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Effects of a sulfhydryl reagent, N-ethylmaleimide , on electrical and mechanical activities of isolated atria
EUROPEAN JOURNAL OF PHARMACOLOGY 7 (1969) 5-13. NORTH-HOLLAND PUBLISHING COMP., AMSTERDAM
EFFECTS OF A SULFHYDRYL REAGENT, N-ETHYLMALEIMIDE, ON ELECTRICAL AND MECHANICAL ACTIVITIES OF ISOLATED ATRIA Noburu TODA and Noriyuki KONISHI
Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto, Japan
Received 13 February 1969
Accepted 26 March 1969
N.TODA and N.KONISHI, Effects of a sulfhydryl reagent, N-ethylmaleimide, on electrical and mechanical activities of isolated atria, European J. Pharmacol. 7 (1969) 5-13. Rabbit sino-atrial node-right atrium preparations, sympathetic nerve-atria preparations and left atrium preparations were used. N-ethylmaleimide (NEM) in concentrations higher than 10"s M produced a dosedependent decrease in atrial rate. At 10-3 M activities the S-A node and right atrium were abolished. The activities were not restored by cysteine and ATP. Transmembrane potentials of S-A nodal pacemaker fibers were affected by 10-3 M NEM. The major changes consisted of a decrease in the slope of diastolic depolarization and an acceleration of repolarization velocity. A tendency for the maximal diastolic potential and the overshoot to decrease were observed. Pacemaker shift frequently occurred. S-A nodal activities were more resistant to NEM than those of the right atrium. Left atrial preparations were driven electrically at different rates. The developed tension-driving rate relationship was not influenced by 10-s M NEM but was measurably depressed by 10.-4 M. The depression of the tension developed at high driving rates (60 to 240 beats/min) was more marked than that at low rates (6 to 30 beats/min). The negative chronotropic response to transmural electrical stimulation applied at the S-A node was slightly reduced in some preparations exposed to 10-7 to 10-s M NEM, but was blocked by 10--4 M. At the same concentration, slowing of atrial rate produced by aeetylcholine was also blocked. The positive chronotropic effect of sympathetic nerve stimulation and of noradrenaline was reduced by 10--4 M NEM. Since the effects of NEM were prevented by preincubation with isomolar concentrations of cysteine, the changes observed were considered to result from an inhibition of the SH groups. Thus, it was concluded that SH groups are involved in the process of contraction and the bioelectrical activity of pacemaker membrane linking automaticity and chronotropic actions of acetylcholine. N-ethylmaleimide Sulfhydryl groups Acetylcholine
1. INTRODUCTION During the past decade N-ethylmaleimide (NEM) has been used as a selective SH alkylating reagent in various field of research. The advantages of this compound as an SH reagent are: (1) high selectivity for SH groups, (2) reaction with only certain accessible SH groups on enzymes, making possible specific metabolic inhibitions, and (3) good penetration into cells (Webb, 1966). However, the use of NEM to investigate the metabolic basis for tissue functions has been very limited. Its only k n o w n pharmacological actions
Noradrenaline Transmembrane potential
are a stimulation of motility of the isolated rat ileum (Goodman and Hiatt, 1964) and a positive inotropic action on the cat papillary muscle (Bennett et al., 1958). However, involvement of the SH groups in cholinergic receptors is suggested from findings that a variety of SH reagents such as lead acetate, iodoacetamide, hydrogen peroxide and NEM depress the mechanical response to acetylcholine of isolated segments of the rat ileum (Goodman and Hiatt, 1964). The experiments presented here are concerned with the study of (1) effects of NEM on bioelectrical functions of S-A nodal pacemaker fibers and on con-
6
N.TODA and N.KON1SHI
tractile force of the isolated left atrium and (2) chronotropic responses to endogenous and exogenous acetylcholine and to noradrenaline.
2. METHODS Fifty-three albino rabbits of either sex, weighing 1.8 to 2.2 kg, were used. Under ether anesthesia the animals were killed by exsanguination from both common carotid arteries. The entire heart was removed and the ventricles were discarded. For investigation of the effects of drugs on pacemaker fibers of the S-A node and on their response to transmural stimulation, conventional atrial preparations were used. The anterior wall of the superior vena cava was opened as described by Paes de Carvalho et al. (1959). The specimen was fixed horizontally between hooks (endocardial surface uppermost) under a resting tension of 300 to 450 mg in the muscle bath of 60 ml capacity in which the nutrient solution was gassed with a mixture of 95% 02-5% CO 2 and was maintained at 30 -+ 0.5°C. Hooks fixing an appendage of the right atrium were connected to the arm lever of a force-displacement transducer (Nihonkoden Kogyo Co.). Sympathetic nerve-atrial preparations obtained by the technique described in an earlier report (Toda and Shimamoto, 1968) were used for studies on changes in chronotropic responses to sympathetic nerve stimulation. The preparation was fixed between hooks in the muscle bath, as described above. For studies on changes in contractile force of the atrium, left atrial preparations were used. Specialized tissues were excluded from the preparation. Artificial electrical stimulation was applied at a constant rate through a pair of stimulating electrode anchoring the cut end of the preparation. Hooks fixing an appendage of the left atrium were connected to the forcedisplacement transducer. Constituents of the nutrient solution were as follows (mM): Na +, 162.1; K +, 5.4; Ca ++, 2.2; C I - , 157.0; HCO3-, 14.9; dextrose, 5.6. All preparations were allowed to equilibrate with the nutrient solution for 60 to 90 min before measurements were taken. Transmembrane potentials were recorded from single pacemaker fibers of the S-A node by the use of a floating microelectrode. Pacemaker fibers were identified from characteristics of the action potential
configuration, particularly the smooth transition from diastolic depolarization to rapid upstroke. The membrane potential was recorded simultaneously on film moving at a rate of 5 cm/sec from a VC-7 oscilloscope (Nihonkodenkogyo Co.) and on a two-channel penwriter (Sanei Sokki Co.). A monopolar silver electrode (0.5 mm diameter) insulated except for the tip was used for transmural electrical stimulation of intracardiac cholinergic and adrenergic nerve fibers innervating the SoA node (Lewartowski, 1963; Vincenzi and West, 1963). The optimal electrode position for the transmural stimulation was shown in the previous report (West and Toda, 1967). The parameters for nerve stimulation were rectangular pulses of 0.1 msec duration and supramaximal (for nerve excitation) inteAsity, applied at frequencies of l, 5, 20 and 100/sec. For stimulation of extracardiac sympathetic nerves, the right postganglionic sympathetic nerve was placed on a bipolar silver electrode which was lifted above the surface of the solution. The nerve was stimulated by a train of rectangular pulses, 1.0 msec duration with supramaximal intensity, applied at frequencies of 1,5 and 20/sec. Left atrial preparations were driven at a constant rate of 60 beats/min by a train of pulses, 3 msec duration and supramaximal intensity (about twice threshold), except when the contractile tensiondriving rate relationship was obtained. In order to obtain the tension-rate relationship, the driving rate (6, 12, 30, 60, 120, 180, 240 beats/min or higher) was raised stepwise from the lowest until the preparation failed to respond to each stimulus. The preparation was driven at each rate until steady state contractions were attained. Electrical stimuli were delivered from a Sanei type ES-103 Z pulse generator. NEM (Wako Chemical Industries) was dissolved in the nutrient solution just before experiments were begun. The drug was added directly to the muscle bath in one of five experimental concentrations, 10 -7, 10 -'6, 10 -5 , 10 --4 or 10 -3 M. Time of exposure to the drug was usually from 30 to 60 min. 1-Cysteine and ATP disodium (Sigma Chemical Co.) were used. Acetylcholine hydrochloride and 1-noradrenaline hydrochloride were applied in cumulative concentrations. Concentrations of the amines were expressed in terms of g/ml of the salts. The doseresponse relationship was obtained prior to and following 20 min of exposure to NEM.
N - E T H Y L M A L E I M I D E ON A T R I A
Parameters of the membrane potential measured in the present study were: cycle length between pacemaker action potentials; maximal diastolic potential; threshold potential; overshoot; 10% and 90% durations; and depolarization time;The same terminology appears in earlier reports (Toda, 1968; Toda and Shimamoto, 1968). The parameters were compared in the same fibers before and after drug action. Mean values of 10 measurements of the cycle length between contractions were obtained. The atrial rate was calculated from the cycle length. Changes in the cycle length or the pacemaker rate produced by transmural stimulation and sympathetic nerve stimulation were obtained when the maximum response was attained. Data were prepared for presentation as means of the absolute values +- standard errors of the means. Comparisons of results were made using the Student's t test.
3. RESULTS 3.1. S-A node The rate of atrial contractions under steady state conditions was not influenced by NEM in concentrations lower than 10 -6 M, but was decreased after 20 min of exposure to 10-5 and 10 --4 M (fig. 1). The mean control value of the atrial rate was 88 -+ 5 beats/ min (n = 22). In 3 out of 22 preparations a moderate increase in rate (8 to 20 beats/min) was produced by
7
10 -5 M NEM. At 10-3 M spontaneous rate was progressively slowed until atrial contractions were completely abolished. The mean time for onset of atrial arrest was 33.3 -+ 5.6 min (n = 22) at which time not only mechanical but electrical activities disappeared from fibers of the right atrium whereas electrical activities of the S-A node were usually maintained. When cysteine (10 -3 M) was preincubated with 10 -3 M NEM, the inhibitory effect of the SH reagent was prevented (open circle in fig. 1). Cysteine alone did not affect atrial rate. In preparations in which atrial activities had been markedly inhibited by high concentrations of NEM, no antagonistic effect of cysteine (10 -3 M) and ATP (10 -3 M) was observed. Atrial activities were not restored by washout of NEM from the bathing solution or by application of noradrenaline in concentrations of 10 -6 to 5 X 10 -6 g/ml in 15 preparations. Transmembrane potentials of S-A nodal pacemaker fibers were not measurably affected in preparations exposed to NEM at lower than 10 --4 M for 30 min. At 10 -3 M the membrane potential was influenced before slowing of the pacemaker rate. Mean values of parameters of the membrane potential re-
OC
-IO"
Table 1 Modification by NEM of parameters of the transmembrane potential of SoA nodal pacemaker fibers.
O O
Control
NEM 10 -3 M*
Parameters measured
(n = 9)
(n = 9)
Cycle length (msec) Maximal diastolic potential (mV) Threshold potential (mV) Overshoot (mV) 90% Duration (msec) 10% Duration (msec) Depolarization time (msec)
800 65 43 13 207 54 15
790 64 44 13 200 44 15
.c
+ 26 + 3 + 3 + 2 + 12 -+ 4 + 2
± 63 + 3 + 3 + 2 + 10 + 4 + 3
n = n u m b e r o f pacemaker fibers and also o f preparations. * Values obtained from preparations exposed to 10 -3 M NEM for 4 to 7 min.
-20-
-50.
NEM concentration ( M )
Fig. 1. Effects o f NEM on atrial rate. Values were obtained after 20 m i n of exposure to NEM. * values obtained after 10 m i n o f exposure. Solid circles = effect o f NEM (n = 22); open circle = effect o f NEM (10 . 3 M) preincubated with 10 .3 M cysteine (n = 3).
8
N.TODA and N.KONISHI tion representative of true pacemaker fibers was converted to that of latent pacemaker fibers after 15 to 30 min o f exposure to NEM. Conversely, in 2 fibers conversion from latent to true pacemaker was observed (I and II in fig. 2). Preincubation with cysteine (10 -3 M) was effective in preventing the membrane effect to NEM.
III
I
I
2 0 0 ms IV
50 mV
Fig. 2. Alteratlons in the membrane potentiai of a S-A nodal fiber induced by 10-s M NEM. Imp'dement into the fiber persisted for 24 rain. I = control; II = 15 min after NEM; III = 24 min after; IV = superimposition of II (presented as A) and III (presented as B). Configuration representative of a latent pacemaker fiber (I) was converted to that of a true pacemaker fiber (II and 1II) after 13 min of exposure to NEM. corded from same pacemaker fibers prior to and following 10 -3 M NEM are tabulated in table 1. Since it was very difficult to keep microelectrodes in the same pacemaker fibers for a long time, especially in the presence of high concentrations o f NEM, changes produced after only 4 to 7 min of exposure to NEM were obtained. As illustrated in table 1 and in fig. 2 IV, the duration of the early plateau phase was shortened. The 10% duration was measurably shortened in 5 out of 9 fibers. The 90% duration was shortened in 3 out of 9 fibers but prolonged in 2 fibers. In 3 instances in which the microelectr0de was held for a long period (15, 20 and 24 min) in one fiber, the longer the exposure to NEM, the more were the 10% and 90% durations shortened. Slowing of the pacemaker rate seen after 5 to 10 min of exposure to 10 -3 M NEM was associated with a reduction of the slope of diastolic depolarization. The threshold potential was not considerably affected, whereas the maximal diastolic potential and the overshoot were decreased (fig. 2). In 3 out of 8 fibers, the configura-
3.2. Contractile force o f the left atrium Left atrial preparations were driven electrically at different rates from 6 to 240 beats/min or higher. The tension developed at 6 to 120 beats/min was a direct function o f driving rates but at 120 to 240 beats/min or higher the tension was an inverse function of the rates; the tension-rate relationship was not significantly affected by 10 -5 M NEM. At 10 --4 M the tension developed at high driving rates (60 to 240 beats/min) was significantly reduced (p < 0.01), the magnitude of the reduction being time-dependent at times up to 40 min. The tension developed at low rates (6 and 12 beats/min) was enhanced after 20 min of exposure to 10 --4 M NEM; at 6 beats/min the enhancement was significant (p < 0.05). The dependency of the contractile tension upon driving rates was markedly decreased by NEM. The results are shown in fig. 3. The maximum driving rate at which alternating contractions, in which the strength of smaller contractions was not less than ~ of that of larger contractions were produced, was compared in control and NEM-added solutions. The mean value was decreased from 350 beats/min (n = 8) to 310 beats/.min (n = 8) by 10 --4 M NEM. 3.3. Chronotropic response to autonomic nerve stimulation Transmural electrical stimulation applied at the S-A node produces negative chronotropic and inotropic effects followed by positive effects, which are thought to be due to the release of endogenous acetylcholine and noradrenaline, respectively (Vincenzi and West, 1963). The negative and positive effects varied directly with frequencies of stimualtion. The negative chronotropic effect of transmural stimulation at 20/sec was moderately reduced in 3 out of 9 preparations but at lO0/sec was reduced in only 2 preparations after 20 min of exposure to 10 -7 M NEM. The reduction was corrected by washout of
N-ETHYLMALEIMIDE ON ATRIA
NEM 10"5M
800-
NEM
" Control
9
10"4M
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Rate of contraction (beats/rain) Fig. 3. Alterations i . the tension-rate relationship of left atria exposed to NEM. Figures in parentheses indicate the n u m b e r o f experiments.
3,000
900"
• Control (9) x NEM 10"7M ( 9 ) o 10"6M (T) IO'SM ( 6 ) 10"4M ( 4 )
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Stimulation frequency ( c / s )
Fig. 4. Effects of NEM on the negative ehronotropic response to transmural electrical stimulation applied at the S-A node. The number of experiments is indicated in parentheses.
6 /"
i
s
2b
16o
Stimulation frequency ( c/$ ) F]~. 5. Effects o f NEM on the positive chronotropic effect o f transmural stimulation. Date here and in fig. 4 were obtained from the same preparations. S y m b o l s in the figure are the same as those indicated in fig. 4.
10
N.TODA and N.KONISH1
•
Control
• Control (13) O NEM 10-6M (9) ;< 10"SM (9) A 10-4 M ( 8 )
l
l
•
120" 10"4M
l
/ ,
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,/" I/X .E 100 E
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Stimulation frequency ( c/s )
//
,b "r
5x~o -7
2x'lo -e
ACh concentration
io-~
5Xi0 "5
( g/ml )
Fig. 6. Modification by NEM of the positive chronotropic effect of sympathetic nerve stimulation. The number of experiments is indicated in parentheses.
Fig. 7. Effects of NEM on the negative chronotropic response to acetyicholine. Figures in parentheses indicate the number of experiments.
NEM. A greater reduction was not produced by 10-6 and 10-5 M in these preparations. At 10-4 M the negative chronotropic effect of nerve stimulation was completely abolished (fig. 4) and was n o t reversed by repeated washouts. The positive chronotropic effect which followed the negative effect of transmural stimulation was not affected by NEM in concentrations lower than 10-5 M. At 10.-4 M NEM the pre-stimulation cycle length between contractions was significantly increased and the positive chronotropic effect of nerve stimulation was considerably reduced (fig. 5). Stimulation of extracardiac, postganglionic sympathetic nerves produced an increase in atrial rate, which was dependent upon frequencies of electrical stimulation. The positive chronotropic effect of sympathetic nerve stimulation was not influenced by NEM at lower than 10-5 M, but was reduced by
10--4 M (fig. 6). The chronotropic effect was not corrected by washout of NEM. 3.4. Chronotropic response to aeetyleholine and noradrenaline The atrial rate was progressively slowed by increasing concentrations of acetylcholine. The concentration-response relationship was not altered by 10--6 or 10-5 M NEM. However, the negative chronotropic effect of acetylcholine was abolished and in 2 out of 8 preparations changed to a positive effect after 20 min of exposure to 10.-4 M NEM. The results are illustrated in fig. 7. The inhibition of the acetylcholine action was not corrected by repeated wash of preparations and by application of cysteine and ATP in concentrations up to 10-3 M. However, when NEM (10 --4 M) was preincubated with 10--4 M cysteine, the inhibitoty effect of the SH reagent was prevented
N-ETHYLMALE1MIDE ON ATRIA
•
Control
× NEM I0-5 M 10-4 M
/ / ~
160
A
140
-g
///
w
120
<"V ..¢ <"
e o
T
a I00
80 ¸
60.
,
0
!.
t
5)(10 "e
2)(10 "8
NA concentration
I0 -7
5x'lo "7
2X'10 "6
( g/ml )
Fig. 8. Effects of NEM on the positive chronotropic response to noradrenaline. Figures in parentheses indicate the number of experiments.
in 3 out of 3 preparations. Noradrenaline in cumulative concentrations produced a dose-related increase in the atrial rate. The concentration-response relationship was not affected by 10 -5 M NEM but was shifted to the right by 10 --4 M (fig. 8). In 2 out of 2 preparations the inhibition of noradrenaline effect was prevented by preincubation of NEM with isomolar concentrations of cysteine.
4. DISCUSSION It is known that certain accessible SH groups of native protein are reactive to NEM, making possible a specific metabolic inhibition (Webb, 1966). According to Skou (1963), the activity of the Na+-K+activated ATPase prepared from ox brain is markedly inhibited after 10 min of exposure to 10 -3 M NEM. Respiration of human erythrocytes is also inhibited some 50% by 1.3 mM NEM (Sheets and Hamilton,
11
1958). At this concentration certain dehydrogenases, electron transport systems, glycolytic and cyclic enzymes are inhibited (summarized by Webb, 1966). At 10 -3 M NEM the electrical activity of S-A nodal pacemaker fibers was influenced. The major changes produced consisted of a decrease in the slope of diastolic depolarization, which resulted in bradycardia and an arrest of S-A nodal activities, and of shortening of the 10% duration. Similar shortening of the action potential duration is observed in fibers of the S-A node, atrium and ventricle exposed to toxic concentrations of ouabain (Toda, unpublished), 2-4-dinitrophenol, sodium azide (De Mello, 1959) and iodoacetate (Kleinfeld et al., 1955). The membrane effect of 2-4-dinitrophenol and iodoacetate is partly corrected by ATP, pyruvate and acetate, whereas that of NEM was not corrected by ATP, cysteine and noradrenaline. Contractibility of the isolated hypodynamic cat papillary muscle is enhanced by 1 mM NEM (Bennett et al., 1958). However, at this and at lower concentrations, NEM exerted not a positive inotropic effect but a negative inotropic effect on left atria isolated from rabbits. It is suggested by Goodman and Hiatt (1964) that the SH groups are involved in the process of contraction of the rat ileum. This would also be the case for atrial contractibility. The SH groups involved would be either part of the muscle protein or components of vital enzyme systems involved in muscle metabolism, or both. NEM inhibits the SH groups of action. However, it seems likely that only a fraction of the SH groups which do not function in polymerization and in ATP-binding react with NEM (Bdrdny et al., 1962; Tonomura and Yoshimura, 1962; Drabikowski and Gergely, 1963). The enhancement of cardiac contractibility produced by cardiac glycosides is thought to be associated with a net loss of K + or a gain of Na + secondary to an impairment of active movements of the ions across membrane (reviewed by Glynn, 1964). NEM causes a loss of K + from duck erythrocytes but, in contrast to other inhibitors, also causes some loss of Na +, this being accompanied by marked suppression of glucose uptake and glycolysis (Tosteson and Johnson, 1957). Respiration of human erythrocytes is also suppressed by NEM (Sheets and Hamilton, 1958). If this is the case for atrial muscle, an inhibition of aerobic metabolism would account for the negative inotropic
12
N.TODA AND N.KONISHI
effect o f NEM. Findings obtained in the present study indicate that atrial fibers are more susceptible to NEM than S-A nodal pacemaker fibers; aerobic metabolism would be expected to participate greater in functions o f contractile tissues than those o f specialized tissues. The negative chronotropic response o f isolated atria to endogenous and exogenous acetylcholine was abolished by 10 -4 M NEM. The inhibitory effect of NEM was prevented by preincubation with cysteine. G o o d m a n and Hiatt (1964) reported that NEM (5.7 × 10 -4 M) prevented acetylcholine-induced contraction o f the isolated rat ileum, at which time 76% o f the SH groups were inhibited. Cardiac glycosides are known to be specific inhibitors of the membrane ATPase, one o f the more important SH enzymes. Toda and West (1966) demonstrated that ouabain produced an augmentation o f the negative chronotropic response o f isolated rabbit atria to endogenous and exogenous acetylcholine. The inverse effect of NEM and ouabain on sensitivity o f the S-A node to acetylcholine would indicate that SH groups but not those of the membrane ATPase are necessary in producing cholinergic responses. The positive chronotropic effect o f endogenous and exogenous noradrenaline was depressed by 10 --4 M NEM. The cardio-stimulation effect o f noradrenaline was blocked in preparations in which atrial activities were abolished by 10 -3 M NEM. However, the inhibitory effect o f NEM on the noradrenaline effect was less marked than that on the acetylcholine effect. It remains to be seen whether the SH groups involved in adrenergic receptor mechanisms are more resistant to NEM or possess a greater safety factor than those in cholinergic mechanisms. It was demonstrated by Komalahiranya and Volle (1963) that the injection o f 0.5 mg o f NEM into the c o m m o n carotid artery produced marked changes in transmission across the cat superior cervical ganglion. The immediate change ( 0 - 1 0 min) produced by NEM is an enhancement o f the postganglionic spike and a depression o f the responses to acetylcholine and KCI. The late change ( 1 0 - 4 5 min) consisted o f an enhancement o f the response to the ganglionic stimulants at a time when the responses to stimulation o f preganglionic nerve was depressed or even abolished. In the present study, however, when preparations were exposed to 10 --4 M NEM for 20 rain, the depression
of the chronotropic response to acetylcholine and to stimulation of intracardiac cholinergic nerves were similar. This was also true in the case o f noradrenaline and sympathetic nerve stimulation. Thus, it is unlikely that in isolated rabbit S-A nodes NEM appreciably affects functions o f cholinergic and adrenergic postganglionic nerve terminals. Thus, it is not known whether reduced sensitivity of S-A nodal pacemaker fibers to the chemical transmitters is due primarily to an inhibition of SH groups in the receptors or to a general inhibition of cell metabolism.
REFERENCES B~ir~iny, M., F.Finkelman and T.Therattil-Antony, 1962, Studies on the bound calcium of actin. Arch. Biochem. Biophys. 98, 28-45. Bennett, D.R., K.S.Andersen, M.V.Andersen, Jr., D.N.Robertson and M.B.Chenoweth, 1958, Structure-activity analysis of the positive inotropic action of conjugated carbonyl compounds on the cat papillary muscle. J. Pharmacol. Exptl. Therap. 122, 489-498. De Mello, W.C., 1959, Metabolism and electrical activity of the heart; action of 2-4-dinitrophenol and ATP, Amer. J. Physiol. 1962, 377-380. Drabikowski, W. and J.Gergely, 1963, The role of sulfhydryl groups in the polymerization and adenosine triphosphatc binding of G-actin, J. Biol. Chem. 238,640--643. Glynn, I.M., 1964, The action of cardiac glycosides on ion movements, Pharmacol. Rev. 16,381-407. Goodman, 1. and R.B.Hiatt, 1964, Chemical factors affecting spontaneous motility of the small intestine in the rat. 1. Sulfhydryl reactants, Biochem. Pharmacol. 13, 871879. Kleinfeld, M., E.Stein, J.Magin and C.E.Kossmann, 1955, The action of iodoacetate on the electrical and mechanical activities of the isolated perfused frog heart, J. Clin. Invest. 34, 1802-1806. Komalahiranya, A. and R.L.Volle, 1963, Alterations of transmission in sympathetic ganglia treated with a sulfhydryl-group inhibitor, N-ethylmaleimide (NEM), J. Pharmacol. Exptl. Therap. 139, 304-311. Lewartowski, B., 1963, Selective stimulation of intra-cardiac postganglionic fibers, Nature, 199, 76-77. Paes de Carvalho, A., W.C.De Mcllo and B.F.Hoffman, 1959, Electrophysiological evidence for specialized fiber types in rabbit atrium, Amer. J. Physiol. 196, 483-488. Sheets, R.F. and H.E.Hamilton, 1958, A reversible effect on the metabolism of human erythrocytes by p-chloromercuribenzoic acid and N-ethyl maleimide, J. Lab. Clin. Med. 52, 138-143. Skou, J.C., 1963, Studies on the Na+ + K+ activated ATP hydrolyzing enzyme system. The role of SH groups,
N-ETHYLMALE1MIDE ON ATRIA Biochem. Biophys. Res. Commun. 10, 79-84. Toda, N., 1968, Influence of sodium ions on the membrane potential of the sino-atrial node in response to sympathetic nerve stimulation, J. Physiol. 196,677-691. Toda, N. and K.Shimamoto, 1968, The influence of sympathetic stimulation on transmembrane potentials in the S-A node, J. Pharmacol. Exptl. Therap. 159, 298-305. Toda, N. and T.C.West, 1966, The influence of ouabain on cholinergic responses in the sinoatrial node, J. Pharmacol. Exptl. Therap. 153, 104-113. Tonomura, Y. and J.Yoshimura, 1962, Binding of p-chloromercuribenzoate to actin, J. Biochem. (Tokyo) 5 1 , 2 5 9 266.
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Tosteson, D.C. and J.Johnson, 1957, The coupling of potassium transport with metabolism in duck red cells, J. Cellular Comp. Physiol. 50, 169-183. Vincenzi, F.F. and T.C.West, 1963, Release of autonomic mediators in cardiac tissue by direct subthreshold electrical stimulation, J. Pharmacol. Exptl. Therap. 141, 185-194. Webb, J.L., 1966, Enzyme and metabolic inhibitors 3, 337-895, Academic Press, New York. West, T.C. and N.Toda, 1967, Response of the A-V node of the rabbit to stimulation of intracardiac cholinergic nerves, Circulation Res. 20, 18-31.
EFFECTS OF A SULFHYDRYL REAGENT, N-ETHYLMALEIMIDE, ON ELECTRICAL AND MECHANICAL ACTIVITIES OF ISOLATED ATRIA Noburu TODA and Noriyuki KONISHI
Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto, Japan
Received 13 February 1969
Accepted 26 March 1969
N.TODA and N.KONISHI, Effects of a sulfhydryl reagent, N-ethylmaleimide, on electrical and mechanical activities of isolated atria, European J. Pharmacol. 7 (1969) 5-13. Rabbit sino-atrial node-right atrium preparations, sympathetic nerve-atria preparations and left atrium preparations were used. N-ethylmaleimide (NEM) in concentrations higher than 10"s M produced a dosedependent decrease in atrial rate. At 10-3 M activities the S-A node and right atrium were abolished. The activities were not restored by cysteine and ATP. Transmembrane potentials of S-A nodal pacemaker fibers were affected by 10-3 M NEM. The major changes consisted of a decrease in the slope of diastolic depolarization and an acceleration of repolarization velocity. A tendency for the maximal diastolic potential and the overshoot to decrease were observed. Pacemaker shift frequently occurred. S-A nodal activities were more resistant to NEM than those of the right atrium. Left atrial preparations were driven electrically at different rates. The developed tension-driving rate relationship was not influenced by 10-s M NEM but was measurably depressed by 10.-4 M. The depression of the tension developed at high driving rates (60 to 240 beats/min) was more marked than that at low rates (6 to 30 beats/min). The negative chronotropic response to transmural electrical stimulation applied at the S-A node was slightly reduced in some preparations exposed to 10-7 to 10-s M NEM, but was blocked by 10--4 M. At the same concentration, slowing of atrial rate produced by aeetylcholine was also blocked. The positive chronotropic effect of sympathetic nerve stimulation and of noradrenaline was reduced by 10--4 M NEM. Since the effects of NEM were prevented by preincubation with isomolar concentrations of cysteine, the changes observed were considered to result from an inhibition of the SH groups. Thus, it was concluded that SH groups are involved in the process of contraction and the bioelectrical activity of pacemaker membrane linking automaticity and chronotropic actions of acetylcholine. N-ethylmaleimide Sulfhydryl groups Acetylcholine
1. INTRODUCTION During the past decade N-ethylmaleimide (NEM) has been used as a selective SH alkylating reagent in various field of research. The advantages of this compound as an SH reagent are: (1) high selectivity for SH groups, (2) reaction with only certain accessible SH groups on enzymes, making possible specific metabolic inhibitions, and (3) good penetration into cells (Webb, 1966). However, the use of NEM to investigate the metabolic basis for tissue functions has been very limited. Its only k n o w n pharmacological actions
Noradrenaline Transmembrane potential
are a stimulation of motility of the isolated rat ileum (Goodman and Hiatt, 1964) and a positive inotropic action on the cat papillary muscle (Bennett et al., 1958). However, involvement of the SH groups in cholinergic receptors is suggested from findings that a variety of SH reagents such as lead acetate, iodoacetamide, hydrogen peroxide and NEM depress the mechanical response to acetylcholine of isolated segments of the rat ileum (Goodman and Hiatt, 1964). The experiments presented here are concerned with the study of (1) effects of NEM on bioelectrical functions of S-A nodal pacemaker fibers and on con-
6
N.TODA and N.KON1SHI
tractile force of the isolated left atrium and (2) chronotropic responses to endogenous and exogenous acetylcholine and to noradrenaline.
2. METHODS Fifty-three albino rabbits of either sex, weighing 1.8 to 2.2 kg, were used. Under ether anesthesia the animals were killed by exsanguination from both common carotid arteries. The entire heart was removed and the ventricles were discarded. For investigation of the effects of drugs on pacemaker fibers of the S-A node and on their response to transmural stimulation, conventional atrial preparations were used. The anterior wall of the superior vena cava was opened as described by Paes de Carvalho et al. (1959). The specimen was fixed horizontally between hooks (endocardial surface uppermost) under a resting tension of 300 to 450 mg in the muscle bath of 60 ml capacity in which the nutrient solution was gassed with a mixture of 95% 02-5% CO 2 and was maintained at 30 -+ 0.5°C. Hooks fixing an appendage of the right atrium were connected to the arm lever of a force-displacement transducer (Nihonkoden Kogyo Co.). Sympathetic nerve-atrial preparations obtained by the technique described in an earlier report (Toda and Shimamoto, 1968) were used for studies on changes in chronotropic responses to sympathetic nerve stimulation. The preparation was fixed between hooks in the muscle bath, as described above. For studies on changes in contractile force of the atrium, left atrial preparations were used. Specialized tissues were excluded from the preparation. Artificial electrical stimulation was applied at a constant rate through a pair of stimulating electrode anchoring the cut end of the preparation. Hooks fixing an appendage of the left atrium were connected to the forcedisplacement transducer. Constituents of the nutrient solution were as follows (mM): Na +, 162.1; K +, 5.4; Ca ++, 2.2; C I - , 157.0; HCO3-, 14.9; dextrose, 5.6. All preparations were allowed to equilibrate with the nutrient solution for 60 to 90 min before measurements were taken. Transmembrane potentials were recorded from single pacemaker fibers of the S-A node by the use of a floating microelectrode. Pacemaker fibers were identified from characteristics of the action potential
configuration, particularly the smooth transition from diastolic depolarization to rapid upstroke. The membrane potential was recorded simultaneously on film moving at a rate of 5 cm/sec from a VC-7 oscilloscope (Nihonkodenkogyo Co.) and on a two-channel penwriter (Sanei Sokki Co.). A monopolar silver electrode (0.5 mm diameter) insulated except for the tip was used for transmural electrical stimulation of intracardiac cholinergic and adrenergic nerve fibers innervating the SoA node (Lewartowski, 1963; Vincenzi and West, 1963). The optimal electrode position for the transmural stimulation was shown in the previous report (West and Toda, 1967). The parameters for nerve stimulation were rectangular pulses of 0.1 msec duration and supramaximal (for nerve excitation) inteAsity, applied at frequencies of l, 5, 20 and 100/sec. For stimulation of extracardiac sympathetic nerves, the right postganglionic sympathetic nerve was placed on a bipolar silver electrode which was lifted above the surface of the solution. The nerve was stimulated by a train of rectangular pulses, 1.0 msec duration with supramaximal intensity, applied at frequencies of 1,5 and 20/sec. Left atrial preparations were driven at a constant rate of 60 beats/min by a train of pulses, 3 msec duration and supramaximal intensity (about twice threshold), except when the contractile tensiondriving rate relationship was obtained. In order to obtain the tension-rate relationship, the driving rate (6, 12, 30, 60, 120, 180, 240 beats/min or higher) was raised stepwise from the lowest until the preparation failed to respond to each stimulus. The preparation was driven at each rate until steady state contractions were attained. Electrical stimuli were delivered from a Sanei type ES-103 Z pulse generator. NEM (Wako Chemical Industries) was dissolved in the nutrient solution just before experiments were begun. The drug was added directly to the muscle bath in one of five experimental concentrations, 10 -7, 10 -'6, 10 -5 , 10 --4 or 10 -3 M. Time of exposure to the drug was usually from 30 to 60 min. 1-Cysteine and ATP disodium (Sigma Chemical Co.) were used. Acetylcholine hydrochloride and 1-noradrenaline hydrochloride were applied in cumulative concentrations. Concentrations of the amines were expressed in terms of g/ml of the salts. The doseresponse relationship was obtained prior to and following 20 min of exposure to NEM.
N - E T H Y L M A L E I M I D E ON A T R I A
Parameters of the membrane potential measured in the present study were: cycle length between pacemaker action potentials; maximal diastolic potential; threshold potential; overshoot; 10% and 90% durations; and depolarization time;The same terminology appears in earlier reports (Toda, 1968; Toda and Shimamoto, 1968). The parameters were compared in the same fibers before and after drug action. Mean values of 10 measurements of the cycle length between contractions were obtained. The atrial rate was calculated from the cycle length. Changes in the cycle length or the pacemaker rate produced by transmural stimulation and sympathetic nerve stimulation were obtained when the maximum response was attained. Data were prepared for presentation as means of the absolute values +- standard errors of the means. Comparisons of results were made using the Student's t test.
3. RESULTS 3.1. S-A node The rate of atrial contractions under steady state conditions was not influenced by NEM in concentrations lower than 10 -6 M, but was decreased after 20 min of exposure to 10-5 and 10 --4 M (fig. 1). The mean control value of the atrial rate was 88 -+ 5 beats/ min (n = 22). In 3 out of 22 preparations a moderate increase in rate (8 to 20 beats/min) was produced by
7
10 -5 M NEM. At 10-3 M spontaneous rate was progressively slowed until atrial contractions were completely abolished. The mean time for onset of atrial arrest was 33.3 -+ 5.6 min (n = 22) at which time not only mechanical but electrical activities disappeared from fibers of the right atrium whereas electrical activities of the S-A node were usually maintained. When cysteine (10 -3 M) was preincubated with 10 -3 M NEM, the inhibitory effect of the SH reagent was prevented (open circle in fig. 1). Cysteine alone did not affect atrial rate. In preparations in which atrial activities had been markedly inhibited by high concentrations of NEM, no antagonistic effect of cysteine (10 -3 M) and ATP (10 -3 M) was observed. Atrial activities were not restored by washout of NEM from the bathing solution or by application of noradrenaline in concentrations of 10 -6 to 5 X 10 -6 g/ml in 15 preparations. Transmembrane potentials of S-A nodal pacemaker fibers were not measurably affected in preparations exposed to NEM at lower than 10 --4 M for 30 min. At 10 -3 M the membrane potential was influenced before slowing of the pacemaker rate. Mean values of parameters of the membrane potential re-
OC
-IO"
Table 1 Modification by NEM of parameters of the transmembrane potential of SoA nodal pacemaker fibers.
O O
Control
NEM 10 -3 M*
Parameters measured
(n = 9)
(n = 9)
Cycle length (msec) Maximal diastolic potential (mV) Threshold potential (mV) Overshoot (mV) 90% Duration (msec) 10% Duration (msec) Depolarization time (msec)
800 65 43 13 207 54 15
790 64 44 13 200 44 15
.c
+ 26 + 3 + 3 + 2 + 12 -+ 4 + 2
± 63 + 3 + 3 + 2 + 10 + 4 + 3
n = n u m b e r o f pacemaker fibers and also o f preparations. * Values obtained from preparations exposed to 10 -3 M NEM for 4 to 7 min.
-20-
-50.
NEM concentration ( M )
Fig. 1. Effects o f NEM on atrial rate. Values were obtained after 20 m i n of exposure to NEM. * values obtained after 10 m i n o f exposure. Solid circles = effect o f NEM (n = 22); open circle = effect o f NEM (10 . 3 M) preincubated with 10 .3 M cysteine (n = 3).
8
N.TODA and N.KONISHI tion representative of true pacemaker fibers was converted to that of latent pacemaker fibers after 15 to 30 min o f exposure to NEM. Conversely, in 2 fibers conversion from latent to true pacemaker was observed (I and II in fig. 2). Preincubation with cysteine (10 -3 M) was effective in preventing the membrane effect to NEM.
III
I
I
2 0 0 ms IV
50 mV
Fig. 2. Alteratlons in the membrane potentiai of a S-A nodal fiber induced by 10-s M NEM. Imp'dement into the fiber persisted for 24 rain. I = control; II = 15 min after NEM; III = 24 min after; IV = superimposition of II (presented as A) and III (presented as B). Configuration representative of a latent pacemaker fiber (I) was converted to that of a true pacemaker fiber (II and 1II) after 13 min of exposure to NEM. corded from same pacemaker fibers prior to and following 10 -3 M NEM are tabulated in table 1. Since it was very difficult to keep microelectrodes in the same pacemaker fibers for a long time, especially in the presence of high concentrations o f NEM, changes produced after only 4 to 7 min of exposure to NEM were obtained. As illustrated in table 1 and in fig. 2 IV, the duration of the early plateau phase was shortened. The 10% duration was measurably shortened in 5 out of 9 fibers. The 90% duration was shortened in 3 out of 9 fibers but prolonged in 2 fibers. In 3 instances in which the microelectr0de was held for a long period (15, 20 and 24 min) in one fiber, the longer the exposure to NEM, the more were the 10% and 90% durations shortened. Slowing of the pacemaker rate seen after 5 to 10 min of exposure to 10 -3 M NEM was associated with a reduction of the slope of diastolic depolarization. The threshold potential was not considerably affected, whereas the maximal diastolic potential and the overshoot were decreased (fig. 2). In 3 out of 8 fibers, the configura-
3.2. Contractile force o f the left atrium Left atrial preparations were driven electrically at different rates from 6 to 240 beats/min or higher. The tension developed at 6 to 120 beats/min was a direct function o f driving rates but at 120 to 240 beats/min or higher the tension was an inverse function of the rates; the tension-rate relationship was not significantly affected by 10 -5 M NEM. At 10 --4 M the tension developed at high driving rates (60 to 240 beats/min) was significantly reduced (p < 0.01), the magnitude of the reduction being time-dependent at times up to 40 min. The tension developed at low rates (6 and 12 beats/min) was enhanced after 20 min of exposure to 10 --4 M NEM; at 6 beats/min the enhancement was significant (p < 0.05). The dependency of the contractile tension upon driving rates was markedly decreased by NEM. The results are shown in fig. 3. The maximum driving rate at which alternating contractions, in which the strength of smaller contractions was not less than ~ of that of larger contractions were produced, was compared in control and NEM-added solutions. The mean value was decreased from 350 beats/min (n = 8) to 310 beats/.min (n = 8) by 10 --4 M NEM. 3.3. Chronotropic response to autonomic nerve stimulation Transmural electrical stimulation applied at the S-A node produces negative chronotropic and inotropic effects followed by positive effects, which are thought to be due to the release of endogenous acetylcholine and noradrenaline, respectively (Vincenzi and West, 1963). The negative and positive effects varied directly with frequencies of stimualtion. The negative chronotropic effect of transmural stimulation at 20/sec was moderately reduced in 3 out of 9 preparations but at lO0/sec was reduced in only 2 preparations after 20 min of exposure to 10 -7 M NEM. The reduction was corrected by washout of
N-ETHYLMALEIMIDE ON ATRIA
NEM 10"5M
800-
NEM
" Control
9
10"4M
l-[
x 240mii: 600 v
(7)//
+oo
(
i
~- 2 0 0
.....
+.+-r-.,
0
+
,+
3'0 ~
,io 'm~o +
,
,+
~
+b
+,+
T
+,
+b ,+o 'm~,o
Rate of contraction (beats/rain) Fig. 3. Alterations i . the tension-rate relationship of left atria exposed to NEM. Figures in parentheses indicate the n u m b e r o f experiments.
3,000
900"
• Control (9) x NEM 10"7M ( 9 ) o 10"6M (T) IO'SM ( 6 ) 10"4M ( 4 )
.// 70C
/ ,(,:"
2,000
,/j,.,
m E
E ,
., l \ . • 1,000-
<£?<,
500'
400" • //
o
;
~
~:o
,60
Stimulation frequency ( c / s )
Fig. 4. Effects of NEM on the negative ehronotropic response to transmural electrical stimulation applied at the S-A node. The number of experiments is indicated in parentheses.
6 /"
i
s
2b
16o
Stimulation frequency ( c/$ ) F]~. 5. Effects o f NEM on the positive chronotropic effect o f transmural stimulation. Date here and in fig. 4 were obtained from the same preparations. S y m b o l s in the figure are the same as those indicated in fig. 4.
10
N.TODA and N.KONISH1
•
Control
• Control (13) O NEM 10-6M (9) ;< 10"SM (9) A 10-4 M ( 8 )
l
l
•
120" 10"4M
l
/ ,
I00
D
,/" I/X .E 100 E
c71/,
,,"
/
/I
c
1.
i E o o
<~. 6o
8(?
"I
60
,
o
Stimulation frequency ( c/s )
//
,b "r
5x~o -7
2x'lo -e
ACh concentration
io-~
5Xi0 "5
( g/ml )
Fig. 6. Modification by NEM of the positive chronotropic effect of sympathetic nerve stimulation. The number of experiments is indicated in parentheses.
Fig. 7. Effects of NEM on the negative chronotropic response to acetyicholine. Figures in parentheses indicate the number of experiments.
NEM. A greater reduction was not produced by 10-6 and 10-5 M in these preparations. At 10-4 M the negative chronotropic effect of nerve stimulation was completely abolished (fig. 4) and was n o t reversed by repeated washouts. The positive chronotropic effect which followed the negative effect of transmural stimulation was not affected by NEM in concentrations lower than 10-5 M. At 10.-4 M NEM the pre-stimulation cycle length between contractions was significantly increased and the positive chronotropic effect of nerve stimulation was considerably reduced (fig. 5). Stimulation of extracardiac, postganglionic sympathetic nerves produced an increase in atrial rate, which was dependent upon frequencies of electrical stimulation. The positive chronotropic effect of sympathetic nerve stimulation was not influenced by NEM at lower than 10-5 M, but was reduced by
10--4 M (fig. 6). The chronotropic effect was not corrected by washout of NEM. 3.4. Chronotropic response to aeetyleholine and noradrenaline The atrial rate was progressively slowed by increasing concentrations of acetylcholine. The concentration-response relationship was not altered by 10--6 or 10-5 M NEM. However, the negative chronotropic effect of acetylcholine was abolished and in 2 out of 8 preparations changed to a positive effect after 20 min of exposure to 10.-4 M NEM. The results are illustrated in fig. 7. The inhibition of the acetylcholine action was not corrected by repeated wash of preparations and by application of cysteine and ATP in concentrations up to 10-3 M. However, when NEM (10 --4 M) was preincubated with 10--4 M cysteine, the inhibitoty effect of the SH reagent was prevented
N-ETHYLMALE1MIDE ON ATRIA
•
Control
× NEM I0-5 M 10-4 M
/ / ~
160
A
140
-g
///
w
120
<"V ..¢ <"
e o
T
a I00
80 ¸
60.
,
0
!.
t
5)(10 "e
2)(10 "8
NA concentration
I0 -7
5x'lo "7
2X'10 "6
( g/ml )
Fig. 8. Effects of NEM on the positive chronotropic response to noradrenaline. Figures in parentheses indicate the number of experiments.
in 3 out of 3 preparations. Noradrenaline in cumulative concentrations produced a dose-related increase in the atrial rate. The concentration-response relationship was not affected by 10 -5 M NEM but was shifted to the right by 10 --4 M (fig. 8). In 2 out of 2 preparations the inhibition of noradrenaline effect was prevented by preincubation of NEM with isomolar concentrations of cysteine.
4. DISCUSSION It is known that certain accessible SH groups of native protein are reactive to NEM, making possible a specific metabolic inhibition (Webb, 1966). According to Skou (1963), the activity of the Na+-K+activated ATPase prepared from ox brain is markedly inhibited after 10 min of exposure to 10 -3 M NEM. Respiration of human erythrocytes is also inhibited some 50% by 1.3 mM NEM (Sheets and Hamilton,
11
1958). At this concentration certain dehydrogenases, electron transport systems, glycolytic and cyclic enzymes are inhibited (summarized by Webb, 1966). At 10 -3 M NEM the electrical activity of S-A nodal pacemaker fibers was influenced. The major changes produced consisted of a decrease in the slope of diastolic depolarization, which resulted in bradycardia and an arrest of S-A nodal activities, and of shortening of the 10% duration. Similar shortening of the action potential duration is observed in fibers of the S-A node, atrium and ventricle exposed to toxic concentrations of ouabain (Toda, unpublished), 2-4-dinitrophenol, sodium azide (De Mello, 1959) and iodoacetate (Kleinfeld et al., 1955). The membrane effect of 2-4-dinitrophenol and iodoacetate is partly corrected by ATP, pyruvate and acetate, whereas that of NEM was not corrected by ATP, cysteine and noradrenaline. Contractibility of the isolated hypodynamic cat papillary muscle is enhanced by 1 mM NEM (Bennett et al., 1958). However, at this and at lower concentrations, NEM exerted not a positive inotropic effect but a negative inotropic effect on left atria isolated from rabbits. It is suggested by Goodman and Hiatt (1964) that the SH groups are involved in the process of contraction of the rat ileum. This would also be the case for atrial contractibility. The SH groups involved would be either part of the muscle protein or components of vital enzyme systems involved in muscle metabolism, or both. NEM inhibits the SH groups of action. However, it seems likely that only a fraction of the SH groups which do not function in polymerization and in ATP-binding react with NEM (Bdrdny et al., 1962; Tonomura and Yoshimura, 1962; Drabikowski and Gergely, 1963). The enhancement of cardiac contractibility produced by cardiac glycosides is thought to be associated with a net loss of K + or a gain of Na + secondary to an impairment of active movements of the ions across membrane (reviewed by Glynn, 1964). NEM causes a loss of K + from duck erythrocytes but, in contrast to other inhibitors, also causes some loss of Na +, this being accompanied by marked suppression of glucose uptake and glycolysis (Tosteson and Johnson, 1957). Respiration of human erythrocytes is also suppressed by NEM (Sheets and Hamilton, 1958). If this is the case for atrial muscle, an inhibition of aerobic metabolism would account for the negative inotropic
12
N.TODA AND N.KONISHI
effect o f NEM. Findings obtained in the present study indicate that atrial fibers are more susceptible to NEM than S-A nodal pacemaker fibers; aerobic metabolism would be expected to participate greater in functions o f contractile tissues than those o f specialized tissues. The negative chronotropic response o f isolated atria to endogenous and exogenous acetylcholine was abolished by 10 -4 M NEM. The inhibitory effect of NEM was prevented by preincubation with cysteine. G o o d m a n and Hiatt (1964) reported that NEM (5.7 × 10 -4 M) prevented acetylcholine-induced contraction o f the isolated rat ileum, at which time 76% o f the SH groups were inhibited. Cardiac glycosides are known to be specific inhibitors of the membrane ATPase, one o f the more important SH enzymes. Toda and West (1966) demonstrated that ouabain produced an augmentation o f the negative chronotropic response o f isolated rabbit atria to endogenous and exogenous acetylcholine. The inverse effect of NEM and ouabain on sensitivity o f the S-A node to acetylcholine would indicate that SH groups but not those of the membrane ATPase are necessary in producing cholinergic responses. The positive chronotropic effect o f endogenous and exogenous noradrenaline was depressed by 10 --4 M NEM. The cardio-stimulation effect o f noradrenaline was blocked in preparations in which atrial activities were abolished by 10 -3 M NEM. However, the inhibitory effect o f NEM on the noradrenaline effect was less marked than that on the acetylcholine effect. It remains to be seen whether the SH groups involved in adrenergic receptor mechanisms are more resistant to NEM or possess a greater safety factor than those in cholinergic mechanisms. It was demonstrated by Komalahiranya and Volle (1963) that the injection o f 0.5 mg o f NEM into the c o m m o n carotid artery produced marked changes in transmission across the cat superior cervical ganglion. The immediate change ( 0 - 1 0 min) produced by NEM is an enhancement o f the postganglionic spike and a depression o f the responses to acetylcholine and KCI. The late change ( 1 0 - 4 5 min) consisted o f an enhancement o f the response to the ganglionic stimulants at a time when the responses to stimulation o f preganglionic nerve was depressed or even abolished. In the present study, however, when preparations were exposed to 10 --4 M NEM for 20 rain, the depression
of the chronotropic response to acetylcholine and to stimulation of intracardiac cholinergic nerves were similar. This was also true in the case o f noradrenaline and sympathetic nerve stimulation. Thus, it is unlikely that in isolated rabbit S-A nodes NEM appreciably affects functions o f cholinergic and adrenergic postganglionic nerve terminals. Thus, it is not known whether reduced sensitivity of S-A nodal pacemaker fibers to the chemical transmitters is due primarily to an inhibition of SH groups in the receptors or to a general inhibition of cell metabolism.
REFERENCES B~ir~iny, M., F.Finkelman and T.Therattil-Antony, 1962, Studies on the bound calcium of actin. Arch. Biochem. Biophys. 98, 28-45. Bennett, D.R., K.S.Andersen, M.V.Andersen, Jr., D.N.Robertson and M.B.Chenoweth, 1958, Structure-activity analysis of the positive inotropic action of conjugated carbonyl compounds on the cat papillary muscle. J. Pharmacol. Exptl. Therap. 122, 489-498. De Mello, W.C., 1959, Metabolism and electrical activity of the heart; action of 2-4-dinitrophenol and ATP, Amer. J. Physiol. 1962, 377-380. Drabikowski, W. and J.Gergely, 1963, The role of sulfhydryl groups in the polymerization and adenosine triphosphatc binding of G-actin, J. Biol. Chem. 238,640--643. Glynn, I.M., 1964, The action of cardiac glycosides on ion movements, Pharmacol. Rev. 16,381-407. Goodman, 1. and R.B.Hiatt, 1964, Chemical factors affecting spontaneous motility of the small intestine in the rat. 1. Sulfhydryl reactants, Biochem. Pharmacol. 13, 871879. Kleinfeld, M., E.Stein, J.Magin and C.E.Kossmann, 1955, The action of iodoacetate on the electrical and mechanical activities of the isolated perfused frog heart, J. Clin. Invest. 34, 1802-1806. Komalahiranya, A. and R.L.Volle, 1963, Alterations of transmission in sympathetic ganglia treated with a sulfhydryl-group inhibitor, N-ethylmaleimide (NEM), J. Pharmacol. Exptl. Therap. 139, 304-311. Lewartowski, B., 1963, Selective stimulation of intra-cardiac postganglionic fibers, Nature, 199, 76-77. Paes de Carvalho, A., W.C.De Mcllo and B.F.Hoffman, 1959, Electrophysiological evidence for specialized fiber types in rabbit atrium, Amer. J. Physiol. 196, 483-488. Sheets, R.F. and H.E.Hamilton, 1958, A reversible effect on the metabolism of human erythrocytes by p-chloromercuribenzoic acid and N-ethyl maleimide, J. Lab. Clin. Med. 52, 138-143. Skou, J.C., 1963, Studies on the Na+ + K+ activated ATP hydrolyzing enzyme system. The role of SH groups,
N-ETHYLMALE1MIDE ON ATRIA Biochem. Biophys. Res. Commun. 10, 79-84. Toda, N., 1968, Influence of sodium ions on the membrane potential of the sino-atrial node in response to sympathetic nerve stimulation, J. Physiol. 196,677-691. Toda, N. and K.Shimamoto, 1968, The influence of sympathetic stimulation on transmembrane potentials in the S-A node, J. Pharmacol. Exptl. Therap. 159, 298-305. Toda, N. and T.C.West, 1966, The influence of ouabain on cholinergic responses in the sinoatrial node, J. Pharmacol. Exptl. Therap. 153, 104-113. Tonomura, Y. and J.Yoshimura, 1962, Binding of p-chloromercuribenzoate to actin, J. Biochem. (Tokyo) 5 1 , 2 5 9 266.
13
Tosteson, D.C. and J.Johnson, 1957, The coupling of potassium transport with metabolism in duck red cells, J. Cellular Comp. Physiol. 50, 169-183. Vincenzi, F.F. and T.C.West, 1963, Release of autonomic mediators in cardiac tissue by direct subthreshold electrical stimulation, J. Pharmacol. Exptl. Therap. 141, 185-194. Webb, J.L., 1966, Enzyme and metabolic inhibitors 3, 337-895, Academic Press, New York. West, T.C. and N.Toda, 1967, Response of the A-V node of the rabbit to stimulation of intracardiac cholinergic nerves, Circulation Res. 20, 18-31.