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Differential distribution of muscarinic receptor subtypes and their regulation by G-protein in rat brain
Gen. Pharmac. Vol. 21, No. 4, pp. 471-476, 1990
0306-3623/90 $3.00 + 0.00 Copyright ~ 1990 Pergamon Press plc
Printed in Great Britain. All rights reserved
DIFFERENTIAL DISTRIBUTION OF MUSCARINIC RECEPTOR SUBTYPES A N D THEIR REGULATION BY G-PROTEIN IN RAT BRAIN JIANN-Wu WEt* and WEN-CHUN HUNG Institute of Neurosciences, National Yang-Ming Medical College, Taipei, Taiwan, Republic of China [Tel. (02) 821-2301] (Received 26 September 1989)
Abstract--l. [~H]quinuclidinylbenzilate ([3H]QNB) binding in rat cerebral and cerebellar synaptosomes had different B,~ values, but similar Kd values. 2. These bindings could be displaced by classic muscarinic agents: pilocarpine (partial agonist), and atropine (antagonist), which both had similar binding affinities in rat cerebral and cerebellar synaptosomes.
3. The new muscarinic M~ selective agents: McN-A-343 (agonist), prienzepine and tribexyphenidyl (antagonists) and higher at~nities for receptor sites in the cerebrum than in the cerebellum. 4. The muscarinic M2 selective agents: carbachol, oxotremorine (agonists), and AF-DX-116 (antagonist) had higher affinities for receptor sites in the cerebellum than in the cerebrum. 5. GPP(NH)p (40/aM) decreased the binding affinities of carbachol and oxotremorine in the cerebellum, but not in the cerebrum. However, it did not decrease the binding affinities of all the antagonists studied in both brain regions. 6. These results reveal that more muscarinic M, sites are present in the cerebrum than in the cerebellum, while the opposite is true for M2 sites. Furthermore, the regulatory role of G-protein on these muscarinic receptor subtypes in the brain is different.
INTRODUCI'ION
MATERIALS AND METHODS
Biochemical studies of labeled ligand binding by the homogenates of the cerebrum and cerebellum of rat brain revealed that these regions contained different proportion of muscarinic receptor subtypes, namely M~ and M 2 (Watson et al., 1982, 1983). Also, many selective cholinergic agents (agonists and antagonists) for M~ and M 2 subtypes have been developed, which have differential effects on several tissue preparations (Hammer et al., 1986). In order to investigate the regulation role of G-protein on these receptor subtypes, these two brain regions and some newly developed selective cholinergic agents in conjugation with classical ones were used in this study. The existence of muscarinic receptor subtypes have been proposed and have become a general concept (Goyal and Rattan, 1978). However, some earlier reports indicated that the regulation role of G-protein on muscarinic receptors in several tissues displayed subtle differences (Berrie et al., 1979; Korn et al., 1983; Dunlap and Brown, 1984). More studies are still needed on this regard under the new concept of the muscarinic receptor subtypes. By the use of well defined subtype enriched regions and selective agents, we believed that more meaningful conclusions could be obtained. For such a purpose, a GTP unhydrolizable compound, Gpp(NH)p was used to reveal the differential property between muscarinic receptor subtypes namely M~ and M2 interacting with either cholinergic agonists or antagonists.
[3H]quinuclidinyl benzilate ([3H]QNB, 42.6Ci/mmol) was purchased from New England Nuclear (Boston, Mass, U.S.A.). The following drugs: atropine sulfate, tribexy-pbenidyl hydrochloride, carbachol, tris-hydroxymethyl-aminomethane (Tris), pilocarpine hydrochloride, oxotremorine, polyethylenimine (50%), bovine serum albumin and Y-guanylyl imidodiphosphate (GPlRNH)p) were obtained from Sigma Chemical Co. (St Louis, Mo., U.S.A.). Other compounds, pirenzepine and AF-DX-116 were gifts from Dr Karl Thomae of Biberach an der Riss, Germany. McN-A-343was a gift from Dr M. Gil De Angeli. Other chemicals of the highest purity were acquired from commercial sources.
*To whom correspondence should be addressed. GP 2,~4--H
471
Preparation of brain synaptosomes from rat cerebral cortex and cerebellar tissues Male Sprague--Dawleyrats with body weight between 250 and 350 g were used for this study. The synaptosomes were prepared from rat brain by homogenization followed by differentialand sucrose gradient centrifugation. The detailed methods for sample preparations from cerebral cortex and from cerebellar tissues were described in previous papers by Wei and Chiang (1985) and Hajos et al. (1984), respectively. These synaptosomes were suspended in a modified Krebs-Ringer bicarbonate buffer for further studies. Routinely, protein content was determined by the method of Lowry et al. (1951), using bovine serum albumin as standard. [ 3H]QNB binding assay Freshly prepared rat cortical and cerebellar synaptosomes were suspended in 10 mM Na-K-phosphate buffer, pH 7.4. The reaction mixture (final vol. 0.5 ml) contained 10mM Na-K-phoshate buffer (pH 7.4), and 0.12nM [3H]QNB (50-60cpm/fmol). Protein concentrations in the assays ranged from 100 to 200/~g. The mixture was preincubated
472
JIANN-WUWE] and WEN-CrluNHUNG
for 5 rain at 25°C, and the reaction was initiated by the addition of [3H]QNB. The reaction continued for 120 min at 25°C. Incubation was also carried out in the reaction mixture containing 10#M atropine sulfate. At the end of reaction, the reaction mixture was diluted 8-fold with icecold 10 mM Na-K-phosphate buffer (pH 7.4). After a quick mix, 4 ml of mixture was filtered through a Whatman glass fiber filter GF/C (1.2 #m) under mild suction. The filters were washed with 20ml (5ml x4) of 10mM Na-Kphosphate buffer (pH 7.4), placed in scintillation vials, dried at 60°C for about 2 hr, covered with 10 ml of scintillation fluid and counted in a liquid scintillation spectrometer. The el~ciency of counting was 35-40%. The specific binding of [3H]QNB was calculated from the differencein counts in the absence and in the presence of 10 pM atropine sulfate. The specific binding ranged from 92 to 97% of the total binding at the [3H]QNB concentrations of 0.06-1.2 nM. Changes made in the assay protocol are specified in the legends of the tables.
Statistical analysis Data were analyzed using the Student's t-test. Results are expressed as mean ± SD. Differences with P < 0.05 were considered significant. RESULTS
Biochemical properties of [3Hj~.NB binding to rat cerebral and cerebellar synaptosomes From saturation analysis of [3H]QNB binding to rat cerebral and cerebellar synaptosomes (Fig. 1), and the Scatchard plot of these data (not shown), it was found that the K~ and Bm,~ binding values were 136 pM and 1800 fmol/mg protein in the cerebral synaptosomes respectively, while the Kd and Bma, binding values were 136 pM and 407 fmol/mg protein in the cerebellar synaptosomes respectively. For the following drug-displacement experiments on the [3H]QNB binding study, some of the reaction parameters were well taken care of in the initial stage of this study in order to get the proper conditions and a reasonable amount of counts in each tube. The protein concentrations used were 50-100 pg of cerebral synaptosomes, and 100-200/ag of cerebeilar synaptosomes in the assay tubes. The incubation time
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Fig. 2. Hill plot of the inhibition of [3H]QNB binding to rat cerebral and cerebeUar muscarinic receptor sites by pilocarpine. B, and B t o t represent [3H]QNB bound in the presence and absence of pilocarpine respectively. The [3H]QNB concentration was 0.12 nM. Cerebrum: ( 3 - - ( 3 without Gpp(NH)p; • • with 40/~M Gpp(NH)p; Cerebellum: A--A without Gpp(NH)p; &• with 40#M Gpp(NH)p.
chosen was 120 min at 25°C in order to get an optimal binding.
Effects of the classical cholinergic agents on the [3H]QNB binding to rat cerebral and cerebellar synaptosomes The partial agonist, piiocarpine, was used to displace the [3H]QNB binding to rat cerebral and cerebellar synaptosomes. The results were shown as the Hill plot in Fig. 2. In the absence of 4 0 p M Gpp(HN)p, the dose--displacement curves of pilocarpine on the cerebral and cerebellar synaptosomes overlapped each other. Even in the presence of 4 0 p M Gpp(NH)p, no shiftment on these dosedisplacement curves were found for these two tissue preparations. The concentration of this agent, which is correspondent to the zero in the vertical axis of this plot (Fig. 2), is equal to the ICso-value. This is the concentration of drug causing 50*/0 reduction in the specific binding. The values obtained from these dose-displacement curves of these two tissue preparations were similar for pilocarpine in the presence of 40/~M Gpp(NH)p or its absence (Table 1). The classical cholinergic antagonist, atropine was used to displace the [3H]QNB binding to rat cerebral and cerebeilar synaptosomes. All the dosedisplacement curves obtained in the presence or absence of 40 # M Gpp(NH)p were squeezed together (Fig. 3). Therefore, drug-affinities (based on the ICs0values) for the receptor sites in the cerebral or cerebellar synaptosomes were similar (Table 2).
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Fig. 1. Saturation analysis of [3H]QNB binding to rat cerebral and cerebellar synaptosomes. Samples were incubated in the Na-K-phosphate buffer as described in the Materials and Methods section, and the amount of the specific [3H]QNB binding with muscarinic receptors was measured. © (3 Cerebrum; • • Cerebellum.
Effects of the M ~ selective cholinergic agents on the [3H]QNB binding to rat cerebral and cerebellar synaptosomes The new Mj selective cholinergic agonist, McN-A343 was used to displace the [JHJQNB binding. Based on the dose--displacement curves, it was found that this compound had a higher affinity for the
Role of G-protein on muscarinic receptor subtypes
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Table I. The IC~0-valuesfor the inhibition of [3H}QNB binding to the muscarinic receptor sites in rat cerebral and cerebellar synaptosomes by cholinergic agonists Cholinergic agonists
Cerebrum
M :selective agonists McN-A-343 +40,aM Gpp(NH)p Carbachol +40pM Gpp(NH)p Oxotremorine +40#M Gpp(NH)p Non-selective. partial agonist Pilocarpine +40/~M Gpp(NH)p
(2.07_+ 0.15)x (2.07 4. 0.15) x (3.09-+ 0.26) x (3.09+0.26) x (5.11 4. 0.25) x (5.11+0.25) x
IC~-values (M) Cerebellum 10-6 10-6 10-~ 10-' 10 ~ 10 5
(I.02 4. 0.26) x 10- ~ (I.02_0.26) x 10 5
(6.11 ± 0.19) x (6.11 + 0.19) x (2.56-+0.30) x (6.47±0.15) x (4.86+0.17) x (9.56 4.0.17) x
10-6 10 .6 10-s 10 -s* 10-6 106"
(I.02 + 0.26) x 10- s (I.02±0.26) x 10-~
The IC~0-values for the agents were determined from each individual dose-displacement curve. The results are expressed as mean 4. SD (n = 4). IC~-value is the concentration of drug causing 50% reduction in the specific binding (in the presence of 0.12 nM of [ ~H]QNB). *Significantly different (P < 0.05) from control (in the absence of Gpp(NH)p).
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Fig. 3. Hill plot of the inhibition of [~H]QNB binding to rat cerebral and cerebellar muscarinic receptor sites by atropine. B i and B~o, represent [3H]QNB bound in the presence and absence of atropine respectively. The [ 3H]QNB concentration was 0.12nM. Cerebrum: O C) without Gpp(NH)p; • • with 4 0 p M Gpp(NH)p; Cerebellum: A /X without Gpp(NH)p; • • with 4 0 p M Gpp(NH)p.
Fig. 4. Hill plot of the inhibition of [3H]QNB binding to rat cerebral and cerebellar muscarinic receptor sites by McN-A343. B, and Bto, represent [~H]QNB bound in the presence and absence of MeN-A-343 respectively. The [3H]QNB concentration was 0.12nM. Cerebrum: C) C) without Gpp(NH)p; • • with 40 p M Gpp(NH)p; Cerebellum: A A without Gpp(NH)p; • • with 40/aM Gpp(NH)p.
r e c e p t o r sites in the c e r e b r u m t h a n in the c e r e b e l l u m (Fig. 4). T h e ICs0-values o f this a g e n t in the cerebral s y n a p t o s o m e s were a b o u t 3-times l o w e r t h a n t h a t in the cerebellar s y n a p t o s o m e s ( T a b l e I). T h e displace-
m e n t ability o f M c N - A - 3 4 3 o n the [ 3 H ] Q N B b i n d i n g by rat cerebral a n d cerebellar s y n a p t o s o m e s w a s the s a m e in the p r e s e n c e o r a b s e n c e o f 40 tt M G p p ( N H ) p (see Fig. 4 a n d T a b l e l).
Table 2. The IC~0-valuesfor the inhibition of [~HJQNB binding to the muscarinic receptor sites in rat cerebral and cerebellar synaptosomes by cholinergic antagonists IC~o-values (M) Cholinergic antagonists Non-selective antagonist Atropine +40pM Gpp(NH)p M :selective antagonists Pirenzepine +40,uM Gpp(NH)p Tribexyphenidyl +40pM Gpp(NH)p M:selective antagonist AF-DX-II6 +40uM Gpp(NH)p
Cerebrum
Cerebellum
(5.13 4.0.44) x 10 -~° (5.13 + 0.44) x 10-I°
(5.13 4.0.44) x 10 -~° (5.13 + 0.44) x 10-I°
(I.08 4. 0.22) x (1.08 4. 0.22) x (4.53 + 0.35) x (4.53 4. 0.35) x
(7.25 + 0.30) x (7.25 4. 0.30) x (2.04 4. 0.24) x (2.04 + 0.24) x
10-6 10 6 10 s 10-s
(I.07 4. 0.18) x 10-6 (I.07_+0.18) × 10 -6
10-6 10 6 10 7 10-7
(7.83 + 0.14) x 10-s (7.83_+0.14)x 10-s
The ICso-valucs for the agents were determined from each individual dose-displacement curve. The results are expressedas mean -+ SD (n = 4). ICs0-valueis the concentration of drug causing 50% reduction in the specific binding (in the presenceof 0.12nM of [~HJQNB).
474
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Fig. 5. Hill plot of the inhibition of [~H]QNB binding to rat cerebral and cerebellar muscarinic receptor sites by pirenzepine. B, and B,o,represent [3H]QNB bound in the presence and absence of pirenzepine respectively. The [3H]QNB concentration was 0.12 nM. Cerebrum: O (3 without Gpp(NH)p; • • with 40 p M Gpp(NH)p; Cerebellum: /k /x without Gpp(NH)p; • • with 40/aM Gpp(NH)p.
Fig. 7. Hill plot of the inhibition of [~H]QNB binding to rat cerebral and cerebellar muscarinic receptor sites by earbachol. Bi and Bto t represent [3H]QNB bound in the presence and absence of carbachol respectively. The concentration of [3H]QNB was 0.12 riM. Cerebrum: (3 . (3 without Gpp(NH)p; • • with 40/~M Gpp(NH)p; Cerebellum: ~, A without Gpp(NH)p; • • with 40/~M Gpp(NH)p.
The new M t selective cholinergic antagonist, pirenzepine was used to displace the [3H]QNB binding. Based on the dose-displacement curves, it was found that pirenzepine had a higher affinity for the receptor sites in the cerebrum than in the cerebellum (Fig. 5). The difference calculated from their ICsovalues obtained (Table 2) was about 6-7-times. Addition of 40/aM Gpp(NH)p did not alter the binding affinities of this compound to the receptor sites in the cerebrum and cerebellum, and no change in the ICs0-values for this agent on these two tissue preparations could be found (Table 2). Another new M~ selective cholinergic antagonist, trihexyphenidyl, was used to displace the [3H]QNB binding. Based on the dose-displacement curves
obtained, it had a higher affinity for receptor sites in the cerebrum than the cerebellum (Fig. 6). The difference, based on the ICso-values, was about 4-5times. The binding affinities of this compound to the receptor sites in the cerebrum and cerebellum were not altered by the addition of 40/~M Gpp(NH)p (Table 2).
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Fig. 6. Hill plot of the inhibition of [3H]QNB binding to rat cerebral and cerebellar muscarinic receptor sites by trihexyphenidyl. B~and Bto t represent [3H]QNB bound in the presence and absence of trihexyphenidyl respectively. The concentration of [~H]QNB was 0.12 nM. Cerebrum: ( 3 - (3 without Gpl~NH)p; • • with 40/~M Gpp(NH)p; Cerebellum: /k /x without Gpp(NH)p; • • with 40 pM Gpp(NH)p.
Effects o f the M 2 selective cholinergic agents on the [JH]QNB binding to rat cerebral and cerebellar synaptosomes The M 2 selective cholinergic agonist, carbachol, was used to displace the [3H]QNB binding to rat cerebral and cerebrellar synaptosomes. Based on the dose-displacement curves obtained, it was found that carbachol had a higher affinity for the receptor sites in the cerebellum than the cerebrum (Fig. 7). The difference was about 10-15-times, based on the ICs0-values obtained in the absence of 40/~M Gpp(NH)p. Since 40 pM Gpp(NH)p caused a significant rightward shift of the dose-displacement curve of carbachol in the cerebellum, but not the cerebrum (Fig. 7), it narrowed the affinity difference between the cerebellum and the cerebrum by a factor of 2-3 (Table 1). Another M 2 selective cholinergic agonist, oxotremorine, had a similar effect as carbachol, except 40/aM GPlNNH)p caused a less extensive rightward shift of dose--displacement curve of carbachol in the cerebellum (Fig. 8 and Table 1). The M2 selective cholinergic antagonist, AFDX-I 16, was used to displace the [3H]QNB binding to the rat cerebral and cerebellar synaptosomes. The dose--displacement curves revealed that it had a higher affinity for the receptor sites in the cerebellum than in the cerebrum (Fig. 9). The difference, based on the ICs0-values obtained, was about 10-15-times in the presence or absence of 4 0 ~ M Gpp(NH)p (Table 2). It also revealed that 4 0 # M Gpp(NH)p did not change the binding affinity of AF-DX-i16
Role of G-protein on muscarihic receptor subtypes
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Fig. 8. Hill plot of the inhibition of [3H]QNB binding to rat cerebral and cerebellar muscarinic receptor sites by oxotremorine. Bi and B~ot represent [3H]QNB bound in the presence and absence of oxotremorine, respectively. The concentration of [3H)QNB was 0.12 nM. Cerebrum: O - O without Gpp(NH)p; • • with 40pM Gpp(NH)p; Cerebellum: Z~ A without Gpp(NH)p; • • with 40/~M Gpp(NH)p. to the receptor sites in either the cerebrum or in the cerebellum. DISCUSSION
The [~H]QNB binding by rat cerebral and cerebellar synaptosomes The binding of the non-selective labeled ligand, [3H]QNB, to rat cerebral and cerebellar synaptosomes reveals that the maximal receptor binding sites are different, whereas the binding affinities of this ligand to those two regions of the brain are still the same. The results imply that the total amount of muscarinic cholinergic receptors in different brain regions are different, but their affinities towards 2
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Fig. 9. Hill plot of the inhibition of [~H]QNB binding to rat cerebral and cerebellar muscarinic receptor sites by AF-DX116. Bi and Btot represent [3H]QNB bound in the presence and absence of AF-DX-116 respectively. The concentration of [3H]QNB was 0.12nM. Cerebrum: O (D without Gpp(NH)p; • • with 40/~ M Gpp(NH)p; Cerebellum: A A without Gpp(NH)p; • • with 40/aM Gpp(NH)p.
475
this non-selective cholinergic labeled ligand used are similar. Up to date, the physiological functions associated with the activation of these receptors in different brain regions still need further investigation. Currently, it is believed that their functions are closely related to memory, learning, arousal, control of movement, etc. (see Nathanson, 1987). Since 1974, the labeled ligand, [3H]QNB, has been proved to be a useful agent in the identification of muscarinic receptor sites in many tissues (Yamamura and Snyder, 1974). However, it does not have the ability to differentiate the receptor subtypes in different tissues. The resolve of this issue relies mainly on the newly developed subtype selective agents, such as pirenzepine (Hammer et al., 1980) or MeNA-343 (Hammer and Giachetti, 1983), to reveal the subtle differences among the receptor subtypes identified.
The displacement of [3H]QNB binding by the classic cholinergic agents The classic cholinergic agents, pilocarpine (partial agonist) and atropine (antagonist), were used to displace [ 3H]QNB binding to the cerebral and cerebellar synaptosomes. No binding affufity difference was found between ligand and receptor interaction in these two brain regions. Even in the presence of 40/zM Gpp(NH)p, the binding affinities of these agents with their receptor sites were still the same. The results are in good agreement with the general notion that the classic cholinergic agents do not have the ability to differentiate the muscarinic receptor subtypes in the tissue (Hulme et al., 1978; Ehlert et al., 1981).
The displacement of [3H]QNB binding by the muscarinic M r selective agents When muscarinic M n selective agents were used to displace [3H]QNB binding to rat cerebral and cerebellar synaptosomes, it was clear that muscarinic M~ selective agonist, MeN-A-343 and antagonists, pirenzepine or trihexyphenidyl had higher affinities for receptor sites in the cerebrum than in the cerebellum to a significant level. This was based on the ICso-values obtained from the dose-displacement curves of [3H]QNB binding to these two brain regions. It is an indication that rat cerebrum contains a greater amount of Mj receptor sites than the cerebellum, which is in good agreement with the results reported by others (Watson et al., 1986). In the presence of 40/~M Gpp(NH)p, it did not significantly alter the binding affinities of these agents: neither agonist, MeN-A-343, nor antagonists, pirenzepine and trihexyphenidyl, toward the receptor sites in the cerebrum or cerebellum. Earlier reports showed that Gpp(NH)p had an ability to decrease the binding affinity of agonist, but not antagonist on the muscarinic M2-receptor enriched tissues such as heart (Barrie et al., 1979; Rosenberger et al., 1979; Wei and Sulakhe, 1979). Our results suggest that the regulation role of G-protein on different muscarinic receptor subtypes is likely to be different.
The displacement of [3H]QNB binding by the muscarinic M2 selective agents When muscarinic M 2 selective agents were used to displace the [aH]QNB binding by rat cerebral and
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JIANN-Wu WEI and WEN-CHUNHUNG
cerebellar synaptosomes. It was found that muscarinic M2 selective agonists, carbachol and oxotremorine, and antagonists, A F - D X - 1 1 6 had higher affinities for receptor sites in the cerebellum than in the cerebrum. This is based on the ICso-values obtained from the dose--displacement curves of [3H]QNB binding to these two brain regions. Clearly, it is an indication that rat cerebellum contains a greater amount of muscarinic M2 receptor sites than the cerebrum. This is in good agreement with the results reported by others (Luthin and Wolfe, 1984; Watson et al., 1983). In the presence of 40/~M G p p ( N H ) p , it had the ability to decrease the binding affinities of muscarinic M 2 selective agonists, carbachol and oxotremorine, to the receptor sites in the cerebellum, but not in the cerebrum. These results are an indication that a G-protein is coupled with muscarinic M2 receptor site in the cerebellum. Similar results were found previously in the cardiac tissue by us (Wei and Sulakhe, 1979). S o m e indications and f u t u r e research work
The results of this study revealed that the newly synthesized muscarinic M~ or M2 selective agents have the ability to differentiate the muscarinic receptor subtypes in rat cerebrum and cerebellum. Furthermore, it seems that more muscarinic M~ receptor sites are present in the cerebrum than in the cerebellum, and the opposite is true for the muscarinic M2 receptor sites. The regulation of G-protein on the muscarinic M t subtype and the M2 subtypes of these two brain regions are likely different. This indication results from the fact that G p p ( N H ) p decreases the binding affinities of muscarinic M2 selective agonists when they interact with the M2 receptor sites; whereas it does not alter the binding affinity o f muscarinic M~ selective agonists when they interact with the M~ receptor sites. The further information on the basic differences between muscarinic receptor subtypes and their coupling to different second messenger systems, such as polyphosphoinositide turnover, cyclic A M P , or ion channel opening are awaiting more studies. Using techniques, such as the cloning of c D N A s and genes for different muscarinic acetylcholine receptors, may provide us with some useful information. Acknowledgement--This work was supported by a NSC grant, 77-0412-B010-14, and a grant from the Clinical Research Center, VGH, Institute of Biomedical Sciences Academia Sinica, Republic of China.
REFERENCES
Berrie C. P., Birdsall J. M., Burgen A. S. V. and Hulme E. C. (1979) Guanine nucleotides modulate muscarinic receptor binding in the heart. Biochem. Biophys. Res. Commun. 87, 1000-1005. Dunlap J. and Brown J. H. (1984) Differences and similarities in muscarinic receptors of rat heart and retina:
effects of agonists, guanine and nueleotides, and N-ethylmaleimide. J. Neurochem. 43, 214-220. Ehlert F. J., Roeske W. R. and Yamamura H. I. (1981) Muscarinic receptor: regulation by guanine nucleotides, ions and N-ethylmaleimide. Fedn Proc. 40, 153-159. Giachetti A., Micheletti R. and Montagna E. (1986)Cardiovascular profile of AF-DX-116, a muscarine M2 receptor antagonist. Life Sci. 38, 1663-1672. Goyal R. K. and Rattan S. (1978) Neurohormonal, hormonal and drug receptors for the lower esophageal sphincter. Gc~stroenterology 74, 598~i19. Hajos F., Tapia R., Wilkin G., Johnson L. and Balaze R. (1974) Subcellular fractionation of rat cerebellum: an electron microscopic and biochemical investigation. 1. Preservation of large fragments of cerebellar glomeruli. Brain Res. 70, 261 279. Hammer R. and Giachetti A. (1983) Muscarinic receptor subtypes: M k and M 2 biochemical and functional characterization. Life Sci. 31, 2991-2998. Hammer R., Berrie C. P., Birdsall N. J. M., Bergen A. S. V. and Hulme E. C. (1980) Pirenzepine distinguishes between different subclasses of muscarinic receptors. Nature 283, 90-92. Hulme E. C., Birdsall N. J. M., Burgen A. S. V. and Mehta P. (1978) The binding of antagonists to brain muscarinic receptors. Molec. Pharmac. 14, 737-750. Korn S. J., Martin M. W. and Harden T. K. (1983) N-ethylmaleimide-induced alteration in the interaction of agonists with muscarinic cholinergic receptors of rat brain. J. Pharmac. Exp. Ther. 224, 118-126. Lowry O. H , Rosebrough N. J., Farr A. L. and Randall R. L. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Luthin G. R. and Wolfe B. B. (1984) Comparison Of ~H-pirenzepine and ~H-quinuclidinyl benzilate binding to muscarinic cholinergic receptor in rat brain. J. Pharmac. Exp. Ther. 228, 648~55. Nathanson N. M. (1987) Molecular properties of the muscarinic acetylcholine receptor. A. Ree. Neurosci. 10, 195-235. Rosenberger L. B., Roeske W. R. and Yamamura H. 1. (1979) The regulation of muscarinic cholinergic receptor by guanine nucleotides in cardiac tissue. Fur. J. Pharmac. 56, 179- 180. Watson M., Roeske W. R. and Yamamura H. 1. (1982) 3H-pirenzepine selectively identifies a high affinity population of muscarinic cholinergic receptors in the rat cerebral cortex. Life Sci. 31, 2019-2023. Watson M., Yamamura H. I. and Roeske W. R. (1983) A unique regulatory profile and regional distribution of ~H-pirenzepine for distinct M~ and M., muscarinic receptor subtypes. Life Sci. 32, 3001 3010. Watson M., Roeske W. R. and Yamamura H. I. (1986) ~H-pirenzepine and 3H-quinuclidinyl benzilate binding to rat cerebral cortical and cardiac muscarinic cholinergic sites. II. Characterization and regulation of antagonist binding to putative muscarinic subtypes. J. Pharmac. Exp. Ther. 237, 419-427. Wei J. W. and Chiang D. H. (1985) Studies of 3H-nitredipine binding and KCl-induced calcium uptake in rat cortical synaptosomes. Gen. Pharmac. 16, 211-216. Wei J. W. and Sulakhe P. V. (1979) Agonist-antagonist interactions with rat atrial muscarinic cholinergic receptor sites: differential regulation by guanine nucleotides. Fur. J. Pharmac. 58, 91-92. Yamamura H. I. and Snyder S. H. (1974) Muscarinic cholinergic binding in rat brain. Proc. Natn. Acad. Sci. U.S.A. 71, 1725--1729.
0306-3623/90 $3.00 + 0.00 Copyright ~ 1990 Pergamon Press plc
Printed in Great Britain. All rights reserved
DIFFERENTIAL DISTRIBUTION OF MUSCARINIC RECEPTOR SUBTYPES A N D THEIR REGULATION BY G-PROTEIN IN RAT BRAIN JIANN-Wu WEt* and WEN-CHUN HUNG Institute of Neurosciences, National Yang-Ming Medical College, Taipei, Taiwan, Republic of China [Tel. (02) 821-2301] (Received 26 September 1989)
Abstract--l. [~H]quinuclidinylbenzilate ([3H]QNB) binding in rat cerebral and cerebellar synaptosomes had different B,~ values, but similar Kd values. 2. These bindings could be displaced by classic muscarinic agents: pilocarpine (partial agonist), and atropine (antagonist), which both had similar binding affinities in rat cerebral and cerebellar synaptosomes.
3. The new muscarinic M~ selective agents: McN-A-343 (agonist), prienzepine and tribexyphenidyl (antagonists) and higher at~nities for receptor sites in the cerebrum than in the cerebellum. 4. The muscarinic M2 selective agents: carbachol, oxotremorine (agonists), and AF-DX-116 (antagonist) had higher affinities for receptor sites in the cerebellum than in the cerebrum. 5. GPP(NH)p (40/aM) decreased the binding affinities of carbachol and oxotremorine in the cerebellum, but not in the cerebrum. However, it did not decrease the binding affinities of all the antagonists studied in both brain regions. 6. These results reveal that more muscarinic M, sites are present in the cerebrum than in the cerebellum, while the opposite is true for M2 sites. Furthermore, the regulatory role of G-protein on these muscarinic receptor subtypes in the brain is different.
INTRODUCI'ION
MATERIALS AND METHODS
Biochemical studies of labeled ligand binding by the homogenates of the cerebrum and cerebellum of rat brain revealed that these regions contained different proportion of muscarinic receptor subtypes, namely M~ and M 2 (Watson et al., 1982, 1983). Also, many selective cholinergic agents (agonists and antagonists) for M~ and M 2 subtypes have been developed, which have differential effects on several tissue preparations (Hammer et al., 1986). In order to investigate the regulation role of G-protein on these receptor subtypes, these two brain regions and some newly developed selective cholinergic agents in conjugation with classical ones were used in this study. The existence of muscarinic receptor subtypes have been proposed and have become a general concept (Goyal and Rattan, 1978). However, some earlier reports indicated that the regulation role of G-protein on muscarinic receptors in several tissues displayed subtle differences (Berrie et al., 1979; Korn et al., 1983; Dunlap and Brown, 1984). More studies are still needed on this regard under the new concept of the muscarinic receptor subtypes. By the use of well defined subtype enriched regions and selective agents, we believed that more meaningful conclusions could be obtained. For such a purpose, a GTP unhydrolizable compound, Gpp(NH)p was used to reveal the differential property between muscarinic receptor subtypes namely M~ and M2 interacting with either cholinergic agonists or antagonists.
[3H]quinuclidinyl benzilate ([3H]QNB, 42.6Ci/mmol) was purchased from New England Nuclear (Boston, Mass, U.S.A.). The following drugs: atropine sulfate, tribexy-pbenidyl hydrochloride, carbachol, tris-hydroxymethyl-aminomethane (Tris), pilocarpine hydrochloride, oxotremorine, polyethylenimine (50%), bovine serum albumin and Y-guanylyl imidodiphosphate (GPlRNH)p) were obtained from Sigma Chemical Co. (St Louis, Mo., U.S.A.). Other compounds, pirenzepine and AF-DX-116 were gifts from Dr Karl Thomae of Biberach an der Riss, Germany. McN-A-343was a gift from Dr M. Gil De Angeli. Other chemicals of the highest purity were acquired from commercial sources.
*To whom correspondence should be addressed. GP 2,~4--H
471
Preparation of brain synaptosomes from rat cerebral cortex and cerebellar tissues Male Sprague--Dawleyrats with body weight between 250 and 350 g were used for this study. The synaptosomes were prepared from rat brain by homogenization followed by differentialand sucrose gradient centrifugation. The detailed methods for sample preparations from cerebral cortex and from cerebellar tissues were described in previous papers by Wei and Chiang (1985) and Hajos et al. (1984), respectively. These synaptosomes were suspended in a modified Krebs-Ringer bicarbonate buffer for further studies. Routinely, protein content was determined by the method of Lowry et al. (1951), using bovine serum albumin as standard. [ 3H]QNB binding assay Freshly prepared rat cortical and cerebellar synaptosomes were suspended in 10 mM Na-K-phosphate buffer, pH 7.4. The reaction mixture (final vol. 0.5 ml) contained 10mM Na-K-phoshate buffer (pH 7.4), and 0.12nM [3H]QNB (50-60cpm/fmol). Protein concentrations in the assays ranged from 100 to 200/~g. The mixture was preincubated
472
JIANN-WUWE] and WEN-CrluNHUNG
for 5 rain at 25°C, and the reaction was initiated by the addition of [3H]QNB. The reaction continued for 120 min at 25°C. Incubation was also carried out in the reaction mixture containing 10#M atropine sulfate. At the end of reaction, the reaction mixture was diluted 8-fold with icecold 10 mM Na-K-phosphate buffer (pH 7.4). After a quick mix, 4 ml of mixture was filtered through a Whatman glass fiber filter GF/C (1.2 #m) under mild suction. The filters were washed with 20ml (5ml x4) of 10mM Na-Kphosphate buffer (pH 7.4), placed in scintillation vials, dried at 60°C for about 2 hr, covered with 10 ml of scintillation fluid and counted in a liquid scintillation spectrometer. The el~ciency of counting was 35-40%. The specific binding of [3H]QNB was calculated from the differencein counts in the absence and in the presence of 10 pM atropine sulfate. The specific binding ranged from 92 to 97% of the total binding at the [3H]QNB concentrations of 0.06-1.2 nM. Changes made in the assay protocol are specified in the legends of the tables.
Statistical analysis Data were analyzed using the Student's t-test. Results are expressed as mean ± SD. Differences with P < 0.05 were considered significant. RESULTS
Biochemical properties of [3Hj~.NB binding to rat cerebral and cerebellar synaptosomes From saturation analysis of [3H]QNB binding to rat cerebral and cerebellar synaptosomes (Fig. 1), and the Scatchard plot of these data (not shown), it was found that the K~ and Bm,~ binding values were 136 pM and 1800 fmol/mg protein in the cerebral synaptosomes respectively, while the Kd and Bma, binding values were 136 pM and 407 fmol/mg protein in the cerebellar synaptosomes respectively. For the following drug-displacement experiments on the [3H]QNB binding study, some of the reaction parameters were well taken care of in the initial stage of this study in order to get the proper conditions and a reasonable amount of counts in each tube. The protein concentrations used were 50-100 pg of cerebral synaptosomes, and 100-200/ag of cerebeilar synaptosomes in the assay tubes. The incubation time
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Fig. 2. Hill plot of the inhibition of [3H]QNB binding to rat cerebral and cerebeUar muscarinic receptor sites by pilocarpine. B, and B t o t represent [3H]QNB bound in the presence and absence of pilocarpine respectively. The [3H]QNB concentration was 0.12 nM. Cerebrum: ( 3 - - ( 3 without Gpp(NH)p; • • with 40/~M Gpp(NH)p; Cerebellum: A--A without Gpp(NH)p; &• with 40#M Gpp(NH)p.
chosen was 120 min at 25°C in order to get an optimal binding.
Effects of the classical cholinergic agents on the [3H]QNB binding to rat cerebral and cerebellar synaptosomes The partial agonist, piiocarpine, was used to displace the [3H]QNB binding to rat cerebral and cerebellar synaptosomes. The results were shown as the Hill plot in Fig. 2. In the absence of 4 0 p M Gpp(HN)p, the dose--displacement curves of pilocarpine on the cerebral and cerebellar synaptosomes overlapped each other. Even in the presence of 4 0 p M Gpp(NH)p, no shiftment on these dosedisplacement curves were found for these two tissue preparations. The concentration of this agent, which is correspondent to the zero in the vertical axis of this plot (Fig. 2), is equal to the ICso-value. This is the concentration of drug causing 50*/0 reduction in the specific binding. The values obtained from these dose-displacement curves of these two tissue preparations were similar for pilocarpine in the presence of 40/~M Gpp(NH)p or its absence (Table 1). The classical cholinergic antagonist, atropine was used to displace the [3H]QNB binding to rat cerebral and cerebeilar synaptosomes. All the dosedisplacement curves obtained in the presence or absence of 40 # M Gpp(NH)p were squeezed together (Fig. 3). Therefore, drug-affinities (based on the ICs0values) for the receptor sites in the cerebral or cerebellar synaptosomes were similar (Table 2).
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Fig. 1. Saturation analysis of [3H]QNB binding to rat cerebral and cerebellar synaptosomes. Samples were incubated in the Na-K-phosphate buffer as described in the Materials and Methods section, and the amount of the specific [3H]QNB binding with muscarinic receptors was measured. © (3 Cerebrum; • • Cerebellum.
Effects of the M ~ selective cholinergic agents on the [3H]QNB binding to rat cerebral and cerebellar synaptosomes The new Mj selective cholinergic agonist, McN-A343 was used to displace the [JHJQNB binding. Based on the dose--displacement curves, it was found that this compound had a higher affinity for the
Role of G-protein on muscarinic receptor subtypes
473
Table I. The IC~0-valuesfor the inhibition of [3H}QNB binding to the muscarinic receptor sites in rat cerebral and cerebellar synaptosomes by cholinergic agonists Cholinergic agonists
Cerebrum
M :selective agonists McN-A-343 +40,aM Gpp(NH)p Carbachol +40pM Gpp(NH)p Oxotremorine +40#M Gpp(NH)p Non-selective. partial agonist Pilocarpine +40/~M Gpp(NH)p
(2.07_+ 0.15)x (2.07 4. 0.15) x (3.09-+ 0.26) x (3.09+0.26) x (5.11 4. 0.25) x (5.11+0.25) x
IC~-values (M) Cerebellum 10-6 10-6 10-~ 10-' 10 ~ 10 5
(I.02 4. 0.26) x 10- ~ (I.02_0.26) x 10 5
(6.11 ± 0.19) x (6.11 + 0.19) x (2.56-+0.30) x (6.47±0.15) x (4.86+0.17) x (9.56 4.0.17) x
10-6 10 .6 10-s 10 -s* 10-6 106"
(I.02 + 0.26) x 10- s (I.02±0.26) x 10-~
The IC~0-values for the agents were determined from each individual dose-displacement curve. The results are expressed as mean 4. SD (n = 4). IC~-value is the concentration of drug causing 50% reduction in the specific binding (in the presence of 0.12 nM of [ ~H]QNB). *Significantly different (P < 0.05) from control (in the absence of Gpp(NH)p).
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Fig. 3. Hill plot of the inhibition of [~H]QNB binding to rat cerebral and cerebellar muscarinic receptor sites by atropine. B i and B~o, represent [3H]QNB bound in the presence and absence of atropine respectively. The [ 3H]QNB concentration was 0.12nM. Cerebrum: O C) without Gpp(NH)p; • • with 4 0 p M Gpp(NH)p; Cerebellum: A /X without Gpp(NH)p; • • with 4 0 p M Gpp(NH)p.
Fig. 4. Hill plot of the inhibition of [3H]QNB binding to rat cerebral and cerebellar muscarinic receptor sites by McN-A343. B, and Bto, represent [~H]QNB bound in the presence and absence of MeN-A-343 respectively. The [3H]QNB concentration was 0.12nM. Cerebrum: C) C) without Gpp(NH)p; • • with 40 p M Gpp(NH)p; Cerebellum: A A without Gpp(NH)p; • • with 40/aM Gpp(NH)p.
r e c e p t o r sites in the c e r e b r u m t h a n in the c e r e b e l l u m (Fig. 4). T h e ICs0-values o f this a g e n t in the cerebral s y n a p t o s o m e s were a b o u t 3-times l o w e r t h a n t h a t in the cerebellar s y n a p t o s o m e s ( T a b l e I). T h e displace-
m e n t ability o f M c N - A - 3 4 3 o n the [ 3 H ] Q N B b i n d i n g by rat cerebral a n d cerebellar s y n a p t o s o m e s w a s the s a m e in the p r e s e n c e o r a b s e n c e o f 40 tt M G p p ( N H ) p (see Fig. 4 a n d T a b l e l).
Table 2. The IC~0-valuesfor the inhibition of [~HJQNB binding to the muscarinic receptor sites in rat cerebral and cerebellar synaptosomes by cholinergic antagonists IC~o-values (M) Cholinergic antagonists Non-selective antagonist Atropine +40pM Gpp(NH)p M :selective antagonists Pirenzepine +40,uM Gpp(NH)p Tribexyphenidyl +40pM Gpp(NH)p M:selective antagonist AF-DX-II6 +40uM Gpp(NH)p
Cerebrum
Cerebellum
(5.13 4.0.44) x 10 -~° (5.13 + 0.44) x 10-I°
(5.13 4.0.44) x 10 -~° (5.13 + 0.44) x 10-I°
(I.08 4. 0.22) x (1.08 4. 0.22) x (4.53 + 0.35) x (4.53 4. 0.35) x
(7.25 + 0.30) x (7.25 4. 0.30) x (2.04 4. 0.24) x (2.04 + 0.24) x
10-6 10 6 10 s 10-s
(I.07 4. 0.18) x 10-6 (I.07_+0.18) × 10 -6
10-6 10 6 10 7 10-7
(7.83 + 0.14) x 10-s (7.83_+0.14)x 10-s
The ICso-valucs for the agents were determined from each individual dose-displacement curve. The results are expressedas mean -+ SD (n = 4). ICs0-valueis the concentration of drug causing 50% reduction in the specific binding (in the presenceof 0.12nM of [~HJQNB).
474
JIANN-WuWE! and WErq-CHuNHUN6 2
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Fig. 5. Hill plot of the inhibition of [~H]QNB binding to rat cerebral and cerebellar muscarinic receptor sites by pirenzepine. B, and B,o,represent [3H]QNB bound in the presence and absence of pirenzepine respectively. The [3H]QNB concentration was 0.12 nM. Cerebrum: O (3 without Gpp(NH)p; • • with 40 p M Gpp(NH)p; Cerebellum: /k /x without Gpp(NH)p; • • with 40/aM Gpp(NH)p.
Fig. 7. Hill plot of the inhibition of [~H]QNB binding to rat cerebral and cerebellar muscarinic receptor sites by earbachol. Bi and Bto t represent [3H]QNB bound in the presence and absence of carbachol respectively. The concentration of [3H]QNB was 0.12 riM. Cerebrum: (3 . (3 without Gpp(NH)p; • • with 40/~M Gpp(NH)p; Cerebellum: ~, A without Gpp(NH)p; • • with 40/~M Gpp(NH)p.
The new M t selective cholinergic antagonist, pirenzepine was used to displace the [3H]QNB binding. Based on the dose-displacement curves, it was found that pirenzepine had a higher affinity for the receptor sites in the cerebrum than in the cerebellum (Fig. 5). The difference calculated from their ICsovalues obtained (Table 2) was about 6-7-times. Addition of 40/aM Gpp(NH)p did not alter the binding affinities of this compound to the receptor sites in the cerebrum and cerebellum, and no change in the ICs0-values for this agent on these two tissue preparations could be found (Table 2). Another new M~ selective cholinergic antagonist, trihexyphenidyl, was used to displace the [3H]QNB binding. Based on the dose-displacement curves
obtained, it had a higher affinity for receptor sites in the cerebrum than the cerebellum (Fig. 6). The difference, based on the ICso-values, was about 4-5times. The binding affinities of this compound to the receptor sites in the cerebrum and cerebellum were not altered by the addition of 40/~M Gpp(NH)p (Table 2).
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Fig. 6. Hill plot of the inhibition of [3H]QNB binding to rat cerebral and cerebellar muscarinic receptor sites by trihexyphenidyl. B~and Bto t represent [3H]QNB bound in the presence and absence of trihexyphenidyl respectively. The concentration of [~H]QNB was 0.12 nM. Cerebrum: ( 3 - (3 without Gpl~NH)p; • • with 40/~M Gpp(NH)p; Cerebellum: /k /x without Gpp(NH)p; • • with 40 pM Gpp(NH)p.
Effects o f the M 2 selective cholinergic agents on the [JH]QNB binding to rat cerebral and cerebellar synaptosomes The M 2 selective cholinergic agonist, carbachol, was used to displace the [3H]QNB binding to rat cerebral and cerebrellar synaptosomes. Based on the dose-displacement curves obtained, it was found that carbachol had a higher affinity for the receptor sites in the cerebellum than the cerebrum (Fig. 7). The difference was about 10-15-times, based on the ICs0-values obtained in the absence of 40/~M Gpp(NH)p. Since 40 pM Gpp(NH)p caused a significant rightward shift of the dose-displacement curve of carbachol in the cerebellum, but not the cerebrum (Fig. 7), it narrowed the affinity difference between the cerebellum and the cerebrum by a factor of 2-3 (Table 1). Another M 2 selective cholinergic agonist, oxotremorine, had a similar effect as carbachol, except 40/aM GPlNNH)p caused a less extensive rightward shift of dose--displacement curve of carbachol in the cerebellum (Fig. 8 and Table 1). The M2 selective cholinergic antagonist, AFDX-I 16, was used to displace the [3H]QNB binding to the rat cerebral and cerebellar synaptosomes. The dose--displacement curves revealed that it had a higher affinity for the receptor sites in the cerebellum than in the cerebrum (Fig. 9). The difference, based on the ICs0-values obtained, was about 10-15-times in the presence or absence of 4 0 ~ M Gpp(NH)p (Table 2). It also revealed that 4 0 # M Gpp(NH)p did not change the binding affinity of AF-DX-i16
Role of G-protein on muscarihic receptor subtypes
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Fig. 8. Hill plot of the inhibition of [3H]QNB binding to rat cerebral and cerebellar muscarinic receptor sites by oxotremorine. Bi and B~ot represent [3H]QNB bound in the presence and absence of oxotremorine, respectively. The concentration of [3H)QNB was 0.12 nM. Cerebrum: O - O without Gpp(NH)p; • • with 40pM Gpp(NH)p; Cerebellum: Z~ A without Gpp(NH)p; • • with 40/~M Gpp(NH)p. to the receptor sites in either the cerebrum or in the cerebellum. DISCUSSION
The [~H]QNB binding by rat cerebral and cerebellar synaptosomes The binding of the non-selective labeled ligand, [3H]QNB, to rat cerebral and cerebellar synaptosomes reveals that the maximal receptor binding sites are different, whereas the binding affinities of this ligand to those two regions of the brain are still the same. The results imply that the total amount of muscarinic cholinergic receptors in different brain regions are different, but their affinities towards 2
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10 -7
10-6
10-5
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Fig. 9. Hill plot of the inhibition of [~H]QNB binding to rat cerebral and cerebellar muscarinic receptor sites by AF-DX116. Bi and Btot represent [3H]QNB bound in the presence and absence of AF-DX-116 respectively. The concentration of [3H]QNB was 0.12nM. Cerebrum: O (D without Gpp(NH)p; • • with 40/~ M Gpp(NH)p; Cerebellum: A A without Gpp(NH)p; • • with 40/aM Gpp(NH)p.
475
this non-selective cholinergic labeled ligand used are similar. Up to date, the physiological functions associated with the activation of these receptors in different brain regions still need further investigation. Currently, it is believed that their functions are closely related to memory, learning, arousal, control of movement, etc. (see Nathanson, 1987). Since 1974, the labeled ligand, [3H]QNB, has been proved to be a useful agent in the identification of muscarinic receptor sites in many tissues (Yamamura and Snyder, 1974). However, it does not have the ability to differentiate the receptor subtypes in different tissues. The resolve of this issue relies mainly on the newly developed subtype selective agents, such as pirenzepine (Hammer et al., 1980) or MeNA-343 (Hammer and Giachetti, 1983), to reveal the subtle differences among the receptor subtypes identified.
The displacement of [3H]QNB binding by the classic cholinergic agents The classic cholinergic agents, pilocarpine (partial agonist) and atropine (antagonist), were used to displace [ 3H]QNB binding to the cerebral and cerebellar synaptosomes. No binding affufity difference was found between ligand and receptor interaction in these two brain regions. Even in the presence of 40/zM Gpp(NH)p, the binding affinities of these agents with their receptor sites were still the same. The results are in good agreement with the general notion that the classic cholinergic agents do not have the ability to differentiate the muscarinic receptor subtypes in the tissue (Hulme et al., 1978; Ehlert et al., 1981).
The displacement of [3H]QNB binding by the muscarinic M r selective agents When muscarinic M n selective agents were used to displace [3H]QNB binding to rat cerebral and cerebellar synaptosomes, it was clear that muscarinic M~ selective agonist, MeN-A-343 and antagonists, pirenzepine or trihexyphenidyl had higher affinities for receptor sites in the cerebrum than in the cerebellum to a significant level. This was based on the ICso-values obtained from the dose-displacement curves of [3H]QNB binding to these two brain regions. It is an indication that rat cerebrum contains a greater amount of Mj receptor sites than the cerebellum, which is in good agreement with the results reported by others (Watson et al., 1986). In the presence of 40/~M Gpp(NH)p, it did not significantly alter the binding affinities of these agents: neither agonist, MeN-A-343, nor antagonists, pirenzepine and trihexyphenidyl, toward the receptor sites in the cerebrum or cerebellum. Earlier reports showed that Gpp(NH)p had an ability to decrease the binding affinity of agonist, but not antagonist on the muscarinic M2-receptor enriched tissues such as heart (Barrie et al., 1979; Rosenberger et al., 1979; Wei and Sulakhe, 1979). Our results suggest that the regulation role of G-protein on different muscarinic receptor subtypes is likely to be different.
The displacement of [3H]QNB binding by the muscarinic M2 selective agents When muscarinic M 2 selective agents were used to displace the [aH]QNB binding by rat cerebral and
476
JIANN-Wu WEI and WEN-CHUNHUNG
cerebellar synaptosomes. It was found that muscarinic M2 selective agonists, carbachol and oxotremorine, and antagonists, A F - D X - 1 1 6 had higher affinities for receptor sites in the cerebellum than in the cerebrum. This is based on the ICso-values obtained from the dose--displacement curves of [3H]QNB binding to these two brain regions. Clearly, it is an indication that rat cerebellum contains a greater amount of muscarinic M2 receptor sites than the cerebrum. This is in good agreement with the results reported by others (Luthin and Wolfe, 1984; Watson et al., 1983). In the presence of 40/~M G p p ( N H ) p , it had the ability to decrease the binding affinities of muscarinic M 2 selective agonists, carbachol and oxotremorine, to the receptor sites in the cerebellum, but not in the cerebrum. These results are an indication that a G-protein is coupled with muscarinic M2 receptor site in the cerebellum. Similar results were found previously in the cardiac tissue by us (Wei and Sulakhe, 1979). S o m e indications and f u t u r e research work
The results of this study revealed that the newly synthesized muscarinic M~ or M2 selective agents have the ability to differentiate the muscarinic receptor subtypes in rat cerebrum and cerebellum. Furthermore, it seems that more muscarinic M~ receptor sites are present in the cerebrum than in the cerebellum, and the opposite is true for the muscarinic M2 receptor sites. The regulation of G-protein on the muscarinic M t subtype and the M2 subtypes of these two brain regions are likely different. This indication results from the fact that G p p ( N H ) p decreases the binding affinities of muscarinic M2 selective agonists when they interact with the M2 receptor sites; whereas it does not alter the binding affinity o f muscarinic M~ selective agonists when they interact with the M~ receptor sites. The further information on the basic differences between muscarinic receptor subtypes and their coupling to different second messenger systems, such as polyphosphoinositide turnover, cyclic A M P , or ion channel opening are awaiting more studies. Using techniques, such as the cloning of c D N A s and genes for different muscarinic acetylcholine receptors, may provide us with some useful information. Acknowledgement--This work was supported by a NSC grant, 77-0412-B010-14, and a grant from the Clinical Research Center, VGH, Institute of Biomedical Sciences Academia Sinica, Republic of China.
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