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A comparison of the antimuscarinic effects of pirenzepine and N-methylatropine on ganglionic and vascular muscarinic receptors in the rat
Life Sciences, Vol. 35, pp. Printed in the U.S.A.
553-560
Pergamon
Press
A COMPARISON OF THE ANTIMUSCARINIC EFFECTS OF PIRENZEPINE AND N-METHYLATROPINE ON GANGLIONIC AND VASCULAR MUSCARINIC RECEPTORS IN THE RAT
J. Wess,
G. Lambrecht,
U. Moser
and E. Mutschler
Faculty of Biochemlstry, Pharmacology and Food Chemlstry, Department of Pharmacology, Unlversity of Frankfurt, Theodor-Stern-Kai 7, D-6000 Frankfurt/M, FRG (Received
in final
form May 25, 1984)
Summary The antlmuscarlnlc properties of plrenzepine and N-methylatropine were evaluated in two intact preparations by measurlng A) the inhlbltion of increase in mean arterlal pressure evoked by McN-A-343 in pithed rats through activat±on of ganglionlc muscarinlc receptors and B) the inhibition of fall in arterlal pressure evoked by methacholine in anaesthetized rats through activation of vascular muscarinic receptors. To characterize the antlmuscarinio potencles of pirenzepine and N-methylatropine, for both antagonlsts doses were calculated that produce a lO-fold shlft to the right of the dose-response curves for A) the pressor response to McN-A-343 (1.v. admlnlstration) in plthed rats (Dlo-P.r.) and B) for the depressor effect to methacholine (l.v. administratlon) in a n a e s t h e t i z e d rats (DlO-an.r.) , respectively. Whereas N-methylatroplne was vlrtually equleffectlve In blocking both muscarinlc responses (DlO-an.r./D]o-P.r. ~ i), plrenzepine, however, was c o n s l d e r a b l y more potent at ganglionic than at vascular muscarinic receptors (DlO-an.r./Dlo-P.r. ~ 16). These data confirm the existence of excitatory ganglionlc muscarlnlc receptors with hlgh afflnlty for pirenzeplne (M~) and provlde evidence for the presence ± of M 2 receptors - receptors which show a low sensltlvity to pirenzeplne on vascular smooth muscle cells. To further characterize the anticholinergic properties of plrenzepine, its effect on the pressor response to DMPP, a nicotinic ganglIonlc stlmu]ant, was investlgated in pithed rats. A high dose of pirenzeplne (1.13 Bmol/kg), given l.v., did not affect nicotinlc gangllonlc transmission.
MuscarlnIc M and M receptor subtypes have been postulated based upon i 2 pharmacological studies on the lower esophageal sphlncter (LES) of the opposum (1,2,3). It was suggested that Mr receptors are located on certain neuronal sites being selectively activate~ by McN-A-343, a potent selective muscarlnlC stimulant of sympathetic ganglia (4-7,11,17), while muscarlnlc receptors in the effector organ (smooth muscle of the LES), selectlvely stimulated by bethanechol, were thought to be of the M 2 type. Indlrect
(8-]2)
as well
as
direct
blndlng
studies
0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press
(13-15)
Ltd.
of plrenzeplne
554
Antimuscarinic
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of P ~ r e n z e p i n e
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suggest that th~s n o n - c l a s s i c a l m u s c a r ~ n ~ c a n t a g o n i s t may d ~ f f e r e n t l a t e these p r o p o s e d subtypes. It could be d e m o n s t r a t e d that h~gh affinity s~tes for p ~ r e n z e p l n e (M. sites) are p r e s e n t at c e r t a i n neuronal s t r u c t u r e s as cerebral I cortex and h l p p o c a m p u s of the rat (8,9,13,Ia) and ~n s y m p a t h e t i c and myenter~c g a n g l l a (10-12,15), whereas b ~ n d l n g sites for p l r e n z e p l n e at effector organs such as heart and ileum have been shown to be p r e d o m i n a n t l y of the low a f f l n l t y type (M 2) (8,9,11,13,14). Thls r e p o r t e d s e l e c t i v i t y of p l r e n z e p l n e has also been c o n f i r m e d ~n f u n c t i o n a l tests (2,3,ii,16-21), a l t h o u g h it has been q u e s t i o n e d r e c e n t l y (22,23). The data c~ted above have led to the f o r m u l a t i o n of o p e r a t i o n a l definitions of subtypes of m u s c a r l n ~ c receptors: The M I r e c e p t o r ~s one which shows a h~gh degree of s e n s l r l v l t y to the a n t a g o n i s t p ~ r e n z e p l n e and the agonlst McN-A-Sa3, and the M 2 r e c e p t o r ~s one which shows a low s e n s ~ t ~ v l t y to p ~ r e n z e p ~ n e and McN-A-943, respectlvely. The alm of the present study was to d e m o n s t r a t e the r e p o r t e d M I selectlvlty of p l r e n z e p l n e and to c h a r a e t e r l z e m u s c a r l n l c receptors located on vascular smooth muscle cells using two intact preparatlons. Accordingly, we have d e t e r m i n e d the a b 1 1 1 t y of p l r e n z e p l n e and N - m e t h y l a t r o p l n e A) to inhlblt the increase in arterial pressure In p i t h e d rats evoked by the s e l e c t l v e M agonlst M c N - A - 3 4 3 (i-7,11,17) and B) to block the decrease in arterla~ pressure in a n a e s t h e t i z e d rats c a u s e d by the m u s c a r l n l c agent methachollnc, respectlvely. M e t h a c h o l l n e was found to be nearly e q u l p o t e n t to a c e t y l c h o l l n e In lowering arterlal blood pressure in anaesthetized rats (29), but a c e t y l c h o l l n e was shown to be more potent by a factor of 18 than m e t h a c h o l l n e in d e p o l a r l z l n g rat s y m p a t h e t i c g a n g l l a by s t i m u l a t i o n of m u s c a r l n l c receptors (28). These data indlcate a certain degree of selectlvlty of m e t h a c h o l i n e and led to our choice of thls a g o n l s t in the a n a e s t h e t i z e d rat. Furthermore, the effect of p l r e n z e p a n e on the p r e s s o r response to DMPP, a nlcotlnlc g a n g l ~ o n l c stlmulant, was i n v e s t ± g a t e d in pithed rats. Our data indicate that p l r e n z e p l n e is a selective MI r e c e p t o r a n t a g o n l s t and that m u s c a r l n l c receptors of the M 2 type are present on v a s c u l a r smooth muscle cells. Methods Drugs' The f o l l o w i n g drugs were used: p l r e n z e p l n e d i h y d r o c h l o r l d e (kindly provided by Dr. R. Hammer, K. Thomae), methachollne chloride (Szgma), N - m e t h y l a t r o p i n e nitrate (Merck), DMPP (Fluka), and M e N - A - 3 4 3 (syntheslzed in our laboratory). All drugs were d i s s o l v e d in sallne (0.9%, w/v) and a d m i n i s t e r e d 1.v. in a volume of O.iml/lOOg. Intravenous p r e t r e a t m e n t wlth a n t a g o n l s t s or sallne (controls) was c a r r i e d out twenty minutes before a d m i n i s t r a t l o n of agonlsts. Thls interval was s e l e c t e d because p r e l i m i n a r y e x p e r i m e n t s showed that after this time the a n t a g o n l s t l c effects of p l r e n z e p l n e and N - m e t h y l a t r o p l n e , respectlvely, were constant durlng the wnole experlment. If not stated otherwlse animals served as thelr own controls. No evidence of b a s e l i n e drlft or d e s e n s l t l z a t l o n was observed, when McN-A-3A3 and methachollne, respeczlvely, were r e p e a t e d l y admlnlstered. The intact a n a e s t h e t l z e d rat: Male, n o r m o t e n s l v e White W l s t a r rats (weight 230-300g) were anaesthetized with pentobarbltone-sodlum (60mg/kg, given 1.p.). The left jugular vein was c a n n u l a t e d for the a d m l n l s t r a t l o n of drugs. Arterial blood p r e s s u r e was m e a s u r e d from the c a n n u l a t e d rlght common c a r o t l d artery by means of a S t a t h a m pressure t r a n s d u c e r c o n n e c t e d to a Helllge
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Antimuscarinic
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a m p l l f l e r and a R1kadenkl recorder. After c a t h e t e r l z a t a o n of the trachea, h e p a r i n (300 I.U./kg) was glven i.v. to prevent c o a g u l a t i o n of the blood. Body t e m p e r a t u r e was kept at 37±I°C throughout the experiment. At the b e g l n n l n g of each experiment, the a n t a g o n l s t s or sallne (controls) were admlnlstered. Then d o s e - r e s p o n s e curves for the v a s o d e p r e s s o r effect to m e t h a c h o l l n e were e s t a b l l s h e d untll a fall of 40-50 mm Hg in mean arterlal pressure was achleved. The plthed rat: Rats were a n a e s t h e t i z e d and p r e p a r e d for blood pressure r e c o r d i n g as d e s c r l b e d above. Addltlonally, both vagi were cut at the neck, and the anlmals were pithed wlth a steel rod. A r t l f l c l a l r e s p l r a t l o n was provlded by means of a B r a u n - M e l s u n g e n pump (Iml/lOOg body welght at a rate of 60 strokes/mln). In each anlmal d o s e - r e s p o n s e curves for the pressor effect of M c N - A - 3 4 3 were e s t a b l l s h e d in the absence and in the presence of the a n t a g o n i s t s twenty minutes after thelr admlnlstratlon. Data Analysls: In the a n a e s t h e t l z e d rats doses of m e t h a c h o l i n e that d e c r e a s e d mean arterial pressure by 30 mm Hg in the absence and in the presence of different doses of plrenzeplne and N-methylatroplne were c a l c u l a t e d from the d o s e - r e s p o n s e curves. Mean dose ratlos were d e t e r m i n e d for each a n t a g o n l s t dose and drawn on a log scale along the ordinate in r e l a t i o n to the log a n t a g o n l s t doses on the absclssa. Stralght llnes were obtained, and from these llnes a n t a g o n i s t doses were estimated, which c o r r e s p o n d to a shift of the control d o s e - r e s p o n s e curve by a factor of i0
(DlO-an.r.-values).
In the plthed rats doses of M e N - A - 3 4 3 that increased mean arterial blood pressure by 60 mm Hg were c a l c u l a t e d from the d o s e - r e s p o n s e curves. D n -p.r.values (antagonlst doses that produce a lO-fold shlft to the r l g h t l ~ f the M c N - A - 3 4 3 control d o s e - r e s p o n s e curve) were d e t e r m l n e d for p l r e n z e p l n e and N - m e t h y l a t r o p l n e as described above. For both a n t a g o n l s t s a ratlo D . _ - a n . r . / D . _ - p . r . was c a l c u l a t e d IU IU be c o n s l d e r e d as a measure of thelr r e c e p t o r selectivity. Statistics: Unless stated p r e s e n t e d as arlthmetic means number of e x p e r l m e n t s (n).
which
may
otherwise, the results of the e x p e r i m e n t s ~ standard error of the mean (S.E.M.) and
are the
Results A n a e s t h e t i z e d rat: The inltlal mean arterial blood pressure amounted to iS4t2 mm Hg (n=42). Intravenous injection of m e t h a c h o l l n e (0.125-1.0 ~g/kg) ellclted a d o s e - r e l a t e d depressor effect. As depicted in Fig. i, p r e t r e a t m e n t with pirenzeplne (2.26-22.61 ~mol/kg) as well as N-methylatroplne (0.027-0.273 ~mo!/kg) produced a d o s e - d e p e n d e n t shift of the d o s e - r e s p o n s e curve for the v a s o d e p r e s s o r effect of m e t h a c h o l l n e to the right. Pithed rat: The r e s t i n g mean arterlal pressure of the p l t h e d rats was 51±1 mm Hg (n=50). The intravenous a d m i n l s t r a t l o n of the selective M I agonlst M c N - A - 3 4 3 (25-400 ~g/kg) e l i c i t e d a d o s e - d e p e n d e n t increase in mean arterial pressure. As shown in Fig. 2, p r e t r e a t m e n t with p l r e n z e p l n e (0.068-0.678 ~mol/kg) or N - m e t h y l a t r o p l n e (0.008-0.082 ~mol/kg) a n t a g o n l z e d the pressor effect caused by McN-A-343, shifting its d o s e - r e s p o n s e curve in a parallel fashlon to the right.
556
Antimuscarinic
Selectivity of Pirenzepine
Vol.
35, No. 5, 1984
N -METHYLATROPINE 50
--40 o~
//
E30 E
~/ ffl
~ m
&, / /
/
T/ ~
&/
N-methylatr
~L
• 0027 2 p mol/k g
o,.
Methachohne ( pg/kgj v ) r,r¢ <
PIRENZEPINE 50
Z <
Z
/
u.l
m ~ ! ~ ezOl~me 0
~mo, I/kg
~m
• 2261 )1
1
,,
~Methachohne ( /Jg/kg,=lv~
FIG. i Antagonzsm by N-methylatropzne (upper panel) and pzrenzepzne (lower panel) of m e t h a c h o l z n e - l n d u e e d vasodepresszon ~n anaesthetzzed rats. Data are presented as mean values ± S.E.M. (n=6). TABLE I A Comparlson of the Antlmuscarlnzc Potencies of Pzrenzepzne and N-Methylatropzne in the Pithed and zn the Anaesthetlzed Rat.
a
Plrenzepzne N-Methylatropzne
b
Dio-an.r.
DIO- p,r.
(umol/kg)
(~mol/kg)
6.75 0.057
0.41
0.044
Dio-an.r./DIo- p.r.
16.5 1.3
aDlo-an.r.: Antagonzst dose that corresponds to a shift of the methacholzne ~@-response curve in the anaesthetlzed rat by a factor of I0. -Dlo-P.r.: A n t a g o n l s t dose that corresponds to a shzft of the McN-A-343 doseresponse curve in the plthed rat by a factor of i0.
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120
Antimuscarinic
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of Pirenzepine
557
N-METHYLATROPINE
100
~6o E ~
60
u,l
/ / / ~ m t /
w
.a/
o. 21)
~, LU
001
/ ~
~
01
~" •
/
~_ /
roI ethylatr • 0oo8 .~o,,~g • 9_027 • 0,082 .."
i
lb McN-A-343 (mg/kg,l.v)
OiC ,¢
zlO01
PIRENZEPINE
601 i control Plrenzepme 0068 pmol/kg 0226 0.678 ,," 001
0'1
Ib McN A-343 (rnglkg,lv)
i
FIG. 2 Antagonism by N-methylatropine (upper panel) and pirenzeplne (lower panel) the M c N - A - 3 4 3 - i n d u c e d pressor response in pithed rats. Data are presented means ± S.E.M. (n=6 for inhibition curves, n=18 for control curves).
of as
In separate experiments the effect of a high dose of pirenzepine (1.13 pmol/kg, i.v.) on the pressor response caused by DMPP (100-500 pmol/kg, i.v.) was investigated in pithed rats, in order to exclude the possibility that the blocking activity of pirenzeplne towards the McN-A-343 pressor response might be due to an antinicotinic action. Rats were divided into two groups (n=7), one group receiving saline (controls), the other one pirenzepine (1.13 ~mol/kg, i.v.). Twenty minutes after the administration of saline or pirenzeplne, respectively, dose-response curves for the pressor effect to DMPP were established. No significant effect (p
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Antimuscarinic
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35, No.
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R e c e p t o r - s e l e c t l v l t y of p i r e n z e p l n e and N - m e t h y l a t r o p l n e : In order to o b t a i n D - v a l u e s for p l r e n z e p l n e and N - m e t h y l a t r o p l n e (see under methods), 1 mean log ~ o s e - r a t l o s were p l o t t e d a g a i n s t the a d m l n l s t e r e d a n t a g o n i s t doses on a l o g a r l t h m e t l c scale (Fig. 3), and s t r a l g h t lines were obtalned. It becomes quite obvious from Fig. 3 that N - m e t h y l a t r o p l n e inhlblts both muscarlnic reponses in the same n a r r o w dose-range, while p ~ r e n z e p l n e is c o n s i d e r ably more potent at g a n g l i o n l c sltes. In order to p r e s e n t q u a n t i t a t i v e data, D l o - v a l u e s for N - m e t h y l a t r o p l n e and p l r e n z e p l n e are listed in Table I. The data c o l l e c t e d in Table I show that p l r e n z e p l n e is more than two orders of m a g n i t u d e less potent than N - m e t h y l a t r o p l n e in the a n a e s t h e t i z e d rat, but the p o t e n c y d i f f e r e n c e in the plthed rat being only somewhat less than one order of magnitude. The D 1 0 - a n . r . / D ~ - p . r . - r a t l o shows that under _ IU the d e s c r l b e d e x p e r i m e n t a l condltlons plrenzeplne ~s about 16 tlmes more potent at g a n g l l o n l c than at v a s c u l a r m u s c a r l n l c r e c e p t o r sites. No such d l f f e r e n c e could be found for the c l a s s i c a l a n t l m u s c a r l n l c agent N - m e t h y l atropine (DlO-an.r./Dlo-P.r. -;1).
2N-METHYLATROPIN E O
PIRENZEPINE
o_
/
fv wJ
0
O
o
9
)005
(105 ' N-Methylatroplne(IJmol/kg) 0 5 '
/ ./
Pirenzepme( pmoll kg)
FIG.3 A c o m p a r i s o n of the a n t i m u s c a r l n i c p o t e n c i e s of N - m e t h y l a t r o p l n e (left panel) and p i r e n z e p i n e (rlght panel) in the pithed rat ( ~ , a n t a g o n i s m against the pressor effect caused by McN-A-343) and in the a n a e s t h e t i z e d rat ( D---43, a n t a g o n i s m against the d e p r e s s o r effect caused by methacholine). Lines are least square estimates, and data are p r e s e n t e d as means ± S.E.M. (n=6). Discussion The e l a s s l f l c a t x o n of m u s c a r i n x c r e c e p t o r s most commonly a p p l i e d is based on the d l s o r l m l n a t l v e p r o p e r t i e s of the m u s c a r l n l c a n t a g o n i s t p i r e n z e p l n e and the m u s c a r l n l e agonlst McN-A-343. The t e r m l n o l o g y Cn p r e s e n t use ~s the following: The M r e c e p t o r shows a hlgh degree of s e n s i t i v i t y to p l r e n z e p l n e and can be s e l e c } i v e l y a c t l v a t e d by McN-A-343, whereas the M 2 r e c e p t o r shows low s e n s i t i v i t y to p i r e n z e p i n e and McN-A-343, r e s p e c t i v e l y (1-3,11-15). On the basis of this r e c e p t o r c l a s s l f l c a t l o n our data confJrm the r e p o r t e d M 1 s e l e c t l v l t y of p l r e n z e p l n e and provide evidence for the presence of M 2 receptor sites on vascular smooth muscle cells. It was c o n f i r m e d that p l r e n z e p i n e in a dose-range from 0 . 0 6 8 - 0 . 6 7 8 ~mol/kg, ~.v., is a potent a n t a g o n i s t of M c N - A - 3 4 3 - z n d u c e d increase in mean arterial pressure in pithed rats (ii), a response, which has been shown to be m e d i a t e d by selective a c t l v a t l o n of m u s c a r l n i c (atroplne-sensltzve) sites in
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m a m m a l i a n sympathetlc ganglia (4,6,7,17). On the contrary, high doses of plrenzepine had to be applled (2.26-22.61 ~mol/kg, 1.v.) to achieve a reductlon of the methachollne-evoked decrease in blood pressure ~n a n a e s t h e t i z e d rats. As the depressor effect to methacholine is thought to be mediated by activatlon of muscarlnic receptors on vascular smooth muscle cells resulting in a g e n e r a l l z e d vasodilatation, the antagonistic effect of pirenzepine is suggested to be a consequence of ~ts Interaction wlth these vascular muscarln~c sites. We could demonstrate that vascular muscarinic receptors, s t i m u l a t e d by exogenously a d m i n i s t e r e d methachollne, show low affinity for plrenzeplne. Therefore, these receptors can be classified to be of the M type, but we cannot exclude the simultaneous presence of M sites on vascula~ 1 smooth muscle cells. These flndlngs are in agreement wlth studies on various other effector organs such as 11eum or heart, which have been shown to contain p r e d o m i n a n t l y muscarlnlc receptors of the M 2 type (8,9,11,13,14, 16-21,24-27). Although one has to keep in m~nd that the presented results are in-vIvo data and p h a r m a c o k i n e t i c considerations do not allow a direct quantlficatlon of affinity differences, the observed selectlvlty of plrenzeplne (DlO-an.r./D._-p.r. ~ 16) agrees well with previous findings in functional in-vltro t e s ~ (16) and binding studies (i0,Ii). Brown et al. ~16) demonstrated a 23-fold higher affinity of pirenzeplne for muscarinic receptors in the rat superior cervical ganglion mediating depolarlzatlon than for those present in the rat ileum causing contraction. Accordingly, competition studies on the binding of trltiated N - m e t h y l s c o p o l a m l n e to ganglionic and atrlal muscarinic receptors showed (ii) that the affinities of plrenzeplne for these two receptor sites differ by a factor of about 30. As expected from pharmacologlcal studies (ii,16,28), no such selectivity difference as described for pirenzepine could be found in our experiments with the classic antimuscarinic drug N-methylatroplne, which was shown to inhibit both muscarinic responses in the same narrow dose-range (0.008-0.273 ~mol/kg, i.v.) y~eldlng a potency ratio of about I. The reported selectivity of plrenzeplne for peripheral ganglia prompted us to investigate whether nicotinic receptors mlght be affected by plrenzeplne, too. Using a hlgh plrenzeplne test dose (1.13 ~mol/kg, i.v.), a dose, which nearly completely abolished the pressor response to McN-A-343, no effect on the pressor response to DMPP could be demonstrated. Thus, plrenzeplne is devold of any antlnlcotlnlC activity. Acknowledgements The authors wish to thank Boehrlnger Ingelhelm KG for financial support of this work and Mrs. Monlka Wagner for fine secretarlal assistance xn the p r e p a r a t i o n of the manuscript. References 1. 2. 3. 4. 5. 6. 7.
R.K. GOYAL and S. RATTAN, Gastroenterol. 74 598-619 (1978). R. GILBERT, S. RATTAN and R.K. GOYAL, Gastroenterol. 84 1166 (1983). S. RATTAN and R.K. GOYAL, Trends Pharmacol. Scl. Suppl. 78-81 (1984). A.P. ROSZKOWSKI, J. Pharmacol. Exp. Ther. 132 156-170 (1961). S. MURAYAMA and K.R. UNNA, J. Pharmacol. Exp. Ther. 140 183-192 (1963). B. LEVY and R.P. AHLQUIST, J. Pharmacol. Exp. Ther. 137 219-228 (1962). A. JONES, B. GOMEZ ALONSO DE LA SIERRA and U. TRENDELENBURG, J. Pharmacol. Exp. Ther. 139 312-320 (1962).
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Antimuscarinic
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8. R. HAMMER, C.P. BERRIE, N.J.M. BIRDSALL, A.S.V. BURGEN and E.C. HULME, Nature 283 90-92 (1980). 9. N.J.M. BIRDSALL, A.S.V. BURGEN, R. HAMMER, E.C. HULME and J. STOCKTON, Scand. J. Gastroenterol. 15 Suppl. 66 1-4 (1980). i0. R. HAMMER, Stand. J. Gastroenterol. 15 Suppl. 66 5-11 (1980). ii. R. HAMMER and A. GIACHETTI, Llfe Sel. 31 2991-2998 (1982). 12. E. MONFERINI and R. HAMMER, 2nd Camerino Symp. on Recent Advances in Receptor Chemlstry, University of Camerlno, CamerIno (Italy), (1983). 13. M. WATSON, W.R. ROESKE and H.I. YAMAMURA, Life SOl. 31 2019-2023 (1982). 14. M. WATSON, H.I. YAMAMURA and W.R. ROESKE, Life Scl. 32 3001-3011 (1983). 15. M. WATSON, W.R. ROESKE, P.C. JOHNSON and H.I. YAMAMURA, Brain Res. 290 179-182 (1984). 16. D.A. BROWN, A. FORWARD and S. MARCH, Brit. J. Pharmacol. 71 362-364
(198o). 17. E. MUTSCHLER and G. LAMBRECHT, Trends Pharmacol. Scl. Suppl. 39-44 (1984). 18. H. KILBINGER, Trends Pharmacol. S c l . S u p p l . 49-52 ( 1 9 8 4 ) . 19. M. PARRY and B.V. HEATHCOTE, L i f e S c l . 31 1465-1471 ( 1 9 8 2 ) . 20. B.I. HIRSCHOWITZ, J. FONG and E. MOLiNA, J. Pharmacol. Exp. Ther. 225 263-268 (1983). 21. P. DEL SOLDATO and F. PAGANI, Pharmacol. Res. Commun. 14 279-287 (1982). 22. I. SZELENYI, Brit. J. Pharmacol. 77 567-569 (1982). 23. P.M. LADURON, J.E. LEYSEN and H. GORISSEN, Arch. Int. Pharmacodyn. Ther. 249 319-321 ( 1 9 8 1 ) . 24. E. PARSONS, T. BUNCE, C. BLAKEMORE and C. RASMUSSEN, Die Behandlung des Ulcus peptlcum mit Plrenzepln, A.L. BLUM and R. HAMMER (Eds.), pp 26-34, Karl Demeter, Munich (1979). 25. R.P. BARLOW, M.P. CAULFIELD, R. KITCHEN, P.M. ROBERTS and J.K. STUBLEY, Brit. J. Pharmaeol. 73 183-184P (1981). 26. H. FUDER, Stand. J. Gastroenterol. 17 Suppl. 72 79-85 (1982). 27. S. HALIM, H. KILBINGER and I. WESSLER, Stand. J. Gastroenterol. 17 Suppl. 72 87-93 (1982). 28. D.A. BROWN, S. FATHERAZI, J. GARTHWAITE and R.D. WHITE, Brit. J. Pharmacol. 70 577-592 (1980). 29. D.F. BIGGS, R.T. COUTTS and D.B. HENDERSON, J. Pharm. Pharmacol. 23 628-629 ( 1 9 7 1 ) .
553-560
Pergamon
Press
A COMPARISON OF THE ANTIMUSCARINIC EFFECTS OF PIRENZEPINE AND N-METHYLATROPINE ON GANGLIONIC AND VASCULAR MUSCARINIC RECEPTORS IN THE RAT
J. Wess,
G. Lambrecht,
U. Moser
and E. Mutschler
Faculty of Biochemlstry, Pharmacology and Food Chemlstry, Department of Pharmacology, Unlversity of Frankfurt, Theodor-Stern-Kai 7, D-6000 Frankfurt/M, FRG (Received
in final
form May 25, 1984)
Summary The antlmuscarlnlc properties of plrenzepine and N-methylatropine were evaluated in two intact preparations by measurlng A) the inhlbltion of increase in mean arterlal pressure evoked by McN-A-343 in pithed rats through activat±on of ganglionlc muscarinlc receptors and B) the inhibition of fall in arterlal pressure evoked by methacholine in anaesthetized rats through activation of vascular muscarinic receptors. To characterize the antlmuscarinio potencles of pirenzepine and N-methylatropine, for both antagonlsts doses were calculated that produce a lO-fold shlft to the right of the dose-response curves for A) the pressor response to McN-A-343 (1.v. admlnlstration) in plthed rats (Dlo-P.r.) and B) for the depressor effect to methacholine (l.v. administratlon) in a n a e s t h e t i z e d rats (DlO-an.r.) , respectively. Whereas N-methylatroplne was vlrtually equleffectlve In blocking both muscarinlc responses (DlO-an.r./D]o-P.r. ~ i), plrenzepine, however, was c o n s l d e r a b l y more potent at ganglionic than at vascular muscarinic receptors (DlO-an.r./Dlo-P.r. ~ 16). These data confirm the existence of excitatory ganglionlc muscarlnlc receptors with hlgh afflnlty for pirenzeplne (M~) and provlde evidence for the presence ± of M 2 receptors - receptors which show a low sensltlvity to pirenzeplne on vascular smooth muscle cells. To further characterize the anticholinergic properties of plrenzepine, its effect on the pressor response to DMPP, a nicotinic ganglIonlc stlmu]ant, was investlgated in pithed rats. A high dose of pirenzeplne (1.13 Bmol/kg), given l.v., did not affect nicotinlc gangllonlc transmission.
MuscarlnIc M and M receptor subtypes have been postulated based upon i 2 pharmacological studies on the lower esophageal sphlncter (LES) of the opposum (1,2,3). It was suggested that Mr receptors are located on certain neuronal sites being selectively activate~ by McN-A-343, a potent selective muscarlnlC stimulant of sympathetic ganglia (4-7,11,17), while muscarlnlc receptors in the effector organ (smooth muscle of the LES), selectlvely stimulated by bethanechol, were thought to be of the M 2 type. Indlrect
(8-]2)
as well
as
direct
blndlng
studies
0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press
(13-15)
Ltd.
of plrenzeplne
554
Antimuscarinic
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suggest that th~s n o n - c l a s s i c a l m u s c a r ~ n ~ c a n t a g o n i s t may d ~ f f e r e n t l a t e these p r o p o s e d subtypes. It could be d e m o n s t r a t e d that h~gh affinity s~tes for p ~ r e n z e p l n e (M. sites) are p r e s e n t at c e r t a i n neuronal s t r u c t u r e s as cerebral I cortex and h l p p o c a m p u s of the rat (8,9,13,Ia) and ~n s y m p a t h e t i c and myenter~c g a n g l l a (10-12,15), whereas b ~ n d l n g sites for p l r e n z e p l n e at effector organs such as heart and ileum have been shown to be p r e d o m i n a n t l y of the low a f f l n l t y type (M 2) (8,9,11,13,14). Thls r e p o r t e d s e l e c t i v i t y of p l r e n z e p l n e has also been c o n f i r m e d ~n f u n c t i o n a l tests (2,3,ii,16-21), a l t h o u g h it has been q u e s t i o n e d r e c e n t l y (22,23). The data c~ted above have led to the f o r m u l a t i o n of o p e r a t i o n a l definitions of subtypes of m u s c a r l n ~ c receptors: The M I r e c e p t o r ~s one which shows a h~gh degree of s e n s l r l v l t y to the a n t a g o n i s t p ~ r e n z e p l n e and the agonlst McN-A-Sa3, and the M 2 r e c e p t o r ~s one which shows a low s e n s ~ t ~ v l t y to p ~ r e n z e p ~ n e and McN-A-943, respectlvely. The alm of the present study was to d e m o n s t r a t e the r e p o r t e d M I selectlvlty of p l r e n z e p l n e and to c h a r a e t e r l z e m u s c a r l n l c receptors located on vascular smooth muscle cells using two intact preparatlons. Accordingly, we have d e t e r m i n e d the a b 1 1 1 t y of p l r e n z e p l n e and N - m e t h y l a t r o p l n e A) to inhlblt the increase in arterial pressure In p i t h e d rats evoked by the s e l e c t l v e M agonlst M c N - A - 3 4 3 (i-7,11,17) and B) to block the decrease in arterla~ pressure in a n a e s t h e t i z e d rats c a u s e d by the m u s c a r l n l c agent methachollnc, respectlvely. M e t h a c h o l l n e was found to be nearly e q u l p o t e n t to a c e t y l c h o l l n e In lowering arterlal blood pressure in anaesthetized rats (29), but a c e t y l c h o l l n e was shown to be more potent by a factor of 18 than m e t h a c h o l l n e in d e p o l a r l z l n g rat s y m p a t h e t i c g a n g l l a by s t i m u l a t i o n of m u s c a r l n l c receptors (28). These data indlcate a certain degree of selectlvlty of m e t h a c h o l i n e and led to our choice of thls a g o n l s t in the a n a e s t h e t i z e d rat. Furthermore, the effect of p l r e n z e p a n e on the p r e s s o r response to DMPP, a nlcotlnlc g a n g l ~ o n l c stlmulant, was i n v e s t ± g a t e d in pithed rats. Our data indicate that p l r e n z e p l n e is a selective MI r e c e p t o r a n t a g o n l s t and that m u s c a r l n l c receptors of the M 2 type are present on v a s c u l a r smooth muscle cells. Methods Drugs' The f o l l o w i n g drugs were used: p l r e n z e p l n e d i h y d r o c h l o r l d e (kindly provided by Dr. R. Hammer, K. Thomae), methachollne chloride (Szgma), N - m e t h y l a t r o p i n e nitrate (Merck), DMPP (Fluka), and M e N - A - 3 4 3 (syntheslzed in our laboratory). All drugs were d i s s o l v e d in sallne (0.9%, w/v) and a d m i n i s t e r e d 1.v. in a volume of O.iml/lOOg. Intravenous p r e t r e a t m e n t wlth a n t a g o n l s t s or sallne (controls) was c a r r i e d out twenty minutes before a d m i n i s t r a t l o n of agonlsts. Thls interval was s e l e c t e d because p r e l i m i n a r y e x p e r i m e n t s showed that after this time the a n t a g o n l s t l c effects of p l r e n z e p l n e and N - m e t h y l a t r o p l n e , respectlvely, were constant durlng the wnole experlment. If not stated otherwlse animals served as thelr own controls. No evidence of b a s e l i n e drlft or d e s e n s l t l z a t l o n was observed, when McN-A-3A3 and methachollne, respeczlvely, were r e p e a t e d l y admlnlstered. The intact a n a e s t h e t l z e d rat: Male, n o r m o t e n s l v e White W l s t a r rats (weight 230-300g) were anaesthetized with pentobarbltone-sodlum (60mg/kg, given 1.p.). The left jugular vein was c a n n u l a t e d for the a d m l n l s t r a t l o n of drugs. Arterial blood p r e s s u r e was m e a s u r e d from the c a n n u l a t e d rlght common c a r o t l d artery by means of a S t a t h a m pressure t r a n s d u c e r c o n n e c t e d to a Helllge
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Antimuscarinic
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a m p l l f l e r and a R1kadenkl recorder. After c a t h e t e r l z a t a o n of the trachea, h e p a r i n (300 I.U./kg) was glven i.v. to prevent c o a g u l a t i o n of the blood. Body t e m p e r a t u r e was kept at 37±I°C throughout the experiment. At the b e g l n n l n g of each experiment, the a n t a g o n l s t s or sallne (controls) were admlnlstered. Then d o s e - r e s p o n s e curves for the v a s o d e p r e s s o r effect to m e t h a c h o l l n e were e s t a b l l s h e d untll a fall of 40-50 mm Hg in mean arterlal pressure was achleved. The plthed rat: Rats were a n a e s t h e t i z e d and p r e p a r e d for blood pressure r e c o r d i n g as d e s c r l b e d above. Addltlonally, both vagi were cut at the neck, and the anlmals were pithed wlth a steel rod. A r t l f l c l a l r e s p l r a t l o n was provlded by means of a B r a u n - M e l s u n g e n pump (Iml/lOOg body welght at a rate of 60 strokes/mln). In each anlmal d o s e - r e s p o n s e curves for the pressor effect of M c N - A - 3 4 3 were e s t a b l l s h e d in the absence and in the presence of the a n t a g o n i s t s twenty minutes after thelr admlnlstratlon. Data Analysls: In the a n a e s t h e t l z e d rats doses of m e t h a c h o l i n e that d e c r e a s e d mean arterial pressure by 30 mm Hg in the absence and in the presence of different doses of plrenzeplne and N-methylatroplne were c a l c u l a t e d from the d o s e - r e s p o n s e curves. Mean dose ratlos were d e t e r m i n e d for each a n t a g o n l s t dose and drawn on a log scale along the ordinate in r e l a t i o n to the log a n t a g o n l s t doses on the absclssa. Stralght llnes were obtained, and from these llnes a n t a g o n i s t doses were estimated, which c o r r e s p o n d to a shift of the control d o s e - r e s p o n s e curve by a factor of i0
(DlO-an.r.-values).
In the plthed rats doses of M e N - A - 3 4 3 that increased mean arterial blood pressure by 60 mm Hg were c a l c u l a t e d from the d o s e - r e s p o n s e curves. D n -p.r.values (antagonlst doses that produce a lO-fold shlft to the r l g h t l ~ f the M c N - A - 3 4 3 control d o s e - r e s p o n s e curve) were d e t e r m l n e d for p l r e n z e p l n e and N - m e t h y l a t r o p l n e as described above. For both a n t a g o n l s t s a ratlo D . _ - a n . r . / D . _ - p . r . was c a l c u l a t e d IU IU be c o n s l d e r e d as a measure of thelr r e c e p t o r selectivity. Statistics: Unless stated p r e s e n t e d as arlthmetic means number of e x p e r l m e n t s (n).
which
may
otherwise, the results of the e x p e r i m e n t s ~ standard error of the mean (S.E.M.) and
are the
Results A n a e s t h e t i z e d rat: The inltlal mean arterial blood pressure amounted to iS4t2 mm Hg (n=42). Intravenous injection of m e t h a c h o l l n e (0.125-1.0 ~g/kg) ellclted a d o s e - r e l a t e d depressor effect. As depicted in Fig. i, p r e t r e a t m e n t with pirenzeplne (2.26-22.61 ~mol/kg) as well as N-methylatroplne (0.027-0.273 ~mo!/kg) produced a d o s e - d e p e n d e n t shift of the d o s e - r e s p o n s e curve for the v a s o d e p r e s s o r effect of m e t h a c h o l l n e to the right. Pithed rat: The r e s t i n g mean arterlal pressure of the p l t h e d rats was 51±1 mm Hg (n=50). The intravenous a d m i n l s t r a t l o n of the selective M I agonlst M c N - A - 3 4 3 (25-400 ~g/kg) e l i c i t e d a d o s e - d e p e n d e n t increase in mean arterial pressure. As shown in Fig. 2, p r e t r e a t m e n t with p l r e n z e p l n e (0.068-0.678 ~mol/kg) or N - m e t h y l a t r o p l n e (0.008-0.082 ~mol/kg) a n t a g o n l z e d the pressor effect caused by McN-A-343, shifting its d o s e - r e s p o n s e curve in a parallel fashlon to the right.
556
Antimuscarinic
Selectivity of Pirenzepine
Vol.
35, No. 5, 1984
N -METHYLATROPINE 50
--40 o~
//
E30 E
~/ ffl
~ m
&, / /
/
T/ ~
&/
N-methylatr
~L
• 0027 2 p mol/k g
o,.
Methachohne ( pg/kgj v ) r,r¢ <
PIRENZEPINE 50
Z <
Z
/
u.l
m ~ ! ~ ezOl~me 0
~mo, I/kg
~m
• 2261 )1
1
,,
~Methachohne ( /Jg/kg,=lv~
FIG. i Antagonzsm by N-methylatropzne (upper panel) and pzrenzepzne (lower panel) of m e t h a c h o l z n e - l n d u e e d vasodepresszon ~n anaesthetzzed rats. Data are presented as mean values ± S.E.M. (n=6). TABLE I A Comparlson of the Antlmuscarlnzc Potencies of Pzrenzepzne and N-Methylatropzne in the Pithed and zn the Anaesthetlzed Rat.
a
Plrenzepzne N-Methylatropzne
b
Dio-an.r.
DIO- p,r.
(umol/kg)
(~mol/kg)
6.75 0.057
0.41
0.044
Dio-an.r./DIo- p.r.
16.5 1.3
aDlo-an.r.: Antagonzst dose that corresponds to a shift of the methacholzne ~@-response curve in the anaesthetlzed rat by a factor of I0. -Dlo-P.r.: A n t a g o n l s t dose that corresponds to a shzft of the McN-A-343 doseresponse curve in the plthed rat by a factor of i0.
Vol.
35, No.
5, 1984
120
Antimuscarinic
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557
N-METHYLATROPINE
100
~6o E ~
60
u,l
/ / / ~ m t /
w
.a/
o. 21)
~, LU
001
/ ~
~
01
~" •
/
~_ /
roI ethylatr • 0oo8 .~o,,~g • 9_027 • 0,082 .."
i
lb McN-A-343 (mg/kg,l.v)
OiC ,¢
zlO01
PIRENZEPINE
601 i control Plrenzepme 0068 pmol/kg 0226 0.678 ,," 001
0'1
Ib McN A-343 (rnglkg,lv)
i
FIG. 2 Antagonism by N-methylatropine (upper panel) and pirenzeplne (lower panel) the M c N - A - 3 4 3 - i n d u c e d pressor response in pithed rats. Data are presented means ± S.E.M. (n=6 for inhibition curves, n=18 for control curves).
of as
In separate experiments the effect of a high dose of pirenzepine (1.13 pmol/kg, i.v.) on the pressor response caused by DMPP (100-500 pmol/kg, i.v.) was investigated in pithed rats, in order to exclude the possibility that the blocking activity of pirenzeplne towards the McN-A-343 pressor response might be due to an antinicotinic action. Rats were divided into two groups (n=7), one group receiving saline (controls), the other one pirenzepine (1.13 ~mol/kg, i.v.). Twenty minutes after the administration of saline or pirenzeplne, respectively, dose-response curves for the pressor effect to DMPP were established. No significant effect (p
558
Antimuscarinic
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of P i r e n z e p i n e
Vol.
35, No.
5, 1984
R e c e p t o r - s e l e c t l v l t y of p i r e n z e p l n e and N - m e t h y l a t r o p l n e : In order to o b t a i n D - v a l u e s for p l r e n z e p l n e and N - m e t h y l a t r o p l n e (see under methods), 1 mean log ~ o s e - r a t l o s were p l o t t e d a g a i n s t the a d m l n l s t e r e d a n t a g o n i s t doses on a l o g a r l t h m e t l c scale (Fig. 3), and s t r a l g h t lines were obtalned. It becomes quite obvious from Fig. 3 that N - m e t h y l a t r o p l n e inhlblts both muscarlnic reponses in the same n a r r o w dose-range, while p ~ r e n z e p l n e is c o n s i d e r ably more potent at g a n g l i o n l c sltes. In order to p r e s e n t q u a n t i t a t i v e data, D l o - v a l u e s for N - m e t h y l a t r o p l n e and p l r e n z e p l n e are listed in Table I. The data c o l l e c t e d in Table I show that p l r e n z e p l n e is more than two orders of m a g n i t u d e less potent than N - m e t h y l a t r o p l n e in the a n a e s t h e t i z e d rat, but the p o t e n c y d i f f e r e n c e in the plthed rat being only somewhat less than one order of magnitude. The D 1 0 - a n . r . / D ~ - p . r . - r a t l o shows that under _ IU the d e s c r l b e d e x p e r i m e n t a l condltlons plrenzeplne ~s about 16 tlmes more potent at g a n g l l o n l c than at v a s c u l a r m u s c a r l n l c r e c e p t o r sites. No such d l f f e r e n c e could be found for the c l a s s i c a l a n t l m u s c a r l n l c agent N - m e t h y l atropine (DlO-an.r./Dlo-P.r. -;1).
2N-METHYLATROPIN E O
PIRENZEPINE
o_
/
fv wJ
0
O
o
9
)005
(105 ' N-Methylatroplne(IJmol/kg) 0 5 '
/ ./
Pirenzepme( pmoll kg)
FIG.3 A c o m p a r i s o n of the a n t i m u s c a r l n i c p o t e n c i e s of N - m e t h y l a t r o p l n e (left panel) and p i r e n z e p i n e (rlght panel) in the pithed rat ( ~ , a n t a g o n i s m against the pressor effect caused by McN-A-343) and in the a n a e s t h e t i z e d rat ( D---43, a n t a g o n i s m against the d e p r e s s o r effect caused by methacholine). Lines are least square estimates, and data are p r e s e n t e d as means ± S.E.M. (n=6). Discussion The e l a s s l f l c a t x o n of m u s c a r i n x c r e c e p t o r s most commonly a p p l i e d is based on the d l s o r l m l n a t l v e p r o p e r t i e s of the m u s c a r l n l c a n t a g o n i s t p i r e n z e p l n e and the m u s c a r l n l e agonlst McN-A-343. The t e r m l n o l o g y Cn p r e s e n t use ~s the following: The M r e c e p t o r shows a hlgh degree of s e n s i t i v i t y to p l r e n z e p l n e and can be s e l e c } i v e l y a c t l v a t e d by McN-A-343, whereas the M 2 r e c e p t o r shows low s e n s i t i v i t y to p i r e n z e p i n e and McN-A-343, r e s p e c t i v e l y (1-3,11-15). On the basis of this r e c e p t o r c l a s s l f l c a t l o n our data confJrm the r e p o r t e d M 1 s e l e c t l v l t y of p l r e n z e p l n e and provide evidence for the presence of M 2 receptor sites on vascular smooth muscle cells. It was c o n f i r m e d that p l r e n z e p i n e in a dose-range from 0 . 0 6 8 - 0 . 6 7 8 ~mol/kg, ~.v., is a potent a n t a g o n i s t of M c N - A - 3 4 3 - z n d u c e d increase in mean arterial pressure in pithed rats (ii), a response, which has been shown to be m e d i a t e d by selective a c t l v a t l o n of m u s c a r l n i c (atroplne-sensltzve) sites in
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m a m m a l i a n sympathetlc ganglia (4,6,7,17). On the contrary, high doses of plrenzepine had to be applled (2.26-22.61 ~mol/kg, 1.v.) to achieve a reductlon of the methachollne-evoked decrease in blood pressure ~n a n a e s t h e t i z e d rats. As the depressor effect to methacholine is thought to be mediated by activatlon of muscarlnic receptors on vascular smooth muscle cells resulting in a g e n e r a l l z e d vasodilatation, the antagonistic effect of pirenzepine is suggested to be a consequence of ~ts Interaction wlth these vascular muscarln~c sites. We could demonstrate that vascular muscarinic receptors, s t i m u l a t e d by exogenously a d m i n i s t e r e d methachollne, show low affinity for plrenzeplne. Therefore, these receptors can be classified to be of the M type, but we cannot exclude the simultaneous presence of M sites on vascula~ 1 smooth muscle cells. These flndlngs are in agreement wlth studies on various other effector organs such as 11eum or heart, which have been shown to contain p r e d o m i n a n t l y muscarlnlc receptors of the M 2 type (8,9,11,13,14, 16-21,24-27). Although one has to keep in m~nd that the presented results are in-vIvo data and p h a r m a c o k i n e t i c considerations do not allow a direct quantlficatlon of affinity differences, the observed selectlvlty of plrenzeplne (DlO-an.r./D._-p.r. ~ 16) agrees well with previous findings in functional in-vltro t e s ~ (16) and binding studies (i0,Ii). Brown et al. ~16) demonstrated a 23-fold higher affinity of pirenzeplne for muscarinic receptors in the rat superior cervical ganglion mediating depolarlzatlon than for those present in the rat ileum causing contraction. Accordingly, competition studies on the binding of trltiated N - m e t h y l s c o p o l a m l n e to ganglionic and atrlal muscarinic receptors showed (ii) that the affinities of plrenzeplne for these two receptor sites differ by a factor of about 30. As expected from pharmacologlcal studies (ii,16,28), no such selectivity difference as described for pirenzepine could be found in our experiments with the classic antimuscarinic drug N-methylatroplne, which was shown to inhibit both muscarinic responses in the same narrow dose-range (0.008-0.273 ~mol/kg, i.v.) y~eldlng a potency ratio of about I. The reported selectivity of plrenzeplne for peripheral ganglia prompted us to investigate whether nicotinic receptors mlght be affected by plrenzeplne, too. Using a hlgh plrenzeplne test dose (1.13 ~mol/kg, i.v.), a dose, which nearly completely abolished the pressor response to McN-A-343, no effect on the pressor response to DMPP could be demonstrated. Thus, plrenzeplne is devold of any antlnlcotlnlC activity. Acknowledgements The authors wish to thank Boehrlnger Ingelhelm KG for financial support of this work and Mrs. Monlka Wagner for fine secretarlal assistance xn the p r e p a r a t i o n of the manuscript. References 1. 2. 3. 4. 5. 6. 7.
R.K. GOYAL and S. RATTAN, Gastroenterol. 74 598-619 (1978). R. GILBERT, S. RATTAN and R.K. GOYAL, Gastroenterol. 84 1166 (1983). S. RATTAN and R.K. GOYAL, Trends Pharmacol. Scl. Suppl. 78-81 (1984). A.P. ROSZKOWSKI, J. Pharmacol. Exp. Ther. 132 156-170 (1961). S. MURAYAMA and K.R. UNNA, J. Pharmacol. Exp. Ther. 140 183-192 (1963). B. LEVY and R.P. AHLQUIST, J. Pharmacol. Exp. Ther. 137 219-228 (1962). A. JONES, B. GOMEZ ALONSO DE LA SIERRA and U. TRENDELENBURG, J. Pharmacol. Exp. Ther. 139 312-320 (1962).
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8. R. HAMMER, C.P. BERRIE, N.J.M. BIRDSALL, A.S.V. BURGEN and E.C. HULME, Nature 283 90-92 (1980). 9. N.J.M. BIRDSALL, A.S.V. BURGEN, R. HAMMER, E.C. HULME and J. STOCKTON, Scand. J. Gastroenterol. 15 Suppl. 66 1-4 (1980). i0. R. HAMMER, Stand. J. Gastroenterol. 15 Suppl. 66 5-11 (1980). ii. R. HAMMER and A. GIACHETTI, Llfe Sel. 31 2991-2998 (1982). 12. E. MONFERINI and R. HAMMER, 2nd Camerino Symp. on Recent Advances in Receptor Chemlstry, University of Camerlno, CamerIno (Italy), (1983). 13. M. WATSON, W.R. ROESKE and H.I. YAMAMURA, Life SOl. 31 2019-2023 (1982). 14. M. WATSON, H.I. YAMAMURA and W.R. ROESKE, Life Scl. 32 3001-3011 (1983). 15. M. WATSON, W.R. ROESKE, P.C. JOHNSON and H.I. YAMAMURA, Brain Res. 290 179-182 (1984). 16. D.A. BROWN, A. FORWARD and S. MARCH, Brit. J. Pharmacol. 71 362-364
(198o). 17. E. MUTSCHLER and G. LAMBRECHT, Trends Pharmacol. Scl. Suppl. 39-44 (1984). 18. H. KILBINGER, Trends Pharmacol. S c l . S u p p l . 49-52 ( 1 9 8 4 ) . 19. M. PARRY and B.V. HEATHCOTE, L i f e S c l . 31 1465-1471 ( 1 9 8 2 ) . 20. B.I. HIRSCHOWITZ, J. FONG and E. MOLiNA, J. Pharmacol. Exp. Ther. 225 263-268 (1983). 21. P. DEL SOLDATO and F. PAGANI, Pharmacol. Res. Commun. 14 279-287 (1982). 22. I. SZELENYI, Brit. J. Pharmacol. 77 567-569 (1982). 23. P.M. LADURON, J.E. LEYSEN and H. GORISSEN, Arch. Int. Pharmacodyn. Ther. 249 319-321 ( 1 9 8 1 ) . 24. E. PARSONS, T. BUNCE, C. BLAKEMORE and C. RASMUSSEN, Die Behandlung des Ulcus peptlcum mit Plrenzepln, A.L. BLUM and R. HAMMER (Eds.), pp 26-34, Karl Demeter, Munich (1979). 25. R.P. BARLOW, M.P. CAULFIELD, R. KITCHEN, P.M. ROBERTS and J.K. STUBLEY, Brit. J. Pharmaeol. 73 183-184P (1981). 26. H. FUDER, Stand. J. Gastroenterol. 17 Suppl. 72 79-85 (1982). 27. S. HALIM, H. KILBINGER and I. WESSLER, Stand. J. Gastroenterol. 17 Suppl. 72 87-93 (1982). 28. D.A. BROWN, S. FATHERAZI, J. GARTHWAITE and R.D. WHITE, Brit. J. Pharmacol. 70 577-592 (1980). 29. D.F. BIGGS, R.T. COUTTS and D.B. HENDERSON, J. Pharm. Pharmacol. 23 628-629 ( 1 9 7 1 ) .