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Species differences in the negative inotropic effect of acetylcholine and soman in rat, guinea pig, and rabbit hearts
Pergamon Press plc
Camp. Biochem.Physiol.Vol. lOOC,No. 3, pp. 591-595,1991 Printed in Great Britain
SPECIES DIFFERENCES IN THE NEGATIVE INOTROPIC EFFECT OF ACETYLCHOLINE AND SOMAN IN RAT, GUINEA PIG, AND RABBIT HEARTS* DONALD M. MAXWELL, ROBERT H. THOMSENand STEVENI. BASKIN U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland, U.S.A. (Received 16 January 1991) Abstract-l. Acetylcholine reduced atria1 contractions by 82.5% in guinea pig, 50.8% in rat, and 41.5% in rabbit. 2. The ECm values for the negative inotropic effect of acetylcholine were 3.3 x lo-’ M in rat and guinea pig atria and 4.1 x 10m6M in rabbit atria. 3. There was no correlation between the species differences in the negative inotropic effect of acetylcholine in atria and the density or affinity of acetylcholinesterase or muscarinic receptors. 4. Inhibition of atria1 acetylcholinesterase with soman reduced the EC% of acetylcholine three-fold in all species, but did not change the maximal inotropic effect of acetylcholine. 5. Species differences in the negative inotropic effect of acetylcholine may be caused by differences in the coupling between myocardial muscarinic receptors and the ion channels that mediate negative inotropy.
INTRODUCTION
The role of acetylcholine as an important regulator of myocardial contractility and heart rate has been established by both physiological and histological studies (See reviews by Higgins et al., 1973; Loffelholz and Pappano, 1985). Acetylcholine produces negative inotropic effects as well as negative chronotropic effects in the intact animal, isolated heart, and muscle bath preparations (Blumenthal et al., 1968). The cardiac effect of acetylcholine is mediated by its binding to myocardial muscarinic acetylcholine receptors (AChR) which are coupled to Na+ and K+ channels that modulate myocardial contractility (North, 1989). Hydrolysis of acetylcholine by acetylcholinesterase (AChE) terminates the action of acetylcholine at the myocardial AChR, but AChE activity can be inhibited with anticholinesterase compounds, which increases the cholinergic effect of acetylcholine in the heart (Doods et al., 1989). Considerable variation has been observed in the cardiovascular response to acetylcholine (Quirion et al., 1979; Ten Eick et al., 1976) and various other cholinergic parameters in different species. For example, there are species differences in the density of myocardial AChR (Wei and Sulakhe, 1978; Fields et al., 1978), the affinity of vascular muscarinic AChR (Yamanaka et al., 1986), myocardial levels of acetylcholine (Brown, 1976; Kilbinger and Loffelholz, 1976; Stanley et al., 1978) and the density of myocardial AChE and other cholinesterases (ChE) (Stanley *The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. In conducting the research described in this report, the investigators-adhered to the “Guide for the Care and Use of Laboratorv Animals”. National Institutes of Health publication 85-23.
et al., 1978). The rat cardiovascular
system is less responsive than other mammals to the in viuo effects of anticholinesterase compounds (Stewart and McKay, 1961; Ligtenstein, 1984), while the guinea pig atria is more responsive than atria of rabbit or rat to antimuscarinic drugs (Li and Mitchelson, 1980) and the neuromuscular blockers (Miura et al., 1981). The purpose of the present study was to analyze the relationship between the species differences in the effect of acetylcholine on myocardial contractility and the species differences in the density and affinity of AChR, AChE, and ChE. Myocardial contractility was evaluated by the inotropic response of isolated atria from several species (i.e., rat, guinea pig and rabbit) whose cardiac responses to acetylcholine, anticholinergic drugs, and anticholinesterase compounds have been reported to vary. The effect of acetylcholine was evaluated in the presence and absence of soman, a potent AChE inhibitor (Andersen et al., 1977; Gray and Dawson, 1987) that has been observed to produce in vivo cardiomyopathy (Singer et al., 1987). METHODS Animals Male Sprague-Dawley rats (Rattus noroeguicus) weighing 200-300 g, Hartley guinea pigs (Curia powellus) weighing 400-500 g, and New Zealand rabbits (Oryctohgus cuniculus) weighing 2.c3.0 kg were obtained from Charles River Laboratories (Wilmington, Ma). Hearts isolated from these animals were rinsed with 0.15 M NaCl and dissected into atria, ventricles, and septum for physiological and biochemical measurements of isolated myocardial regions. Materials Acetylcholine bromide, acetvl-b-methvlcholine, and Triton-X-100 were purchased from Sigma Chemical Co. (St Louis, MO). [3H]-quinuclidinylbenzilate (13 mCi/mmol) and [‘HI-acetylcholine (56 mCi/mmole) were purchased 591
592
D. M. MAXWELL et al. Table 1. Regional differences in myocardial muscarinic acetylcholine receptors
100
90 T
s
80
.t; z
70 60
er 5
50 40 30 20 i
w 0
Muscarinic receptor density (pmol/g tissue)* Rabbit Left atrium Right atrium Septum Left ventricle Right ventricle
10-c o!
-14
:
:
-12
:
:
-10
:
:
-a
:
:
-6
log ACh Concentration
:
:
-4
:
I
-2
(M)
Fig. 1. Effect of acetylcholine in electrically stimulated contractions of left atria from rabbits (O), rats (A) and guinea pigs (0) expressed as percent of maximal contro1 contraction. Values are means + S.E. (n = 6). from New England Nuclear (Boston, Ma). [‘4C]-acetylb-methylcholine (57 mCi/mmole) was synthesized by Amersham (Arlington Heights, IL). Soman (methyl pinacolylphosphonofluoridate) was obtained from the Chemical Research, Development and Engineering Center (Aberdeen Proving Ground, MD). All other chemicals were reagent grade. Physiological measurements The inotropic responses of isolated left atria and ventricles were measured by the method of Levy (1971). Atria or ventricles were mounted on stimulating electrodes under OSg tension in baths containing well oxygenated (95% 0,/j% CO,) Krebs-Henseleit solution that was prepared as described by Winegard and Shanes (1962). The KrebsHenseleit solution contained 27.2 mM NaHCO,, 118.0 mM KCI, l.OmM KH,PO,, 1.2mM MgSO,, 2SmM CaCl,, and 11.1 mM glucose at pH 7.4. Tissues were maintained at 32°C and allowed to equilibrate in the bath for 30 min prior to the beginning of stimulation at 1 Hz using a square wave of Smsec duration at a voltage 10% above threshold (l-5 V). An FT-03 Grass force transducer was used to record force as an index of mechanical activity. The first derivative of force was obtained electronicaIly and was used as a measure of inotropy. Solutions of acetylcholine or soman were added sequentially to the tissue bath in order of increasing concentration with a wash between each test concentration. Receptor binding measurements Heart regions were prepared for measurement of muscarinic receptors by homogenization in 9 volumes of 0.32 M sucrose by using a Polytron homogenizer (Brinkman Instru100 90
80
-4
-1
log ACh Concentration
I
-3
(M)
Fig. 2. Dose-response of negative inotropic effect of acetylcholine in left atria of rabbits (O), rats (A) and guinea pigs (0). Response to acetylcholine is expressed as percent of maximal negative inotropic effect from Fig. 1. EC, for acetylchoiine is shown by (. . -).
14.8 * 10.6 + 5.9 + 7.0 + 6.3 +
Rat
0.q 0.4t 0.2tt 0.2tz 0.2tj
28.2 f 21.3 + 15.2 + 5.7 a 16.0 +
1.5 1.4 I.31 0.81 1.7$
Guinea pig 19.8 * l8,9 f 13.6 + 14.3 + 12.8 k
0.6 0.9 0.73 0.5# 0.6$
*Values represent [‘HI-QNBbinding at pH 7.4 (37°C). Values are means f. S.D. (n = 6). TSignificant difference from same region in rat or guinea pig (P < 0.05). jSignificant difference from left or right atrium of same species (P < 0.05).
ments, Westbury, NY). The homogenates were filtered through cheesecloth and the eluates were assayed for muscarinic AChR by the method of Fields et al. (1978) using [3H]-quinuclidinylbenzilate (QNB) as receptor ligand. Enzyme measurements Heart regions were prepared for measurement of AChE and ChE by homogenization in 9 volumes of 0.1 M phosphate buffer containing 0.15 M NaCl by using a Polytron homogenizer. After Polytron homogenization each heart homogenate received an amount of Triton X-100 equal to 1% of its volume and was homogenized again using a Potter-Elvehjem homogenizer, These homogenates were centrifuged at 30,OOOgfor 30 min at &6”C, and the resultant supernates were assayed for enzyme activity. AChE and ChE activity were measured by the method of Siakotos et al. (1969) using either [‘4C]-acetyl-~-methylcholine, a specific substrate for AChE, or [‘HI-acetylcholine which estimated the rate of acetylcholine hydrolysis by all types of ChE. Dafa analysis Scatchard analysis of binding data, analysis of MichaelisMenten equations for enzyme kinetics, and analysis of the graded dose-response relationship of physiological data were performed using the computer programs of Tallarida and Murray (1987). Significant differences in physiological and biochemical measurements were analyzed by the Newman-Keuls test (Hicks, 1982). Differences were considered significant if P < 0.05.
RESULTS
Preliminary experiments indicated that ventricles were much less responsive to acetylcholine than were atria for all species. While inotropy in atria from some species was maximally reduced by as much as 83% by acetylcholine, ventricles were reduced by < 10%. Therefore, further evaluation of the dose-response of acetylcholine on myocardial inotropy was performed in left atria. The negative inotropic response of left atria varied in both the magnitude of maximal response and in the acetylcholine concentration necessary to produce 50% of the maximal response (ECU,,). The maximal reduction of electrically stimulated atria1 contractions was 82.5% in guinea pigs, 50.8% in rats, and 41.5% in rabbits (Fig. 1). The EC,, for the negative inotropic response to acetylcholine was 3.3 x lo-‘M in rats and guinea pigs and 4.1 x 10m6M in rabbits (Fig. 2). The regional and species variations in the muscarinic AChR binding is shown in Table 1. The density of receptors was greater in atria than in septum or ventricles in all species. The density of receptors in all
Species differences in negative inotropy Table 2. Regional diflarenccs in myocardial acetylcholinesterase Acetylcholinesterase Rabbit Left atrium Right atrium Septum Left ventricle Right ventricle
0.19 * 0.23 + 0.11 * 0.05 f 0.13 *
activity (pmol/min/g Rat
o.ost 0.03t 0.02tt o.olt$ 0.01tz
0.53 f 0.58 * 0.30 * 0.14 * 0.30 *
0.04 0.05 0.02$ 0.02$ 0.02%
tissue)*
Guinea pig 0.47 +- 0.03 0.77 f 0.05 0.54 f 0.05 0.18 f 0.03% 0.39 f 0.03$
*Values represent hydrolysis of acetyl-B-methyl choline at 37°C (pH 7.4). Values are means + S.D. (n = 6). tSignificant difference from same region in rat or guinea pig (P < 0.05). SSignificant difference from left or right atrium of same species (P < 0.05).
regions was about 2-fold greater in rats and guinea pigs than in rabbits. The variation in AChE is shown in Table 2. The AChE activity was generally greater in atria than in ventricles or septum, which was similar to the regional distribution of AChR. The level of AChE activity in all regions was 2-3 times greater in rats and guinea pigs than in rabbits, which correlated with the species variation in AChR. The variation in ChE activity is shown in Table 3. The pattern of activity generally paralleled that of AChE and AChR (i.e. atria > ventricles = septum and guinea pig = rat > rabbit), except that the rat had a much greater level of ChE activity than either of the other species. The affinities of AChR and AChE in myocardial tisssue of rats, guinea pigs and rabbits are shown in Table 4. The affinity of ChE for acetylcholine was not determined because the ChE activity is a composite result of several enzymes (i.e., AChE, butyrylcholinesterase, propionylcholinesterase) that have different intrinsic affinities. The affinities of AChR for ‘[HI-QNB were not significantly different among these species. The affinities of AChE for acetyl-bmethylcholine in different species were also not significantly different. Soman, by itself, produced no changes in myocardial contractility at concentrations from 10m9 to 10m4M (data not shown), although concentrations > 10e6 M produced complete AChE inhibition (Fig. 3). The ECSO for the negative inotropic response to acetylcholine was reduced 3-fold in atria that had been previously exposed to 2 x 10V6M soman, in comparison to control atria that were not exposed to soman (Table 5). The magnitude of the maximal inotropic response to acetylcholine was not altered by prior exposure to soman (data not shown).
593
Table 4. Aflinitias of AChE and AChR from whole heart homogenates AChE* (mW 0.15_+0.04 0.12 f 0.03 0.17 f 0.04
Species Rat Guinea pig Rabbit
AChRt WI) 27_+ 10 30* 9 32 f 7
lK,,, of acetyl-fi-methylcholine
at pH 7.4 (37°C). Values are expressed as means + S.E. (n = 3). tApparent K, of [3H]-QNB at pH 7.4 (37°C). Values are expressed as means + S.E. (n = 3). DISCUSSION The negative inotropic response of atria to acetylcholine was much greater than the response of ventricles. This regional variation in response to acetylcholine was similar to previous observations (Higgins et al., 1973; Loffelholz and Pappano, 1985) that compared atria1 and ventricular responses to acetylcholine. The greater responsiveness of atria to acetylcholine is correlated with histochemical evidence that atria have a greater degree of cholinergic innervation than do ventricles (Loffelholz and Pappano, 1985), and by observations that atria have a greater density of biochemical markers for cholinergic innervation such as AChR (Table 1), AChE (Table 2), and ChE (Table 3). Species variation in the negative inotropic response to acetylcholine was evident in both the maximal response and the EC%. The order of maximal inotropic response was guinea pig > rat > rabbit, while the order of EC,, was guinea pig = rat < rabbit. However, species differences in biochemical markers of cholinergic innervation did not correlate with species differences in the ECU,, or the maximal negative inotropic response to acetylcholine. AChE and ChE are usually considered to be present in excess at cholinergic synapses, and therefore the density of AChE or ChE would not necessarily be expected to correlate with species differences in choline+ response. However, receptor density has been observed to influence the magnitude of the negative inotropic response of atria to agonists. In a comparison of the negative inotropy produced by acetylcholine and adenosine, the greater effect of acetylcholine was found to be correlated with the greater receptor density of AChR vs adenosine receptors (Linden et a[., 1985); and in our experiments the greater response of atria vs ventricles also correlated with the greater AChR density in atria vs ventricles. Nevertheless, species differences in the density of
Table 3. Regional differences in myocardial cholinesterase Cholinesterase activity (pmole/min/g Rabbit Left atrium Right atrium Septum Left ventricle Right ventricle
0.68 * 0.80 k 0.34 * 0.34 * 0.42 k
Rat
0.05t 0.09t 0.03Tf 0.04Tf 0.03tt
*Values represent hydrolysis Values are means k S.D. tsignificant difference from (P < 0.05). $Signno~,fference from < .
4.02 + 0.15 5.13 f 0.30 2.01 k0.17f 1.93+0.18f 2.62 + 0.15$
tissue)*
Guinea pig 1.24kO.12
1.95f 0.08 0.91 f 0.081 0.64 * 0.05$ 1.06 f 0.05$
of acetylcholine at pH 7.4 (37°C). (n = 6). same region in rat or guinea pig left or right atrium of same species
-11
-10
-9
log Somon
-8
-7
Concentration
-6
-5
(M)
Fig. 3. Effect of soman on AChE in heart of rabbits (O), rats (A) and guinea pigs (0). Heart slices were incubated for 5 min at 32°C and rinsed before AChE measurements.
594 Table 5.
D.M. MAXWELL~~ al. EC-
of acetvlcholine for nenative inotrouv of atria
Control CUM>
Soman* fUM)
Rat
0.33f 0.05
0.12* o.ost
2.75
Guinea pig Rabbit
0.33 i: 0.03 4.12 i: 0.08
0.~0~0~~~ 1.36 f 0.09t
3.30 3.03
ControIk.oman
*ECUS of acetylcholine after 5 min incubation with 2 x 10m6M soman. tSignificant difference from control (P < 0.05).
AChR (Table 3) were not correlated with the species differences in maximal negative inotropy in response to a~etylcholine. Although there was a great difference in the EQ,,for acetylcholine to produce negative inotropic effects in atria from rats and guinea pigs (3.3 x lo-‘M) vs rabbits (4.1 x 10s6 M), there was no significant species variation in the affinity of the AChR mediating the inotropic action of acetylcholine or the AChE catalyzing its hydrolysis (Table 4). Inasmuch as the muscarinic choline&c receptors measured by QNB binding are thought to consist of several subtypes of differing affinities (Birdsall et al., 1983; Hulme et al., 1990), it could be argued that species variation in agonist ECU,,may be reflected in species differences of a subtype of muscarinic receptor. However, 80% of the cardiac muscarinic receptors are of a single subtype (Closse et al., 1987), which reduces the likelihood of this explanation. Soman produced no negative (i.e., inhibitory) inotropic effect in atria. This observation in cardiac muscle was in sharp contrast to previous studies of excitatory effects in muscle, where sornan at similar concentrations (10m6M) produced enhanced response to electrical stimulation in striated (Smith et al., 1981) as well as smooth muscle (Adler et al., 1987). This difference between the responsiveness of inhibitory and excitatory cholinergic synapses to soman may indicate a fundamental difference in the neurophysiolo~c~ mechanisms of these synapses (North, 1989). Soman has been reported to bind to cholinergic receptors in heart as a result of its displacement of the muscarinic ligand cis-methyldioxolane in heart homogenates (Silveira et al., 1990). Although these cismethyldioxolane binding sites had a high affinity for soman (K, = 8 x lo-” M) and other organophosphorus compounds, Silveira and co-workers did not provide a functional correlation between binding affinity and any physiological effect of organophosphorus compounds in heart. Inasmuch as soman at concentrations much higher ( 10m9M to 10m4M) than the soman Kn for these receptors produced no negative inotropy with functionally intact atria (i.e., atria responsive to acetylcholine) in our experiments, we concluded that these high affinity binding sites for soman were probably non-functional. The absence of a direct effect of soman on myocardial contractions suggests that the in uivo cardiomyopathy that has been observed with soman (Singer et al., 1987) is the result of excessive vagal stimulation and not a direct effect of soman on cardiac tissue. Soman did produce an effect on the acetylcholine dose-response curve for negative inotropy. A consistent 3-fold decrease in the acetylcholine EC,, without alteration of the maximal response occurred in all species in the presence of 2 x 10m6M soman (i.e.,
where AChE was completely inhibited). Physostig mine and BW 284C51, a specific AChE inhibitor, have been previously demonstrated to produce a similar reduction in the a~tyl~holine EC, in rat atria (Slavikova and Hlavickova, 1982; Doods et al., 1989). This suggests that AChR were maximally occupied in both the presence and absence of AChE inhibition and that the species differences in maximal response were not the result of different fractional responses due to species variation in the ability of AChE to hydrolyze a~tyI~holine. The most likely explanation for the species differences in maximal negative inotropy is that the coupling of AChR to the ion channels that mediate negative inotropy may differ between species. Alterations in the coupling of receptors and ion channels have already been proposed as explanations for other physiolo~c~ phenomena that produce reductions in maximal physiolo~cal response, such as desensitization of cholinergic responses to acetylcholine and developmental changes in cholinergic responses (Nathanson, 1989). This explanation for species differences in maximal negative inotropy is supported by the observation that the same pattern of species variation in maximal negative inotropy that occurred with a~tylcholine (guinea pig > rat > rabbit) has been observed with adenosine, another agonist that produces negative inotropy (Belardinelli et al., 1982). Furthermore, antagonists of negative inotropy, such as stercuronium (Li and Mitchelson, 1980) or B-bungarotoxin (Miura et al., 198 1), also produce the same pattern of species variation in maximal inotropic response. The EC, of agonists or antagonists for the negative inotropic response is determined by their affinities for the muscarinic receptor, but the magnitude of the response is determined by the coupling of the agonist or antagonist receptor complex to the ion channels that produce the negative inotropic response (Linden et al., 1985; North, 1989). In heart both Ca2+ and K+ currents are involved in negative inotropy and both ion channels are coupled to the muscarinic receptor by guanine nucleotide binding proteins (Gproteins) (Kurachi et al., 1986). All of the protein components of negative inotropy-muscarinic recep tors, G-proteins, ion channels-exhibit heterogeneity in their amino acid sequences in different species (Hulme et al., 1990). The expression of this heterogeneity as species differences in the coupling arrangement of the receptor/G-protein/ion channel complex is probably a functional response to the physiological requirements imposed by the variation in the rate of the cardiac de~la~~tion/hyperpola~zation cycle (Lepeschkin, 1965), which is known to vary as heart rate varies between species (Poupa and Brix, 1984). REFERENCES Adler M., Reutter S. A., Moore D. H. and Filbert M. G. (1987) Actions of soman on isolated tracheal smooth muscle. In Cellular rmd ~otecufar Basis of C~oI~erg~c Function (Edited by Dowdall M. J. and Hawthorne J. N.) pp. 582-597. Ellis Horwood, Chichester. Andersen R. A., Aaraas I., Gaare F. and Fonnum F. (1977) Inhibition of acetylcholinesterase from different compounds, carbaspecies by organophosphorus mates and methylsulfonylfluoride. Gen. Pharmacol. 8, 331-334.
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Hicks C. W. (1982) Fundamental Concepts in the Design of Experiments, pp. 8696. Holt, Rinehart and Winston, New York. Higgins C. B., Vatner S. F. and Braunwald E. (1973) Parasympathetic control of the heart. Pharmacol. Rev. 25, 119-155. Hulme E. C., Birdsall N. J. M. and Buckley N. J. (1990) Muscarinic receptor subtypes. Ann. Rev. Pharmacol. Toxicol. 30, 633673.
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Singer A. W., Jaax N. K., Graham J. S. and McLeod C. G. (1987) Cardiomyopathy in soman and sarin intoxicated rats. Toxicol. Lett. 36, 243-249. Slavikova J., Vlk J. and Hlavickova V. (1982) Acetylcholinesterase and butyrylcholinesterase activity in the atria of the heart of adult albino rats. Physiol. Bohemoslov. 31, 407414.
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Camp. Biochem.Physiol.Vol. lOOC,No. 3, pp. 591-595,1991 Printed in Great Britain
SPECIES DIFFERENCES IN THE NEGATIVE INOTROPIC EFFECT OF ACETYLCHOLINE AND SOMAN IN RAT, GUINEA PIG, AND RABBIT HEARTS* DONALD M. MAXWELL, ROBERT H. THOMSENand STEVENI. BASKIN U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland, U.S.A. (Received 16 January 1991) Abstract-l. Acetylcholine reduced atria1 contractions by 82.5% in guinea pig, 50.8% in rat, and 41.5% in rabbit. 2. The ECm values for the negative inotropic effect of acetylcholine were 3.3 x lo-’ M in rat and guinea pig atria and 4.1 x 10m6M in rabbit atria. 3. There was no correlation between the species differences in the negative inotropic effect of acetylcholine in atria and the density or affinity of acetylcholinesterase or muscarinic receptors. 4. Inhibition of atria1 acetylcholinesterase with soman reduced the EC% of acetylcholine three-fold in all species, but did not change the maximal inotropic effect of acetylcholine. 5. Species differences in the negative inotropic effect of acetylcholine may be caused by differences in the coupling between myocardial muscarinic receptors and the ion channels that mediate negative inotropy.
INTRODUCTION
The role of acetylcholine as an important regulator of myocardial contractility and heart rate has been established by both physiological and histological studies (See reviews by Higgins et al., 1973; Loffelholz and Pappano, 1985). Acetylcholine produces negative inotropic effects as well as negative chronotropic effects in the intact animal, isolated heart, and muscle bath preparations (Blumenthal et al., 1968). The cardiac effect of acetylcholine is mediated by its binding to myocardial muscarinic acetylcholine receptors (AChR) which are coupled to Na+ and K+ channels that modulate myocardial contractility (North, 1989). Hydrolysis of acetylcholine by acetylcholinesterase (AChE) terminates the action of acetylcholine at the myocardial AChR, but AChE activity can be inhibited with anticholinesterase compounds, which increases the cholinergic effect of acetylcholine in the heart (Doods et al., 1989). Considerable variation has been observed in the cardiovascular response to acetylcholine (Quirion et al., 1979; Ten Eick et al., 1976) and various other cholinergic parameters in different species. For example, there are species differences in the density of myocardial AChR (Wei and Sulakhe, 1978; Fields et al., 1978), the affinity of vascular muscarinic AChR (Yamanaka et al., 1986), myocardial levels of acetylcholine (Brown, 1976; Kilbinger and Loffelholz, 1976; Stanley et al., 1978) and the density of myocardial AChE and other cholinesterases (ChE) (Stanley *The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. In conducting the research described in this report, the investigators-adhered to the “Guide for the Care and Use of Laboratorv Animals”. National Institutes of Health publication 85-23.
et al., 1978). The rat cardiovascular
system is less responsive than other mammals to the in viuo effects of anticholinesterase compounds (Stewart and McKay, 1961; Ligtenstein, 1984), while the guinea pig atria is more responsive than atria of rabbit or rat to antimuscarinic drugs (Li and Mitchelson, 1980) and the neuromuscular blockers (Miura et al., 1981). The purpose of the present study was to analyze the relationship between the species differences in the effect of acetylcholine on myocardial contractility and the species differences in the density and affinity of AChR, AChE, and ChE. Myocardial contractility was evaluated by the inotropic response of isolated atria from several species (i.e., rat, guinea pig and rabbit) whose cardiac responses to acetylcholine, anticholinergic drugs, and anticholinesterase compounds have been reported to vary. The effect of acetylcholine was evaluated in the presence and absence of soman, a potent AChE inhibitor (Andersen et al., 1977; Gray and Dawson, 1987) that has been observed to produce in vivo cardiomyopathy (Singer et al., 1987). METHODS Animals Male Sprague-Dawley rats (Rattus noroeguicus) weighing 200-300 g, Hartley guinea pigs (Curia powellus) weighing 400-500 g, and New Zealand rabbits (Oryctohgus cuniculus) weighing 2.c3.0 kg were obtained from Charles River Laboratories (Wilmington, Ma). Hearts isolated from these animals were rinsed with 0.15 M NaCl and dissected into atria, ventricles, and septum for physiological and biochemical measurements of isolated myocardial regions. Materials Acetylcholine bromide, acetvl-b-methvlcholine, and Triton-X-100 were purchased from Sigma Chemical Co. (St Louis, MO). [3H]-quinuclidinylbenzilate (13 mCi/mmol) and [‘HI-acetylcholine (56 mCi/mmole) were purchased 591
592
D. M. MAXWELL et al. Table 1. Regional differences in myocardial muscarinic acetylcholine receptors
100
90 T
s
80
.t; z
70 60
er 5
50 40 30 20 i
w 0
Muscarinic receptor density (pmol/g tissue)* Rabbit Left atrium Right atrium Septum Left ventricle Right ventricle
10-c o!
-14
:
:
-12
:
:
-10
:
:
-a
:
:
-6
log ACh Concentration
:
:
-4
:
I
-2
(M)
Fig. 1. Effect of acetylcholine in electrically stimulated contractions of left atria from rabbits (O), rats (A) and guinea pigs (0) expressed as percent of maximal contro1 contraction. Values are means + S.E. (n = 6). from New England Nuclear (Boston, Ma). [‘4C]-acetylb-methylcholine (57 mCi/mmole) was synthesized by Amersham (Arlington Heights, IL). Soman (methyl pinacolylphosphonofluoridate) was obtained from the Chemical Research, Development and Engineering Center (Aberdeen Proving Ground, MD). All other chemicals were reagent grade. Physiological measurements The inotropic responses of isolated left atria and ventricles were measured by the method of Levy (1971). Atria or ventricles were mounted on stimulating electrodes under OSg tension in baths containing well oxygenated (95% 0,/j% CO,) Krebs-Henseleit solution that was prepared as described by Winegard and Shanes (1962). The KrebsHenseleit solution contained 27.2 mM NaHCO,, 118.0 mM KCI, l.OmM KH,PO,, 1.2mM MgSO,, 2SmM CaCl,, and 11.1 mM glucose at pH 7.4. Tissues were maintained at 32°C and allowed to equilibrate in the bath for 30 min prior to the beginning of stimulation at 1 Hz using a square wave of Smsec duration at a voltage 10% above threshold (l-5 V). An FT-03 Grass force transducer was used to record force as an index of mechanical activity. The first derivative of force was obtained electronicaIly and was used as a measure of inotropy. Solutions of acetylcholine or soman were added sequentially to the tissue bath in order of increasing concentration with a wash between each test concentration. Receptor binding measurements Heart regions were prepared for measurement of muscarinic receptors by homogenization in 9 volumes of 0.32 M sucrose by using a Polytron homogenizer (Brinkman Instru100 90
80
-4
-1
log ACh Concentration
I
-3
(M)
Fig. 2. Dose-response of negative inotropic effect of acetylcholine in left atria of rabbits (O), rats (A) and guinea pigs (0). Response to acetylcholine is expressed as percent of maximal negative inotropic effect from Fig. 1. EC, for acetylchoiine is shown by (. . -).
14.8 * 10.6 + 5.9 + 7.0 + 6.3 +
Rat
0.q 0.4t 0.2tt 0.2tz 0.2tj
28.2 f 21.3 + 15.2 + 5.7 a 16.0 +
1.5 1.4 I.31 0.81 1.7$
Guinea pig 19.8 * l8,9 f 13.6 + 14.3 + 12.8 k
0.6 0.9 0.73 0.5# 0.6$
*Values represent [‘HI-QNBbinding at pH 7.4 (37°C). Values are means f. S.D. (n = 6). TSignificant difference from same region in rat or guinea pig (P < 0.05). jSignificant difference from left or right atrium of same species (P < 0.05).
ments, Westbury, NY). The homogenates were filtered through cheesecloth and the eluates were assayed for muscarinic AChR by the method of Fields et al. (1978) using [3H]-quinuclidinylbenzilate (QNB) as receptor ligand. Enzyme measurements Heart regions were prepared for measurement of AChE and ChE by homogenization in 9 volumes of 0.1 M phosphate buffer containing 0.15 M NaCl by using a Polytron homogenizer. After Polytron homogenization each heart homogenate received an amount of Triton X-100 equal to 1% of its volume and was homogenized again using a Potter-Elvehjem homogenizer, These homogenates were centrifuged at 30,OOOgfor 30 min at &6”C, and the resultant supernates were assayed for enzyme activity. AChE and ChE activity were measured by the method of Siakotos et al. (1969) using either [‘4C]-acetyl-~-methylcholine, a specific substrate for AChE, or [‘HI-acetylcholine which estimated the rate of acetylcholine hydrolysis by all types of ChE. Dafa analysis Scatchard analysis of binding data, analysis of MichaelisMenten equations for enzyme kinetics, and analysis of the graded dose-response relationship of physiological data were performed using the computer programs of Tallarida and Murray (1987). Significant differences in physiological and biochemical measurements were analyzed by the Newman-Keuls test (Hicks, 1982). Differences were considered significant if P < 0.05.
RESULTS
Preliminary experiments indicated that ventricles were much less responsive to acetylcholine than were atria for all species. While inotropy in atria from some species was maximally reduced by as much as 83% by acetylcholine, ventricles were reduced by < 10%. Therefore, further evaluation of the dose-response of acetylcholine on myocardial inotropy was performed in left atria. The negative inotropic response of left atria varied in both the magnitude of maximal response and in the acetylcholine concentration necessary to produce 50% of the maximal response (ECU,,). The maximal reduction of electrically stimulated atria1 contractions was 82.5% in guinea pigs, 50.8% in rats, and 41.5% in rabbits (Fig. 1). The EC,, for the negative inotropic response to acetylcholine was 3.3 x lo-‘M in rats and guinea pigs and 4.1 x 10m6M in rabbits (Fig. 2). The regional and species variations in the muscarinic AChR binding is shown in Table 1. The density of receptors was greater in atria than in septum or ventricles in all species. The density of receptors in all
Species differences in negative inotropy Table 2. Regional diflarenccs in myocardial acetylcholinesterase Acetylcholinesterase Rabbit Left atrium Right atrium Septum Left ventricle Right ventricle
0.19 * 0.23 + 0.11 * 0.05 f 0.13 *
activity (pmol/min/g Rat
o.ost 0.03t 0.02tt o.olt$ 0.01tz
0.53 f 0.58 * 0.30 * 0.14 * 0.30 *
0.04 0.05 0.02$ 0.02$ 0.02%
tissue)*
Guinea pig 0.47 +- 0.03 0.77 f 0.05 0.54 f 0.05 0.18 f 0.03% 0.39 f 0.03$
*Values represent hydrolysis of acetyl-B-methyl choline at 37°C (pH 7.4). Values are means + S.D. (n = 6). tSignificant difference from same region in rat or guinea pig (P < 0.05). SSignificant difference from left or right atrium of same species (P < 0.05).
regions was about 2-fold greater in rats and guinea pigs than in rabbits. The variation in AChE is shown in Table 2. The AChE activity was generally greater in atria than in ventricles or septum, which was similar to the regional distribution of AChR. The level of AChE activity in all regions was 2-3 times greater in rats and guinea pigs than in rabbits, which correlated with the species variation in AChR. The variation in ChE activity is shown in Table 3. The pattern of activity generally paralleled that of AChE and AChR (i.e. atria > ventricles = septum and guinea pig = rat > rabbit), except that the rat had a much greater level of ChE activity than either of the other species. The affinities of AChR and AChE in myocardial tisssue of rats, guinea pigs and rabbits are shown in Table 4. The affinity of ChE for acetylcholine was not determined because the ChE activity is a composite result of several enzymes (i.e., AChE, butyrylcholinesterase, propionylcholinesterase) that have different intrinsic affinities. The affinities of AChR for ‘[HI-QNB were not significantly different among these species. The affinities of AChE for acetyl-bmethylcholine in different species were also not significantly different. Soman, by itself, produced no changes in myocardial contractility at concentrations from 10m9 to 10m4M (data not shown), although concentrations > 10e6 M produced complete AChE inhibition (Fig. 3). The ECSO for the negative inotropic response to acetylcholine was reduced 3-fold in atria that had been previously exposed to 2 x 10V6M soman, in comparison to control atria that were not exposed to soman (Table 5). The magnitude of the maximal inotropic response to acetylcholine was not altered by prior exposure to soman (data not shown).
593
Table 4. Aflinitias of AChE and AChR from whole heart homogenates AChE* (mW 0.15_+0.04 0.12 f 0.03 0.17 f 0.04
Species Rat Guinea pig Rabbit
AChRt WI) 27_+ 10 30* 9 32 f 7
lK,,, of acetyl-fi-methylcholine
at pH 7.4 (37°C). Values are expressed as means + S.E. (n = 3). tApparent K, of [3H]-QNB at pH 7.4 (37°C). Values are expressed as means + S.E. (n = 3). DISCUSSION The negative inotropic response of atria to acetylcholine was much greater than the response of ventricles. This regional variation in response to acetylcholine was similar to previous observations (Higgins et al., 1973; Loffelholz and Pappano, 1985) that compared atria1 and ventricular responses to acetylcholine. The greater responsiveness of atria to acetylcholine is correlated with histochemical evidence that atria have a greater degree of cholinergic innervation than do ventricles (Loffelholz and Pappano, 1985), and by observations that atria have a greater density of biochemical markers for cholinergic innervation such as AChR (Table 1), AChE (Table 2), and ChE (Table 3). Species variation in the negative inotropic response to acetylcholine was evident in both the maximal response and the EC%. The order of maximal inotropic response was guinea pig > rat > rabbit, while the order of EC,, was guinea pig = rat < rabbit. However, species differences in biochemical markers of cholinergic innervation did not correlate with species differences in the ECU,, or the maximal negative inotropic response to acetylcholine. AChE and ChE are usually considered to be present in excess at cholinergic synapses, and therefore the density of AChE or ChE would not necessarily be expected to correlate with species differences in choline+ response. However, receptor density has been observed to influence the magnitude of the negative inotropic response of atria to agonists. In a comparison of the negative inotropy produced by acetylcholine and adenosine, the greater effect of acetylcholine was found to be correlated with the greater receptor density of AChR vs adenosine receptors (Linden et a[., 1985); and in our experiments the greater response of atria vs ventricles also correlated with the greater AChR density in atria vs ventricles. Nevertheless, species differences in the density of
Table 3. Regional differences in myocardial cholinesterase Cholinesterase activity (pmole/min/g Rabbit Left atrium Right atrium Septum Left ventricle Right ventricle
0.68 * 0.80 k 0.34 * 0.34 * 0.42 k
Rat
0.05t 0.09t 0.03Tf 0.04Tf 0.03tt
*Values represent hydrolysis Values are means k S.D. tsignificant difference from (P < 0.05). $Signno~,fference from < .
4.02 + 0.15 5.13 f 0.30 2.01 k0.17f 1.93+0.18f 2.62 + 0.15$
tissue)*
Guinea pig 1.24kO.12
1.95f 0.08 0.91 f 0.081 0.64 * 0.05$ 1.06 f 0.05$
of acetylcholine at pH 7.4 (37°C). (n = 6). same region in rat or guinea pig left or right atrium of same species
-11
-10
-9
log Somon
-8
-7
Concentration
-6
-5
(M)
Fig. 3. Effect of soman on AChE in heart of rabbits (O), rats (A) and guinea pigs (0). Heart slices were incubated for 5 min at 32°C and rinsed before AChE measurements.
594 Table 5.
D.M. MAXWELL~~ al. EC-
of acetvlcholine for nenative inotrouv of atria
Control CUM>
Soman* fUM)
Rat
0.33f 0.05
0.12* o.ost
2.75
Guinea pig Rabbit
0.33 i: 0.03 4.12 i: 0.08
0.~0~0~~~ 1.36 f 0.09t
3.30 3.03
ControIk.oman
*ECUS of acetylcholine after 5 min incubation with 2 x 10m6M soman. tSignificant difference from control (P < 0.05).
AChR (Table 3) were not correlated with the species differences in maximal negative inotropy in response to a~etylcholine. Although there was a great difference in the EQ,,for acetylcholine to produce negative inotropic effects in atria from rats and guinea pigs (3.3 x lo-‘M) vs rabbits (4.1 x 10s6 M), there was no significant species variation in the affinity of the AChR mediating the inotropic action of acetylcholine or the AChE catalyzing its hydrolysis (Table 4). Inasmuch as the muscarinic choline&c receptors measured by QNB binding are thought to consist of several subtypes of differing affinities (Birdsall et al., 1983; Hulme et al., 1990), it could be argued that species variation in agonist ECU,,may be reflected in species differences of a subtype of muscarinic receptor. However, 80% of the cardiac muscarinic receptors are of a single subtype (Closse et al., 1987), which reduces the likelihood of this explanation. Soman produced no negative (i.e., inhibitory) inotropic effect in atria. This observation in cardiac muscle was in sharp contrast to previous studies of excitatory effects in muscle, where sornan at similar concentrations (10m6M) produced enhanced response to electrical stimulation in striated (Smith et al., 1981) as well as smooth muscle (Adler et al., 1987). This difference between the responsiveness of inhibitory and excitatory cholinergic synapses to soman may indicate a fundamental difference in the neurophysiolo~c~ mechanisms of these synapses (North, 1989). Soman has been reported to bind to cholinergic receptors in heart as a result of its displacement of the muscarinic ligand cis-methyldioxolane in heart homogenates (Silveira et al., 1990). Although these cismethyldioxolane binding sites had a high affinity for soman (K, = 8 x lo-” M) and other organophosphorus compounds, Silveira and co-workers did not provide a functional correlation between binding affinity and any physiological effect of organophosphorus compounds in heart. Inasmuch as soman at concentrations much higher ( 10m9M to 10m4M) than the soman Kn for these receptors produced no negative inotropy with functionally intact atria (i.e., atria responsive to acetylcholine) in our experiments, we concluded that these high affinity binding sites for soman were probably non-functional. The absence of a direct effect of soman on myocardial contractions suggests that the in uivo cardiomyopathy that has been observed with soman (Singer et al., 1987) is the result of excessive vagal stimulation and not a direct effect of soman on cardiac tissue. Soman did produce an effect on the acetylcholine dose-response curve for negative inotropy. A consistent 3-fold decrease in the acetylcholine EC,, without alteration of the maximal response occurred in all species in the presence of 2 x 10m6M soman (i.e.,
where AChE was completely inhibited). Physostig mine and BW 284C51, a specific AChE inhibitor, have been previously demonstrated to produce a similar reduction in the a~tyl~holine EC, in rat atria (Slavikova and Hlavickova, 1982; Doods et al., 1989). This suggests that AChR were maximally occupied in both the presence and absence of AChE inhibition and that the species differences in maximal response were not the result of different fractional responses due to species variation in the ability of AChE to hydrolyze a~tyI~holine. The most likely explanation for the species differences in maximal negative inotropy is that the coupling of AChR to the ion channels that mediate negative inotropy may differ between species. Alterations in the coupling of receptors and ion channels have already been proposed as explanations for other physiolo~c~ phenomena that produce reductions in maximal physiolo~cal response, such as desensitization of cholinergic responses to acetylcholine and developmental changes in cholinergic responses (Nathanson, 1989). This explanation for species differences in maximal negative inotropy is supported by the observation that the same pattern of species variation in maximal negative inotropy that occurred with a~tylcholine (guinea pig > rat > rabbit) has been observed with adenosine, another agonist that produces negative inotropy (Belardinelli et al., 1982). Furthermore, antagonists of negative inotropy, such as stercuronium (Li and Mitchelson, 1980) or B-bungarotoxin (Miura et al., 198 1), also produce the same pattern of species variation in maximal inotropic response. The EC, of agonists or antagonists for the negative inotropic response is determined by their affinities for the muscarinic receptor, but the magnitude of the response is determined by the coupling of the agonist or antagonist receptor complex to the ion channels that produce the negative inotropic response (Linden et al., 1985; North, 1989). In heart both Ca2+ and K+ currents are involved in negative inotropy and both ion channels are coupled to the muscarinic receptor by guanine nucleotide binding proteins (Gproteins) (Kurachi et al., 1986). All of the protein components of negative inotropy-muscarinic recep tors, G-proteins, ion channels-exhibit heterogeneity in their amino acid sequences in different species (Hulme et al., 1990). The expression of this heterogeneity as species differences in the coupling arrangement of the receptor/G-protein/ion channel complex is probably a functional response to the physiological requirements imposed by the variation in the rate of the cardiac de~la~~tion/hyperpola~zation cycle (Lepeschkin, 1965), which is known to vary as heart rate varies between species (Poupa and Brix, 1984). REFERENCES Adler M., Reutter S. A., Moore D. H. and Filbert M. G. (1987) Actions of soman on isolated tracheal smooth muscle. In Cellular rmd ~otecufar Basis of C~oI~erg~c Function (Edited by Dowdall M. J. and Hawthorne J. N.) pp. 582-597. Ellis Horwood, Chichester. Andersen R. A., Aaraas I., Gaare F. and Fonnum F. (1977) Inhibition of acetylcholinesterase from different compounds, carbaspecies by organophosphorus mates and methylsulfonylfluoride. Gen. Pharmacol. 8, 331-334.
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