Muscarinic M2 receptors in bovine tracheal smooth muscle: discrepancies between binding and function

European Journal of Pharmacology, 153 (1988) 73-82

73

Elsevier EJP 50414

Muscarinic M 2 receptors in bovine tracheal smooth muscle: discrepancies between binding and function A d F. R o f f e l

1,2,.

C a r o l i n a R.S. E l z i n g a 1,2, R o n a l d G . M . V a n A m s t e r d a m a, R o k u s A. D e Z e e u w 2 a n d J o h a n Z a a g s m a 1

I Department of Pharmacology and Therapeutics and 2 Department of Analytical Chemistry and Toxicology, University of Groningen, Antonius Deusinglaan 2, 9713 A W Groningen, The Netherlands

Received 4 March 1988, revised MS received 13 May 1988, accepted 7 June 1988

Previous work showing that AF-DX 116, a cardioselective muscarinic antagonist in functional experiments, does not discriminate between muscarinic receptors in bovine cardiac and tracheal membranes has been extended. In addition to AF-DX 116 we used the muscarinic antagonists, atropine, pirenzepine, 4-DAMP methobromide, gallamine, hexahydrosiladifenidol and methoctramine, in radioligand binding experiments on bovine cardiac left ventricular and tracheal smooth muscle membranes. The functional antagonism of the methacholine-induced contraction of bovine tracheal smooth muscle strips was also evaluated. An excellent correlation was found for all compounds between the binding affinities for muscarinic receptors in cardiac and tracheal smooth muscle membranes; moreover, the affinities found in cardiac membranes correspond with the pA 2 values reported for atrial preparations of rat and guinea pig. However, significant and occasionally marked discrepancies were found between binding and functional affinities of these muscarinic antagonists on bovine tracheal smooth muscle. Smooth muscle (tracheal); Ventricle (left); Muscarinic receptor subtypes; (Binding vs. functional experiments, Bovine)

1. Introduction

Various subtypes of the muscarinic receptor have been identified over the past years. Pirenzepine was the first compound to distinguish between muscarinic receptors in different tissues and organs in both binding and functional studies ( H a m m e r et al., 1980). This led to the subdivision of these receptors into the M 1 subtype, mainly found in sympathetic ganglia and the central

* To whom all correspondence should be addressed: Dept. of Pharmacology and Therapeutics, University of Groningen, A. Deusinglaan 2, 9713 AW Groningen, The Netherlands.

nervous system, and the M 2 subtype, found in the peripheral effector organs of the parasympathetic nervous system and also in parts of the central nervous system. However, it is now clear that the M 2 muscarinic receptors are not a homogeneous class and recent reports suggest that the same applies to the M 1 receptors (Lambrecht et al., in p r e s s ) . M 2 receptors in cardiac and smooth muscle preparations can be distinguished functionally by the use of antagonists such as A F - D X 116 (Micheletti et al., 1987), gallamine (Clark and Mitchelson, 1976), himbacine (Gilani and Cobbin, 1986) and methoctramine (Melchiorre et al., 1987), which are all cardioselective, and by 4 - D A M P methobromide (Barlow et al., 1976) and hexahydrosiladifenidol (Mutschler and Lambrecht,

0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

74 1984), which are selective for smooth muscle (for a review see Eglen and Whiting, 1986). On the basis of these results Giachetti and coworkers have proposed that cardiac muscarine receptors should be classified as M 2 and smooth muscle receptors as M 3 (Giachetti and Micheletti, in press). Others have suggested that cardiac muscarinic receptors should be termed M2A (Eglen and Whiting, 1986) or M2~ receptors (Lambrecht et al., in press) and the smooth muscle receptors M2B or M2~. Recent results show that A F - D X 116 has an even lower potency on muscarinic receptors in exocrine glands than on those in smooth muscle (cf. H a m m e r et al., 1986; Micheletti et al., 1987; Doods et al., 1987). Therefore, a subdivision of the M 2 muscarinic receptors into M 2 (heart), M 3 (exocrine glands) and M 4 (smooth muscle) seems justified and has been proposed by Batink et al. (1987). Until now these different subclassifications have not been corroborated by binding studies in smooth muscle. While studying the muscarinic receptor in bovine tracheal smooth muscle membranes we found that A F - D X 116 binds with equally high affinity to these receptors as to those in bovine cardiac left ventricular membranes (Roffel et al., 1987). Similar observations were reported previously for the neuromuscular blocking agents, gallamine and pancuronium (Choo et al., 1985; N e d o m a et al., 1985), although it should be realized that these two compounds also possess allosteric properties towards muscarinic receptors (Dunlap and Brown, 1983; Stockton et al., 1983). We aimed to compare the binding properties of a group of five antagonists that discriminate between M 2 receptors and of atropine and pirenzepine in bovine cardiac and tracheal smooth muscle membranes, and to compare these data with their abilities to antagonize the methacholine-induced contraction of tracheal smooth muscle. It was found that besides gallamine and A F - D X 116, 4 - D A M P methobromide, hexahydrosiladifenidol and methoctramine also do not discriminate between bovine cardiac and tracheal smooth muscle muscarinic receptors in binding experiments. However, a marked discrepancy was observed with all selective antagonists between their binding and functional potencies on tracheal smooth muscle.

2. Materials and methods

2.1. Membrane preparation Fresh bovine tracheas were obtained from the slaughterhouse and transported to the laboratory in ice-cold buffer (20 m M Tris-HCl p H 7.0, 1.0 m M EDTA, 0.1 m M phenylmethylsulfonyl fluoride). The tracheal muscle was dissected and after the mucosa and serosal connective tissue had been carefully removed, the smooth muscle layer was minced with a scalpel and homogenized with 10 volumes of buffer using a Polytron PT10-35 equipped with a PTA 10S probe (3 × 10 s, setting 8, 30 s intervals) at 4 ° C . The homogenate was centrifuged for 20 rain at 5 000 X g and the supernatant was recentrifuged for 60 rain at 70000 × g. The resulting pellet was resuspended in 50 mM sodium phosphate buffer p H 7.4. Aliquots were frozen in liquid N 2 and stored for up to 2 months at - 80 ° C. Left ventricular muscle from fresh bovine hearts was minced with a tissue press and homogenized with a Virtis homogenizer (4 x 10 s, 20000 rpm, 30 s intervals) at 4 ° C . A purified sarcolemmal preparation was isolated from the homogenate according to the differential centrifugation procedure described by Velema and Zaagsma (1981), but 0.15 M KC1 was used instead of 0.6 M KC1 in the final step of the contractile protein extraction. The final pellet was resuspended in phosphate buffer and stored as described above. Membrane protein was measured by the method of Lowry et al. (1951) with BSA as the standard.

2.2. Binding experiments M e m b r a n e protein, approximately 150 /~g (trachea) or 350 /~g (heart), was mixed with [3H]dexetimide (0.2 10.0 nM in saturation experiments, 3.0 nM in inhibition experiments) and displacing antagonists in the concentrations indicated in a final volume of 550 ~1 of 50 mM sodium phosphate buffer (pH 7.4). Unlabeled dexetimide (2 /~M) was used to define non-specific binding in saturation experiments. Incubations in duplicate (30 min, 37 ° C) were terminated by the addition of 4 ml of ice-cold buffer, followed by

75 filtration through W h a t m a n G F / B filters. The tubes were rinsed twice with 4 ml of ice-cold buffer which was then filtered. After the addition of 4 ml of Aqualuma Plus ~ and shaking for 2 h, the filters were counted for radioactivity in a Packard Tri-Carb 4450 liquid scintillation counter.

2.3. Contraction experiments Fresh bovine tracheas were transported to the laboratory in ice-cold Krebs-Henseleit (KH) buffer solution pregassed with 95% 02-5% CO2; p H 7.4. The tracheal muscle was dissected carefully and smooth muscle strips (12 × 3 mm) were prepared free of mucosa and connective tissue in K H solution gassed with 95% 02-5% CO 2 at room temperature. These strips were used either immediately or the next day. In the latter case, they were kept at 2 4 ° C (which was shown in separate experiments to be an optimal temperature) in a system in which they were continuously superfused (flow 3.9 m l / m i n ) with gassed K H solution. N o differences were detected in experiments conducted on fresh or stored tissues. Smooth muscle strips were mounted in 20-ml organ baths (KH, 37 o C) under isotonic recording with a preload of 0.5 g. After a 100-min equilibration period strips were precontracted twice with methacholine (0.1, 1, 10 and 0.1, 1, 10, 100/~M with washing periods of 60 min) before construction of the control dose-response curve. After a 60-rain washing period, one or two consecutive dose-response curves were constructed in the presence of antagonist (with a 30 min preincubation), the concentration of antagonist being at least 10 times higher in the second curve than in the first one. In each experiment two out of eight smooth muscle strips received no antagonist and served as controls for the decrease of sensitivity that occurred with time; accordingly, antagonist-induced shifts of the dose-response curves to the right were corrected for these (minor) spontaneous shifts. Each antagonist was tested in four experiments on different days, thus yielding four independent Schild plots.

2.4. Data analysis Binding parameters were calculated with the 1981 modification of the L I G A N D program of

Munson and Rodbard (1980), which includes a model of allosteric interactions as described by Thakur and DeLisi (1978). A two-site binding model was preferred over a one-site binding model when a significant reduction in the residual sum of squares was obtained (F-test, P < 0.05). Competitive antagonism in the contraction experiments was assessed according to Arunlakshana and Schild (1959). pA 2 values were calculated for each concentration of antagonist when the slope of the plot did not differ significantly from unity according to: p A 2 = - l o g { [ a n t a g o n i s t ] / ( D R - 1 ) } and the mean value (_+ S.E.M.) was calculated (MacKay, 1978).

2.5. Materials [3H]Dexetimide (17 C i / m m o l ) was purchased and unlabeled dexetimide was a gift from Janssen Pharmaceutica (Beerse, Belgium); A F - D X 116 and pirenzepine were gifts from Dr. Karl Thomae G m b H (Biberach an der Riss, FRG); 4 - D A M P methobromide was a gift from Dr. R.B. Barlow (Bristol, UK); hexahydrosiladifenidol was a gift from Dr. G. Lambrecht (Frankfurt am Main, FRG); methoctramine was a gift from Dr. C. Melchiorre (Camerino, Italy). Aqualuma Plus ® was purchased from L u m a c / 3 M (Schaesberg, The Netherlands). All other chemicals were of reagent grade.

3. Results

3.1. Binding experiments Muscarinic receptors in bovine tracheal and left ventricular membranes were first compared in saturation experiments with [3H]dexetimide as the radioligand. The incubation time used (30 min) was found in separate experiments to allow full equilibration (data not shown). [3H]Dexetimide bound to a single population of sites in both preparations although there were differences in the receptor density (left ventricle: p K d = 8.74 + 0 . 0 3 , Bma x = 845 _+ 127 f m o l / m g protein (means _+ S.E.M of six m e m b r a n e batches); trachea: p K d =

76 TABLE 2

9.12 4- 0.04, B m a x = 2161 4- 397 fmol/mg protein (means + S.E.M. of four membrane batches)). Figure 1 shows the concentration-dependent inhibition of [3Hldexetimide binding to cardiac and tracheal smooth muscle membranes by the different antagonists. All compounds displaced specific [3Hldexetimide binding completely in both organs except gallamine (83% in cardiac membranes, 84% in tracheal membranes). The competition curves were steep and could be adequately described by a one binding site model for atropine, 4-DAMP methobromide, hexahydrosiladifenidol and pirenzepine in both preparations and for AF-DX 116, gallamine and methoctramine in the heart. In tracheal membranes however AF-DX 116, gallamine and methoctramine gave shallow or biphasic competition curves which, as expected, could be fitted significantly better assuming a two-site model (P < 0.005 for AF-DX 116 and gallamine, P < 0.05 for methoctramine). Fitting the gallamine displacement curve to a two-site model with allosteric interactions did not yield a significantly better fit (F-test, P > 0.05). Binding parameters of all compounds are given in table 1.

Functional potencies of selective muscarinic antagonists on bovine tracheal smooth muscle, calculated as described in methods (data analysis). pA 2 Atropine Pirenzepine A F - D X 116 4 - D A M P methobromide Gallamine Hexahydrosiladifenidol Methoctramine

8.96±0.06 6.92 +_0.08 6.30±0.07 9.03 ± 0.05 4.25 7.45 ± 0.05 6.45

Slope a (25) b (30) (25) (20) (24) ~ (17) (27) c

1.16±0.1l 1.10 ± 0.05 1.06±0.02 1.12 ± 0.06 0.71 ± 0.04 d 1.18 _+0.08 0.79 ± 0.06 ~

M e a n s ± S.E.M. from four independent Schild plots: b means ±S.E.M. ( n u m b e r of determinations); c intercept of Schild plot having a slope smaller than one; d significantly different from unity: P < 0.01 (two-tailed Student's t-test); e significantly different from unity: P < 0.05 (two-tailed Student's t-test).

concentration-dependent fashion and over a large concentration range. None of the compounds (including gallamine and methoctramine) depressed the maximum contraction even at the highest concentrations used. The slopes of the Schild plots did not differ significantly from unity except for gallamine and methoctramine, the slopes of which were significantly smaller than one. Schild plots

3.2. Contraction experiments All antagonists tested shifted the methacholine dose-response curve to the right in a parallel and

TABLE 1 Binding parameters calculated from the competition curves depicted in fig. 1. The parameters for the statistically better fit are shown (see Data analysis). Trachea

Atropine Pirenzepine A F - D X 116 4-DAMP methobromide Gallamine Hexahydrosiladifenidol Methoctramine

Heart

pK i

%R ~

pK i

%R

8.52 ± 0.05 (4) b 6.37 ± 0.01 (5) 7.38 ± 0.03 (6) 5.57 ± 0.10

100 100 74 ± 1 26 ± 1

8.20 ± 0.04 (4) 6.22 ± 0.01 (3) 7.03 _ 0.04 (3)

100 100 100

8.03 ± 0.04 (4) 7.14 ± 0.12 (4) 4.42_+0.14

100 68 ± 2 32±2

7.80 ± 0.05 (3) 6.54 ± 0.06 (3)

100 100

6.76 ± 0.12 (5) 7.81 ± 0.09 (5) 5.38±0.11

100 83 ± 2 17±2

6.41 _+0.04 (3) 7.45 _+0.05 (4)

100 100

a Percentage of binding sites with the affinity shown; b means_+ S.E.M. (number of determinations).

77

oo~~ ~ , ~ - - ~

,oo

~\.

i

~ so

oX

soJ

\,o

I

-O-C,-O--

oJ

O

0-1 i

-10

-11

-g

-8

-7

-6 Lo 9 (ATR)

- 0

-5

-9

1

-8

i

-6

r

-5

r

-4

L o 9 (4-DAM P)

so \o\©~O ~o~

i

-7

%

~

oA e

~

i

-9

-8

-7

r

n

-6

i

-5

i

n

-9

-4 -3 Lo~j (AF-DX 116)

i

-8

i

-7

i

-6

i

i

-5

-4

i

-3

Log (HHSiO)

1o%

100

"<\ so_l •

"0-

o] -9

-8

-7

-6

-5

-4

-3

1 -I0

i -9

i -8

i -7

r -6

J - 5

i -4

Lo 9 (Methoc)

L o 9 (GAL L)

lOO_]~_~_~.v_e.~,~o *

._

56

oJ i

-9

u

-8

i

-7

i

-6

i

-5

i

-4

Lo 9 IPZ)

i

-3

Fig. 1. Inhibition of total [3H]dexetimide binding to bovine tracheal ( ~ ) and left ventricular (O) membranes by atropine (ATR), pirenzepine (PZ), AF-DX 116, 4-DAMP methobromide, gallamine (GALL), hexahydrosiladifenidol (HHSiD) and methoctramine (Methoc). Solid lines represent computer fitted curves according to a one-binding site model (heart, all compounds; trachea, atropine, pirenzepine, 4-DAMP methobromide and hexahydrosiladifenidol) or a two-binding site model (trachea, AF-DX 116, gallamine and methoctramine). Data points represent means of three to six experiments, each performed in duplicate.

78

.... !

Pz +

AF-OXIle

/

i ¢t

Log IDcug)

OAMp

4

HHSIO

~

M*tho~

CALt

i

6

$

4

3

Fig. 2. Schild plots for the antagonism of the methacholine-induced contraction of bovine tracheal smooth muscle strips by atropine (ATR), pirenzepine (PZ) and AF-DX 116 (upper panel) and 4-DAMP methobromide, gallamine (GALL), hexahydrosiladifenidol (HHSiD) and methoctramine (Methoc) (lower panel). Data points represent means _+S.E.M. of three to five determinations; regression lines were calculated with all individual data points.

are shown in fig. 2; the slopes of these plots as well as pA 2 values are given in table 2.

4. Discussion

4.1. Binding experiments The results of our binding studies with seven muscarinic antagonists (one non-selective and six selective) in bovine cardiac left ventricular membranes can be compared with those of binding studies in the rat, guinea pig and rabbit heart (Hammer and Giachetti, 1982; Berrie et al., 1983; Hammer, 1985; Hammer et al., 1986; Baudiere et

al,, 1987; Closse et al., 1987; Doods et al., 1987): the cardiac pK i values for atropine, pirenzepine, AF-DX 116, 4-DAMP methobromide and hexahydrosiladifenidol are consistent for different species. The same is true for methoctramine: Giachetti et al. (1988) reported a pK~ value of 7.45 in the rat which is exactly the same as that in the cow (table 1). Gallamine has been shown to interact with muscarinic receptors in the heart in an allosteric fashion, slowing the dissociation kinetics of the radioligand (Dunlap and Brown, 1983; Stockton et al., 1983). Some authors have reported this to appear in competition binding studies (Dunlap and Brown, 1983; Nedoma et al., 1985); others, however, do not observe deviation from simple competitive binding behaviour (Choo et al., 1985a; Closse et al., 1987). In our studies the [3H]dexetimide/gallamine competition curves showed a monophasic, homogeneous character; allosteric behaviour only appeared as a higher level of non-displaceable binding (fig. 1). Therefore we compared our one-site-fit pKi value (6.54) with that of others and found that it correlated well with those reported by Choo et al. (1985) (6.41, guinea pig) and Closse et al. (1987) (6.74, rat). In conclusion, the binding affinities in bovine cardiac membranes of all seven muscarinic antagonists correlate very well with those reported for small laboratory animals such as the rat and guinea pig. Comparison of our binding results of bovine trachea with those of smooth muscle from the literature shows that the p K i values for atropine, pirenzepine, 4-DAMP methobromide and hexahydrosiladifenidol are also consistent between different organs of different species (cf. Hammer et al., 1980; Choo and Mitchelson, 1985; Yang et al., 1986; Batra, 1987; Baudiere et al., 1987; Giraldo et al., in press; Madison et al., 1987; Rothberg et al., 1987). The binding affinities of gallamine for guinea pig (6.81, Choo et al., 1985) and rat ileum (6.51, Nedoma et al., 1985), based on a one-site model, are quite similar to our one-site-fit pK~ value (6.76 + 0.03) for gallamine in bovine tracheal smooth muscle• However, a significantly improved fit was obtained when we assumed a two-site model, yielding a pK i value of 7.14 (table 1) for the major population of binding sites (68%). It

79 might be argued that the gallamine displacement curves should not be fitted according to a noncooperative two-site model as this drug is wellknown for its negative cooperative interactions with muscarinic receptors. However, fitting these curves according to a two binding site model with allosteric interactions as described by Thakur and DeLisi (1978) did not yield an improved fit. This does not mean that gallamine does not exhibit allosteric interactions in this system but merely that it is not possible to conclude this from the data. Finally, A F - D X 116 and methoctramine also showed heterogeneous binding behaviour in bovine tracheal smooth muscle membranes. This has been reported for A F - D X 116 in bovine trachea (Roffel et al., 1987) and guinea pig ileum (Giraldo et al., 1987); the affinities and relative numbers of high (Mz-like)- and low (M3-1ike)-affinity sites correlate well between both organs. In conclusion, as in the heart, the binding affinities of these muscarinic antagonists to bovine tracheal smooth muscle

90.

/ a 80,

50

70

7

~

44

04

30 50/

70.

O2 60. I 40

1 50

i 60

l

ZO

v 80

/ 90

PKi,heart / PA2,trachea

Fig. 3. Correlation between the binding affinities (pKi) of atropine (1), pirenzepine (2), AF-DX 116 (3), 4-DAMP methobromide (4), gallamine (5), hexahydrosiladifenidol (6) methoctramine (7) and [3H]dexetimide (8) for muscarinic receptors in cardiac and tracheal smooth muscle membranes (filled symbols) and between the binding affinity (pKi) and functional affinity (pA2) in tracheal smooth muscle (open symbols). The high-affinity pK i values for AF-DX 116, gallamine and methoctramine were used. The correlation coefficient of the linear regression analysis between the two binding parameters was 0.9898.

membranes agree very well with those reported in literature for smooth muscle. The p K i values for all antagonists on bovine cardiac and tracheal smooth muscle membranes showed an excellent correlation (fig. 3; r = 0.9898, P < 0.001). Thus, from binding studies there is no reason to regard the M2-type binding sites in these two organs as being different muscarinic receptor subtypes. Similar findings have been reported very recently for guinea pig heart and ileum, with [3H]N-methylscopolamine as the radioligand (Michel and Whiting, 1987).

4.2. Contraction experiments The pA 2 values that we found using bovine tracheal smooth muscle (table 2) agree very well with those reported for guinea pig and rat smooth muscle preparations (ileum, trachea, bladder and taenia) (Batink et al., 1987; Eglen et al., 1987; Lambrecht et al., in press; Micheletti et al., 1987). We obtained a slope of significantly less than one for the Schild plot for gallamine, which could point to allosteric behaviour in bovine tracheal smooth muscle. In preliminary experiments we found that gallamine slowed the dissociation of [3H]dexetimide from m e m b r a n e s of this tissue (results not shown); this property might well account for a slope lower than unity if it results in an increased association time of the agonist with the receptor and thus in a diminished rightward shift of the dose-response curve. As a consequence the pA 2 values for gallamine decrease with increasing concentrations of the antagonist, e.g. a pA 2 value of 4.14 _+ 0.15 (n = 5) at 100/~M but of 3.77 _+ 0.08 (n = 4 ) at 3 m M in our experiments. The pA 2 values reported for rat ileum (4.40; N e d o m a et al., 1985), guinea pig trachea (4.41; Eglen et al., 1987) and guinea pig ileum (4.73 at 110/~M; Clark and Mitchelson, 1976) show similar low affinities for gallamine in smooth muscle. Finally, our results with methoctramine do not agree completely with those reported earlier. Melchiorre et al. (1987), who introduced this compound, reported simple competitive behaviour in guinea pig atria (pA 2 value of 8.13) and in rat ileum and bladder (pA 2 value of 5.69 and 5.70, respectively). However, methoctramine has a slope

80 significantly less than unity in bovine tracheal smooth muscle (fig. 2), the pA 2 value ranging from 6.20 _+ 0.06 (n = 17) at 3-30 /~M to 5.84 +_ 0.08 (n = 10) at 100-300 /IM (significantly different: P < 0 . 0 0 5 , two-tailed Student's t-test). In agreement with this observation, methoctramine has been shown to slow the dissociation of [3H]N-methylscopolamine in cardiac membranes (Giachetti et al., 1988), indicating an allosteric behaviour like that of gallamine. 4.3. Comparison of binding and contraction experirnen ts The pA 2 and high-affinity pK i values of all antagonists for bovine tracheal smooth muscle have been compared and showed significant and occasionally large discrepancies between the binding and functional parameters of selective antagonists. These discrepancies are opposite for the cardio-selective and the smooth muscle-selective antagonists, the binding potency being higher than the functional potency for AF-DX 116, gallamine and methoctramine but lower for 4-DAMP methobromide and hexahydrosiladifenidol. Evidently there is no correlation between the binding and functional potencies of these antagonists in tracheal smooth muscle. It should be mentioned that these observations are in contrast with findings on cardiac muscarine receptors where binding and functional potency estimates for rat and guinea pig atria correlate well (Batink et al., 1987; Giachetti et al., 1988). This in turn implies that the binding affinities in bovine cardiac membranes found in this study, which agree with the pK i values for rat and guinea pig cardiac membranes and thus with the pA2 values for rat and guinea pig atria (vide supra), will correlate with the functional affinities in bovine heart. We also found some discrepancies between the binding and functional affinities of atropine and pirenzepine for tracheal smooth muscle muscarinic receptors. Although this has been observed by others (Choo et al., 1985; Hammer, 1985), it has not led to a different classification of tracheal smooth muscle muscarinic receptors based on the results of binding and functional experiments as is the case with the M2-selective antagonists.

Although discrepancies between the binding and functional affinities of gallamine, pancuronium and 4-DAMP methobromide to smooth muscle tissues have been reported (Choo et al., 1985; Choo and Mitchelson, 1985), a logical explanation does not seem to be at hand. Choo et al. (1985) suggested that the allosteric properties of gallamine and pancuronium might be involved in this behaviour. However, this explanation cannot account for all the results presented here since AF-DX 116, 4-DAMP methobromide and hexahydrosiladifenidol, all antagonists of the competitive type, show the same behaviour. Nedoma et al. (1985) suggested that homogenization of smooth muscle might bring about dramatic changes in the affinities of gallamine and related compounds for muscarinic receptors in this tissue. Although this possibility cannot be ruled out, the striking correlation between the binding affinities of all antagonists on cardiac and tracheal smooth muscle membranes makes it unlikely that non-specific changes in the nature of the receptors occur during homogenization of the tissues and subsequent isolation of membranes. Future studies with binding experiments in intact tissue or isolated cells and functional experiments with tissue fragments or isolated membranes (measuring antagonism of methacholine-induced phosphoinositide metabolism or adenylate cyclase inhibition) might shed light on the relationship between the binding pqtencies and functional potencies of these antagonists in tracheal smooth muscle, Giraldo et al. (1987) have suggested recently that the low-affinity site for AF-DX 116 might represent the functional receptor. There are, however, some problems with this interpretation. In the first place, when the low-affinity p K i values for AF-DX 116, gallamine and methoctramine are compared, there are still significant discrepancies between the binding and functional affinities (cf. tables 1 and 2). Secondly, the low-affinity binding sites for AF-DX 116, gallamine and methoctramine only account for a minority of the total population of binding sites (26, 32 and 17%, respectively). In line with the suggestion of Giraldo et al. (1987) one of the referees proposed that there could be two populations of binding sites in tracheal smooth muscle membranes, the majority

81 (70-80%) having cardiac (M2) binding properties a n d the r e m a i n d e r h a v i n g s m o o t h m u s c l e ( M 4 ) b i n d i n g p r o p e r t i e s , r e p r e s e n t i n g the f u n c t i o n a l rec e p t o r . A s s u m i n g t h a t t h e r a d i o l i g a n d [3H]dexe t i m i d e has s o m e s e l e c t i v i t y for t h e m i n o r p o p u l a t i o n of M 4 - t y p e sites, w h i c h in n o r m a l s a t u r a t i o n e x p e r i m e n t s is n o t d e t e c t a b l e , this m o d e l w o u l d p r e d i c t t h a t the b i n d i n g s e l e c t i v i t y ( p K i , H / p K i , L ) for the c a r d i o - s e l e c t i v e a n t a g o n i s t s ( A F - D X 116, g a l l a m i n e a n d m e t h o c t r a m i n e ) w o u l d d e c r e a s e to s o m e extent, w h i l e the s m o o t h m u s c l e s e l e c t i v e antagonists (4-DAMP methobromide and hexahydrosiladifenidol) would maintain exhibiting m o n o p h a s i c i n h i b i t i o n curves. H o w e v e r , as disc u s s e d a b o v e , the d i s c r e p a n c y b e t w e e n the p K i v a l u e s for b i n d i n g to t h e m a j o r p o p u l a t i o n o f M 2-type sites a n d t h e f u n c t i o n a l p A 2 v a l u e s w o u l d remain. In conclusion, we have shown that muscarinic a n t a g o n i s t s t h a t h a v e b e e n r e p o r t e d to f u n c t i o n ally d i s c r i m i n a t e b e t w e e n c a r d i a c a n d s m o o t h muscle preparations do not discriminate between bovine cardiac and tracheal smooth muscle memb r a n e s in b i n d i n g studies. A s a c o n s e q u e n c e , marked discrepancies occur between the binding a n d f u n c t i o n a l p o t e n c i e s of t h e s e c o m p o u n d s in bovine tracheal smooth muscle. Since allosteric p r o p e r t i e s o f t h e a n t a g o n i s t s c a n b e r u l e d o u t as a g e n e r a l m e c h a n i s m , a s a t i s f y i n g e x p l a n a t i o n is n o t yet available.

Acknowledgements This work was supported by the Netherlands Asthma Foundation (NAF Grant 84.24). Drs. K. Ensing, H. Meurs and N.C. Punt are thanked for valuable discussions. We are grateful to Janssen Pharmaceutica (Beerse, Belgium) for the gift of dexetimide, to Dr. Karl Thomae GmBH (Biberach an der Kiss, FRG) for gifts of AF-DX 116 and pirenzepine, and to Dr. R.B. Barlow (Bristol, UK), Dr. G. Lambrecht (Frankfurt am Main, FRG) and Dr. C. Melchiorre (Camerino, Italy) for gifts of 4-DAMP methobromide, hexahydrosiladifenidol and methoctramine, respectively.

References Arunlakshana, O. and H.O. Schild, 1959, Some quantitative uses of drug antagonists, Br. J. Pharmacol. Chemother. 14, 48.

Barlow, R.B., K.J. Berrie, P.A.M. Glenton, N.M. Nikolaou and K.S. Soh, 1976, A comparison of affinity constants for muscarine-sensitive acetylcholine receptors in guinea-pig atrial pacemaker cells at 29 ° C and in ileum at 29°C and 37°C, Br. J. Pharmacol. 58, 613. Batink, H.D., D. Davidesko, H.N. Doods, K.J. Van Charldorp, A. De Jonge and P.A. Van Zwieten, 1987, Subdivision of Mz-receptors into three types, Br. J. Pharmacol. 90, 81P. Batra, S., 1987, Comparison of muscarinic acetylcholine binding in the urinary bladder and submandibular gland of the rabbit, European J. Pharmacol. 138, 83. Baudiere, B., E. Monferini, E. Giraldo, H. Ladinsky and J.P. Bali, 1987, Characterization of the muscarinic receptor subtype in isolated gastric fundic cells of the rabbit, Biochem. Pharmacol. 36, 2957. Berrie, C.P., N.J.M. Birdsall, A.S.V. Burgen and E.C. Hulme, 1983, The binding properties of muscarinic receptors in the rat lacrimal gland: comparison with the cerebral cortex and myocardium, Br. J. Pharmacol. 78, 67P. Choo, L.K., E. Leung and F. Mitchelson, 1985, Failure of gallamine and pancuronium to inhibit selectively ( - ) [3H]quinuclidinyl benzilate binding in guinea-pig atria, Can. J. Physiol. Pharmacol. 63, 200. Choo, L.K. and F.J. Mitchelson, 1985, Comparison of the affinity constant of some muscarinic receptor antagonists with their displacement of [3H]quinuclidinyl benzilate binding in atrial and ileal longitudinal muscle of the guinea-pig, J. Pharm. Pharmacol. 37, 656. Clark, A.L. and F. Mitchelson, 1976, The inhibitory effect ot gallamine on muscarinic receptors, Br. J. Pharmacol. 58, 323. Closse, A., H. Bittiger, D. Langenegger and A. Wanner, 1987, Binding studies with [3H]cis-methyldioxolane in different tissues; Under certain conditions [3H]cis-methyldioxolane labels preferentially but not exclusively agonist high affinity states of muscarinic M z receptors, Naunyn-Schmiedeb. Arch. Pharmacol. 335, 372. Doods, H.N., M.-J. Mathy, D. Davidesko, K.J. Van Charldorp, A. De Jonge and P.A. Van Zwieten, 1987, Selectivity of muscarinic antagonists in radioligand and in vivo experiments for the putative M~, M 2 and M 3 receptors, J. Pharmacol. Exp. Ther. 242, 257. Dunlap, J. and J.H. Brown, 1983, Heterogeneity of binding sites on cardiac muscarinic receptors induced by the neuromuscular blocking agents gallamine and pancuronium, Mol. Pharmacol. 24, 15. Eglen, R.M., B.A. Kenny, A.D. Michel and R.L. Whiting, 1987, Muscarinic activity of McN-A-343 and its value in muscarinic receptor classification, Br. J. Pharmacol. 90, 693. Eglen, R.M. and R.L. Whiting, 1986, Muscarinic receptor subtypes: a critique of the current classification and a proposal for a working nomenclature, J. Auton. Pharmacol. 5, 323. Giachetti, A., E. Giraldo, C. Melchiorre, R. Micheletti and H. Ladinsky, 1988, Further characterization of methoctramine interaction with muscarinic receptors, Trends Pharmacol.

82 Sci. 9 (Supplement Subtypes of Muscarinic Receptors III), 87. Giachetti, A. and R. Micheletti, Pharmacology of selective muscarinic agents, Proc. 6th Camerino-Noordwijkerhout Symp. Recent Advances Receptor Chemistry (Elsevier/ North-Holland Biomedical, Amsterdam) (in press). Gilani, S.A.H. and L.B. Cobbin, 1986, The cardio-selectivity of himbacine: a muscarine receptor antagonist, NaunynSchmiedeb. Arch. Pharmacol. 332, 16. Giraldo, E., E. Monferini, H. Ladinsky and R. Hammer, 1987, Muscarinic receptor heterogeneity in guinea pig intestinal smooth muscle: binding studies with AF-DX 116, European J. Pharmacol. 141,475. Giraldo, E., M.A. Vigano, H. Ladinsky and R. Hammer, Muscarinic receptor characterization in guinea pig ileum longitudinal smooth muscle, Proc. 6th Camerino-Noordwijkerhout Symp. Recent Advances Receptor Chemistry (Elsevier/North-Holland Biomedical, Amsterdam) (in press). Hammer, R., 1985, Differenzierung von muskarinischen Rezeptoren durch Bindungsstudien, in: Rezeptoren und Nerv~3se Versorgung des Bronchopulmonalen Systems (Verlag Gideon & Reuss, Mi.inchen) p. 110. 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 M2; 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. Lambrecht, G., U. Moser, G. Gmelin, R. Tacke and E. Mutschler, Heterogeneity in muscrinic receptors: evidence from pharmacological and electrophysiological studies with selective antagonists, in: Muscarinic Cholinergic Mechanisms, eds. S. Cohen and M. Sokolovsky (Freund Publishing House Ltd., London, Tel-Aviv) (in press). Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Mackay, D., 1978, How should values of pA 2 and affinity constants for pharmacological competitive antagonists be estimated?, J. Pharm. Pharmacol. 30, 312. Madison, J.M., C.A. Jones, M. Tom-Moy and J.K. Brown, 1987, Affinities of pirenzepine for muscarinic cholinergic

receptors in membranes isolated from bovine tracheal mucosa and smooth muscle, Am. Rev. Respir. Dis. 135, 719. Melchiorre, C., A. Cassinelli and W. Quaglia, 1987, Differential blockade of muscarinic receptor subtypes by polymethylene tetramines. Novel class of selective antagonists of cardiac M-2 muscarinic receptors, J. Med. Chem. 30, 201. Michel, A.D. and R.L. Whiting, 1987, Direct binding studies on ileal and cardiac muscarinic receptors, Br. J. Pharmacol. 92, 755. Micheletti, R., E. Montagna and A. Giachetti, 1987, AF-DX 116, a cardioselective muscarinic antagonist, J. Pharmacol. Exp. Ther. 241, 628. Munson, R.J. and D. Rodbard, 1980, LIGAND: a versatile computerized approach for characterization of ligand-binding systems, Anal. Biochem. 107, 220. Mutschler, E. and G. Lambrecht, 1984, Selective muscarinic agonists and antagonists in functional tests, Trends Pharmacol. Sci. 5 (Supplement Subtypes of Muscarinic Receptors I), 39. Nedoma, J., N.A. Dorofeeva, S. Tucek, S.A. Shelkovnikov and A.F. Danilov, 1985, Interaction of the neuromuscular blocking drugs alcuronium, decamethonium, gallamine, pancuronium, ritebronium, tercuronium and d-tubocurarine with muscarinic acetylcholine receptors in the heart and ileum, Naunyn-Schmiedeb. Arch. Pharmacol. 329. 176. Roffel, A.F., W.G. In "t Hout, R.A. De Zeeuw and J. Zaagsma, 1987, The M 2 selective antagonist AF-DX 116 shows high affinity for muscarine receptors in bovine tracheal membranes, Naunyn-Schmiedeb. Arch. Pharmacol. 335, 593. Rothberg, K.G., P.L. Morris and J.S. Douglas, 1987, Characterization of cholinergic muscarinic receptors in cow tracheal muscle membranes, Biochem. Pharmacol. 36, 1687. Stockton, J.M., N.J.M. Birdsall, A.S.V. Burgen and E.C. Hulme, 1983, Modification of the binding properties of muscarinic receptors by gallamine, Mol. Pharmacol. 23, 551. Thakur, A.K. and C. DeLisi, 1978, Theory of ligand binding to heterogeneous receptor populations: characterization of the free-energy distribution function, Biopolymers 17, 1075. Velema, J. and J. Zaagsma, 1981, Purification and characterization of cardiac sarcolemma and sarcoplasmatic reticulum from rat ventricle muscle, Arch. Biochem. Biophys. 212, 678. Yang, C.M., J.M. Farley and T.M. Dwyer, 1986, Biochemical characteristics of muscarinic cholinoceptors in swine tracheal smooth muscle, J. Auton. Pharmacol. 6, 15.