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Antimuscarinic action of methoctramine, a new cardioselective M-2 muscarinic receptor antagonist, alone and in combination with atropine and gallamine
European Journal of Pharmacology, 144 (1987) 117-124
117
Elsevier EJP 50028
Antimuscarinic action of methoctramine, a new cardioselective M-2 muscarinic receptor antagonist, alone and in combination with atropine and gallamine C a r l o M e l c h i o r r e *, Piero Angeli, G i i n t e r L a m b r e c h t 1, E r n s t M u t s c h l e r 1, M a r i a T. Picchio a n d Jiirgen W e s s Department of Chemical Sciences, Universityof Camerino, 62032 Camerino (MC), Italy and I Faculty of Biochemistry, Pharmacy and Food Chemistry, Department of Pharmacology, University of Frankfurt/M, Theodor-Stern-Kai 7, Geb. 75 A, D-6000 Frankfurt/M, F.R.G. Received 6 July 1987, revised MS received 17 August 1987, accepted 15 September 1987
The antimuscarinic effects of methoctramine (N,N'-bis[6-[(2-methoxybenzyl)amino]hexyl]-l,8-octanediamine tetrahydrochloride) were investigated in vitro in isolated paced left (force) and spontaneously beating right (force and rate) atria of guinea pigs as well as ileum of guinea pig and rat. Methoctramine was a potent competitive antagonist of M-2 muscarinic receptors in myocardium and pacemaker cells over a wide range of concentrations. The pA 2 values ranged from 7.74 to 7.93. They were not significantly different in the two cardiac preparations and were independent of the agonist used (muscarine and carbachol). A combination of methoctramine with atropine resulted in addition of the dose ratios for left atria, which is expected for two antagonists interacting competitively with the same receptor site. In contrast, a combination of methoctramine with gallamine produced a less than additive shift of the dose-response curve for carbachol, confirming that gallamine acts as an allosteric antagonist at cardiac muscarinic receptors. Methoctramine was 54 to 132-fold less potent in ileal than in atrial preparations (pA 2 values ranging from 5.81 to 6.20) which makes it the most cardioselective antimuscarinic agent now available. A combination of methoctramine with atropine gave a slight supra-additive antagonism on guinea pig ileum, which suggests that methoctramine interacts to some extent with a second independent site. These results strongly reinforce the view that M-2 muscarinic receptors are not a homogeneous population. Polymethylene tetraamines; Methoctramine; Atropine; Gallamine; M-2 muscarinic receptor subtypes
1. Introduction
There has been a resurgence of interest in muscarinic receptors mainly as a consequence of the discovery of drugs which act preferentially on muscarinic receptor subtypes and of the therapeutic potential of these agents. Based essentially on the discriminatory properties of the novel antimuscarinic drug pirenzepine, two major subclasses of muscarinic receptors (M-1 and M-2) were postulated ( H a m m e r et al., 1980; H a m m e r and * To whom all correspondence should be addressed.
Giachetti, 1982). The M-1 type (displaying high affinity to pirenzepine) is located primarily in the central nervous system and autonomic ganglia whereas the M-2 type (showing low affinity to pirenzepine) is present mainly in peripheral effectot organs and in certain parts of the CNS. A number of authors have confirmed this subclassification and there are several recent reviews of this area of research (BirdsaU and Hulme, 1985; 1987; Eglen and Whiting, 1986; Nilvebrant, 1986). Moreover, recent cloning studies have revealed that the muscarinic M-1 receptor of the cerebral cortex and the M-2 receptor of the heart represent
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
118
distinct gene products and have different amino acid sequences (Kubo et al., 1986a,b; Peralta et al., 1987). Owing to the availability of selective ligands, there is increasing evidence to suggest that M-2 muscarinic receptors can be subdivided further. The first indication that M-2 receptors are not a homogeneous population was the finding that gallamine, a neuromuscular blocking agent, has greater affinity for muscarinic receptors in atrium than for those in ileum (Riker and Wescoe, 1951; Clark and Mitchelson, 1976). More recently, certain muscarinic antagonists such as 4-DAMP (Barlow et al., 1976) and hexahydro-sila-difenidol (Lambrecht et al., 1984; 1987; Mutschler and Lambrecht 1984; Lambrecht and Mutschler, 1985) were shown to possess a higher affinity for muscarinic receptors in smooth muscle organs than for those in atrium, whereas the reverse is true for AF-DX 116, an analogue of pirenzepine (Hammer et al., 1986; Giachetti et al., 1986; Micheletti et al., 1987), and himbacine (Anwar-ul et al., 1986). Moreover, structure-activity relationship studies of tetraamines related to benextramine, the prototype of a class of irreversible a-adrenoreceptor antagonists with cholinergic blocking activity also (Melchiorre et al., 1978; Benfey et al., 1979; Melchiorre and Belleau, 1981; Melchiorre, 1981), have led to the discovery of methoctramine (N,N'bis[6-[(2-methoxybenzyl)amino]hexyl]-l,8-octanediamine tetrahydrochloride; fig. 1) which was shown to display an unprecedented selectivity toward cardiac M-2 muscarinic receptors (Melchiorre et al., 1987). We now report on further in vitro experiments with methoctramine in atrial and ileal preparations derived from different species (guinea pig and rat) and with different agonists. In order to verify the mechanism of action of methoctramine, antagonist combination experiments were carried out with the classical competitive antagonist atropine and the allosteric antimuscarinic agent gallamine (Clark and Mitchelson, 1976; Stockton et al., 1983). The data were analyzed quantitatively according to well established pharmacological criteria, namely Schild analysis (Arunlakshana and Schild, 1959; Tallarida et al., 1979) and assessment of interactions between antagonists according to the dose-ratio method (Paton and Rang, 1965).
(•)--CH2NH(CH2)6 NH(CH2)8NH(CH2)6NHCH 2--~
OCH3
4HCl
CH30
Fig. 1. Structural formula of methoctrarnine.
2. Materials and methods
2.1. General considerations Guinea pigs (200-300 g) or rats (150-200 g) were killed by cervical dislocation and the organs required were set up rapidly under 1 g tension in 20 ml organ baths containing physiological salt solution (PSS) kept at the appropriate temperature (see below) and aerated with 5% CO2-95%O2. The composition of PSS was as follows (mM): NaCI 118, KC1 4.7, MgSO4. 7H20 1.18, CaC12 2.52, KH2PO 4 1.18, NaHCO 3 23.8, glucose 11.7. Doseresponse curves were obtained by cumulative addition of the agonist (Van Rossum, 1963). The concentration of agonist in the organ bath was increased approximately 3-fold at each step, with each addition being made only after the response to the previous addition had attained a maximal level and remained steady. Contractions were recorded by means of a force transducer connected to a two-channel Gemini polygraph. 2.2. Guinea pig left atria The heart of male guinea pigs was rapidly removed and the right and left atria were separated out. The left atria were mounted in PSS at 30 °C and stimulated through platinum electrodes by square-wave pulses (1 ms, 1Hz, 4-7V). Inotropic activity was recorded isometrically. The tissues were equilibrated for 1 h, and cumulative dose-response curves were obtained for muscarine (0.01-3/~M) or carbachol (0.01-3 /~M). Following incubation with the antagonist for 30 (methoctramine) or 60 rain (atropine), a new dose-response curve was obtained for the agonist. In combination experiments (simultaneous administration) with methoctramine and atropine (0.1 /~M) or gallamine (0.1 mM), the contact time for antagonists was always 60 min and the agonist
119 used was carbachol. The end of the methoctramine effect was determined by the following procedure, m control dose-response curve for carbachol was obtained, followed by a curve in the presence of methoctramine (0.1 /tM). The antagonist was then removed from the bath and a dose-response curve to carbachol was again obtained after a washing period of 60 min. The start of the methoctramine effect was determined by exposure of the atria to methoctramine (58.5 nM) for 30 and 60 min, respectively, before a second dose-response curve for carbachol was obtained.
centrations and each concentration was tested four to eight times. Dissociation constants (pA 2 values) were estimated from Schild plots (Arunlakshana and Schild, 1959) constrained to slope -1.0, as required by the theory (Tallarida et al., 1979). When this method was used, it was always verified that the experimental data generated a line whose derived slope was not significantly different from unity (P > 0.05). The data are presented as means + S.E. of n experiments. Differences between mean values were tested for significance by Student'ttest.
2.3. Spontaneously beating guinea pig right atria
2.6. Drugs
Spontaneously beating right atria were suspended in PSS at 30 ° C. Heart rate and size of contractions were recorded isometrically using muscarine (0.01-3 /~M) as agonist. The contact time was 30 min for methoctramine and 60 min for atropine.
2.4. Rat and guinea pig ileum Portions of terminal ileum (length 20 mm) were taken at about 5 cm from the ileum-cecum junction and mounted in PSS at 37°C. Tension changes were recorded isotonically. The tissues were equilibrated for 30 min and dose-response curves for furmethide (0.01-3 /xM) or carbachol (0.01-10 /~M) were obtained at 30 min intervals, the first curve being discarded and th~ second taken as control. Following incubation with the antagonist for 30 (methoctramine) or 60 min (atropine), a new dose-response curve was obtained for the agonist. In combination experiments (simultaneous administration) on guinea pig ileum with methoctramine (10 and 100 t~M) and atropine (0.1/~M), carbachol was used as agonist and the contact time for the antagonists was 60 min. The start of methoctramine action was determined by exposing guinea pig ileum to methoctramine (3.15/IM) for 30 and 60 rain, respectively, before a second dose-response curve was obtained for carbachol.
2.5. Determination of dissociation constants Dose ratios at the ECs0 values of the agonists were calculated at three to five antagonist con-
All drugs used, ( _+)-muscarine chloride (Sigma), carbachol (carbamoylcholine chloride, Fluka), furmethide (furtrethonium methiodide) and methoctramine tetrahydrochloride (synthesized in one of our laboratories), atropine sulphate (Merck) and gallamine triethiodide (kindly supplied by Dr. A. Giachetti), were dissolved in double-distilled water.
3. Results
3.1. Left atria Methoctramine (0.1-10 /~M) antagonized potently the negative inotropic effects induced by muscarine or carbachol in electrically stimulated atria. There was a dose-dependent parallel shift to the right of the agonist dose-response curves without either basal tension or maximum response being affected (shown for carbachol in fig. 2). This antagonism was reversible, since 60 min washing of the tissues after incubation with methoctramine (0.1 /~M) brought the ECs0 value for carbachol back to the control value. Furthermore, the maximum effect of methoctramine (58.5 nM) was reached within 30 rnin contact time with a dose ratio of 6.10 + 1.20 (4) since the extent of antagonism following 60 min incubation (DR = 5.95 _ 0.7 (4)) was not significantly different (P > 0.05). Irrespective of the agonist used, linear Schild plots were obtained and the slopes were not significantly different from unity (table 1). The carbachol-induced responses were antagonized by
120 % of Carbachol Effect
./i--/i
,/?,,
0
0. / . / . / , 7 , / I I * / 8
7
6
5
4
log M Carloachol
Fig. 2. Antagonism of carbachol-induced negative inotropic effect in guinea pig left atria. Dose:response curves for carbachol were obtained before ( I ) and after exposure to 0.1 (ll), 0.3 (A), l(1), 3 (1~) and 1 0 / * M ( . ) methoctramine for 30 min. Each point is the mean -t-S.E. of four to eight observations. Ordinate: percentage inhibition of the myocardial force of contraction. Abscissa: - l o g M of carbachol concentration.
methoctramine with a pA 2 value of 7.93 + 0.09, that closely resembled the pA 2 value (7.77 + 0.07) obtained with muscarine as the agonist (fig. 4, table 1). Under the same experimental conditions atropine was about one order of magnitude more potent than methoctramine, the pA2 values amounting to 8.97 _+0.11 and 9.13_ 0.07 against muscarine and carbachol, respectively (table 1).
3.1.1. Antagonism by methoctramine and atropine or gallamine in combination The mutual competition between methoctramine and atropine or gallamine was investigated on the basis of dose-ratio analysis (Paton and Rang, 1965). The experimental dose ratios of the combination of methoctramine (3 and 10 #M) with atropine (0.1 #M) amounted to 462 and 767, respectively, and were not significantly different from those expected for a combination of two competitive antagonists (495 and 653) (table 2). On the other hand, a combination of methoctramine (3 #M) and gallamine (0.1 mM) produced a dose ratio (188) which was significantly lower (P < 0.01) than the value expected (325) for two competitive antagonists (table 2). 3.2. Right atria In spontaneously beating right atria, methoctramine (0.1-3 /*M) caused a dose-dependent inhibition of the negative chronotropic and inotropic effects induced by muscarine without modifying basal rate or tension. The Schild plots were linear and the slopes of the regression lines were not significantly different from unity (fig. 4, table 1). A pA 2 value of 7.73 _ 0.12 was found at muscarinic receptors in pacemaker cells, which was not significantly different (P > 0.05) from the values obtained at myocardial receptors in left (see 3.1.) and right atria (7.74 + 0.09) (table 1).
TABLE 1 Antagonist affinities at guinea pig ileal and atrial muscarinic receptors. Atropine
Preparation a
Methoctramine Concentration (/*M)
pA 2 b
Slope c
pA 2 b
Slope c
Left atrium d Left atrium c Right atrium d (force) (rate) Ileum r Ileum e Rat ileum f
0.1, 0.3, 1, 3 0.1, 0.3, 1, 3, 10
7.77 5:0.07 7.93 _+0.09
1.128 _ 0.09 0.945 _+0.06
8.97 + 0.11 9.13 + 0.07
1.07 _+0.08 1.09 ± 0.07
0.1, 0.3, 1, 3 0.1, 0.3, 1, 3 3, 10, 30 3, 10, 30, 100, 300 10, 30, 100
7.745:0.09 7.73 _ 0.12 6.00-/-0.07 6.205:0.06 5.81 + 0.03
1.12 _+0.14 1.08 _+0.14 0.99 +0.10 0.98 5:0.06 1.067 5:0.106
8.95 +0.09 9.05 + 0.10 8.89-1-0.10 8.915:0.11 8.93 5:0.07
1.01 -t-0.03 1.02 + 0.08 1.05+_0.05 1.045:0.03 1.02 5:0.05
a Tissues were from guinea pigs unless otherwise indicated, b Each p A 2 value was obtained from 12 to 27 dose ratios at the indicated concentrations for methoctramine and at 0.01, 0.1 and 1 # M concentrations for atropine and was determined by the method of constrained plot (Tallarida et al., 1979). Results are presented as the means +S.E. c Slopes were estimated from Schild plots (Arunlakshana and Schild, 1959) and were not significantly different (P > 0.05) from unity, d The agonist was muscarine, e The agonist was carbachol, f The agonist was furmethide.
121 TABLE 2 Dose ratios obtained with methoctramine, atropine and gallamine alone and in combination on muscarinic receptors of guinea pig left atrium using carbachol as agonist. Each dose ratio (DR) value is the mean + S.E. and the number of observations is given in parentheses. Methoctramine concentration (/~M)
Methoctramine dose ratio (DR1)
Atropine (0.1 #M) dose ratio (DR2)
Experimental combination dose ratio
Expected combination dose ratio (DR 1 + D R 2 - 1)
3
213 +_84 (n = 5)
283 4- 60 (n = 5)
462 + 66 a (n = 8)
495
10
371 + 64 (n = 4)
767 + 93 a (n = 6)
653
188 + 32 b (n = 7)
325
Gallamine (0.1 mM) dose ratio (DR 2) 3
207 +_38 (n = 7)
119 + 14 (n = 6)
a Not significantly different (P > 0.05) from expected combination dose ratio for two competitive antagonists, b Significantly different (P < 0.01) from expected combination dose ratio for two competitive antagonists.
3.3. Ileum
Methoctramine (3-300 #M) caused a dosed e p e n d e n t inhibition of f u r m e t h i d e - and carbachol-induced contractions of guinea pig and rat ileum. The agonist dose-response curves were displaced in a parallel fashion with no changes in the maximum response (shown for carbachol in the guinea pig ileum in fig. 3). The methoctramine
% of C a r b a c h o l 100
,o
log (DR - 1) 3
Effect • ~
III ° '''''''~ Ill
•
•
~0
Z,/L//,/ . / ..," , / , / zt,/ ,l
(3.15 #M) effect in guinea pig ileum started rapidly since equilibrium conditions were reached within 30 min of incubation. The dose ratios following 30 or 60 min contact time were 5.88 + 0.73 (4) and 5.97 + 0.65 (4), respectively, and were not significantly different (P > 0.05). The data derived from Schild analysis point to competitive antagonism
,7 ,jl//"
2
1
7
6
5
4
- log M M e t h o c t r a m i n e
8 - Log M C a r b a c h o l
Fig. 3. Effect of methoctramine treatment on carbachol-induced contractions of guinea pig ileum. Dose-response curves to carbachol were obtained before (o) and after exposure to 3 (n), 10 (A), 30 (,i), 100 (1)) and 300 #M ( 0 ) methoctramine for 30 rain. Each point is the mean +S.E. of four to seven observations. Ordinate: percentage of maximal response elicited by carbachol. Abscissa: - log M of carbachol concentration.
Fig. 4. Schild plot for the antagonism between muscarine (atria) or carbachol (ileum) and methoctramine at cardiac and ileal muscarinic receptors in guinea pig right atrium (rate, O), paced left atrium (force, m) and ileum (A). The points are the means of four to eight experiments and the S.E.s are indicated. Ordinate: log ( D R - 1) where D R = ECs0 ratio measured from the displacement of the agonist concentration-response curves by methoctramine. Abscissa: - l o g M concentration of methoctramine.
122 TABLE 3 Dose ratios obtained with methoctramine and atropine alone and in combination on muscarinic receptors of guinea pig ileum using carbachol as agonist. Each dose ratio (DR) value is the mean q-S.E, and the number of observations is given in parentheses. Methoctramine concentration (/tM)
Methoctramine dose ratio (DR 1)
Atropine (0.1 b t M ) dose ratio (DR2)
Experimental combination dose ratio
Expected combination dose ratio (DR 1+ DR 2 -1)
10
14± 1.9 (n = 5)
191 :i: 16 (n = 12)
409±40 a (n = 8)
204
100
96 ± 10 (n = 5)
617 ± 57 a (n = 9)
286
a significantly different (P < 0.01) from expected combination dose ratio for two competitive antagonists.
(fig. 4, table 1). Irrespective of agonist a n d species used, the p A 2 values were similar, r a n g i n g from 5.81 + 0.03 to 6.20 + 0.06. Atropine, with p A 2 values a r o u n d 8.9, was a b o u t three orders of m a g n i tude more p o t e n t t h a n m e t h o c t r a m i n e in ileal preparations.
3. 3.1. Antagonism by methoctramine and atropine in combination G u i n e a pig ileal p r e p a r a t i o n s were exposed s i m u l t a n e o u s l y to m e t h o c t r a m i n e (10 a n d 100 ttM) a n d a t r o p i n e (0.1 /~M). C o m b i n a t i o n dose ratios (409 a n d 617) were o b t a i n e d which were signific a n t l y higher (P < 0.01) t h a n those expected for a
TABLE 4 Selectivity ratios of methoctramine expressed as the antilog of the difference between the pA 2 values for atrium and ileum muscarinic receptors. Preparations a
Selectivity ratio
Left atrium/ileumb Left atrium/ileum¢ Left atrium/rat ileum a Right atrium (force)/ileum b Right atrium (force)/rat ileum b Right atrium (force)/rat ileum b Left atrium (force)/right atrium (rate) t
59 54 132 55 91 166 e 1.1
a Tissues were from guinea pigs unless otherwise indicated, b The agonists were muscarine and furmethide in the atrium and ileum, respectively, c The agonist was carbachol in both preparations, d The agonists were carbacbol and furmethide in the atrium and ileum, respectively, e Value obtained by recalculation of the pA 2 values reported for guinea pig atria and rat ileum by Melchiorre et al. (1987) by the constrained plot method, f The agonist was muscarine in both preparations.
c o m b i n a t i o n of two pure competitive antagonists (204 a n d 286) (table 3).
3.4. Methoctramme selectivity T a b l e 4 shows the selectivity ratios for m e t h o c t r a m i n e . It is evident that the affinity of m e t h o c t r a m i n e to cardiac muscarinic M-2 receptors is a b o u t two orders of m a g n i t u d e higher than that to M-2 receptors in ileal smooth muscle.
4. Discussion T h e m u s c a r i n i c M-2 receptors in cardiac tissues a n d smooth muscle organs have b e e n claimed to b e l o n g to different subclasses (Clark a n d Mitchelson, 1976; Barlow et al., 1976; L a m b r e c h t et al., 1984; Mutschler a n d Lambrecht, 1984; L a m b r e c h t a n d Mutschler, 1985; A n w a r - u l et al., 1986; Micheletti et al., 1987) d e n o t e d as cardiac M - 2 a a n d s m o o t h muscle M-2fl type ( L a m b r e c h t et al., 1987). This heterogeneity has recently been confirmed b y the d i s c r i m i n a t i n g properties of the cardioselective a n t i m u s c a r i n i c agent methoctram i n e (Melchiorre et al., 1987). W e have n o w further investigated the a n t i m u s c a r i n i c effects of m e t h o c t r a m i n e in atrial a n d ileal p r e p a r a t i o n s derived from different species a n d using different agonists. I n addition, a n t a g o n i s t c o m b i n a t i o n experiments were carried out with a t r o p i n e (a classical competitive antagonist) a n d gallamine ( a n allosteric a n t i m u s c a r i n i c agent at cardiac m u s c a r i n i c receptors) in order to verify the m e c h a n i s m of action of m e t h o c t r a m i n e .
123 Methoctramine was an apparently simple competitive antagonist in both atrial and ileal preparations since it produced antagonism that was reversible and surmountable over a wide range of concentrations (figs. 2-4) and its apparent affinity in each tissue appeared to be independent of the concentration used. Thus, Schild analysis of the data yielded slopes which were not significantly different from unity. Furthermore, dissociation constants and slopes were independent of both agonist and species (table 1). The affinity of methoctramine for cardiac muscarinic M-2a receptors in both pacemaker and myocardium cells was much higher than that observed at M-2fl receptors in the ileum (tables 1 and 4). Methoctramine displayed a selectivity ratio ranging from 54 to 132 (table 4), which is in agreement with results previously reported (Melchiorre et al., 1987). Thus, the selectivity profile of methoctramine is analogous to that of A F - D X 116 (Giachetti et al., 1986; Hammer et al., 1986; Micheletti et al., 1987), but it must be emphasised that the cardioselectivity of methoctramine is much greater. The mutual competition between methoctramine and atropine or gallamine was investigated in electrically stimulated left atria and in ileum according to the dose ratio method (Paton and Rang, 1965). As shown in table 2, in the atria a combination of methoctramine and atropine resulted in dose ratios equal to the sum of the dose ratios produced by the two antagonists when tested alone. This suggests strongly that methoctramine and atropine compete mutually with the same receptor site. Further evidence for the mechanism of action of methoctramine came from the observation that a combination of methoctramine and gallamine resulted in a less than additive antagonistic effect (table 2). This indicates that the two antagonists interact with two different but interdependent sites of the cardiac muscarinic receptor. These results are very similar to those reported for a combination of atropine and gallamine (Clark and Mitchelson, 1976). Experiments performed in guinea pig ileum with a combination of methoctramine and atropine gave results which were not totally consistent with a competitive mode of action of methoctramine (ta-
ble 3). In fact, the combination dose ratios were somewhat greater than expected (table 3), but these results are not consistent with the multiplication of dose ratios which would be expected if the two antagonists interacted exclusively at independent sites. In summary, it was demonstrated that methoctramine is a potent competitive antimuscarinic agent at atrial muscarinic M-2a receptors controlling heart rate and force of contractions whereas it is about two orders of magnitude less potent at ileal muscarinic M-2fl receptors. Thus, methoctramine is the most cardioselective antimuscarinic agent known today and represents a highly useful tool for the subclassification of muscarinic receptors in the periphery as well as in the central nervous system. In addition, it may have therapeutic potential in the treatment of sinus bradycardia without producing concomitant side-effects on other parasympathetically innervated organs.
Acknowledgement Supported by the Ministry of Public Education (Rome).
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124 Egien, R.M. and R.L. Whiting, 1986, Muscarinic receptor subtypes: A critique of the current classification and a proposal for a working nomenclature, J. Autonom. Pharmacol. 5, 323. Giachetti, A., R. Micheletti and E. Montagna, 1986, Cardioselective profile of AF-DX 116, a muscarine M-2 receptor antagonist, Life Sci. 38, 1663. Hammer, R., C.P. Berrie, N.J.M. Birdsall, A.S.V. Burgen and E.C. Hulme, 1980, Pirenzepine distinguishes between different subclasses of muscarinic receptors, Nature 283, 90. Hammer R. and A. Giachetti, 1982, Muscarinic receptor subtypes: M 1 and M 2. Biochemical and functional characterization, Life Sci. 31, 2991. Hammer, R., E. Giraldo, G.B. Schiavi, E. Monferini and H. Ladinsky, 1986, Binding profile of a novel cardioselective muscarine receptor antagonist, AF-DX 116, to membranes of peripheral tissues and brain in the rat, Life Sci. 38, 1653. Kubo, T., K. Fukuda, A. Mikami, A. Maeda, H. Takahashi, M. Mishina, T. Haga, K. Haga, A. Ichiyama, K. Kangawa, M. Kojima, H. Matsuo, T. Hirose and S. Numa, 1986a, Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetyichohne receptor, Nature 323, 411. Kubo, T., A. Maeda, K. Sugimoto, I. Akiba, A. Mikami, H. Takahashi, T. Haga, K. Haga, A. Ichiyama, K. Kangawa, H. Matsuo, T. Hirose and S. Numa, 1986b, Primary structure of porcine cardiac muscarinic acetylcholine receptor deduced from the cDNA sequence, FEBS Lett. 209, 367. Lambrecht, G., U. Moser, E. Mutschler, J. Wess, H. Linoh, M. Strecker and R. Tacke, 1984, Hexahydro-sila-difenidol: a selective antagonist on ileal muscarinic receptors, NaunynSchmiedeb. Arch. Pharmacol. 325 (Suppl.), R62. Lambrecht, G. and E. Mutschler, 1985, Selective inhibition of muscarinic receptors in intestinal smooth muscle, in: Muscarinic Receptor Subtypes in the GI Tract, eds. G. Lux and E.E. Daniel (Springer-Verlag, Berlin) p. 20. Lambrecht, G., E. Mutschler, U. Moser, J. Riotte, M. Wagner, J. Wess, G. Gmelin, R. Tacke and H. Zilch, 1987, Heterogeneity in muscarinic receptors: Evidence from pharmacological and electrophysiological studies with selective antagonists, in: Muscarinic Cholinergic Mechanisms, eds. S. Cohen and M. Sokolowsky (Freund Publishing House Ltd., Tel-Aviv, London) p. 245. Melchiorre, C., 1981, Tetramine disulfides: a new tool in alpha-adrenergic pharmacology, Trends Pharmacol. Sci. 2, 209.
Melchiorre, C. and B. Belleau, 1981, Recent developments in structure-activity relationships among inhibitors of the adrenergic alpha-receptor, in: Adrenoceptors and Catecholamine Action, Vol. 1, ed. G. Kunos (John Wiley Inc., New York) p. 181. Melchiorre, C., A. Cassinelli and W. Quaglia, 1987, Differential blockade of muscarinic receptor subtypes by polymethylene tetraamines. Novel class of selective antagonists of cardiac M-2 muscarinic receptors, J. Med. Chem. 30, 201. Melchiorre, C., M.S. Yong, B.G. Benfey and B. Belleau, 1978, Molecular properties of the adrenergic alpha-receptor. II. Optimum covalent inhibition by two different prototypes of polyamine disulfides, J. Med. Chem. 21, 1126. Micheletti, R., E. Montagna and A. Giachetti, 1987, AF-DX 116, a cardioselective muscarinic antagonist, J. Pharmacol. Exp. Ther. 240, 2505. Mutschler, E. and G. Lambrecht, 1984, Selective muscarinic agonists and antagonists in functional tests, Trends Pharmacol. Sci. 5 (Suppl.), 39. Nilvebrant, L., 1986, On the muscarinic receptors in the urinary bladder and the putative subclassification of muscarinic receptors, Acta Pharmacol. Toxicol. 59 (Suppl. I), 3. Paton, W.D.M. and H.P. Rang, 1965, The uptake of atropine and related drugs by intestinal smooth muscle of the guinea pig in relation to acetylcholine receptors, Proc. R. Soc. London Ser. B 163, 1. Peralta, E.G., J.W. Winslow, G.L. Peterson, D.H. Smith, A. Ashkenazi, J. Ramachandran, M.I. Schimerlik and D.J. Capon, 1987, Primary structure and biochemical properties of an M 2 muscarinic receptor, Science 236, 600. Riker, W.F. and W.C. Weskoe, 1951, The pharmacology of flaxedil with observations on certain analogues, Ann. N.Y. Acad. Sci. 54, 373. Stockton, J.M., N.J.M. Birdsall, A.V.S. Burgen and E.C. Hulme, 1983, Modification of the binding properties of muscarinic receptors by gallamine, Mol. Pharmacol. 23, 551. Tallarida, R.J., A. Cowan and M.W. Adler, 1979, pA 2 and receptor differentiation: a statistical analysis of competitive antagonism, Life Sci. 25, 637. Van Rossum, J.M., 1963, Cumulative dose-response curves. Techniques for the making of dose-response curves in isolated organs and the evaluation of drug parameters, Arch. Int. Pharmacodyn. Ther. 143, 299.
117
Elsevier EJP 50028
Antimuscarinic action of methoctramine, a new cardioselective M-2 muscarinic receptor antagonist, alone and in combination with atropine and gallamine C a r l o M e l c h i o r r e *, Piero Angeli, G i i n t e r L a m b r e c h t 1, E r n s t M u t s c h l e r 1, M a r i a T. Picchio a n d Jiirgen W e s s Department of Chemical Sciences, Universityof Camerino, 62032 Camerino (MC), Italy and I Faculty of Biochemistry, Pharmacy and Food Chemistry, Department of Pharmacology, University of Frankfurt/M, Theodor-Stern-Kai 7, Geb. 75 A, D-6000 Frankfurt/M, F.R.G. Received 6 July 1987, revised MS received 17 August 1987, accepted 15 September 1987
The antimuscarinic effects of methoctramine (N,N'-bis[6-[(2-methoxybenzyl)amino]hexyl]-l,8-octanediamine tetrahydrochloride) were investigated in vitro in isolated paced left (force) and spontaneously beating right (force and rate) atria of guinea pigs as well as ileum of guinea pig and rat. Methoctramine was a potent competitive antagonist of M-2 muscarinic receptors in myocardium and pacemaker cells over a wide range of concentrations. The pA 2 values ranged from 7.74 to 7.93. They were not significantly different in the two cardiac preparations and were independent of the agonist used (muscarine and carbachol). A combination of methoctramine with atropine resulted in addition of the dose ratios for left atria, which is expected for two antagonists interacting competitively with the same receptor site. In contrast, a combination of methoctramine with gallamine produced a less than additive shift of the dose-response curve for carbachol, confirming that gallamine acts as an allosteric antagonist at cardiac muscarinic receptors. Methoctramine was 54 to 132-fold less potent in ileal than in atrial preparations (pA 2 values ranging from 5.81 to 6.20) which makes it the most cardioselective antimuscarinic agent now available. A combination of methoctramine with atropine gave a slight supra-additive antagonism on guinea pig ileum, which suggests that methoctramine interacts to some extent with a second independent site. These results strongly reinforce the view that M-2 muscarinic receptors are not a homogeneous population. Polymethylene tetraamines; Methoctramine; Atropine; Gallamine; M-2 muscarinic receptor subtypes
1. Introduction
There has been a resurgence of interest in muscarinic receptors mainly as a consequence of the discovery of drugs which act preferentially on muscarinic receptor subtypes and of the therapeutic potential of these agents. Based essentially on the discriminatory properties of the novel antimuscarinic drug pirenzepine, two major subclasses of muscarinic receptors (M-1 and M-2) were postulated ( H a m m e r et al., 1980; H a m m e r and * To whom all correspondence should be addressed.
Giachetti, 1982). The M-1 type (displaying high affinity to pirenzepine) is located primarily in the central nervous system and autonomic ganglia whereas the M-2 type (showing low affinity to pirenzepine) is present mainly in peripheral effectot organs and in certain parts of the CNS. A number of authors have confirmed this subclassification and there are several recent reviews of this area of research (BirdsaU and Hulme, 1985; 1987; Eglen and Whiting, 1986; Nilvebrant, 1986). Moreover, recent cloning studies have revealed that the muscarinic M-1 receptor of the cerebral cortex and the M-2 receptor of the heart represent
0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
118
distinct gene products and have different amino acid sequences (Kubo et al., 1986a,b; Peralta et al., 1987). Owing to the availability of selective ligands, there is increasing evidence to suggest that M-2 muscarinic receptors can be subdivided further. The first indication that M-2 receptors are not a homogeneous population was the finding that gallamine, a neuromuscular blocking agent, has greater affinity for muscarinic receptors in atrium than for those in ileum (Riker and Wescoe, 1951; Clark and Mitchelson, 1976). More recently, certain muscarinic antagonists such as 4-DAMP (Barlow et al., 1976) and hexahydro-sila-difenidol (Lambrecht et al., 1984; 1987; Mutschler and Lambrecht 1984; Lambrecht and Mutschler, 1985) were shown to possess a higher affinity for muscarinic receptors in smooth muscle organs than for those in atrium, whereas the reverse is true for AF-DX 116, an analogue of pirenzepine (Hammer et al., 1986; Giachetti et al., 1986; Micheletti et al., 1987), and himbacine (Anwar-ul et al., 1986). Moreover, structure-activity relationship studies of tetraamines related to benextramine, the prototype of a class of irreversible a-adrenoreceptor antagonists with cholinergic blocking activity also (Melchiorre et al., 1978; Benfey et al., 1979; Melchiorre and Belleau, 1981; Melchiorre, 1981), have led to the discovery of methoctramine (N,N'bis[6-[(2-methoxybenzyl)amino]hexyl]-l,8-octanediamine tetrahydrochloride; fig. 1) which was shown to display an unprecedented selectivity toward cardiac M-2 muscarinic receptors (Melchiorre et al., 1987). We now report on further in vitro experiments with methoctramine in atrial and ileal preparations derived from different species (guinea pig and rat) and with different agonists. In order to verify the mechanism of action of methoctramine, antagonist combination experiments were carried out with the classical competitive antagonist atropine and the allosteric antimuscarinic agent gallamine (Clark and Mitchelson, 1976; Stockton et al., 1983). The data were analyzed quantitatively according to well established pharmacological criteria, namely Schild analysis (Arunlakshana and Schild, 1959; Tallarida et al., 1979) and assessment of interactions between antagonists according to the dose-ratio method (Paton and Rang, 1965).
(•)--CH2NH(CH2)6 NH(CH2)8NH(CH2)6NHCH 2--~
OCH3
4HCl
CH30
Fig. 1. Structural formula of methoctrarnine.
2. Materials and methods
2.1. General considerations Guinea pigs (200-300 g) or rats (150-200 g) were killed by cervical dislocation and the organs required were set up rapidly under 1 g tension in 20 ml organ baths containing physiological salt solution (PSS) kept at the appropriate temperature (see below) and aerated with 5% CO2-95%O2. The composition of PSS was as follows (mM): NaCI 118, KC1 4.7, MgSO4. 7H20 1.18, CaC12 2.52, KH2PO 4 1.18, NaHCO 3 23.8, glucose 11.7. Doseresponse curves were obtained by cumulative addition of the agonist (Van Rossum, 1963). The concentration of agonist in the organ bath was increased approximately 3-fold at each step, with each addition being made only after the response to the previous addition had attained a maximal level and remained steady. Contractions were recorded by means of a force transducer connected to a two-channel Gemini polygraph. 2.2. Guinea pig left atria The heart of male guinea pigs was rapidly removed and the right and left atria were separated out. The left atria were mounted in PSS at 30 °C and stimulated through platinum electrodes by square-wave pulses (1 ms, 1Hz, 4-7V). Inotropic activity was recorded isometrically. The tissues were equilibrated for 1 h, and cumulative dose-response curves were obtained for muscarine (0.01-3/~M) or carbachol (0.01-3 /~M). Following incubation with the antagonist for 30 (methoctramine) or 60 rain (atropine), a new dose-response curve was obtained for the agonist. In combination experiments (simultaneous administration) with methoctramine and atropine (0.1 /~M) or gallamine (0.1 mM), the contact time for antagonists was always 60 min and the agonist
119 used was carbachol. The end of the methoctramine effect was determined by the following procedure, m control dose-response curve for carbachol was obtained, followed by a curve in the presence of methoctramine (0.1 /tM). The antagonist was then removed from the bath and a dose-response curve to carbachol was again obtained after a washing period of 60 min. The start of the methoctramine effect was determined by exposure of the atria to methoctramine (58.5 nM) for 30 and 60 min, respectively, before a second dose-response curve for carbachol was obtained.
centrations and each concentration was tested four to eight times. Dissociation constants (pA 2 values) were estimated from Schild plots (Arunlakshana and Schild, 1959) constrained to slope -1.0, as required by the theory (Tallarida et al., 1979). When this method was used, it was always verified that the experimental data generated a line whose derived slope was not significantly different from unity (P > 0.05). The data are presented as means + S.E. of n experiments. Differences between mean values were tested for significance by Student'ttest.
2.3. Spontaneously beating guinea pig right atria
2.6. Drugs
Spontaneously beating right atria were suspended in PSS at 30 ° C. Heart rate and size of contractions were recorded isometrically using muscarine (0.01-3 /~M) as agonist. The contact time was 30 min for methoctramine and 60 min for atropine.
2.4. Rat and guinea pig ileum Portions of terminal ileum (length 20 mm) were taken at about 5 cm from the ileum-cecum junction and mounted in PSS at 37°C. Tension changes were recorded isotonically. The tissues were equilibrated for 30 min and dose-response curves for furmethide (0.01-3 /xM) or carbachol (0.01-10 /~M) were obtained at 30 min intervals, the first curve being discarded and th~ second taken as control. Following incubation with the antagonist for 30 (methoctramine) or 60 min (atropine), a new dose-response curve was obtained for the agonist. In combination experiments (simultaneous administration) on guinea pig ileum with methoctramine (10 and 100 t~M) and atropine (0.1/~M), carbachol was used as agonist and the contact time for the antagonists was 60 min. The start of methoctramine action was determined by exposing guinea pig ileum to methoctramine (3.15/IM) for 30 and 60 rain, respectively, before a second dose-response curve was obtained for carbachol.
2.5. Determination of dissociation constants Dose ratios at the ECs0 values of the agonists were calculated at three to five antagonist con-
All drugs used, ( _+)-muscarine chloride (Sigma), carbachol (carbamoylcholine chloride, Fluka), furmethide (furtrethonium methiodide) and methoctramine tetrahydrochloride (synthesized in one of our laboratories), atropine sulphate (Merck) and gallamine triethiodide (kindly supplied by Dr. A. Giachetti), were dissolved in double-distilled water.
3. Results
3.1. Left atria Methoctramine (0.1-10 /~M) antagonized potently the negative inotropic effects induced by muscarine or carbachol in electrically stimulated atria. There was a dose-dependent parallel shift to the right of the agonist dose-response curves without either basal tension or maximum response being affected (shown for carbachol in fig. 2). This antagonism was reversible, since 60 min washing of the tissues after incubation with methoctramine (0.1 /~M) brought the ECs0 value for carbachol back to the control value. Furthermore, the maximum effect of methoctramine (58.5 nM) was reached within 30 rnin contact time with a dose ratio of 6.10 + 1.20 (4) since the extent of antagonism following 60 min incubation (DR = 5.95 _ 0.7 (4)) was not significantly different (P > 0.05). Irrespective of the agonist used, linear Schild plots were obtained and the slopes were not significantly different from unity (table 1). The carbachol-induced responses were antagonized by
120 % of Carbachol Effect
./i--/i
,/?,,
0
0. / . / . / , 7 , / I I * / 8
7
6
5
4
log M Carloachol
Fig. 2. Antagonism of carbachol-induced negative inotropic effect in guinea pig left atria. Dose:response curves for carbachol were obtained before ( I ) and after exposure to 0.1 (ll), 0.3 (A), l(1), 3 (1~) and 1 0 / * M ( . ) methoctramine for 30 min. Each point is the mean -t-S.E. of four to eight observations. Ordinate: percentage inhibition of the myocardial force of contraction. Abscissa: - l o g M of carbachol concentration.
methoctramine with a pA 2 value of 7.93 + 0.09, that closely resembled the pA 2 value (7.77 + 0.07) obtained with muscarine as the agonist (fig. 4, table 1). Under the same experimental conditions atropine was about one order of magnitude more potent than methoctramine, the pA2 values amounting to 8.97 _+0.11 and 9.13_ 0.07 against muscarine and carbachol, respectively (table 1).
3.1.1. Antagonism by methoctramine and atropine or gallamine in combination The mutual competition between methoctramine and atropine or gallamine was investigated on the basis of dose-ratio analysis (Paton and Rang, 1965). The experimental dose ratios of the combination of methoctramine (3 and 10 #M) with atropine (0.1 #M) amounted to 462 and 767, respectively, and were not significantly different from those expected for a combination of two competitive antagonists (495 and 653) (table 2). On the other hand, a combination of methoctramine (3 #M) and gallamine (0.1 mM) produced a dose ratio (188) which was significantly lower (P < 0.01) than the value expected (325) for two competitive antagonists (table 2). 3.2. Right atria In spontaneously beating right atria, methoctramine (0.1-3 /*M) caused a dose-dependent inhibition of the negative chronotropic and inotropic effects induced by muscarine without modifying basal rate or tension. The Schild plots were linear and the slopes of the regression lines were not significantly different from unity (fig. 4, table 1). A pA 2 value of 7.73 _ 0.12 was found at muscarinic receptors in pacemaker cells, which was not significantly different (P > 0.05) from the values obtained at myocardial receptors in left (see 3.1.) and right atria (7.74 + 0.09) (table 1).
TABLE 1 Antagonist affinities at guinea pig ileal and atrial muscarinic receptors. Atropine
Preparation a
Methoctramine Concentration (/*M)
pA 2 b
Slope c
pA 2 b
Slope c
Left atrium d Left atrium c Right atrium d (force) (rate) Ileum r Ileum e Rat ileum f
0.1, 0.3, 1, 3 0.1, 0.3, 1, 3, 10
7.77 5:0.07 7.93 _+0.09
1.128 _ 0.09 0.945 _+0.06
8.97 + 0.11 9.13 + 0.07
1.07 _+0.08 1.09 ± 0.07
0.1, 0.3, 1, 3 0.1, 0.3, 1, 3 3, 10, 30 3, 10, 30, 100, 300 10, 30, 100
7.745:0.09 7.73 _ 0.12 6.00-/-0.07 6.205:0.06 5.81 + 0.03
1.12 _+0.14 1.08 _+0.14 0.99 +0.10 0.98 5:0.06 1.067 5:0.106
8.95 +0.09 9.05 + 0.10 8.89-1-0.10 8.915:0.11 8.93 5:0.07
1.01 -t-0.03 1.02 + 0.08 1.05+_0.05 1.045:0.03 1.02 5:0.05
a Tissues were from guinea pigs unless otherwise indicated, b Each p A 2 value was obtained from 12 to 27 dose ratios at the indicated concentrations for methoctramine and at 0.01, 0.1 and 1 # M concentrations for atropine and was determined by the method of constrained plot (Tallarida et al., 1979). Results are presented as the means +S.E. c Slopes were estimated from Schild plots (Arunlakshana and Schild, 1959) and were not significantly different (P > 0.05) from unity, d The agonist was muscarine, e The agonist was carbachol, f The agonist was furmethide.
121 TABLE 2 Dose ratios obtained with methoctramine, atropine and gallamine alone and in combination on muscarinic receptors of guinea pig left atrium using carbachol as agonist. Each dose ratio (DR) value is the mean + S.E. and the number of observations is given in parentheses. Methoctramine concentration (/~M)
Methoctramine dose ratio (DR1)
Atropine (0.1 #M) dose ratio (DR2)
Experimental combination dose ratio
Expected combination dose ratio (DR 1 + D R 2 - 1)
3
213 +_84 (n = 5)
283 4- 60 (n = 5)
462 + 66 a (n = 8)
495
10
371 + 64 (n = 4)
767 + 93 a (n = 6)
653
188 + 32 b (n = 7)
325
Gallamine (0.1 mM) dose ratio (DR 2) 3
207 +_38 (n = 7)
119 + 14 (n = 6)
a Not significantly different (P > 0.05) from expected combination dose ratio for two competitive antagonists, b Significantly different (P < 0.01) from expected combination dose ratio for two competitive antagonists.
3.3. Ileum
Methoctramine (3-300 #M) caused a dosed e p e n d e n t inhibition of f u r m e t h i d e - and carbachol-induced contractions of guinea pig and rat ileum. The agonist dose-response curves were displaced in a parallel fashion with no changes in the maximum response (shown for carbachol in the guinea pig ileum in fig. 3). The methoctramine
% of C a r b a c h o l 100
,o
log (DR - 1) 3
Effect • ~
III ° '''''''~ Ill
•
•
~0
Z,/L//,/ . / ..," , / , / zt,/ ,l
(3.15 #M) effect in guinea pig ileum started rapidly since equilibrium conditions were reached within 30 min of incubation. The dose ratios following 30 or 60 min contact time were 5.88 + 0.73 (4) and 5.97 + 0.65 (4), respectively, and were not significantly different (P > 0.05). The data derived from Schild analysis point to competitive antagonism
,7 ,jl//"
2
1
7
6
5
4
- log M M e t h o c t r a m i n e
8 - Log M C a r b a c h o l
Fig. 3. Effect of methoctramine treatment on carbachol-induced contractions of guinea pig ileum. Dose-response curves to carbachol were obtained before (o) and after exposure to 3 (n), 10 (A), 30 (,i), 100 (1)) and 300 #M ( 0 ) methoctramine for 30 rain. Each point is the mean +S.E. of four to seven observations. Ordinate: percentage of maximal response elicited by carbachol. Abscissa: - log M of carbachol concentration.
Fig. 4. Schild plot for the antagonism between muscarine (atria) or carbachol (ileum) and methoctramine at cardiac and ileal muscarinic receptors in guinea pig right atrium (rate, O), paced left atrium (force, m) and ileum (A). The points are the means of four to eight experiments and the S.E.s are indicated. Ordinate: log ( D R - 1) where D R = ECs0 ratio measured from the displacement of the agonist concentration-response curves by methoctramine. Abscissa: - l o g M concentration of methoctramine.
122 TABLE 3 Dose ratios obtained with methoctramine and atropine alone and in combination on muscarinic receptors of guinea pig ileum using carbachol as agonist. Each dose ratio (DR) value is the mean q-S.E, and the number of observations is given in parentheses. Methoctramine concentration (/tM)
Methoctramine dose ratio (DR 1)
Atropine (0.1 b t M ) dose ratio (DR2)
Experimental combination dose ratio
Expected combination dose ratio (DR 1+ DR 2 -1)
10
14± 1.9 (n = 5)
191 :i: 16 (n = 12)
409±40 a (n = 8)
204
100
96 ± 10 (n = 5)
617 ± 57 a (n = 9)
286
a significantly different (P < 0.01) from expected combination dose ratio for two competitive antagonists.
(fig. 4, table 1). Irrespective of agonist a n d species used, the p A 2 values were similar, r a n g i n g from 5.81 + 0.03 to 6.20 + 0.06. Atropine, with p A 2 values a r o u n d 8.9, was a b o u t three orders of m a g n i tude more p o t e n t t h a n m e t h o c t r a m i n e in ileal preparations.
3. 3.1. Antagonism by methoctramine and atropine in combination G u i n e a pig ileal p r e p a r a t i o n s were exposed s i m u l t a n e o u s l y to m e t h o c t r a m i n e (10 a n d 100 ttM) a n d a t r o p i n e (0.1 /~M). C o m b i n a t i o n dose ratios (409 a n d 617) were o b t a i n e d which were signific a n t l y higher (P < 0.01) t h a n those expected for a
TABLE 4 Selectivity ratios of methoctramine expressed as the antilog of the difference between the pA 2 values for atrium and ileum muscarinic receptors. Preparations a
Selectivity ratio
Left atrium/ileumb Left atrium/ileum¢ Left atrium/rat ileum a Right atrium (force)/ileum b Right atrium (force)/rat ileum b Right atrium (force)/rat ileum b Left atrium (force)/right atrium (rate) t
59 54 132 55 91 166 e 1.1
a Tissues were from guinea pigs unless otherwise indicated, b The agonists were muscarine and furmethide in the atrium and ileum, respectively, c The agonist was carbachol in both preparations, d The agonists were carbacbol and furmethide in the atrium and ileum, respectively, e Value obtained by recalculation of the pA 2 values reported for guinea pig atria and rat ileum by Melchiorre et al. (1987) by the constrained plot method, f The agonist was muscarine in both preparations.
c o m b i n a t i o n of two pure competitive antagonists (204 a n d 286) (table 3).
3.4. Methoctramme selectivity T a b l e 4 shows the selectivity ratios for m e t h o c t r a m i n e . It is evident that the affinity of m e t h o c t r a m i n e to cardiac muscarinic M-2 receptors is a b o u t two orders of m a g n i t u d e higher than that to M-2 receptors in ileal smooth muscle.
4. Discussion T h e m u s c a r i n i c M-2 receptors in cardiac tissues a n d smooth muscle organs have b e e n claimed to b e l o n g to different subclasses (Clark a n d Mitchelson, 1976; Barlow et al., 1976; L a m b r e c h t et al., 1984; Mutschler a n d Lambrecht, 1984; L a m b r e c h t a n d Mutschler, 1985; A n w a r - u l et al., 1986; Micheletti et al., 1987) d e n o t e d as cardiac M - 2 a a n d s m o o t h muscle M-2fl type ( L a m b r e c h t et al., 1987). This heterogeneity has recently been confirmed b y the d i s c r i m i n a t i n g properties of the cardioselective a n t i m u s c a r i n i c agent methoctram i n e (Melchiorre et al., 1987). W e have n o w further investigated the a n t i m u s c a r i n i c effects of m e t h o c t r a m i n e in atrial a n d ileal p r e p a r a t i o n s derived from different species a n d using different agonists. I n addition, a n t a g o n i s t c o m b i n a t i o n experiments were carried out with a t r o p i n e (a classical competitive antagonist) a n d gallamine ( a n allosteric a n t i m u s c a r i n i c agent at cardiac m u s c a r i n i c receptors) in order to verify the m e c h a n i s m of action of m e t h o c t r a m i n e .
123 Methoctramine was an apparently simple competitive antagonist in both atrial and ileal preparations since it produced antagonism that was reversible and surmountable over a wide range of concentrations (figs. 2-4) and its apparent affinity in each tissue appeared to be independent of the concentration used. Thus, Schild analysis of the data yielded slopes which were not significantly different from unity. Furthermore, dissociation constants and slopes were independent of both agonist and species (table 1). The affinity of methoctramine for cardiac muscarinic M-2a receptors in both pacemaker and myocardium cells was much higher than that observed at M-2fl receptors in the ileum (tables 1 and 4). Methoctramine displayed a selectivity ratio ranging from 54 to 132 (table 4), which is in agreement with results previously reported (Melchiorre et al., 1987). Thus, the selectivity profile of methoctramine is analogous to that of A F - D X 116 (Giachetti et al., 1986; Hammer et al., 1986; Micheletti et al., 1987), but it must be emphasised that the cardioselectivity of methoctramine is much greater. The mutual competition between methoctramine and atropine or gallamine was investigated in electrically stimulated left atria and in ileum according to the dose ratio method (Paton and Rang, 1965). As shown in table 2, in the atria a combination of methoctramine and atropine resulted in dose ratios equal to the sum of the dose ratios produced by the two antagonists when tested alone. This suggests strongly that methoctramine and atropine compete mutually with the same receptor site. Further evidence for the mechanism of action of methoctramine came from the observation that a combination of methoctramine and gallamine resulted in a less than additive antagonistic effect (table 2). This indicates that the two antagonists interact with two different but interdependent sites of the cardiac muscarinic receptor. These results are very similar to those reported for a combination of atropine and gallamine (Clark and Mitchelson, 1976). Experiments performed in guinea pig ileum with a combination of methoctramine and atropine gave results which were not totally consistent with a competitive mode of action of methoctramine (ta-
ble 3). In fact, the combination dose ratios were somewhat greater than expected (table 3), but these results are not consistent with the multiplication of dose ratios which would be expected if the two antagonists interacted exclusively at independent sites. In summary, it was demonstrated that methoctramine is a potent competitive antimuscarinic agent at atrial muscarinic M-2a receptors controlling heart rate and force of contractions whereas it is about two orders of magnitude less potent at ileal muscarinic M-2fl receptors. Thus, methoctramine is the most cardioselective antimuscarinic agent known today and represents a highly useful tool for the subclassification of muscarinic receptors in the periphery as well as in the central nervous system. In addition, it may have therapeutic potential in the treatment of sinus bradycardia without producing concomitant side-effects on other parasympathetically innervated organs.
Acknowledgement Supported by the Ministry of Public Education (Rome).
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