European Journal of Pharmacology, 179 (1990) 159-165 159 Elsevier

FJP 51236

Selective antagonists provide evidence that M 1 muscarinic receptors may mediate carbachol-induced drinking in the rat

1 2 2 3 Carlo Polidori , Maurizio Massi I, Günter Lambrecht , Ernst Mutschier , Reinhold Tacke and Carlo Melchiorre 4 1 Institute of Pharmacology, University of Camerino, 1-62032 Camerino ( MC). ltaly, 1 Institute of Pharmacology, University of Frankfurt, Theodor-Stern-Kai 7, D-6000 Frankfurt/ M. F.R.G., 3 Institute of lnorganic: Chemistry, University of Karlsruhe, Engesserstr., D-7500 Karlsruhe, F.R.G.• and 4 Department of Pharmaceurical Sciences, Universily of Bologna, Via Belmeloro 6, 1-40116 Bologna, lta/y

Reccived 7 December 1989, accepted 9 January 1990

The present study served to investigate the ability of seven selective muscarinic antagonists to inhibit carbachol-in­ duced drinking in the rat. The muscarinic antagonists were given by intracerebroventricular (i.c.v.) injection 1 min before the i.c.v. injection of carbachol (1 p.gjrat). The M 2 antagonist, methoctramine, was inactive up to 80.3 nmoljrat. The M3 antagonist, p-fluoro-hexahydro-sila-difenidol, elicited a modest (42%) but statistically significant inhibition of drinking only at 80 nmoljrat. On the other band, the selective M1 antagonists, (R)-trihexyphenidyl, o-methoxy-sila-hexocyclium and pirenzepine, produced a marked and dose-dependent inhibition of carbachol-induced drinking, their 1050 values being 0.51. 7.36 and 9.31 nmoljrat. Also the M1/M 3 antagonists, 4-diphenylacetoxy-N­ methylpiperidine methiodide and hexahydro-sila-difen.idol, were potent inhibitors of carbachol-induced drinking, their

I 0 50 values (0.28 and 11.09 nmoljrat) being related to their pA 2 values for M1 receptors in rabbi t vas deferens. These data suggest that carbachol-induced drinking may be mediated by activation of muscarinic M1 receptors.

Carbachol-induced drinking; Muscarinic receptor antagonists; Muscarinic receptor subtypes;

Muscarinic M1 receptors; Musearlnie M2 receptors; Muscarinic M3 receptors

1. Introduction muscanmc receptors. On this basis, these recep­ tors have been classified into at least three sub­ The first indication of muscarinic receptor het­ types: M (Hammer and Giachetti, 1982), M erogeneity was derived from studies on agonist 1 2 (M , Mutschier et al., 1988; M A, Eglen and binding (Birdsall et al., 1980). However, agonist 20 2 Whiting, 1986) and M (M ß, Mutschier et al., binding curves are difficult to interpret in terms of 3 2 1988; M28, Eglen and Whiting, 1982) according to receptor subtypes, owing to the complexity of Doods et al. (1987). Pirenzepine shows higher agonist-receptor interaction, involving both differ­ affinity for M1 receptors than for the M2 and M 3 ent affinity states and coupling proteins. The dis­ types (Hammer and Giachetti, 1982), methoctra­ covery of antimuscarinic compounds which are mine displays high selectivity for the M 2 type selective for certain tissues or organs provided a (Melchiorre et al., 1987; Melchiorre, 1988; in press; more reliable approach to the subclassification of Michel and Whiting, 1988), whereas hexahydro­ sila-difenidol and 4-diphenylacetoxy-N-methyl­

Correspondence to: C. Melchiorre, Departmenl of Phanna­ piperidine methiodide (4-DAMP) show higher af­ ceulicaJ Sciences, University of Bologna, Via Belmeloro 6, finity for M 3 and M 1 types than for M 2 receptors 40126 Bologna, ltaly. (Eitze and Figala, 1988; Mutschier et al., 1988;

0014-2999/90/$03.50 ® 1990 Elsevier Science Publishers B.V. (Biomedical Division) 160

Barlow et al., 1976). More recently, Lambrecht et dihydro-11-[( 4-methyl-l-piperazinyl)-acetyl]-6H­ al. (1988a, b, c) have developed the highly selec­ pyrido[2,3-b](1.4)benzodiazepin-6-one} dihydro­

tive M 3 muscarinic antagonist, p-fluoro-hexahy­ chloride and (R)-trihexyphenidyl hydrochloride dro-sila-difenidol, as weil as the very potent and were kindly supplied by Dr. A. Giachetti and Dr.

selective M1 muscarinic antagonists, o-methoxy­ A.J. Aasen, respectively; methoctramine {N .N '­ sila-hexocyclium and (R)-trihexyphenidyl. bis[6-[[(2-methoxybenzyl]amino]hexyl}-1,8-octane­ The centrat administration of acetylcholine diamine tetrahydrochloride, 4-DAMP ( 4-diphenyl­ agonists not only has several effects on the regu­ acetoxy-N-methylpiperidine methiodide), hexa­ lation of body fluids and of blood pressure in the hydro-sila-difenidol [cyclohexylphenyl(3-piperi­ rat: Stimulation of water intake, inhibition of salt donopropyl) silanol hydrochloride], p-fluoro­ appetite, release of vasopressin but in addition, hexahydro-sila-difenidol [cyclohexy(4-fluoro­ produces a neuregenie hypertensive response (for phenyl)(3-piperidinopropyl)silanol] and o-meth­ review see Fitzsimons, 1979; Simpson, 1986). The oxy-sila-hexocyclium ( 4-{ [cyclohexylhydroxy(2- understanding of the functional relationships be­ methoxyphenyl)silyl]methyl} -1, 1-dimethylpipera­ tween these effects can only be understood if the zinium methyl sulfate) were all synthesized in our receptor subtype involved is known. Selective laboratories. pharmacological manipulations of these functions also can only be carried out with knowledge of the 2.3. Surgery and intracranial injections subtype of receptor involved in each of the above All the animals were anesthetized (Equithesin, mentioned effects. On the basis of the available 3 mljkg body weight i.p.) and fitted by Stereotaxie evidence that drinking induced by acetylcholine surgery with a stainless-steel cannula (o.d. 600 agonists is mediated by muscarinic receptors p.m) aimed at 1 mm above the lateral ventricle. (Fitzsimons, 1979). and taking advantage of the The guide cannula was attached to the skull by variety of selective muscarinic antagonists men­ means of jewelry screws and dental acrylic ce­ tioned above. we designed the present study to ment. The rats were aiJowed one week to recover investigate the subtype of muscarinic receptor in­ from surgery before testing began. volved in carbachol-induced drinking. An earlier The drugs to be tested were dissolved in sterile investigation bad provided indications that M 2 isotonic saline, with the exception of hexahydro­ receptors may not be involved (Massi et al., 1989) sila-difenidol and p-fluoro-hexahydro-sila-difen­ in the response. idol that were dissolved in distilled water. being slightly soluble in isotonic saline. The drugs were 2. Materials and methods given by intracerebroventricular (i.c.v.) injection through a stainless-steel injector (o.d. 300 p.m) 2.1. Anima/s temporarily inserted into the guide cannula and protruding 2 mm beyond the cannula tip. This Male albino Wistar rats (Charles River, Calco technical expedient was adopted in order to re­ Corno, Italy) averaging 250-275 g at the beginning duce the possibility of back-flow of the solutions of the experiments were housed individually in a into the guide cannula. All the drugs were given in temperature-controlled room on a 12: 12 Iight­ a constant volume of 1 p.l, except hexahydro-sila­ dark cycle. Food in pellets (MILL, Morini, Reggio difenidol and p-fluoro-hexahydro-sila-difenidol Emilia, ltaly) and tap water were available ad that were given in a 2-1-ll volume. libitum. 2.4. Experimental pro<:edure 2.2. Drugs 2.4.1. Effect of muscarinic antagonists on carbachol­ The following drugs were used: carbachol induced drinking (carbamoylcholine chloride) (Merck), (Ile 5 ]angio­ Carbachol {lp.gjrat) was administered by i.c.v. tensin II (Peninsula Lab. Inc. ); pirenzepine {5,11- injection to water-replete rats 1 min after the i.c.v. 161 administration of a muscanmc antagonist or of 100 vehicle (controls). After carbachol injection, the animals were retumed to their home cages where water and food were freely available. Water intake was recorded over a 2-h period of observation. For each antagonist, the experiment was carried out 50 according to a 'within subjecf design, in which each rat received all the doses of antagonists as weil as the vehicle used for the antagonist itself. The rats were tested twice a week. Different groups of animals were used for the different antagonists.

0.1 10 2. 4. 2. Effect of muscarinic antagonists on angioten­ sin 11-induced drinking Fig. 1. Percent inhibition induced by differ~nt doses of 4- (Ilc 5]Angiotensin II was given by i.c.v. injection DAM P, ( R)-trihexyphenidyl (THP). o-methoxy-sila-hexo­ cyclium (o-MeSiHC), pirenzepine (PZ). hexahydro-sila-difen­ to water-replete rats at the dose of 100 ngjrat. idol (HHSiD), p-fluoro-hexahydro-sila-difenidol (pFHHSiD) Again, muscarinic antagonists or simple vehicle and methoctrarnine (Metho) of drinking induced by carbachol were administered i.c.v. 1 min before (lle5)angio­ (1 JLg/rat). The values are means ± S.F..M. for 6-10 subjects. tensin II administration. Water intake was re­ The inhibitions reported were determined at 120 min after corded for 60 min after the second i.c.v. injection. carbachol administration. Difference from controls (vehicle + carbachol): • P < 0.05; •• P < 0.01; where not indicated, the Only one dose of antagonist, the highest tested difference from controls was not statistically significant. versus carbachol-induced drink.ing, was used. Each animal was also tested with the appropriate vehlcle. ranged between 8 and 12 ml of water;rat in the different experimental groups. 2. 5. Statistical analysis Figure 1 shows the dose-response relationships for the inhibitory effect of the muscarinic The data are reported as means ± S.E.M. Stat­ antagonists on carbachol-induced drinking. To istical analysis of data for each compound tested compare the effects of these antagonists. water was done by multifactorial analysis of variance intake was expressed as percent of that of the (repeated measures) to check the overall signifi­ controls and doses are reported in nmoljrat. cance of drug treatment. Planned pairwise com­ The potency (expressed as -log 10 ) of the parisons were carried out by means of t-tests. 50 seven antagonists tested to inhibit carbachol-in­ Statistical significance was set at P < 0.05. duced drinking is reported in table 1. In the range of doses tested, none of the muscarinic antagonists evoked evident behavioural alterations that might 3. Results have prevented the animal from drinking. As seen in previous experiments (Massi et al.,

3. 1. Effect of muscarinic antagonists on carbacho/­ 1989), the selective M 2 antagonist, methoctra­ induced drinking mine, produced a very slight inhibition of carbachol-induced drinking, in the range of doses Carbachol, given by pulse i.c. v. injection at a from 3.3 to 80.3 nmoljrat (2.5-60 p.gjrat). The dose of 1 p.gjrat, induced copious drinking within overall analysis of variance revealed the absence 1 or 2 min following injection. Most of the dipso­ of a statistically significant drug effect, together genic response was expressed in the first 60-min with the absence of drug-time interaction. In the period. However, additional drinkingwas also de­ present study 60 p.g/rat was the highest dose of tected in several animals in the 2nd h of observa­ methoctramine tested, as we observed that admin­ tion. The overall water intake in the 2-h period istration of 100 p.gjrat plus carbachol 1 p.g pro- 162

TABLE 1

Comparison of -log 1050 values (with 95% conridence Iimits (c.l.)) of muscarinic antagonists tested on carbachol-induced drinking. with their pA 2 values at M1 (rabbit vas deferens), M 2 (guinea-pig left atria) and M3 (guinea-pig or rat ileum) receptors. Data taken from • Eltze and Figala (1988), b Melchiorre et al. (1987), c Lambrecht et al. (1988a). d Lambrecht et al. (1988c). c Lambrecht et al. 1 (1988b), r Eltze (1988), and Eglen and Whiting (1986). h Binding affinities (pKi values) at M1 /M 2 /M 3 receptors, (R)-tri­ hexyphenidyl: 9.0j7.7j8.1; 4-DAMP: 9.1/8.1/8.9 (Waelbroeck et al., 1988; 1989)

Antagonist -log 1050 pA2 (95% c.l.) Mt M2 MJ Methoctramine < 7.1 6.85. 8.13 h 5.69 b p-Fluoro-hexahydro-sila-difenidol < 7.1 6.68 c 6.01 c 7.84 c Pirenzepine 8.03 (7.63; 8.44) 8.24c 6.82 c 6.88 c Hexahydro-sila-difenidol 7.96 (7.41; 8.50) 7.92 c 6.53 c 7.96 c o-Methoxy-sila-hexocycü um 8.13 (7.98; 8.28) 8.31 d 6.41 d 6.96 d (R)-Trihexyphenidyl h 9.29 (7.90; 10.68) 10.11 c 8.15 e 8.68 c 4-0AMP h 9.55 (9.19; 9.90) 9.12 r 7.90 I 9.0 I

duced heavy prostration~ followed by episodes of was tested in the range of doses of 0.1, 1 and 10 intense locomotor Stimulation often ending in ro­ nmoljrat (0.03-3.37 1-Lg/rat). The inhibitory effect tations along the rat's longitudinal axis. The Mr at the lowest dose tested was significant up to 60 selective antagonist, p-fluoro-hexahydro-sila-di­ min following carbachol injection. The higher do­ fenidol. evoked a slight and statistically not sig­ ses significantly inhibited drinking throughout the nificant inhibition of carbachol-induced drinking 2-h period of observation. The 1050 of the drug at doses up to 40 nmoljrat (15.4 J.Lgfrat)- A was 0.51 nmol (95% c.l.: 0.021-12.51). The M1- significant drug effect was detected only in re­ selective antagonist, o-methoxy-sila-hexocyclium, sponse to 80 nmoljrat (F(1,7) = 14.27; P < 0.01); was about 10 times less potent than (R)-tri­ pairwise comparisons showed that the inhibitory hexyphenidyl. lt did not evoke significant inhibi­ effect was significant throughout the 2-h period of tion of drinking at the dose of 1 nmoljrat (0.47 observation. Even at this dose, however, the in­ J.Lg) but markedly suppressed drinking at 10 and hibition was less than 50%. Higher doses were not tested because of problems related to drug solubil­ ity. On the other band, the five other muscarinic antagonists tested evoked a marked suppression of carbachol-induced drinking, the mostpotent being 12

4-DAMP and (R)·trihexyphenidyl. The overall 10 ..::.. analyis of variance revealed a powerful effect of e -a 4-DAMP in the range of doses tested (F(3,15) = 1.11 lliC 6.93; P < 0.005). Following the lowest dose of 0.06 ....~ e nmoljrat (31 ngjrat) the intake of treated rats ~ 4 wa~. lower, but statistically indistinguishable from Ge 1.11 that of the controls_ The inhibitory effect. on the ~ 2 other band, was very pronounced and statistically ~ significant at the dose of 0.276 and 1.10 nmoljrat. 0 0 15 30 120 The time course of the drinking inhibition induced TIME. min by 4-DAMP is shown in fig. 2. The 10 of the Fig. 2. Timecourse of drinking inducd by carbachol (1 #'g/rat) 50 following pretreatment with isotonic saline (ISO) or with dif­ drug on carbachol-induced drinking was 0.288 ferent doses (nmoljrat) of 4-DAMP. Difference from controls: nmol (95% confidence Iimits (c.l.): 0.127-0.650). • P < 0.05: • • P < 0.01; where not indicated the difference The selective M1 antagonist. (R)-trihexyphenidyl, from controls was not statistically significant. 163

100 nmoljrat. The 1050 of o-methoxy-sila-hexo­ after i.c.v. administration of each of the seven cyclium was 7.36 nmol (95% c.l.: 5.19-10.45). The muscarinic antagonists tested was very similar and

M 1-selective antagonist, pirenzepine, was slightly statistically indistinguishable from that of control less potent than o-methoxy-sila-hexocyclium. In rats receiving vehicle only (table 2). the range of doses tested, 1.17-94.2 nmol/rat (0.5- 40 pgjrat), it produced a highly significant effect (F(4,20) = 36.64; P < 0.0001). The effect was sig­ 4. Discussion nificant throughout the 2-h period of observation even at the dose of 1.17 nmol. The I 0 50 of It is important to emphasize first that none of pirenzepine was 9.31 nmol (95% c.l.: 3.66-23.67). the musearlnie antagonists tested produced a sig­ Finally. hexahydro-sila-difenidol proved to be es­ nificant inhibition of [Ile5 ]angiotensin 11-induced sentially as potent as pirenzepine, its 1050 being drinking, even at the highest doses employed, 11.09 nmol (95% c.l.: 3.15-39.03). versus carbachol-induced drinking (table 2). This finding provides evidence that the inhibition of 3.2. Effect of muscarinic antagonists on angiotensin carbachol-induced drinking that we observed can­ 1/-induced drinking not be related to general behavioural impairment induced by the drugs. but is indeed the expression To exclude the possibility that the muscarinic of a selective interaction with the cholinergic antagonists tested might have inhibited carbachol­ mechanism controlling drinking. induced drinking by producing a behavioural im­ Concerning the characterization of the receptor pairment, the same drugs were tested on angio­ subtype involved in carbachol-induced drinking, tensin-induced drinking. For each drug we tested the present results suggest that M1 receptors, rather the highest dose used versus carbachol-induced than other subtypes of muscarinic receptors, ap­ drinking. Angiotensin I I evoked a very prompt parently mediate the response. The possibility that dipsogenie response, within 15-60 s. Most of the M 2 receptor are involved is ruled out by the drinking occurred in the first 15 min after i.c.v. inactivity of the M 2-selective muscarinic antago­ injection and was usually complete in the first 30 nist, methoctramine, in our test. This finding is in min in most of the rats treated. The water intake keeping with our previous results (Massi et al., 1989), as weil as with the report of Schiavone et al.

(1989) who used the M2 antagonist. AF-DX 116. TABLE 2 Moreover, the selective M 3 antagonist. p-fluoro­ Cumulative water intake in 60 min following i.c.v. injection of hexahydro-sila-difenidol, slightly inhibited drink­ (lle5)angiotensin II (100 ngjrat) in rats pretreated with ing only at the very high dose of 80 nmoljrat, musearlnie antagonists (doses in nmoljrat) or with simple arguing against the involvement of an M 3 recep­ vehicle tor. Finally. the observation that, as reported in Antagonist (dose) Water intake (mljrat) a table I. the five antagonists that inhibited Vehicle Antagonist carbachol-induced drinking effectively showed a good correlation between inhibitory potency in Methoctramine (80.3) 16.0±2.1 21.2 ± 1.6 p-Fluoro-hexahydro-sila- our test and affinity for the M1 receptor in rabbit difenidol (80) 15.5 ± 1.2 17.2 ± 2.1 vas deferens is direct evidence in favour of the Pirenzepine (94.2) 14.2± 1.6 16.6 ± 1.6 mediation of the effect by an M 1 receptor. In fact: Hexahydro-sila-di fenidol (a) pirenzepine, hexahydro-sila-difenidol and o­ (108.6) 17.0±2.0 14.2± 3.5 methoxy-sila-hexocyclium have pA values for M o-Methox y-sila-hexocyclium 2 1 (100) 16.1 ± 2.8 15.1±1.6 receptors clearly higher than those of methoctra­ ( R)-Trihexyphenidyl (1 0) 14.1 ± 3.2 17.2± 1.0 mine and of p-fluoro-hexahydro-sila-difenidol, 4-DAMP (1.1) 17.1 ±2.5 15.2± 2.0 which were clearly less potent to inhibit a The difference between vehicle- and antagonist-treated rats carbachol-induced drinking; (b) pirenzepine, never reached statistical significance. hexahydro-sila-difenidol and o-methoxy-sila- 164

hexocyclium, which have very similar pA 2 values p-fluoro-hexahydro-sila-difenidol, which gave (from 7.92 to 8.31), proved to be essentially clear-cut support to our hypothesis of the involve­ equipotent in ouT test; (c) 4-DAMP and (R)-tri­ ment of M1 receptors in cholinergic-induced hexyphenidyl, which have pA 2 values foT M 1 Te­ drinking. ceptors higheT than those of the pTeviously men­ tioned antagonists, weTe also moTe potent in ouT test. lndeed, 4-DAMP pToved to be slightly more Acknowledgements potent than (R)-tTihexyphenidyl although its affin­ ity foT M1 Teceptors is about one order of magni­ Supported by a grant from Regione Marche ( Ricerca Sani­ tude loweT in functional studies (table 1). How­ taria. 1986). Wc would like to thank Dr. A. Giachetti (Menarini. ever, in binding experiments using NB-OK 1 cells, Florence) and Dr. A.J. Aasen (University of Oslo) for gifts of pirenzepine and (R)-trihexyphenidyl, respectivcly. 4-DAMP and (R)-trihexyphenidyl competed equipotently with N-methylscopolamine at M 1 re­ ceptors (pKi = 9.1 and 9.0, respectively) (Wael­ References broeck et al., 1988; 1989). The pA 2 values for M 2 and M 3 receptors of the seven antagonists tested Barlow, R.B., K.J. Berry, P.A.M. Glcnton, N.M. Nikolaou and aTe not related to theiT potency for inhibition of K.S. Soh, 1976, A comparison of affinity constants for carbachol-induced drinking (table 1). muscarine-sensitive acetylcholinc receptors in guinea pig The conclusion from the present study is not atrial pacemaker cells al 29°C andin ileum at 29°C and consistent with that drawn from a previous one 37°C, Br. J. Pharmacol. SR, 613. Birdsall, N.J.M .• A.S.V. Burgen, R. Hammer, E.C. Hulme and (Hagan et al., 1987) which suggested that M 2 J. Stockton, 1980. Pirenzepine, a Iigand with original bind­ andjor M 3 receptors would mediate cholinergic ing properties to muscarinic receptors, Scand. J. Gastroen­ drinking in the rat. Indeed, the results in this terol. 15 (Suppl. 66). 1. report are not strictly comparable to ours, since Doods, H.N., M.-J. Mathy, D. Davidesko. K.J. Van Charldorp. these authors injected theiT substances diTectly A. Oe Jonge and P.A. Van Zwieten, 1987, Selcctivity of musearlnie antagonists in radioligand and in vivo experi­ into the lateral hypothalamus adjacent to the peri­ mcnts for the putative M1• M2 and M 3 receptors, J. fornical Tegion, while our drugs were injected into Pharmacol. f.xp. Ther. 242, 257. the ventricular space. PTobably, following i.c.v. Eglen. R.M. and R.L. Whiting. 1986, Muscarinic rcceptor injection, drugs Teach only to a slight extent the subtypes: A critique of the current classification and a lateral hypothalamus while they can act at other proposal for a working nomenclaturc, J. Autonom. Pharmacol. 5, 323. sites such as the subfornical oTgan, which is very Elt:r.e, M .. 1988, Muscarinic M1- and M 2-reccptors mediating sensitive to the dipsogenie action of carbachol opposite cffects on neuromuscular transmission in rabbit and, also is easily accessible fTom the ventTicular vas dcferens, European J. Pharmacol. 151, 205. space. HoweveT, the different conclusion reached Eltze, M. and V. Figala. 1988, Affinity and selectivity of might have been a consequence of differences in biperidcn enantiomcrs for muscarinic rcceptor subtypes, European J. Pharmacol. 158, 11. pharmacological tools employed. In fact, Hagan et Fitzsimons, J.T., 1979, The Physiology of Thirst and Sodium al. (1987) based their conclusions largely on the Appetite (Cambridge University Press, Cambridge). findings obtained with muscarinic agonists, while Hagan, J.J., J.A.D.M. Tonnaer and C.L.E. Brockkamp. 1987, we used only antagonists. Our choice was related Cholinergic Stimulation of drinking from the lateral hypo­ thalamus: indications for M muscarinic rcceptor mcdia­ to the fact that putative M agonists exhihit low 2 1 tion. Pharmacol. Biochem. Behav. 26. 771. efficacy and behave as partial agonists in a range Hammer, R. and A. Giacheni, 1982, Muscarinic recepor suh­ of pharmacological and biochemical tests. Dif­ types: M1 and !'vt 2 • Biochemical and functional characteri­ ferences in efficacy due to variations in intrinsic zation, Life Sei. 31, 2991. activity or efficiency of receptor coupling, may Lambrecht, G., R. Feifel, B. Forth, C. Strohmann, R. Tacke give a false impression of Teceptor specificity. and E. Mutschler, 1988a, p-Fiuoro-hex.ahydro-sila-difen­ idol: the first M 2 p-sel~tive muscarinic antagonist. Europe­ MoTeover, our study involved a greater variety of an J. Pharmacol. 152. 193. antagonists, including the most recent and selec­ Lamhrecht, G., R. Feifel, U. Moser, A.J. Aasen, M. tive M 2 and M 3 antagonists, methoctramine and Waelbroeck. J. Cristophe and E. Mutschler, 1988b, Stereo- 165

selcctivity of the enantiomers of trihexyphenidyl and its Mutschler. E., U. Moser. J. Wess and G. Lambrecht, 19RR, methiodide at muscarinic reccptor subtypes, European J. ~ew approaches to the subclassification of muscarinic re­ Pharmacol. 155, 167. ceptors, in: Rccent Advanccs in Receptor Chemistry, eds. Lambrecht, G., G. Gmelin, K. Rafeiner, C. Strohmann. R. C. Melchiorre and M. Giannella (Eiscvier Scicnce Pub­ Tacke and E. Mutschler. 1988c, o-Methoxy-sila-hexo­ lishers B.V., Amsterdam) p. 195.

cyclium: a new quaternary M 1-selectivc muscarinic antago­ Schiavone, A., A. Prudentino and R. !>Aicheletti, 1989, nist, Europcan J. Pharmacol. 151. 155. Muscarinic reccptor subtypes involved in bethanechol-in­ Massi, M .. C. Polidori. and C. Melchiorre. 1989, Methoctra­ duced watcr intakc in thc rat, J. Pharm. Pharmacol. 41, 71.

mine, a selective M 2,. musearlnie receptor antagonist, does Simpson, J.B., 1986, The nature and localization of central not inhihit carhachol-induced drinking in thc rat. European rcceptor systems, in: The Physiology of Thirst and Sodium J. Pharmacol. l 63. 387. Appetite, eds. G. De Caro. A.N. Epstcin and M. Massi Melchiorre. C.. 1988, Polymethylene tetramines: a new gencra­ (Plenum Press. New York) p. 309. tion of selective muscarinic antagonists, Trends Pharmacol. Waclbroeck, M., J. Camus, M. Tastenoy and J. Christophe, Sei. 9, 216. 1988, 80% of musearlnie receptors expressed by thc N 8-0K Mclchiorre. C.. Polymethylene tetraamines: a novcl class of 1 human ncuroblastoma cell line show high affinity for

cardioselectivc M 2-antagonists, Med. Res. Rev. (in press). pirenzepine and are comparable to rat hippocampus M1 Melchiorre, C., A. Cassinelli and W. Quaglia, 1987. Differen­ receptors, FEBS Let t. 226, 28 7. tial blockadc of muscarinic receptor subtypes hy polymeth­ Waelbrocck, ~.. M. Tastenoy, J. Camus, R. Feifel. E. ylene tetraamines. Novel class of sclectivc antagonists of Mutschler. C. Strohmann, R. Tacke, G. Lambrecht and J. cardiac M-2 muscarinic rcceptors. J. Med. Chem. 30, 201. Christophc. Stercoselectivity of the interaction of muscarinic Michel. A.D. and R.L. Whiting, 1988, Methoctramine. a poly­ antagonists with their reccptors. Trends Pharmacol. Sei. 10 methylcne tctraaminc, differentiales three suhtypcs of (Suppl.) p. 65. muscarinic receptor in direct bindig studies, European J. Pharmacol. 145. 61.