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The effect of subtype-selective muscarinic receptor antagonists on the cholinergic current in motoneurons of the lobster cardiac ganglion
87
Brain Research, 552 (1991) 87-92 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116687S BRES 16687
The effect of subtype-selective muscarinic receptor antagonists on the cholinergic current in motoneurons of the lobster cardiac ganglion Joseph E. Freschi Department of Neurology, Emory University School of Medicine, Atlanta, GA 30322 (U.S.A.) (Accepted 8 January 1991)
Key words: Muscarinic receptor; Muscarinic subtype; Neuron; Voltage clamp; Invertebrate; Crustacean; Lobster
Muscarinic agonists evoke a voltage-dependent inward current in motoneurons of the lobster cardiac ganglion. In this study, a number of drugs, known to show muscarinic receptor subtype selectivity in mammals, were used to determine the pharmacological profile of the muscarinic receptor on lobster motoneurons. The neurons were held under voltage-clamp, and various concentrations of the antagonists were applied in the presence of 1 mM methacholine. From competition curves plotting agonist-induced current against antagonist concentration, the inhibitor affinity constant and the slope factor were determined. The rank order of potencies of antagonists having an effect was: atropine > pirenzepine > 4-DAMP > methoctramine > HHSiD = (R)-HHD > (S)-HHD. Neither AF-DX 116 nor gallamine were effective at concentrations as high as 10 mM. The Ml-selective agonist McN-A-343 had no effect. Although this crustacean muscarinic receptor resembles the mammalian M 1 muscarinic receptor because of its relatively high affinity for pirenzepine, the rank order of other subtype-specific antagonists does not otherwise resemble that of any of the pharmacologically defined muscarinic receptors in mammals. It may be preferable, therefore, to use a term such as 'pirenzepine-sensitive' muscarinic receptor rather than M~ or 'Ml-like' for invertebrate muscarinic receptors with pharmacological characteristics like those reported here.
INTRODUCTION The
muscarinic
MATERIALS AND METHODS receptor
is t h o u g h t
to
be
highly
c o n s e r v e d a m o n g i n v e r t e b r a t e and v e r t e b r a t e species 29'
33.
N e v e r t h e l e s s , t h e r e h a v e yet to b e p u b l i s h e d studies in
which n e w e r s u b t y p e - s e l e c t i v e a n t a g o n i s t s are u s e d to compare
the
pharmacological
profile o f i n v e r t e b r a t e
m u s c a r i n i c r e c e p t o r s with t h o s e o f v e r t e b r a t e s . The discovery of invertebrate muscarinic receptors with a r e l a t i v e l y high affinity for p i r e n z e p i n e has led i n v e s t i g a t o r s to classify t h e s e r e c e p t o r s as t h e M 1 s u b t y p e o r as ' M l - l i k e '5't9. H o w e v e r , e x p e r i m e n t s with a w i d e r s e l e c t i o n of s u b t y p e - s e l e c t i v e a n t a g o n i s t s s h o w that d e m onstration
of
high
pirenzepine
affinity m a y
not
be
sufficient to u n e q u i v o c a l l y i d e n t i f y t h e M 1 s u b t y p e 32. We recently described a muscarinic receptor-activated i n w a r d c u r r e n t in m o t o n e u r o n s o f t h e l o b s t e r cardiac g a n g l i o n ~3. T h i s v o l t a g e - d e p e n d e n t cation c o n d u c t a n c e is p r e d o m i n a n t l y s e l e c t i v e for s o d i u m ions. In this p a p e r I report
the results of e x p e r i m e n t s in w h i c h I u s e d a
n u m b e r o f m u s c a r i n i c antagonists, which s h o w selectivity for v a r i o u s m a m m a l i a n m u s c a r i n i c r e c e p t o r s u b t y p e s , to d e f i n e an a n t a g o n i s t p o t e n c y p r o f i l e for this c r u s t a c e a n receptor.
The methods of dissection, ligation of individual motoneuron axons, and voltage-clamping with two microelectrodes have been published 13'x4'23'24. Individual neurons were voltage-clamped with two microelectrodes filled with 3 M KCI. Drugs were perfused through a 2-ml recording chamber in which the solution was 90% exchanged in about 10 s at a maximum flow rate of about 40 ml/min23. To test the effect of various muscarinic antagonists, competition experiments were performed using 1 mM methacholine (MeCh) as the agonist. Antagonists were applied at various concentrations and allowed to incubate for 20-60 min. The effect of different incubation times was tested for each antagonist. Each antagonist was studied in a minimum of 3 different experiments on different days. In each experiment the current evoked by 1 mM MeCh in the presence of antagonist was normalized as a percent of the control MeCh response in the absence of antagonist. The results from all the experiments with a given antagonist were averaged. Data from MeCh concentration-response experiments were fit to the logistic equation y = Ymax [xn/(xn+ECson)], where y is the response; Ymax is the maximum response at infinite MeCh concentration; x is the concentration of MeCh; ECso is the value of x at which the response is 0.5 of maximum; and n is the slope factor (pseudo Hill coefficient)8. From competition experiments, assuming bimolecular competitive interaction, antagonist concentration-response data were fit to a modification of the above logistic equation, Y = Ym~x/[l+(x/1Cso)n], where Ym~xis the control response to MeCh in the absence of antagonist, and 1C5o is the concentration of antagonist that causes a 50% inhibition of the control response to MeCh. The inhibitor affinity constant K i was calculated by correct-
Correspondence: J.E. Freschi, Department of Neurology, 401 Woodruff Memorial Research Building, Emory University School of Medicine, P.O. Drawer V, Atlanta, GA 30322, U.S.A.
88 ing the IC5o values for the agonist concentration, according to the equation K i = ICso/(I+[MeCh]/ECso)4. The assumption of bimolecular competition was based on experiments in which antagonist inhibition was overcome by increasing the MeCh concentration until a response similar to the maximum control response was obtained. This is shown explicitly for pirenzepine in Fig. 5. In other cases, instead of a parallel shift of the complete dose-response curve, the response to 10 mM MeCh was obtained in the presence of antagonist and compared to a maximal control MeCh response (without antagonist). Curve-fitting was performed by a non-linear, least-squares routine using the Marquardt-Levenberg modification of the Gauss-Newton algorithm (RS/1, BBN Software Products). Pirenzepine, methoctramine, 4-DAMP (4-diphenylacetoxy-Nmethyl piperidine methiodide), and McN-A-343 (4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium) were purchased from Research Biochemicals Inc. Methacholine, atropine and gallamine were bought from Sigma Chemical Co. Enantiomers of hexahydro-difenidol ((R)-(-)- and (S)-(+)-HHD) and hexahydrosila-difenidol (HHSiD) were provided by Drs. G. Lambrecht and R. Tacke, University of Karlsruhe, West Germany. AF-DX116 ((11[[2(diethylamino)methyl]- 1-piperidinyl]acetyl]5,11 -dihydro-6H-pyrido[2, 3-b][1,4]benzodiazepine 6-)one) was supplied by Boehringer-Ingelheim Pharmaceuticals.
RESULTS
Methacholine dose-response relationship T h e r e s p o n s e s to d i f f e r e n t c o n c e n t r a t i o n s of M e C h w e r e o b t a i n e d b o t h by a p p l y i n g c u m u l a t i v e c o n c e n t r a tions and by a p p l y i n g single c o n c e n t r a t i o n s f o l l o w e d by complete washing out between there
was no d e s e n s i t i z a t i o n
applications.
o r run
down
Because e x c e p t at
c o n c e n t r a t i o n s g r e a t e r t h a n 1 m M (Fig. 1A), the results were comparable. MeCh caused a concentration-depend e n t i n w a r d c u r r e n t in v o l t a g e - c l a m p e d m o t o n e u r o n s . In s o m e n e u r o n s the t h r e s h o l d d o s e for d e t e c t i o n of the c u r r e n t was a b o u t 10 # M a n d s a t u r a t i o n was r e a c h e d at doses a r o u n d 5 m M .
The dose-response
c u r v e for 8
d i f f e r e n t e x p e r i m e n t s is s h o w n in Fig. 1. In two of the 8 e x p e r i m e n t s , c u m u l a t i v e c o n c e n t r a t i o n s w e r e used. T h e
EC5o and
slope f a c t o r c a l c u l a t e d f r o m t h e s e d a t a w e r e ,
r e s p e c t i v e l y , 0.45 m M and 1.08. W e did not study the d o s e - r e s p o n s e r e l a t i o n s h i p for o t h e r agonists, but b o t h m u s c a r i n e a n d p i l o c a r p i n e , in
A
c o n c e n t r a t i o n s of 1 - 5 m M , c a u s e d r e s p o n s e s of t h e s a m e
[METHACHOLINE] (raM)
o r d e r of m a g n i t u d e as did M e C h . A r e c o l i n e , h o w e v e r , at the s a m e c o n c e n t r a t i o n s , was less e f f e c t i v e in e v o k i n g t h e 0.01
0.05
0.1
0.2
0.5
ATROPINE
lnA[ 100 s 1
3
5
10 Control
100 s
B 160 140
- T
10 rain
~
"
~
120 ,,,.,v W t" O ~L
100 80 60
20 rnln
40 20
-5
-4
-3
-2
40 rain
Log [ M e t h a c h o l i n e ] (M)
Fig. 1. Dose-response relationship between MeCh concentration and voltage-clamped membrane current. A: representative inward currents evoked by different concentrations of MeCh on a ligated motoneuron in normal lobster saline. Concentration in mmol/liter is indicated above each current trace. For each trace, wash-out was started when the response reached a steady maximum or first began to decline. Note desensitization at the concentrations greater than 1 mM. Vh = -42 mV. B: plot of MeCh concentration against inward current amplitude. Eight different experiments were averaged, and the means and standard deviations are shown. In each experiment the currents were normalized to the amplitude evoked by 1 mM MeCh, which was arbitrarily assigned the value of 100%.
Wash
~
'
:
"
~
"
Fig. 2. Effect of different antagonist incubation times. An unligated motoneuron was voltage-clamped at -48 mV and incubated in Saline containing 0.3 #M TTX, 5 mM Mn 2+, and 0.1/~M atropinel The response to 1 mM MeCh was then tested at the times indicated to the left of each trace. The control is the response to 1 mM MeCh before addition of antagonist. Wash indicates the response to MeCh 30 min after wash-out of the antagonist.
89 cholinergic current; and similar concentrations of McNA-343 were completely without effect (data not shown).
Effect of differing incubation times If insufficient time is allowed for equilibrium of ligand binding to the muscarinic receptors, the binding affinity of the ligand is incorrectly calculated, and the equations described in Methods are invalid 9. Fig. 2 shows an experiment in which the effect of 0.1/tM atropine was determined after 10, 20 and 40 min of incubation. The inhibition seen at 10 min was not further increased at 20 and 40 min. We found that for all the antagonists studied, a steady-state of inhibition was reached before 30 min.
Competition experiments Atropine. As is the case for mammalian muscarinic receptors, the subtype-unselective antagonists showed a higher affinity than did the various drugs that reportedly distinguish muscarinic receptor subtypes. Both scopolamine and atropine effectively inhibited the MeChinduced current at doses below 0.1/~M. Atropine was studied in detail. Some representative examples of the reduction in agonist-evoked current at various concen-
[ATROPINE] (pM)
A
CONTROL
-
-
0.01
~
'
"
~
0.t
'
t
~
trations of atropine are shown in Fig. 3A. The results of 8 different experiments were averaged and are shown in the competition curve of Fig. 4. The K i determined from this plot was 20 nM, and the slope factor was 0.73. Of the antagonists studied in this manner, only atropine showed a slope factor significantly different from 1 (Table I). Pirenzepine. This drug was the first, and has been the most widely used, selective Ml-receptor antagonist 6'18'28. Of the potentially selective antagonists studied in these experiments, pirenzepine had the greatest potency in inhibiting the cholinergic current in lobster cardiac ganglion motoneurons. Representative examples of the reduction of cholinergic current at different concentrations are shown in Fig. 3B. The averaged results of 6 experiments are plotted in the competition curve of Fig. 4. The K i was 0.43/~M and the slope factor was 1.02. As discussed in the Methods section, a maximum response to MeCh could be obtained in the presence of pirenzepine if the concentration of MeCh were sufficiently large. In the experiment shown in Fig. 5, pirenzepine, 1 /~M, caused a right shift of the MeCh dose-response curve. The maximum response in the presence of pirenzepine was comparable to that obtained in its absence. 4-DAMP. 4-DAMP binds with relatively high affinity to all muscarinic receptor subtypes but is most potent at M 3 receptors 2'3"7"2°'28'3°,where it binds with 50-100 times greater affinity than does pirenzepine. At Ml-receptors,
WASH
l nAL_ ~ J - ~ 100 s
B
CONTROL .
.
.
.
0.1 .
.
1
10
80
!,o
[4-DAMP] (pM)
CONTROL
H-EiD
WASH
.
100 s
C
•
100
[PIRENZEPINE] (pM)
4O 1
10
s0
~
\ \\ \\
2o 0
D
[HHSlD](pM) CONTROL
1
10
100
I
I
I
I
I
I
I
-9
-8
-7
-6
-5
-4
-3
WASH Log [Antagonist] (M)
Fig. 3. Examples of inhibition of MeCh-induced inward current by various concentrations of antagonists. In each case, a voltageclamped motoneuron was incubated in saline containing different concentrations of antagonist, indicated above each trace, for 30 min. The effect of 1 mM MeCh was then tested. Each experiment was done on a different cell, the clamped membrane potentials of which were (in mV): A, -50; B, -54; C, -42; D , - 4 5 .
Fig. 4. Competition curves of atropine (ATR), pirenzepine (PZ), 4-DAMP (DAMP), HHSiD, (R)-HHD, and methoctramine (MCT) plotted against the response to 1 mM MeCh. The response in the presence of an antagonist was normalized to the response in the absence of the antagonist. Each point is the mean value of several experiments (details in text). Since only 3 (R)-HHD concentrations were studied, a least-squares analysis was not done on these data, and the smooth curve represents the fit only to the HHSiD data.
90 however, 4-DAMP affinity is only about 5-10 times greater 7,32. As shown in the examples in Fig. 3C, on lobster motoneurons 4-DAMP was an effective inhibitor of the cholinergic current. As shown in the competition curve (Fig. 4), however, 4-DAMP was half as potent as pirenzepine (Ki, 0.8/~M; slope factor; 1.09). Analogues of hexahydodifenidol. Lambrecht and coworkers 1°'1kss'34 have shown that these drugs are useful tools in subclassifying the mammalian muscarinic receptor. Both racemic H H D and HHSiD have high affinity for both the MI- and M3-receptors but low affinity for M2-receptors. Furthermore, the enantiomers of H H D show stereoselectivity, the (R)-enantiomers being more potent than the (S)-enantiomers. In Fig. 3D, the effect of different concentrations of HHSiD on the cholinergic current in lobster motoneurons is shown. The average of 4 experiments is plotted in the competition curve of Fig. 4. To study the effects of (R)- and (S)-HHD, 3 concentrations were examined in 3 different experiments. Although insufficient to construct complete competition curves, the results were averaged and, for the (R)-enantiomer, shown in relationship to the HHSiD curve (Fig. 4). The (R)-enantiomer of H H D was about as effective as HHSiD in inhibiting the agonistinduced current. On the other hand, the (S)-enantiomer was considerably less potent (not shown), although there was insufficient data to allow quantification of the difference. For HHSiD, the K i was 8.3 pM and the slope factor was 0.99. The averaged values for (R)-HHD superimpose on the HHSiD competition curve of Fig. 4; therefore, the K i value is assumed to be the same. Mammalian M2-selective antagonists. In mammals, both methoctramine and AF-DX 116 show high selectivity for the Ms-receptor ~6's°'s6. In the lobster cardiac ganglion, methoctramine was nearly twice as potent as H H D analogues in inhibiting the cholinergic current (Fig.
1.4!
1.2 o
c tO C
1.0
•
Control
0.8
•
PZ, 1 pM
o o,
0.6
[I:
0.4
/
/
~" ,/
tO
0.2 0.0
-$
I
I
I
-4
-3
-2
Log [MeCh] ( I ) Fig. 5. Competitive antagonism of MeCh response by pirenzepine (PZ). Inward current is plotted against MeCh concentration in saline with and without 1 a M PZ.
4), but 10-fold less potent than pirenzepine. The Ki for methoctramine was 4.3 a M and the slope factor was 0.88. Both AF-DX 116 and the non-competitive inhibitor gallamine were ineffective at 10 mM, the highest concentration tested.
Summary and comparison to mammalian muscarinic receptors. The results of all the competition experiments are summarized in Table I. Since invertebrates generally require higher concentrations of agonists and antagonists than do mammals for effects at a given receptor I~, the results were normalized as a ratio of K i antagonist/Ki atropine and compared to the values published for mammalian muscarinic receptor subtypes. Only those drugs having an effect at concentrations less than 10 mM are listed. As summarized in the table and from the results, the rank order of potencies was: atropine > pirenzepine > 4-DAMP > methoctramine > HHSiD = (R)-HHD > > (S)-HHD (> AF-DX 1t6, gallamine). DISCUSSION
TABLE I
Some pharmacological properties of the muscarinic receptor on motoneurons of the lobster cardiac ganglion Drug
Slope factor
Ki (t~M)
K i antagonist/K i atropine Lobster
Mammalian MzM2Ms
Atropine Pirenzepine 4-DAMP Methoetramine HHSiD (R)-HHD
0.73 1.02 1.09 0.88 0.99 -
0.02 0.43 0.8 4.3 8.3 8.3
1 21.5 40 215 415 415
1 10 1 50 10 2
1 100 1.6 1.6 100 33
1 50 2.5 100 7.5 2.5
The concentrations of muscarinic agonists and antagonists used in these experiments are similar to those needed to see effects at the muscarinic receptors of a number of different invertebrates, including molluscs s9, i n s e c t s 5"17A9'27, and c r u s t a c e a n s ~s'25. When the potencies of putative selective antagonists used in these experiments were normalized relative to the potency of atropine, only pirenzepine showed an order of potency similar to that seen in mammals. Therefore, the lobster muscarinic receptor cannot be directly compared with any subtype of vertebrate muscarinic receptor. In vertebrates, M 1 receptors show a greater sensRivity to pirenzepine and analogues of hexahydrodefinidol than do M s receptors 6"1°'al. Vertebrate M s receptors, on the other
91 hand, show a higher sensitivity to m e t h o c t r a m i n e and A F - D X 11616'2°'26. V e r t e b r a t e M 3 receptors are characterized by a lower sensitivity to pirenzepine than to 4 - D A M P and H H D analogues (especially p-fluoro-hexahydro.sila.difenidol)7,11,16,21,30,31. A l t h o u g h the relatively g r e a t e r sensitivity of the lobster m o t o n e u r o n muscarinic r e c e p t o r to pirenzepine and 4 - D A M P suggest a similarity to the m a m m a l i a n M 1 receptor, the low potency of the H H D analogues is quite unlike their effect on the v e r t e b r a t e M 1 receptor. E v e n less potent, however, were those drugs that, in m a m m a l s , show a relatively high selectivity t o w a r d the M 2 receptor. T h e r e have been a few studies in invertebrates in which the Ml-selective drug pirenzepine was used to characterize muscarinic receptors. In those studies, the IC50 o r K i values for a t r o p i n e and pirenzepine at ' M l - l i k e ' r e c e p t o r s were similar to those r e p o r t e d here 5'19'29. T h e r e are presently no o t h e r studies in invertebrates in which a range of muscarinic r e c e p t o r - s u b t y p e antagonists was used to c o m p a r e the i n v e r t e b r a t e r e c e p t o r with the 3 subtypes of m a m m a l i a n muscarinic receptor. Recently, Tietz and coworkers 32 r e p o r t e d that postjunctional muscarinic receptors in chicken atria show a high affinity for
pirenzepine, but the rank o r d e r of potencies for o t h e r selective antagonists was different from that seen at M 1, M 2 or M 3 receptors. In addition, the Ml-selective agonist MeN-A-343 was only a p o o r partial agonist in this p r e p a r a t i o n . Thus, they showed that a high affinity for pirenzepine was not sufficient to unequivocally identify a M 1 receptor. T h e results from the p r e s e n t e x p e r i m e n t s on the lobster cardiac ganglion lead to a similar conclusion: Despite the relatively high affinity for pirenzepine, the r e c e p t o r does not otherwise r e s e m b l e any of the muscarinic receptors pharmacologically classified in mammals. Until molecular biological studies d e m o n s t r a t e the structural sequence homologies b e t w e e n i n v e r t e b r a t e and v e r t e b r a t e muscarinic receptors, it m a y be p r e f e r a b l e to use the term 'pirenzepine-sensitive' muscarinic r e c e p t o r r a t h e r than M 1 o r ' M l - l i k e ' for i n v e r t e b r a t e muscarinic receptors with pharmacological profiles like that r e p o r t e d here.
Acknowledgements. This work was supported by NIH Grant NS22628. I thank Dr. K. Minneman for advise during the experimental phase of the project and for his comments on the manuscript.
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27
28
mann, C., Tacke, R., Rodrigues de Miranda, J.E, Christophe, J. and Mutschler, E., Pharmacology of hexahydro-difenidol, hexahydro-sila-difenidol and related selective muscarinic antagonists, Trends Pharmacol. Sci., Suppl., 10 (1989) 60-64. Livengood, D.R., Coupling ratio of the Na-K pump in the lobster cardiac ganglion, J. Gen. Physiol., 82 (1983) 853-874. Livengood, D.R. and Kusano, K., Evidence for an electrogenic sodium pump in follower cells of the lobster cardiac ganglion, J. Neurophysiol., 35 (1972) 170-186. Marder, E. and Eisen, J.S., Electrically coupled pacemaker neurons respond differently to the same physiological inputs and neurotransmitters, J. Neurophysiol., 51 (1984) 1362-1373. Melchiorre, C., Angeli, P., Lambrecht, G., Mutschler, E., Picchio, M.T. and Wess, J., Antimuscarinic action of methoctramine, a new cardioselective M-2 muscarinic receptor antagonist, alone and in combination with atropine and gallamine, Eur. J. Pharmacol., 144 (1987) 117-124. Meyer, M.R. and Reddy, G.R., Muscarinic and nicotinic cholinergic binding sites in the terminal abdominal ganglion of the cricket (Acheta domesticus), J. Neurochem., 45 (1985) 1101-1112. Mitchelson, E, Muscarinic receptor differentiation, Pharmacol. Ther., 37 (1988) 357-423.
29 Murray, T.E and Mpitsos, G.J., Evidence for heterogeneity of muscarinic receptors in the mollusc Pleurobranckaea, Brain Res. Bull. 21 (1988) 181-190. 30 Pitier, T.A. and Alger, B.E., Evidence of the M 3 muscarinic receptor subtype in hippocampal neurons, Soc. Neurosci. Abstr., 15 (1989) 1303. 31 Quirion, R., Araujo, D.M. and Lapchak, P., Binding characteristics of 3H-4DAMP, a putative muscarinic-M 3 antagonist, in rat brain, Soc. Neurosci. Abstr., 15 (1989) 1302. 32 Tietz, H., Lindmar, R., Loffelholz, K., Strohmann, C., Tacke, R., Lambrecht, G. and Mutschler, E., High affinity pirenzepine receptors in chicken atria are different from M 1 type, Trends Pharmacol. Sci., Suppl. 10., (1989) 107. 33 Venter, J.C., Eddky, B., Hall, L.M. and Fraser, C.M., Monoclonal antibodies detect the conservation of muscarinic cholinergic receptor structure from Drosophila to human brain and detect possible structural homology with fl-adrenergic receptors, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 272-276. 34 Waelbroeck, M., Tastenoy, M., Camus, J.. Feifel, R., Mutschler, E., Strohmann, C., Tacke, R., Lambrecht, G. and Christophe, J., Stereoselectivity of the interaction of muscarinic antagonists with their receptors, Trends PharmacoL Sci., Suppl., 10 (1989) 65-69.
Brain Research, 552 (1991) 87-92 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116687S BRES 16687
The effect of subtype-selective muscarinic receptor antagonists on the cholinergic current in motoneurons of the lobster cardiac ganglion Joseph E. Freschi Department of Neurology, Emory University School of Medicine, Atlanta, GA 30322 (U.S.A.) (Accepted 8 January 1991)
Key words: Muscarinic receptor; Muscarinic subtype; Neuron; Voltage clamp; Invertebrate; Crustacean; Lobster
Muscarinic agonists evoke a voltage-dependent inward current in motoneurons of the lobster cardiac ganglion. In this study, a number of drugs, known to show muscarinic receptor subtype selectivity in mammals, were used to determine the pharmacological profile of the muscarinic receptor on lobster motoneurons. The neurons were held under voltage-clamp, and various concentrations of the antagonists were applied in the presence of 1 mM methacholine. From competition curves plotting agonist-induced current against antagonist concentration, the inhibitor affinity constant and the slope factor were determined. The rank order of potencies of antagonists having an effect was: atropine > pirenzepine > 4-DAMP > methoctramine > HHSiD = (R)-HHD > (S)-HHD. Neither AF-DX 116 nor gallamine were effective at concentrations as high as 10 mM. The Ml-selective agonist McN-A-343 had no effect. Although this crustacean muscarinic receptor resembles the mammalian M 1 muscarinic receptor because of its relatively high affinity for pirenzepine, the rank order of other subtype-specific antagonists does not otherwise resemble that of any of the pharmacologically defined muscarinic receptors in mammals. It may be preferable, therefore, to use a term such as 'pirenzepine-sensitive' muscarinic receptor rather than M~ or 'Ml-like' for invertebrate muscarinic receptors with pharmacological characteristics like those reported here.
INTRODUCTION The
muscarinic
MATERIALS AND METHODS receptor
is t h o u g h t
to
be
highly
c o n s e r v e d a m o n g i n v e r t e b r a t e and v e r t e b r a t e species 29'
33.
N e v e r t h e l e s s , t h e r e h a v e yet to b e p u b l i s h e d studies in
which n e w e r s u b t y p e - s e l e c t i v e a n t a g o n i s t s are u s e d to compare
the
pharmacological
profile o f i n v e r t e b r a t e
m u s c a r i n i c r e c e p t o r s with t h o s e o f v e r t e b r a t e s . The discovery of invertebrate muscarinic receptors with a r e l a t i v e l y high affinity for p i r e n z e p i n e has led i n v e s t i g a t o r s to classify t h e s e r e c e p t o r s as t h e M 1 s u b t y p e o r as ' M l - l i k e '5't9. H o w e v e r , e x p e r i m e n t s with a w i d e r s e l e c t i o n of s u b t y p e - s e l e c t i v e a n t a g o n i s t s s h o w that d e m onstration
of
high
pirenzepine
affinity m a y
not
be
sufficient to u n e q u i v o c a l l y i d e n t i f y t h e M 1 s u b t y p e 32. We recently described a muscarinic receptor-activated i n w a r d c u r r e n t in m o t o n e u r o n s o f t h e l o b s t e r cardiac g a n g l i o n ~3. T h i s v o l t a g e - d e p e n d e n t cation c o n d u c t a n c e is p r e d o m i n a n t l y s e l e c t i v e for s o d i u m ions. In this p a p e r I report
the results of e x p e r i m e n t s in w h i c h I u s e d a
n u m b e r o f m u s c a r i n i c antagonists, which s h o w selectivity for v a r i o u s m a m m a l i a n m u s c a r i n i c r e c e p t o r s u b t y p e s , to d e f i n e an a n t a g o n i s t p o t e n c y p r o f i l e for this c r u s t a c e a n receptor.
The methods of dissection, ligation of individual motoneuron axons, and voltage-clamping with two microelectrodes have been published 13'x4'23'24. Individual neurons were voltage-clamped with two microelectrodes filled with 3 M KCI. Drugs were perfused through a 2-ml recording chamber in which the solution was 90% exchanged in about 10 s at a maximum flow rate of about 40 ml/min23. To test the effect of various muscarinic antagonists, competition experiments were performed using 1 mM methacholine (MeCh) as the agonist. Antagonists were applied at various concentrations and allowed to incubate for 20-60 min. The effect of different incubation times was tested for each antagonist. Each antagonist was studied in a minimum of 3 different experiments on different days. In each experiment the current evoked by 1 mM MeCh in the presence of antagonist was normalized as a percent of the control MeCh response in the absence of antagonist. The results from all the experiments with a given antagonist were averaged. Data from MeCh concentration-response experiments were fit to the logistic equation y = Ymax [xn/(xn+ECson)], where y is the response; Ymax is the maximum response at infinite MeCh concentration; x is the concentration of MeCh; ECso is the value of x at which the response is 0.5 of maximum; and n is the slope factor (pseudo Hill coefficient)8. From competition experiments, assuming bimolecular competitive interaction, antagonist concentration-response data were fit to a modification of the above logistic equation, Y = Ym~x/[l+(x/1Cso)n], where Ym~xis the control response to MeCh in the absence of antagonist, and 1C5o is the concentration of antagonist that causes a 50% inhibition of the control response to MeCh. The inhibitor affinity constant K i was calculated by correct-
Correspondence: J.E. Freschi, Department of Neurology, 401 Woodruff Memorial Research Building, Emory University School of Medicine, P.O. Drawer V, Atlanta, GA 30322, U.S.A.
88 ing the IC5o values for the agonist concentration, according to the equation K i = ICso/(I+[MeCh]/ECso)4. The assumption of bimolecular competition was based on experiments in which antagonist inhibition was overcome by increasing the MeCh concentration until a response similar to the maximum control response was obtained. This is shown explicitly for pirenzepine in Fig. 5. In other cases, instead of a parallel shift of the complete dose-response curve, the response to 10 mM MeCh was obtained in the presence of antagonist and compared to a maximal control MeCh response (without antagonist). Curve-fitting was performed by a non-linear, least-squares routine using the Marquardt-Levenberg modification of the Gauss-Newton algorithm (RS/1, BBN Software Products). Pirenzepine, methoctramine, 4-DAMP (4-diphenylacetoxy-Nmethyl piperidine methiodide), and McN-A-343 (4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium) were purchased from Research Biochemicals Inc. Methacholine, atropine and gallamine were bought from Sigma Chemical Co. Enantiomers of hexahydro-difenidol ((R)-(-)- and (S)-(+)-HHD) and hexahydrosila-difenidol (HHSiD) were provided by Drs. G. Lambrecht and R. Tacke, University of Karlsruhe, West Germany. AF-DX116 ((11[[2(diethylamino)methyl]- 1-piperidinyl]acetyl]5,11 -dihydro-6H-pyrido[2, 3-b][1,4]benzodiazepine 6-)one) was supplied by Boehringer-Ingelheim Pharmaceuticals.
RESULTS
Methacholine dose-response relationship T h e r e s p o n s e s to d i f f e r e n t c o n c e n t r a t i o n s of M e C h w e r e o b t a i n e d b o t h by a p p l y i n g c u m u l a t i v e c o n c e n t r a tions and by a p p l y i n g single c o n c e n t r a t i o n s f o l l o w e d by complete washing out between there
was no d e s e n s i t i z a t i o n
applications.
o r run
down
Because e x c e p t at
c o n c e n t r a t i o n s g r e a t e r t h a n 1 m M (Fig. 1A), the results were comparable. MeCh caused a concentration-depend e n t i n w a r d c u r r e n t in v o l t a g e - c l a m p e d m o t o n e u r o n s . In s o m e n e u r o n s the t h r e s h o l d d o s e for d e t e c t i o n of the c u r r e n t was a b o u t 10 # M a n d s a t u r a t i o n was r e a c h e d at doses a r o u n d 5 m M .
The dose-response
c u r v e for 8
d i f f e r e n t e x p e r i m e n t s is s h o w n in Fig. 1. In two of the 8 e x p e r i m e n t s , c u m u l a t i v e c o n c e n t r a t i o n s w e r e used. T h e
EC5o and
slope f a c t o r c a l c u l a t e d f r o m t h e s e d a t a w e r e ,
r e s p e c t i v e l y , 0.45 m M and 1.08. W e did not study the d o s e - r e s p o n s e r e l a t i o n s h i p for o t h e r agonists, but b o t h m u s c a r i n e a n d p i l o c a r p i n e , in
A
c o n c e n t r a t i o n s of 1 - 5 m M , c a u s e d r e s p o n s e s of t h e s a m e
[METHACHOLINE] (raM)
o r d e r of m a g n i t u d e as did M e C h . A r e c o l i n e , h o w e v e r , at the s a m e c o n c e n t r a t i o n s , was less e f f e c t i v e in e v o k i n g t h e 0.01
0.05
0.1
0.2
0.5
ATROPINE
lnA[ 100 s 1
3
5
10 Control
100 s
B 160 140
- T
10 rain
~
"
~
120 ,,,.,v W t" O ~L
100 80 60
20 rnln
40 20
-5
-4
-3
-2
40 rain
Log [ M e t h a c h o l i n e ] (M)
Fig. 1. Dose-response relationship between MeCh concentration and voltage-clamped membrane current. A: representative inward currents evoked by different concentrations of MeCh on a ligated motoneuron in normal lobster saline. Concentration in mmol/liter is indicated above each current trace. For each trace, wash-out was started when the response reached a steady maximum or first began to decline. Note desensitization at the concentrations greater than 1 mM. Vh = -42 mV. B: plot of MeCh concentration against inward current amplitude. Eight different experiments were averaged, and the means and standard deviations are shown. In each experiment the currents were normalized to the amplitude evoked by 1 mM MeCh, which was arbitrarily assigned the value of 100%.
Wash
~
'
:
"
~
"
Fig. 2. Effect of different antagonist incubation times. An unligated motoneuron was voltage-clamped at -48 mV and incubated in Saline containing 0.3 #M TTX, 5 mM Mn 2+, and 0.1/~M atropinel The response to 1 mM MeCh was then tested at the times indicated to the left of each trace. The control is the response to 1 mM MeCh before addition of antagonist. Wash indicates the response to MeCh 30 min after wash-out of the antagonist.
89 cholinergic current; and similar concentrations of McNA-343 were completely without effect (data not shown).
Effect of differing incubation times If insufficient time is allowed for equilibrium of ligand binding to the muscarinic receptors, the binding affinity of the ligand is incorrectly calculated, and the equations described in Methods are invalid 9. Fig. 2 shows an experiment in which the effect of 0.1/tM atropine was determined after 10, 20 and 40 min of incubation. The inhibition seen at 10 min was not further increased at 20 and 40 min. We found that for all the antagonists studied, a steady-state of inhibition was reached before 30 min.
Competition experiments Atropine. As is the case for mammalian muscarinic receptors, the subtype-unselective antagonists showed a higher affinity than did the various drugs that reportedly distinguish muscarinic receptor subtypes. Both scopolamine and atropine effectively inhibited the MeChinduced current at doses below 0.1/~M. Atropine was studied in detail. Some representative examples of the reduction in agonist-evoked current at various concen-
[ATROPINE] (pM)
A
CONTROL
-
-
0.01
~
'
"
~
0.t
'
t
~
trations of atropine are shown in Fig. 3A. The results of 8 different experiments were averaged and are shown in the competition curve of Fig. 4. The K i determined from this plot was 20 nM, and the slope factor was 0.73. Of the antagonists studied in this manner, only atropine showed a slope factor significantly different from 1 (Table I). Pirenzepine. This drug was the first, and has been the most widely used, selective Ml-receptor antagonist 6'18'28. Of the potentially selective antagonists studied in these experiments, pirenzepine had the greatest potency in inhibiting the cholinergic current in lobster cardiac ganglion motoneurons. Representative examples of the reduction of cholinergic current at different concentrations are shown in Fig. 3B. The averaged results of 6 experiments are plotted in the competition curve of Fig. 4. The K i was 0.43/~M and the slope factor was 1.02. As discussed in the Methods section, a maximum response to MeCh could be obtained in the presence of pirenzepine if the concentration of MeCh were sufficiently large. In the experiment shown in Fig. 5, pirenzepine, 1 /~M, caused a right shift of the MeCh dose-response curve. The maximum response in the presence of pirenzepine was comparable to that obtained in its absence. 4-DAMP. 4-DAMP binds with relatively high affinity to all muscarinic receptor subtypes but is most potent at M 3 receptors 2'3"7"2°'28'3°,where it binds with 50-100 times greater affinity than does pirenzepine. At Ml-receptors,
WASH
l nAL_ ~ J - ~ 100 s
B
CONTROL .
.
.
.
0.1 .
.
1
10
80
!,o
[4-DAMP] (pM)
CONTROL
H-EiD
WASH
.
100 s
C
•
100
[PIRENZEPINE] (pM)
4O 1
10
s0
~
\ \\ \\
2o 0
D
[HHSlD](pM) CONTROL
1
10
100
I
I
I
I
I
I
I
-9
-8
-7
-6
-5
-4
-3
WASH Log [Antagonist] (M)
Fig. 3. Examples of inhibition of MeCh-induced inward current by various concentrations of antagonists. In each case, a voltageclamped motoneuron was incubated in saline containing different concentrations of antagonist, indicated above each trace, for 30 min. The effect of 1 mM MeCh was then tested. Each experiment was done on a different cell, the clamped membrane potentials of which were (in mV): A, -50; B, -54; C, -42; D , - 4 5 .
Fig. 4. Competition curves of atropine (ATR), pirenzepine (PZ), 4-DAMP (DAMP), HHSiD, (R)-HHD, and methoctramine (MCT) plotted against the response to 1 mM MeCh. The response in the presence of an antagonist was normalized to the response in the absence of the antagonist. Each point is the mean value of several experiments (details in text). Since only 3 (R)-HHD concentrations were studied, a least-squares analysis was not done on these data, and the smooth curve represents the fit only to the HHSiD data.
90 however, 4-DAMP affinity is only about 5-10 times greater 7,32. As shown in the examples in Fig. 3C, on lobster motoneurons 4-DAMP was an effective inhibitor of the cholinergic current. As shown in the competition curve (Fig. 4), however, 4-DAMP was half as potent as pirenzepine (Ki, 0.8/~M; slope factor; 1.09). Analogues of hexahydodifenidol. Lambrecht and coworkers 1°'1kss'34 have shown that these drugs are useful tools in subclassifying the mammalian muscarinic receptor. Both racemic H H D and HHSiD have high affinity for both the MI- and M3-receptors but low affinity for M2-receptors. Furthermore, the enantiomers of H H D show stereoselectivity, the (R)-enantiomers being more potent than the (S)-enantiomers. In Fig. 3D, the effect of different concentrations of HHSiD on the cholinergic current in lobster motoneurons is shown. The average of 4 experiments is plotted in the competition curve of Fig. 4. To study the effects of (R)- and (S)-HHD, 3 concentrations were examined in 3 different experiments. Although insufficient to construct complete competition curves, the results were averaged and, for the (R)-enantiomer, shown in relationship to the HHSiD curve (Fig. 4). The (R)-enantiomer of H H D was about as effective as HHSiD in inhibiting the agonistinduced current. On the other hand, the (S)-enantiomer was considerably less potent (not shown), although there was insufficient data to allow quantification of the difference. For HHSiD, the K i was 8.3 pM and the slope factor was 0.99. The averaged values for (R)-HHD superimpose on the HHSiD competition curve of Fig. 4; therefore, the K i value is assumed to be the same. Mammalian M2-selective antagonists. In mammals, both methoctramine and AF-DX 116 show high selectivity for the Ms-receptor ~6's°'s6. In the lobster cardiac ganglion, methoctramine was nearly twice as potent as H H D analogues in inhibiting the cholinergic current (Fig.
1.4!
1.2 o
c tO C
1.0
•
Control
0.8
•
PZ, 1 pM
o o,
0.6
[I:
0.4
/
/
~" ,/
tO
0.2 0.0
-$
I
I
I
-4
-3
-2
Log [MeCh] ( I ) Fig. 5. Competitive antagonism of MeCh response by pirenzepine (PZ). Inward current is plotted against MeCh concentration in saline with and without 1 a M PZ.
4), but 10-fold less potent than pirenzepine. The Ki for methoctramine was 4.3 a M and the slope factor was 0.88. Both AF-DX 116 and the non-competitive inhibitor gallamine were ineffective at 10 mM, the highest concentration tested.
Summary and comparison to mammalian muscarinic receptors. The results of all the competition experiments are summarized in Table I. Since invertebrates generally require higher concentrations of agonists and antagonists than do mammals for effects at a given receptor I~, the results were normalized as a ratio of K i antagonist/Ki atropine and compared to the values published for mammalian muscarinic receptor subtypes. Only those drugs having an effect at concentrations less than 10 mM are listed. As summarized in the table and from the results, the rank order of potencies was: atropine > pirenzepine > 4-DAMP > methoctramine > HHSiD = (R)-HHD > > (S)-HHD (> AF-DX 1t6, gallamine). DISCUSSION
TABLE I
Some pharmacological properties of the muscarinic receptor on motoneurons of the lobster cardiac ganglion Drug
Slope factor
Ki (t~M)
K i antagonist/K i atropine Lobster
Mammalian MzM2Ms
Atropine Pirenzepine 4-DAMP Methoetramine HHSiD (R)-HHD
0.73 1.02 1.09 0.88 0.99 -
0.02 0.43 0.8 4.3 8.3 8.3
1 21.5 40 215 415 415
1 10 1 50 10 2
1 100 1.6 1.6 100 33
1 50 2.5 100 7.5 2.5
The concentrations of muscarinic agonists and antagonists used in these experiments are similar to those needed to see effects at the muscarinic receptors of a number of different invertebrates, including molluscs s9, i n s e c t s 5"17A9'27, and c r u s t a c e a n s ~s'25. When the potencies of putative selective antagonists used in these experiments were normalized relative to the potency of atropine, only pirenzepine showed an order of potency similar to that seen in mammals. Therefore, the lobster muscarinic receptor cannot be directly compared with any subtype of vertebrate muscarinic receptor. In vertebrates, M 1 receptors show a greater sensRivity to pirenzepine and analogues of hexahydrodefinidol than do M s receptors 6"1°'al. Vertebrate M s receptors, on the other
91 hand, show a higher sensitivity to m e t h o c t r a m i n e and A F - D X 11616'2°'26. V e r t e b r a t e M 3 receptors are characterized by a lower sensitivity to pirenzepine than to 4 - D A M P and H H D analogues (especially p-fluoro-hexahydro.sila.difenidol)7,11,16,21,30,31. A l t h o u g h the relatively g r e a t e r sensitivity of the lobster m o t o n e u r o n muscarinic r e c e p t o r to pirenzepine and 4 - D A M P suggest a similarity to the m a m m a l i a n M 1 receptor, the low potency of the H H D analogues is quite unlike their effect on the v e r t e b r a t e M 1 receptor. E v e n less potent, however, were those drugs that, in m a m m a l s , show a relatively high selectivity t o w a r d the M 2 receptor. T h e r e have been a few studies in invertebrates in which the Ml-selective drug pirenzepine was used to characterize muscarinic receptors. In those studies, the IC50 o r K i values for a t r o p i n e and pirenzepine at ' M l - l i k e ' r e c e p t o r s were similar to those r e p o r t e d here 5'19'29. T h e r e are presently no o t h e r studies in invertebrates in which a range of muscarinic r e c e p t o r - s u b t y p e antagonists was used to c o m p a r e the i n v e r t e b r a t e r e c e p t o r with the 3 subtypes of m a m m a l i a n muscarinic receptor. Recently, Tietz and coworkers 32 r e p o r t e d that postjunctional muscarinic receptors in chicken atria show a high affinity for
pirenzepine, but the rank o r d e r of potencies for o t h e r selective antagonists was different from that seen at M 1, M 2 or M 3 receptors. In addition, the Ml-selective agonist MeN-A-343 was only a p o o r partial agonist in this p r e p a r a t i o n . Thus, they showed that a high affinity for pirenzepine was not sufficient to unequivocally identify a M 1 receptor. T h e results from the p r e s e n t e x p e r i m e n t s on the lobster cardiac ganglion lead to a similar conclusion: Despite the relatively high affinity for pirenzepine, the r e c e p t o r does not otherwise r e s e m b l e any of the muscarinic receptors pharmacologically classified in mammals. Until molecular biological studies d e m o n s t r a t e the structural sequence homologies b e t w e e n i n v e r t e b r a t e and v e r t e b r a t e muscarinic receptors, it m a y be p r e f e r a b l e to use the term 'pirenzepine-sensitive' muscarinic r e c e p t o r r a t h e r than M 1 o r ' M l - l i k e ' for i n v e r t e b r a t e muscarinic receptors with pharmacological profiles like that r e p o r t e d here.
Acknowledgements. This work was supported by NIH Grant NS22628. I thank Dr. K. Minneman for advise during the experimental phase of the project and for his comments on the manuscript.
REFERENCES 1 Barker, D.L., Murray, T.E, Siebenaller, J.F. and Mpitsos, G.J. Characterization of muscarinic cholinergic receptors in the crab nervous system, J. Neurochem., 46 (1986) 583-588. 2 Barlow, R.B., Berry, K.J., Glenton, P.A.M., Nikolaou, N.M. and Soh, K.S., A comparison of affinity constants for muscarinesensitive acetylcholine receptors in guinea-pig atrial pacemaker cells at 29 °C and in ileum at 29 °C and 37 °C, Br. J. Pharmacol., 58 (1976) 613-620. 3 Barlow, R.B., Burston, K.N. and Vis, A., Three types of muscarinic receptors?, Br. J. Pharmacol., 68 (1980) 141-142P. 4 Bennett, J.P. and Yamamura, H.I., Neurotransmitter, hormone, or drug receptor binding methods. In H.I. Yamamura, S.J. Enna and M.J. Kuhar (Eds.), Neurotransmitter receptor binding, Raven, New York, 1985, pp. 61-89. 5 Benson, J.A., Ml-like muscarinic receptors mediate cholinergic activation of an inward current in isolated neuronal somata from locust thoracic ganglia, Soc. Neurosci. Abstr., 15 (1989) 365. 6 Birdsall, N.J.M. and Huime, E.C., Muscarinic receptor subclasses, Trends Pharmacol. Sci., 4 (1983) 1-4. 7 Brown, D.A., Forward, A. and Marsh, S., Antagonist discrimination between ganglionic and ileal muscarinic receptors, Br. J. Pharmacol., 71 (1980)362-364. 8 De Lean, A.P., Munson, P.J. and Rodbard, D., Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose-response curves, Am. J. Physiol., 235 (1978) E97-E102. 9 Ehlert, EJ., Roeske, W.R. and Yamamura, H.I., Muscarinic receptor [3H]ligand binding methods. In P.J. Marangos, R.M. Cohen and I.C. Campbell (Eds.), Brain Receptor Methodologies, Academic, 1984, pp. 339-355. 10 Eltze, M., Gmelin, G., Wess, J., Strohmann, C., Tacke, R., Mutschler, E. and Lambrecht, G., Presynaptic muscarinic receptors mediating inhibition of neurogenic contractions in rabbit vas deferens are of the ganglionic Mr-type, Eur. J. Pharmacol., 158 (1988) 233-242. 11 Feifel, R., Wagner-Roder, M., Strohmann, C., Tacke, R.,
12 13 14 15 16 17 18 19 20
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