European Journal of Pharmacology, 52 (1978) 235--238 © Elsevier/North-Holland Biomedical Press
235
Short c o m m u n i c a t i o n R E G I O N A L AND S U B C E L L U L A R D I ST R I B U T IO N OF MYOCARDIAL MUSCARINIC CHOLINERGIC RECEPTORS JIANN-WU WEI and PRAKASH V. SULAKHE * Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Canada, S7N OWO Received 22 August 1978, accepted 12 September 1978
J.-W. WEI and P.V. SULAKHE, Regional and subcellular distribution of myocardial muscarinic cholinergic receptors, European J. Pharmacol. 52 (1978) 235--238. Regional and subcellular distributions of muscarinic cholinergic receptors were investigated in the myocardium of commonly used laboratory animals. The density of receptor sites (expressed in terms of either pmol/g protein or pmol/g tissue), amongst the regions examined, was found much higher in right and left atrium in the case of rat and rabbit whereas for the guinea pig and dog, the distribution was diffuse. However, irrespective of the species and/or region studies, the microsomal fraction, amongst the subcellular fractions, showed the highest enrichment of receptors. Muscarinic cholinergic receptors
Regional distribution
1. I n t r o d u c t i o n The negative c h r o n o t r o p i c and inotropic effects o f vagal stimulation and acetylcholine on the m y o c a r d i u m are well d o c u m e n t e d (see review by Higgins et al., 1973). Further, the receptors involved in these actions o f acetylcholine have been classified as muscarinic cholinergic. Biochemical studies of identification and properties of these receptors in different regions of the m y o c a r d i u m are of great significance since such studies would enable correlations of r e c e p t o r densities and affinities with physiological and pharmacological actions o f cholinergic agents on this organ. [3H]-Quinuclidinyl benzilate (QNB), a p o t e n t muscarinic antagonist, has proven useful in identification of muscarinic receptors in membranes o f m a n y tissues (Yamamura and Snyder, 1974). We r epor t here some interesting differences in the regional distribution o f muscarinic r e c e p t o r sites in the m y o c a r d i u m * All correspondence and reprint requests should be sent to: Dr. P.V. Sulakhe at the above address.
Subcellular distribution
Heart
of c o m m o n l y used laboratory animals. The density of r e c e p t o r sites (in terms of pmol/g tissue), amongst the regions examined, was f o u n d m uch higher in right and left atrium in the case of rat and rabbit whereas for the guinea pig and dog, t he distribution was diffuse. Further, when expressed as percentage of total cardiac receptors, atria from rat and rabbit cont ai ned 43 to 53% whereas guinea pig and dog atria contained only 25 to 27%. However, the microsomal fraction, am onst the subcellular fractions isolated from atrial or ventricular homogenates o f each species, showed t he highest enrichment of r e c e p t o r sites (in terms o f fm ol / m g protein).
2. Materials and m e t h o d s 2.1. Materials
[3H]-Quinuclidinyl benzilate (29.4 Ci/mmol) was purchased from New England Nuclear, Canada. All ot her chemicals were of highest purity available commercially.
236
2.2. Tissue preparation for regional distribution The myocardium [rat (200--300 g), guinea pig (300--400 g), rabbit (3--4 kg) and dog (10--12 kg)], freed of connective tissue, great vessels, epi- and endocardium, blood and fat, was separated into various regions. For rat, rabbit and guinea pig, sinoatrial node and atrioventricular node were present in the sample of right atrium used; for dog, the nodes were dissected free of the right atrial tissue and the atrium and the separated nodes were used. The separated regions were thoroughly washed, blotted, weighed and then cut into fine pieces. The minced tissues were separately homogenized at 4°C in 1 0 v o l (based on tissue weight) of 10 mM imidazoleHC1 (pH 7.8) in a Polytron PT-10 homogenizer (10 sec × 3 with waiting of 30 sec in between bursts; setting 8). The homogenates, after filtration through double layer of cheesecloth, were used in the binding assay.
2.3. Subcellular fractionation In some experiments, atrial or ventricular homogenates were subjected to differential centrifugation as described earlier by Ma et al. (1978) and the subcellular fractions were used in the binding assay.
2.4. Binding assay The assay mixture ( 0 . 1 5 m l ) contained 50 mM Tris-HC1, pH 7.8, 10 mM MgC12, 5 nM [3H]QNB (15--20 cpm per fmole) and 0.15--0.25 mg protein. Reactions were carried o u t for 10 min at 37°C. All other details were as described by Yamamura and Snyder (1974). Specific binding was determined from the difference between counts obtained in the absence and presence of 1 pM atropine sulphate; 85 to 90% of the total binding (without atropine in assay) was found to be specific in every case. Elsewhere, it will be reported that specific [3H]QNB binding to sites in homogenates or the subcellular fractions displays the affinity and stereospecificity expected of mus-
J.-W. WEI, P.V. SULAKHE carinic cholinergic receptors described by Yamamura and Snyder (1974) in other tissue preparations. 3. Results and discussion Results presented in table I show that left atrium and right atrium of rat and rabbit had greater density of muscarinic receptors compared to other regions o f the myocardimn: Interestingly, even though sinoatrial node and atrioventricular node were present in the right atrial tissue, left atrium exhibited greater density than right atrium. This is in agreement with the recent abstract by Fields et al. (1977) concerning regional distribution in the rabbit heart. Unexpectedly, however, the patterns of regional distribution of QNB binding sites were diffuse in the case of guinea pig and dog. Nevertheless, the results of table 1 clearly show that ventricles contain substantial amounts of muscarinic receptor sites, a finding that was obtained with each species examined. When expressed as pmole/g of myocardium (or g of either ventricle), the rank order for receptor densities was rat > guinea pig > dog > rabbit; when expressed as pmole/g of either atrium, the rank order was r a t > rabbit > guinea pig = dog. Table 1 also shows that within ventricles, distribution of receptor density was similar; ventricular tissue from the apical and base regions displayed rather similar densities. Interestingly, when expressed as percentage of total cardiac receptors, atria contained 43--53% of sites in the case of rat and rabbit whereas atria from guinea pig and dog contained only 25--27%. In other words, for the latter species, ventricles contained even greater percentage of total cardiac receptors than atria. The regional differences reported here in densities of the receptor sites are not due to the differences in the affinities of the receptor sites towards QNB or in the rates of binding, since under the assay conditions used (i.e. 5 nM QNB and 10 min incubation at 37°C), maximal binding was reached in every case. When atrial homogenates were fractionated
CARDIAC MUSCARINIC RECEPTORS
237
TABLE 1 Regional d i s t r i b u t i o n o f m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s in t h e m y o c a r d i u m o f d i f f e r e n t species. Regions used were: r i g h t a t r i u m ( R A ) , left a t r i u m (LA), v e n t r i c u l a r s e p t u m , r i g h t v e n t r i c l e base ( R V B ) a n d apical ( R V A ) regions, left ventricle base ( L V B ) a n d apical ( L V A ) regions. E x c e p t for dog, sinoatrial (SA) a n d a t r i o v e n tricular ( A V ) n o d e s were p r e s e n t in t h e r i g h t atrial tissue used. In t h e case of dog, b i n d i n g values for SA a n d A V n o d e s were 116.5 + 3.0 a n d 130.7 + 2.7 respectively. Results are e x p r e s s e d in t e r m s of p m o l e [ 3 H ] Q N B b o u n d / g p r o t e i n ; values in p a r e n t h e s e s r e p r e s e n t p m o l e Q N B b o u n d / g tissue. All results are m e a n s + S.E. o f 3--6 experiments. Region
Rat
Rabbit
G u i n e a pig
Dog
RA
2 0 0 . 0 + 4.2 (22.7 -+ 0.5)
98.9 + 1.6 ( 1 0 . 2 -+ 0.2)
1 7 6 . 6 + 6.9 (9.9 +- 0.4)
1 0 8 . 4 + 2.0 (7.2 + 0.5)
LA
2 4 7 . 9 + 1.2 (27.8 -+ 0.2)
164.5 -+ 3.0 (14.3 -+ 0.3)
170.7 + 1.9 (10.3 -+ 0.1)
148.1 + 6.5 (9.5 + 0.4)
Septum
113.9 + 2.7 (16.7 +- 0.4)
42.7 + 0.9 (5.6 + 0.1)
1 9 2 . 3 + 5.7 (17.6 + 0.5)
87.2 + 1.7 (10.2 + 0.2)
RVB
1 2 2 . 4 -+ 0.6 (16.9 -+ 0.1)
50.3 + 0.8 (6.1 + 0.1)
156.7 + 4.3 ( 1 2 . 0 + 0.3)
1 3 9 . 3 + 6.0 (12.5 -+ 0.5)
RVA
113.9 + 3.0 (15.4 -+ 0.4)
36.2 + 1.8 (4.8 -+ 0.2)
1 5 1 . 6 + 2.1 (7.8 + 0.1)
163.7 + 3.7 (17.7 + 0.4)
LVB
1 0 9 . 2 +- 5.1 (16.6 -+ 0.8)
39.8 + 1.6 (6.0 + 0.2)
1 5 1 . 0 + 1.1 (15.3 + 0.1)
100.7 + 5.8 (10.4 + 0.6)
LVA
120.0 -+ 3.4 (17.5 -+ 0.5)
36.2 + 0.9 (5.3 + 0.1)
1 5 2 . 9 + 4.3 (15.3 + 0.4)
109.3 + 0.7 (10.6 -+ 0.1)
(see table 2), the heavy microsomal fraction (40,000 × g residue) showed the highest specific activity for [3H]QNB binding and con-
tained 15 (guinea pig), 19 (rat), 28 (rabbit) and 22% (dog) of the total homogenate binding activity (homogenate activity = 100%). The
TABLE 2 Subcellular d i s t r i b u t i o n of m u s c a r i n i c cholinergic r e c e p t o r s in a t r i a of d i f f e r e n t species. Atrial h o m o g e n a t e s were c e n t r i f u g e d as described in t h e Materials a n d m e t h o d s s e c t i o n (also see t a b l e 1 ) a n d t h e f r a c t i o n s assayed for [ 3 H ] Q N B binding. Results are e x p r e s s e d as f m o l / m g p r o t e i n a n d are means_+ S.E. of 3 d e t e r m i n a t i o n s . In each case, t h e 1 0 0 , 0 0 0 X g -- s u p e r n a t a n t f r a c t i o n failed to s h o w a n y d e t e c t a b l e b i n d i n g activity ( n o t s h o w n in table). T h e p a t t e r n s of s u b c e l l u l a r d i s t r i b u t i o n for v e n t r i c u l a r h o m o g e n a t e s were similar t o t h o s e s h o w n here for atrial h o m o g e n a t e s a n d h e n c e are n o t i n c l u d e d in this table. Fraction
Rat
Rabbit
G u i n e a pig
Dog
Nuclear-myofibrillar (1,000 x g--residue)
3 4 3 . 3 +_ 12.8
1 6 0 . 8 _+ 8.6
2 3 8 . 8 +_ 9.0
80.3 _+ 3.4
Mitochondrial ( 1 0 , 0 0 0 x g -- residue)
6 6 7 . 2 +_ 33.7
3 7 4 . 2 _+ 17.0
3 0 5 . 6 _+ 10.2
3 0 6 . 7 _+ 23.5
Heavy m i c r o s o m a l (40,000 x g-- residue)
9 6 5 . 5 _+ 23.9
8 6 5 . 3 +- 16.0
578.7 +_ 19.1
3 9 9 . 6 _+ 19.3
Light m i c r o s o m a l ( 1 0 0 , 0 0 0 × g -- r e s i d u e )
1 8 0 . 0 +_ 14.4
3 6 0 . 0 +- 16.2
2 8 2 . 4 _+ 21.8
1 5 4 . 3 _+ 11.3
238
nuclear-myofibrillar fraction (1,000 X g residue) contained about 40--60% of the total binding activity, the mitochondrial-lysosomal fraction, about 17--20%, and the light microsomal fraction, 2--4%; the soluble fraction (100,000 X g supernatant) did not contain any detectable binding activity. Similar patterns of subceltular distribution were also observed for ventricular tissue (results not shown). These findings demonstrate that muscarinic receptors are membrane-bound and are present most likely in the plasma membrane. This view is supported by our previous observation showing an enrichment of [3H]atropine binding activity in the plasma membrane fraction of dog and guinea pig atria (Ma et al., 1978). It would be of interest to see whether or not patterns of distribution of acetylcholine and its synthetic enzyme, choline acetyl transferase show species-related regional differences as we have noted for the QNB binding sites. Nevertheless, our results support the view (see Higgins et al., 1973 ) that acetylcholine directly influences ventricular performance by interacting with the ventricular muscarinic receptors. The finding of St. Louis and Sulakhe (1976) that cardiac ventricular guanylate cyclase activity is stimulated by acetylcholine and inhibited by atropine further supports this view.
J.-W. WEI, P.V. SULAKHE
Acknowledgements This work was supported by a grant from the Saskatchewan Heart Foundation. J.-W. Wei is a post. doctoral research fellow of the Canadian Heart Foundation.
References Fields, J.Z., W.R. Roeske, E. Morkin and H.I. Yamamura, 1977, Biochemical demonstration of muscarinic cholinergic receptors in mammalian heart, Federation Proc. 36, 979 (abstract). Higgins, C.B., S.F. Vatner and E. Braunwald, 1973, Parasympathetic control of the heart, Pharmacol. Rev. 25,119. Ma, S.K., P.V. Sulakhe and N.L.K. Leung, 1978, Binding of [3H] atropine by cardiac plasma membrane-enriched fractions, in: Recent Advances in Studies on Cardiac Structure and Metabolism, Vol. 11, eds. T. Kobayashi, T. Sano and N.S. Dhalla (University Park Press, Baltimore) p. 249. St. Louis, P.J. and P.V. Sulakhe, 1976, Adenylate cyclase, guanylate cyclase and cyclic nucleotide phosphodiesterases of guinea pig cardiac sarcolemma, Biochem. J. 158, 535. Yamamura, H.I. and S.H. Snyder, 1974, Muscarinic cholinergic receptor binding in the longitudinal muscle of the guinea pig ileum with [3H] quinuclidinyl benzilate, Mol. Pharmacol. 10,861.
235
Short c o m m u n i c a t i o n R E G I O N A L AND S U B C E L L U L A R D I ST R I B U T IO N OF MYOCARDIAL MUSCARINIC CHOLINERGIC RECEPTORS JIANN-WU WEI and PRAKASH V. SULAKHE * Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Canada, S7N OWO Received 22 August 1978, accepted 12 September 1978
J.-W. WEI and P.V. SULAKHE, Regional and subcellular distribution of myocardial muscarinic cholinergic receptors, European J. Pharmacol. 52 (1978) 235--238. Regional and subcellular distributions of muscarinic cholinergic receptors were investigated in the myocardium of commonly used laboratory animals. The density of receptor sites (expressed in terms of either pmol/g protein or pmol/g tissue), amongst the regions examined, was found much higher in right and left atrium in the case of rat and rabbit whereas for the guinea pig and dog, the distribution was diffuse. However, irrespective of the species and/or region studies, the microsomal fraction, amongst the subcellular fractions, showed the highest enrichment of receptors. Muscarinic cholinergic receptors
Regional distribution
1. I n t r o d u c t i o n The negative c h r o n o t r o p i c and inotropic effects o f vagal stimulation and acetylcholine on the m y o c a r d i u m are well d o c u m e n t e d (see review by Higgins et al., 1973). Further, the receptors involved in these actions o f acetylcholine have been classified as muscarinic cholinergic. Biochemical studies of identification and properties of these receptors in different regions of the m y o c a r d i u m are of great significance since such studies would enable correlations of r e c e p t o r densities and affinities with physiological and pharmacological actions o f cholinergic agents on this organ. [3H]-Quinuclidinyl benzilate (QNB), a p o t e n t muscarinic antagonist, has proven useful in identification of muscarinic receptors in membranes o f m a n y tissues (Yamamura and Snyder, 1974). We r epor t here some interesting differences in the regional distribution o f muscarinic r e c e p t o r sites in the m y o c a r d i u m * All correspondence and reprint requests should be sent to: Dr. P.V. Sulakhe at the above address.
Subcellular distribution
Heart
of c o m m o n l y used laboratory animals. The density of r e c e p t o r sites (in terms of pmol/g tissue), amongst the regions examined, was f o u n d m uch higher in right and left atrium in the case of rat and rabbit whereas for the guinea pig and dog, t he distribution was diffuse. Further, when expressed as percentage of total cardiac receptors, atria from rat and rabbit cont ai ned 43 to 53% whereas guinea pig and dog atria contained only 25 to 27%. However, the microsomal fraction, am onst the subcellular fractions isolated from atrial or ventricular homogenates o f each species, showed t he highest enrichment of r e c e p t o r sites (in terms o f fm ol / m g protein).
2. Materials and m e t h o d s 2.1. Materials
[3H]-Quinuclidinyl benzilate (29.4 Ci/mmol) was purchased from New England Nuclear, Canada. All ot her chemicals were of highest purity available commercially.
236
2.2. Tissue preparation for regional distribution The myocardium [rat (200--300 g), guinea pig (300--400 g), rabbit (3--4 kg) and dog (10--12 kg)], freed of connective tissue, great vessels, epi- and endocardium, blood and fat, was separated into various regions. For rat, rabbit and guinea pig, sinoatrial node and atrioventricular node were present in the sample of right atrium used; for dog, the nodes were dissected free of the right atrial tissue and the atrium and the separated nodes were used. The separated regions were thoroughly washed, blotted, weighed and then cut into fine pieces. The minced tissues were separately homogenized at 4°C in 1 0 v o l (based on tissue weight) of 10 mM imidazoleHC1 (pH 7.8) in a Polytron PT-10 homogenizer (10 sec × 3 with waiting of 30 sec in between bursts; setting 8). The homogenates, after filtration through double layer of cheesecloth, were used in the binding assay.
2.3. Subcellular fractionation In some experiments, atrial or ventricular homogenates were subjected to differential centrifugation as described earlier by Ma et al. (1978) and the subcellular fractions were used in the binding assay.
2.4. Binding assay The assay mixture ( 0 . 1 5 m l ) contained 50 mM Tris-HC1, pH 7.8, 10 mM MgC12, 5 nM [3H]QNB (15--20 cpm per fmole) and 0.15--0.25 mg protein. Reactions were carried o u t for 10 min at 37°C. All other details were as described by Yamamura and Snyder (1974). Specific binding was determined from the difference between counts obtained in the absence and presence of 1 pM atropine sulphate; 85 to 90% of the total binding (without atropine in assay) was found to be specific in every case. Elsewhere, it will be reported that specific [3H]QNB binding to sites in homogenates or the subcellular fractions displays the affinity and stereospecificity expected of mus-
J.-W. WEI, P.V. SULAKHE carinic cholinergic receptors described by Yamamura and Snyder (1974) in other tissue preparations. 3. Results and discussion Results presented in table I show that left atrium and right atrium of rat and rabbit had greater density of muscarinic receptors compared to other regions o f the myocardimn: Interestingly, even though sinoatrial node and atrioventricular node were present in the right atrial tissue, left atrium exhibited greater density than right atrium. This is in agreement with the recent abstract by Fields et al. (1977) concerning regional distribution in the rabbit heart. Unexpectedly, however, the patterns of regional distribution of QNB binding sites were diffuse in the case of guinea pig and dog. Nevertheless, the results of table 1 clearly show that ventricles contain substantial amounts of muscarinic receptor sites, a finding that was obtained with each species examined. When expressed as pmole/g of myocardium (or g of either ventricle), the rank order for receptor densities was rat > guinea pig > dog > rabbit; when expressed as pmole/g of either atrium, the rank order was r a t > rabbit > guinea pig = dog. Table 1 also shows that within ventricles, distribution of receptor density was similar; ventricular tissue from the apical and base regions displayed rather similar densities. Interestingly, when expressed as percentage of total cardiac receptors, atria contained 43--53% of sites in the case of rat and rabbit whereas atria from guinea pig and dog contained only 25--27%. In other words, for the latter species, ventricles contained even greater percentage of total cardiac receptors than atria. The regional differences reported here in densities of the receptor sites are not due to the differences in the affinities of the receptor sites towards QNB or in the rates of binding, since under the assay conditions used (i.e. 5 nM QNB and 10 min incubation at 37°C), maximal binding was reached in every case. When atrial homogenates were fractionated
CARDIAC MUSCARINIC RECEPTORS
237
TABLE 1 Regional d i s t r i b u t i o n o f m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s in t h e m y o c a r d i u m o f d i f f e r e n t species. Regions used were: r i g h t a t r i u m ( R A ) , left a t r i u m (LA), v e n t r i c u l a r s e p t u m , r i g h t v e n t r i c l e base ( R V B ) a n d apical ( R V A ) regions, left ventricle base ( L V B ) a n d apical ( L V A ) regions. E x c e p t for dog, sinoatrial (SA) a n d a t r i o v e n tricular ( A V ) n o d e s were p r e s e n t in t h e r i g h t atrial tissue used. In t h e case of dog, b i n d i n g values for SA a n d A V n o d e s were 116.5 + 3.0 a n d 130.7 + 2.7 respectively. Results are e x p r e s s e d in t e r m s of p m o l e [ 3 H ] Q N B b o u n d / g p r o t e i n ; values in p a r e n t h e s e s r e p r e s e n t p m o l e Q N B b o u n d / g tissue. All results are m e a n s + S.E. o f 3--6 experiments. Region
Rat
Rabbit
G u i n e a pig
Dog
RA
2 0 0 . 0 + 4.2 (22.7 -+ 0.5)
98.9 + 1.6 ( 1 0 . 2 -+ 0.2)
1 7 6 . 6 + 6.9 (9.9 +- 0.4)
1 0 8 . 4 + 2.0 (7.2 + 0.5)
LA
2 4 7 . 9 + 1.2 (27.8 -+ 0.2)
164.5 -+ 3.0 (14.3 -+ 0.3)
170.7 + 1.9 (10.3 -+ 0.1)
148.1 + 6.5 (9.5 + 0.4)
Septum
113.9 + 2.7 (16.7 +- 0.4)
42.7 + 0.9 (5.6 + 0.1)
1 9 2 . 3 + 5.7 (17.6 + 0.5)
87.2 + 1.7 (10.2 + 0.2)
RVB
1 2 2 . 4 -+ 0.6 (16.9 -+ 0.1)
50.3 + 0.8 (6.1 + 0.1)
156.7 + 4.3 ( 1 2 . 0 + 0.3)
1 3 9 . 3 + 6.0 (12.5 -+ 0.5)
RVA
113.9 + 3.0 (15.4 -+ 0.4)
36.2 + 1.8 (4.8 -+ 0.2)
1 5 1 . 6 + 2.1 (7.8 + 0.1)
163.7 + 3.7 (17.7 + 0.4)
LVB
1 0 9 . 2 +- 5.1 (16.6 -+ 0.8)
39.8 + 1.6 (6.0 + 0.2)
1 5 1 . 0 + 1.1 (15.3 + 0.1)
100.7 + 5.8 (10.4 + 0.6)
LVA
120.0 -+ 3.4 (17.5 -+ 0.5)
36.2 + 0.9 (5.3 + 0.1)
1 5 2 . 9 + 4.3 (15.3 + 0.4)
109.3 + 0.7 (10.6 -+ 0.1)
(see table 2), the heavy microsomal fraction (40,000 × g residue) showed the highest specific activity for [3H]QNB binding and con-
tained 15 (guinea pig), 19 (rat), 28 (rabbit) and 22% (dog) of the total homogenate binding activity (homogenate activity = 100%). The
TABLE 2 Subcellular d i s t r i b u t i o n of m u s c a r i n i c cholinergic r e c e p t o r s in a t r i a of d i f f e r e n t species. Atrial h o m o g e n a t e s were c e n t r i f u g e d as described in t h e Materials a n d m e t h o d s s e c t i o n (also see t a b l e 1 ) a n d t h e f r a c t i o n s assayed for [ 3 H ] Q N B binding. Results are e x p r e s s e d as f m o l / m g p r o t e i n a n d are means_+ S.E. of 3 d e t e r m i n a t i o n s . In each case, t h e 1 0 0 , 0 0 0 X g -- s u p e r n a t a n t f r a c t i o n failed to s h o w a n y d e t e c t a b l e b i n d i n g activity ( n o t s h o w n in table). T h e p a t t e r n s of s u b c e l l u l a r d i s t r i b u t i o n for v e n t r i c u l a r h o m o g e n a t e s were similar t o t h o s e s h o w n here for atrial h o m o g e n a t e s a n d h e n c e are n o t i n c l u d e d in this table. Fraction
Rat
Rabbit
G u i n e a pig
Dog
Nuclear-myofibrillar (1,000 x g--residue)
3 4 3 . 3 +_ 12.8
1 6 0 . 8 _+ 8.6
2 3 8 . 8 +_ 9.0
80.3 _+ 3.4
Mitochondrial ( 1 0 , 0 0 0 x g -- residue)
6 6 7 . 2 +_ 33.7
3 7 4 . 2 _+ 17.0
3 0 5 . 6 _+ 10.2
3 0 6 . 7 _+ 23.5
Heavy m i c r o s o m a l (40,000 x g-- residue)
9 6 5 . 5 _+ 23.9
8 6 5 . 3 +- 16.0
578.7 +_ 19.1
3 9 9 . 6 _+ 19.3
Light m i c r o s o m a l ( 1 0 0 , 0 0 0 × g -- r e s i d u e )
1 8 0 . 0 +_ 14.4
3 6 0 . 0 +- 16.2
2 8 2 . 4 _+ 21.8
1 5 4 . 3 _+ 11.3
238
nuclear-myofibrillar fraction (1,000 X g residue) contained about 40--60% of the total binding activity, the mitochondrial-lysosomal fraction, about 17--20%, and the light microsomal fraction, 2--4%; the soluble fraction (100,000 X g supernatant) did not contain any detectable binding activity. Similar patterns of subceltular distribution were also observed for ventricular tissue (results not shown). These findings demonstrate that muscarinic receptors are membrane-bound and are present most likely in the plasma membrane. This view is supported by our previous observation showing an enrichment of [3H]atropine binding activity in the plasma membrane fraction of dog and guinea pig atria (Ma et al., 1978). It would be of interest to see whether or not patterns of distribution of acetylcholine and its synthetic enzyme, choline acetyl transferase show species-related regional differences as we have noted for the QNB binding sites. Nevertheless, our results support the view (see Higgins et al., 1973 ) that acetylcholine directly influences ventricular performance by interacting with the ventricular muscarinic receptors. The finding of St. Louis and Sulakhe (1976) that cardiac ventricular guanylate cyclase activity is stimulated by acetylcholine and inhibited by atropine further supports this view.
J.-W. WEI, P.V. SULAKHE
Acknowledgements This work was supported by a grant from the Saskatchewan Heart Foundation. J.-W. Wei is a post. doctoral research fellow of the Canadian Heart Foundation.
References Fields, J.Z., W.R. Roeske, E. Morkin and H.I. Yamamura, 1977, Biochemical demonstration of muscarinic cholinergic receptors in mammalian heart, Federation Proc. 36, 979 (abstract). Higgins, C.B., S.F. Vatner and E. Braunwald, 1973, Parasympathetic control of the heart, Pharmacol. Rev. 25,119. Ma, S.K., P.V. Sulakhe and N.L.K. Leung, 1978, Binding of [3H] atropine by cardiac plasma membrane-enriched fractions, in: Recent Advances in Studies on Cardiac Structure and Metabolism, Vol. 11, eds. T. Kobayashi, T. Sano and N.S. Dhalla (University Park Press, Baltimore) p. 249. St. Louis, P.J. and P.V. Sulakhe, 1976, Adenylate cyclase, guanylate cyclase and cyclic nucleotide phosphodiesterases of guinea pig cardiac sarcolemma, Biochem. J. 158, 535. Yamamura, H.I. and S.H. Snyder, 1974, Muscarinic cholinergic receptor binding in the longitudinal muscle of the guinea pig ileum with [3H] quinuclidinyl benzilate, Mol. Pharmacol. 10,861.
