to each vial and counted 24 b later by liquid scintillation spectrometry. Efficiency of counting was 20~. Values for saline treated animals are the mean 4 S.E.M. for 4 rats, while individual values for 2 atropine pretreated rats are presented. Brain regions
Corpus striatum Cerebral cortex Hippocampus Hypothalamus Medulla-pons Cerebellum
/3H/QNB accumulated (counts~rain~rag tissue) Saline treated
Atropine treated (50 mg/kg, i.m.)
621 ~ 16 526 ! 20 504 I 14 191 ~ 8 159 [ 5 92 !: 5
164; 148 146; 136 178:178 106; 105 109; 117 101: 90
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Fig. 2. Saturability of [3H]QNB accumulation with increasing amounts of [3HIQNB in 3 brain regions of the rat. Rats were injected with various concentrations of [aH]QNB intravenously via the tail vein as in Fig. 1 and sacrificed after I b. Each point represents the mean of 3-8 animals which varied by no more than 10-15~.
in the corpus striatum and hippocampus there is no reduction in [3H]QNB levels between 2.5 rain and 4 h. Between 4 and 24 h. [3H]QNB levels in the corpus striatum and hippocampus decline about 25 o . Highest [3H]QNB accumulation occurs in the corpus striatum, whose values are abouc 25-30~., greater than those of the hippocampus between I and 4 h (Figs. 1-3). The order of [3H]QNB accumulation in lvro in 6 discrete areas of the rat brain is as follows: corpus striatum > cerebral cortex :~ hippocampus .~ hypothalamus midbrain ports > cerebellum (Table I). This closely resembles the regional distribution of [3H]QNB binding in vitro °-°. Specific [aH]QNB binding to membrane fragments of brain tissue 07 vitro is a saturable process with a half-maximal binding value of about 0.1-0.4 nM "°. To determine whether [3H]QNB binding in vivo is saturable, we rejected rats intravenously with 2.8-70 nmoles of [3H]QNB. Corpora striata, hippocampi and cerebella were assayed for accumulated [3H]QNB 60 min after injection (Fig. 2). [3H]QNB accumulation is clearly saturable in the corpus striatu m and hippocampus. In the hippocampus there is a plateau of binding between 42 and 70 nmoles of injected [3H]QNB. In the corpus striatum, although [aH]QNB accumulation is saturating it does not completely plateau at these doses. This is not unexpected, because values presented are for total binding without subtracting nonspecific QNB accumulation. [3H]QNB binding in the cerebellum is much less than in the other two brain regions being only one-sixth the binding in corpus striatum. Cerebellar [3H]QNB accumulation saturates to a plateau between 42 and 70 nmoles of injected [3H]QNB. If it is assumed that the amount of [aH]QNB accumulated in the various brain regions at a dose of 42 nmoles approximately reflects the maximal number o f atropinedisplaceable QNB binding sites, then the calculated numbers of muscarinic sites for the corpus striatum and hippocampus respectively are about 280 and 190 pmoles/g of wet weight (values obtained from Fig. 31. Because a major portion of cerebellar
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173
TABLE 11 TISSUE RADIOACTIVITY ASSOCIATED WITH THE PARTICULATE
FRACTION FROM RAT BRAIN
Rats were pretreated with either saline or atropine (50 mg/kg, i.m.) 30 rain prior to intravenous injection of [3H]QNB (28 nmoles) via the tail vein and sacrificed after 1 h. A 10~ homogenate of the cerebral cortex was prepared in 0.05 M Na,K-phosphate buffer (pH 7.4) and then centrifuged at 20,000 ;~ g for 20 rain. An aliquot of the supernatant and of the buffered reconstituted pellet was counted in a Tricarb liquid scintillation spectrometer at an efficiency of 30%. Experiment
Treatment
1 2 3 4
Tissue radioactivity (counts/rain × 10 3//i'aetion)
saline saline atropine atropine
Supernatant
Pellet
42 28 36 31
234 191 27 28
Q N B b i n d i n g might not involve muscarinic receptors, such a calculation has not been made for this brain region. The n u m b e r of Q N B b i n d i n g sites determined in vitro in whole brain homogenates is a b o u t 65 pmoles/g wet weight of tissue 2°, which is only 2 5 - 3 0 % of the n u m b e r o f sites calculated from in vivo experiments, i n previous experiments with m e m b r a n e fragments of the brain, some portions o f the tissue are discarded so that values for the total n u m b e r of sites may be underestimated. In the present study, since approximately 80 % of the tissue radioactivity is associated with the particulate fraction after homogenization a n d centrifugation, the above values for the corpus striatum a n d h i p p o c a m p u s may be overestimated (Table ll). If [~H]QNB a c c u m u l a t i o n in various brain regions involves muscarinic receptor sites, it should be possible to prevent this b i n d i n g by prior t r e a t m e n t with potent (A) 3H-QNB I28nmoleS,ilV )
(B) 3H-QNB I42 nmoles ,iVl)
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Fig. 3. Effect of atropine pretreatment on [3H]QNB accumulation in 3 brain regions of the rat. Different doses of atropine were administered intramuscularly 30 rain prior to intravenous injections of [:~H]QNB. Rats were sacrificed after 1 h and treated as stated in Fig. 1. Each histogram represents the mean of at least 4 animals which varied by no more than 10-15%.
174
S H O R T ( :OM M t:*\ I( 'A IIt)!'-;S
muscarinic antagonists. Accordingly, rats were injected either intraperitoneally o, intramuscularly with a variety of drugs 30 rain before receiving an intravenous injection of [3H]QNB. They were killed 60 rain later and their corpora striat~.,, hippo~ campi and cerebella assayed for [3H]QNB (Fig. 3). As little as ! mg/kg atropine reduces [3H]QNB accumulation in the corpus striatum and hippocampus by about 30-35 ~. Higher doses of atropine produce a progressively greater inhibitio~ so that with 25 mg/kg of atropine [aH]QNB accumulation in the corpus striatmn and hippocampus is reduced by 7 0 - 8 0 ~ . At the three doses examined (1. 5 or ?5 mg/kg~ atropine fails to reduce [3H]QNB accumulation in the cerebellum, suggesting that cerebellar [3H]QNB accumulation may not involve binding to muscarinic receptor.~ even though it is saturable. Rats receiving 42 nmoles of [3H]QNB intravenously after pretreatment with 25 or 50 mg/kg of atropine show similar regional pattern., o l QN B binding although some differences are detected (Fig. 3B). Whereas 25 mg/kg of atropine reduces striatal binding after a 28 nmole [3H]QNB dose by 80'};, with the higher [~H]QNB dose (42 nmoles), the same amount of atropine lowers QN B binding b\. on l~ 60 9/oo.Similarly, while with the lower dose of [3H]QNB, 25 mg/kg of atropine reduces hippocampal binding by 70 ~, at a higher dose of [3H]QNB. the same amount of atropine lowers QNB binding by only 40'},(,. Increasing the atropine dose from 25 mg/kg to 50 mg/kg produces a further decline in [~H]QNB (42 nmoles) binding m the corpus striatum and hippocampus. However. even at 50mg/kg, atropine fails ~o lower [aH]QNB binding in the cerebellum The fact that the binding of higher doses of [3H]QNB is less susceptible to inhibition by atropine indicates that the effect of atropine on QNB binding is competitive and can be overcome by increasing amounts of QNB. To determine whether the influence of atropine on QNB binding represents a selective interaction with muscarinic receptors, rats were pretreated with either 5 mg/kg of mecamylamine, a nicotinic cholinergic antagonist, 50 mg/kg of phenobarbital, or 50 mg/kg of L-dihydroxyphenylatanine (L-dopa~, all intraperitoneally. After 30 rain rats received [ZH]QNB (28 nmotes) intravenously, were killed after 60 rain and their corpora striata, hypothalami, midbrain-pons, cerebral cortices, hippocampi and cerebella assayed for [3H]QNB. None of these drug treatments affect [:~H]QNB accumulation in any of the 6 brain regions examined, although signs of sedation are observed with pentobarbital and piloerection occurs with L-DOPA administration. In some animals, a variety of peripheral tissues were assayed for [:~H]QNB accumulation. [aH]QNB accumulation in the heart is reduced 80 o.,,, by pretreatmem with 5 mg/kg of atropine. By contrast [3H]QNB accumulation in the rat ileum. diaphragm, kidney, pineal and lung are unaffected by pretreatment with this dose of atropine. More extensive studies are required to assess whether the [aH]QNB in the heart indeed represent selective binding to muscarinic cholinerg~c receptors. In summary, several lines of evidence indicate that [aH]QNB accumulation in the brain predominantly involves muscarinic cholinergic receptor sites. The relative amounts of QNB binding in the brain regions examined parallels their relative number of muscarinic cholinergic receptor sites determined bv in vitro [3H]QNB binding studies 19,z° and neurophysiologic investigations ~°-12. QNB accumulation in vivo
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175
can be prevented by relatively low doses of atropine but not by a nicotinic cholinergic a n t a g o n i s t or noncholinergic drugs. The a p p a r e n t maximal n u m b e r of b i n d i n g sites for QN B in the corpus striatum a n d h i p p o c a m p u s appears to be within an order of m a g n i t u d e of the n u m b e r of sites calculated from in ritro studies. The very slow decline of [aH]QNB levels in the corpus striatum and h i p p o c a m p u s indicates that Q N B binds tightly to the muscarinic receptor. This avid b i n d i n g has made feasible the a u t o r a d i o g r a p h i c localization of [3H]QNB in rat brain after i n t r a v e n o u s injection ( K u h a r a n d Y a m a m u r a , in preparation). Supported by U S P H S G r a n t s DA-00266, a grant of the J o h n A. H a r f o r d F o u n d a t i o n , N I M H R S D A A w a r d MH-33128 to S.H.S. a n d N I M H Special Research Fellowship MH-54777 to H.1.Y. We t h a n k Mr. James Clark for technical assistance.
1 ALBANUS, L., Central and peripheral effects of anticholinergic compounds, Acta pharmacol.
toxicol. (Kbk.), 28 (1970) 305-326. 2 ALBANUS, L., HAMMARSTROM, L., SUNDWALL, A., ULLBERG, S., AND VANGBO, B., Distribution and metabolism of '~H-atropine in mice, Acta physiol, scand., 73 (1968) 447 456. 3 ALBANUS, L., SUNDWALL, A., VANGBO, B., AND WINBLADH, B., The fate of atropine in the dog, Acta pharmacol, toxicol. (Kbk.), 26 (1968) 571-582. 4 BELD,A. J., XYt~ARIENS,E. J., Stereospecific binding as a tool in attempts to localize and isolate muscarinic receptors. Part II. Binding of ( ~)-Benzetimide, (--)-Benzetimide and atropine to a fraction from bovine tracheal smooth muscle and to bovine caudate nucleus, Europ. J. Pharmacol., 25 (1974) 203-209. 5 BURGEN, A. S. V., HILLY, C. R., AND YOUNG, J. M., The binding of 3H-propylbenzilylcholine mustard by longitudinal muscle strips from guinea pig small intestine, Brit. J. Pharmacol., 50 (1974) 145-15 I. 6 BURGEN, A. S. V., H1LEY, C. R., AND YOUNG, J. M., The properties of muscarinic receptors in mammalian cerebral cortex, Brit. J. Pharmacol., 51 (1974) 279-285. 7 FARROW,J. T., AND O'BRIEN, R. D., Binding of atropine and muscarone to rat brain fractions and its relation to the acetylcholine receptor, Molec. Pharmacol., 9 (1973) 33-40. 8 FEWTRELL,C., AND RANG,H. P., Distribution of bound 3H-benzilylcholine mustard in subcellular fractions of smooth muscle from guinea pig ileum, Brit. J. Pharmacol., 43 (1971) 417P-418P. 9 H~LE¥,C. R., AND BORGEN,A. S. V., The distribution of muscarinic receptor sites in the nervous system of the dog, J. Neurockem., 22 (1974) 159-162. 10 KRNJEVIC, K., Actions of drugs on single neurones in the cerebral cortex, Brit. Med. Bull., 21 (1965) 10-14. I 1 KRNJEVIC, K., AND PHILLIS, J. W., Pharmacological properties of acetylcholine sensitive cells in the cerebral cortex, J. Physiol. (Lond.), 166 (1963) 328-350. 12 MCLENNAN, H., AND YORK, D. H., Cholinergic mechanisms in the caudate nucleus, J. Physiol. (Lond.), 187 (1966) 163-175. 13 MEYERnOFFER,A., Absolute configuration of 3-quinuclidinyl benzilate and the behavioral effect in the dog of the optical isomers, J. Med. Chem., 15 0972) 994-995. 14 PATON, W. D. M., AND RANG, H. P., The uptake of atropine and related drugs by intestinal smooth muscle of the guinea pig in relation to acetylcholine receptors, Proc. roy. Soc. B, 1633 (1965) I 44. 15 SCHt.EIVER, L. S., AND ELDEFRAWl.M. E., Identification of the nicotinic and muscarinic acetylcholine receptors in subcellular fractions of mouse brain, Neuropkarmacol., 13 (1974) 53-63. 16 SOUDIJN,W., VANWIJNGAARDEN,I., AND ARtENS, E. J., Dexetimide, a useful tool in acetylcholine receptor localization, Europ. J. Pharmacol., 24 (1973) 43-48. 17 WINr~LADH,B, The fate of atropine in the puppy, Acta pharmacol, toxicol. (Kbk.), 32 (1973) 46-64. 18 WITTER, A., SLANGEN, J. L,, AND TERPSTRA, G. K., Distribution of :3H-methylatropine in rat brain, Neuropkarmacol., 12 (1973) 835-841.
19 YAMAMURA,H. i., KUHAR, M. J., GREENBERG, D., AND SNYDIER,S. H., MuscarinJ~: ct~oli~lerg~c receptor binding: Regional distribution in monkey brain, Brain Research, 66 (1974) 54I 546. 20 YAMAMURA,H. 1., AND SNYDER, S. H., Muscarinic cholinergic binding in rat brairL Pr¢,c. Jzc:r Acad Sci. (Wash.), 71 (1974) 1725-1729. 21 YAMAMURA,H. I., AND SNYDER,S. H., Muscarinic cholinergic receptor binding in the iongitudinai muscle of the guinea pig ileum with aH-quinuclidinyl benzilate, Molec. Pharmacol.~ (1974). in press;
Brain Research, 80 (19;4i i )i) 17(', Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
In vivo identification of muscarinic cholinergic receptor binding in rat brain
H E N R Y I. Y A M A M U R A ,
M I C H A E L J. K U H A R AND S O L O M O N H. S N Y D E R
Departments of Pharmacology and Experimental Therapeutics, and Psychiatry and the Behavioral Sciences, Baltimore, Md. 21205 (U.S.A.) (Accepted July 23rd, 1974)
Muscarinic cholinergic receptor binding has been identified biochemically in vitro by the binding of cholinergic agonists 7,'5 and antagonists 4-9.14.t~.19 ~. 3-Quinuclidinyl benzilate ( Q N B ) i s a potent central muscarinic antagonist1, ':~. Using [SH]QNB. we have identified its binding selectively to muscarinic cholinergic receptor sites in mammalian brain 19,2° and guinea pig ileum '~1. Specific [aH]QNB binding in the brain and guinea pig ileum in ~,itro is displaced by muscarinic cholinergic agonists and antagonists m proportion to their pharmacological potency but is not affected by nicotinic cholinergic and other classes of drugs '~0,21. In the monkey brain. [;~H]QN B binding is highest in the extrapyramidal areas tputamen and caudate nucleus ~. which contain the highest acetylcholine levels in the brain, is intermediate in the hippocampus and lowest in the cerebellum and white matter areas in which only negligible binding of [3H]QNB associated specifically with muscarlnic receptor can be identifiedl.~ We sought to identify muscarinic cholinergic receptor binding in vivo in order to evaluate the feasibility of light autoradiographic localization of [3H]QNB and to evaluate alterations in receptor binding after pharmacological and physiological manipulations. Several workers have measured the accumulation of cholinergic drugs in mammalian brain, but most of the accumulation observed was rather evenly distributed throughout the brain 2,3,IT,Is. We here report the accumulation of [3H]QNB by various regions of rat brain in a fashion which indicates that QNB selectively labels the muscarinic cholinergic receptor in vivo. Male Sprague-Dawley rats. 150-200 g, were anesthetized with ether and each received a rapid injection, via the tail vein. of [aH]QNB (4 Ci/mmole) in saline. Rats received various concentrations of [3H]QNB in 0.3 ml saline and were sacrificed at different time intervals. Rats were decapitated, their brains and peripheral tissues rapidly removed and placed in vials containing 1 ml of NEN solubilizer tProtosol) and incubated overnight at 37 ~C. Each vial contained 15 ml Hvdromix I Yorktown Research, New York) fluor and was counted in a Packard Tri-Carb Model No. 3375 by liquid scintillation spectroscopy with an efficiency of 20 ~,. In vivo metabolism of [3H]QNB was determined in samples in which rats received 28 nmoles of [aH]QNB and were sacrificed 2 h after injection. A small portion of the brain was dissected and homogenized in 3 ml of 0.2 °/o,acetic acid in 96 "/~,,ethanol, left
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Fig. I. Time course of [aH]QNB accumulation into various regions of rat brain. Rats were given 60/;Ci of [:3HIQNB (4 Ci/mmole) intravenously via the tail vein in 0.3 ml of saline and sacrificed after various time intervals. Brain regions were placed in scintillation vials containing 1 ml of NEN solubilizer (Protosol) and incubated at 37 'C until solubilized. Each point represents the mean of at least 3 animals which varied by no more than 10-15~o. at a m b i e n t temperature for 30 min and centrifuged at 20,000 × g For 20 rain. A n aliquot of the s u p e r n a t a n t was c h r o m a t o g r a p h e d on Silica-gel with fluorescent indicator with authentic [aH]QNB in two solvent systems: heptane t o l u e n e - d i e t h y l a m i n e (2:1:1 and 4:1:1) and n - b u t a n o l - a c e t i c acid-water (4:1:1). Radiolabeled Q N B extracted from the brain homogenate migrated as a single peak with authentic [3H]QN B and unlabeled Q N B in both solvent systems. [3H]QNB is accumulated rapidly by the regions of the rat brain examined with peak levels in all regions at 2.5 rain, the first time p o i n t that we measured. In the cerebellum, [3H]QNB levels then fall so rapidly that by 10 rain they are only a b o u t half of peak values. Thereafter cerebellar Q N B levels decline to values a b o u t one-fifth of peak levels after 60 min a n d then remain c o n s t a n t for up to 24 h (Fig. 1). By contrast,
TABLE 1 REGIONAL
ACCUMULATION
OF
[3H]QNB I N
SALINE
AND
ATROPINE
PRETREATED
RAT
BRAIN
Rats were pretreated 30 rain either with saline or atropine (50 mg/kg, i,m.) and then injected with 28 nmoles of [3H]QNB (4 Ci/mmole) in a 0.3 ml volume via the tail vein and sacrificed 1 h after [3H]QNB injection. Each tissue was placed in a vial containing I ml of Protosol and incubated for 48 h at 37 ~C. Fifteen ml of Hydromix fluor was added to each vial and counted 24 b later by liquid scintillation spectrometry. Efficiency of counting was 20~. Values for saline treated animals are the mean 4 S.E.M. for 4 rats, while individual values for 2 atropine pretreated rats are presented. Brain regions
Corpus striatum Cerebral cortex Hippocampus Hypothalamus Medulla-pons Cerebellum
/3H/QNB accumulated (counts~rain~rag tissue) Saline treated
Atropine treated (50 mg/kg, i.m.)
621 ~ 16 526 ! 20 504 I 14 191 ~ 8 159 [ 5 92 !: 5
164; 148 146; 136 178:178 106; 105 109; 117 101: 90
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Fig. 2. Saturability of [3H]QNB accumulation with increasing amounts of [3HIQNB in 3 brain regions of the rat. Rats were injected with various concentrations of [aH]QNB intravenously via the tail vein as in Fig. 1 and sacrificed after I b. Each point represents the mean of 3-8 animals which varied by no more than 10-15~.
in the corpus striatum and hippocampus there is no reduction in [3H]QNB levels between 2.5 rain and 4 h. Between 4 and 24 h. [3H]QNB levels in the corpus striatum and hippocampus decline about 25 o . Highest [3H]QNB accumulation occurs in the corpus striatum, whose values are abouc 25-30~., greater than those of the hippocampus between I and 4 h (Figs. 1-3). The order of [3H]QNB accumulation in lvro in 6 discrete areas of the rat brain is as follows: corpus striatum > cerebral cortex :~ hippocampus .~ hypothalamus midbrain ports > cerebellum (Table I). This closely resembles the regional distribution of [3H]QNB binding in vitro °-°. Specific [aH]QNB binding to membrane fragments of brain tissue 07 vitro is a saturable process with a half-maximal binding value of about 0.1-0.4 nM "°. To determine whether [3H]QNB binding in vivo is saturable, we rejected rats intravenously with 2.8-70 nmoles of [3H]QNB. Corpora striata, hippocampi and cerebella were assayed for accumulated [3H]QNB 60 min after injection (Fig. 2). [3H]QNB accumulation is clearly saturable in the corpus striatu m and hippocampus. In the hippocampus there is a plateau of binding between 42 and 70 nmoles of injected [3H]QNB. In the corpus striatum, although [aH]QNB accumulation is saturating it does not completely plateau at these doses. This is not unexpected, because values presented are for total binding without subtracting nonspecific QNB accumulation. [3H]QNB binding in the cerebellum is much less than in the other two brain regions being only one-sixth the binding in corpus striatum. Cerebellar [3H]QNB accumulation saturates to a plateau between 42 and 70 nmoles of injected [3H]QNB. If it is assumed that the amount of [aH]QNB accumulated in the various brain regions at a dose of 42 nmoles approximately reflects the maximal number o f atropinedisplaceable QNB binding sites, then the calculated numbers of muscarinic sites for the corpus striatum and hippocampus respectively are about 280 and 190 pmoles/g of wet weight (values obtained from Fig. 31. Because a major portion of cerebellar
SHORT COMMUNICATIONS
173
TABLE 11 TISSUE RADIOACTIVITY ASSOCIATED WITH THE PARTICULATE
FRACTION FROM RAT BRAIN
Rats were pretreated with either saline or atropine (50 mg/kg, i.m.) 30 rain prior to intravenous injection of [3H]QNB (28 nmoles) via the tail vein and sacrificed after 1 h. A 10~ homogenate of the cerebral cortex was prepared in 0.05 M Na,K-phosphate buffer (pH 7.4) and then centrifuged at 20,000 ;~ g for 20 rain. An aliquot of the supernatant and of the buffered reconstituted pellet was counted in a Tricarb liquid scintillation spectrometer at an efficiency of 30%. Experiment
Treatment
1 2 3 4
Tissue radioactivity (counts/rain × 10 3//i'aetion)
saline saline atropine atropine
Supernatant
Pellet
42 28 36 31
234 191 27 28
Q N B b i n d i n g might not involve muscarinic receptors, such a calculation has not been made for this brain region. The n u m b e r of Q N B b i n d i n g sites determined in vitro in whole brain homogenates is a b o u t 65 pmoles/g wet weight of tissue 2°, which is only 2 5 - 3 0 % of the n u m b e r o f sites calculated from in vivo experiments, i n previous experiments with m e m b r a n e fragments of the brain, some portions o f the tissue are discarded so that values for the total n u m b e r of sites may be underestimated. In the present study, since approximately 80 % of the tissue radioactivity is associated with the particulate fraction after homogenization a n d centrifugation, the above values for the corpus striatum a n d h i p p o c a m p u s may be overestimated (Table ll). If [~H]QNB a c c u m u l a t i o n in various brain regions involves muscarinic receptor sites, it should be possible to prevent this b i n d i n g by prior t r e a t m e n t with potent (A) 3H-QNB I28nmoleS,ilV )
(B) 3H-QNB I42 nmoles ,iVl)
CORPUS STRIATUM
CORPUS 5TRLATUM
300
~.:
~
o
HIPPOCAMPUS
[~
HIPPOCAMPUS
zoo
~E ~-
200
CER£BELLUM
o
~-~ ~P ~
ATROPIN F ( mglk 9 , Lm )
CEREBELLUM
r--1 R25 c--1 O 50 ATROP INE (mg/kg ,Jim )
Fig. 3. Effect of atropine pretreatment on [3H]QNB accumulation in 3 brain regions of the rat. Different doses of atropine were administered intramuscularly 30 rain prior to intravenous injections of [:~H]QNB. Rats were sacrificed after 1 h and treated as stated in Fig. 1. Each histogram represents the mean of at least 4 animals which varied by no more than 10-15%.
174
S H O R T ( :OM M t:*\ I( 'A IIt)!'-;S
muscarinic antagonists. Accordingly, rats were injected either intraperitoneally o, intramuscularly with a variety of drugs 30 rain before receiving an intravenous injection of [3H]QNB. They were killed 60 rain later and their corpora striat~.,, hippo~ campi and cerebella assayed for [3H]QNB (Fig. 3). As little as ! mg/kg atropine reduces [3H]QNB accumulation in the corpus striatum and hippocampus by about 30-35 ~. Higher doses of atropine produce a progressively greater inhibitio~ so that with 25 mg/kg of atropine [aH]QNB accumulation in the corpus striatmn and hippocampus is reduced by 7 0 - 8 0 ~ . At the three doses examined (1. 5 or ?5 mg/kg~ atropine fails to reduce [3H]QNB accumulation in the cerebellum, suggesting that cerebellar [3H]QNB accumulation may not involve binding to muscarinic receptor.~ even though it is saturable. Rats receiving 42 nmoles of [3H]QNB intravenously after pretreatment with 25 or 50 mg/kg of atropine show similar regional pattern., o l QN B binding although some differences are detected (Fig. 3B). Whereas 25 mg/kg of atropine reduces striatal binding after a 28 nmole [3H]QNB dose by 80'};, with the higher [~H]QNB dose (42 nmoles), the same amount of atropine lowers QN B binding b\. on l~ 60 9/oo.Similarly, while with the lower dose of [3H]QNB, 25 mg/kg of atropine reduces hippocampal binding by 70 ~, at a higher dose of [3H]QNB. the same amount of atropine lowers QNB binding by only 40'},(,. Increasing the atropine dose from 25 mg/kg to 50 mg/kg produces a further decline in [~H]QNB (42 nmoles) binding m the corpus striatum and hippocampus. However. even at 50mg/kg, atropine fails ~o lower [aH]QNB binding in the cerebellum The fact that the binding of higher doses of [3H]QNB is less susceptible to inhibition by atropine indicates that the effect of atropine on QNB binding is competitive and can be overcome by increasing amounts of QNB. To determine whether the influence of atropine on QNB binding represents a selective interaction with muscarinic receptors, rats were pretreated with either 5 mg/kg of mecamylamine, a nicotinic cholinergic antagonist, 50 mg/kg of phenobarbital, or 50 mg/kg of L-dihydroxyphenylatanine (L-dopa~, all intraperitoneally. After 30 rain rats received [ZH]QNB (28 nmotes) intravenously, were killed after 60 rain and their corpora striata, hypothalami, midbrain-pons, cerebral cortices, hippocampi and cerebella assayed for [3H]QNB. None of these drug treatments affect [:~H]QNB accumulation in any of the 6 brain regions examined, although signs of sedation are observed with pentobarbital and piloerection occurs with L-DOPA administration. In some animals, a variety of peripheral tissues were assayed for [:~H]QNB accumulation. [aH]QNB accumulation in the heart is reduced 80 o.,,, by pretreatmem with 5 mg/kg of atropine. By contrast [3H]QNB accumulation in the rat ileum. diaphragm, kidney, pineal and lung are unaffected by pretreatment with this dose of atropine. More extensive studies are required to assess whether the [aH]QNB in the heart indeed represent selective binding to muscarinic cholinerg~c receptors. In summary, several lines of evidence indicate that [aH]QNB accumulation in the brain predominantly involves muscarinic cholinergic receptor sites. The relative amounts of QNB binding in the brain regions examined parallels their relative number of muscarinic cholinergic receptor sites determined bv in vitro [3H]QNB binding studies 19,z° and neurophysiologic investigations ~°-12. QNB accumulation in vivo
SHORT COMMUNICATIONS
175
can be prevented by relatively low doses of atropine but not by a nicotinic cholinergic a n t a g o n i s t or noncholinergic drugs. The a p p a r e n t maximal n u m b e r of b i n d i n g sites for QN B in the corpus striatum a n d h i p p o c a m p u s appears to be within an order of m a g n i t u d e of the n u m b e r of sites calculated from in ritro studies. The very slow decline of [aH]QNB levels in the corpus striatum and h i p p o c a m p u s indicates that Q N B binds tightly to the muscarinic receptor. This avid b i n d i n g has made feasible the a u t o r a d i o g r a p h i c localization of [3H]QNB in rat brain after i n t r a v e n o u s injection ( K u h a r a n d Y a m a m u r a , in preparation). Supported by U S P H S G r a n t s DA-00266, a grant of the J o h n A. H a r f o r d F o u n d a t i o n , N I M H R S D A A w a r d MH-33128 to S.H.S. a n d N I M H Special Research Fellowship MH-54777 to H.1.Y. We t h a n k Mr. James Clark for technical assistance.
1 ALBANUS, L., Central and peripheral effects of anticholinergic compounds, Acta pharmacol.
toxicol. (Kbk.), 28 (1970) 305-326. 2 ALBANUS, L., HAMMARSTROM, L., SUNDWALL, A., ULLBERG, S., AND VANGBO, B., Distribution and metabolism of '~H-atropine in mice, Acta physiol, scand., 73 (1968) 447 456. 3 ALBANUS, L., SUNDWALL, A., VANGBO, B., AND WINBLADH, B., The fate of atropine in the dog, Acta pharmacol, toxicol. (Kbk.), 26 (1968) 571-582. 4 BELD,A. J., XYt~ARIENS,E. J., Stereospecific binding as a tool in attempts to localize and isolate muscarinic receptors. Part II. Binding of ( ~)-Benzetimide, (--)-Benzetimide and atropine to a fraction from bovine tracheal smooth muscle and to bovine caudate nucleus, Europ. J. Pharmacol., 25 (1974) 203-209. 5 BURGEN, A. S. V., HILLY, C. R., AND YOUNG, J. M., The binding of 3H-propylbenzilylcholine mustard by longitudinal muscle strips from guinea pig small intestine, Brit. J. Pharmacol., 50 (1974) 145-15 I. 6 BURGEN, A. S. V., H1LEY, C. R., AND YOUNG, J. M., The properties of muscarinic receptors in mammalian cerebral cortex, Brit. J. Pharmacol., 51 (1974) 279-285. 7 FARROW,J. T., AND O'BRIEN, R. D., Binding of atropine and muscarone to rat brain fractions and its relation to the acetylcholine receptor, Molec. Pharmacol., 9 (1973) 33-40. 8 FEWTRELL,C., AND RANG,H. P., Distribution of bound 3H-benzilylcholine mustard in subcellular fractions of smooth muscle from guinea pig ileum, Brit. J. Pharmacol., 43 (1971) 417P-418P. 9 H~LE¥,C. R., AND BORGEN,A. S. V., The distribution of muscarinic receptor sites in the nervous system of the dog, J. Neurockem., 22 (1974) 159-162. 10 KRNJEVIC, K., Actions of drugs on single neurones in the cerebral cortex, Brit. Med. Bull., 21 (1965) 10-14. I 1 KRNJEVIC, K., AND PHILLIS, J. W., Pharmacological properties of acetylcholine sensitive cells in the cerebral cortex, J. Physiol. (Lond.), 166 (1963) 328-350. 12 MCLENNAN, H., AND YORK, D. H., Cholinergic mechanisms in the caudate nucleus, J. Physiol. (Lond.), 187 (1966) 163-175. 13 MEYERnOFFER,A., Absolute configuration of 3-quinuclidinyl benzilate and the behavioral effect in the dog of the optical isomers, J. Med. Chem., 15 0972) 994-995. 14 PATON, W. D. M., AND RANG, H. P., The uptake of atropine and related drugs by intestinal smooth muscle of the guinea pig in relation to acetylcholine receptors, Proc. roy. Soc. B, 1633 (1965) I 44. 15 SCHt.EIVER, L. S., AND ELDEFRAWl.M. E., Identification of the nicotinic and muscarinic acetylcholine receptors in subcellular fractions of mouse brain, Neuropkarmacol., 13 (1974) 53-63. 16 SOUDIJN,W., VANWIJNGAARDEN,I., AND ARtENS, E. J., Dexetimide, a useful tool in acetylcholine receptor localization, Europ. J. Pharmacol., 24 (1973) 43-48. 17 WINr~LADH,B, The fate of atropine in the puppy, Acta pharmacol, toxicol. (Kbk.), 32 (1973) 46-64. 18 WITTER, A., SLANGEN, J. L,, AND TERPSTRA, G. K., Distribution of :3H-methylatropine in rat brain, Neuropkarmacol., 12 (1973) 835-841.
19 YAMAMURA,H. i., KUHAR, M. J., GREENBERG, D., AND SNYDIER,S. H., MuscarinJ~: ct~oli~lerg~c receptor binding: Regional distribution in monkey brain, Brain Research, 66 (1974) 54I 546. 20 YAMAMURA,H. 1., AND SNYDER, S. H., Muscarinic cholinergic binding in rat brairL Pr¢,c. Jzc:r Acad Sci. (Wash.), 71 (1974) 1725-1729. 21 YAMAMURA,H. I., AND SNYDER,S. H., Muscarinic cholinergic receptor binding in the iongitudinai muscle of the guinea pig ileum with aH-quinuclidinyl benzilate, Molec. Pharmacol.~ (1974). in press;