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Electrophysiological Studies on the Mechanism of Electrically Induced Sustained Rhythmic Activity in the Rabbit Right Atrium TAKEHIKO SAITO, MOTOHIRO OTOGURO, AND TETSU MATSUBARA
SUMMARY Right atrial preparations with no spontaneous activity were isolated from rabbit hearts. Action potentials with specific oscillatory after potentials were recorded from some fibers in the upper pectinate muscle and in the pectinate muscle along the crista terminalis. The amplitude and rate of rise of the oscillatory potential increased as the stimulus interval decreased. In 12 of 35 preparations in Tyrode's solution, sustained rhythmic activity resulted from the depolarizing phase of the enhanced oscillatory potential. The action potential during sustained rhythmic activity was characterized by slow diastolic depolarization, as in the sinoatrial (SA) node. A single premature stimulus or a train of stimuli sometimes caused an acceleration of the rate of excitation rather than its suppression. Sustained rhythmic activity was maintained at a low resting potential and ceased spontaneously when the slope of the slow diastolic depolarization decreased and the maximum diastolic potential increased. Stimulation just after termination of sustained rhythmic activity neither increased the amplitude of the oscillatory potential nor initiated further sustained rhythmic activity. Initiation of new sustained rhythmic activity required a period of quiescence before electrical stimulation. These results suggest that the sustained rhythmic activity described in this paper results not from reentry but, rather, from spontaneous generation of action potentials by the atrial fibers having oscillatory afterpotentials.
IT IS WELL KNOWN that premature impulses arising at certain times during the cardiac cycle may induce a burst of rapid repetitive activity (sustained rhythmic activity). Such activity has been assumed to result from either automatic ectopic pacemakers or reentry.1"" Sustained rhythmic activity which is initiated and terminated by single premature impulses generally has been thought to result from reentry. However, recent reports indicate that some atrial and ventricular specialized fibers can develop sustained ectopic rhythms that are initiated and sometimes terminated by premature impulses.7"1'2 So far, such sustained rhythmic activity has been observed mainly as the result of drugs such as catecholamines or digitalis and abnormal ionic environments such as a low sodium solution. The rhythms differ from those of automatic fibers, because they sometimes result from oscillatory afterpotentials that are enhanced by a series of extrinsic stimuli. Recently, in the rabbit right atrium with no spontaneous activity in Tyrode's solution, Tanaka et al.l:l found fibers that showed action potentials having oscillatory afterpotentials. In a preliminary report, they stated that such fibers respond to an increase in the rate of electrical stimulation with an increase in the amplitude of oscillatory afterpotentials and become spontaneously active in Tyrode's solution.14 From the Department of Physiology, St. Marianna University School of Medicine, Kawasaki (Dr. T. Saito) and the Department of Internal Medicine, Tokyo Medical College, Tokyo, Japan (Dr. M. Otoguro and Dr. T. Matsubara). This study was supported in part by a research grant from the Japan Research Promoter Society for Cardiovascular Disease. A preliminary report of this work was presented at the Fifty-First General Meeting of the Physiological Society of Japan, 1974. Address for reprints: Dr. Takehiko Saito, Department of Physiology, St. Marianna University School of Medicine, 2095, Sugao Takatsu-ku Kawasaki, Japan. Received January 24, 1977; accepted for publication September 8, 1977.
In the experiment reported here, sustained rhythmic activity was induced in the rabbit right atrium when the preparation was stimulated at a proper stimulus interval after a certain period of quiescence. The experiment was initiated to investigate whether such activity is caused by ectopic automatic pacemakers or by reentry. Methods Rabbits weighing 2-3 kg were anesthetized with urethan (1 g/kg administered intravenously). The entire heart was removed quickly and placed in a Lucite bath perfused with Tyrode's solution of the following composition (miu): NaCl, 137; KC1, 2.6; CaCl2, 1.8; MgCl2, 0.5; NaHCO;t, 11.7; NaH2PO4, 3.2; glucose, 5.6. Subsequent dissection was performed in this Tyrode's solution, which was equilibrated with a gas mixture of 95% O2 and 5% CO2 and maintained at a temperature of about 32°C. The ventricles and left atrium were removed and discarded. The endocardial surface of the right atrium was exposed by an incision through the free wall at the atrioventricular groove, extending along the anterior border of the right atrial appendage and through the anterior wall of the superior vena cava, as described in detail by Paes de Carvalho et al.15 The right atrium, which showed spontaneous activity, was transferred to a tissue bath having a volume of 20 ml and was superfused with oxygenated Tyrode's solution. After pinning the preparation with the endocardial surface up to a cork block in the tissue bath, its appearance was like that shown schematically in Figure 1 (upper diagram). Further dissection was made along the dashed line, L, in the figure in order to remove the sinoatrial node, atrioventricular node, and crista terminalis. The part of the right atrium which remained in the tissue bath after the dissection and which usually did not show spontaneous activity is schematically shown in Figure 1 (lower diagram). The right atrial preparation was pinned
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diastole. Both potentials were recorded either independently or simultaneously on a continuously moving film at a slow speed (5-10 mm/sec). Results Action Potential with an Oscillatory Afterpotential
pectinate muscle along crista terminalis
FIGURE 1 Schematic representation of the rabbit right atrial preparation and transmembrane action potentials recorded from two different type of fibers. In the upper diagram, the right and lower portions of the dashed line, L, indicated that the crista terminalis, SA node, A V node, and ventricle, were discarded. The remaining right atrial myocardium, which usually did not show any spontaneous activity in Tyrode's solution, is shown in the lower diagram. The approximate location of each recording site is indicated on the lower diagram of the preparation. The preparation was stimulated electrically every 5 seconds. In records a and b, the lower traces are of the same cell as in the upper traces but at higher gain than the upper, in order to display the terminal repolarization phase. Time and voltage calibrations for the upper and lower traces are indicated in record b. SVC: superior vena cava, IVC: inferior vena cava, CT: crista terminalis, SA: sinoatrial node, CS: coronary sinus, A VV: atrioventricular valve.
under slight tension to a cork block in the tissue bath. It was left unstimulated for several minutes in Tyrode's solution at a temperature of about 36°C. Some preparations started to fire spontaneously at a regular slow rate during the rest period. In order to find a pacemaker region in such preparations, mechanical stimuli were given to them by a fine needle. The pacemaker region that showed a rate change with a touch of a needle was cut off. Unless spontaneous activity appeared, the preparation was stimulated electrically with a concentric metal electrode (diameter 1 mm) consisting of copper inside and stainless steel outside. Rectangular current pulses of 0.5msec duration and twice threshold intensity were applied to the preparation from a pulse generator with an isolation unit (NIHON KOHDEN MSE-3R). Transmembrane action potentials of single fibers were recorded by floating microelectrodes filled with 3 M KG (tip resistance of about 30 Mfl). The potential was led to different vertical axes of a dual beam oscilloscope (NIHON KOHDEN VC-7A) via two DC amplifiers of different gain. In most experiments, the upper beam, which was at an appropriate gain and sweep speed to display the entire action potential on the oscilloscope screen, was triggered by stimulus pulses. The lower beam was set at high gain and always was positioned as a stationary spot at the lower left corner on the oscilloscope screen to record changes in potential during terminal repolarization and
Right atrial preparations that were quiescent in Tyrode's solution were driven electrically. In 12 of 35 preparations, sustained rhythmic activity was induced at a certain stimulus rate. This activity persisted for a while and ceased spontaneously. The phenomenon was induced regularly in the same preparation as long as the stimulation was applied after a period of quiescence. Transmembrane action potentials were recorded from many sites in these preparations. It was possible to recognize at least two types of atrial fibers in terms of the time courses of their afterpotentials. Figure 1 shows action potentials recorded from two different fibers in the same preparation and the approximate sites at which the recordings were made. The preparation was stimulated electrically with a stimulus interval of 5 seconds. The upper and lower traces of each record were obtained from the same cell but with different amplifications. In record a, the action potential showed a long-lasting afterpotential which decayed to the resting potential. The action potentials with such an afterpotential commonly were recorded in the atrial roof muscle fibers and in most of the pectinate muscle fibers.1" The resting and action potential amplitudes measured for 18 pectinate muscle fibers with this type of afterpotential were 81 ± 9 mV and 92 ± 11 mV (mean ± SD), respectively, at a stimulus interval of 1 second. The duration at 50% repolarization was 48 ± 11 msec under the same stimulus conditions. In record b, the action potential was recorded from a fiber of the upper pectinate muscle. The action potential of this fiber did not terminate with a simple return to the resting potential but, rather, was followed by a secondary depolarization. The amplitude of this delayed afterpotential, which was defined as the difference between the afterdepolarization at its peak and resting potential, was about 6 mV. The afterpotential decayed from its peak to the resting potential with a time course of over 2 seconds. Action potentials with this specific afterpotential were obtained mostly from some of the upper pectinate muscle fibers and the pectinate muscle fibers along the crista terminalis. The amplitude of the oscillatory afterpotential varied greatly from fiber to fiber. Some fibers showed only afterhyperpolarization without being followed by afterdepolarization. The resting and action potential amplitudes measured from pectinate muscle fibers with oscillatory afterpotentials of more than 5 mV were 73 ± 7 mV and 80 ± 10 mV (mean ± SD), respectively, at a stimulus interval of 1 second. The duration at 50% repolarization was 72 ± 16 msec (mean ± SD). Initiation and Termination of Sustained Rhythmic Activity Figure 2 shows changes in the oscillatory afterpotentials of single driven impulses interposed at various intervals
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ELECTRICALLY INDUCED SUSTAINED RHYTHMIC ACTIVITY/Sairo el al.
a !
b
d FIGURE 2 Effects of single driven impulses interposed at various moments in the cardiac cycle. The preparation was stimulated electrically every 6 seconds. Action potentials were recorded from a fiber in the upper pectinate muscle. The records were taken at a high gain and slow speed in order to show the terminal repolarization phase. Thus, most of the action potential is off scale. Record a shows the control responses. A single driven impulse was applied at 2 seconds (b), I second (c), and 0.5 second (d) after one of the control response. In record d, nondriven impulses were initiated. Time and voltage calibrations are indicated in record c.
after regularly driven action potentials. The action potentials were obtained from a single fiber in the upper pectinate muscle. In record a, each action potential was followed by an oscillatory afterpotential of about 5 mV amplitude which occurred long after completion of the preceding afterpotential. When a single test stimulus was applied at an interval of about 2 seconds after the control response in record b, a driven action potential occurred at a slightly less negative level of membrane potential than that seen before the preceding action potential, because it arose from the terminal repolarization phase of the preceding afterpotential. This action potential was followed by a slightly enhanced afterpotential; the slope and amplitude of the secondary depolarization were increased. As a test stimulus was applied at about 1 second in record c, a driven action potential occurred at the peak of the preceding afterpotential. The action potential was followed first by a hyperpolarization and subsequently by a depolarization. The slope and amplitude of this depolarization were greater than those of the control response. If we look only at the events that occur between the maximum afterhyperpolarization and peak of the afterdepolarization, we can see a slow diastolic depolarization that is very similar to that seen in automatic fibers. In record d, a further decrease in the stimulus interval to 500 msec resulted in a further enhancement of the oscillatory afterpotential. Then the fiber became spontaneously active. The rate of sustained rhythmic activity increased during the first few beats and became regular. This sustained
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rhythmic activity continued for about 20 seconds at the rate of about 3 cycles/sec. When the upper pectinate muscle was dissected off, sustained rhythmic activity stopped and was not again induced by electrical stimulation. Figure 3 shows changes in the action potentials and their afterpotentials elicited by trains of stimuli at constant rates. A train of 10 successive stimuli at rates between 0.5 and 2 cycles/sec was applied every 50 seconds. The first action potentials following the long quiescent period were characterized by a prominent spike potential with a low-level plateau (see upper traces in each record). The terminal repolarization phase of the low-level plateau was followed by the oscillatory afterpotential (see lower traces in each record). Subsequent repetitive stimulation produced a gradual prolongation of the spike duration and a cumulative loss of the maximum diastolic potential. Shorter stimulus intervals caused more pronounced changes in the plateau duration and afterpotential (record a-c). Changes in the plateau duration depending on the rate of stimulation have been discussed in relation to the potassium conductance of the membrane.l:)-17-1S The amplitude of the oscillatory afterpotentials that followed the last driven impulses of a train of 10 successive stimuli was about 4.5 mV in record a, 6 mV in record b, and 10 mV
FIGURE 3 Effect of changes in the stimulus interval on the amplitude of the oscillatory afterpotential. A train of electrical stimuli at intervals of 2 seconds (a), 1.5 seconds (b), 1 second (c), and 0.5 second (d) were applied on a previously quiescent fiber. A ction potentials were recorded from a fiber in the upper pectinate muscle. Quiescent periods between each record were kept constant at 50 seconds. The upper beam was triggered by stimulus pulses and swept at the proper speed to see the action potential profile. The lower beam remained as a stationary spot at the lower left corner on the oscilloscope screen and was deflected vertically at higher gain. Both were photographed simultaneously on continuously moving film at a slow speed. The gain of the lower traces is so large that most of the action potential proper is off scale. In record d, sustained rhythmic activity was induced after the fourth driven impulse (arrow). Time and voltage calibrations were indicated in record c for the upper traces and in record d for the lower traces.
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in record c. When the fiber was driven at a stimulus interval of 500 msec in record d, it became spontaneously active after the fourth impulse. Then electrical stimuli were stopped immediately. Not all of the spontaneous action potentials were seen in the upper trace, because the upper beam was triggered only by the stimulus pulses. The action potentials during sustained rhythmic activity are quite different in appearance from those obtained by electrical stimulation. They are characterized by a slow upstroke, low amplitude with rounded peak, absence of overshoot, and slow diastolic depolarization. A fiber that had a resting potential of -75 mV before electrical stimulation showed a lower take-off potential of about - 5 5 mV and a maximum diastolic potential of about -66 mV during sustained rhythmic activity. Sustained rhythmic activity, which had a rate of about 2 cycles/sec, persisted for about 10 seconds and ceased spontaneously. The last nondriven action potential was followed by a subthreshold depolarization of about 15 mV which failed to initiate the next action potential. Figure 4 shows sustained rhythmic activity arising from the enhanced oscillatory afterpotential. In record a, 10 successive stimuli at a stimulus interval of 2 seconds were applied. The slope and amplitude of the oscillatory potential increased gradually during the first four stimuli and decreased during subsequent stimuli. In this case, the firing level for each driven action potential gradually shifted in the depolarizing direction during a sequence of stimulation, whereas the maximum diastolic potential was little changed because of the enhancement of the afterhyperpolarization. The afterpotentials of the third and fourth impulses arose to their peaks with an upward concavity and decayed to the diastolic potential in two steps, first rapidly to a given level and then slowly to the maximum diastolic potential. They seem to be comparable to a local response evoked by a subthreshold pulse in a quiescent Purkinje fiber.19'20 In record b, four successive stimuli at a stimulus interval of 1 second were applied, and sustained rhythmic activity was induced after the third impulse (arrow). The fourth
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stimulus, applied during the slow diastolic depolarization of the nondriven action potential (triangle), caused an increase in the slope of slow diastolic depolarization with a consequent increase in the rate of excitation. The fiber that had a resting potential of -72 mV before electrical stimulation showed a take-off potential of about - 6 0 raV and a maximum diastolic potential of about - 6 7 mV during sustained rhythmic activity. Sustained rhythmic activity at 4 cycles/sec persisted for about 15 seconds and ceased spontaneously with a slight increase in the maximum diastolic potential and a decrease in the slope of slow diastolic depolarization. The action potentials during sustained rhythmic activity were similar to those of a spontaneously active fiber such as that of the SA node. The last nondriven action potential was followed by a subthreshold depolarization of about 15 mV which had a hump during repolarization and decayed to the resting potential in several seconds. Sustained rhythmic activity usually showed constant rates of 1-4 cycles/sec and ceased spontaneously. Initiation of additional sustained rhythmic activity required a few stimuli after a certain period of quiescence. In 2 of 12 preparations, a spontaneous repetition of sustained rhythmic activity was observed. Figure 5 shows an example of such activity. In record a, sustained rhythmic activity at about 2 cycles/sec appeared after the second driven impulse (arrow). Subsequent spontaneous activity caused a progressive decrease in the maximum diastolic potential. A decrease in maximum diastolic potential of about 16 mV resulted in a decrease in amplitude of the action potentials with a notch on their upstrokes. The rate of spontaneous activity was decreased. Each action potential initiated at a low membrane potential was followed by a subthreshold depolarization (lower trace in the middle part of record a). Such activity persisted for about 30 seconds and terminated in oscillations. The maximum diastolic potential increased gradually before spontaneous activity appeared. After quiescence lasting a few seconds, a burst of impulses resumed with the appearance of oscillations of increasing amplitude and again was suppressed at the level of maximum decrease of the diastolic potential (very end of record a). This cycle repeated itself for about 2 minutes (record b). We did not attempt to record responses simultaneously from fibers in
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FIGURE 4 Oscillatory afterpotentials leading to sustained rhythmic activity. A train of electrical stimuli at intervals of 2 seconds (a) and I second (b) were applied to a previously quiescent fiber. Action potentials were recorded from a fiber in the upper pectinate muscle close to the crista terminalis. The recording method is the same as that in Figure 3. In record a, subthreshold depolarization appeared on the first few impulses and was gradually suppressed during subsequent impulses. In record b, sustained rhythmic activity appeared after the third impulse (arrow). A single stimulus applied on the slow diastolic depolarization of a nondriven impulse (triangle) caused acceleration of the rate of spontaneous activity. Time and voltage calibrations for the upper and lower traces are indicated in record b.
FIGURE 5 Spontaneous repetition of a burst of discharges. Action potentials were recorded from a fiber in the pectinate muscle along the crista terminalis. The recording method is same as that in Figure 3. In record a, sustained rhythmic activity appeared after the second impulse (arrow). Record b is a continuation of record a. Spontaneous activity persisted for about 3 minutes and ceased abruptly. Time and voltage calibrations were indicated in record a for the upper traces and in record b for the lower traces.
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ELECTRICALLY INDUCED SUSTAINED RHYTHMIC ACTIVITY/Saito et al. different areas of the atrium. It is not clear whether the low amplitude action potentials initiated at the low maximum diastolic potential are able to propagate and excite the whole preparation. A similar spontaneous repetition of the burst of impulses also has been recorded from rabbit atrial preparations superfused with aconitine.21'22 Figure 6 shows changes in the oscillatory afterpotential during the recovery phase after termination of sustained rhythmic activity. In record a, sustained rhythmic activity occurred after the second impulse following a long quiescent period. The rate of excitation increased gradually over the first few beats. The maximum rate of excitation was about 4 cycles/sec. Sustained rhythmic activity persisted for about 20 seconds and ceased spontaneously. Single stimuli were applied at 15 (b), 30 (c), and 60 (d) seconds during the quiescent period. The slope and amplitude of the afterdepolarization were greatly suppressed following shorter quiescent periods and gradually increased after progressively longer quiescent periods. Repetitive stimulation just after termination of sustained rhythmic activity neither potentiated the afterpotential nor initiated sustained rhythmic activity. After about 1 minute of quiescence, the same stimulus conditions elicited the same rhythmic activity as that shown in record a. This phenomenon was recorded from four other fibers. The fiber showing a more rapid rate of spontaneous activity had a tendency to cause a stronger suppression of the oscillation after termination of spontaneous activity. Effects of Electrical Stimulation on Sustained Rhythmic Activity
Once sustained rhythmic activity had been triggered by external stimuli, it persisted for a while and terminated spontaneously after a slight decrease in the rate of excitation. A slight decrease in the slope of slow diastolic depolarization and an increase in the amplitude of the maximum diastolic potential, associated with the reduction of the rate of excitation, were observed before termination of spontaneous activity. Figure 7 shows the effects of premature stimuli on the rate of sustained rhythmic activity. Premature impulses were induced at various intervals during the cardiac cycle. In records a
FIGURE 6 Suppression and recovery of the oscillatory afterpotential. Action potentials were recorded from a fiber in the upper pectinate muscle. The gain is so high that only the lower part of the action potentials is seen. Sustained rhythmic activity appeared after second driven impulses and persisted for about 20 seconds (a). A single driven impulse was applied at 15 seconds (b), 30 seconds (c), and 60 seconds (d) after termination of sustained rhythmic activity. Time and voltage calibrations were indicated in record a.
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FIGURE 7 Effect of electrically driven premature impulses on the rate of sustained rhythmic activity. Action potentials were recorded from fibers in the pectinate muscle very close to the crista terminalis. The gain is so high that only the lower part of the action potentials is seen. In each record, onset of sustained rhythmic activity and application of the premature stimuli were indicated by arrows and triangles, respectively. Records a and b were obtained from same cell. In record a, single premature stimuli were applied in two different cardiac cycles during sustained rhythmic activity. In record b, a train of stimuli al an interval of 1 second was applied during sustained rhythmic activity. In record c, single premature stimuli were applied in two different cardiac cycles and just after termination of sustained rhythmic activity. Time and voltage calibrations for records a and b are indicated in record a.
and c, single premature impulses were elicited both early and late in the cardiac cycle. Each premature impulse was followed by a slight hyperpolarization of the membrane, but it was unsuccessful in terminating sustained rhythmic activity. Premature impulses elicited during the first several beats of sustained rhythmic activity caused a noticeable increase in the rate of excitation. In record b, eight successive premature impulses at a rate of 1 cycle/sec were applied during sustained rhythmic activity. They also did not terminate the spontaneous activity. In record c, a single premature impulse just after termination of the spontaneous rhythm was followed by a large afterhyperpolarization with suppression of the subsequent afterdepolarization. When electrical stimulation at rates greatly in excess of the rate of sustained rhythmic activity (overdrive) was applied to the atrial preparations, some resumed beating spontaneously after a short quiescent period and others immediately resumed spontaneity just after the external stimuli were interrupted. Figure 8 shows a complex effect of overdrive on spontaneous activity. In record a, the first 11 responses are the control rhythmic activity at about 2 cycles/sec. Then repetitive stimulation at a rate of 4 cycles/sec was applied for about 30 seconds, as indicated by a horizontal line below the responses. When stimuli were interrupted, the spontaneous activity immediately occurred at almost same rate as that of the control (record b); yet, this rhythm slowed abruptly within several impulses. The rate of this slow rhythm returned gradually to that of the control. This phenomenon was different from the overdrive suppression observed in the normal cardiac
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FIGURE 8 Effect of fast drive on the spontaneously active preparation. The period of electrical driving at a rate of 4 cycles/sec is indicated by the horizontal line below responses in records a and b. Time and voltage calibrations were indicated in record c.
pacemaker. Abrupt slowing of the rate of excitation and the appearance of a hump in the slow diastolic depolarization might suggest a pacemaker shift with a partial conduction block. Discussion On the basis of anatomical differences and characteristics of the transmembrane action potentials, several types of fibers have been identified in the mammalian right atrium.15'23"26 Paes de Carvalho et al.15 found that the rabbit right atrium contains several distinct types of fibers. One fiber of particular interest, located in the SA ring bundle running parallel to the crista terminalis on the venous border, is characterized by action potentials with a prominent plateau and, sometimes, an inherent slow diastolic depolarization. This bundle gives at least one major branch to the upper pectinate muscle. Hogan and Davis26 found specialized conducting fibers, so-called atrial plateau fibers, along the caval border of the crista terminalis and along the free-running strand in the canine right atrium. Those fibers seem to be comparable to the SA ring bundle fibers and their branches in the rabbit atrium. In the present experiments, action potentials with oscillatory afterpotentials were recorded from some fibers in the upper pectinate muscles and in the basal region of the pectinate muscles along the crista terminalis. The amplitude of the afterpotentials varied greatly from fiber to fiber in the same preparation and from preparation to preparation. The action potentials recorded from fibers several hundred microns away from the particular fiber with the oscillatory afterpotential usually were not followed by any oscillatory afterpotentials. The plateau duration of the action potential in that particular fiber was longer than that of the ordinary pectinate muscle. Although we have not observed the exact distribution of these particular fibers, the electrical characteristics of the fibers and their approximate location suggest the possibility that they may be branches of the SA ring bundle or be transitional fibers between the SA ring bundle and ordinary pectinate muscle.
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It generally has been assumed that spontaneous or evoked premature impulses may initiate or terminate only reentrant rhythms. Macro reentrant circuits involving the atrium, AV node, and ventricular specialized conduction system have been demonstrated in the rabbit heart by Janse et al.27 and Wit et al.28 In small segments of the rabbit left atrium with no anatomical obstacle, Allessie et al.29 reported that the application of a single adequately timed stimulus can ir : ; and always terminate tachycardia. Based on results of multiple microelectrode recording, they suggested that such a tachycardia is maintained by reentrant circuits. In the present experiments, sustained rhythmic activity was induced in the previously quiescent atrial fiber only with the aid of a series of a few external stimuli. The following evidence suggests that such rhythmic activity results not from reentry, but rather from spontaneous generation of action potentials by the specific type of fiber described above: 1. The oscillatory afterpotential in a particular fiber was enhanced by repetitive stimulation at an appropriate interval. The enhanced oscillation brought the membrane to a less negative level, and then sustained rhythmic activity resulted from the depolarizing phase of the oscillatory afterpotential. The initial phase of sustained rhythmic activity usually increased in rate during the first several beats and this was accompanied by an increase in the slope of the slow diastolic depolarization. This would not be observed in reentrant rhythms but is observed sometimes in automatic pacemaker rhythms. A number of investigators have, described the spontaneous activity arising from the specific afterpotentials in many kinds of preparations under the action of several agents. Examples of this sort of behavior of the membrane are found in atrial fibers during exposure to acetylstrophanthidin,8 aconitine,22 and Ba2+.30 Hashimoto and Moe8 demonstrated that strophanthidin-induced spontaneous activity did not appear in the ordinary muscle fibers, but in the atrial specialized fibers of the dog heart as well as in the Purkinje fibers.9 2. A slightly decreased slope of the slow diastolic depolarization and an increased magnitude of the maximum diastolic potential were observed just before termination of sustained rhythmic activity. The oscillatory potential was markedly suppressed just after termination of the spontaneous activity and the initiation of new sustained rhythmic activity required a certain period of quiescence which is associated with recovery of the oscillatory potentials. These facts may suggest that the oscillatory potential is not only related to the initiation of sustained rhythmic activity, but also to termination of this activity. 3. A single stimulus or repetitive stimuli applied during sustained rhythmic activity sometimes caused an acceleration of the rate of excitation rather than its suppression. This would not be observed in reentrant rhythms. Recently, sustained rhythmic activity, that can be initiated and terminated by a premature impulse and that is probably not reentrant, has been demonstrated in Purkinje fibers exposed to sodium-free solution" and in mitral valve of the monkey heart exposed to catecholamines.12 When automatic fibers are stimulated electrically at a rate
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ELECTRICALLY INDUCED SUSTAINED RHYTHMIC ACTIVITY/Saito et al. more rapid than their spontaneous rates, a phenomenon known as overdrive suppression occurs.31 In the present preparations, the effect of overdrive on sustained rhythmic activity appeared to be complex. When stimuli were interrupted suddenly, overdrive suppression was observed in some cases, but in other cases rhythmic activity resumed at a rate similar to the control and occasionally was suppressed after several beats. We have not made a detailed study of this phenomenon in the present paper. Recently, however, Vassalle et al.32 carefully studied the effects of overdrive on idioventricular pacemakers in dogs with complete atrioventricular block. They found that overdrive suppression and overdrive excitation can be present at the same time and antagonism between them causes various different effects of overdrive on idioventricular pacemakers. A small action potential with a slow rate of depolarization has a tendency to develop conduction block and is known as a possible cause of cardiac arrhythmias.2"6 Wit et al.33 reported various degrees of conduction block of ectopic impulses formed in the mitral valve. In the present experiment, an ectopic pacemaker usually showed a higher rate of discharge than that of normal pacemakers. The characteristics of the transmembrane potentials included a low amplitude of the resting and action potentials and a low action potential upstroke velocity. Since these factors are favorable to failure of propagation of impulses, it is possible that a spontaneous repetition of the burst of impulses described in Figure 5 may result from a combination of conduction block and regular ectopic rhythm, although we do not have any data concerning the propagation, or block, of the low amplitude responses. So far there is little information on the nature of the mechanisms underlying the oscillatory afterpotential in the atrial specific fiber. Hashimoto and Moe8 reported that a transient depolarization which is associated with strophanthidin-induced automaticity is suppressed by an elevation of the external potassium concentration or application of acetylcholine. Some of the spontaneously active preparations obtained in the present experiment became quiescent when the external potassium concentration was increased from 2.6 mM to 5.2 mM (unpublished experiment). Ferrier and Moe34 suggested that the transient depolarization of the Purkinje fiber under the influence of strophanthidin may be caused by Ca2+ influx. Lederer and Tsien35 demonstrated a voltage-dependent inward current which is responsible for strophanthidininduced transient depolarization of the Purkinje fiber. Once sustained rhythmic activity is triggered in a particular fiber, the configuration of the spontaneous action potentials is quite similar to that of action potentials recorded from a spontaneously active fiber such as that of the SA node. The exact level of membrane potential from which the spontaneous activity is triggered is uncertain and difficult to define, because the action potentials recorded in the present experiment were not always recorded from the site of impulse origin itself. However, spontaneous action potentials usually were elicited at a membrane potential less than —60 mV, brought about by the enhanced oscillatory afterpotential, and maintained at a low diastolic membrane potential between —70 and —60 mV. Recently, evidence has accumulated which
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indicates that spontaneous action potentials also may occur in a number of normally quiescent fibers, when the resting potential is reduced by an application of depolarizing currents.36'37 Action potentials occurring at low membrane potentials are insensitive to tetrodotoxin. The ionic basis for the action potentials during sustained rhythmic activity in the present experiment is uncertain. However, the membrane activity in the particular fibers described here seems to consist of at least two distinct and different mechanisms depending on different levels of membrane potential. This has already been suggested for other cardiac fibers.6-12> 36~39 In conclusion, the results of this study have shown the existence of a particular fiber in the rabbit atrium which has action potentials with an oscillatory afterpotential. It seems possible that the enhancement and suppression of the oscillatory afterpotential of the above-mentioned fiber play an important role in the initiation and termination of sustained rhythmic activity. In addition to the development of ectopic pacemaker function, several other factors such as local block of conduction or irregular interaction of activity among adjacent fibers might be able to produce more serious arrhythmias. Acknowledgments The authors thank Professor W. H. Miller, Department of Ophthalmology and Visual Science, Yale University School of Medicine, and Professor J. Toyoda, Department of Physiology, St. Marianna University School of Medicine, for their critical reading of the manuscript. Thanks are also due to Y. Takada for her valuable assistance in the preparation of the manuscript.
References 1. Trautwein W: Generation and conduction of impulses in the heart as affected by drugs. Pharmacol Rev IS: 277-332, 1963 2. Hoffman BF, Cranefield PF: The physiological basis of cardiac arrhythmias. Am J Med 37: 670-684, 1964 3. Watanabe Y, Dreifus LS: Newer concepts in the genesis of cardiac arrhythmias. Am Heart J 76: 114-135, 1968 4. Cranefield PF, Wit AL, Hoffman BF: Genesis of cardiac arrhythmias. Circulation 47: 190-204, 1973 5. Wit AL, Rosen MR, Hoffman BF: Electrophysiology and pharmacology of cardiac arrhythmias: II. Relationship of normal and abnormal electrical activity of cardiac fibers to the genesis of arrhythmias. Am Heart J 88: 515-524, 1974 6. Cranefield PF: The conduction of the cardiac impulse; the slow response and cardiac arrhythmias. New York, Futura, 1975 7. Davis LD: Effect of changes in cycle length on diastolic depolarization produced by ouabain in canine Purkinje fibers. Circ Res 32: 206214, 1973 8. Hashimoto K, Moe GK: Transient depolarizations induced by acetylstrophanthidin in specialized tissue of dog atrium and ventricle. Circ Res 32:618-624,1973 9. Ferrier GR, Saunders JH, Mendez C: A cellular mechanism for the generation of ventricular arrhythmias by acetylstrophanthidin. Circ Res 32: 600-609, 1973 10. Rosen MR, Gelband H, Merker C, Hoffman BF: Mechanisms of digitalis toxicity: Effects of ouabain on phase four of canine Purkinje fiber transmembrane potentials. Circulation 47: 681-689, 1973 11. Cranefield PF, Aronson RS: Initiation of sustained rhythmic activity by single propagated action potentials in canine cardiac Purkinje fibers exposed to sodium-free solution or to ouabain. Circ Res 34: 477-481, 1974 12. Wit AL, Cranefield PF: Triggered activity in cardiac muscle fibers of the simian mitral valve. Circ Res 38: 85-98, 1976 13. Tanaka I, Tosaka T, Saito K, Shin-mura H, Saito T: Changes in the configuration of the rabbit atrial action potential after various period of rest. Jap J Physiol 17: 487-504, 1967 14. Saito T, Otoguro M, Kobayashi F, Matsubara T: Automatic activity in rabbit atrium. J Physiol SocJap38: 325, 1974 15. Paes de Carvalho A, de Mello WC, Hoffman BF: Electrophysiological evidence for specialized fiber types in rabbit atrium. Am J Physiol 196:483-488, 1959
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16. Tanaka I, Saito T: Changes in duration of plateau phase and afterdepolarization of the rabbit atrial action potential by repetitive excitation. J Physiol Soc Jap 29: 272-273, 1967 17. Lewartowski B, Czarnecka M, Zielinski A: Adaptation pattern of cellular action potentials of rabbit atria to the changes in rate and rhythm of stimulation. Acta Physiol Acad Sci Hung 36: 335-341, 1969 18. Saito T: Changes in a train of action potentials in the rabbit atrium after a rest period: Effects of various external media. Jap J Physiol 21:251-263, 1971 19. Trautwein W, Kassebaum DG: On the mechanism of spontaneous impulse generation in the pacemaker of the heart. J Gen Physiol 45: 317-330,1961 20. Aronson RS, Cranefield PF: The effect of resting potential on the electrical activity of canine cardiac Purkinje fibers exposed to Na-free solution or to ouabain. Pfluegers Arch 347: 101-116, 1974 21. Goto M, Tamai T, Yanaga T: Studies on the appearance and termination of aconitine-induced atrial fibrillation with microelectrodes. Jap J Physiol 13: 196-207, 1963 22. Azuma K, Iwane H, Ibukiyama C, Watabe Y, Shin-mura H, lwaoka M, Wakatsuki T, Saito K, Shimizu K, Takada S, Yasui N: Experimental studies on aconitine-induced atrial fibrillation with microelectrodes. Israel J Med Sci 5: 470-474, 1969 23. Horibe H: Studies on the spread of the right atrial activation by means of intracellular microelectrode. Jap Circ J 25: 583-593, 1961 24. Miyauchi A: Electrical events in specialized muscle fibers of a mammalian right atrium. Jap Heart J 3: 357-372, 1962 25. Sano T, Yamagishi S: Spread of excitation from the sinus node. Circ Res 16: 423-430, 1965 26. Hogan PM, Davis LD: Evidence for specialized fibers in the canine right atrium. Circ Res 23: 387-396, 1968 27. Janse MJ, van Capelle FJL, Freud GE, Durrer D: Circus movement within the AV node as a basis for supraventricular tachycardia as shown by multiple microelectrode recording in the isolated rabbit
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heart. Circ Res 28: 403-414, 1971 28. Wit AL, Goldreyer BN, Damato AN: An in vitro model of paroxysmal supraventricular tachycardia. Circ Res 43: 862-875, 1971 29. Allessie MA, Bonke FIM, Schopman FJG: Circus movement in rabbit atrial muscle as a mechanism of tachycardia. Circ Res 43: 5462,1973 30. Toda N: Barium-induced automaticity in relation to calcium ions and norepinephrine in the rabbit left atrium. Circ Res 27: 45-57, 1970 31. Lu HH, Lange G, Brooks CMcC: Factors controlling pacemaker action in cells of the sinoatrial node. Circ Res 17: 460-471, 1965 32. Vassalle M, Cummins M, Castro C, Stuckey JH: The relationship between overdrive suppression and overdrive excitation in ventricular pacemaker in dogs. Circ Res 38: 367-374, 1976 33. Wit AL, Fenoglio JJ, Wagner BM, Bassett AL: Electrophysiological properties of cardiac muscle in the anterior mitral valve leaflet and the adjacent atrium in the dog. Possible implications for the genesis of atrial dysrhythmias. Circ Res 32: 731-745, 1973 34. Ferrier GR, Moe GK: Effect of calcium on acetylstrophanthidininduced transient depolarizations in canine Purkinje tissue. Circ Res 33:508-515, 1973 35. Lederer WJ, Tsien RW: Transient inward current underlying arrhythmogenic effects of cardiotonic steroids in Purkinje fibers. J Physiol 263: 73-100, 1976 36. Brown HF, Noble SJ: Membrane currents underlying delayed rectification and pacemaker activity in frog atrial muscle. J Physiol (Lond) 204:717-736, 1969 37. Katzung BG: Effects of extracellular calcium and sodium on depolarization-induced automaticity in guinea pig papillary muscle. Circ Res 37:118-127, 1975 38. Hauswirth O, Noble D, Tsien RW: The mechanism of oscillatory activity at low membrane potentials in cardiac Purkinje fibers. J Physiol (Lond) 200: 255-265, 1969 39. lmanishi S: Calcium-sensitive discharges in canine Purkinje fibers. Jap J Physiol 21: 443-463, 1971
Mechanism of Suppression of Renin Secretion by Clonidine in the Dog PETER L. NOLAN AND IAN A.
REID
SUMMARY The mechanism by which clonidine suppresses renin secretion was investigated in pentobarbitalanesthetized dogs in which renal perfusion pressure was controlled by means of an aortic clamp. Clonidine (30 /ig/ kg, iv) lowered mean arterial pressure (MAP) from 124 ± 8 to 104 ± 4 mm Hg (P < 0.01) and reduced plasma renin activity (PRA) to 32 ± 4% of the control value (P < 0.01) after 60 minutes. Ganglion blockade with pentolinium (3 mg/kg, im) decreased MAP from 148 ± 7 to 117 ± 3 mm Hg (P < 0.01) and reduced PRA to 55 ± 13% of the control value (P < 0.05) after 45 minutes. Pentolinium converted the hypotension produced by clonidine to hypertension (108 ± 9 to 146 ± 10 mm Hg at 60 minutes, P < 0.05) and abolished the suppression of PRA (105 ± 14% of control at 60 minutes, P > 0.05). In a further series of experiments, the effects of oxymetazoline, an a-adrenergic receptor agonist which is closely related to clonidine but which does not cross the blood brain barrier, were studied. Oxymetazoline (10 /ig/kg, iv) increased MAP from 127 ± 3 to 154 ± 2 mm Hg (P < 0.01) and elevated PRA to 176 ± 22% of the control value (P < 0.02) after 30 minutes. A higher dose of oxymetazoline (30 pg/kg) increased MAP from 129 ± 10 to 161 ± 9 mm Hg (P < 0.05) and increased PRA to 256 ± 37% of control (P < 0.05) after 30 minutes. Taken together, these data support the hypothesis that the inhibition of renin secretion by clonidine results from a centrally mediated decrease in sympathetic neural activity.
SEVERAL INVESTIGATORS have reported that the hypotension and bradycardia produced by clonidine are accompanied by a reduction in renin secretion.1"6 Considerable evidence suggests that the clonidine-induced decreases in blood pressure and heart rate are effected by a From the Department of Physiology, University of California, San Francisco, California. Supported by U.S. Public Health Service Grant AM 06704. Dr. Peter L. Nolan is a Research Fellow of the Bay Area Heart Research Committee. Dr. Ian A. Reid is the Recipient of Research Career Development Award HL 00104.
centrally mediated decrease in sympathetic tone,7 although peripheral actions to increase afferent baroreceptor activity8 and impair sympathetic neurotransmission9'10 may also contribute. The suppression of renin secretion by clonidine also appears to result from decreased sympathetic activity. It is known that renal denervation decreases renin secretion4 Address for reprints: Dr. Peter L. Nolan, Department of Physiology, S-762, University of California, San Francisco, California 94143. Received April 25, 1977; accepted for publication August 31, 1977.
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Electrophysiological studies on the mechanism of electrically induced sustained rhythmic activity in the rabbit right atrium. T Saito, M Otoguro and T Matsubara Circ Res. 1978;42:199-206 doi: 10.1161/01.RES.42.2.199 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1978 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/42/2/199.citation
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Electrophysiological Studies on the Mechanism of Electrically Induced Sustained Rhythmic Activity in the Rabbit Right Atrium TAKEHIKO SAITO, MOTOHIRO OTOGURO, AND TETSU MATSUBARA
SUMMARY Right atrial preparations with no spontaneous activity were isolated from rabbit hearts. Action potentials with specific oscillatory after potentials were recorded from some fibers in the upper pectinate muscle and in the pectinate muscle along the crista terminalis. The amplitude and rate of rise of the oscillatory potential increased as the stimulus interval decreased. In 12 of 35 preparations in Tyrode's solution, sustained rhythmic activity resulted from the depolarizing phase of the enhanced oscillatory potential. The action potential during sustained rhythmic activity was characterized by slow diastolic depolarization, as in the sinoatrial (SA) node. A single premature stimulus or a train of stimuli sometimes caused an acceleration of the rate of excitation rather than its suppression. Sustained rhythmic activity was maintained at a low resting potential and ceased spontaneously when the slope of the slow diastolic depolarization decreased and the maximum diastolic potential increased. Stimulation just after termination of sustained rhythmic activity neither increased the amplitude of the oscillatory potential nor initiated further sustained rhythmic activity. Initiation of new sustained rhythmic activity required a period of quiescence before electrical stimulation. These results suggest that the sustained rhythmic activity described in this paper results not from reentry but, rather, from spontaneous generation of action potentials by the atrial fibers having oscillatory afterpotentials.
IT IS WELL KNOWN that premature impulses arising at certain times during the cardiac cycle may induce a burst of rapid repetitive activity (sustained rhythmic activity). Such activity has been assumed to result from either automatic ectopic pacemakers or reentry.1"" Sustained rhythmic activity which is initiated and terminated by single premature impulses generally has been thought to result from reentry. However, recent reports indicate that some atrial and ventricular specialized fibers can develop sustained ectopic rhythms that are initiated and sometimes terminated by premature impulses.7"1'2 So far, such sustained rhythmic activity has been observed mainly as the result of drugs such as catecholamines or digitalis and abnormal ionic environments such as a low sodium solution. The rhythms differ from those of automatic fibers, because they sometimes result from oscillatory afterpotentials that are enhanced by a series of extrinsic stimuli. Recently, in the rabbit right atrium with no spontaneous activity in Tyrode's solution, Tanaka et al.l:l found fibers that showed action potentials having oscillatory afterpotentials. In a preliminary report, they stated that such fibers respond to an increase in the rate of electrical stimulation with an increase in the amplitude of oscillatory afterpotentials and become spontaneously active in Tyrode's solution.14 From the Department of Physiology, St. Marianna University School of Medicine, Kawasaki (Dr. T. Saito) and the Department of Internal Medicine, Tokyo Medical College, Tokyo, Japan (Dr. M. Otoguro and Dr. T. Matsubara). This study was supported in part by a research grant from the Japan Research Promoter Society for Cardiovascular Disease. A preliminary report of this work was presented at the Fifty-First General Meeting of the Physiological Society of Japan, 1974. Address for reprints: Dr. Takehiko Saito, Department of Physiology, St. Marianna University School of Medicine, 2095, Sugao Takatsu-ku Kawasaki, Japan. Received January 24, 1977; accepted for publication September 8, 1977.
In the experiment reported here, sustained rhythmic activity was induced in the rabbit right atrium when the preparation was stimulated at a proper stimulus interval after a certain period of quiescence. The experiment was initiated to investigate whether such activity is caused by ectopic automatic pacemakers or by reentry. Methods Rabbits weighing 2-3 kg were anesthetized with urethan (1 g/kg administered intravenously). The entire heart was removed quickly and placed in a Lucite bath perfused with Tyrode's solution of the following composition (miu): NaCl, 137; KC1, 2.6; CaCl2, 1.8; MgCl2, 0.5; NaHCO;t, 11.7; NaH2PO4, 3.2; glucose, 5.6. Subsequent dissection was performed in this Tyrode's solution, which was equilibrated with a gas mixture of 95% O2 and 5% CO2 and maintained at a temperature of about 32°C. The ventricles and left atrium were removed and discarded. The endocardial surface of the right atrium was exposed by an incision through the free wall at the atrioventricular groove, extending along the anterior border of the right atrial appendage and through the anterior wall of the superior vena cava, as described in detail by Paes de Carvalho et al.15 The right atrium, which showed spontaneous activity, was transferred to a tissue bath having a volume of 20 ml and was superfused with oxygenated Tyrode's solution. After pinning the preparation with the endocardial surface up to a cork block in the tissue bath, its appearance was like that shown schematically in Figure 1 (upper diagram). Further dissection was made along the dashed line, L, in the figure in order to remove the sinoatrial node, atrioventricular node, and crista terminalis. The part of the right atrium which remained in the tissue bath after the dissection and which usually did not show spontaneous activity is schematically shown in Figure 1 (lower diagram). The right atrial preparation was pinned
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diastole. Both potentials were recorded either independently or simultaneously on a continuously moving film at a slow speed (5-10 mm/sec). Results Action Potential with an Oscillatory Afterpotential
pectinate muscle along crista terminalis
FIGURE 1 Schematic representation of the rabbit right atrial preparation and transmembrane action potentials recorded from two different type of fibers. In the upper diagram, the right and lower portions of the dashed line, L, indicated that the crista terminalis, SA node, A V node, and ventricle, were discarded. The remaining right atrial myocardium, which usually did not show any spontaneous activity in Tyrode's solution, is shown in the lower diagram. The approximate location of each recording site is indicated on the lower diagram of the preparation. The preparation was stimulated electrically every 5 seconds. In records a and b, the lower traces are of the same cell as in the upper traces but at higher gain than the upper, in order to display the terminal repolarization phase. Time and voltage calibrations for the upper and lower traces are indicated in record b. SVC: superior vena cava, IVC: inferior vena cava, CT: crista terminalis, SA: sinoatrial node, CS: coronary sinus, A VV: atrioventricular valve.
under slight tension to a cork block in the tissue bath. It was left unstimulated for several minutes in Tyrode's solution at a temperature of about 36°C. Some preparations started to fire spontaneously at a regular slow rate during the rest period. In order to find a pacemaker region in such preparations, mechanical stimuli were given to them by a fine needle. The pacemaker region that showed a rate change with a touch of a needle was cut off. Unless spontaneous activity appeared, the preparation was stimulated electrically with a concentric metal electrode (diameter 1 mm) consisting of copper inside and stainless steel outside. Rectangular current pulses of 0.5msec duration and twice threshold intensity were applied to the preparation from a pulse generator with an isolation unit (NIHON KOHDEN MSE-3R). Transmembrane action potentials of single fibers were recorded by floating microelectrodes filled with 3 M KG (tip resistance of about 30 Mfl). The potential was led to different vertical axes of a dual beam oscilloscope (NIHON KOHDEN VC-7A) via two DC amplifiers of different gain. In most experiments, the upper beam, which was at an appropriate gain and sweep speed to display the entire action potential on the oscilloscope screen, was triggered by stimulus pulses. The lower beam was set at high gain and always was positioned as a stationary spot at the lower left corner on the oscilloscope screen to record changes in potential during terminal repolarization and
Right atrial preparations that were quiescent in Tyrode's solution were driven electrically. In 12 of 35 preparations, sustained rhythmic activity was induced at a certain stimulus rate. This activity persisted for a while and ceased spontaneously. The phenomenon was induced regularly in the same preparation as long as the stimulation was applied after a period of quiescence. Transmembrane action potentials were recorded from many sites in these preparations. It was possible to recognize at least two types of atrial fibers in terms of the time courses of their afterpotentials. Figure 1 shows action potentials recorded from two different fibers in the same preparation and the approximate sites at which the recordings were made. The preparation was stimulated electrically with a stimulus interval of 5 seconds. The upper and lower traces of each record were obtained from the same cell but with different amplifications. In record a, the action potential showed a long-lasting afterpotential which decayed to the resting potential. The action potentials with such an afterpotential commonly were recorded in the atrial roof muscle fibers and in most of the pectinate muscle fibers.1" The resting and action potential amplitudes measured for 18 pectinate muscle fibers with this type of afterpotential were 81 ± 9 mV and 92 ± 11 mV (mean ± SD), respectively, at a stimulus interval of 1 second. The duration at 50% repolarization was 48 ± 11 msec under the same stimulus conditions. In record b, the action potential was recorded from a fiber of the upper pectinate muscle. The action potential of this fiber did not terminate with a simple return to the resting potential but, rather, was followed by a secondary depolarization. The amplitude of this delayed afterpotential, which was defined as the difference between the afterdepolarization at its peak and resting potential, was about 6 mV. The afterpotential decayed from its peak to the resting potential with a time course of over 2 seconds. Action potentials with this specific afterpotential were obtained mostly from some of the upper pectinate muscle fibers and the pectinate muscle fibers along the crista terminalis. The amplitude of the oscillatory afterpotential varied greatly from fiber to fiber. Some fibers showed only afterhyperpolarization without being followed by afterdepolarization. The resting and action potential amplitudes measured from pectinate muscle fibers with oscillatory afterpotentials of more than 5 mV were 73 ± 7 mV and 80 ± 10 mV (mean ± SD), respectively, at a stimulus interval of 1 second. The duration at 50% repolarization was 72 ± 16 msec (mean ± SD). Initiation and Termination of Sustained Rhythmic Activity Figure 2 shows changes in the oscillatory afterpotentials of single driven impulses interposed at various intervals
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a !
b
d FIGURE 2 Effects of single driven impulses interposed at various moments in the cardiac cycle. The preparation was stimulated electrically every 6 seconds. Action potentials were recorded from a fiber in the upper pectinate muscle. The records were taken at a high gain and slow speed in order to show the terminal repolarization phase. Thus, most of the action potential is off scale. Record a shows the control responses. A single driven impulse was applied at 2 seconds (b), I second (c), and 0.5 second (d) after one of the control response. In record d, nondriven impulses were initiated. Time and voltage calibrations are indicated in record c.
after regularly driven action potentials. The action potentials were obtained from a single fiber in the upper pectinate muscle. In record a, each action potential was followed by an oscillatory afterpotential of about 5 mV amplitude which occurred long after completion of the preceding afterpotential. When a single test stimulus was applied at an interval of about 2 seconds after the control response in record b, a driven action potential occurred at a slightly less negative level of membrane potential than that seen before the preceding action potential, because it arose from the terminal repolarization phase of the preceding afterpotential. This action potential was followed by a slightly enhanced afterpotential; the slope and amplitude of the secondary depolarization were increased. As a test stimulus was applied at about 1 second in record c, a driven action potential occurred at the peak of the preceding afterpotential. The action potential was followed first by a hyperpolarization and subsequently by a depolarization. The slope and amplitude of this depolarization were greater than those of the control response. If we look only at the events that occur between the maximum afterhyperpolarization and peak of the afterdepolarization, we can see a slow diastolic depolarization that is very similar to that seen in automatic fibers. In record d, a further decrease in the stimulus interval to 500 msec resulted in a further enhancement of the oscillatory afterpotential. Then the fiber became spontaneously active. The rate of sustained rhythmic activity increased during the first few beats and became regular. This sustained
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rhythmic activity continued for about 20 seconds at the rate of about 3 cycles/sec. When the upper pectinate muscle was dissected off, sustained rhythmic activity stopped and was not again induced by electrical stimulation. Figure 3 shows changes in the action potentials and their afterpotentials elicited by trains of stimuli at constant rates. A train of 10 successive stimuli at rates between 0.5 and 2 cycles/sec was applied every 50 seconds. The first action potentials following the long quiescent period were characterized by a prominent spike potential with a low-level plateau (see upper traces in each record). The terminal repolarization phase of the low-level plateau was followed by the oscillatory afterpotential (see lower traces in each record). Subsequent repetitive stimulation produced a gradual prolongation of the spike duration and a cumulative loss of the maximum diastolic potential. Shorter stimulus intervals caused more pronounced changes in the plateau duration and afterpotential (record a-c). Changes in the plateau duration depending on the rate of stimulation have been discussed in relation to the potassium conductance of the membrane.l:)-17-1S The amplitude of the oscillatory afterpotentials that followed the last driven impulses of a train of 10 successive stimuli was about 4.5 mV in record a, 6 mV in record b, and 10 mV
FIGURE 3 Effect of changes in the stimulus interval on the amplitude of the oscillatory afterpotential. A train of electrical stimuli at intervals of 2 seconds (a), 1.5 seconds (b), 1 second (c), and 0.5 second (d) were applied on a previously quiescent fiber. A ction potentials were recorded from a fiber in the upper pectinate muscle. Quiescent periods between each record were kept constant at 50 seconds. The upper beam was triggered by stimulus pulses and swept at the proper speed to see the action potential profile. The lower beam remained as a stationary spot at the lower left corner on the oscilloscope screen and was deflected vertically at higher gain. Both were photographed simultaneously on continuously moving film at a slow speed. The gain of the lower traces is so large that most of the action potential proper is off scale. In record d, sustained rhythmic activity was induced after the fourth driven impulse (arrow). Time and voltage calibrations were indicated in record c for the upper traces and in record d for the lower traces.
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in record c. When the fiber was driven at a stimulus interval of 500 msec in record d, it became spontaneously active after the fourth impulse. Then electrical stimuli were stopped immediately. Not all of the spontaneous action potentials were seen in the upper trace, because the upper beam was triggered only by the stimulus pulses. The action potentials during sustained rhythmic activity are quite different in appearance from those obtained by electrical stimulation. They are characterized by a slow upstroke, low amplitude with rounded peak, absence of overshoot, and slow diastolic depolarization. A fiber that had a resting potential of -75 mV before electrical stimulation showed a lower take-off potential of about - 5 5 mV and a maximum diastolic potential of about -66 mV during sustained rhythmic activity. Sustained rhythmic activity, which had a rate of about 2 cycles/sec, persisted for about 10 seconds and ceased spontaneously. The last nondriven action potential was followed by a subthreshold depolarization of about 15 mV which failed to initiate the next action potential. Figure 4 shows sustained rhythmic activity arising from the enhanced oscillatory afterpotential. In record a, 10 successive stimuli at a stimulus interval of 2 seconds were applied. The slope and amplitude of the oscillatory potential increased gradually during the first four stimuli and decreased during subsequent stimuli. In this case, the firing level for each driven action potential gradually shifted in the depolarizing direction during a sequence of stimulation, whereas the maximum diastolic potential was little changed because of the enhancement of the afterhyperpolarization. The afterpotentials of the third and fourth impulses arose to their peaks with an upward concavity and decayed to the diastolic potential in two steps, first rapidly to a given level and then slowly to the maximum diastolic potential. They seem to be comparable to a local response evoked by a subthreshold pulse in a quiescent Purkinje fiber.19'20 In record b, four successive stimuli at a stimulus interval of 1 second were applied, and sustained rhythmic activity was induced after the third impulse (arrow). The fourth
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stimulus, applied during the slow diastolic depolarization of the nondriven action potential (triangle), caused an increase in the slope of slow diastolic depolarization with a consequent increase in the rate of excitation. The fiber that had a resting potential of -72 mV before electrical stimulation showed a take-off potential of about - 6 0 raV and a maximum diastolic potential of about - 6 7 mV during sustained rhythmic activity. Sustained rhythmic activity at 4 cycles/sec persisted for about 15 seconds and ceased spontaneously with a slight increase in the maximum diastolic potential and a decrease in the slope of slow diastolic depolarization. The action potentials during sustained rhythmic activity were similar to those of a spontaneously active fiber such as that of the SA node. The last nondriven action potential was followed by a subthreshold depolarization of about 15 mV which had a hump during repolarization and decayed to the resting potential in several seconds. Sustained rhythmic activity usually showed constant rates of 1-4 cycles/sec and ceased spontaneously. Initiation of additional sustained rhythmic activity required a few stimuli after a certain period of quiescence. In 2 of 12 preparations, a spontaneous repetition of sustained rhythmic activity was observed. Figure 5 shows an example of such activity. In record a, sustained rhythmic activity at about 2 cycles/sec appeared after the second driven impulse (arrow). Subsequent spontaneous activity caused a progressive decrease in the maximum diastolic potential. A decrease in maximum diastolic potential of about 16 mV resulted in a decrease in amplitude of the action potentials with a notch on their upstrokes. The rate of spontaneous activity was decreased. Each action potential initiated at a low membrane potential was followed by a subthreshold depolarization (lower trace in the middle part of record a). Such activity persisted for about 30 seconds and terminated in oscillations. The maximum diastolic potential increased gradually before spontaneous activity appeared. After quiescence lasting a few seconds, a burst of impulses resumed with the appearance of oscillations of increasing amplitude and again was suppressed at the level of maximum decrease of the diastolic potential (very end of record a). This cycle repeated itself for about 2 minutes (record b). We did not attempt to record responses simultaneously from fibers in
2 IK
FIGURE 4 Oscillatory afterpotentials leading to sustained rhythmic activity. A train of electrical stimuli at intervals of 2 seconds (a) and I second (b) were applied to a previously quiescent fiber. Action potentials were recorded from a fiber in the upper pectinate muscle close to the crista terminalis. The recording method is the same as that in Figure 3. In record a, subthreshold depolarization appeared on the first few impulses and was gradually suppressed during subsequent impulses. In record b, sustained rhythmic activity appeared after the third impulse (arrow). A single stimulus applied on the slow diastolic depolarization of a nondriven impulse (triangle) caused acceleration of the rate of spontaneous activity. Time and voltage calibrations for the upper and lower traces are indicated in record b.
FIGURE 5 Spontaneous repetition of a burst of discharges. Action potentials were recorded from a fiber in the pectinate muscle along the crista terminalis. The recording method is same as that in Figure 3. In record a, sustained rhythmic activity appeared after the second impulse (arrow). Record b is a continuation of record a. Spontaneous activity persisted for about 3 minutes and ceased abruptly. Time and voltage calibrations were indicated in record a for the upper traces and in record b for the lower traces.
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ELECTRICALLY INDUCED SUSTAINED RHYTHMIC ACTIVITY/Saito et al. different areas of the atrium. It is not clear whether the low amplitude action potentials initiated at the low maximum diastolic potential are able to propagate and excite the whole preparation. A similar spontaneous repetition of the burst of impulses also has been recorded from rabbit atrial preparations superfused with aconitine.21'22 Figure 6 shows changes in the oscillatory afterpotential during the recovery phase after termination of sustained rhythmic activity. In record a, sustained rhythmic activity occurred after the second impulse following a long quiescent period. The rate of excitation increased gradually over the first few beats. The maximum rate of excitation was about 4 cycles/sec. Sustained rhythmic activity persisted for about 20 seconds and ceased spontaneously. Single stimuli were applied at 15 (b), 30 (c), and 60 (d) seconds during the quiescent period. The slope and amplitude of the afterdepolarization were greatly suppressed following shorter quiescent periods and gradually increased after progressively longer quiescent periods. Repetitive stimulation just after termination of sustained rhythmic activity neither potentiated the afterpotential nor initiated sustained rhythmic activity. After about 1 minute of quiescence, the same stimulus conditions elicited the same rhythmic activity as that shown in record a. This phenomenon was recorded from four other fibers. The fiber showing a more rapid rate of spontaneous activity had a tendency to cause a stronger suppression of the oscillation after termination of spontaneous activity. Effects of Electrical Stimulation on Sustained Rhythmic Activity
Once sustained rhythmic activity had been triggered by external stimuli, it persisted for a while and terminated spontaneously after a slight decrease in the rate of excitation. A slight decrease in the slope of slow diastolic depolarization and an increase in the amplitude of the maximum diastolic potential, associated with the reduction of the rate of excitation, were observed before termination of spontaneous activity. Figure 7 shows the effects of premature stimuli on the rate of sustained rhythmic activity. Premature impulses were induced at various intervals during the cardiac cycle. In records a
FIGURE 6 Suppression and recovery of the oscillatory afterpotential. Action potentials were recorded from a fiber in the upper pectinate muscle. The gain is so high that only the lower part of the action potentials is seen. Sustained rhythmic activity appeared after second driven impulses and persisted for about 20 seconds (a). A single driven impulse was applied at 15 seconds (b), 30 seconds (c), and 60 seconds (d) after termination of sustained rhythmic activity. Time and voltage calibrations were indicated in record a.
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FIGURE 7 Effect of electrically driven premature impulses on the rate of sustained rhythmic activity. Action potentials were recorded from fibers in the pectinate muscle very close to the crista terminalis. The gain is so high that only the lower part of the action potentials is seen. In each record, onset of sustained rhythmic activity and application of the premature stimuli were indicated by arrows and triangles, respectively. Records a and b were obtained from same cell. In record a, single premature stimuli were applied in two different cardiac cycles during sustained rhythmic activity. In record b, a train of stimuli al an interval of 1 second was applied during sustained rhythmic activity. In record c, single premature stimuli were applied in two different cardiac cycles and just after termination of sustained rhythmic activity. Time and voltage calibrations for records a and b are indicated in record a.
and c, single premature impulses were elicited both early and late in the cardiac cycle. Each premature impulse was followed by a slight hyperpolarization of the membrane, but it was unsuccessful in terminating sustained rhythmic activity. Premature impulses elicited during the first several beats of sustained rhythmic activity caused a noticeable increase in the rate of excitation. In record b, eight successive premature impulses at a rate of 1 cycle/sec were applied during sustained rhythmic activity. They also did not terminate the spontaneous activity. In record c, a single premature impulse just after termination of the spontaneous rhythm was followed by a large afterhyperpolarization with suppression of the subsequent afterdepolarization. When electrical stimulation at rates greatly in excess of the rate of sustained rhythmic activity (overdrive) was applied to the atrial preparations, some resumed beating spontaneously after a short quiescent period and others immediately resumed spontaneity just after the external stimuli were interrupted. Figure 8 shows a complex effect of overdrive on spontaneous activity. In record a, the first 11 responses are the control rhythmic activity at about 2 cycles/sec. Then repetitive stimulation at a rate of 4 cycles/sec was applied for about 30 seconds, as indicated by a horizontal line below the responses. When stimuli were interrupted, the spontaneous activity immediately occurred at almost same rate as that of the control (record b); yet, this rhythm slowed abruptly within several impulses. The rate of this slow rhythm returned gradually to that of the control. This phenomenon was different from the overdrive suppression observed in the normal cardiac
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CIRCULATION RESEARCH
204 i : ! i
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FIGURE 8 Effect of fast drive on the spontaneously active preparation. The period of electrical driving at a rate of 4 cycles/sec is indicated by the horizontal line below responses in records a and b. Time and voltage calibrations were indicated in record c.
pacemaker. Abrupt slowing of the rate of excitation and the appearance of a hump in the slow diastolic depolarization might suggest a pacemaker shift with a partial conduction block. Discussion On the basis of anatomical differences and characteristics of the transmembrane action potentials, several types of fibers have been identified in the mammalian right atrium.15'23"26 Paes de Carvalho et al.15 found that the rabbit right atrium contains several distinct types of fibers. One fiber of particular interest, located in the SA ring bundle running parallel to the crista terminalis on the venous border, is characterized by action potentials with a prominent plateau and, sometimes, an inherent slow diastolic depolarization. This bundle gives at least one major branch to the upper pectinate muscle. Hogan and Davis26 found specialized conducting fibers, so-called atrial plateau fibers, along the caval border of the crista terminalis and along the free-running strand in the canine right atrium. Those fibers seem to be comparable to the SA ring bundle fibers and their branches in the rabbit atrium. In the present experiments, action potentials with oscillatory afterpotentials were recorded from some fibers in the upper pectinate muscles and in the basal region of the pectinate muscles along the crista terminalis. The amplitude of the afterpotentials varied greatly from fiber to fiber in the same preparation and from preparation to preparation. The action potentials recorded from fibers several hundred microns away from the particular fiber with the oscillatory afterpotential usually were not followed by any oscillatory afterpotentials. The plateau duration of the action potential in that particular fiber was longer than that of the ordinary pectinate muscle. Although we have not observed the exact distribution of these particular fibers, the electrical characteristics of the fibers and their approximate location suggest the possibility that they may be branches of the SA ring bundle or be transitional fibers between the SA ring bundle and ordinary pectinate muscle.
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It generally has been assumed that spontaneous or evoked premature impulses may initiate or terminate only reentrant rhythms. Macro reentrant circuits involving the atrium, AV node, and ventricular specialized conduction system have been demonstrated in the rabbit heart by Janse et al.27 and Wit et al.28 In small segments of the rabbit left atrium with no anatomical obstacle, Allessie et al.29 reported that the application of a single adequately timed stimulus can ir : ; and always terminate tachycardia. Based on results of multiple microelectrode recording, they suggested that such a tachycardia is maintained by reentrant circuits. In the present experiments, sustained rhythmic activity was induced in the previously quiescent atrial fiber only with the aid of a series of a few external stimuli. The following evidence suggests that such rhythmic activity results not from reentry, but rather from spontaneous generation of action potentials by the specific type of fiber described above: 1. The oscillatory afterpotential in a particular fiber was enhanced by repetitive stimulation at an appropriate interval. The enhanced oscillation brought the membrane to a less negative level, and then sustained rhythmic activity resulted from the depolarizing phase of the oscillatory afterpotential. The initial phase of sustained rhythmic activity usually increased in rate during the first several beats and this was accompanied by an increase in the slope of the slow diastolic depolarization. This would not be observed in reentrant rhythms but is observed sometimes in automatic pacemaker rhythms. A number of investigators have, described the spontaneous activity arising from the specific afterpotentials in many kinds of preparations under the action of several agents. Examples of this sort of behavior of the membrane are found in atrial fibers during exposure to acetylstrophanthidin,8 aconitine,22 and Ba2+.30 Hashimoto and Moe8 demonstrated that strophanthidin-induced spontaneous activity did not appear in the ordinary muscle fibers, but in the atrial specialized fibers of the dog heart as well as in the Purkinje fibers.9 2. A slightly decreased slope of the slow diastolic depolarization and an increased magnitude of the maximum diastolic potential were observed just before termination of sustained rhythmic activity. The oscillatory potential was markedly suppressed just after termination of the spontaneous activity and the initiation of new sustained rhythmic activity required a certain period of quiescence which is associated with recovery of the oscillatory potentials. These facts may suggest that the oscillatory potential is not only related to the initiation of sustained rhythmic activity, but also to termination of this activity. 3. A single stimulus or repetitive stimuli applied during sustained rhythmic activity sometimes caused an acceleration of the rate of excitation rather than its suppression. This would not be observed in reentrant rhythms. Recently, sustained rhythmic activity, that can be initiated and terminated by a premature impulse and that is probably not reentrant, has been demonstrated in Purkinje fibers exposed to sodium-free solution" and in mitral valve of the monkey heart exposed to catecholamines.12 When automatic fibers are stimulated electrically at a rate
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ELECTRICALLY INDUCED SUSTAINED RHYTHMIC ACTIVITY/Saito et al. more rapid than their spontaneous rates, a phenomenon known as overdrive suppression occurs.31 In the present preparations, the effect of overdrive on sustained rhythmic activity appeared to be complex. When stimuli were interrupted suddenly, overdrive suppression was observed in some cases, but in other cases rhythmic activity resumed at a rate similar to the control and occasionally was suppressed after several beats. We have not made a detailed study of this phenomenon in the present paper. Recently, however, Vassalle et al.32 carefully studied the effects of overdrive on idioventricular pacemakers in dogs with complete atrioventricular block. They found that overdrive suppression and overdrive excitation can be present at the same time and antagonism between them causes various different effects of overdrive on idioventricular pacemakers. A small action potential with a slow rate of depolarization has a tendency to develop conduction block and is known as a possible cause of cardiac arrhythmias.2"6 Wit et al.33 reported various degrees of conduction block of ectopic impulses formed in the mitral valve. In the present experiment, an ectopic pacemaker usually showed a higher rate of discharge than that of normal pacemakers. The characteristics of the transmembrane potentials included a low amplitude of the resting and action potentials and a low action potential upstroke velocity. Since these factors are favorable to failure of propagation of impulses, it is possible that a spontaneous repetition of the burst of impulses described in Figure 5 may result from a combination of conduction block and regular ectopic rhythm, although we do not have any data concerning the propagation, or block, of the low amplitude responses. So far there is little information on the nature of the mechanisms underlying the oscillatory afterpotential in the atrial specific fiber. Hashimoto and Moe8 reported that a transient depolarization which is associated with strophanthidin-induced automaticity is suppressed by an elevation of the external potassium concentration or application of acetylcholine. Some of the spontaneously active preparations obtained in the present experiment became quiescent when the external potassium concentration was increased from 2.6 mM to 5.2 mM (unpublished experiment). Ferrier and Moe34 suggested that the transient depolarization of the Purkinje fiber under the influence of strophanthidin may be caused by Ca2+ influx. Lederer and Tsien35 demonstrated a voltage-dependent inward current which is responsible for strophanthidininduced transient depolarization of the Purkinje fiber. Once sustained rhythmic activity is triggered in a particular fiber, the configuration of the spontaneous action potentials is quite similar to that of action potentials recorded from a spontaneously active fiber such as that of the SA node. The exact level of membrane potential from which the spontaneous activity is triggered is uncertain and difficult to define, because the action potentials recorded in the present experiment were not always recorded from the site of impulse origin itself. However, spontaneous action potentials usually were elicited at a membrane potential less than —60 mV, brought about by the enhanced oscillatory afterpotential, and maintained at a low diastolic membrane potential between —70 and —60 mV. Recently, evidence has accumulated which
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indicates that spontaneous action potentials also may occur in a number of normally quiescent fibers, when the resting potential is reduced by an application of depolarizing currents.36'37 Action potentials occurring at low membrane potentials are insensitive to tetrodotoxin. The ionic basis for the action potentials during sustained rhythmic activity in the present experiment is uncertain. However, the membrane activity in the particular fibers described here seems to consist of at least two distinct and different mechanisms depending on different levels of membrane potential. This has already been suggested for other cardiac fibers.6-12> 36~39 In conclusion, the results of this study have shown the existence of a particular fiber in the rabbit atrium which has action potentials with an oscillatory afterpotential. It seems possible that the enhancement and suppression of the oscillatory afterpotential of the above-mentioned fiber play an important role in the initiation and termination of sustained rhythmic activity. In addition to the development of ectopic pacemaker function, several other factors such as local block of conduction or irregular interaction of activity among adjacent fibers might be able to produce more serious arrhythmias. Acknowledgments The authors thank Professor W. H. Miller, Department of Ophthalmology and Visual Science, Yale University School of Medicine, and Professor J. Toyoda, Department of Physiology, St. Marianna University School of Medicine, for their critical reading of the manuscript. Thanks are also due to Y. Takada for her valuable assistance in the preparation of the manuscript.
References 1. Trautwein W: Generation and conduction of impulses in the heart as affected by drugs. Pharmacol Rev IS: 277-332, 1963 2. Hoffman BF, Cranefield PF: The physiological basis of cardiac arrhythmias. Am J Med 37: 670-684, 1964 3. Watanabe Y, Dreifus LS: Newer concepts in the genesis of cardiac arrhythmias. Am Heart J 76: 114-135, 1968 4. Cranefield PF, Wit AL, Hoffman BF: Genesis of cardiac arrhythmias. Circulation 47: 190-204, 1973 5. Wit AL, Rosen MR, Hoffman BF: Electrophysiology and pharmacology of cardiac arrhythmias: II. Relationship of normal and abnormal electrical activity of cardiac fibers to the genesis of arrhythmias. Am Heart J 88: 515-524, 1974 6. Cranefield PF: The conduction of the cardiac impulse; the slow response and cardiac arrhythmias. New York, Futura, 1975 7. Davis LD: Effect of changes in cycle length on diastolic depolarization produced by ouabain in canine Purkinje fibers. Circ Res 32: 206214, 1973 8. Hashimoto K, Moe GK: Transient depolarizations induced by acetylstrophanthidin in specialized tissue of dog atrium and ventricle. Circ Res 32:618-624,1973 9. Ferrier GR, Saunders JH, Mendez C: A cellular mechanism for the generation of ventricular arrhythmias by acetylstrophanthidin. Circ Res 32: 600-609, 1973 10. Rosen MR, Gelband H, Merker C, Hoffman BF: Mechanisms of digitalis toxicity: Effects of ouabain on phase four of canine Purkinje fiber transmembrane potentials. Circulation 47: 681-689, 1973 11. Cranefield PF, Aronson RS: Initiation of sustained rhythmic activity by single propagated action potentials in canine cardiac Purkinje fibers exposed to sodium-free solution or to ouabain. Circ Res 34: 477-481, 1974 12. Wit AL, Cranefield PF: Triggered activity in cardiac muscle fibers of the simian mitral valve. Circ Res 38: 85-98, 1976 13. Tanaka I, Tosaka T, Saito K, Shin-mura H, Saito T: Changes in the configuration of the rabbit atrial action potential after various period of rest. Jap J Physiol 17: 487-504, 1967 14. Saito T, Otoguro M, Kobayashi F, Matsubara T: Automatic activity in rabbit atrium. J Physiol SocJap38: 325, 1974 15. Paes de Carvalho A, de Mello WC, Hoffman BF: Electrophysiological evidence for specialized fiber types in rabbit atrium. Am J Physiol 196:483-488, 1959
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16. Tanaka I, Saito T: Changes in duration of plateau phase and afterdepolarization of the rabbit atrial action potential by repetitive excitation. J Physiol Soc Jap 29: 272-273, 1967 17. Lewartowski B, Czarnecka M, Zielinski A: Adaptation pattern of cellular action potentials of rabbit atria to the changes in rate and rhythm of stimulation. Acta Physiol Acad Sci Hung 36: 335-341, 1969 18. Saito T: Changes in a train of action potentials in the rabbit atrium after a rest period: Effects of various external media. Jap J Physiol 21:251-263, 1971 19. Trautwein W, Kassebaum DG: On the mechanism of spontaneous impulse generation in the pacemaker of the heart. J Gen Physiol 45: 317-330,1961 20. Aronson RS, Cranefield PF: The effect of resting potential on the electrical activity of canine cardiac Purkinje fibers exposed to Na-free solution or to ouabain. Pfluegers Arch 347: 101-116, 1974 21. Goto M, Tamai T, Yanaga T: Studies on the appearance and termination of aconitine-induced atrial fibrillation with microelectrodes. Jap J Physiol 13: 196-207, 1963 22. Azuma K, Iwane H, Ibukiyama C, Watabe Y, Shin-mura H, lwaoka M, Wakatsuki T, Saito K, Shimizu K, Takada S, Yasui N: Experimental studies on aconitine-induced atrial fibrillation with microelectrodes. Israel J Med Sci 5: 470-474, 1969 23. Horibe H: Studies on the spread of the right atrial activation by means of intracellular microelectrode. Jap Circ J 25: 583-593, 1961 24. Miyauchi A: Electrical events in specialized muscle fibers of a mammalian right atrium. Jap Heart J 3: 357-372, 1962 25. Sano T, Yamagishi S: Spread of excitation from the sinus node. Circ Res 16: 423-430, 1965 26. Hogan PM, Davis LD: Evidence for specialized fibers in the canine right atrium. Circ Res 23: 387-396, 1968 27. Janse MJ, van Capelle FJL, Freud GE, Durrer D: Circus movement within the AV node as a basis for supraventricular tachycardia as shown by multiple microelectrode recording in the isolated rabbit
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heart. Circ Res 28: 403-414, 1971 28. Wit AL, Goldreyer BN, Damato AN: An in vitro model of paroxysmal supraventricular tachycardia. Circ Res 43: 862-875, 1971 29. Allessie MA, Bonke FIM, Schopman FJG: Circus movement in rabbit atrial muscle as a mechanism of tachycardia. Circ Res 43: 5462,1973 30. Toda N: Barium-induced automaticity in relation to calcium ions and norepinephrine in the rabbit left atrium. Circ Res 27: 45-57, 1970 31. Lu HH, Lange G, Brooks CMcC: Factors controlling pacemaker action in cells of the sinoatrial node. Circ Res 17: 460-471, 1965 32. Vassalle M, Cummins M, Castro C, Stuckey JH: The relationship between overdrive suppression and overdrive excitation in ventricular pacemaker in dogs. Circ Res 38: 367-374, 1976 33. Wit AL, Fenoglio JJ, Wagner BM, Bassett AL: Electrophysiological properties of cardiac muscle in the anterior mitral valve leaflet and the adjacent atrium in the dog. Possible implications for the genesis of atrial dysrhythmias. Circ Res 32: 731-745, 1973 34. Ferrier GR, Moe GK: Effect of calcium on acetylstrophanthidininduced transient depolarizations in canine Purkinje tissue. Circ Res 33:508-515, 1973 35. Lederer WJ, Tsien RW: Transient inward current underlying arrhythmogenic effects of cardiotonic steroids in Purkinje fibers. J Physiol 263: 73-100, 1976 36. Brown HF, Noble SJ: Membrane currents underlying delayed rectification and pacemaker activity in frog atrial muscle. J Physiol (Lond) 204:717-736, 1969 37. Katzung BG: Effects of extracellular calcium and sodium on depolarization-induced automaticity in guinea pig papillary muscle. Circ Res 37:118-127, 1975 38. Hauswirth O, Noble D, Tsien RW: The mechanism of oscillatory activity at low membrane potentials in cardiac Purkinje fibers. J Physiol (Lond) 200: 255-265, 1969 39. lmanishi S: Calcium-sensitive discharges in canine Purkinje fibers. Jap J Physiol 21: 443-463, 1971
Mechanism of Suppression of Renin Secretion by Clonidine in the Dog PETER L. NOLAN AND IAN A.
REID
SUMMARY The mechanism by which clonidine suppresses renin secretion was investigated in pentobarbitalanesthetized dogs in which renal perfusion pressure was controlled by means of an aortic clamp. Clonidine (30 /ig/ kg, iv) lowered mean arterial pressure (MAP) from 124 ± 8 to 104 ± 4 mm Hg (P < 0.01) and reduced plasma renin activity (PRA) to 32 ± 4% of the control value (P < 0.01) after 60 minutes. Ganglion blockade with pentolinium (3 mg/kg, im) decreased MAP from 148 ± 7 to 117 ± 3 mm Hg (P < 0.01) and reduced PRA to 55 ± 13% of the control value (P < 0.05) after 45 minutes. Pentolinium converted the hypotension produced by clonidine to hypertension (108 ± 9 to 146 ± 10 mm Hg at 60 minutes, P < 0.05) and abolished the suppression of PRA (105 ± 14% of control at 60 minutes, P > 0.05). In a further series of experiments, the effects of oxymetazoline, an a-adrenergic receptor agonist which is closely related to clonidine but which does not cross the blood brain barrier, were studied. Oxymetazoline (10 /ig/kg, iv) increased MAP from 127 ± 3 to 154 ± 2 mm Hg (P < 0.01) and elevated PRA to 176 ± 22% of the control value (P < 0.02) after 30 minutes. A higher dose of oxymetazoline (30 pg/kg) increased MAP from 129 ± 10 to 161 ± 9 mm Hg (P < 0.05) and increased PRA to 256 ± 37% of control (P < 0.05) after 30 minutes. Taken together, these data support the hypothesis that the inhibition of renin secretion by clonidine results from a centrally mediated decrease in sympathetic neural activity.
SEVERAL INVESTIGATORS have reported that the hypotension and bradycardia produced by clonidine are accompanied by a reduction in renin secretion.1"6 Considerable evidence suggests that the clonidine-induced decreases in blood pressure and heart rate are effected by a From the Department of Physiology, University of California, San Francisco, California. Supported by U.S. Public Health Service Grant AM 06704. Dr. Peter L. Nolan is a Research Fellow of the Bay Area Heart Research Committee. Dr. Ian A. Reid is the Recipient of Research Career Development Award HL 00104.
centrally mediated decrease in sympathetic tone,7 although peripheral actions to increase afferent baroreceptor activity8 and impair sympathetic neurotransmission9'10 may also contribute. The suppression of renin secretion by clonidine also appears to result from decreased sympathetic activity. It is known that renal denervation decreases renin secretion4 Address for reprints: Dr. Peter L. Nolan, Department of Physiology, S-762, University of California, San Francisco, California 94143. Received April 25, 1977; accepted for publication August 31, 1977.
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Electrophysiological studies on the mechanism of electrically induced sustained rhythmic activity in the rabbit right atrium. T Saito, M Otoguro and T Matsubara Circ Res. 1978;42:199-206 doi: 10.1161/01.RES.42.2.199 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1978 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571
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