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Methoctramine, a cardioselective antagonist: muscarinic receptor mediating prostaglandin synthesis in isolated rabbit heart
European Journal of Pharmacology, 192 (1991) 63-70
63
© 1991 ElsevierScience Publishers B.V. (BiomedicalDivision) 0014-2999/91/$03.50 ADONIS 001429999100068P EJP 51649
Methoctramine, a cardioselective antagonist: muscarinic receptor mediating prostaglandin synthesis in isolated rabbit heart N e e l a m Jaiswal a n d K a f a i t U. M a l i k Department of Pharmacology, College of Medicine, The University of Tennessee at Memphis, Memphis, TN 38163, U.S.A.
Received 28 May 1990, revisedMS received27 August 1990, accepted 9 October 1990 The antimuscarinic properties of the methoctramine with high selectivity for cardiac muscarinic M 2 receptors were investigated on cholinergically induced changes in prostaglandin (PG) synthesis and mechanical function in the isolated perfused rabbit heart. Acetylcholine (ACh)- and arecaidine propargyl ester (APE)-induced increases in PG synthesis were significantly attenuated by methoctramine in a concentration-dependent manner, Methoctramine at a low concentration of 0.1/tM potentiated ACh-induced PG synthesis, which was blocked by simultaneous infusion of hexahydro-sila-difenidol (HHSiD), a M,, receptor antagonist. Methoctramine produced an additive effect with HHSiD in diminishing the ACh- or APE-induced PG synthesis. Methoctramine displayed a potent antagonistic activity at M 2 receptors that mediate the decrease in heart rate and increase in coronary perfusion pressure in isolated perfused rabbit heart. Methoctramine also minimized ACh- and APE-induced decrease in developed tension. In contrast, at 0.1-0.75/~M it exhibited no antagonistic activity at vascular muscarinic receptors (M3) mediating vasodilation in response to ACh or APE. These data suggest that methoctramine has a high affinity for cardiac M 2 receptors mediating increases in PG output and coronary perfusion pressure as well as decrease in heart rate and developed tension and has a low affinity for M 3 receptors mediating coronary vasodilator response. Methoctramine; Muscarinic receptor subtypes; Cardioselectivity; Cardiac muscarinic M2a receptors; Prostaglandin synthesis I. Introduction A large number of pharmacological and biochemical studies have shown that there are subclasses of muscarinic receptors (Hammer and Giachetti, 1982; Birdsall and Hulme, 1983; Harden et al., 1986). Evidence from direct binding studies has indicated that there are at least two subtypes of muscarinic receptors distinguished by their affinity for the antagonist pirenzepine (Hammer et al., 1980). The M 1 receptor subtype (displaying high affinity to pirenzepine) is located primarily in the central nervous system and autonomic ganglia, whereas the M 2 subtype (showing low affinity to pirenzepine) is present mainly in effector cells in peripheral organs such as the heart and ileum and in certain parts of the central nervous system (Hammer and Giachetti, 1982). Several lines of evidence from functional and biochemical studies have indicated that ileal and atrial muscarinic receptors differ from each other (Mutschler and Lambrecht, 1984; Eglen and Whiting, 1986; Gilani and Cobbin, 1986; Giachetti et al., 1986; Melchiorre et al., 1987). These observations
Correspondence to: K.U. Malik, Department of Pharmacology,College of Medicine, 874 Union Avenue, Crowe No. 100, Memphis, TN 38163, U.S.A.
have led to further classification of muscarinic receptors into M1, M z and M 3 receptors (Lambrecht et al., 1987; Birdsall et al., 1987; Doods et al., 1987; Mutschler et al., 1987). Our recent studies in the isolated rabbit heart suggest that prostaglandin (PG) synthesis in the rabbit heart elicited by acetylcholine (ACh) receptor agonists is mediated via activation of M2~ (M2) as well as MEB (M3) receptors. Supporting this proposal, we have shown that PG synthesis elicited by ACh and arecaidine propargyl ester (APE) (Mutschler and Lambrecht, 1984) was attenuated by 11-[2-[(diethylamino)methyl]-l-piperidinyl]acetyl]-5,11-dihydro-6-H-pyrido-[2,3,-b][1,4]-benzodiazepine-6-one (AF-DX116) (a selective M E receptor antagonist) (Hammer et al., 1986) and by hexahydrosila-difenidol (HHSiD) (having a greater affinity for ileal, tracheal, and urinary bladder muscarinic receptors than for atrial muscarinic receptors and called M 3 receptor antagonist) (Jaiswal and Malik, 1988; Jaiswal et al., 1988). The recent discovery that methoctramine, a polymethylene tetramine compound, is about 270 times more potent in inhibiting stimulation of cardiac muscarinic receptors than in antagonizing muscarinic receptor-stimulated contractions in smooth muscle (Melchiorre et al., 1987) suggests that this compound is a selective muscarinic M 2 receptor antagonist. The present study was undertaken to determine the selectivity
64 of methoctramine for M 2 muscarinic receptors involved in the action of ACh receptor agonists on cardiac PG synthesis and changes in mechanical function in the isolated rabbit heart.
2. Materials and methods
Male New Zealand White rabbits weighing 1.0-2.0 kg (Myrtle's Rabbitry, Thompson's Station, TN) were used in all experiments. After anesthesia with i.v. sodium pentobarbital, 30 mg/kg (Abbot Laboratory, North Chicago, IL), the abdomen was opened and heparin (1000 U/kg) administered in the vena cava. After thoractomy the heart was quickly removed, placed into a warmed chamber (37 o C), and perfused at a rate of 18 ml/min with a Harvard peristaltic pump (Harvard Apparatus Co., Inc., MiUis, MA) according to the technique of Langendorff with oxygenated (95 % 02-5 % CO 2) Krebs-Henseleit buffer of the following composition (millimolar): 114 NaC1, 4.7 KC1, 1.2 KH2PO4, 25.0 NaHCO3, 2.5 CaC12, 1.2 MgSO4 and 5.5 glucose. The coronary perfusion pressure was measured with a Statham pressure transducer (P2306; Statham Medical Instruments, Los Angeles, CA) attached to the aortic cannula. Myocardial tension was measured with a Grass FT.03C force-displacement transducer (Grass Instruments Co., Quincy, MA) connected to a stainless steel hook attached to the left ventricular apex. The transducer was adjusted to provide a diastolic tension of 2 g. The heart rate was measured with a tachometer triggered by the ventricular contractions. The experimental period began after 20 min had elapsed. At this time, any heart that failed to develop at least 8 g of systolic tension was discarded. 2.1. Experimental protocol Hearts were perfused for 20 rain without any experimental intervention. Perfusate was then collected for 1 rain before the injection of a bolus dose of drug into the arterial circuit. As there was a 35-45 s delay between the time the injection was made and the time the agonist reached the heart, 1 rain was allowed to elapse before a second 1-min perfusate collection was made. Four experimental protocols were followed. 2.1.1. Protocol 1 The first series of experiments was performed to investigate the effects of different doses (1-10 nmol) of ACh and APE on PG output and mechanical responses of the heart. McN-A-343, a Ml-selective agonist, was not used in this series of experiments because we had demonstrated in a previous study that it does not alter the basal PG synthesis as well as mechanical responses of the heart at a concentration at which ACh and APE produced significant effects (Jaiswal and Malik, 1988).
After a 20-min stabilization period, ACh or APE was administered in a random order as a bolus at 10-rain intervals for up to 120 min of perfusion of the heart. Drugs were administered between 20-120 min of perfusion of the heart because the basal output of PGs was found to be stable during this period. Moreover, heart rate, myocardial tension and coronary perfusion pressure also remained stable during the time course of the experiment. A second series of experiments was performed to investigate the effects of methoctramine on PG output and on the mechanical responses of the heart. The experimental protocol consisted four or five periods, separated by 15-rain intervals; ACh (3 nmol), APE (3 nmol), arachidonic acid (33 nmol), or the respective vehicle, contained in 100 #1 saline (0.9% NaC1), was administered as a bolus during all periods. After collecting the perfusate samples and measuring cardiac parameters during administration of the above drugs in the first period, the heart was infused with the M E muscarinic receptor antagonist methoctramine at a lower dose in the second period and with higher doses in consecutive periods. 2.1.2. Protocol 2 This series of experiments was performed to determine the effects of combined administration of 0.1 or 0.75 #M methoctramine with different doses of HHSiD on PG output and on changes in mechanical function of the heart produced by 3 nmol ACh. In a previous study, we had shown that HHSiD, a M3-selective antagonist, significantly reduced ACh- or APE-induced PG synthesis in a concentration-dependent manner and produced an additive effect with the M 2 receptor blocker AF-DX 116 (Jaiswal and Malik, 1988). We therefore investigated the effects of methoctramine combined with different concentrations of HHSiD. The experimental protocol consisted of five periods, separated by 15-min intervals. ACh or its vehicle was administered as a bolus in all five periods. After administering ACh in the first period, 0.1 or 0.75 /~M methoctramine was infused in the second period, and then 0.1 or 0.75 #M methoctramine plus 0.01, 0.05, or 0.1/tM HHSiD in the third, fourth, and fifth periods, respectively. 2.1.3. Protocol 3 This series of experiments was performed to determine the effects of methoctramine (1.0 #M) and the cyclooxygenase inhibitor indomethacin (2.8 #M) on PG output elicited by administration of a bolus injection of ACh (3 nmol), APE (3 nmol), isoproterenol (0.4 nmol), or arachidonic acid (33 nmol) as described in the first series of experiments. In all four series, perfusate samples were collected for l-rain periods immediately before and after administration of drugs and were then frozen ( - 2 0 ° C) for later PG determination.
65 n=6
2.2. Determination of PG
VEH
METHOCTRAMINE 0.1
6-keto-PGFl~ concentration was determined in the perfusate sample by radioimmunoassay Briefly, 100 /~1 of sample was mixed with 2 000-3 000 cpm of tracer plus the appropriate concentration of antibody. The tracer and antibody were prepared in a buffer consisting of (g/l) 1.0 NaN 3, 9.0 NaC1, 6.8 KH2PO4, 26.1 KzHPO4 26 and 2.0 gelatin. The tubes were vortexed and incubated overnight at 4 ° C. Bound tracer was separated from free tracer by adding 1.0 ml of dextran-coated charcoal to each tube, and radioactivity was determined by liquid scintillation spectroscopy. The antibody for 6-keto-PGFl~ was provided by Dr. C. Leffler of the Department of Physiology and Biophysics, University of Tennessee. Cross-reactivity of the 6-keto-PGF1, antibody was less than 0.1% with thromboxane B2, and 13,14-dihydro-15-keto-PGE 2 and less than 0.5% with PGE 2 and PGF2~.
2.3. Drugs ACh, atropine, arachidonic acid and indomethacin were purchased from Sigma Chemical Co., St. Louis, MO. Methoctramine was purchased from Research Biochemicals, Inc., Natick, MA. APE and HHSiD were gifts from Dr. G. Lambrecht (Pharmakologisches Institut, Frankfurt F.R.G.).
2.4. Analysis of data The results are expressed as means + S.E.M. The data were analyzed by analysis of variance, and the differences between means were determined by Student's t-test for paired observations. The null hypothesis was rejected at P < 0.05. The basal PG output represents the amount of PG in samples collected for l-rain periods immediately before drug administration and is expressed as ng/min of immunoreactive 6-keto-PGFl~. Although the basal PG output during the course of the experiment remained constant in each heart, it was variable among different hearts. Therefore, the rise in PG output ehcited by various agents was expressed as percent increase above basal. The pA 2 value for methoctramine was presented as log (antagonist)/(dose r a t i o - 1), and the mean values were calculated as described by Mackay (1978).
3. Results
ACh at a dose of 1-10 nmol significantly enhanced the output of 6-keto-PGFl~ in a dose-dependent manner
0.75
1.0
300 -
~
200
~
100
CL
""
*t 0 Basal 6-Keto-PGFt~ (ng/min)
2.33_+ 0.64
1.97+ 0.90
2.39_+ 2.63_+ 0.28 0.62
2.80_+ 0.94
Fig. 1. Effect of of methoctramine on the output of 6-keto-POF],~ elicited by acetylcholine (ACh, 3 nmol) in the isolated perfused rabbit heart. A C h was injected as a bolus in a volume of 0.1 rnl into the aortic cannula. Data are expressed as means_+ S.E. * Denotes values significantly different from the basal level, whereas -t denotes a significant difference between the values in the presence of methoctramine and vehicle (VEH) (P < 0.05).
(range 42 + 5 to 250 + 21% above basal level). Methoctramine at 0.1/~M potentiated the effect of ACh (3 nmol) in increasing 6-keto-PGFt~ output in the rabbit heart (from 100 + 9 to 232 + 34% above basal). The effect of ACh in increasing 6-keto-PGFl~ output was not potentiated or altered at 0.5 #M methoctramine, but it was reduced at 0.75 /~M methoctramine; increasing the methoctramine concentration to 1.0 #M abolished the effect of ACh in increasing 6-keto-PGFl~ output. Methoctramine displayed an IC50 of 0.73 + 0.025 /~M and a pA 2 of 7.21 __+0.14 for inhibiting ACh-induced PG synthesis (tables 1 and 2). TABLE 1 Comparison of the IC5o of methoctramine for antagonizing the increase in 6-keto-PFGx,~ output and the decreases in heart rate and changes in coronary perfusion pressure (CPP) elicited by 3 nmol of A C h or APE. Values are means 5: S.E. Parameters
6-keto-PGF1, , Heart rate CPP (decrease) CPP (increase)
ICs0 (/~M) ACh
APE
0.73 + 0.025 0.09 + 0.003 3.13 5:0.62
0.59 5:0.13 0.14 5:0.015 1.30 5:0.20 0.23 5:0.04
TABLE 2 Affinity (PA2) of methoctramine for inhibiting the increase in 6-ketoPFG1, , output and the decreases in heart rate and changes in coronary perfusion pressure (CPP) elicited by 3 nmol of A C h or APE. Values are means + S.E. Parameters
3.1. Effect of methoctramine on 6-keto-PGFl~ synthesis ellicited by A Ch (fig. 1)
0.5
(p.M)
6-keto-PGF m Heart rate CPP (decrease) CPP (increase)
pA 2 values fo methoctramine ACh
APE
7.21 + 0.14 7.90 5:0.16 5.34 ___0.09
7.69 5:0.43 7.83 + 0.12 6.01 5:0.17 7.73 5:0.16
66 n=6
VEH
METHOCTRAMINE 0.1
° >e
0.75
VEH
1.0
METHOCTRAMINE
/I.tM)
0.1
0.5
0.75
1.0
145+5
165+7
160+ 2
153+5
•
2°° f
6
0.5
(laM)
100t -7- ~
01-
Basal
6-Keto-PGF
(ng/min) let
1.65±
0.42
3.20+-
1.20
2.84±
0.49
2.84+-
0.27
2.17+-
0.41
Fig. 2. Effect of methoctramine on the output of 6-keto-PGF]a elicited by arecaidine propargyl ester (APE, 3 nmol) in the isolated perfused rabbit heart. APE was injected as a bolus in a volume of 0.1 ml into the aortic cannula. Data are expressed as means+ S.E. * Denotes values significantly different from the basal level, whereas t denotes a significant difference between the values in the presence of methoctramine and vehicle (VEH) (P < 0.05).
-20
Basal Heart Rate (bpm)
160+7
o-~
Basal Developed Tension
21+__2
20i-1
19+~2 21±1
16±4
(g)
3.2. Effect of methoctramine on 6-keto-PGFl~ synthesis elicited by APE (fig. 2) to
-10
o_ a. v
-20
~:
A P E (1-5 n m o l ) increased in a d o s e - d e p e n d e n t m a n n e r the o u t p u t of 6 - k e t o - P G F l ~ (range 62 + 7 to 180 + 15% a b o v e b a s a l level). A d m i n i s t r a t i o n o f 3 n m o l A P E significantly i n c r e a s e d the o u t p u t of 6 - k e t o - P G F l ~ (102 + 23% a b o v e basal, n = 5; P < 0.05); this effect was not altered in the presence of 0.1 o r 0.5 ~tM m e t h o c t r a m i n e . A t higher c o n c e n t r a t i o n s of 0.75 or 1.0
ACh (nmol) 1.0
3.0
5.0
,°° I-
v
>ID
°f
. 69+7
55--+6 70+-7
61 +-_7 55+-7
Fig. 4. Effects of methoctramine on heart rate (beats per minute
(bpm)), developed tension (g), and coronary perfusion pressure (nun Hg) in the isolated perfused rabbit heart elicited by ACh (3 nmol). ACh was injected as a bolus in a volume of 0.1 ml into the aortic cannula. Data are expressed as means+ S.E. * Denotes values significantly different from the basal level, whereas 1 denotes a significant difference between the values in the presence of methoctramine and vehicle (VEH) (P < 0.05).
10.0
ca. 75E :~ v 50
~c
Basal Pedusion Pressure (ram Hg)
10 E 2°
C
/~M, m e t h o c t r a m i n e a b o l i s h e d the 6 - k e t o - P G F l ~ o u t p u t induced by APE.
3.3. Effect of methoctramine on cardiac parameters of the heart induced by ACh (fig. 3) A C h (1-10 n m o l ) significantly d e c r e a s e d the h e a r t rate a n d m y o c a r d i a l d e v e l o p e d tension in a d o s e - d e p e n d e n t f a s h i o n (fig. 3a a n d b). A C h (1 n m o l ) p r o d u c e d a significant d e c r e a s e in c o r o n a r y p e r f u s i o n pressure (15 + 2 m m Hg, n = 6; P < 0.05). I n c r e a s i n g the d o s e of A C h to 3 n m o l d i d n o t p r o d u c e a n y further decrease in c o r o n a r y p e r f u s i o n pressure. A higher dose of 10 n m o l A C h p r o d u c e d a b i p h a s i c effect, a decrease in pressure f o l l o w e d b y a n i n c r e a s e in c o r o n a r y p e r f u s i o n pressure
(fig. 3c). &-- ~
60
40
~
2o
o_ Fig. 3. Effects of A C h on heart rate (beats per minute (bpm)), developed tension (g), and coronary perfusion pressure (ram Hg) in the isolated perfused rabbit heart. A C h was injected as a bolus in a volume of 0.1 ml into the aortic cannula.
I n f u s i o n of m e t h o c t r a m i n e at a low c o n c e n t r a t i o n of 0.1 # M m i n i m i z e d the d e c r e a s e in h e a r t rate p r o d u c e d b y 3 n m o l A C h . I n c r e a s i n g the c o n c e n t r a t i o n of m e t h o c t r a m i n e to 0 . 5 - 1 . 0 / ~ M a b o l i s h e d the d e c r e a s e in h e a r t rate c a u s e d b y 3 n m o l A C h (fig. 4). T h e A C h - i n d u c e d d e c r e a s e in d e v e l o p e d t e n s i o n (2 +__0.5 g, n = 6, P < 0 . 0 5 ) was significantly r e d u c e d at 0.1 # M a n d
67
abolished at 0.5/~M methoctramine. Methoctramine at 0.1-0.75/~M did not alter the fall in coronary perfusion pressure produced by ACh, but minimized it at 1.0/~M, which may be due to its non-selective effect at this concentration.
3.4. Effects of methoctramine on actions of A P E on cardiac parameters of the heart APE at a dose of 1-5 nmol produced a significant, dose-dependent decrease in heart rate and developed tension (fig. 5a and b). APE at a dose of 1 nmol produced a biphasic effect on coronary perfusion pressure, a decrease followed by an increase in coronary perfusion pressure (fig. 5c). Increasing the dose of APE to 3 nmol did not produce any further change in coronary perfusion pressure. Infusion of methoctramine at a low concentration of 0.1-1 # M minimized in a concentration-dependent manner the decrease in heart rate produced by APE (3 nmol) (fig. 6). The APE-induced decrease in developed tension (3 + 1, n = 6; P < 0.05) was not altered by 0.1 /zM methoctramine, but was abolished by 0.5-1.0/~M of methoctramine (fig. 6). The secondary increase, but not the initial decrease, in coronary perfusion pressure caused by APE was inhibited by methoctramine (0.1-0.75/~M) in a concentration-dependent fashion. Methoctramine at a higher con-
APE (nmol) 1.0
3.0
5.0
100p
--r
50 b
l¸ "~ =
20~
Q-O 0.--
~
lO
N~-
o
-~
> ~
C
t~
¢. ~ 6 0
'°f
VEH
-
METHOCTRAMINE (pM_) 0.1 0.5 0.75 1.0
-5o
- oo° E
Basal Heart Rate (bpm)
151± 7
137± 7
153± 7
21 +_2
20L-_1
19+_2
161_+ 1
155_+ 5
"10
_o.~
Basat Developed Tension (g)
21 _+1
16_+4
3O 1
*
1"
1"
v.15[ n
-30 Basal Perfusion Pressure (rnm Hg)
62_.~7 55_+6 70+7
61_+7
55_+7
Fig. 6. Effects of methoctramine on heart rate (beats per minute (bpm)), developed tension (g), and coronary perfusion pressure ( m m Hg) in the isolated perfused rabbit heart elicited by APE (3 nmol). APE was injected as a bolus in a volume of 0.1 ml into the aortic cannula. Data are expressed as means + S.E. * Denotes values significantly different from the basal level, whereas t denotes a significant difference between the values in the presence of methoctramine and vehicle (VEH) (P < 0.05).
centration (1.0 /~M) minimized the decrease as well as the increase in coronary perfusion pressure induced by APE, probably due to its non-selective effect at this concentration. The IC50 and affinity estimate of methoctramine for APE-induced changes in the mechanical parameters of the heart are shown in tables 1 and 2. Methoctramine was much more potent in reducing the effect of APE in decreasing heart rate or increasing coronary perfusion pressure (pA 2 of 7.83 + 0.12 and 7.73 _+ 0.16, respectively) than in decreasing coronary perfusion pressure (pA 2 of 6.01 + 0.17).
3.5. Effects of combined admin&tration of methoctramine (M e antagonist) with different concentrations of HHSiD (M 3 antagonist) on the action of ACh on 6-keto-PGFl~ output (table 3)
g EE40 Q) O_
20
Fig. 5. Effects of different doses of APE on heart rate (beats per minute (bpm)), developed tension (g), and coronary perfusion pressure ( m m Hg) in the isolated perfused rabbit heart. A C h was injected as a bolus in a volume of 0.1 ml into the aortic cannula.
H H S i D (0.01-1.0 #M), a M 3 receptor antagonist, reduced the output of 6-keto-PGFl~ caused by ACh in a concentration-dependent manner (Jaiswal et al., 1988). The effect of ACh in increasing 6-keto-PGFl~ output was significantly enhanced during infusion of 0.1 /~M
68 TABLE 3 Effects of methoctramine (0.1/~M) + H H S i D (0.01-0.1/~M) on 6-ketoP F G l a synthesis elicited by A C h (3 nmol). Values are means ± S.E. of five to seven experiments. Drug
6-keto-PGFl~
ACh A C h + methoctramine (0.1 #M) A C h + H H S i D (0.01/~M) ACh+HHSiD(0.05#M) A C h + H H S i D (0.1/~M)
Basal (ng/min)
Increase (% above basal)
1.47±0.14 2.02±0.27 1.31±0.15 1.20+0.11 2.14+0.23
51± 9 105±18 42± 5 17+ 6 2+ 2
a a,b a,c a,c c
a Values significantly different from basal, b Significant difference between the values in the presence of methoctramine and ACh. c Significant different between the values in the presence of methoctramine + HHSiD from that obtained with methoctramine.
methoctramine (fig. 1). A simultaneous infusion of HHSiD at 0.01 #M, which by itself did not alter the ACh-induced 6-keto-PGFl~ output (82 + 17 and 7 0 _ 19% above basal in the absence and presence of 0.01 /~M of HHSiD, respectively; n = 5), inhibited the potentiating effect of methoctramine on ACh (table 3). A further increase in the concentration of HHSiD to 0.05 and 0.1 ~tM abolished the 6-keto-PGF~ output produced by ACh. Methoctramine at a concentration of 0.75 /~M significantly reduced the ACh-induced PG output (fig. 1). A simultaneous infusion of 0.05 /~M HHSiD produced an additive effect in attenuating PG output (data not shown). 3.6. Effects of combined administration of methoctramine and HHSiD on actions of A Ch on mechanical parameters of the heart (table 4) We have shown in a previous study (Jaiswal et al., 1988) that HHSiD (0.01-0.1 /~M) did not alter the TABLE 4
decrease in heart rate, but attenuated the decrease in developed tension and fall in coronary perfusion pressure produced by ACh. Methoctramine a M 2 receptor antagonist, significantly reduced the decrease in heart rate and developed tension caused by ACh, and these changes were not altered by simultaneous infusion of HHSiD (0.01-0.1 #M). The effect of HHSiD in attenuating the ACh-induced fall in coronary perfusion pressure was not altered in the presence of methoctramine. 3. 7. Effects of methoctramine on the actions of isoproterenol and arachidonic acid A bolus injection of isoproterenol (0.4 nmol) into the heart significantly increased the 6-keto-PGFl~ output (114 + 13% above basal), heart rate by 122 + 6 beats/min, and myocardial developed tension by 10.5 + 1.8 g and decreased the coronary perfusion pressure by 11+ 3 mm Hg. The changes in PG output and mechanical function of the heart produced by isoproterenol were not affected by the muscarinic receptor antagonist methoctramine (data not shown). Administration of arachidonic acid (33 nmol) produced a 4- to 5-fold increase in 6-keto-PGF1, ~ output (from 3.7 + 0.8 to 13.6 + 1.1 ng/min) (P < 0.05, n = 5) and a decrease in coronary perfusion pressure (10 + 4 mm Hg) without altering the basal heart rate or developed tension. The basal as well as arachidonic acid-induced 6-keto-PGFl~ output and the decrease in coronary perfusion pressure was not altered by methoctramine (data not shown). Indomethacin (2.8 t~M), a cyclooxygenase inhibitor, reduced the basal and abolished the ACh-, APE-, isoproterenol- and arachidonic acid-induced PG output without altering the cardiac responses (data not shown).
4. Discussion
Effect of methoctramine (0.1 ~ M ) + H H S i D (0.01-0.1/~M) on mechanical parameters of the heart elicited by A C h (3 nmol). Values are means 5-S.E. of five to even experiments. Drug
Heart rate (bpm)
Developed Perfusion pressure tension (g) (ram Hg)
ACh (Basal) A C h + methoctramine (Basal) A C h + HHSiD (0.01/~M) (Basal) A C h + HHSiD (0.05 tzM) (Basal) A C h + HHSiD (0.1/zM) (Basal)
-16±1 a (167 + 7) - 5 ± 1 a,b (118±10) -5±1 a (155 ± 3) -5±1 a (142±2) -5+1 a (132±9)
-2±1 ~ (17 ± 1) 1± 1 (18±1) 0 (17 + 1) 0 (17±1) 0 (17±1) -
-
-21+4 a (64 ± 4) - 25 + 3 a (74±2) - 1 6 ± 2 a'c (66 + 3) - 1 0 ± 2 a'c (68±1) - 6 ± 2 ax (73+3)
a Values significantly different from basal, b Significant difference between the values in the presence of methoctramine and ACh. c Significant difference between the values in the presence of m e t h o c t r a m i n e + HHSiD from that obtained with methoctramine.
Our recent studies in the isolated rabbit heart (Jaiswal and Malik, 1988; Jaiswal et al., 1988) indicate that: (1) the increase in cardiac PG synthesis and the decrease in myocardial developed tension elicited by cholinergic stimuli are mediated via activation of M 2, as well as M 3 muscarinic receptors because AF-DX 116, a M 2 receptor antagonist, and HHSiD, a M 3 receptor antagonist, attenuated the PG output elicited by ACh or APE; (2) the decrease in heart rate and the increase in coronary perfusion pressure is due to activation of M z receptors because AF-DX 116, but not HHSiD, inhibited the decrease in heart rate and the increase in coronary perfusion pressure elicited by ACh or APE; and (3) the decrease in coronary perfusion pressure caused by ACh or APE was selectively blocked by HHSiD, but not by AF-DX 116, suggesting that it is due to activation of
69 M 3 receptors. The present study, which was undertaken to determine the antimuscafinic effects of the cardioselective M2 receptor antagonist methoctramine, indicates that this agent is highly selective for cardiac M 2 receptors. In the isolated rabbit heart, methoctramine at 0.1 /tM potentiated the effect of ACh in enhancing 6-ketoPGFI~ output. This effect of methoctramine was similar to that of the M 2 receptor antagonist AF-DX 116 (Jaiswal and Malik, 1988; Jaiswal et al., 1988). Since the potentiating effect of methoctramine on ACh-induced 6-keto-PGF~, output was significantly reduced by the simultaneous infusion of H H S i D (M 3 antagonist), it would appear that ACh has a higher affinity for M 3 receptors; thus, the blockade of a small population of M2 receptors with a lower c o n c e n t r a t i o n of methoctramine probably allowed the expression of a greater effect of ACh on PG output through activation of M 3 receptors. Moreover, H H S i D showed an additive effect with methoctramine in blocking the increase in 6-keto-PGFl~ output induced by ACh. The selectivity of methoctramine in attenuating ACh- and APE-induced PG synthesis was indicated by our finding that methoctramine did not alter the increase in 6-keto-PGF~ output induced by the non-cholinergic agent isoproterenol or by arachidonic acid. The mechanism by which ACh and APE enhanced PG output might be due to either an augmented mobilization of arachidonic acid from the phospholipid stores or to an increase in cyclooxygenase activity. Our finding that the metabolism of exogenous arachidonic acid to 6-keto-PGFl~ was unaffected by methoctramine suggests that stimulation of PG synthesis does not involve activation of cyclooxygenase, but is most likely due to increased release of arachidonic acid from tissue lipids. Although PGs are synthesized by various cell types in the heart, the conversion of arachidonic acid to 6-keto-PGFl~ occurs predominantly in endothelial cells of the coronary vessels in the heart (Wennmalm, 1979). In view of these observations and the demonstration that coronary vessels contain both M 2 and M 3 receptors, it is possible that the major site of PG synthesis ellicited by cholinergic stimuli is in the coronary vessels. The finding that methoctramine, but not HHSiD, a M 3 receptor antagonist, abolished the decrease in heart rate produced by ACh or APE suggest that bradycardia produced by cholinergic stimuli is mediated via activation of a distinct subtype of muscarinic receptor, most likely M 2. A corollary of this conclusion is that methoctramine is a selective cardiac M 2 receptor antagonist. The decreases in developed tension produced by ACh and APE appeared to be due to activation of both M 2 and M 3 receptors, because they were minimized by both methoctramine and HHSiD. That methoctramine selectively blocks M e receptors in the rabbit heart was also indicated by our finding that the decrease in coronary perfusion pressure produced by
ACh or APE that is due to stimulation of M 3 receptors and is blocked by H H S i D (Jaiswal et al., 1988) was not altered by methoctramine. Moreover, APE produced a biphasic effect on coronary perfusion pressure, an initial fall followed by a rise in coronary perfusion pressure; and only the later component of the response that has been reported to be due to stimulation of M 2 receptors (insensitive to blockade by pirenzepine or HHSiD) (Jaiswal an Malik, 1988; Jaiswal et al., 1988) was attenuated by methoctramine. Methoctramine has also been reported to display 100 times greater affinity for the muscarinic receptor involved in the action of cholinergic stimuli in producing negative inotropic and chronotropic effects in guinea pig atria than for that involved in mediating tracheal contraction (Giraldo et al., 1988). Methoctramine was also a more potent muscarinic receptor antagonist for the bradycardiac effect than for the increases in bladder tone, salivary secretion, and hypotension produced by muscarinic agents in anesthetized cats (Giraldo et al., 1988). In a direct binding study, methoctramine also exhibited a higher affinity for cardiac (M2) than for exocrine gland muscarinic receptors (M3) and a 16-fold higher affinity for M 2 than for M~ receptors (Michel and Whiting, 1988). In conclusion, this study demonstrates that methoctramine is a cardioselective M 2 receptor antagonist. Moreover, the results of this study in conjunction with our previous study (Jaiswal et al., 1988) suggest that the effect of ACh receptor agonists in promoting 6-ketoP G F ~ synthesis and in producing a decrease in myocardial developed tension is due to activation of M 2 and M 3 receptors. The decrease in heart rate and the increase in coronary perfusion pressure are mediated via activation of M 2 receptors, whereas the decrease in coronary perfusion pressure is mediated via M 3 receptors. The pharmacological profile of methoctramine was similar to that of AF-DX 116, a cardioselective M 2 receptor antagonist (Jaiswal and Malik, 1988; Jaiswal et al., 1988; Hammer et al., 1986; Giachetti et al., 1986; Micheletti et al., 1987; Duckies et al., 1987). Methoctramine has been reported to exhibit a 10-fold higher degree of cardioselectivity than AF-DX 116 (Giachetti et al., 1986; Micheletti et al., 1987).
Acknowledgements The authors thank Anne Estes and Bruce January for technical assistance and various pharmaceutical companies (see Materials and methods) for providing various drugs.
References Birdsall, N.J.M., C.A.M. Curtis, P. Eveleigh,E.C. Huime, E.K. Pedder, D. Poyner, J.M. Stockton and M. Wheatly, 1987, Muscarinic receptor subclasses, in: eds. M.J. Dowdall and J.N. Hawthorne,
70 Cellular and Molecular Basis of Cholinergic Function (Ellis Horwood Ltd., Chichester, England) p. 46. Birdsall, N.J.M. and E.C. Hulme, 1983, Muscarinic receptor subclasses, Trends Pharmacol. Sci. 4, 459. Doods, H.N., M.J. Mathy, D. Davidesko, K.J. Van Charldorp, A. De Jonge and P.A. Van Zwieten, 1987, Selectivity of muscarinic antagonists in radioligand and in vivo experiments for the putative M1, M2 and M3 receptors, J. Pharmacol. Exp. Ther. 242, 257. Duckles, S.P., H.I. Yamamura and V. Lee, 1987, AF-DX 116 discriminates between muscarinic M2 receptors of the heart and vasculature, Life Sci. 40, 1507. Eglen, R.M. and R.L. Whiting, 1986, Differential affinities of muscarinic antagonists at ileal and atrial receptors, Br. J. Pharmacol. 87 (Suppl.), 33P. Giachetti, A., R. Micheletti and E. Montagana, 1986, Cardioselective profile of AF-DX 116, a muscarinic M2 receptor antagonist, Life Sci. 38, 1663. Gilani, A.S. and L.B. Cobbin, 1986, The cardio-selectivity of himbiacine: a muscarinic receptor antagonist, Naunynn-Sehmiedeb. Arch. Pharmacol. 332, 16. Giraldo, E., R. Michelleti, E. Montagna, A. Giachetti, M.A. Vigano, H. Ladinsky and C. Melchoirre, 1988, Binding and functional characterization of the cardioselective muscarinic antagonist methoctramine, J. Pharmacol. Exp. Ther. 244, 1016. Hammer, R., C.P. Berrie, N.J.M. Birdsall, A.S.V. Burgen and E.C. Hulme, 1980, Pirenzepine distinguishes between different subclasses of muscarinic receptors, Nature (London) 283, 90. Hammer, R. and A. Giachetti, 1982, Muscarinic receptor subtypes: M-1 and M-2, Biochemical and functional characterization, Life Sci. 31, 2991. Hammer, R., E. Giraldo, G.B. Schiavi, E. Monferini and H. Ladinsky, 1986, Binding profile of a novel cardioselective muscarinic receptor antagonist, AF-DX 116, to membranes of peripheral tissues and brain in the rats, Life Sci. 38, 1653. Harden, T.K., L.I. Tanner, M.W. Martin, N. Nakahata, A.R. Hughes, J.R. Hepler, T. Evans, S.B. Masters and J.H. Brown, 1986, Characteristics of the two biochemical responses to stimulation of muscarinic cholinergic receptors, Trends Pharmacol. Sci. (Suppl.), 14.
Jaiswal, N., G. Lambrecht, E. Mutschler and K.U. Malik, 1988, Contribution of M2, and M2fl muscarinic receptors to the action of cholinergic stimuli on prostaglandin synthesis and mechanical function in the isolated rabbit heart, J. Pharmacol. Exp. Ther. 247, 104. Jaiswal, N. and K.U. Malik, 1988, Prostaglandin synthesis elicited by cholinergic stimuli is mediated by activation of M2 muscarinic receptors in rabbit heart, J. Pharmacol. Exp. Ther. 245, 59. Lambrecht, G., U. Moser, J. Riotte, M. Wagner, J. Wess and E. Mutschler, 1987, Heterogeneity in muscarinic receptors: evidence from pharmacological and electrophysiological studies with selective antagonists, in: International Symposium on Muscarinic Cholinergic Mechanisms, eds. S. Cohen and M. Sokolovsky, (TelAviv) p. 245. MacKay, D., 1978, How should values of pA 2 and affinity constants for pharmacological competetive antagonists be estimated, J. Pharm. Pharmacol. 30, 312. Melchiorre, C., A. Cassinelli and W. Quaglia, 1987, Differential blockade of muscarinic receptor subtypes by polymethylene tetraamines. Novel class of selective antagonists of cardiac M-2 muscarinic receptors, J. Med. Chem. 30, 201. Michel, A.D. and R.L. Whiting, 1988, Methoctramine, a polymethylene tetraamine, differentiates three subtypes of muscarinic receptors in direct binding studies, European J. Pharmacol. 145, 61. Micheletti, R., E. Montagna and A. Giachetti, 1987, AF-DX 116, a cardioselective muscarinic antagonist, J. Pharmacol. Exp. Ther. 241,628. Mutschler, E., G. Gmelin, U. Moser, J. Wess and G. Lambrecht, 1987, Structure-activity relationships of drugs acting selectively at different muscarinic receptors, in: Pharmacology, International Congress Series NO, 750, eds. M.J. Rand and C. Raber (Excerpta Medica (Elsevier), Amsterdam) p. 67. Mutschler, E. and G. Lambrecht, 1984, Selective muscarinic agonists and antagonists in functional tests, Trends Pharmacol. Sci. 5 (Suppl.), 39. Wennmalm, A., 1979, Prostaglandin-mediated inhibition of noradrenalin release. VI. On the intra-cardiac source of prostraglandins released from isolated rabbit heart, Acta Physiol. Scand. 105, 254.
63
© 1991 ElsevierScience Publishers B.V. (BiomedicalDivision) 0014-2999/91/$03.50 ADONIS 001429999100068P EJP 51649
Methoctramine, a cardioselective antagonist: muscarinic receptor mediating prostaglandin synthesis in isolated rabbit heart N e e l a m Jaiswal a n d K a f a i t U. M a l i k Department of Pharmacology, College of Medicine, The University of Tennessee at Memphis, Memphis, TN 38163, U.S.A.
Received 28 May 1990, revisedMS received27 August 1990, accepted 9 October 1990 The antimuscarinic properties of the methoctramine with high selectivity for cardiac muscarinic M 2 receptors were investigated on cholinergically induced changes in prostaglandin (PG) synthesis and mechanical function in the isolated perfused rabbit heart. Acetylcholine (ACh)- and arecaidine propargyl ester (APE)-induced increases in PG synthesis were significantly attenuated by methoctramine in a concentration-dependent manner, Methoctramine at a low concentration of 0.1/tM potentiated ACh-induced PG synthesis, which was blocked by simultaneous infusion of hexahydro-sila-difenidol (HHSiD), a M,, receptor antagonist. Methoctramine produced an additive effect with HHSiD in diminishing the ACh- or APE-induced PG synthesis. Methoctramine displayed a potent antagonistic activity at M 2 receptors that mediate the decrease in heart rate and increase in coronary perfusion pressure in isolated perfused rabbit heart. Methoctramine also minimized ACh- and APE-induced decrease in developed tension. In contrast, at 0.1-0.75/~M it exhibited no antagonistic activity at vascular muscarinic receptors (M3) mediating vasodilation in response to ACh or APE. These data suggest that methoctramine has a high affinity for cardiac M 2 receptors mediating increases in PG output and coronary perfusion pressure as well as decrease in heart rate and developed tension and has a low affinity for M 3 receptors mediating coronary vasodilator response. Methoctramine; Muscarinic receptor subtypes; Cardioselectivity; Cardiac muscarinic M2a receptors; Prostaglandin synthesis I. Introduction A large number of pharmacological and biochemical studies have shown that there are subclasses of muscarinic receptors (Hammer and Giachetti, 1982; Birdsall and Hulme, 1983; Harden et al., 1986). Evidence from direct binding studies has indicated that there are at least two subtypes of muscarinic receptors distinguished by their affinity for the antagonist pirenzepine (Hammer et al., 1980). The M 1 receptor subtype (displaying high affinity to pirenzepine) is located primarily in the central nervous system and autonomic ganglia, whereas the M 2 subtype (showing low affinity to pirenzepine) is present mainly in effector cells in peripheral organs such as the heart and ileum and in certain parts of the central nervous system (Hammer and Giachetti, 1982). Several lines of evidence from functional and biochemical studies have indicated that ileal and atrial muscarinic receptors differ from each other (Mutschler and Lambrecht, 1984; Eglen and Whiting, 1986; Gilani and Cobbin, 1986; Giachetti et al., 1986; Melchiorre et al., 1987). These observations
Correspondence to: K.U. Malik, Department of Pharmacology,College of Medicine, 874 Union Avenue, Crowe No. 100, Memphis, TN 38163, U.S.A.
have led to further classification of muscarinic receptors into M1, M z and M 3 receptors (Lambrecht et al., 1987; Birdsall et al., 1987; Doods et al., 1987; Mutschler et al., 1987). Our recent studies in the isolated rabbit heart suggest that prostaglandin (PG) synthesis in the rabbit heart elicited by acetylcholine (ACh) receptor agonists is mediated via activation of M2~ (M2) as well as MEB (M3) receptors. Supporting this proposal, we have shown that PG synthesis elicited by ACh and arecaidine propargyl ester (APE) (Mutschler and Lambrecht, 1984) was attenuated by 11-[2-[(diethylamino)methyl]-l-piperidinyl]acetyl]-5,11-dihydro-6-H-pyrido-[2,3,-b][1,4]-benzodiazepine-6-one (AF-DX116) (a selective M E receptor antagonist) (Hammer et al., 1986) and by hexahydrosila-difenidol (HHSiD) (having a greater affinity for ileal, tracheal, and urinary bladder muscarinic receptors than for atrial muscarinic receptors and called M 3 receptor antagonist) (Jaiswal and Malik, 1988; Jaiswal et al., 1988). The recent discovery that methoctramine, a polymethylene tetramine compound, is about 270 times more potent in inhibiting stimulation of cardiac muscarinic receptors than in antagonizing muscarinic receptor-stimulated contractions in smooth muscle (Melchiorre et al., 1987) suggests that this compound is a selective muscarinic M 2 receptor antagonist. The present study was undertaken to determine the selectivity
64 of methoctramine for M 2 muscarinic receptors involved in the action of ACh receptor agonists on cardiac PG synthesis and changes in mechanical function in the isolated rabbit heart.
2. Materials and methods
Male New Zealand White rabbits weighing 1.0-2.0 kg (Myrtle's Rabbitry, Thompson's Station, TN) were used in all experiments. After anesthesia with i.v. sodium pentobarbital, 30 mg/kg (Abbot Laboratory, North Chicago, IL), the abdomen was opened and heparin (1000 U/kg) administered in the vena cava. After thoractomy the heart was quickly removed, placed into a warmed chamber (37 o C), and perfused at a rate of 18 ml/min with a Harvard peristaltic pump (Harvard Apparatus Co., Inc., MiUis, MA) according to the technique of Langendorff with oxygenated (95 % 02-5 % CO 2) Krebs-Henseleit buffer of the following composition (millimolar): 114 NaC1, 4.7 KC1, 1.2 KH2PO4, 25.0 NaHCO3, 2.5 CaC12, 1.2 MgSO4 and 5.5 glucose. The coronary perfusion pressure was measured with a Statham pressure transducer (P2306; Statham Medical Instruments, Los Angeles, CA) attached to the aortic cannula. Myocardial tension was measured with a Grass FT.03C force-displacement transducer (Grass Instruments Co., Quincy, MA) connected to a stainless steel hook attached to the left ventricular apex. The transducer was adjusted to provide a diastolic tension of 2 g. The heart rate was measured with a tachometer triggered by the ventricular contractions. The experimental period began after 20 min had elapsed. At this time, any heart that failed to develop at least 8 g of systolic tension was discarded. 2.1. Experimental protocol Hearts were perfused for 20 rain without any experimental intervention. Perfusate was then collected for 1 rain before the injection of a bolus dose of drug into the arterial circuit. As there was a 35-45 s delay between the time the injection was made and the time the agonist reached the heart, 1 rain was allowed to elapse before a second 1-min perfusate collection was made. Four experimental protocols were followed. 2.1.1. Protocol 1 The first series of experiments was performed to investigate the effects of different doses (1-10 nmol) of ACh and APE on PG output and mechanical responses of the heart. McN-A-343, a Ml-selective agonist, was not used in this series of experiments because we had demonstrated in a previous study that it does not alter the basal PG synthesis as well as mechanical responses of the heart at a concentration at which ACh and APE produced significant effects (Jaiswal and Malik, 1988).
After a 20-min stabilization period, ACh or APE was administered in a random order as a bolus at 10-rain intervals for up to 120 min of perfusion of the heart. Drugs were administered between 20-120 min of perfusion of the heart because the basal output of PGs was found to be stable during this period. Moreover, heart rate, myocardial tension and coronary perfusion pressure also remained stable during the time course of the experiment. A second series of experiments was performed to investigate the effects of methoctramine on PG output and on the mechanical responses of the heart. The experimental protocol consisted four or five periods, separated by 15-rain intervals; ACh (3 nmol), APE (3 nmol), arachidonic acid (33 nmol), or the respective vehicle, contained in 100 #1 saline (0.9% NaC1), was administered as a bolus during all periods. After collecting the perfusate samples and measuring cardiac parameters during administration of the above drugs in the first period, the heart was infused with the M E muscarinic receptor antagonist methoctramine at a lower dose in the second period and with higher doses in consecutive periods. 2.1.2. Protocol 2 This series of experiments was performed to determine the effects of combined administration of 0.1 or 0.75 #M methoctramine with different doses of HHSiD on PG output and on changes in mechanical function of the heart produced by 3 nmol ACh. In a previous study, we had shown that HHSiD, a M3-selective antagonist, significantly reduced ACh- or APE-induced PG synthesis in a concentration-dependent manner and produced an additive effect with the M 2 receptor blocker AF-DX 116 (Jaiswal and Malik, 1988). We therefore investigated the effects of methoctramine combined with different concentrations of HHSiD. The experimental protocol consisted of five periods, separated by 15-min intervals. ACh or its vehicle was administered as a bolus in all five periods. After administering ACh in the first period, 0.1 or 0.75 /~M methoctramine was infused in the second period, and then 0.1 or 0.75 #M methoctramine plus 0.01, 0.05, or 0.1/tM HHSiD in the third, fourth, and fifth periods, respectively. 2.1.3. Protocol 3 This series of experiments was performed to determine the effects of methoctramine (1.0 #M) and the cyclooxygenase inhibitor indomethacin (2.8 #M) on PG output elicited by administration of a bolus injection of ACh (3 nmol), APE (3 nmol), isoproterenol (0.4 nmol), or arachidonic acid (33 nmol) as described in the first series of experiments. In all four series, perfusate samples were collected for l-rain periods immediately before and after administration of drugs and were then frozen ( - 2 0 ° C) for later PG determination.
65 n=6
2.2. Determination of PG
VEH
METHOCTRAMINE 0.1
6-keto-PGFl~ concentration was determined in the perfusate sample by radioimmunoassay Briefly, 100 /~1 of sample was mixed with 2 000-3 000 cpm of tracer plus the appropriate concentration of antibody. The tracer and antibody were prepared in a buffer consisting of (g/l) 1.0 NaN 3, 9.0 NaC1, 6.8 KH2PO4, 26.1 KzHPO4 26 and 2.0 gelatin. The tubes were vortexed and incubated overnight at 4 ° C. Bound tracer was separated from free tracer by adding 1.0 ml of dextran-coated charcoal to each tube, and radioactivity was determined by liquid scintillation spectroscopy. The antibody for 6-keto-PGFl~ was provided by Dr. C. Leffler of the Department of Physiology and Biophysics, University of Tennessee. Cross-reactivity of the 6-keto-PGF1, antibody was less than 0.1% with thromboxane B2, and 13,14-dihydro-15-keto-PGE 2 and less than 0.5% with PGE 2 and PGF2~.
2.3. Drugs ACh, atropine, arachidonic acid and indomethacin were purchased from Sigma Chemical Co., St. Louis, MO. Methoctramine was purchased from Research Biochemicals, Inc., Natick, MA. APE and HHSiD were gifts from Dr. G. Lambrecht (Pharmakologisches Institut, Frankfurt F.R.G.).
2.4. Analysis of data The results are expressed as means + S.E.M. The data were analyzed by analysis of variance, and the differences between means were determined by Student's t-test for paired observations. The null hypothesis was rejected at P < 0.05. The basal PG output represents the amount of PG in samples collected for l-rain periods immediately before drug administration and is expressed as ng/min of immunoreactive 6-keto-PGFl~. Although the basal PG output during the course of the experiment remained constant in each heart, it was variable among different hearts. Therefore, the rise in PG output ehcited by various agents was expressed as percent increase above basal. The pA 2 value for methoctramine was presented as log (antagonist)/(dose r a t i o - 1), and the mean values were calculated as described by Mackay (1978).
3. Results
ACh at a dose of 1-10 nmol significantly enhanced the output of 6-keto-PGFl~ in a dose-dependent manner
0.75
1.0
300 -
~
200
~
100
CL
""
*t 0 Basal 6-Keto-PGFt~ (ng/min)
2.33_+ 0.64
1.97+ 0.90
2.39_+ 2.63_+ 0.28 0.62
2.80_+ 0.94
Fig. 1. Effect of of methoctramine on the output of 6-keto-POF],~ elicited by acetylcholine (ACh, 3 nmol) in the isolated perfused rabbit heart. A C h was injected as a bolus in a volume of 0.1 rnl into the aortic cannula. Data are expressed as means_+ S.E. * Denotes values significantly different from the basal level, whereas -t denotes a significant difference between the values in the presence of methoctramine and vehicle (VEH) (P < 0.05).
(range 42 + 5 to 250 + 21% above basal level). Methoctramine at 0.1/~M potentiated the effect of ACh (3 nmol) in increasing 6-keto-PGFt~ output in the rabbit heart (from 100 + 9 to 232 + 34% above basal). The effect of ACh in increasing 6-keto-PGFl~ output was not potentiated or altered at 0.5 #M methoctramine, but it was reduced at 0.75 /~M methoctramine; increasing the methoctramine concentration to 1.0 #M abolished the effect of ACh in increasing 6-keto-PGFl~ output. Methoctramine displayed an IC50 of 0.73 + 0.025 /~M and a pA 2 of 7.21 __+0.14 for inhibiting ACh-induced PG synthesis (tables 1 and 2). TABLE 1 Comparison of the IC5o of methoctramine for antagonizing the increase in 6-keto-PFGx,~ output and the decreases in heart rate and changes in coronary perfusion pressure (CPP) elicited by 3 nmol of A C h or APE. Values are means 5: S.E. Parameters
6-keto-PGF1, , Heart rate CPP (decrease) CPP (increase)
ICs0 (/~M) ACh
APE
0.73 + 0.025 0.09 + 0.003 3.13 5:0.62
0.59 5:0.13 0.14 5:0.015 1.30 5:0.20 0.23 5:0.04
TABLE 2 Affinity (PA2) of methoctramine for inhibiting the increase in 6-ketoPFG1, , output and the decreases in heart rate and changes in coronary perfusion pressure (CPP) elicited by 3 nmol of A C h or APE. Values are means + S.E. Parameters
3.1. Effect of methoctramine on 6-keto-PGFl~ synthesis ellicited by A Ch (fig. 1)
0.5
(p.M)
6-keto-PGF m Heart rate CPP (decrease) CPP (increase)
pA 2 values fo methoctramine ACh
APE
7.21 + 0.14 7.90 5:0.16 5.34 ___0.09
7.69 5:0.43 7.83 + 0.12 6.01 5:0.17 7.73 5:0.16
66 n=6
VEH
METHOCTRAMINE 0.1
° >e
0.75
VEH
1.0
METHOCTRAMINE
/I.tM)
0.1
0.5
0.75
1.0
145+5
165+7
160+ 2
153+5
•
2°° f
6
0.5
(laM)
100t -7- ~
01-
Basal
6-Keto-PGF
(ng/min) let
1.65±
0.42
3.20+-
1.20
2.84±
0.49
2.84+-
0.27
2.17+-
0.41
Fig. 2. Effect of methoctramine on the output of 6-keto-PGF]a elicited by arecaidine propargyl ester (APE, 3 nmol) in the isolated perfused rabbit heart. APE was injected as a bolus in a volume of 0.1 ml into the aortic cannula. Data are expressed as means+ S.E. * Denotes values significantly different from the basal level, whereas t denotes a significant difference between the values in the presence of methoctramine and vehicle (VEH) (P < 0.05).
-20
Basal Heart Rate (bpm)
160+7
o-~
Basal Developed Tension
21+__2
20i-1
19+~2 21±1
16±4
(g)
3.2. Effect of methoctramine on 6-keto-PGFl~ synthesis elicited by APE (fig. 2) to
-10
o_ a. v
-20
~:
A P E (1-5 n m o l ) increased in a d o s e - d e p e n d e n t m a n n e r the o u t p u t of 6 - k e t o - P G F l ~ (range 62 + 7 to 180 + 15% a b o v e b a s a l level). A d m i n i s t r a t i o n o f 3 n m o l A P E significantly i n c r e a s e d the o u t p u t of 6 - k e t o - P G F l ~ (102 + 23% a b o v e basal, n = 5; P < 0.05); this effect was not altered in the presence of 0.1 o r 0.5 ~tM m e t h o c t r a m i n e . A t higher c o n c e n t r a t i o n s of 0.75 or 1.0
ACh (nmol) 1.0
3.0
5.0
,°° I-
v
>ID
°f
. 69+7
55--+6 70+-7
61 +-_7 55+-7
Fig. 4. Effects of methoctramine on heart rate (beats per minute
(bpm)), developed tension (g), and coronary perfusion pressure (nun Hg) in the isolated perfused rabbit heart elicited by ACh (3 nmol). ACh was injected as a bolus in a volume of 0.1 ml into the aortic cannula. Data are expressed as means+ S.E. * Denotes values significantly different from the basal level, whereas 1 denotes a significant difference between the values in the presence of methoctramine and vehicle (VEH) (P < 0.05).
10.0
ca. 75E :~ v 50
~c
Basal Pedusion Pressure (ram Hg)
10 E 2°
C
/~M, m e t h o c t r a m i n e a b o l i s h e d the 6 - k e t o - P G F l ~ o u t p u t induced by APE.
3.3. Effect of methoctramine on cardiac parameters of the heart induced by ACh (fig. 3) A C h (1-10 n m o l ) significantly d e c r e a s e d the h e a r t rate a n d m y o c a r d i a l d e v e l o p e d tension in a d o s e - d e p e n d e n t f a s h i o n (fig. 3a a n d b). A C h (1 n m o l ) p r o d u c e d a significant d e c r e a s e in c o r o n a r y p e r f u s i o n pressure (15 + 2 m m Hg, n = 6; P < 0.05). I n c r e a s i n g the d o s e of A C h to 3 n m o l d i d n o t p r o d u c e a n y further decrease in c o r o n a r y p e r f u s i o n pressure. A higher dose of 10 n m o l A C h p r o d u c e d a b i p h a s i c effect, a decrease in pressure f o l l o w e d b y a n i n c r e a s e in c o r o n a r y p e r f u s i o n pressure
(fig. 3c). &-- ~
60
40
~
2o
o_ Fig. 3. Effects of A C h on heart rate (beats per minute (bpm)), developed tension (g), and coronary perfusion pressure (ram Hg) in the isolated perfused rabbit heart. A C h was injected as a bolus in a volume of 0.1 ml into the aortic cannula.
I n f u s i o n of m e t h o c t r a m i n e at a low c o n c e n t r a t i o n of 0.1 # M m i n i m i z e d the d e c r e a s e in h e a r t rate p r o d u c e d b y 3 n m o l A C h . I n c r e a s i n g the c o n c e n t r a t i o n of m e t h o c t r a m i n e to 0 . 5 - 1 . 0 / ~ M a b o l i s h e d the d e c r e a s e in h e a r t rate c a u s e d b y 3 n m o l A C h (fig. 4). T h e A C h - i n d u c e d d e c r e a s e in d e v e l o p e d t e n s i o n (2 +__0.5 g, n = 6, P < 0 . 0 5 ) was significantly r e d u c e d at 0.1 # M a n d
67
abolished at 0.5/~M methoctramine. Methoctramine at 0.1-0.75/~M did not alter the fall in coronary perfusion pressure produced by ACh, but minimized it at 1.0/~M, which may be due to its non-selective effect at this concentration.
3.4. Effects of methoctramine on actions of A P E on cardiac parameters of the heart APE at a dose of 1-5 nmol produced a significant, dose-dependent decrease in heart rate and developed tension (fig. 5a and b). APE at a dose of 1 nmol produced a biphasic effect on coronary perfusion pressure, a decrease followed by an increase in coronary perfusion pressure (fig. 5c). Increasing the dose of APE to 3 nmol did not produce any further change in coronary perfusion pressure. Infusion of methoctramine at a low concentration of 0.1-1 # M minimized in a concentration-dependent manner the decrease in heart rate produced by APE (3 nmol) (fig. 6). The APE-induced decrease in developed tension (3 + 1, n = 6; P < 0.05) was not altered by 0.1 /zM methoctramine, but was abolished by 0.5-1.0/~M of methoctramine (fig. 6). The secondary increase, but not the initial decrease, in coronary perfusion pressure caused by APE was inhibited by methoctramine (0.1-0.75/~M) in a concentration-dependent fashion. Methoctramine at a higher con-
APE (nmol) 1.0
3.0
5.0
100p
--r
50 b
l¸ "~ =
20~
Q-O 0.--
~
lO
N~-
o
-~
> ~
C
t~
¢. ~ 6 0
'°f
VEH
-
METHOCTRAMINE (pM_) 0.1 0.5 0.75 1.0
-5o
- oo° E
Basal Heart Rate (bpm)
151± 7
137± 7
153± 7
21 +_2
20L-_1
19+_2
161_+ 1
155_+ 5
"10
_o.~
Basat Developed Tension (g)
21 _+1
16_+4
3O 1
*
1"
1"
v.15[ n
-30 Basal Perfusion Pressure (rnm Hg)
62_.~7 55_+6 70+7
61_+7
55_+7
Fig. 6. Effects of methoctramine on heart rate (beats per minute (bpm)), developed tension (g), and coronary perfusion pressure ( m m Hg) in the isolated perfused rabbit heart elicited by APE (3 nmol). APE was injected as a bolus in a volume of 0.1 ml into the aortic cannula. Data are expressed as means + S.E. * Denotes values significantly different from the basal level, whereas t denotes a significant difference between the values in the presence of methoctramine and vehicle (VEH) (P < 0.05).
centration (1.0 /~M) minimized the decrease as well as the increase in coronary perfusion pressure induced by APE, probably due to its non-selective effect at this concentration. The IC50 and affinity estimate of methoctramine for APE-induced changes in the mechanical parameters of the heart are shown in tables 1 and 2. Methoctramine was much more potent in reducing the effect of APE in decreasing heart rate or increasing coronary perfusion pressure (pA 2 of 7.83 + 0.12 and 7.73 _+ 0.16, respectively) than in decreasing coronary perfusion pressure (pA 2 of 6.01 + 0.17).
3.5. Effects of combined admin&tration of methoctramine (M e antagonist) with different concentrations of HHSiD (M 3 antagonist) on the action of ACh on 6-keto-PGFl~ output (table 3)
g EE40 Q) O_
20
Fig. 5. Effects of different doses of APE on heart rate (beats per minute (bpm)), developed tension (g), and coronary perfusion pressure ( m m Hg) in the isolated perfused rabbit heart. A C h was injected as a bolus in a volume of 0.1 ml into the aortic cannula.
H H S i D (0.01-1.0 #M), a M 3 receptor antagonist, reduced the output of 6-keto-PGFl~ caused by ACh in a concentration-dependent manner (Jaiswal et al., 1988). The effect of ACh in increasing 6-keto-PGFl~ output was significantly enhanced during infusion of 0.1 /~M
68 TABLE 3 Effects of methoctramine (0.1/~M) + H H S i D (0.01-0.1/~M) on 6-ketoP F G l a synthesis elicited by A C h (3 nmol). Values are means ± S.E. of five to seven experiments. Drug
6-keto-PGFl~
ACh A C h + methoctramine (0.1 #M) A C h + H H S i D (0.01/~M) ACh+HHSiD(0.05#M) A C h + H H S i D (0.1/~M)
Basal (ng/min)
Increase (% above basal)
1.47±0.14 2.02±0.27 1.31±0.15 1.20+0.11 2.14+0.23
51± 9 105±18 42± 5 17+ 6 2+ 2
a a,b a,c a,c c
a Values significantly different from basal, b Significant difference between the values in the presence of methoctramine and ACh. c Significant different between the values in the presence of methoctramine + HHSiD from that obtained with methoctramine.
methoctramine (fig. 1). A simultaneous infusion of HHSiD at 0.01 #M, which by itself did not alter the ACh-induced 6-keto-PGFl~ output (82 + 17 and 7 0 _ 19% above basal in the absence and presence of 0.01 /~M of HHSiD, respectively; n = 5), inhibited the potentiating effect of methoctramine on ACh (table 3). A further increase in the concentration of HHSiD to 0.05 and 0.1 ~tM abolished the 6-keto-PGF~ output produced by ACh. Methoctramine at a concentration of 0.75 /~M significantly reduced the ACh-induced PG output (fig. 1). A simultaneous infusion of 0.05 /~M HHSiD produced an additive effect in attenuating PG output (data not shown). 3.6. Effects of combined administration of methoctramine and HHSiD on actions of A Ch on mechanical parameters of the heart (table 4) We have shown in a previous study (Jaiswal et al., 1988) that HHSiD (0.01-0.1 /~M) did not alter the TABLE 4
decrease in heart rate, but attenuated the decrease in developed tension and fall in coronary perfusion pressure produced by ACh. Methoctramine a M 2 receptor antagonist, significantly reduced the decrease in heart rate and developed tension caused by ACh, and these changes were not altered by simultaneous infusion of HHSiD (0.01-0.1 #M). The effect of HHSiD in attenuating the ACh-induced fall in coronary perfusion pressure was not altered in the presence of methoctramine. 3. 7. Effects of methoctramine on the actions of isoproterenol and arachidonic acid A bolus injection of isoproterenol (0.4 nmol) into the heart significantly increased the 6-keto-PGFl~ output (114 + 13% above basal), heart rate by 122 + 6 beats/min, and myocardial developed tension by 10.5 + 1.8 g and decreased the coronary perfusion pressure by 11+ 3 mm Hg. The changes in PG output and mechanical function of the heart produced by isoproterenol were not affected by the muscarinic receptor antagonist methoctramine (data not shown). Administration of arachidonic acid (33 nmol) produced a 4- to 5-fold increase in 6-keto-PGF1, ~ output (from 3.7 + 0.8 to 13.6 + 1.1 ng/min) (P < 0.05, n = 5) and a decrease in coronary perfusion pressure (10 + 4 mm Hg) without altering the basal heart rate or developed tension. The basal as well as arachidonic acid-induced 6-keto-PGFl~ output and the decrease in coronary perfusion pressure was not altered by methoctramine (data not shown). Indomethacin (2.8 t~M), a cyclooxygenase inhibitor, reduced the basal and abolished the ACh-, APE-, isoproterenol- and arachidonic acid-induced PG output without altering the cardiac responses (data not shown).
4. Discussion
Effect of methoctramine (0.1 ~ M ) + H H S i D (0.01-0.1/~M) on mechanical parameters of the heart elicited by A C h (3 nmol). Values are means 5-S.E. of five to even experiments. Drug
Heart rate (bpm)
Developed Perfusion pressure tension (g) (ram Hg)
ACh (Basal) A C h + methoctramine (Basal) A C h + HHSiD (0.01/~M) (Basal) A C h + HHSiD (0.05 tzM) (Basal) A C h + HHSiD (0.1/zM) (Basal)
-16±1 a (167 + 7) - 5 ± 1 a,b (118±10) -5±1 a (155 ± 3) -5±1 a (142±2) -5+1 a (132±9)
-2±1 ~ (17 ± 1) 1± 1 (18±1) 0 (17 + 1) 0 (17±1) 0 (17±1) -
-
-21+4 a (64 ± 4) - 25 + 3 a (74±2) - 1 6 ± 2 a'c (66 + 3) - 1 0 ± 2 a'c (68±1) - 6 ± 2 ax (73+3)
a Values significantly different from basal, b Significant difference between the values in the presence of methoctramine and ACh. c Significant difference between the values in the presence of m e t h o c t r a m i n e + HHSiD from that obtained with methoctramine.
Our recent studies in the isolated rabbit heart (Jaiswal and Malik, 1988; Jaiswal et al., 1988) indicate that: (1) the increase in cardiac PG synthesis and the decrease in myocardial developed tension elicited by cholinergic stimuli are mediated via activation of M 2, as well as M 3 muscarinic receptors because AF-DX 116, a M 2 receptor antagonist, and HHSiD, a M 3 receptor antagonist, attenuated the PG output elicited by ACh or APE; (2) the decrease in heart rate and the increase in coronary perfusion pressure is due to activation of M z receptors because AF-DX 116, but not HHSiD, inhibited the decrease in heart rate and the increase in coronary perfusion pressure elicited by ACh or APE; and (3) the decrease in coronary perfusion pressure caused by ACh or APE was selectively blocked by HHSiD, but not by AF-DX 116, suggesting that it is due to activation of
69 M 3 receptors. The present study, which was undertaken to determine the antimuscafinic effects of the cardioselective M2 receptor antagonist methoctramine, indicates that this agent is highly selective for cardiac M 2 receptors. In the isolated rabbit heart, methoctramine at 0.1 /tM potentiated the effect of ACh in enhancing 6-ketoPGFI~ output. This effect of methoctramine was similar to that of the M 2 receptor antagonist AF-DX 116 (Jaiswal and Malik, 1988; Jaiswal et al., 1988). Since the potentiating effect of methoctramine on ACh-induced 6-keto-PGF~, output was significantly reduced by the simultaneous infusion of H H S i D (M 3 antagonist), it would appear that ACh has a higher affinity for M 3 receptors; thus, the blockade of a small population of M2 receptors with a lower c o n c e n t r a t i o n of methoctramine probably allowed the expression of a greater effect of ACh on PG output through activation of M 3 receptors. Moreover, H H S i D showed an additive effect with methoctramine in blocking the increase in 6-keto-PGFl~ output induced by ACh. The selectivity of methoctramine in attenuating ACh- and APE-induced PG synthesis was indicated by our finding that methoctramine did not alter the increase in 6-keto-PGF~ output induced by the non-cholinergic agent isoproterenol or by arachidonic acid. The mechanism by which ACh and APE enhanced PG output might be due to either an augmented mobilization of arachidonic acid from the phospholipid stores or to an increase in cyclooxygenase activity. Our finding that the metabolism of exogenous arachidonic acid to 6-keto-PGFl~ was unaffected by methoctramine suggests that stimulation of PG synthesis does not involve activation of cyclooxygenase, but is most likely due to increased release of arachidonic acid from tissue lipids. Although PGs are synthesized by various cell types in the heart, the conversion of arachidonic acid to 6-keto-PGFl~ occurs predominantly in endothelial cells of the coronary vessels in the heart (Wennmalm, 1979). In view of these observations and the demonstration that coronary vessels contain both M 2 and M 3 receptors, it is possible that the major site of PG synthesis ellicited by cholinergic stimuli is in the coronary vessels. The finding that methoctramine, but not HHSiD, a M 3 receptor antagonist, abolished the decrease in heart rate produced by ACh or APE suggest that bradycardia produced by cholinergic stimuli is mediated via activation of a distinct subtype of muscarinic receptor, most likely M 2. A corollary of this conclusion is that methoctramine is a selective cardiac M 2 receptor antagonist. The decreases in developed tension produced by ACh and APE appeared to be due to activation of both M 2 and M 3 receptors, because they were minimized by both methoctramine and HHSiD. That methoctramine selectively blocks M e receptors in the rabbit heart was also indicated by our finding that the decrease in coronary perfusion pressure produced by
ACh or APE that is due to stimulation of M 3 receptors and is blocked by H H S i D (Jaiswal et al., 1988) was not altered by methoctramine. Moreover, APE produced a biphasic effect on coronary perfusion pressure, an initial fall followed by a rise in coronary perfusion pressure; and only the later component of the response that has been reported to be due to stimulation of M 2 receptors (insensitive to blockade by pirenzepine or HHSiD) (Jaiswal an Malik, 1988; Jaiswal et al., 1988) was attenuated by methoctramine. Methoctramine has also been reported to display 100 times greater affinity for the muscarinic receptor involved in the action of cholinergic stimuli in producing negative inotropic and chronotropic effects in guinea pig atria than for that involved in mediating tracheal contraction (Giraldo et al., 1988). Methoctramine was also a more potent muscarinic receptor antagonist for the bradycardiac effect than for the increases in bladder tone, salivary secretion, and hypotension produced by muscarinic agents in anesthetized cats (Giraldo et al., 1988). In a direct binding study, methoctramine also exhibited a higher affinity for cardiac (M2) than for exocrine gland muscarinic receptors (M3) and a 16-fold higher affinity for M 2 than for M~ receptors (Michel and Whiting, 1988). In conclusion, this study demonstrates that methoctramine is a cardioselective M 2 receptor antagonist. Moreover, the results of this study in conjunction with our previous study (Jaiswal et al., 1988) suggest that the effect of ACh receptor agonists in promoting 6-ketoP G F ~ synthesis and in producing a decrease in myocardial developed tension is due to activation of M 2 and M 3 receptors. The decrease in heart rate and the increase in coronary perfusion pressure are mediated via activation of M 2 receptors, whereas the decrease in coronary perfusion pressure is mediated via M 3 receptors. The pharmacological profile of methoctramine was similar to that of AF-DX 116, a cardioselective M 2 receptor antagonist (Jaiswal and Malik, 1988; Jaiswal et al., 1988; Hammer et al., 1986; Giachetti et al., 1986; Micheletti et al., 1987; Duckies et al., 1987). Methoctramine has been reported to exhibit a 10-fold higher degree of cardioselectivity than AF-DX 116 (Giachetti et al., 1986; Micheletti et al., 1987).
Acknowledgements The authors thank Anne Estes and Bruce January for technical assistance and various pharmaceutical companies (see Materials and methods) for providing various drugs.
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