Muscarinic M1- and M2-receptors mediating opposite effects on neuromuscular transmission in rabbit vas deferens

European Journal of Pharmacology, 151 (1988) 205-221

205

Elsevier EJP 50322

Muscarinic M i- and M 2-receptors mediating opposite effects on neuromuscular transmission in rabbit vas deferens M a n f r i d Eltze Department of Pharmacology, Byk Gulden Lomberg Chemische Fabrik GmbH, D-7750 Konstanz, F.R.G.

Received 16 November 1987, revised MS received 28 January 1988, accepted 5 April 1988

Twitch contractions of the rabbit vas deferens elicited by electrical field stimulation were inhibited by tetrodotoxin, guanethidine, bretylium and a,fl-methylene-ATP but were unaffected by hexamethonium, physostigmine, 1,1-dimetbyl-4-phenylpiperazinium and prazosin, suggesting that they resulted from ATP released following postganglionic sympathetic nerve stimulation. McN-A-343 inhibited but carbachol and several other muscarinic agonists potentiated the twitch contractions; these effects were not modified by hexamethonium or physostigmine. Muscarinic agonists had no effect on the tension in unstimulated organs whereas contractions elicited by ATP, noradrenaline and KCI were potentiated by carbachol but remained unaffected by McN-A-343. The responses of the twitch contractions to McN-A-343 and carbachol were inhibited to different degrees by antimuscarinic drugs: the affinity (pA2) of atropine, secoverine and himbacine against MeN-A-343 and carbachol was similar. However, pirenzepine, telenzepine, trihexyphenidyl, dicyclomine and hexahydro-sila-difenidol displayed preferential antagonism of the responses to McNA-343 whereas the converse was true for AF-DX 116 and gallamine. The highly significant correlation between the pA 2 values obtained for 10 antagonists against carbachol responses in rabbit vas deferens and rat left atrium suggests that the receptors may be similar. The data support the presence of a presynaptic Ml-receptor mediating inhibition and a postsynaptic, cardiac-like M2-receptor responsible for enhancing neurogenic contractions in rabbit vas deferens. Muscarinic M 1 receptors; Muscarinic

M2

receptors; Antimuscarinic drugs; Vas deferens; Left atrium (Rabbit, Rat)

1. Introduction Presynaptic muscarinic receptors have been reported to exist in a variety of tissues (Westfall, 1977; Starke, 1981) and have been studied extensively in mammalian heart (LiSffelholz, 1979; Muscholl, 1980), guinea-pig ileum (Mutschler and Lambrecht, 1984; Kilbinger and Nafziger, 1985; N o r t h et al., 1985) and the rabbit ear artery (Rand and Varma, 1971; Choo et al., 1985; 1986). The function of presynaptic muscarinic receptors is to modulate the output of neurotransmitters such as noradrenaline and dopamine in addition to that of acetylcholine itself (Kilbinger, 1985). The ability of acetylcholine to enhance nerve stimulation-induced twitch contraction has been

demonstrated in both rat ( G r a h a m et al., 1968; Liao and Freer, 1983) and guinea-pig vas deferens (Sjtistrand, 1973). However, the pharmacological properties and localization of the receptors involved have not yet been studied in detail. It has been suggested that the cholinergic enhancement of twitch contraction of electrical field-stimulated rat vas deferens may be mediated by muscarinic receptors present on presynaptic nerve terminals (Liao and Freer, 1983). This explanation has been questioned recently and a postsynaptic effect has been postulated (Ballejo et al., 1987). The potentiation arises from enhancement of 'non-adrenergic' transmission rather than of adrenergic, A T P being the transmitter mediating the a - a d r e n ~ e p t o r - r e sistant component of the biphasic contraction of

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

206 this organ (Sneddon et al., 1984; Lee, 1985). ATP also mediates the twitch phase of the contraction in the vas deferens of guinea-pig (Sneddon and Burnstock, 1984; Kirkpatrick and Burnstock, 1987) and rabbit (Sneddon et al., 1984). There is pharmacological evidence that the smooth muscle of the guinea-pig vas deferens is additionally innervated by cholinergic fibres (Birmingham, 1966). Exogenous acetylcholine has been found to exert a dual muscarinic effect, consisting of presynaptic inhibition of noradrenaline release and postsynaptic enhancement of the nerve stimulation-induced contraction (Stj~irne, 1975). The possibility of additional cholinergic innervation has also been shown by field stimulation of the rat vas deferens, which causes release of acetylcholine (Knoll et al., 1972). However the actions of acetylcholine agonists on neurogenic contractions of the rabbit vas deferens have not been investigated. The aim of the present study was therefore to determine (a) whether muscarinic modulation of neurotransmission occurs in this tissue and if so, (b) to classify the possible receptor subtypes involved and, (c) to determine their location. The actions of muscarinic agonists which stimulate preferentially either muscarinic M~-receptors, e.g. McN-A-343 (Goyal and Rattan, 1978; Hammer and Giachetti, 1982) or Mz-receptors, e.g. carbachol (Goyal and Rattan, 1978) and muscarinic antagonists with a preferential affinity for M~-receptors, pirenzepine (Hammer et al., 1980), telenzepine (Eltze et al., 1985; Doods et al., 1987; Tonnaer et al., 1987), trihexyphenidyl and dicyclomine (Giachetti et al., 1986a) were investigated. Further studies were performed with antagonists that have a preferential affinity to cardiac M2-receptors e.g. AF-DX 116 (Giachetti et al., 1986b), gallamine (Birdsall and Hulme, 1985) and himbacine (Anwar-ul et al., 1986) or to smooth muscle M2-receptors, e.g. 4-diphenylacetoxy-N-methylpiperidine methiodide (4DAMP; Barlow and Shepherd, 1985) and hexahydrosila-difenidol (Lambrecht et al., 1984; Mutschler and Lambrecht, 1984). These data were compared with those obtained from experiments on the rat isolated left atrium. The results now presented suggest that modulatory presynaptic muscarinic Ma- and postsynaptic

Mz-receptors are located in the rabbit vas deferens and that they are concerned with the inhibition or enhancement of neurogenic contractions, respectively.

2. Materials and methods 2.1. Organ bath-suspended rabbit vas deferens

Male New Zealand white rabbits (2.5-3.0 kg) were killed by exsanguination after the animals had been anaesthetized with pentobarbital sodium (60 m g / k g i.v.) and the vasa deferentia were removed. The organs were carefully dissected free of surrounding tissue and were divided into four segments, two prostatic portions of 1 cm and two epididymal portions of approximately 1.5 cm length. Each tissue was folded in two and fixed vertically by means of a hook-shaped platinum electrode and a cotton thread connected to a forcedisplacement transducer in a 10 ml water-jacketed organ bath. A second platinum ring electrode was placed at the top of the bathing fluid which consisted of (mM): NaCI 118.0, KC1 4.7, CaC12 • 2HzO 2.5, M g S O 4 . 7 H 2 0 0.6, K H 2 P O 4 1.2, N a H C O 3 25.0 and glucose 11.1; 10 - 6 M yohimbine was included to block a2-adrenoceptors. This solution was maintained at 31°C and was continuously bubbled with 95% 02-5% CO 2. The tension of the preparations was set at 0.75 g and they were left to equilibrate for 30 min before the continuous field stimulation (0.5 ms, 30 V, 0.05 Hz) was started. The contractions of eight preparations run in parallel were measured isometrically (K-30, Hugo Sachs Elektronik) and recorded on multichannel recorders (Kipp and Zonen, BD 9). 2.1.1. Response curves to agonists Cumulative concentration-response curves to muscarinic receptor agonists were obtained following the stabilization of the contractile response to electrical field stimulation. Agonists were added directly to the bath and the concentration was increased as soon as there appeared a stable response to the previous concentration.

207 The apparent potency of an agonist was expressed by its - l o g ECs0 value, i.e. the - l o g of the molar concentration that had an effect equal to 50% of the individual maximal effect. The maximal response was related to carbachol (relative maximal effect = 1.00) tested on the same organ as a reference.

2.1.2. Effect of antagonists Four preparations were used in any one experiment to determine reproducible concentration-response curves for McN-A-343 (10 - 7 - 2 x 10 -6 M) and four for carbachol (10 - s - 5 × 10 -7 M). The agonist concentration was raised in steps of 0.3 log units. Subsequent curves were then obtained at 45 mm intervals, the antagonist being added 15 rain prior to the addition of an agonist. The antagonist concentration was also increased so that the effects of more than one concentration could be measured on one organ. The inverse procedure was employed in one case (McN-A-343 against telenzepine). Agonist concentration-response curves were obtained and single concentrations of the agonist were applied after a wash-out period and step-wise increasing antagonist concentrations were then added to reverse the effect of the agonist. Approximately 1 h was required before apparent equilibrium was reached. The ECs0 values of MeN-A-343 in the presence of telenzepine were thus determined from the shifts of individual concentration-response curves produced by two or three successive additions of telenzepine. Schild plots for competitive antagonism were made from the dose ratios (x) of the agonist obtained for different antagonist concentrations [B] to estimate the pA 2 value with confidence limits and the slope of the regression line from each individual organ segment (Arunlakshana and Schild, 1959). The regression line for all organs was fitted by a conventional least squares method (Waud and Parker, 1971). The point estimator (mean) and its 95% confidence limits were calculated on the log-transformed scale. A second approach was used in which the slopes of the regression lines in the Schild plots were constrained to 1. The use of the constrained slope is more consistent with the competitive theory with connects

pA 2 with - l o g K B, provided that the slope does not differ significantly from unity (Tallarida et al., 1979). In those cases where the slope of the Schild plot differs significantly from unity, pA 2 values determined from constrained regression lines should be regarded as a purely experimental quantity. Statistical significance was assumed when P ~<0.05.

2.2. Rat isolated left atrium The affinity of drugs to cardiac muscarinic receptors was determined on electrically driven left atrium of the rat by means of the displacement of concentration-response curves for the negative inotropic effect of carbachol as described in detail by Eltze et al. (1985). Antagonist affinity (pA 2) was determined as described above.

2.3. Drugs Telenzepine dihydrochloride, 4,9-dihydro-3methyl-4-[(4-methyl-l-piperaTJ nyl)-acetyl]-10Hthieno-[3,4-b][1,5]benzodiazepin-10-one (Byk Gulden); pirenzepine dihydrochloride (Boehringer Ingelheim); guanethidine sulfate (Ciba); bretylium tosylate (Wellcome); pilocarpine chloride (Merck); prazosin (Pfizer); trihexyphenidyl hydrochloride (Serva); oxotremorine sesquifumarate (Ega-Chemie); AHR-602, N-benzyl-3-pyrrolidyl acetate methobromide (A.H. Robins, Richmond, USA); secoverine hydrochloride (Philips-Duphar); McNA-343, 4-[m-chlorophenylcarbamoyloxy]-2-butynyltrimethylammonium chloride (RBI, Wayland, USA); dicyclomine hydrochloride (Merrell Dow); RS-86, spiro(l-methyl-4-piperidyl)-N-ethyisuccinimide hydrobromide (Sandoz). Isoarecoline hydrochloride, arecaidine propargyl ester hydrobromide (APE) and hexahydro-sila-difenidol hydrochloride (HHSiD) were the generous gifts of Prof. G. Lambrecht, University of Frankfurt, Germany. Himbacine hydrochloride was kindly donated by Prof. W.C. Taylor, Department of Organic Chemistry, University of Sydney, Australia. 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide and AF-DX 116, l l-(2-[(diethylamino)methyl]-lpiperidinylacetyl)-5,-11-dihydro-6H-pyrido-(2.3-b)(1.4)-benzodiazepin-6-one) were synthesized in our

208 chemistry d e p a r t m e n t . All other purchased from Sigma (Munich).

drugs

were

3. Results 3.1. Field-stimulated rabbit vas deferens

the contractions. O n l y higher c o n c e n t r a t i o n s of D M P P (10 -4 M) caused a tonic c o n t r a c t i o n which could be almost completely i n h i b i t e d by prior a d d i t i o n of 10 -5 M prazosin. This could have resulted from the release of n o r a d r e n a l i n e from a site not further investigated in this study. T h e results of these experiments are s u m m a r i z e d in table 1.

3.1.1. Characterization of the neurogenic contraction Electrical field s t i m u l a t i o n of the organ baths u s p e n d e d r a b b i t vas deferens elicited i n d i v i d u a l phasic c o n t r a c t i o n s of the ' r a p i d twitch' type which were reproducible for more than 6 h. T e t r o d o t o x i n (3 x 10 -8 - 10 -7 M), g u a n e t h i d i n e (10 -6 - 10- 5 M) a n d b r e t y l i u m (2 x 10-5 M) caused a dose-dep e n d e n t i n h i b i t i o n of the twitch contractions, which was not seen with the ganglion blocking agent h e x a m e t h o n i u m (10 -6 - 3 x 10 - 4 M). This f i n d i n g supports the c o n c l u s i o n that the contractions resulted from postganglionic adrenergic nerve s t i m u l a t i o n a n d can therefore be considered as n e u r o g e n i c responses. T h e absence of nicotinic receptor activation was c o n f i r m e d by the inability of the ganglion s t i m u l a n t , 1,1-dimethyl-4-phenylp i p e r a z i n i u m ( D M P P , 10 -6 - 10 -5 M), to modify

TABLE 1 (A) The effect of different drugs on electrically induced twitch contractions of the rabbit vas deferens. (B) Carbachol-induced potentiation of contractions elicited by KCI, noradrenaline (NA) or ATP is unstimulated preparations. The values are given as the means 5: S.E.M. for 6-8 experiments in each case.

3.1.2. Effect of antagonists on neurogenic contractions T h e selective a t - a d r e n o c e p t o r a n t a g o n i s t , prazosin (10 - 8 - 10 -6 M), had no detectable effect on n e u r o g e n i c contractions. Low c o n c e n t r a tions (3 x 10 -8 - 3 x 10 -7 M) of the stable a n a logue of a d e n o s i n e 5 ' - t r i p h o s p h a t e , a , f l - m e t h y l e n e - A T P slightly p o t e n t i a t e d the c o n t r a c t i o n s ; this p o t e n t i a t i o n could be blocked by prior a d d i t i o n of prazosin (10 -6 M). Higher c o n c e n t r a t i o n s of a,flm e t h y l e n e - A T P (10 -6 - 2 x 10 -6 M) almost completely abolished the twitch response in all segments of the organ (table 1). This suggests that A T P is released by nerve s t i m u l a t i o n a n d elicits the contractile response u n d e r these experimental conditions.

3.1.3. Effect of muscarinic agonists on the neurogenic contractions McN-A-343 ( 1 0 - 7 - 2 x 10 ..6 M) caused a d o s e - d e p e n d e n t i n h i b i t i o n of the twitch response. The i n h i b i t i o n stabilized 5 m i n after each successive c o n c e n t r a t i o n a n d was readily reversible on washing (fig. 1). The four p o r t i o n s of each vas

(A) Electrically stimulated preparations Drug

Concentration

% inhibition

Tetrodotoxin Guanethidine Bretylium Hexamethonium Physostigmine DMPP Prazosin

3 x l 0 -s M 2.5 x 10- 6 M 2 x 10- 5 M 3 x 10-4 M 10 -6 M 10 -5 M 10 - 6 M 10 - 6 M

80:1:9 50 + 8 85 4-7 No inhibition No inhibition No inhibition No inhibition 81 -t-4

a,fl-Methylene-ATP

(B) Unstimulated preparations Combination of drugs

% potentiation

KCI (25 mM) after carbachol (10 - 7 M) 180=1=11 NA (10- 5 M) after carbachol (5 × 1 0 - 8 M) 101 + 11 ATP (10 -3 M) after carbachol (5 × 10 -8 M) 83+14

t Carbachol~

I MeN-A-343

Fig. 1. Representative tracing showung the potentiating effect of carbachol (10 - 8 - 2 × 10 -7 M) and the inhibitory effect of McN-A-343 (10 .-7- 2 × 10 -6 M) on contractions induced by field stimulation in a prostatic portion of the rabbit vas deferens. Arrows indicate the period of drug administration and washout.

209

Rabbitvas deferens Car bach 0._._..~[

200"

¢o

150.

~§ -o =

/ I

/~didyn~31

end

~'~,

I00

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Fig. 2. Concentration-response curves of the potentiating effect of carbachol (above) and the irdaibitory effect of McN-A-343 (below) on contractions to field stimulation in the epididymal (r-I) and the prostatic end (©) of the rabbit vas deferens. The results are means ±S.E.M. for 12 separate experiments with 6 rabbits.

deferens used showed a steady increase in sensitivity to McN-A-343 from the prostatic ( - l o g ECs0 = 6.08; 5.65 - 6.50) to the epididymal end of the organ ( - l o g EC~0 = 6.25; 5.92 - 6.58; fig. 2). This difference was significant (means with 95% confidence limits, n = 21; P ~< 0.01, two-sided). Carbachol (10 -8 - 5 × 10 -7 M) caused a concentration-dependent potentiation of the twitch contraction in response to field stimulation with a maximal effect in most cases at 2 x 10 -7 M (figs. 1 and 2). Higher concentrations (10 -6 M) reduced twitch amplitude to levels lower than those recorded in the control solution. The enhancement of contractile responses was more pronounced in the epididymal end, the maximal potentiation (144%) being 3.3 times higher than that obtained in the prostatic end (fig. 2), although the force developed in the control solution was greater with the prostatic (1.35 + 0.07 g) than with the epididymal (0.84 + 0.09 g) organ ends (means + S.E.M., n = 40). The - l o g ECs0 for carbachol, however, did not differ significantly between the epididymal (7.33; 7.08 - 7.65) and the prostatic (7.17; 6.82 7.52) organ ends (means with 95% confidence limits, n = 12). All the other muscarinic agonists tested produced a rapid and marked potentiation of the twitch response of the rabbit vas deferens. The

Rabb)tvasdeferens

i,oo

I

",o

< i

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~

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g

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Fig. 3. Concentration-response curves for the potentiating effect of muscarinic agonists on contractions of the field-stimulated rabbit vas deferens. All responses are expressed as a percentage of the maximal response to carbachol tested on the same organ. The results are means + S.D. for 15-32 muscles prepared from 8 animals for carbachol and 4 animals for all other drugs.

210 TABLE 2 Apparent potency of muscarinic receptor agonists to enhance contractions of the field-stimulated, isolated rabbit vas deferens. ECs0 values were calculated from single dose-response curves related to their individual maximal effect. Responses were related to the concentration-response curve of carbachol with its maximal effect at 2× 10 - 7 M (relative maximal effect =

1.00).

Drug

- log EC~0 (M) median (95% confidence limits)

Relative maximal effect mean -1-S.D.

n

APE Oxotremorine dI-Muscarine Carbachol Arecoline RS-86 Bethanechol Pilocarpine Isoarecoline AHR-602

7.88 (7.51, 8.26) 7.84 (7.44, 8.25) 7.67(7.14, 8.19) 7.34 (6.97, 7.71) 6.86 (6.51, 7.22) 6.41 (5.99, 6.82) 5.76 (5.24, 6.29) 5.59(5.18,6.01) 5.42 (5.14, 5.70) 4.07 (3.87, 4.32)

0.77 ±0.21 0.75 +0.12 1.03 + 0.22 1.00 1.03 + 0.23 1.01 +0.18 1.35 + 0.24 1.25+0.17 1.40+0.29 0.72+0.12

15 16 16 32 16 15 16 16 16 16

p o t e n t i a t i n g effect was d e m o n s t r a b l e at threshold c o n c e n t r a t i o n s of 3 x 10 -9 M from A P E a n d o x o t r e m o r i n e , 1 0 - 8 M for c a r b a c h o l , d l - m u s c a r i n e a n d a c e t y l c h o l i n e whereas c o n c e n t r a t i o n s of 3 x 10 -8 M arecoline, 10 -7 M RS-86, 3 x 10 -7 M b e t h a n e c h o l , 1 0 - 6 M p i l o c a r p i n e and isoarecoline a n d 10 -5 M A H R - 6 0 2 were necessary to elicit any d e t e c t a b l e p o t e n t i a t i o n (fig. 3). W i t h the exception of acetyicholine, which o n l y caused a short-lasting effect, all o t h e r d r u g s p r o d u c e d a m o r e sustained p o t e n t i a t i o n . T h e t w i t c h - e n h a n c i n g effect of all the m u s c a r i n i c agonists m e n t i o n e d was readily reversible on washing. T h e d a t a are s u m m a r i z e d in table 2. N o n e of the agonists tested had a n y effect on the baseline tension o f the organs at c o n c e n t r a tions which m a r k e d l y inhibited o r p o t e n t i a t e d the twitch c o n t r a c t i o n s (not illustrated).

3.1.4. Site of action of muscarinic agonist-induced inhibition or potentiation of contractions T h e influence o f various drugs on M c N - A - 3 4 3 i n h i b i t i o n a n d c a r b a c h o l p o t e n t i a t i o n was investigated in o r d e r to d e t e r m i n e the localization of the different m u s c a r i n i c a g o n i s t - i n d u c e d effects on n e u r o g e n i c contractions. A t r o p i n e ( 1 0 - 9 _ 1 0 - ~ M) d o s e d e p e n d e n t l y a n t a g o n i z e d M c N - A - 3 4 3 inhibition and carbachol potentiation whereas

h e x a m e t h o n i u m (3 x 10 -.4 M), p h y s o s t i g m i n e (10 -6 M) a n d p r a z o s i n (10 -6 M) h a d no effect on either the basal twitch r e s p o n s e ( t a b l e 1) or the inhibition d u e to M c N - A - 3 4 3 a n d the p o t e n t i a t i o n d u e to c a r b a c h o l (not illustrated). T h e s e two agonists h a d no o b s e r v a b l e effect on u n s t i m u l a t e d p r e p a r a t i o n s ( n o t illustrated). C a r b a c h o i (5 × 1 0 - ~ 2 x 10 -7 M) p o t e n t i a t e d the c o n t r a c t i o n s elicited in u n s t i m u l a t e d p r e p a r a t i o n s by exogenous KCI (25 mM), n o r a d r e n a l i n e (10 -5 M) a n d A T P (5 x 10 - 4 - 10 -3 M; table 1) whereas M c N A-343 (10 - 6 - 3 x 10 --6 M) did n o t m o d i f y the responses to these m y o t r o p i c agents (not illustrated).

3.1.5. Interaction between McN-A-343 and carbachol T h e p o s s i b i l i t y that M c N - A - 3 4 3 a n d c a r b a c h o l s t i m u l a t e s e p a r a t e receptors was tested b y e x a m i n ing the effect of one agonist in the presence of the other. C o n c e n t r a t i o n s of M c N - A - 3 4 3 (6 x 10 7 M) which d e p r e s s e d twitch c o n t r a c t i o n s by a p p r o x i m a t e l y 50% had no influence on the s h a p e of the s u b s e q u e n t c a r b a c h o l d o s e - r e s p o n s e curve. Similarly, the response to M c N - A - 3 4 3 r e m a i n e d unaltered in p r e p a r a t i o n s in which c o n t r a c t i o n a m p l i t u d e had been e n h a n c e d by 10 .7 M c a r b a c h o l (not illustrated).

3.2. Effect of antagonists T h r e s h o l d c o n c e n t r a t i o n s of 2 x 10 -8 M p i r e n z e p i n e caused parallel shifts to the right of c o n c e n t r a t i o n - r e s p o n s e curves to M c N - A - 3 4 3 but 30-fold higher c o n c e n t r a t i o n s (6 x 1 0 - 7 M) o f the a n t a g o n i s t were necessary for a similar degree of d i s p l a c e m e n t of c a r b a c h o l c o n c e n t r a t i o n - r e s p o n s e curves. T h e m a x i m a l effect of c a r b a c h o l was even p o t e n t i a t e d b y this selective M l - a n t a g o n i s t (fig. 4). T h e slopes of the regression lines for b o t h agonists in the Schild plots d i d not differ significantly from unity (table 3) b u t the p A 2 value for p i r e n z e p i n e against M c N - A - 3 4 3 (7.64) was 1.45 log units higher than against c a r b a c h o l (6.19), i n d i c a t i n g a preferential i n h i b i t i o n of the response to M c N - A - 3 4 3 . T h e converse was true for the cardioselective M 2 - a n t a g o n i s t A F - D X 116, which was a s t r o n g e r a n t a g o n i s t of the response to c a r b a c h o l t h a n to M c N - A - 3 4 3 (fig. 5). T h e difference in p A 2 values

211 %

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verse procedure was used in the latter case. Increasing antagonist concentrations were added to organs in which the twitch contraction had been previously inhibited by a single concentration of McN-A-343. The parallel displacement of MeNA-343 concentration-response curves by telenzepine yielded a pA 2 value of 8.61 with linear regression line but the slope (1.49) was significantly different from unity. It can be concluded from the difference in pA 2 values (1.22 log units) for telenzepine against both agonists that telenzepine inhibits the response to MeN-A-343 preferentially. Among the other muscarinic antagonists investigated, trihexyphenidyl, dicyclomine, hexahydro-sila-difenidol and 4-DAMP also inhibited Rabbit vasdeferens

%

° 8,~

i

6

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p Pifenzep,ne

Fig. 4. Cumulative concentration-respons¢ curve for M e N - A 343 (top panel) and carbachol (middle panel) in the rabbit vas

6

,o.S J

deferens in the presence of plrenzepine added 15 rain before the agonist (o, control curve). The figures on the curves refer to - log molar concentrations of pirenzepine. Each point is the mean ± S.D. from a typical experiment with 4 organ segments. Ordinate: ~ of maximal agonist control response. Lower panel: Schild plot for the antagonism by pirenzepine of McN-A-343induced inhibition (r-l) and carbachol-induced potentiation (<3) of contraction. Each point is the mean±S.D, for 16 experiments.

as c a l c u l a t e d f r o m S c h i l d p l o t s was 0.55. G a l l a m i n e also g e n e r a t e d a p A 2 v a l u e t h a t w a s 0.46 log units higher against carbachol than against McNA - 3 4 3 (fig. 7 a n d t a b l e 3). T e l e n z e p i n e (3 × 10 - s - 10 - 6 M ) was a c o m p e titive a n t a g o n i s t o f c a r b a c h o l w i t h a s l o p e for the r e g r e s s i o n line n o t s i g n i f i c a n t l y d i f f e r e n t f r o m u n i t y (fig. 6). T h e effects o f c o n c e n t r a t i o n s o f c a r b a c h o l h i g h e r t h a n 2 × 10 - 7 M w e r e p o t e n t i a t e d b y this M ~ - s e l e c t i v e a n t i m u s c a r i n i c drug. T e n - f o l d l o w e r c o n c e n t r a t i o n s o f t e l e n z e p i n e (3 × 10 - 9 - 5 × 10 - 9 M ) s h i f t e d t h e c o n c e n t r a t i o n - r e s p o n s e c u r v e s for M e N - A - 3 4 3 to t h e r i g h t b u t h i g h e r c o n c e n t r a t i o n s w e r e f o u n d to b e ins u r m o u n t a b l e b y t h e agonist. T h e r e f o r e , a n in-

65

5.5 ,

p McN-A-3/, 3

50"

0

=

~

~.o

8

?

6

5

p Car t~chot

SY

p AF-DX 116 Fig. 5. Cumulative concentration-response curves for M c N - A 343 (top panel) and cazbachol (middle panel) in the rabbit vas

defercns in the presence of AF-DX 116 added 15 rain before the asonist (e, control curve). The figures on the curves refer to - log molar concentrations of AF-DX 116. Each point is the mean ± S.D. from a typical experiment with 4 organ segments. Ordinate: % of maximal agonist control response. Lower panel: Schiid plot for the antagonism by AF-DX 116 of McN-A-343induced inhibition (17) and carbachol-induced potentiation (o) of contraction. Each point is the mcan±S.D, for 16 and 12 experiments, respectively.

212 TABLE 3 Affinities for antagonists at muscarinic receptors in rabbit vas deferens, related to inhibition of either McN-A-343-induced decrease or carbachol-induced potentiation of contraction in response to field stimulation, respectively, pA 2 values (mean with 95% confidence limits) and slopes of the regression (mean :t: S.D.) were calculated from Schild plots and from regression lines whose slopes were constrained to 1 as required by the theoretical model of competitive antagonism (mean + S.E.M.). Drug

Agonist

Selectivity a

McN-A-343

Atropine Secoverine Telenzepine Dicyclomine Pirenzepine AF-DX 116 Trihexyphenidyl Gallamine 4-DAMP Himbacine HHSiD

Carbachol

ratio

pA 2

Slope

n

pA 2

Slope

n

9.16 (8.22, 10.21) 9.46 + 0.09 8.48 (7.75, 9.27) 8.60±0.11 8.61 (7.76, 9.33) 8.86 + 0.06 7.68 (7.25, 8.20) 7.70 _+0.08 7.64 (7.13, 8.19) 7.79 ± 0.10 6.84(6.39,7.30) 6.85 + 0.10 8.79 (8.31, 9.30) 9.02 -t-0.09 5.00 (4.61, 5.42) 5.02 5:0.07 9.06(8.62, 9.52) 9.12 -+0.09 7.94(7.53, 8.38) 8.05 + 0.10 7.88 (7.42, 8.38) 7.92 + 0.07

1.69+0.20 b 1.00 1.18-+0.05 1.00 1.49-+0.05 b 1.00 1.03+0.06 1.00 1.22 + 0.09 1.00 1.03±0.03 1.00 1.25 -+0.03 b 1.00 1.02_+0.05 1.00 1.14+0.02 1.00 1.16-t-0.03 1.00 1.09-t-0.03 1.00

12

8.72 (8.00, 9.50) 9.05 -+0.11 8.41 (7.82, 9.04) 8.49+0.11 7.39 (7.10, 7.75) 7.51 + 0.09 6.17 (5.60, 6.78) 6.15 + 0.10 6.19 (5.93, 6.46) 6.23 + 0.08 7.39(6.85,7.89) 7.40 ± 0.10 7.27 (6.95, 7.62) 7.44 4- 0.08 5.46 (4.96, 6.01) 5.43 + 0.08 8.10(7.81, 8.39) 8.16 + 0.10 8.07 (7.59, 8.56) 8.06 + 0.09 6.71 (6.04, 7.45) 6.54 4- 0.07

1.68_+0.02 b 1.00 1.095-0.10 1.00 1.19_+0.04 1.00 1.02+0.09 1.00 1.07 + 0.04 1.130 1.00+0.04 1.00 1.20+ 0.04 ~' 1.00 0.91 +0.02 1.00 1.08+0.02 1.00 1.00_+0.08 1.00 0.86+0.07 1.00

16

12 20 16 16 16 12 19 12 12 19

12 14 12 16 12 12 12 12 12 19

2.8 2.6 1.2 1.3 16.6 22.4 32.4 35.5 28.2 36.3 0.29 0.28 33.1 38.0 0.35 0.39 9.1 9.1 0.74 0.98 14.8 24.0

" Ant±log of the difference between the pA 2 values against McN-A-343 and carbachol, b Slope significantly different from unity (P ~<0.05).

the McN-A-343 response preferentially. The diff e r e n c e in t h e p A 2 v a l u e s o f t h e s e a n t a g o n i s t s a g a i n s t b o t h a g o n i s t s i n d i c a t e s a 33-, 32-, 15- a n d 9-fold selectivity for receptors stimulated by M c N - A - 3 4 3 , r e s p e c t i v e l y , w h e r e a s a t r o p i n e , secoverine and himbacine proved to be nearly e q u i p o t e n t a g a i n s t b o t h a g o n i s t s (fig. 7 a n d t a b l e

i n v o l v e d h a v i n g t h e s l o p e s o f all r e g r e s s i o n fines c o n s t r a i n e d t o 1, i r r e s p e c t i v e o f t h e b e s t fit. T h e pA/values thus become a better estimation of the a f f i n i t y c o n s t a n t s ; t h e v a l u e s o b t a i n e d a r e inc l u d e d in t a b l e 3.

3).

3.2.1. Assessment of selectivity of antagonists for muscarinic receptors

None of the antimuscarinics tested had any effect on the control twitch contractions at concentrations that antagonized either McN-A-343 inhibition or carbachol potentiation. S e v e r a l a n t a g o n i s t s , e.g. a t r o p i n e a n d t e l e n z e p i n e , h a d s t e e p s l o p e s in t h e S c h i l d p l o t w h e n M c N - A - 3 4 3 w a s u s e d as t h e a g o n i s t . A s e c o n d approach, designed to better assess the pA 2 values related to a theoretical competitive antagonism,

T h e d i f f e r e n t d e g r e e t o w h i c h M~, c a r d i o s e l e c tive M 2 or unselective antimuscarinic drugs block M e N - A - 3 4 3 r e s p o n s e s p r e f e r e n t i a l l y in r e l a t i o n t o c a r b a c h o l r e s p o n s e s o f t h e r a b b i t v a s d e f e r e n s is s h o w n in fig. 8. D i c y c l o m i n e , t r i h e x y p h e n i d y l a n d pirenzepine showed a near equal selectivity with a f a c t o r o f 36 t o 38 f o r a p r e f e r e n t i a l a f f i n i t y to Ml-receptors followed by hexahydro-sila-difenidol with a factor of 24 and telenzepine with a factor

213

%

Rabbit vas deferens I00-

3.3. Comparison of antimuscarinic drugs as antagonists of carbachoi in rabbit vas deferens and

Telenzepine://~

rat left atrium

o

~

~

~ p McN-A-343

I0050-

0

75 7

$

.)

6.5

6 p ~oJ'bachol

2.0-

In order to further specify the subtype of muscarinic receptors involved in the potentiating effect of carbachol in the rabbit vas deferens, the present results were compared with data obtained from a cardiac tissue. Least-squares regression analysis was used for the purpose. Table 4 summ a r i z e s t h e pA 2 values for different antimuscarinics from constrained Schild plots relating to carbachol-induced negative inotropy of rat left atrium. A highly significant positive correlation was calculated between the pA 2 values for ten antimuscarinic drugs acting on rabbit vas deferens

x

--Rabbd

1.0

1.0¸ o

vas deferens

20

~ /.

~

,~,

÷

. o

p Telenzepine

Fig. 6. Top panel: cumulative concentration-response curve for MeN-A-343 in the rabbit was deferens (O, control curve) and curves obtained by adding increasing concentrations of telenzepine ( 3 x 1 0 - 9 - 2 x 1 0 - s M) to single organ segments in which the contractions had been reduced previously by fixed concentrations of MeN-A-M3 ( 5 x 1 0 - 7 - S X 1 0 - s M). The control curve is from a typical experiment with 6 organ segments from one animal. Middle panel: cumulative concentration-response curves for carbachol in the presence of telenzepine added 15 mm before the agonist (e, control curve). The figures on the curves refer to - l o g molar concentrations of telenzepine. Each point is the mean+S.D, from a typical experiment with 4 organ segments. Lower panel: Schild plot for the antagonism by telenzepine of McN-A-343-induced inhibition (rt) and carbachoi-induced potentiation (o) of contraction. Each point is the mean+S.D, for 20 and 14 experiments, respectively.

of 22. However, the value for telenzepine is probably underestimated because of the long time needed to reach equilibrium with this drug at the Ml-receptor. 4-DAMP was also M:selective by a factor of 9. Atropine, socoverine and himbacine proved to be non-selective. AF-DX 116 and gallamine showed weak but definite selectivity for M2-receptors in this tissue.

Z/

1.0

x

0 20.

HlmbOcl/

10 0

0: Goomo.// ck

Fig. 7. Schild plots for the antagonism by trihexyphenidyl,. dicyclomine, himbacine and gaUamine of McN-A-343-induced inhibition (n) and carbachol-induced potentiation ( o ) of contractions in rabbit was deferens. Each point is the mean + S.D. for n experiments given in table 3.

214 Rabbit vas deferens

Antilo( of c, pA lOO

THP 30

Pit

Dic HHSiD Tel

t.-D

Atr

n

Sec

Fq

H-UI

0.3.

AF- DX

Fig. 8. Selectivity ratios of various muscarinic receptor antagonists of M 1- and M2-receptors in the rabbit vas deferens (THP = trihexypbenidyl, Pir = pirenzepine, Dic = dicyclomine, HHSiD = hexahydro-sila-difenidol, Tel = telenzepine, 4-D = 4-DAMP, A t r = atropine, Sec = secoverine, Him = himbacine, Gall = gallamine, AF-DX = AF-DX 116). MeN-A-343 and carbachol were employed as preferential stimulants of M l- and M2-receptors, respectively. Selectivity was expressed as the antilog of the difference in pA 2 values of one antagonist against the two agonists (table 3, calculations for constrained slope = 1).

and the pA 2 values from rat left atrium (r = 0.979,

0.1 o s

~0

SlOp 108 f 9>Tus

the muscarinic receptors mediating enhancement of neurogenic contractions by carbachol in rabbit vas deferens are predominantly of the cardiac M2_subtype"

:t .~ = ,~ "=

4. Discussion

The present investigation showed that, with the exception of the muscarinic agonist, MeN-A-343, all the other cholinomimetics tested were more or less preferential for M2-receptors, e.g. APE, carbachol and arecoline, and potentiated the contractions caused by field stimulation of the isolated rabbit vas deferens. Both inhibition by MeN-A-343 and potentiation by carbachol appeared to be mediated by muscarinic receptors

~

o

I

s

o

, 6

, ~ pA 2

b

10

Rat left atr)um

Fig. 9. Correlation of pA 2 values for different anttmuscarinic drugs listed in table 4 and derived from the inhibition of carbachol-induced negative inotropy on rat left atrium versus antagonism of the carbachol-potentiating effect on rabbit vas deferens (tables 3 and 4, calculations for constrained slope = 1).

215 TABLE 4 Drug antagonism at muscafinic receptors of rat left atrium related to carbachol-induced decrease in tension development. pA 2 values (mean with 95~$ confidence limits) and slopes of the regression (mean 5: S.D.) were calculated from Schild plots and from regression lines whose slope were constrained to 1 as required by the theoretical model of competitive antagonism (mean 5: S.E.M.). Drug

pA 2

Slope

Atropine

8.86 (8.74, 8.99) a 8.92+0.11 7.32 (7.13, 7.42) • 7.32 + 0.09 6.46 (6.33, 6.61) a 6.34 + 0.10 7.89 (7.36, 8.38) 7.86 + 0.10 5.66 (5.30, 5.96) 5.645:0.10 6.63 (6.19, 7.11) 6.46 + 0.09 7.12 (6.79, 7.46) 7.00 + 0.09 6.43 (6.12, 6.77) 6.32 + 0.09 7.45 (7.06, 7.86) 7.365:0.11 8.32 (7.94, 8.70) 8.20 5:0.08

1.10 + 0.09 1.00 0.995:0.10 1.00 0.89+0.11 1.00 0.97 + 0.03 1.00 0.96 + 0.03 1.00 0.80±0.02 1.00 0.85 ±0.04 1.00 0.85 5:0.05 1.00 0.87 5:0.02 1.00 0.87 5:0.05 1.00

Telenzepine Pirenzepine 4-DAMP Gallamine Dicyclomine A F - D X 116 HHSiD Trihexyphenidyl Himbacine

n 5-11 6-11 7-9 8 8 b

8

b

8

b

7

b

8 11

• Data taken from Eltze et al. (1985). b Slope significantly different from unity (P ~ 0.05).

since they could be antagonized competitively by atropine. Hexamethonium and physostigmine failed to modify the effects of McN-A-343 and carbachol. Therefore a ganglionic site of action or an indirect mechanism releasing endogenous acetylcholine can be excluded, at least for the effects of McN-A-343 and carbachol. These two agonists had no observable contractile effect on unstimulated organs. McN-A-343 did not modify the contractions elicited in the unstimulated preparation by KCI, noradrenaline and ATP added exogenously whereas carbachol potentiated the responses of all myotropic agents. Therefore, it is most likely that McN-A-343 acted presynaptically to modulate transmitter release whereas the major site of action of carbachol seems to be postsynaptic. The reduction of neurotransmitter release by acetylcholine results in most tissues, from an interaction with presynaptic muscarinic receptors

(Starke, 1981). The opposite effect, i.e. increased neurotransmitter release, is generally seen after nicotinic receptor activation (LiSffelholz, 1979). However, in the experiments presented here, carbachol and McN-A-343 produced no contraction of the unstimulated rabbit vas deferens. The possibility of an interaction of these agonists with nicotinic receptors is also not supported by the observation that a high concentration of the gan# i o n stimulant, DMPP (10 -4 M), was necessary to elicit a tonic contraction susceptible to blockade by prazosin. The potentiation by carbachol of the contractions to field stimulation was more pronounced in the epididymal end of the organ, an effect which also has been observed in rat vas deferens (Lee, 1985). Whether this effect can be explained by the weaker basal contractility observed in this segment of the organ cannot yet be explained. Transmission in the epididymal segment of the rat vas deferens is predominantly adrenergic (McGrath, 1978; MacDonald and McGrath, 1980). Theoretically, the potentiating effect of carbachol in the rabbit vas deferens could result from an additional release of noradrenaline that further enhances the twitch response. However, prazosin did not alter the potentiating effect of carbachol in either the epididymal or prostatic end of the vas deferens. The potency of agonists to enhance twitch contractions in the rabbit vas deferens preparation was, in general, similar to that observed at peripheral M2-receptors, e.g. rat ileum, oxotremorine > muscarine > pilocarpine > AHR-602 (Brown et al., 1980) and rat bladder, oxotremorine > muscarine > carbachol (Grana et al., 1987) or guinea-pig left atrium, carbachol > bethanechol > pilocarpine (Eglen et al., 1987). APE and oxotremorine, although acting as partial agonists related to carbachol, were the strongest potentiating agonists in the rabbit vas deferens, their relative potency being similar to that found in rabbit ear artery, oxotremorine = APE > carbachoi > pilocarpine > AHR-602 (Choo et al., 1986). The difference in potency between APE and carbachol in rabbit vas deferens (-log ECs0 = 7.88 vs. 7.34) was similar to that reported for rat atrium (7.72 vs. 7.06) by Mutschler and Lambrecht (1984).

216

Although RS-86 has been described as being even more selective in binding experiments for M~-receptors than the putative Ml-agonist, McNA-343 (Tonnaer et al., 1987), it only caused potentiation of twitch contraction. In addition to McN-A-343 (Goyai and Rattan, 1978; Hammer and Giachetti, 1982; Mutschler and Lambrecht, 1984), pilocarpine (Van Charldorp et al., 1985; Caulfield and Stubley, 1982) and AHR-602 (Franko et al., 1963) have been suggested as preferential Ml-agonists, at least in vivo experiments. However, neither pilocarpine nor AHR-602 inhibited the twitch contractions of the rabbit vas deferens. The explanation for this observation could be that either their selectivity is not great enough or their efficacy is too low to stimulate M~-receptors in this preparation. In addition, the effective receptor reserve (Kenakin, 1986) is frequently higher in vivo than in vitro and agonists with low efficacy, such as pilocarpine and AHR602 appear to be selective (Eglen and Whiting, 1986). These discrepancies could also be explained by the fact that the rabbit vas deferens has two different muscarinic receptors mediating opposite actions. The maximal enhancement of contractions by one agonist does not reflect its true 'intrinsic activity' because it must be assumed that there is concomitant stimulation of McN-A-343sensitive, inhibitory muscarinic receptors at higher concentrations, which may attenuate the potentiation. Therefore, only the term 'relative maximal effect' was used for the agonists. Similarly, the ECso values calculated from these concentrationresponse curves do not precisely reflect the affinities of the agonists to a distinct receptor but can only be regarded as an approximate measure of potency. Several lines of evidence suggest that the effects of McN-A-343 and particularly carbachol are mediated by different muscarinic receptors. (1) The most convincing evidence is that the two have opposite effects. Furthermore, McN-A-343 had no influence on the shape of the following carbachol concentration-response curve and vice versa. (2) According to receptor theory, receptor subtypes must be postulated to exist if one antagonist exhibits different affinities against various agonists (Furchgott, 1972). The present investigation pro-

vides, by means of this approach, convincing evidence that the muscarinic receptors in rabbit vas deferens are heterogenous. The clearest evidence for postulating different muscarinic receptor subtypes was obtained with selective Ml-antagonists, e.g. pirenzepine (Hammer et al., 1980; Watson et al., 1982), dicyclomine, trihexyphenidyl (Giachetti et al., 1986a) and telenzepine (Eltze et al., 1985: Doods et al., 1987; Tonnaer et al., 1987) which differentiate muscarinic receptor subtypes through their markedly higher affinity for the Ml-receptor. These drugs show a 22- to 38-fold greater ability to antagonize McN-A-343-induced inhibition of twitch contraction than to affect the carbachol responses in rabbit vas deferens. Alternatively, AF-DX 116 and the neuromuscular blocking agent gallamine appear to be useful pharmacological tools since they preferentially antagonize functional responses mediated by carbachol at the cardiac M2-subtype. Drugs like atropine and secoverine show no selectivity. Their small affinity difference indicates the known inability of these compounds to discriminate between muscarinic receptor subtypes. The same is true for himbacine. The affinity difference calculated from pA 2 values of known antimuscarinics therefore justifies the classification of the receptors involved as M 1 and M 2•

Interestingly, an increase in the maximal response to carbachol occurred after rightward displacement of the concentration-response curves by Ml-receptor antagonists, e.g. pirenzepine, telenzepine and dicyclomine. The enhancement may be explained by the blockade of inhibitory M~-receptots activated by carbachoi concentrations higher than 2-5 × 10 -7 M which, under these conditions can no longer counteract the potentiation. Most of the antagonists tested against both agonists generated slopes in the Schild plots not significantly different from unity. The regression lines were straight for at least 1.5 log units, comprising four different antagonist concentrations. However, atropine exhibited steep slopes against both agonists. The same was true for telenzepine against McNA-343 and to a lesser degree for trihexyphenidyl against both agonists. It remains unclear in the case of the unselective drug, atropine what factor(s) disturbed the simple 1:1 relation of the agonist

217

and antagonist in competing for muscarinic receptors. The unselective nature of the antagonist was probably not responsible, since neither secoverine nor himbacine showed a similar slope deviation. However insufficient equilibration time could account for this phenomenon. If the interaction of the drugs with their respective receptors is ratelimiting in this particular tissue, lower concentrations of antagonist would require a longer time to reach equilibrium than higher concentrations (Kenakin, 1982). The effect of telenzepine was found to be insurmountable by MeN-A-343 when administered before the agonist. Therefore, the reverse procedure was employed to reverse the effect of the agonist. More than 1 h was required even under these conditions to arrive at an apparent equilibrium of telenzepine and a stepwise reversal of the agonist effect. Thus, the anomalous kinetic behaviour of telenzepine at M~-receptors may be responsible for the steep slope of the Schild plot. This has also been shown in radioligand receptor binding studies, where the binding of [3H]telenzepine at the M~-receptor slowly reached equilibrium at 30°C ( - 2 h) and dissociation from this receptor was also remarkably slow (t~/2 = 140 rain at 30 o C; Sehudt et al., 1988). In the present experiments, the heterogeneous receptor population could disturb simple competitive antagonism, possibly leading to mixed responses. There is no reason to assume that such an effect influenced pA 2 determinations of antagonists against both agonists, since most of the antagonists investigated had Schild plot with slopes not significantly different from unity. However, this situation seems to dominate if the agonist, carbachol, after stimulation of M2-receptors at lower concentrations, activates M~-receptors at high concentrations. The inhibition of the latter interaction by selective M~-antagonists leads to potentiation of the control response to carbachol. Recent studies with the M2-antagonist AF-DX 116 suggest that the M2-muscarinic receptors are a heterogenous group of receptors rather than a single subtype (Hammer et al., 1986; Giachetti et al., 1986b). AF-DX 116 has high affinity to M2-receptors in the heart, low affinity to those in exotrine glands and medium affinity in smooth muscle

(Batink et al., 1986). Its M2(cardiac)/M1 selectivity is poor, ranging from 1.6 (Whiting et al., 1987) to 6.8 (Hammer et al., 1986). Similarly, himbacine and gallamine clearly differentiate between cardiac and smooth muscle M2-receptors, both drugs showing an approximately 10-fold higher affinity to cardiac receptors (Anwar-ul et al., 1986; Clark and Mitchelson, 1976). The inverse is true for the smooth muscle selective muscarinic antagonists, 4-DAMP (Barlow et al., 1976) and hexahydrosila-difenidol (Lambrecht et al., 1984; Mutschler and Lambrecht, 1984). Gallamine has been shown to discriminate between pre- and postjunctional muscarinic receptors in the canine saphenous vein, displaying a 25-fold higher affinity for the prejunctional receptor classified as M 2 than to the postjunctional M~-receptor to which pirenzepine has a 40-fold higher affinity (O'Rourke and Vanhoutte, 1987). Thus, the higher affinity of gallamine for M 2 than for M~-receptors in the rabbit vas deferens supports the view of its selectivity. It should not be forgotten that gallamine, besides its possible allosteric interaction with the muscarinic agonist binding sites also blocks nicotinic receptors and a variety of ionic channels (for a recent review, see Mitchelson, 1987). Secoverine was an equally potent antagonist of responses to McN-A-343 and carbachol, confirming both the results obtained with this drug in the rabbit ear artery (Choo et al., 1986) and the data on postsynaptic muscarinic receptors in the guinea-pig left atrium, rat bladder and guinea-pig or rat ileum (Choo and Mitchelson, 1985). Secoverine has been shown to be selective for muscarinic receptors in intestinal smooth muscle compared to those in glandular tissue (Zwagemakers and Claassen, 1981) but it does not discriminate between M1- and Mz-receptors in binding experiments (Tonnaer et al., 1987; Nilvebrant and Sparf, 1986). The presumed smooth muscle M2-selective antagonist 4-DAMP was 9-fold more potent against the responses to MeN-A-343 as compared to those to carbachol. This finding is in accordance with binding studies on rat cortical membranes in which this drug exhibited selectivity for the pirenzepine-sensitive binding site as compared

218

to the myocardium (Berrie et al., 1983; Tonnaer et al., 1987). The same holds true for hexahydrosila-difenidol, which discriminates between M1and cardiac M2-binding sites with a factor of 17 (Ladinsky et al., 1987). The potency of 10 muscarinic antagonists to inhibit the carbachol potentiating effect in rabbit vas deferens proved to be linearly correlated with the activity of the drugs to inhibit negative inotropy in rat left atrium. These results we now obtained strongly indicate that the receptors mediating these particular responses are similar and support the hypothesis that a cardiac-like M2-receptor subtype mediates the action of carbachol in rabbit vas deferens. There is pharmacological evidence suggesting that the motor innervation of the rabbit vas deferens is not solely adrenergic. ATP and noradrenaline are released as co-transmitters from the sympathetic nerves of the vas deferens of several species (Sneddon et al., 1984). The initial short-lasting twitch contraction is not antagonized by the a~-adrenoceptor antagonist, prazosin, but is susceptible to blockade by the P2-purinoceptor antagonist, ANAPP 3. Prazosin has the opposite effect in that it reduces only the secondary tonic response to motor nerve stimulation (Sneddon et al., 1984). This was also evident from the present study as the stable ATP analogue a,fl-methyleneATP abolished the neurogenic twitch contractions in the rabbit vas deferens. Thus, it is probable that muscarinic receptor agonists exert their potentiating effect on twitch contractions in this organ through enhancement of 'non-adrenergic' transmission, as has been shown in the rat vas deferens (Lee, 1985). The present results suggest that both presynaptically located M 1- and postsynaptically located M2-receptors regulate non-adrenergic transmission in rabbit vas deferens. The ability of carbachol to potentiate not only the contractions elicited by ATP but also the responses to noradrenaline and KCI added exogenously to unstimulated preparations, points to a more generalized increase in postsynaptic sensitivity. This increase could possibly occur via facilitation of the depolarization of the postsynaptic membrane, as has been suggested for the guinea-pig vas deferens (SjiSstrand, 1973).

The nature of the muscarinic receptor subtype involved in the inhibition of noradrenaline release has been studied in the perfused rat heart (Fuder et al., 1982) and the rabbit ear artery (Choo et al., 1986). Also pirenzepine displayed a relatively low affinity to prejunctional muscarinic receptors in the rat heart as well as in the canine saphenous vein (O'Rourke and Vanhoutte, 1987), thus suggesting the presence of an M2-subtype. The pA 2 values for pirenzepine showed that inhibition of acetylcholine and noradrenaline release in the submucous plexus neurones of the guinea-pig ileum was also due to activation of presynaptic M2-receptors (Surprenant, 1986). The presence of presynaptically located inhibitory Ml-receptors in the rabbit ear artery (Choo et al., 1986) has been suggested. Such receptors could be of physiological importance, as discussed by Wanke et al. (1987). The results of the present investigation with rabbit vas deferens are an additional example of the existence of functional Ml-muscarinic receptors on adrenergic nerve terminals inhibiting transmitter release. In summary, the rabbit vas deferens preparation is an excellent model in which muscarinic M land M2-receptor selectivity of antagonists can be determined concomitantly with different agonists. Thus, it is a unique system in which Ml-receptors seem to be located on adrenergic nerve terminals and M2-receptors on a postsynaptic site. These receptors mediate opposite effects on neurotransmission.

Acknowledgements The author gratefully acknowledges the skillful technical assistance of Mrs. H. K6nig, Miss E. Helmlinger, Miss C. Biirkle, Mrs. B. Ullrich and Miss. S. Conzelmann. He also wishes to thank Dr. M. Galvan for improving the English and Mrs. G. Langenstein for carefully typing the manuscript.

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