In vivo recording of electrical activity of canine tracheal smooth muscle T. KONDO, K. TAMURA, K. ONOE, Departments of Medicine and Physiology, Isehara, Kunugawa 259-l 1, Japan
H. TAKAHIRA, Y. OHTA, AND H. YAMABAYASHI School of Medicine, Tokai University,
KONDO, T.,K. TAMURA, K. ONOE, H. TAKAHIRA,Y.OHTA, AND H. YAMABAYASHI. In vivo recording of electricalactivity of canine tracheal smooth muscle. J. Appl. Physiol. 72(l): 135-142, 1992.-Electrical activity of the tracheal smooth muscle was studied usingextracellular bipolar electrodesin 37decerebrate, paralyzed, and mechanically ventilated dogs. A spontaneous oscillatory potential that consistedof a slow sinusoidalwave of 0.57 t 0.13 (SD) H z mean frequency but lacked a fast spike component was recorded from 15 dogs.Lung collapseaccomplishedby bilateral pneumothoraxes evoked or augmentedthe slow potentials that were associatedwith an increasein tracheal muscle contraction in 26 dogs. This suggeststhat the inputs from the airway mechanoreceptorsreflexly activate the tracheal smooth musclecells. Bilateral vagal transection abolishedboth the spontaneousand the reflexly evoked slowwaves and provided relaxation of the tracheal smoothmuscle.Electrical stimulation of the distal nerve with a train pulse (0.5 ms, l-30 Hz) evoked slow-waveoscillatory potentials accompanied by a contraction of the tracheal smoothmusclein all the experimental animals. Our observationsin this in vivo study confirm that the electrical activity of tracheal smooth muscleconsists of slow oscillatory potentials and that tracheal contraction is at least partly coupled to the slow-wave activity of the smooth muscle.
slow wave was similar to the membrane potential reported in the tracheal smooth muscle of guinea pigs both in vivo and in vitro (21). Studies of ferret (6) and cow (4) tracheal smooth muscle showed that acetylcholine released from the tracheal nerve terminals evoked postsynaptic excitatory potentials. Augmentation of acetylcholine release also evoked slow membrane oscillatory potential with corresponding smooth muscle contraction (4). The results of our studies suggest that such slow waves exist in the intact dog, provided that the tracheal smooth muscle is vagally innervated and that in vivo recordings of the muscle can be made. In this study, we have attempted to record electrical activity from the tracheal smooth muscle of the decerebrate dog. METHODS
Experiments were performed in 37 adult mongrel dogs weighing 5.0-12.8 kg. Anesthesia was induced by the intramuscular injection of ketamine hydrochloride (10 mg/ kg). Surgical anesthesia was maintained with the intravenous administration of thiamylal sodium (total amount 5-10 mg/kg). The carotid arteries were bilaterally ligated autonomic regulation; extracellular recording; pharmaco- below the bifurcation of external and internal carotid armechanical coupling; excitation-contraction coupling; shv teries. The tracheal branch arteries were preserved to wave maintain tracheal circulation. The vagus nerves were exposed bilaterally at the high cervical region and were kept warm and moist during the operative procedures. THE ELECTRICAL ACTIVITY of the airway smooth muscle An endotracheal tube was inserted orally, and the tip was varies by animal species and with differen .t experimental positioned just below the vocal cords. The animals were conditions (28). Most of the p lrevious i.nvestigations made decerebrate to eliminate the possible side effects of studying tracheal smooth muscle have utilized only in anesthesia on smooth muscle and on neural activity durvitro methods of intracellular recordings. As a result ing recording. The brain stem was transected at the rosthere is little information on in vivo tracheal smooth tral borders of superior colliculi, and both cerebral hemimuscle activity. The studies on isolated tracheal muscle spheres were removed by suction. The experiments were from cow and dog have shown that the muscle is electriperformed with the dog in the supine position on a heatcally and mechanically quiescent in physiological solu- ing pad. All animals were paralyzed with pancuronium tion (11,13,24,27). In contrast, isolated tracheal smooth bromide and mechanically ventilated with 100% oxygen. muscle of the guinea pig in Krebs solution exhibits spon- To prevent atelectasis due to ventilation with 100% oxytaneous oscillatory electrical potentials (16) associated gen, we periodically inflated the lungs by a resuscitation with muscle contraction (21). Similar membrane potenbag. A tracheostomy was made on the thoracic trachea tial fluctuations are observed in other species of animals approximately four rings above the bifurcation. Mechanwhen acetylcholine (11) tetrae thylammonium (11, ‘13, ical ventilation was then maintained via the tracheoswas made by a small 27), or histamine (1 1) is’ added to the solution or when tomy opening. A left pneumothorax glucose is removed from the solution (2 ). incision in the chest wall or in the mediastinum. CathA recent study of human bronchial smooth muscle eters were inserted into the femoral artery and vein for continuous monitoring of arterial blood pressure and addemonstrated spontaneous membrane oscillatory potentials in 90% of the isolated tissues (10). This spontaneous ministration of intravenous fluids, respectively. Samples Ul61-7567/92 $2.00Copyright 0 1992the American Physiological Society
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136
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respirator
FIG. 1. Experimental preparations. Left: group I. Surgical operation was not done on trachea. Exact location of tip of electrode-delivering needle (Etr) was observed using fiber-optic bronchoscopy. A balloon-tipped catheter, which measures changes in tracheal dimension as pressure changes (Ptr), was inserted through thoracic tracheostomy. Right: group II. Anterior wall of trachea was widely opened. An isometric force transducer was tied to smooth muscle.
for arterial blood gas analysis were obtained from the arterial catheter. The electrodes used for all recordings were a pair of silver-silver chloride wires (0.13 mm OD). The wires were insulated with Teflon except for the distal 5 mm, which were hook shaped. To record electrical activity and tension of the tracheal smooth muscle, we divided the animals into two groups. Ingroup I (n = l3), the recording electrode was delivered by a hook-shaped 22-gauge hypodermic needle. The needle was percutaneously introduced into the tracheal lumen at the level of the midcervical region (Fig. 1). The precise position of the needle tip was confirmed by fiberoptic bronchoscopy (BF-B3, Olympus, Tokyo, Japan). Under fiberscopic observation, the introducing needle was positioned onto the posterior wall of the trachea. The needle was then removed, leaving the noninsulated tip of the electrode in the muscle layer. Two wires were introduced into the tracheal smooth muscle, 3-5 mm apart. Changes in the tracheal caliber due to smooth muscle contraction were measured as pressure changes in an air-filled balloon placed in the tracheal lumen. A balloon-tip catheter (15 ml maximum capacity; Fig. 1) was inserted through the thoracic tracheostomy opening (Fig. 1, left), and the tip was placed 2 cm below the recording site. In group II (n = Z4), the cervical trachea was incised along the midline from the third to the ninth cartilaginous ring. The ventral half of the rings was removed, leaving -10% of the cartilaginous rings to which the tra-
cheal smooth muscle was attached. The recording electrode was delivered via a 25gauge hypodermic needle and implanted into the tracheal smooth muscle layer under direct observation. The force developed by the tracheal smooth muscle was measured by an isometric force transducer (TBGllT, Nihon Kohden, Tokyo, Japan) tied to the smooth muscle with silk threads (Fig. 1, right). The electrical signals from the tracheal smooth muscle were filtered with a high-pass filter (time constant of 0.3 s) and then amplified and recorded on a chart recorder. In two of the group II animals, the electrical activity was recorded both with and without frequency filtration. In another two of the group II animals, the electrical activity was recorded with a monopolar electrode. The respiratory airflow was measured by a hot-wire flow meter (RF-L, Minato Medical, Osaka, Japan) placed between the respirator and endotracheal tube. The experimental protocol consisted of the following: 1) Spontaneous activity of the tracheal smooth muscle was recorded with the vagus nerves intact. 2) Transient atelectasis was induced by discontinuation of mechanical ventilation. 3) Lungs were then reinflated with 400 ml of air while still off the ventilator. 4) A positive end-expiratory pressure (PEEP) of 10 cmH,O was applied. 5) Both vagus nerves were then transected bilaterally at a high cervical level. 6) After careful dissection of the vagus nerves, the distal end of the right vagus nerve was mounted onto a pair of platinum electrodes, which was subsequently connected to a stimulator isolation unit. The right vagus nerve was then stimulated by a train of
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TRACHEAL 200mmHg 0
IB
0
I*/“---.
BP
100cmH20
Ptr
ELECTRICAL
5OpV
Etr 0
exp. Flow in lmin
I
2. Spontaneous electrical activity of tracheal smooth muscle. Traces are blood pressure (BP), endotracheal balloon pressure (Ptr), electrical activity of tracheal smooth muscle (Etr), and respiratory flow (Flow). Record from a group I dog. FIG.
square pulses (duration 0.5 ms, frequency l-50 Hz, intensity 0.5-50 V). The electrical stimulation was repeated at intervals of 2-3 min. 7) Fifty milligrams of methacholine chloride were administered intratracheally to stimulate the cholinergic receptors on the smooth muscle directly. At the end of each experiment, an autopsy was performed on each animal to confirm that the tips of the recording electrodes were properly implanted in the tracheal smooth muscle. To exclude the possibility of false impulses due to esophageal smooth muscle activity, we measured the electrical and mechanical activities after a proximal esophagectomy in two dogs. RESULTS
The arterial blood gas values of the 35 dogs before initiation of the experimental protocol were pH 7.37 t 0.06, PCO, 33.0 +- 7.5 Torr, and PO, 404 t 101 (SD) Torr. Systolic arterial blood pressure was maintained > 120 mmHg during the experiments. Spontaneous electrical activity with intact uagi. With the vagus nerv *es intact, spontaneous electrical activity was recorded from the tracheal smooth muscle in 6 of the 12 (50%) animals in group I (Fig. 2) and in 8 of the 23 (35%) animals in group 11. This spontaneous activity consisted of slow oscillatory potentials with an average maximum frequency of 0.57 t- 0.13 Hz (n = 10). The maximum amplitudes ranged from 20 to 140 ,uV. In all animals, no fast spike components were observed. In some dogs rhythmic contracti .ons of the trachea were found to be synchronized with the mean arterial blood pressure changes (Fig. 2). The waxing and waning of the slow potentials occurred in phase with the tracheal contraction rhythm. In two of the group II animals, the recording was done while the animals’ blood gases were extremely abnormal. When the dog experienced a respiratory acidosis, i.e., arterial blood pH between 7.16 and 7.20, the tracheal smooth muscle contracted and electrical activity was
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prominent. When the same animal experienced a respiratory alkalosis, i.e., arterial blood pH between 7.52 and 7.60, the tracheal smooth muscle relaxed. In the relaxed state the electrical activity was still observed, but the amplitude of the activity was usually very low. Effects of lung collapse and inflution. To test the effects of a vagally mediated reflex on the slow-wave potential and to eliminate the possibility of mechanical influence due to artificial respiration, we transiently disconnected the ventilator. When respiration was terminated, the lungs collapsed to a level less than residual volume (RV), and the tracheal smooth muscles were found to contract gradually in all experimental animals. With this maneuver, slow-wave potentials developed in 11 (85%) of the group I animals and 15 (63%) of the group II animals (Fig. 2). The highest frequency of the observed waves corresponded to progressive increases in smooth muscle tensile force development. The maximum frequencies attained were 0.65 t 0.08 Hz in the group I animals and 0.60 -t 0.10 in the group II animals. The difference was not statistically significant (P < 0.01, t test). The slowwave oscillations continued during the entire period of tracheal smooth muscle contraction. With the resumption of artificial respiration, both the increase in the tracheal tension and the slow-wave activity disappeared or became periodic for a few seconds, then disappeared. Because the dogs were ventilated with 100% oxygen, it was possible to discontinue ventilation for short durations without provoking hypoxia. During these periods, the lungs were alternatively inflated and deflated by a large syringe. When the lungs were inflated, the tracheal smooth muscle relaxed and the electrical activity was suppressed (Fig. 3). When the lungs were deflated, the tracheal smooth muscle contracted and continuous electrical activity appeared. During delivery of a PEEP, the tracheal smooth muscle relaxed and the slow-wave potentials were suppressed. When PEEP was discontinued, the tracheal smooth muscle again contracted and the slow waves soon resumed. Effects of bilateral vugotomy. Ligation of the vagus nerves transiently enhanced the spontaneous slow-wave potential. Bilateral transection of the vagus nerves eliminated both the spontaneous oscillatory waves and the tracheal muscle tone (Fig. 4). A two- or threefold increase or decrease in tidal volume produced no electrical activity. In addition, cessation of mechanical ventilation did not induce a reflex contraction of the tracheal smooth muscle. Effect of vagus nerve stimulation. Stimulation of the distal end of the transected vagus nerve with train square pulses (10 s) evoked a phasic contraction of the tracheal smooth muscle in all dogs (n = 33). The slow-wave potentials were similar in morphology to the spontaneous potentials and were invariably associated with a phasic muscle contraction (Fig. 5). Immediately after the onset of the electrical stimulation, an oscillatory electrical potential developed. This activity preceded the onset of tracheal muscle contraction by -0.8 ms. After the initial two or three large-amplitude slow waves, the amplitude and frequency of the evoked slow waves decreased gradually. In 17 of the 33 animals, the evoked slow waves dis-
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138
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200mmHg
BP contract
Etrc
2OPV 01
m
COLlAPSE
I-------I 1NFLATION
30 set
FIG. 3. Effects of lung inflation and deflation on tracheal smooth muscle. Traces are BP, force developed by tracheal smooth muscle (Ftr), electrical activity of rostra1 and caudal tracheal smooth muscle (Etrr and Etrc, respectively). Electrical activities are recorded from 2 parts of tracheal smooth muscle -1.5 cm apart. Lungs were periodically inflated while mechanical ventilation was discontinued. Record from a group 11 dog.
appeared at peak muscle contraction. Another phase of oscillatory activity appeared in the relaxation phase in 16 of 33 of the dogs. The amplitude of this late activity tended to be lower than that of the early oscillatory waves. Figure 6 shows the changes in oscillatory waves and tracheal muscle contraction associated with different stimulus intensities. The amplitudes of the oscillatory waves were three or four times greater in monopolar than in bipolar recordings. The threshold for the development of the electrical activity was - 1.0 V, which is similar to the threshold for the development of a contractile force (n = 7). Further increase in stimulus intensity produced correspondingly greater peak amplitudes and increased phasic contractions. The frequency of the oscillatory
waves also tended to increase with increased stimulus intensity. Figure 7 shows the relationship between stimulus intensity and contractile force when the distal end of the transected right vagus nerve was stimulated (n = 7). The contractile force was measured as the difference between the peak of a phasic contraction and the force immediately before the stimulation, i.e., an active tension (Z3), and then expressed as percentage of the observed con-
2OOmg
BP
clI-centract
t Ptr
b
2ossc
FIG.
4. A record before (k#) and after (right) vagotomy.
FIG. 5. After bilateral vagotomy, distal end of right vagus was stimulated with pulse train (frequency 30 Hz, duration 0.5 ms or 10 s). Traces are BP, Ftr, and raw (REtr) and filtered (Etr) electrical activity of the tracheal smooth muscle. Vagal stimulation with 20-V intensity developed a phasic contraction of tracheal smooth muscle and an oscillatory electrical activity. Stimulation of cardiac branch of vagus nerve caused bradycardia and consequent hypotension.
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139
ACTIVITY
2oornlnH~
BP 0
I
so9 Ftr
*
.
, 1ov
2.5 u
l.OU
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10 SW FIG. 6. Experimental condition is similar to that in Fig. 5, but recordings are done with a monopolar Right vagus nerve was stimulated with 30-Hz pulse train.
traction to the contraction stimulated at 30 V. When comparing the force-stimulation curve generated by the air-filled balloon method with that generated by the isometric force transducer, we noted no significant difference. The shapes of the curves achieved using both methods were similar, and maximum contraction was attained at 30 V for both force-measuring methods. Accordingly, we regarded the results of the two methods as equivalent. A constant level of tracheal contraction was maintained by applying a continuous low-frequency (l-6 Hz) stimulus to the vagus nerve (20). During this contraction, oscillatory electrical activity developed (Fig. 8). In Fig. 8, the amplitude of the slow wave fluctuated during continuous stimulation. The slow-wave activity oscillated or gradually faded when smooth muscle tone was sustained by vagal stimulation. The excitatory effect of vagal nerve stimulation was blocked by the administration of intravenous atropine sulfate (0.5 mg). %
lUO-
l
4
&
4
I I
1
SO-
I 1
0’
11 2
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10 20 5UV FIG. 7. Stimulus intensity (log voltage, Ordinate) vs. active force (%force, abscissa; it = 7). Distal end of the transected right vagus nerve was stimulated with train pulses (30 Hz, 0.5 ms, 10 s). Bars, SE.
electrode.
Effect of methacholine. Intratracheal administration of methacholine (50 mg) caused marked tracheal constriction and slow-wave potentials in five of seven dogs from both groups (Fig. 9). The amplitude and frequency of electrical activity were similar to the activity observed spontaneously in an intact animal. The amplitudes were greatest at the onset of airway contraction. They remained at a lower level for several minutes and finally faded away, even in the presence of sustained tracheal contraction. Resection of the esophagus. After resection of the cervical esophagus, the spontaneous electrical activity still developed in the two animals examined. When the right vagus nerve was stimulated with a pulse train of supramaximal intensity, the tracheal smooth muscle cont ratted and a slow-wave potential similar to those seen with an intact esophagus developed (Fig. 10). DISCUSSION
Electrical activity of the tracheal smooth muscle. The major findings in the present investigation demonstrate the capability of in vivo recording of electrical activity from canine tracheal smooth muscle and the slow-wave characteristics of the recorded electrical activity. In 1975, Akasaka et al. (I) reported in vivo recordings of electrical activity in dogs. In their report, the electrical activity was comprised of action potentials, and the discharge pattern had no relationship to respiratory phase. It remains to be investigated whether slow-wave potentials or action potentials are more reliable for the demonstration of in vivo electrical activity of the canine tracheal smooth muscle. As described below, most of the recent in vitro studies suggest that electrical activity of airway smooth muscle is comprised of slow-wave potentials. Canine tracheal smooth muscle has been described as an intermediate type of single-unit and multiunit groups according to Bozler’s categorization (3). The muscle has scarce innervation (11, 22, 27), gap junctions are rare (27), and there are no action potentials. Some investiga-
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140
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REtr
ACTIVITY
A’SAif t
FIG. 8. After bilateral vagotomy, distal end of right vagus was continuously stimulated with pulse train (6 Hz, 0.5 ms, 5 V).
200mmHg
ZOOcmH20
Ptr
MCh
injection
BP
0
1°r Ftr 0 2cwv
Etr 0
insp.
2Osec. I I FIG. 9. Intratracheal administration of 50 mg methacholine (MCh) evoked oscillatory electrical activity and tracheal contraction. Ptr, Etr, and Flow are as in Fig. 2.
tors have reported that canine tracheal smooth muscle has a steady membrane potential in Krebs solution (7, 27). An increase in membrane potential produces tracheal contraction, but such polarization never evokes oscillatory wave potentials (7). On the other hand, some investigators using intracellular electrodes (2, 12) have recorded slow oscillatory membrane potentials. Our findings suggest that the slow oscillatory waves we recorded originate directly from the tracheal smooth muscle. The frequency of the slow waves obtained from group I (trachea opened) and from group II (trachea closed) animals was not significantly different. This finding strongly suggests that the characteristics of the slow wave were independent of the mechanical condition of the tracheal smooth muscle. The experiment utilizing a proximal esophagectomy further reinforces our consideration. The augmentation of the slow-wave activity by lung deflation or vagus nerve stimulation was followed by the contraction of the tracheal smooth muscle. Moreover, bilateral transection of the vagus nerves not only abolished the spontaneous slow-wave activity but also decreased the resting tone of the tracheal smooth muscle. These findings suggest that both the slow oscillatory wave and the contraction of tracheal smooth muscle are
10s
10. After dissection of cervical esophagus, right vagus was stimulated with 30-Hz 30-V pulse train. FIG.
nerve
controlled by vagal efferents. The possibility that mechanical intervention induced slow-wave potentials was disproved by withdrawal of ventilatory support from the paralyzed dog and observation of an enhancement of the slow-wave potentials. The onset of evoked slow waves produced by vagal stimulation always preceded the onset of the tracheal muscle contraction. Finally, the characteristics of the slow-wave potential of the dog trachea were similar to those reported in other animals both in vivo and in vitro. For example, the mean maximum frequency of the slow wave in our study was 0.57 t 0.13 Hz; this was within the range of the wave frequency of other studies on several species, i.e., 0.5-1.4 Hz (2, 10, 12, 14, 21). Spontaneous electrical activity was recorded in 40% of the animals; in some dogs, slow-wave potentials developed in phase with the rhythm of the tracheal phasic contractions. This finding, however, does not necessarily indicate an absolute requirement of electrical activity for contraction of the tracheal muscle because the tracheal smooth muscle maintains a resting tension without developing electrical activity. Phasic contraction of tracheal smooth muscle has been reported in previous literature (15, 26). In our study the phasic contraction of tracheal smooth muscle correlated with the phasic change in mean arterial blood pressure as seen in Fig. 2, and both
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changes were abolished by vagotomy. Presumably, these findings suggest that both rhythms arose from the same central architecture. Sullivan et al. (26) observed phasic contraction only during sleep associated with rapid eye movement in the intact dog. Thus phasic contraction might not occur under normal conditions. Because our animals underwent major surgery and because insertion of the needle electrodes to the tissue might release inflammatory mediators, the electrical activity observed in this study could have been evoked by experimental intervention. If such intervention has occurred, however, the changes in electrical activity seen with changes in the experimental conditions suggest that experimental intervention should bias only the provocation of the electrical activity. We speculate that augmentation of electrical activity during the contraction phase must be under the command of the vagal efferents because vagotomy eliminated both phasic contractions and electrical activity. Vagus nerve stimulation or atelectasis evoked slow-wave potentials in all the animals studied. With the intact peritracheal innervation, a tonic release of acetylcholine was maintained at the motor endplate. Presumably, under these conditions, electrical activity could be easily evoked in the tracheal smooth muscle. Role of the vagus nerve. It has been suggested that the contraction and relaxation of tracheal smooth muscle induced by lung deflation and inflation are regulated by a reflex arising from pulmonary mechanoreceptors (17,29, 30). In our study the slow-wave activity developed immediately after cessation of ventilatory support; conversely, the activity was suppressed when the lungs were inflated with a bolus of air. These findings indicate that development of the oscillatory electrical activity is mediated via a vagal reflex arising from pulmonary mechanoreceptars. The reduction of slow-wave potentials with the application of PEEP further reinforces the reflex hypothesis. Hypercapnia may also play some role in the generation of slow waves and associated smooth muscle contraction. Reports in the literature have shown that hypercapnia increases the contraction of tracheal smooth muscle of anesthetized cats (17, 25) and dogs (19). We found that the oscillatory waves persisted for several seconds after a period of transient apnea, suggesting that apnea-induced hypercapnia may influence slow-wave formation and tracheal smooth muscle tone. Abolition of the oscillatory wave and the resting tension of tracheal smooth muscle by transection of the vagus nerves is consistent with the knowledge that the tracheal smooth muscle maintains a resting tension by continuous vagal efferent tone (20). The oscillatory wave was always followed by a contraction of the tracheal smooth muscle. However, in 58% of the experimental animals, the tracheal smooth muscle maintained its resting tension even if the oscillatory electrical waves were not observed. This finding suggests that oscillatory electrical activity is not necessary for the maintenance of a resting tension in tracheal smooth muscle, Electrical stimulation of the distal end of the vagus nerve consistently generated oscillatory potentials and phasic contractions. This indicates that the synchronized excitation of tracheal smooth muscle by the vagal release of acetylcholine significantly influences the gener-
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ation of slow oscillatory potentials. The force developed by the tracheal smooth muscle increased concomitantly with increases in stimulus intensity and frequency. Kannan et al. (12) have reported that acetylcholine provokes oscillatory activity and spontaneous contraction in isolated canine tracheal smooth muscle. They speculated that the changes in conductance underlying these oscillations represent the periodic opening of Ca2+ channels by acetylcholine (8). Induction of oscillatory potentials by the intratracheal administration of methacholine and total inhibition of stimulus-evoked potential by atropine support the Ca2+ channel hypothesis. Physiologicul significance of the electrical activity. Dog tracheal smooth muscle has been found to contract without oscillatory membrane potentials. This phenomenon has been noted in the late phase of continuous stimulation and in the case of intratracheal administration of methacholine. The absence of oscillatory potentials in the face of muscle contraction supports a hypothesis that voltage-dependent Ca2+ channels and decreases in membrane potential are not primarily involved in generating acetylcholine-induced contraction in the canine tracheal muscle (5). Electromechanical coupling is reported to be involved in acetylcholine-induced contraction of the ferret tracheal smooth muscle (14, 18). However, the maximum contribution of the electromechanical coupling is only -30% in acetylcholine-induced contraction of the dog’s tracheal smooth muscle, and this mechanism occurs only when the concentration of acetylcholine is >10-16 M in the organ bath (9). It has been reported, in isolated tracheal smooth muscle of ferret (14) and dog (12), that electrical oscillation of the membrane potential occurred with depolarization by the addition of acetylcholine to the organ bath solution. From these reports we may speculate that development of a slow wave reflects a decrease in membrane potential of the smooth muscle cell. In conclusion, we have shown that the electrical activity of slow oscillatory waves can be recorded in vivo from canine tracheal smooth muscle. The development of slow waves is invariably associated with tracheal contraction, and both the slow waves and tracheal contraction are mediated via vagal efferents. Tracheal contraction, however, may occur without slow waves, The development of the slow wave may indicate a decrease in membrane potential of the tracheal smooth muscle. This study was partly supported by Tokai University School of Medicine Research Aid. Address for reprint requests: T. Kondo, Div. of Pulmonology, Dept. of Medicine, School of Medicine, Tokai University, Isehara, Kanagawa 25941, Japan. Received 29 May 1990; accepted in final form 14 August 1991. REFERENCES AKASAKA, K.,K. KoNNo,Y.ONO,S. MUE, CABE, M. KUMAGAI, AND T. ISE. A new electrode for electromyographic study of bronchial smooth muscle. Tohoku J. hp. Med. 117: 49-54, 1975. BOSE, R., AND D. BOSE. Excitation-contraction coupling in multiunit tracheal smooth muscle during metabolic depletion: induction of rhythmicity. Am. J. Physiol. 233 (Cell Physiol. 2): C8-C13, 1977. BOZLER,
E. Action potential and conduction of excitation in muscle. Biol. Symp. 3: 95-109, 1940.
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CAMERON, A. R., AND C. T. KIRKPATRICK. A study of excitatory neuromuscular transmission in the bovine trachea. J. physiol. Lond. 270: 733-745,1977, COBURN, R. F. Electromechanical coupling in canine trachealis muscle: acetylcholine contractions. Am. J. Physiol. 236 (CeZZPhysioz. 5): c177-Cl84,1979. COBURN, R. F. Neural coordination of excitation of ferret trachealis muscle, Am. J. Physiol. 246 (CkZZPhysiol. 15): C459-466, 1984. COBURN, R. F., AND T. YAMAGUCHI. Membrane potential-dependent and -independent tension in the canine tracheal muscle. J. Pharmacol. Exp. Ther. 201: 276-284,1977. DANIEL, E. E. Control of airway smooth muscle. In: The Airways: Neural Control in Health and Disease, edited by M. A. Kaliner and P. J. Barnes. New York: Dekker, 1988, p. 485-521. (Lung Biol. Health Dis. Ser.) FARLEY, J. M., AND P. R. MILES. Role of depolarization in acetylcholine-induced contractions of dog trachealis muscle. J. Pharmacot. Exp. Ther. 201: 119-205, 1977. ITO, Y., AND T. YOSHITOMI. Autoregulation of acetylcholine release from vagus nerve terminals through activation of muscarinic receptors in the dog trachea. Br. J. Pharmacol. 93: 636-646,1989. KANNAN, M. S., AND E. E. DANIEL. Structural and functional study of control of canine tracheal smooth muscle. Am. J. Physiol. 238 (Cell Physiol. 7): C27--C33, 1980. KANNAN, M. S., L. P. JAGER, E. E. DANIEL, AND R. E. GARFIELD. Effects of 4-aminopyridine and tetraethylammonium chloride on the electrical activity and cable properties of canine tracheal smooth muscle. J. Phurmacol. &p. Ther. 227: 706-715, 1983. KIRKPATRICK, C. T. Excitation and contraction in bovine tracheal smooth muscle, J. Physiol. Lond. 244: 263-281, 1975. LEE, H. K., AND C. G. MURLAS. Electromechanical effects of leukotriene D4 on ferret tracheal muscle and its muscarinic responsiveness. Lung 167: 173-185,1989. LOOFBOUROW, G. N., W. B. WOOD, AND I. L. BAIRD. Tracheal constriction in the dog. Am. J. Physiol. 191: 411-415, 1957. MCCAIG, D. I., AND J. F. SOUHRADA. Alteration of electrophysiological properties of airway smooth muscle from sensitized guineapigs. Respir. Physiol. 41: 49-60, 1980. MITCHELL, R. A., D. A. HERBERT, AND D. G. BAKER. Inspiratory
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STEPHENS, N. L, Structure and mechanical properties. Am. Rev. Respir. Dis. 136: W-7, 1987. 24. STEPHENS, N. L., AND E. KROGER. Effect of hypoxia on airway smooth muscle mechanics and electrophysiology. J. AppZ. Physiol. 23.
28: 630-635,197O.
STROHL, K. P., M. P. NORCIA, A. D. WOLIN, M. A. Hmru, E. LUNTEREN, AND E. D. DEAL. Nasal and tracheal responses to chemical and somatic afferent stimulation in anesthetized cats. J. Appl. Physiol. 65: 870-877, 1988. 26. SULLIVAN, C. E., N. ZAMEL, L. F. KOZAR, E. MURPHY, AND E. A. PHILLIPSON. Regulation of airway smooth muscle tone in sleeping dogs. Am. Rev. Respir. Dis. 119: 87-99, 1979. 27. SUZUKI, H., K. MORITA, AND H. KURIYAMA. Innervation and properties of the smooth muscle of the dog trachea. Jpn. J. Physiol. 26: 25.
303-320,1976.
TOMITA, T. Electrical properties of airway smooth muscle. In: Airway Smooth Muscle in HeaZth and Disease, edited by R. F. Coburn. New York: Plenum, 1989, p. 151-167. 29. WI~)DICOMBE, J. G. Action potentials in parasympathetic and sympathetic efferent fibres to the trachea and lungs of dogs and cats. J. Physiol. Land. 186: 56-88, 1966. 30. WIDDICOMBE, J. G., AND J. A. NADEL. Reflex effects of lung inflation on tracheal volume. J. AppZ. Physiol. 18: 681-686, 1963. 28.
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H. TAKAHIRA, Y. OHTA, AND H. YAMABAYASHI School of Medicine, Tokai University,
KONDO, T.,K. TAMURA, K. ONOE, H. TAKAHIRA,Y.OHTA, AND H. YAMABAYASHI. In vivo recording of electricalactivity of canine tracheal smooth muscle. J. Appl. Physiol. 72(l): 135-142, 1992.-Electrical activity of the tracheal smooth muscle was studied usingextracellular bipolar electrodesin 37decerebrate, paralyzed, and mechanically ventilated dogs. A spontaneous oscillatory potential that consistedof a slow sinusoidalwave of 0.57 t 0.13 (SD) H z mean frequency but lacked a fast spike component was recorded from 15 dogs.Lung collapseaccomplishedby bilateral pneumothoraxes evoked or augmentedthe slow potentials that were associatedwith an increasein tracheal muscle contraction in 26 dogs. This suggeststhat the inputs from the airway mechanoreceptorsreflexly activate the tracheal smooth musclecells. Bilateral vagal transection abolishedboth the spontaneousand the reflexly evoked slowwaves and provided relaxation of the tracheal smoothmuscle.Electrical stimulation of the distal nerve with a train pulse (0.5 ms, l-30 Hz) evoked slow-waveoscillatory potentials accompanied by a contraction of the tracheal smoothmusclein all the experimental animals. Our observationsin this in vivo study confirm that the electrical activity of tracheal smooth muscleconsists of slow oscillatory potentials and that tracheal contraction is at least partly coupled to the slow-wave activity of the smooth muscle.
slow wave was similar to the membrane potential reported in the tracheal smooth muscle of guinea pigs both in vivo and in vitro (21). Studies of ferret (6) and cow (4) tracheal smooth muscle showed that acetylcholine released from the tracheal nerve terminals evoked postsynaptic excitatory potentials. Augmentation of acetylcholine release also evoked slow membrane oscillatory potential with corresponding smooth muscle contraction (4). The results of our studies suggest that such slow waves exist in the intact dog, provided that the tracheal smooth muscle is vagally innervated and that in vivo recordings of the muscle can be made. In this study, we have attempted to record electrical activity from the tracheal smooth muscle of the decerebrate dog. METHODS
Experiments were performed in 37 adult mongrel dogs weighing 5.0-12.8 kg. Anesthesia was induced by the intramuscular injection of ketamine hydrochloride (10 mg/ kg). Surgical anesthesia was maintained with the intravenous administration of thiamylal sodium (total amount 5-10 mg/kg). The carotid arteries were bilaterally ligated autonomic regulation; extracellular recording; pharmaco- below the bifurcation of external and internal carotid armechanical coupling; excitation-contraction coupling; shv teries. The tracheal branch arteries were preserved to wave maintain tracheal circulation. The vagus nerves were exposed bilaterally at the high cervical region and were kept warm and moist during the operative procedures. THE ELECTRICAL ACTIVITY of the airway smooth muscle An endotracheal tube was inserted orally, and the tip was varies by animal species and with differen .t experimental positioned just below the vocal cords. The animals were conditions (28). Most of the p lrevious i.nvestigations made decerebrate to eliminate the possible side effects of studying tracheal smooth muscle have utilized only in anesthesia on smooth muscle and on neural activity durvitro methods of intracellular recordings. As a result ing recording. The brain stem was transected at the rosthere is little information on in vivo tracheal smooth tral borders of superior colliculi, and both cerebral hemimuscle activity. The studies on isolated tracheal muscle spheres were removed by suction. The experiments were from cow and dog have shown that the muscle is electriperformed with the dog in the supine position on a heatcally and mechanically quiescent in physiological solu- ing pad. All animals were paralyzed with pancuronium tion (11,13,24,27). In contrast, isolated tracheal smooth bromide and mechanically ventilated with 100% oxygen. muscle of the guinea pig in Krebs solution exhibits spon- To prevent atelectasis due to ventilation with 100% oxytaneous oscillatory electrical potentials (16) associated gen, we periodically inflated the lungs by a resuscitation with muscle contraction (21). Similar membrane potenbag. A tracheostomy was made on the thoracic trachea tial fluctuations are observed in other species of animals approximately four rings above the bifurcation. Mechanwhen acetylcholine (11) tetrae thylammonium (11, ‘13, ical ventilation was then maintained via the tracheoswas made by a small 27), or histamine (1 1) is’ added to the solution or when tomy opening. A left pneumothorax glucose is removed from the solution (2 ). incision in the chest wall or in the mediastinum. CathA recent study of human bronchial smooth muscle eters were inserted into the femoral artery and vein for continuous monitoring of arterial blood pressure and addemonstrated spontaneous membrane oscillatory potentials in 90% of the isolated tissues (10). This spontaneous ministration of intravenous fluids, respectively. Samples Ul61-7567/92 $2.00Copyright 0 1992the American Physiological Society
135
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136
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respirator
FIG. 1. Experimental preparations. Left: group I. Surgical operation was not done on trachea. Exact location of tip of electrode-delivering needle (Etr) was observed using fiber-optic bronchoscopy. A balloon-tipped catheter, which measures changes in tracheal dimension as pressure changes (Ptr), was inserted through thoracic tracheostomy. Right: group II. Anterior wall of trachea was widely opened. An isometric force transducer was tied to smooth muscle.
for arterial blood gas analysis were obtained from the arterial catheter. The electrodes used for all recordings were a pair of silver-silver chloride wires (0.13 mm OD). The wires were insulated with Teflon except for the distal 5 mm, which were hook shaped. To record electrical activity and tension of the tracheal smooth muscle, we divided the animals into two groups. Ingroup I (n = l3), the recording electrode was delivered by a hook-shaped 22-gauge hypodermic needle. The needle was percutaneously introduced into the tracheal lumen at the level of the midcervical region (Fig. 1). The precise position of the needle tip was confirmed by fiberoptic bronchoscopy (BF-B3, Olympus, Tokyo, Japan). Under fiberscopic observation, the introducing needle was positioned onto the posterior wall of the trachea. The needle was then removed, leaving the noninsulated tip of the electrode in the muscle layer. Two wires were introduced into the tracheal smooth muscle, 3-5 mm apart. Changes in the tracheal caliber due to smooth muscle contraction were measured as pressure changes in an air-filled balloon placed in the tracheal lumen. A balloon-tip catheter (15 ml maximum capacity; Fig. 1) was inserted through the thoracic tracheostomy opening (Fig. 1, left), and the tip was placed 2 cm below the recording site. In group II (n = Z4), the cervical trachea was incised along the midline from the third to the ninth cartilaginous ring. The ventral half of the rings was removed, leaving -10% of the cartilaginous rings to which the tra-
cheal smooth muscle was attached. The recording electrode was delivered via a 25gauge hypodermic needle and implanted into the tracheal smooth muscle layer under direct observation. The force developed by the tracheal smooth muscle was measured by an isometric force transducer (TBGllT, Nihon Kohden, Tokyo, Japan) tied to the smooth muscle with silk threads (Fig. 1, right). The electrical signals from the tracheal smooth muscle were filtered with a high-pass filter (time constant of 0.3 s) and then amplified and recorded on a chart recorder. In two of the group II animals, the electrical activity was recorded both with and without frequency filtration. In another two of the group II animals, the electrical activity was recorded with a monopolar electrode. The respiratory airflow was measured by a hot-wire flow meter (RF-L, Minato Medical, Osaka, Japan) placed between the respirator and endotracheal tube. The experimental protocol consisted of the following: 1) Spontaneous activity of the tracheal smooth muscle was recorded with the vagus nerves intact. 2) Transient atelectasis was induced by discontinuation of mechanical ventilation. 3) Lungs were then reinflated with 400 ml of air while still off the ventilator. 4) A positive end-expiratory pressure (PEEP) of 10 cmH,O was applied. 5) Both vagus nerves were then transected bilaterally at a high cervical level. 6) After careful dissection of the vagus nerves, the distal end of the right vagus nerve was mounted onto a pair of platinum electrodes, which was subsequently connected to a stimulator isolation unit. The right vagus nerve was then stimulated by a train of
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TRACHEAL 200mmHg 0
IB
0
I*/“---.
BP
100cmH20
Ptr
ELECTRICAL
5OpV
Etr 0
exp. Flow in lmin
I
2. Spontaneous electrical activity of tracheal smooth muscle. Traces are blood pressure (BP), endotracheal balloon pressure (Ptr), electrical activity of tracheal smooth muscle (Etr), and respiratory flow (Flow). Record from a group I dog. FIG.
square pulses (duration 0.5 ms, frequency l-50 Hz, intensity 0.5-50 V). The electrical stimulation was repeated at intervals of 2-3 min. 7) Fifty milligrams of methacholine chloride were administered intratracheally to stimulate the cholinergic receptors on the smooth muscle directly. At the end of each experiment, an autopsy was performed on each animal to confirm that the tips of the recording electrodes were properly implanted in the tracheal smooth muscle. To exclude the possibility of false impulses due to esophageal smooth muscle activity, we measured the electrical and mechanical activities after a proximal esophagectomy in two dogs. RESULTS
The arterial blood gas values of the 35 dogs before initiation of the experimental protocol were pH 7.37 t 0.06, PCO, 33.0 +- 7.5 Torr, and PO, 404 t 101 (SD) Torr. Systolic arterial blood pressure was maintained > 120 mmHg during the experiments. Spontaneous electrical activity with intact uagi. With the vagus nerv *es intact, spontaneous electrical activity was recorded from the tracheal smooth muscle in 6 of the 12 (50%) animals in group I (Fig. 2) and in 8 of the 23 (35%) animals in group 11. This spontaneous activity consisted of slow oscillatory potentials with an average maximum frequency of 0.57 t- 0.13 Hz (n = 10). The maximum amplitudes ranged from 20 to 140 ,uV. In all animals, no fast spike components were observed. In some dogs rhythmic contracti .ons of the trachea were found to be synchronized with the mean arterial blood pressure changes (Fig. 2). The waxing and waning of the slow potentials occurred in phase with the tracheal contraction rhythm. In two of the group II animals, the recording was done while the animals’ blood gases were extremely abnormal. When the dog experienced a respiratory acidosis, i.e., arterial blood pH between 7.16 and 7.20, the tracheal smooth muscle contracted and electrical activity was
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prominent. When the same animal experienced a respiratory alkalosis, i.e., arterial blood pH between 7.52 and 7.60, the tracheal smooth muscle relaxed. In the relaxed state the electrical activity was still observed, but the amplitude of the activity was usually very low. Effects of lung collapse and inflution. To test the effects of a vagally mediated reflex on the slow-wave potential and to eliminate the possibility of mechanical influence due to artificial respiration, we transiently disconnected the ventilator. When respiration was terminated, the lungs collapsed to a level less than residual volume (RV), and the tracheal smooth muscles were found to contract gradually in all experimental animals. With this maneuver, slow-wave potentials developed in 11 (85%) of the group I animals and 15 (63%) of the group II animals (Fig. 2). The highest frequency of the observed waves corresponded to progressive increases in smooth muscle tensile force development. The maximum frequencies attained were 0.65 t 0.08 Hz in the group I animals and 0.60 -t 0.10 in the group II animals. The difference was not statistically significant (P < 0.01, t test). The slowwave oscillations continued during the entire period of tracheal smooth muscle contraction. With the resumption of artificial respiration, both the increase in the tracheal tension and the slow-wave activity disappeared or became periodic for a few seconds, then disappeared. Because the dogs were ventilated with 100% oxygen, it was possible to discontinue ventilation for short durations without provoking hypoxia. During these periods, the lungs were alternatively inflated and deflated by a large syringe. When the lungs were inflated, the tracheal smooth muscle relaxed and the electrical activity was suppressed (Fig. 3). When the lungs were deflated, the tracheal smooth muscle contracted and continuous electrical activity appeared. During delivery of a PEEP, the tracheal smooth muscle relaxed and the slow-wave potentials were suppressed. When PEEP was discontinued, the tracheal smooth muscle again contracted and the slow waves soon resumed. Effects of bilateral vugotomy. Ligation of the vagus nerves transiently enhanced the spontaneous slow-wave potential. Bilateral transection of the vagus nerves eliminated both the spontaneous oscillatory waves and the tracheal muscle tone (Fig. 4). A two- or threefold increase or decrease in tidal volume produced no electrical activity. In addition, cessation of mechanical ventilation did not induce a reflex contraction of the tracheal smooth muscle. Effect of vagus nerve stimulation. Stimulation of the distal end of the transected vagus nerve with train square pulses (10 s) evoked a phasic contraction of the tracheal smooth muscle in all dogs (n = 33). The slow-wave potentials were similar in morphology to the spontaneous potentials and were invariably associated with a phasic muscle contraction (Fig. 5). Immediately after the onset of the electrical stimulation, an oscillatory electrical potential developed. This activity preceded the onset of tracheal muscle contraction by -0.8 ms. After the initial two or three large-amplitude slow waves, the amplitude and frequency of the evoked slow waves decreased gradually. In 17 of the 33 animals, the evoked slow waves dis-
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138
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ACTIVITY
200mmHg
BP contract
Etrc
2OPV 01
m
COLlAPSE
I-------I 1NFLATION
30 set
FIG. 3. Effects of lung inflation and deflation on tracheal smooth muscle. Traces are BP, force developed by tracheal smooth muscle (Ftr), electrical activity of rostra1 and caudal tracheal smooth muscle (Etrr and Etrc, respectively). Electrical activities are recorded from 2 parts of tracheal smooth muscle -1.5 cm apart. Lungs were periodically inflated while mechanical ventilation was discontinued. Record from a group 11 dog.
appeared at peak muscle contraction. Another phase of oscillatory activity appeared in the relaxation phase in 16 of 33 of the dogs. The amplitude of this late activity tended to be lower than that of the early oscillatory waves. Figure 6 shows the changes in oscillatory waves and tracheal muscle contraction associated with different stimulus intensities. The amplitudes of the oscillatory waves were three or four times greater in monopolar than in bipolar recordings. The threshold for the development of the electrical activity was - 1.0 V, which is similar to the threshold for the development of a contractile force (n = 7). Further increase in stimulus intensity produced correspondingly greater peak amplitudes and increased phasic contractions. The frequency of the oscillatory
waves also tended to increase with increased stimulus intensity. Figure 7 shows the relationship between stimulus intensity and contractile force when the distal end of the transected right vagus nerve was stimulated (n = 7). The contractile force was measured as the difference between the peak of a phasic contraction and the force immediately before the stimulation, i.e., an active tension (Z3), and then expressed as percentage of the observed con-
2OOmg
BP
clI-centract
t Ptr
b
2ossc
FIG.
4. A record before (k#) and after (right) vagotomy.
FIG. 5. After bilateral vagotomy, distal end of right vagus was stimulated with pulse train (frequency 30 Hz, duration 0.5 ms or 10 s). Traces are BP, Ftr, and raw (REtr) and filtered (Etr) electrical activity of the tracheal smooth muscle. Vagal stimulation with 20-V intensity developed a phasic contraction of tracheal smooth muscle and an oscillatory electrical activity. Stimulation of cardiac branch of vagus nerve caused bradycardia and consequent hypotension.
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139
ACTIVITY
2oornlnH~
BP 0
I
so9 Ftr
*
.
, 1ov
2.5 u
l.OU
3OY
10 SW FIG. 6. Experimental condition is similar to that in Fig. 5, but recordings are done with a monopolar Right vagus nerve was stimulated with 30-Hz pulse train.
traction to the contraction stimulated at 30 V. When comparing the force-stimulation curve generated by the air-filled balloon method with that generated by the isometric force transducer, we noted no significant difference. The shapes of the curves achieved using both methods were similar, and maximum contraction was attained at 30 V for both force-measuring methods. Accordingly, we regarded the results of the two methods as equivalent. A constant level of tracheal contraction was maintained by applying a continuous low-frequency (l-6 Hz) stimulus to the vagus nerve (20). During this contraction, oscillatory electrical activity developed (Fig. 8). In Fig. 8, the amplitude of the slow wave fluctuated during continuous stimulation. The slow-wave activity oscillated or gradually faded when smooth muscle tone was sustained by vagal stimulation. The excitatory effect of vagal nerve stimulation was blocked by the administration of intravenous atropine sulfate (0.5 mg). %
lUO-
l
4
&
4
I I
1
SO-
I 1
0’
11 2
1
5
8
I
10 20 5UV FIG. 7. Stimulus intensity (log voltage, Ordinate) vs. active force (%force, abscissa; it = 7). Distal end of the transected right vagus nerve was stimulated with train pulses (30 Hz, 0.5 ms, 10 s). Bars, SE.
electrode.
Effect of methacholine. Intratracheal administration of methacholine (50 mg) caused marked tracheal constriction and slow-wave potentials in five of seven dogs from both groups (Fig. 9). The amplitude and frequency of electrical activity were similar to the activity observed spontaneously in an intact animal. The amplitudes were greatest at the onset of airway contraction. They remained at a lower level for several minutes and finally faded away, even in the presence of sustained tracheal contraction. Resection of the esophagus. After resection of the cervical esophagus, the spontaneous electrical activity still developed in the two animals examined. When the right vagus nerve was stimulated with a pulse train of supramaximal intensity, the tracheal smooth muscle cont ratted and a slow-wave potential similar to those seen with an intact esophagus developed (Fig. 10). DISCUSSION
Electrical activity of the tracheal smooth muscle. The major findings in the present investigation demonstrate the capability of in vivo recording of electrical activity from canine tracheal smooth muscle and the slow-wave characteristics of the recorded electrical activity. In 1975, Akasaka et al. (I) reported in vivo recordings of electrical activity in dogs. In their report, the electrical activity was comprised of action potentials, and the discharge pattern had no relationship to respiratory phase. It remains to be investigated whether slow-wave potentials or action potentials are more reliable for the demonstration of in vivo electrical activity of the canine tracheal smooth muscle. As described below, most of the recent in vitro studies suggest that electrical activity of airway smooth muscle is comprised of slow-wave potentials. Canine tracheal smooth muscle has been described as an intermediate type of single-unit and multiunit groups according to Bozler’s categorization (3). The muscle has scarce innervation (11, 22, 27), gap junctions are rare (27), and there are no action potentials. Some investiga-
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140
TRACHEAL
ELECTRICAL
REtr
ACTIVITY
A’SAif t
FIG. 8. After bilateral vagotomy, distal end of right vagus was continuously stimulated with pulse train (6 Hz, 0.5 ms, 5 V).
200mmHg
ZOOcmH20
Ptr
MCh
injection
BP
0
1°r Ftr 0 2cwv
Etr 0
insp.
2Osec. I I FIG. 9. Intratracheal administration of 50 mg methacholine (MCh) evoked oscillatory electrical activity and tracheal contraction. Ptr, Etr, and Flow are as in Fig. 2.
tors have reported that canine tracheal smooth muscle has a steady membrane potential in Krebs solution (7, 27). An increase in membrane potential produces tracheal contraction, but such polarization never evokes oscillatory wave potentials (7). On the other hand, some investigators using intracellular electrodes (2, 12) have recorded slow oscillatory membrane potentials. Our findings suggest that the slow oscillatory waves we recorded originate directly from the tracheal smooth muscle. The frequency of the slow waves obtained from group I (trachea opened) and from group II (trachea closed) animals was not significantly different. This finding strongly suggests that the characteristics of the slow wave were independent of the mechanical condition of the tracheal smooth muscle. The experiment utilizing a proximal esophagectomy further reinforces our consideration. The augmentation of the slow-wave activity by lung deflation or vagus nerve stimulation was followed by the contraction of the tracheal smooth muscle. Moreover, bilateral transection of the vagus nerves not only abolished the spontaneous slow-wave activity but also decreased the resting tone of the tracheal smooth muscle. These findings suggest that both the slow oscillatory wave and the contraction of tracheal smooth muscle are
10s
10. After dissection of cervical esophagus, right vagus was stimulated with 30-Hz 30-V pulse train. FIG.
nerve
controlled by vagal efferents. The possibility that mechanical intervention induced slow-wave potentials was disproved by withdrawal of ventilatory support from the paralyzed dog and observation of an enhancement of the slow-wave potentials. The onset of evoked slow waves produced by vagal stimulation always preceded the onset of the tracheal muscle contraction. Finally, the characteristics of the slow-wave potential of the dog trachea were similar to those reported in other animals both in vivo and in vitro. For example, the mean maximum frequency of the slow wave in our study was 0.57 t 0.13 Hz; this was within the range of the wave frequency of other studies on several species, i.e., 0.5-1.4 Hz (2, 10, 12, 14, 21). Spontaneous electrical activity was recorded in 40% of the animals; in some dogs, slow-wave potentials developed in phase with the rhythm of the tracheal phasic contractions. This finding, however, does not necessarily indicate an absolute requirement of electrical activity for contraction of the tracheal muscle because the tracheal smooth muscle maintains a resting tension without developing electrical activity. Phasic contraction of tracheal smooth muscle has been reported in previous literature (15, 26). In our study the phasic contraction of tracheal smooth muscle correlated with the phasic change in mean arterial blood pressure as seen in Fig. 2, and both
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TRACHEAL
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changes were abolished by vagotomy. Presumably, these findings suggest that both rhythms arose from the same central architecture. Sullivan et al. (26) observed phasic contraction only during sleep associated with rapid eye movement in the intact dog. Thus phasic contraction might not occur under normal conditions. Because our animals underwent major surgery and because insertion of the needle electrodes to the tissue might release inflammatory mediators, the electrical activity observed in this study could have been evoked by experimental intervention. If such intervention has occurred, however, the changes in electrical activity seen with changes in the experimental conditions suggest that experimental intervention should bias only the provocation of the electrical activity. We speculate that augmentation of electrical activity during the contraction phase must be under the command of the vagal efferents because vagotomy eliminated both phasic contractions and electrical activity. Vagus nerve stimulation or atelectasis evoked slow-wave potentials in all the animals studied. With the intact peritracheal innervation, a tonic release of acetylcholine was maintained at the motor endplate. Presumably, under these conditions, electrical activity could be easily evoked in the tracheal smooth muscle. Role of the vagus nerve. It has been suggested that the contraction and relaxation of tracheal smooth muscle induced by lung deflation and inflation are regulated by a reflex arising from pulmonary mechanoreceptors (17,29, 30). In our study the slow-wave activity developed immediately after cessation of ventilatory support; conversely, the activity was suppressed when the lungs were inflated with a bolus of air. These findings indicate that development of the oscillatory electrical activity is mediated via a vagal reflex arising from pulmonary mechanoreceptars. The reduction of slow-wave potentials with the application of PEEP further reinforces the reflex hypothesis. Hypercapnia may also play some role in the generation of slow waves and associated smooth muscle contraction. Reports in the literature have shown that hypercapnia increases the contraction of tracheal smooth muscle of anesthetized cats (17, 25) and dogs (19). We found that the oscillatory waves persisted for several seconds after a period of transient apnea, suggesting that apnea-induced hypercapnia may influence slow-wave formation and tracheal smooth muscle tone. Abolition of the oscillatory wave and the resting tension of tracheal smooth muscle by transection of the vagus nerves is consistent with the knowledge that the tracheal smooth muscle maintains a resting tension by continuous vagal efferent tone (20). The oscillatory wave was always followed by a contraction of the tracheal smooth muscle. However, in 58% of the experimental animals, the tracheal smooth muscle maintained its resting tension even if the oscillatory electrical waves were not observed. This finding suggests that oscillatory electrical activity is not necessary for the maintenance of a resting tension in tracheal smooth muscle, Electrical stimulation of the distal end of the vagus nerve consistently generated oscillatory potentials and phasic contractions. This indicates that the synchronized excitation of tracheal smooth muscle by the vagal release of acetylcholine significantly influences the gener-
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ation of slow oscillatory potentials. The force developed by the tracheal smooth muscle increased concomitantly with increases in stimulus intensity and frequency. Kannan et al. (12) have reported that acetylcholine provokes oscillatory activity and spontaneous contraction in isolated canine tracheal smooth muscle. They speculated that the changes in conductance underlying these oscillations represent the periodic opening of Ca2+ channels by acetylcholine (8). Induction of oscillatory potentials by the intratracheal administration of methacholine and total inhibition of stimulus-evoked potential by atropine support the Ca2+ channel hypothesis. Physiologicul significance of the electrical activity. Dog tracheal smooth muscle has been found to contract without oscillatory membrane potentials. This phenomenon has been noted in the late phase of continuous stimulation and in the case of intratracheal administration of methacholine. The absence of oscillatory potentials in the face of muscle contraction supports a hypothesis that voltage-dependent Ca2+ channels and decreases in membrane potential are not primarily involved in generating acetylcholine-induced contraction in the canine tracheal muscle (5). Electromechanical coupling is reported to be involved in acetylcholine-induced contraction of the ferret tracheal smooth muscle (14, 18). However, the maximum contribution of the electromechanical coupling is only -30% in acetylcholine-induced contraction of the dog’s tracheal smooth muscle, and this mechanism occurs only when the concentration of acetylcholine is >10-16 M in the organ bath (9). It has been reported, in isolated tracheal smooth muscle of ferret (14) and dog (12), that electrical oscillation of the membrane potential occurred with depolarization by the addition of acetylcholine to the organ bath solution. From these reports we may speculate that development of a slow wave reflects a decrease in membrane potential of the smooth muscle cell. In conclusion, we have shown that the electrical activity of slow oscillatory waves can be recorded in vivo from canine tracheal smooth muscle. The development of slow waves is invariably associated with tracheal contraction, and both the slow waves and tracheal contraction are mediated via vagal efferents. Tracheal contraction, however, may occur without slow waves, The development of the slow wave may indicate a decrease in membrane potential of the tracheal smooth muscle. This study was partly supported by Tokai University School of Medicine Research Aid. Address for reprint requests: T. Kondo, Div. of Pulmonology, Dept. of Medicine, School of Medicine, Tokai University, Isehara, Kanagawa 25941, Japan. Received 29 May 1990; accepted in final form 14 August 1991. REFERENCES AKASAKA, K.,K. KoNNo,Y.ONO,S. MUE, CABE, M. KUMAGAI, AND T. ISE. A new electrode for electromyographic study of bronchial smooth muscle. Tohoku J. hp. Med. 117: 49-54, 1975. BOSE, R., AND D. BOSE. Excitation-contraction coupling in multiunit tracheal smooth muscle during metabolic depletion: induction of rhythmicity. Am. J. Physiol. 233 (Cell Physiol. 2): C8-C13, 1977. BOZLER,
E. Action potential and conduction of excitation in muscle. Biol. Symp. 3: 95-109, 1940.
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142 4.
5. 6. 7.
8.
9.
10. 11. 12.
13. 14.
15.
16. 17.
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