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Are muscarinic receptors in the central and peripheral nervous system different?
European Journal o f Pharmacology, 30 ( 197 5) 360--363 © North-Holland Publishing Company
Short communication ARE MUSCARINIC RECEPTORS IN THE CENTRAL AND P E R I P H E R A L N E R V O U S SYSTEM D I F F E R E N T ? Arie J. BELD, Sylvia VAN DEN HOVEN, Alfonsus C. WOUTERSE and Maria A.P. ZEGERS
Department o f Pharmacology, University o f Nijmegen, The Netherlands Received 29 November 1974, accepted 20 December 1974
A.J. BELD, S. VAN DEN HOVEN, A.C. WOUTERSE and M.A.P. ZEGERS, Are muscarinic receptors in the central and peripheral nervous system different?, European J. Pharmacol. 30 (1975) 360--363. The concentration-dependent binding of atropine-3H to membrane fractions from bovine tracheal muscle, parotid gland and caudate nucleus, measured by equilibrium dialysis, revealed the presence of virtually identical high affinity binding sites in all three tissues. Sch 1000 and Sch 1178, geometrical isomers of N-isopropylatropine bromide with a large potency ratio as antimuscarinics, inhibited atropine binding identically in all three tissues. Differences in properties of muscarinic receptors in these tissues are either non-existent or too small to be detected by the applied techniques. Bovine tracheal muscle, parotid gland, caudate nucleus Muscarinic receptor Sch 1000
1. Introduction Muscarinic agonists and antagonists affect smooth muscle, the central nervous system, the heart, the eye and exocrine glands through muscarinic receptors. The question arises whether these receptors are all the same or can be divided into different types, analogous to ~1 and ~2 type adrenergic receptors and H1 and H2 type histaminergic receptors. With the objective of differentiating between muscarinic receptors of various origin, the parameters of the action of antagonists have been determined by means of several biological methods. Barlow et al. {1972) measured affinity constants of muscarinic antagonists in the ileum, bronchial muscle and iris of the guinea pig. Inch et al. (1973) determined central and peripheral activities of antiacetylcholine drugs in the central and peripheral nervous system. Both studies led to the same
Affinity constants Atropine binding Sch 1178
conclusion, that antimuscarinic drugs do not differ in their intrinsic potency to inhibit central or peripheral muscarinic receptors. We have looked at the problem from a different perspective by calculating the affinity constants of atropine for muscarinic receptors in bovine tracheal muscle, parotid gland and caudate nucleus. The data were obtained from determinations of direct binding of tritium-labeled atropine to crude membrane fractions prepared from these tissues. The results, which show only minor differences between the affinity constants in the different tissues, lead to the conclusion that muscarinic receptors throughout the organism are identical as far as the experimental method can discern. We also measured the inhibition of atropine binding to these tissue fractions by Sch 1000 and Sch 1178, geometrical isomers of Nisopropyl-atropine bromide. Given by inhalation, Sch 1000 is a potent bronchodilator of
A.J. Beld et al., Central and peripheral muscarinic receptors
the anticholinergic type and has almost no side effects on heart rate, salivary secretion and central activity (Engelhardt and Wick, 1957; Ulmer et al., 1973; Pappius and Salorinne, 1973; Minette et al., 1973). However, when injected intravenously in humans Sch 1000 is a strong inhibitor of acetyl-fl-methylcholine-induced parotid secretion (Diamant et al., 1974). In our experiments Sch 1000 inhibited atropine binding in tracheal muscle, parotid gland and caudate nucleus in the same way, confirming the conclusion of Diamant, that Sch 1000 is a general and potent anticholinergic of the muscarinic type. The lack of side effects when the, drug is given by inhalatiorz must be asc~ibe~ to distributibn phenomena'and not to 'diffe'r$nt affinities for muscarinic receptors in various tissues. Sch 1178, a compound with much weaker antimuscarinic properties (Wick, 1974, personal communication) also inhibited atropine binding in all three tissues identically, but at a much higher dose level.
2. Materials and methods 2.1. Materials
Atropine-3H (spec. act. 400 mCi/mmol) was purchased from The Radiochemical Centre, England. Sch 1000 and Sch 1178 were obtained from C.H. Boehringer Sohn, Ingelheim, Germany through the courtesy of Mr. J. Siebelink. Sch 1000 is the quaternary Nisopropyl derivative of atropine with the isopropyl group endo to the three atom bridge of the tricyclic tropinol skeleton. In Sch 1178 the isopropyl group is in the opposite position. 2.2. Target tissues
Bovine tracheae, brains and parotid glands were obtained from the abattoir. The brains and parotid glands were transported to the laboratory in an ice bath; the tracheae in cold (0--4°C) Krebs--Henseleit solution. The tra-
361
cheal muscle and parotid glands were separated from adhering non-target tissue as much as possible. 2. 3. Homogenization
The homogenization of the tracheal muscle and parotid glands and the preparation of the microsomal pellets were the same as described previously for tracheal muscle (Beld and Ari~ns, 1974). Caudate nuclei were removed from the brains and homogenized in Krebs-Henseleit solution to a final tissue concentration of 10% in a Potter teflon-glass homogenizer. 2. 4. Binding experiments
The microsomal pellets of tracheal muscle and parotid glands were resuspended in Krebs--Henseleit by Potter homogenization to an original tissue concentration of either 0.50 or 1.00 g/ml. For the caudate nuclei the 10% homogenate was used as such in the binding experiments. The equilibrium dialysis binding and calculation of free and bound concentrations were essentially as described earlier (Beld and Ari~ns, 1974). All the binding experiments were carried out at room temperature.
3. Results and discussion The concentration-dependent binding of atropine-3H (1.0--28.0 nmol/1) to preparations of bovine tracheal muscle, parotid gland and caudate nucleus is depicted in fig. 1. Atropine was used in preference to other cholinergic ligands, because it shows almost no unspecific binding in the concentration range measured, as judged by the shape of the binding curves and the Scatchard plots (not shown) where there axe only slight deviations from linearity at the highest concentrations. Other anticholinergics show (Beld and Ari~ns, 1974) or could show considerable unspecific binding.
A.J. Beld et al., Central and peripheral muscarinic receptors
362
Table 1
Atropine-H3 p Moles bound/~ tissue
Binding parameters for atropine -3 H in bovine tracheal muscle, parotid gland and caudate nucleus. Affinity constants mol/l -+ S.E. Binding capacities mol/g tissue (wet weight) -+ S.E.
2_0
,5
:J:
.S:
s/f
10
/
~0
25. 10- 9 M
Atropine- H3 (free)
Atropine H3 p Moles bound,/~ tissue
.... Caudate nucleus
200
150 ,f!. 100
/
50
"
/
At ropine- H3 (Free)
At ropine-H 3 pMoles bound/~ tissue Parot id .~land 20
15
/
10-
,f"
/ 0
5
10
15
2'0
~5.10 -9 M
At rc~ine-H3 (free)
Fig. 1. Binding curves of atropine -3H to bovine tracheal muscle, parotid gland and caudate nucleus ( 1 . 0 - - 2 8 . 0 nmol/1).
Tissue
Affinity constant
Tracheal muscle
2.96 + 0 . 2 3 X 10 -9
19.9 -+ 0.6 X 10-12
Caudate 1.89 + 0.07 X 10- 9 nucleus
1 6 7 . 8 + 2.4 X 10-12
Parotid gland
2.09 + 0.07 X 10- 9
Binding capacity
17.5 + 0.2 X 10-12
The binding curves can be fitted to a model of one single saturable binding site. Binding parameters (affinity constants and binding capacities) were calculated from the binding data by a computerized curve fitting procedure, which is a least squares fitting method, based on a gradient method for the non-linear pm'ameters and a linear regression method for the linear parameters, originally developed by G. Fast (University Computer Centre, Nijmegen). The corresponding standard errors of the parameters were calculated in the last iteration with the usual equations applied to the quasi-linearization (table 1). Attempts to fit the binding curves to a model consisting of a saturable high affinity binding site and a linear distribution of low affinity binding sites revealed only minimal contribution from those sites. No improvement of the goodness of fit was obtained with this model. The affinity constants for parotid gland and caudate nucleus are equal within the experimental error, while the value for tracheal muscle deviates significantly by the same criteria. This difference, however is so small, that it cannot be taken as conclusive evidence for differences in receptor properties in parotid gland and caudate nucleus on one hand and in smooth muscle on the other. For the time being, we cannot exclude the possibility that with other radioactively labeled cholinergic ligands the differences in affinity con-
A.J. Beld et al., Central and peripheral muscarinic receptors Atropine -H3 bound °/o o f c o n t r o l
100 A~•
• " ~ .
• o •
Parotid o~tand Caudate nucleus Tracheal smooth muscle
363
doses, as could be expected from its lower potency as an antimuscarinic. Acknowledgement
1178
m-m
io'-8
TO'-6
The authors are indebted to Drs. M.A. van 't Hof for his advice in the mathematical treatment of the data. lo'-4 ,
Sch 1000/Sch |178
Fig. 2. Inhibition of atropine-3H binding in bovine tracheal muscle, parotid gland and caudate nucleus by Sch 1000 and Sch 1178. Bound atropine is expressed as a percentage of binding in the controls. Free concontrations of atropine -3H ranged from 10 to 12 nmol/ 1, corresponding to approximately 80% receptor saturation in the controls. Points are the mean of duplicates.
stants are larger and hence differences in receptor properties can be substantiated. Further experiments will be performed in our laboratory to investigate this possibility. The affinity constants of Sch 1000 and Sch 1178 could n o t be measured directly, because the c o m p o u n d s were n o t at our disposal in a labeled form of sufficiently high specific activity. Therefore we measured the inhibition of atropine binding by increasing concentrations of both compounds. As can be seen in fig. 2. Sch 1000 inhibits atropine binding in all three organs to the same extent within the experimental error. From this observation, the conclusion can be drawn that Sch 1000 is a potent antimuscarinic c o m p o u n d with no selectivity of action as far as its binding to receptors in different tissues is concerned. Reported seleetivity of pharmacological action must therefore be ascribed its manner of application form and/or distribution. Sch 1178 also inhibits atrol~ine binding identically in the three different tissues, b u t at much higher
References Barlow, R.B., F.M. Franks and J.D.M. Pearson, 1972, A comparison of the affinities of antagonists for acetylcholine receptors in the ileum, bronchial muscle and iris of the guinea pig, Brit. J. Pharmacol. 46, 300. Beld, A.J. and E.J. Arii~ns, 1974, Stereospecific binding as a tool in attempts to localize and isolate muscarinic receptors, European J. Pharmacol. 25, 203. Diamant, H., T. Ekstrand and J. Rydnert, 1974, Der anticholinergische Effekt yon Sch 1000 auf die Parotissekretion w~ihrend Stimulierung mit Acetyl#-methylcholin, Arzneim. Forsch. 24, 573. Engelhardt, A. and H. Wick, 1957, Beziehungen zwischen Konstitution und Wirkung bei Tropeinen und ihren quart/iren Derivaten, Arzneim. Forsch. 7,217. Inch, T.D., D.M. Green and P.B.J. Thompson, 1973, The central and peripheral activities of anti-acetylcholine drugs. Some concepts of practical relevance, J. Pharm. Pharmacol. 25, 359. Minette, A., M. Marcq, M. Bruninx, B. Spaas and G. van Hove, 1973, R~sultats d'une recherche sur les effets ventilatoires et associ~s du Sch 1000 en a~rosol chez 54 patients atteints de bronchoobstruction r6versible, Revue de l'Institut d'Hygi~ne des Mines Hasselt (Belgique) 28, 22. Pappius, H. and Y. Salorinne, 1973, Comparative trial of a new anticholinergic bronchodilator, Sch 1000, and salbutamol in chronic bronchitis, Brit. Med. J. 4, 134. Ulmer, W.T., J. Dorsch, J. Iravani, K.-G. Schiiler, G. Stempel and E. Vastag, 1973, Anticholinergika als Bronchodilatatoren. Beitrag zur Frage der Bedeutung yon Acetylcholin als Bronchokonstriktorische Transmittersubstanz bei Atemwegsobstruktionen, Arzneim. Forsch. 23, 468.
Short communication ARE MUSCARINIC RECEPTORS IN THE CENTRAL AND P E R I P H E R A L N E R V O U S SYSTEM D I F F E R E N T ? Arie J. BELD, Sylvia VAN DEN HOVEN, Alfonsus C. WOUTERSE and Maria A.P. ZEGERS
Department o f Pharmacology, University o f Nijmegen, The Netherlands Received 29 November 1974, accepted 20 December 1974
A.J. BELD, S. VAN DEN HOVEN, A.C. WOUTERSE and M.A.P. ZEGERS, Are muscarinic receptors in the central and peripheral nervous system different?, European J. Pharmacol. 30 (1975) 360--363. The concentration-dependent binding of atropine-3H to membrane fractions from bovine tracheal muscle, parotid gland and caudate nucleus, measured by equilibrium dialysis, revealed the presence of virtually identical high affinity binding sites in all three tissues. Sch 1000 and Sch 1178, geometrical isomers of N-isopropylatropine bromide with a large potency ratio as antimuscarinics, inhibited atropine binding identically in all three tissues. Differences in properties of muscarinic receptors in these tissues are either non-existent or too small to be detected by the applied techniques. Bovine tracheal muscle, parotid gland, caudate nucleus Muscarinic receptor Sch 1000
1. Introduction Muscarinic agonists and antagonists affect smooth muscle, the central nervous system, the heart, the eye and exocrine glands through muscarinic receptors. The question arises whether these receptors are all the same or can be divided into different types, analogous to ~1 and ~2 type adrenergic receptors and H1 and H2 type histaminergic receptors. With the objective of differentiating between muscarinic receptors of various origin, the parameters of the action of antagonists have been determined by means of several biological methods. Barlow et al. {1972) measured affinity constants of muscarinic antagonists in the ileum, bronchial muscle and iris of the guinea pig. Inch et al. (1973) determined central and peripheral activities of antiacetylcholine drugs in the central and peripheral nervous system. Both studies led to the same
Affinity constants Atropine binding Sch 1178
conclusion, that antimuscarinic drugs do not differ in their intrinsic potency to inhibit central or peripheral muscarinic receptors. We have looked at the problem from a different perspective by calculating the affinity constants of atropine for muscarinic receptors in bovine tracheal muscle, parotid gland and caudate nucleus. The data were obtained from determinations of direct binding of tritium-labeled atropine to crude membrane fractions prepared from these tissues. The results, which show only minor differences between the affinity constants in the different tissues, lead to the conclusion that muscarinic receptors throughout the organism are identical as far as the experimental method can discern. We also measured the inhibition of atropine binding to these tissue fractions by Sch 1000 and Sch 1178, geometrical isomers of Nisopropyl-atropine bromide. Given by inhalation, Sch 1000 is a potent bronchodilator of
A.J. Beld et al., Central and peripheral muscarinic receptors
the anticholinergic type and has almost no side effects on heart rate, salivary secretion and central activity (Engelhardt and Wick, 1957; Ulmer et al., 1973; Pappius and Salorinne, 1973; Minette et al., 1973). However, when injected intravenously in humans Sch 1000 is a strong inhibitor of acetyl-fl-methylcholine-induced parotid secretion (Diamant et al., 1974). In our experiments Sch 1000 inhibited atropine binding in tracheal muscle, parotid gland and caudate nucleus in the same way, confirming the conclusion of Diamant, that Sch 1000 is a general and potent anticholinergic of the muscarinic type. The lack of side effects when the, drug is given by inhalatiorz must be asc~ibe~ to distributibn phenomena'and not to 'diffe'r$nt affinities for muscarinic receptors in various tissues. Sch 1178, a compound with much weaker antimuscarinic properties (Wick, 1974, personal communication) also inhibited atropine binding in all three tissues identically, but at a much higher dose level.
2. Materials and methods 2.1. Materials
Atropine-3H (spec. act. 400 mCi/mmol) was purchased from The Radiochemical Centre, England. Sch 1000 and Sch 1178 were obtained from C.H. Boehringer Sohn, Ingelheim, Germany through the courtesy of Mr. J. Siebelink. Sch 1000 is the quaternary Nisopropyl derivative of atropine with the isopropyl group endo to the three atom bridge of the tricyclic tropinol skeleton. In Sch 1178 the isopropyl group is in the opposite position. 2.2. Target tissues
Bovine tracheae, brains and parotid glands were obtained from the abattoir. The brains and parotid glands were transported to the laboratory in an ice bath; the tracheae in cold (0--4°C) Krebs--Henseleit solution. The tra-
361
cheal muscle and parotid glands were separated from adhering non-target tissue as much as possible. 2. 3. Homogenization
The homogenization of the tracheal muscle and parotid glands and the preparation of the microsomal pellets were the same as described previously for tracheal muscle (Beld and Ari~ns, 1974). Caudate nuclei were removed from the brains and homogenized in Krebs-Henseleit solution to a final tissue concentration of 10% in a Potter teflon-glass homogenizer. 2. 4. Binding experiments
The microsomal pellets of tracheal muscle and parotid glands were resuspended in Krebs--Henseleit by Potter homogenization to an original tissue concentration of either 0.50 or 1.00 g/ml. For the caudate nuclei the 10% homogenate was used as such in the binding experiments. The equilibrium dialysis binding and calculation of free and bound concentrations were essentially as described earlier (Beld and Ari~ns, 1974). All the binding experiments were carried out at room temperature.
3. Results and discussion The concentration-dependent binding of atropine-3H (1.0--28.0 nmol/1) to preparations of bovine tracheal muscle, parotid gland and caudate nucleus is depicted in fig. 1. Atropine was used in preference to other cholinergic ligands, because it shows almost no unspecific binding in the concentration range measured, as judged by the shape of the binding curves and the Scatchard plots (not shown) where there axe only slight deviations from linearity at the highest concentrations. Other anticholinergics show (Beld and Ari~ns, 1974) or could show considerable unspecific binding.
A.J. Beld et al., Central and peripheral muscarinic receptors
362
Table 1
Atropine-H3 p Moles bound/~ tissue
Binding parameters for atropine -3 H in bovine tracheal muscle, parotid gland and caudate nucleus. Affinity constants mol/l -+ S.E. Binding capacities mol/g tissue (wet weight) -+ S.E.
2_0
,5
:J:
.S:
s/f
10
/
~0
25. 10- 9 M
Atropine- H3 (free)
Atropine H3 p Moles bound,/~ tissue
.... Caudate nucleus
200
150 ,f!. 100
/
50
"
/
At ropine- H3 (Free)
At ropine-H 3 pMoles bound/~ tissue Parot id .~land 20
15
/
10-
,f"
/ 0
5
10
15
2'0
~5.10 -9 M
At rc~ine-H3 (free)
Fig. 1. Binding curves of atropine -3H to bovine tracheal muscle, parotid gland and caudate nucleus ( 1 . 0 - - 2 8 . 0 nmol/1).
Tissue
Affinity constant
Tracheal muscle
2.96 + 0 . 2 3 X 10 -9
19.9 -+ 0.6 X 10-12
Caudate 1.89 + 0.07 X 10- 9 nucleus
1 6 7 . 8 + 2.4 X 10-12
Parotid gland
2.09 + 0.07 X 10- 9
Binding capacity
17.5 + 0.2 X 10-12
The binding curves can be fitted to a model of one single saturable binding site. Binding parameters (affinity constants and binding capacities) were calculated from the binding data by a computerized curve fitting procedure, which is a least squares fitting method, based on a gradient method for the non-linear pm'ameters and a linear regression method for the linear parameters, originally developed by G. Fast (University Computer Centre, Nijmegen). The corresponding standard errors of the parameters were calculated in the last iteration with the usual equations applied to the quasi-linearization (table 1). Attempts to fit the binding curves to a model consisting of a saturable high affinity binding site and a linear distribution of low affinity binding sites revealed only minimal contribution from those sites. No improvement of the goodness of fit was obtained with this model. The affinity constants for parotid gland and caudate nucleus are equal within the experimental error, while the value for tracheal muscle deviates significantly by the same criteria. This difference, however is so small, that it cannot be taken as conclusive evidence for differences in receptor properties in parotid gland and caudate nucleus on one hand and in smooth muscle on the other. For the time being, we cannot exclude the possibility that with other radioactively labeled cholinergic ligands the differences in affinity con-
A.J. Beld et al., Central and peripheral muscarinic receptors Atropine -H3 bound °/o o f c o n t r o l
100 A~•
• " ~ .
• o •
Parotid o~tand Caudate nucleus Tracheal smooth muscle
363
doses, as could be expected from its lower potency as an antimuscarinic. Acknowledgement
1178
m-m
io'-8
TO'-6
The authors are indebted to Drs. M.A. van 't Hof for his advice in the mathematical treatment of the data. lo'-4 ,
Sch 1000/Sch |178
Fig. 2. Inhibition of atropine-3H binding in bovine tracheal muscle, parotid gland and caudate nucleus by Sch 1000 and Sch 1178. Bound atropine is expressed as a percentage of binding in the controls. Free concontrations of atropine -3H ranged from 10 to 12 nmol/ 1, corresponding to approximately 80% receptor saturation in the controls. Points are the mean of duplicates.
stants are larger and hence differences in receptor properties can be substantiated. Further experiments will be performed in our laboratory to investigate this possibility. The affinity constants of Sch 1000 and Sch 1178 could n o t be measured directly, because the c o m p o u n d s were n o t at our disposal in a labeled form of sufficiently high specific activity. Therefore we measured the inhibition of atropine binding by increasing concentrations of both compounds. As can be seen in fig. 2. Sch 1000 inhibits atropine binding in all three organs to the same extent within the experimental error. From this observation, the conclusion can be drawn that Sch 1000 is a potent antimuscarinic c o m p o u n d with no selectivity of action as far as its binding to receptors in different tissues is concerned. Reported seleetivity of pharmacological action must therefore be ascribed its manner of application form and/or distribution. Sch 1178 also inhibits atrol~ine binding identically in the three different tissues, b u t at much higher
References Barlow, R.B., F.M. Franks and J.D.M. Pearson, 1972, A comparison of the affinities of antagonists for acetylcholine receptors in the ileum, bronchial muscle and iris of the guinea pig, Brit. J. Pharmacol. 46, 300. Beld, A.J. and E.J. Arii~ns, 1974, Stereospecific binding as a tool in attempts to localize and isolate muscarinic receptors, European J. Pharmacol. 25, 203. Diamant, H., T. Ekstrand and J. Rydnert, 1974, Der anticholinergische Effekt yon Sch 1000 auf die Parotissekretion w~ihrend Stimulierung mit Acetyl#-methylcholin, Arzneim. Forsch. 24, 573. Engelhardt, A. and H. Wick, 1957, Beziehungen zwischen Konstitution und Wirkung bei Tropeinen und ihren quart/iren Derivaten, Arzneim. Forsch. 7,217. Inch, T.D., D.M. Green and P.B.J. Thompson, 1973, The central and peripheral activities of anti-acetylcholine drugs. Some concepts of practical relevance, J. Pharm. Pharmacol. 25, 359. Minette, A., M. Marcq, M. Bruninx, B. Spaas and G. van Hove, 1973, R~sultats d'une recherche sur les effets ventilatoires et associ~s du Sch 1000 en a~rosol chez 54 patients atteints de bronchoobstruction r6versible, Revue de l'Institut d'Hygi~ne des Mines Hasselt (Belgique) 28, 22. Pappius, H. and Y. Salorinne, 1973, Comparative trial of a new anticholinergic bronchodilator, Sch 1000, and salbutamol in chronic bronchitis, Brit. Med. J. 4, 134. Ulmer, W.T., J. Dorsch, J. Iravani, K.-G. Schiiler, G. Stempel and E. Vastag, 1973, Anticholinergika als Bronchodilatatoren. Beitrag zur Frage der Bedeutung yon Acetylcholin als Bronchokonstriktorische Transmittersubstanz bei Atemwegsobstruktionen, Arzneim. Forsch. 23, 468.