Excitation-contraction coupling in multiunit tracheal smooth muscle during metabolic depletion: induction of rhythmicity RATNA
BOSE AND DEEPAK
Department Winnipeg,
BOSE
of Pharmacology and Therapeutics, Manitoba, Canada R3E OW3
BOSE, RATNA, AND DEEPAK BOSE. Excitation-contraction coupling in multiunit tracheal smooth muscle during metaboZic depletion: induction of rhythmicity. Am. J. Physiol.
233(l): CS-C13, 1977or Am. J. Physiol.: Cell Physiol. Z(1): CS-C13, 1977.- Multiunit canine tracheal smooth muscle responded to carbachol with graded depolarization and tonic contraction. The sameconcentration of carbachol, &er metabolic depletion by substrate removal, producedrhythmic contractions and action potentials. Similar mechanical effects were also observed with acetylcholine or histamine. These effects were reversed by reintroducing glucoseor p-hydroxybutyrate, but not by 3-0-methylglucose, which is not metabolized; hence,the structural requirements for glucose,per se, or any osmotic effect were ruled out. Sensitivity to extracellular Ca2+was increased. A Ca2+-influx blocker, D-600, in low concentration (2 x 10e8M) abolished the rhythmic contractions without affecting the tonic contraction. Progressivemetabolic depletion in presenceof carbachol led to fluctuations in membrane potential with a crest of depolarization and appearance of action potentials, each of which resulted in a small contraction. Many of the small contractions partially fusedto form the major rhythmic contractions which appearedat a frequency of oneper minute. Rhythmicity could not be producedby increasing extracellular K+ concentration (20-120mM) in presenceof atropine (10m7M), but instead a tonic contraction occurred. These results suggest changesin excitation-contraction coupling mechanismwith agonists like acetylcholine, carbachol, or histamine during substrate deprivation. trachealis; histamine; glucose-freemedium
COUPLING is the sequence of events which, after an effective stimulus, results in shortening and/or tension development in the muscle. The detailed mechanism of excitation-contraction coupling in smooth muscle and how it is linked to cell metabolism remains to be determined. Bozler (6) has classified smooth muscle into multiunit nonpropagating types and single-unit, rhythmic muscles where cell-tocell propagation exists. The former respond with graded de$larization to agonists, while the latter exhibit spike electrogenesis. Somlyo and Somlyo (22, 23) have suggested the probable existence of a continuous spectrum of fibers with tonic and phasic activity. Rhythmic mechanical activity can be induced in certain multiunit muscles under special circumstances by agonists that stimulate the muscle directly (3,14), or by EXCITATION-CONTRACTION
Faculty
of Medicine,
University
of Manitoba,
treatment with pharmacological agents such as TEA (tetraethylammonium) (15, 17, 19), or by denervation (5). While st u d ying the biochemical changes associated with metabolic depletion of canine trachealis muscle, oscillatory behavior was observed (4). The present study was undertaken to establish if there was a link between the metabolic status and the single-unit type behavior of the multiunit dog trachealis. There are several reports of “peristaltic-like waves” in vivo, in trachea and bronchi of dogs (7,21,18). A cholinergic motor-control mechanism has been attributed to these (18). We report here the reversible induction of rhythmic contractions associated with electrical spikes on removal of glucose in a muscle which normally responds to exogenous chemical stimuli with graded depolarization and tonic contraction. METHODS
Trachea were obtained from 7- to 20.kg dogs which were anesthetized with 35 mg/kg pentobarbital. The tracheal m .uscle was di ssected out from the dorsal surface of the trachea and equilibrated in Krebs-Henseleit solution (in mM: NaCI-118, KC1 4.7, KH,PO, 1.4, NaHCO, 25, MgS04 1.2, CaCl, 2.5, glucose 11) at 37”C, during which period it was stimulated with a programmable stimulator which delivered 60-Hz sinusoidal pulses for 12 s at intervals of 5 min. The stimuli were delivered through platinum-plate electrodes placed parallel to the muscle strip. The muscle was stretched to optimum length (I+,, the length at which maximum active tension developed in response to electrical stimulation), and then exposed to 2 x low7 M carbachol in the presence or absence of glucose in the physiological solution. After 2-4 h in glucose-free solution, the carbacholtreated muscle strips exhibited stable regular rhythmic contractions which continued for several hours. Both continuous flow and fixed volume (10 ml) techniques have been used. In fixed volume experiments, bathing solution was changed after 60 min, if not earlier, or 0.5 mM glucose added every 90 min to ensure adequate supply of substrate. Paired strips were left in glucose containing Krebs-Henseleit solution for the same length of time. Isometric tension was recorded with a FT-03 transducer. Floating glass microelectrodes with a suspending con-
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ductor (courtesy of Drs. E. A. Kroeger and J. M. Marshall) were used for recording intracellular electrical activity. The microelectrodes were drawn from glass capillary tubes (1 mm OD, 0.5 mm ID) prefilled with glass fibers (Omegadot, Frederick Haer). The tip was filled with 2.7 M KC1 by capillary action and the rest back-filled with a syringe. Electrodes of 25-50 M resistance were used. Both electrical and mechanical activities were recorded on a Hewlett Packard 141 B oscilloscope as well as on a Brush Mark 280 recorder, through a Grass P-16 or Transidyne neuroprobe preamplifier. Sucrose-gap technique was also used for continuous recording of the membrane potential changes. Channels were made in a Perspex block so that a tracheal muscle strip was covered by free-flowing sucrose and Krebs solutions (1.8 x 20 mM). The solutions in the channel were left at 34OC. Muscle strips were equilibrated with and without glucose Krebs solution and tested with 2 x low7 M carbachol to see if a stable rhythmicity could be observed, at which time the muscle strips were quickly frozen in liquid nitrogen for ATP and CP estimations. Adenosine triphosphate (ATP) and creatine phosphate (CP) were measured by a coupled enzyme assay described previously (4). Data were analyzed by oneway classification analysis of variance. Multiple comparisons were made with Duncan’s new multiple-range test.
DEPLETION
c9
LKCL
4
0
80
mM
[J
OG
v
‘“ISTAMINE
OG
IO-‘M
+G L ‘ACETYLCHOLINE
lO+M
OG
FIG. 2. Isometric tension trace of a pair of TSM strips, one of which was in glucose-free medium (OG) up until stable rhythmicity appeared with carbachol and other remained in glucose-containing medium (+ G). After removing carbachol, various agonists (KCl, histamine, acetylcholine) were added separately, as indicated, to same pair of strips.
RESULTS
Effect of glucose removal on mechanical activity. Tracheal muscle, like other multiunit muscles, responds with a tonic contraction when exposed to 80 mM K+, acetylcholine, carbachol, or histamine, and this contraction is maintained as long as there is glucose and O2 in the medium (Fig. 1). However, when glucose was absent, the carbachol-stimulated strips went through a period of irregular contractions, which became regular at a frequency of 1.11 t 0.34 per minute (mean t SE) (n = 65) and lasted for l-5 h. At this stage the removal of carbachol and the addition of either histamine (low5 M) or acetylcholine (lo+ M) also produced rhythmic contractions. Addition of lo-120 mM K+ did not produce rhythmic contractions in presence of atropine (10B7 M). Instead, a tonic contraction was observed (Fig. 2). The rhythmically contracting trachea responded with a myogenic response when stretched by 1820% of I+, (Fig. 3).
FIG. 1. Isometric tension trace of a pair of tracheal smooth muscle (TSM) strips. After a l-h equilibration in normal Krebs-Henseleit solution at 37”C, carbachol (2 x 10m7M) was added as shown. Top trace in glucose-free medium (OG) and bottom trace in 11 mM glucose containing (+G) medium. Numbers between panels denote time intervals (hours). Both solutions were gassed with 95% oxygen and 5% carbon dioxide.
C
FIG. 3. Effect of quick stretch (18% of I+,) applied at (-) to a TSM strip which was A, rhythmically contracting in presence of 2 x 10B7 M carbachol in glucose-free medium; B , tonically contracted by 2 x low7 M carbachol in presence of glucose; C, relaxed in absence of glucose; and D , relaxed in presence of glucose. Myogenic response was seen only in A.
Myogenic response has been shown to occur only in single-unit muscles (8). In order to establish if these changes are due to alterations in the ATP concentration, addition of substrate and/or changing to conditions which are expected to alter the concentration of cellular ATP should change the nature of contraction. As shown in Fig. 4, a nonmetabolizable form of sugar, 3-O-methylglucose (10 mM), did not alter the contractions, whereas addition of phydroxybutyrate (10 mM), a substrate which would lead to the production of ATP, only through the mitochondria, abolished rhythmicity. Similarly, glucose (10 mM) rapidly increased tension and abolished phasic mechan-
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Cl0
R. BOSE AND 0. BOSE
ical activity. Anoxia produced with 95% Nz and 5% CO, after the addition of glucose was unable to maintain the tonic contraction, and oscillation reappeared. Reintroduction of oxygen again abolished rhythmicity. Since there are only two known sources of ATP in the cell, glycolysis and mitochondrial oxidative phosphorylation, and since P-hydroxybutyrate oxidation, but not glycolysis alone, was able to support the tonic contraction, it appears that mitochondrial ATP was required to sustain a tonic contraction produced by histamine or acetylcholine. Rhythmicity was also observed under anoxic or hypoxic (Paz < 100 mm Hg) conditions in muscles which had never been deprived of substrate, in presence of carbachol (lo+ M), but the oscillations were slower (0.53 per minute; n = 7) (Fig. 4, lower and last part of upper traces). Rhythmicity could not be observed in the absence of an agonist. The muscle strip could be repeatedly washed and exposed to carbachol (10e7 M), and stable rhythmicity could be observed each time. Atropine (10D7 M) abolished the rhythmicity induced by carbachol, but not that due to histamine. The possibility that a significant component of the effects of carbachol and other agonists, under condition of glucose deprivation, was by release of neurotransmitters from neural elements in the tissue seems unlikely since rhythmicity was not affected by the addition of either 3 x low6 M tetrodotoxin (which can abolish nerve action potential without affecting smooth muscle), 10m7M sotalol, a P-adrenergic receptor blocker, and 2 x 10m6M phentolamine, an a-adrenergic receptor blocker. Tissue ATP concentration and effect of extracellular ATP. In order to confirm that tissue levels of ATP and creatine phosphate (CP) decreased in the absence of glucose, we have measured the ATP and CP concentrations. As shown in Fig. 5, the ATP concentration decreased in the glucose-free medium from 1.1 to 0.5 mM, 2 min
4. Isometric tension trace of TSM strips in presence of 2 x M carbachol. Top trace: a nonmetabolizable sugar, 34-methylglucose (MeGI, 10 mM), added as indicated to glucose-free medium, and then 10 mM glucose was added at Gl. Asker a stable tonic response in presence of glucose, anoxia was produced with a 95% N, and 5% CO, mixture. Middle trace: 10 mM P-hydroxybutyrate (BOHB) was added to a tracheal strip which was rhythmically contracting with carbachol in absence of glucose. Bottom trace: anoxic TSM strip made to contract rhythmically with carbachol in presence of glucose. 95% OZ, 5% CO, introduced at 0,. FIG.
10e7
1.0 mM 0.5
0
+G Ic
OG Ic. ATP
OG
+G Ic
Ic CP
FIG. 5. ATP and creatine phosphate (CP) concentrations in tracheal strips in normal (+G) or glucose-free (OG) solution. One group from each contained 2 x low7 M carbachol (C). Vertical lines denote standard error values and horizontal bars denote a statistically significant difference (P < 0.05), as determined by Duncan’s new multiple-range test. n = 4-8 in each case.
and CP concentration decreased from 1.4 to 0.38 mM. In the presence of carbachol, the reduction in ATP concentration was greater (reduced from 1.2 to 0.29 mM). Since ATP has been shown to enter the soleus muscle (9), we decided to see if extracellular ATP had any effect on glucose-deprived muscles. Figure 6 shows the effects of various adenine nucleotides and nucleosides on the carbachol contractions. One interpretation of these results is that the return of tonic contraction is due to the ribose moiety in the nucleoside, which probably breaks down to form ribose l-phosphate, and with the help of hexose monophosphate shunt and glycolytic enzymes, provides fuel to Krebs cycle, and then produces ATP. Hence, ATP, ADP, AMP, adenine, inosine, but not adenine and sodium pyrophosphate, were able to produce tonic contractions (in 8 of 11 strips) following various degrees of relaxations. Iodoacetate (1 mM), known to inhibit glycolysis, blocked the tonic contraction with ATP and other nucleosides (seen in the absence of glucose) without affecting the relaxant action. Adenine group of the ATP appears to be responsible for the relaxant action at these concentrations (2 mM). The fact that K+ (80-120 mM) produced a sustained contraction suggested that ATP concentration was not varying in the vicinity of the contractile proteins. The possibility of K+ causing greater influx of Ca was ruled out by the use of a small amount of K+ (20 mM) in the presence of atropine (10e7 M), when a tonic contraction was also observed. These observations suggest that membrane properties were changed under conditions of metabolic depletion leading to alterations in excitationcontraction coupling. Effect of glucose removal on electrical activity. Intracellular electrical recording (Fig. 7A) showed a burst of spikes associated with each rhythmic contraction, which appears as partially fused tetanus (Fig. 8A) in glucose-free medium with carbachol. Oscillations in membrane potential, resulting in very slight oscillations of the tonic contraction, appeared long before the rhythmicity was observed (Fig. 7B). The maximum amplitude of spikes observed with rhythmic contractions were 20 mV and the average duration 0.75 t 0.29 s amplitude. On (mean t SE; n = 11) at half maximum returning glucose (10 mM), the membrane potential decreased (Fig. 9) and the membrane went through a
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Cl1
AMP
t ADENINE
INOSINE
4 IODOACETATE
10 0[
‘\
-
iINOSINE
4
Na Pi-Pi
,
4 min.
OL
#
b
2 min.
FIG. 6. Isometric tension trace with 2 x lo-’ M carbachol of a pair of TSM strips, one of which was in glucose-free medium (lower one of each pair) and other in glucose-containing medium; 2 mM of test
20
r
A
B
compounds (except iodoacetate, cated. A 10mV
1 mM) added to medium as indi-
I 10 sec.
FIG. 7. A: intracellular microelectrodes record (top) and tension (bottom) traces of a TSM strip made rhythmically active in glucosefree medium with lo-’ M carbachol present. B: intracellular microelectrode record showing oscillations with slight changes in tension of a tonically contracted (12 g) TSM strip after 30 min in glucose-free medium with carbachol present. Time scale five times faster than in A.
series of fast oscillations which decreased and disappeared. Sucrose-gap technique yielded similar results when a single gap was used (Fig. 8). On starting the second gap, the rhythmicity disappeared. As shown in Fig. 8A, each action potential resulted in a small contraction, many of which fused to form a big contraction. The membrane depolarized 7-15 mV before the oscillations in membrane potential were seen. In contrast, in presence of glucose, both membrane potential and tension were very stable following the initial graded depolarization [Fig. 8B and C) and oscillations, when observed, never exceeded 3 mV in amplitude. Effect of extracellular calcium (Ca). Since Ca2+ has a central role in excitation-contraction coupling, the effects of Ca addition, deprivation, and addition of D-600, a compound which has been shown to block the Ca current in cardiac muscles, were studied. Figure 10
C 1OmV
c mv&
+G
FIG. 8. A: electrical (top) and tension (bottom) traces obtained from a typical single sucrose-gap experiment with a TSM strip made to contract rhythmically in glucose-free medium (OG) with 10v7 M carbachol. B: electrical (top) and tension (bottom) traces obtained from a typical single sucrose-gap experiment. Sucrose solution (8%) had 10 mM glucose, and Krebs- Henseleit solution contained glucose and 10m7M carbachol. Initial graded depolarization and contraction with introduction of carbachol. C: stable membrane potential and tension seen after 2 h in carbachol.
shows that D-600 at lo-* M concentration blocked the rhythmicity due to carbachol of the strips in glucose-free medium without appreciably affecting the tonic contraction of the paired strip in glucose-containing medium. Higher concentrations (10-5 M) of D-600 relaxed the tonically contracted muscle. At this higher concentration, both rhythmicity and tone were abolished in tra-
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R. BOSE AND D. BOSE
the Ca2+ increased above 2.5 mM. This effect of high Ca may be due to membrane stabilization and needs to be investigated in greater detail. These observations also suggest differences in excitation-contraction coupling under conditions of metabolic deprivation. DISCUSSION FIG. 9. Intracellular electrical record (top) and isometric tension trace showing effect of adding 10 mM glucose (Gl) to a rhythmically contracting TSM strip. 4 D-600 i
5 0I &I
21clO-~M 2 mine
5 Z :
I
5 c
O
5 0
I
FIG. 10. Effect of D-600, added as indicated to TSM strips which were tonically contracted with carbachol (2 x 10s7 M) in presence of glucose (top and bottom trace) or in absence of glucose (middle trace).
A
5
6
9z if 10
5
I
II
VCa 1mM
2 min.
11. A: effect of cumulatively adding 0.5 mM EGTA (pH 7.0) at various points indicated (A) to TSM strips made to contract with carbachol (2 x 100~ M) in presence of glucose (+G) or in absence of glucose (OG). B: effect of cumulatively adding 1 mM Ca at various points indicated (v) to medium containing 2.5 mM Ca. FIG.
cheal strips in glucose-free solution. This further suggested that alterations in Ca movement had occurred under conditions of ATP depletion. Removing extracellular Ca in the medium completely abolished rhythmicity, whereas it decreased the tonic contraction only by 35%. Figure 1lA shows the effects of EGTA on tonic and rhythmic contractions and, here again, the rhythmic strips show greater sensitivity to Ca removal. Similarly, differences in the responses of the tonic and rhythmic contractions were observed when Ca was increased above 2.5 mM already present in the Krebs solution. Figure 1lB shows that increase in Ca, up to a concentration of 5 mM, increased the amplitude of contractions above which it did not have any effect. Very high concentration of Ca (9 mM) caused desynchronization of the contractions. In contrast, the tonically contracted tracheal strip in glucose relaxed as
In smooth muscle, the exact nature of excitationcontraction coupling and how it is affected by metabolic depletion is far from understood. Trachealis muscle is an electrically quiescent muscle, responding with graded and sustained depolarization in response to exogenous acetylcholine or histamine. Rabbit carotid artery and sheep carotid artery are also normally electrically quiescent but show action potentials when exposed to TEA (19) or cyanide (14), respectively. We have been able to produce rhythmicity in canine trachealis muscle by metabolic depletion as well as by parasympathetic denervation (unpublished observation) or TEA. The denervated trachealis also required carbachol for initiating rhythmic contractions. Whether a decreased level of ATP may be at least partly responsible for the production of rhythmicity in the latter cases (TEA and denervation) remains to be seen. Denervation of vas deferens leading to decreased level of ATP has been reported earlier (25). In a normally single-unit muscle (taenia coli), glucose was shown to function as a brake on the frequency of its spontaneous discharge, which persisted for hours after the mechanical response was abolished by glucose removal (1). Mechanisms by which tonic contraction is sustained only when ATP produced by mitochondria is available, but not with glycolysis alone, are not understood. This process of ATP production may be intimately coupled to other membrane processes. Why the mitochondria (or its ATP) are required for the tonic contraction with histamine, acetylcholine, and carbachol, and not to the same extent by K-induced tonic contraction, is not understood. Oscillation in heart mitochondria (at one per minute) has been demonstrated in vitro by measuring changes in transmittance, H+, K+, respiration rate, and morphology (11). ADP was found to have an important role in these oscillations. Gradmann and Slayman (12) have reported on an oscillating electrogenic pump in the poky mutants of Neurospora crassa, which have defective mitochondria. Rhythmicity in the presence of glucose and anoxia was much slower in frequency and amplitude and sometimes (6 of 13) not observed in fresh muscles. It is possible that, in these instances, the ATP concentration was insufficiently low to permit oscillation. However, even under these conditions, the tonic contraction was consistently reduced to 15.8 t 5% (n = S), whereas under similar conditions, the contraction with 80 mM KC1 decreased only to 63.9 t 6% (n = 8). ATP concentrations under hypoxic or anoxic conditions have not been determined in the tracheal muscle. NADH and NADPH concentrations under glucose-free normoxic conditions are expected to be low compared to anoxic conditions, and if the difference in rhythmicity under these two conditions is in any way related to this factor (ratio of reduced to oxidized form of nucleotides), it remains to be seen. Another factor for decreased
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rhythmicity in anoxic muscles could be the intracellular pH which is expected to decrease with lactate production. Acidosis has been shown to block Ca2+ influx (24). The nonmetabolizable analogue of glucose did not affect rhythmicity; therefore, structural requirement for glucose per se did not exist for the tonic contraction. This also rules out any osmotic effects due to glucose lack. Decreased ATP levels could alter the membrane characteristic by altering the Ca2+ bound to the membrane, thereby affecting Na conductance and directly increasing excitability (2). Histamine, under normoxic conditions with glucose present, did not produce slow waves or spikes but, on metabolically depleting the muscle, oscillations both in membrane potential and contractions were observed. Transmembrane flux of Ca2+ was found to be important in studies with rabbit aorta when the muscle was depolarized with K+ or when metabolically depleted (24). Our results also show similar changes in Ca sensitivity with metabolically depleted strips depending more on external Ca2+. It is possible that some internal store of Ca2+, which is normally used for maintaining a tonic contraction with carbachol, is depleted and unavailable for contraction. Whether this source is mitochondria, sarcoplasmic reticulum, or plasma membrane remains to be determined. Electron microscopic examination shows the presence of nexuses and also other types of junctions (R. Bose, unpublished observation) which may play a role in the conduction of impulses (10, 13, 23) and which may be affected by metabolic depletion. Demonstration of ac-
Cl3
DEPLETION
tion potentials across a single 8-mm sucrose gap shows that conduction is occurring in these muscles. However, since with double-sucrose gap rhythmicity disappeared, it is possible that enough pacemakers are not present, and carbachol produced the pacemakers which are masked under normal conditions and unmasked with glucose deprivation. It has been suggested that stable membranes may be labilized to produce action potentials (22). Our data support this view. The pulsatile mechanical activity during hypoxia may greatly aid the distribution of ventilation to the alveoli. To summarize our results, glucose deprivation leads to changes in electrical and mechanical properties of the tracheal muscle strips which normally show a multiunit behavior but, under conditions of metabolic deprivation, show a single-unit behavior. Sensitivity to extracellular Ca2+ is changed with decreased ATP levels and, hence, the study of mechanisms by which ATP alters the electrical properties and fluxes of ions becomes necessary; however, it is possible that processes associated with ATP synthesis, rather than the ATP molecule itself, may be responsible for the changes in excitation-contraction coupling. We extend sincere thanks to Dr. H. Yamaguchi for his design and generous help with the sucrose-gap apparatus. Expert technical assistance of Mrs. T. Chau is greatly appreciated. This research was supported by a grant from the Medical Research Council of Canada (MA-4339) to D. Bose and a fellowship to R. Bose from the Canadian Heart Foundation. Received for publication
13
July 1976.
REFERENCES J., S. G. R. H&BERG, AND A. R. TIMMS. The effect of removing and readmitting glucose on the electrical and mechanical activity and glucose and glycogen content of intestinal smooth muscle from the taenia coli of the guinea pig. Acta
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5. BOSE, D., AND I. R. INNES. Induction of rhythmicity in normally quiescent smooth muscle. J. Phurm. PhurmacoZ. 26: 59-61, 1974. 6. BOZLER, E. Conduction, automaticity and tonus of visceral muscles. Experientiu 4: 213-218, 1948. 7. BIJLLOWA, J. G. M., AND C. GOTTLIEB. Roentgen-ray studies of bronchial function. Am. J. Med. Sci. 160: 98-107, 1920. 8. BURNSTOCK, G., AND C. L. PRESSER. Response of smooth muscles to quick stretch; relation of stretch to conduction. Am. J. PhysioZ. 198: 921-925, 1960. 9. CHAUDRY, I. H., AND 320-326, 1970. 10. GABELLA, G.
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C. T. Excitation and contraction in bovine tracheal smooth muscle. J. Physiol., London 244: 263-281, 1975. KNULL, H. R., AND D. BOSE. Reversibility of mechanical and biochemical changes in smooth muscle due to anoxia and substrate depletion. Am. J. PhysioZ. 229: 329-333, 1975. KROEGER, E. A., AND N. L. STEPHENS. Effect of tetraethylammonium on tonic airway smooth muscle: initiation of phasic electrical activity. Am. J. PhysioZ. 228: 633-636, 1975. LOOFBOURROW, G. N., W. B. WOOD, AND I. L. BAIRD. Tracheal constriction in the dog. Am. J. Physiol. 191: 411-415, 1957. MEKATA, F. Electrophysiological studies of the smooth muscle cell membrane of the rabbit common carotid artery. J. Gen.
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studies by lanthanum influx. CircuZation Res. 30: 44-54, 1972. 25. WESTFALL, D. P., R. I. F. LEE, AND R. E. STITZEL. Morphological and biochemical changes in supersensitive smooth muscle. Federation
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BOSE AND DEEPAK
Department Winnipeg,
BOSE
of Pharmacology and Therapeutics, Manitoba, Canada R3E OW3
BOSE, RATNA, AND DEEPAK BOSE. Excitation-contraction coupling in multiunit tracheal smooth muscle during metaboZic depletion: induction of rhythmicity. Am. J. Physiol.
233(l): CS-C13, 1977or Am. J. Physiol.: Cell Physiol. Z(1): CS-C13, 1977.- Multiunit canine tracheal smooth muscle responded to carbachol with graded depolarization and tonic contraction. The sameconcentration of carbachol, &er metabolic depletion by substrate removal, producedrhythmic contractions and action potentials. Similar mechanical effects were also observed with acetylcholine or histamine. These effects were reversed by reintroducing glucoseor p-hydroxybutyrate, but not by 3-0-methylglucose, which is not metabolized; hence,the structural requirements for glucose,per se, or any osmotic effect were ruled out. Sensitivity to extracellular Ca2+was increased. A Ca2+-influx blocker, D-600, in low concentration (2 x 10e8M) abolished the rhythmic contractions without affecting the tonic contraction. Progressivemetabolic depletion in presenceof carbachol led to fluctuations in membrane potential with a crest of depolarization and appearance of action potentials, each of which resulted in a small contraction. Many of the small contractions partially fusedto form the major rhythmic contractions which appearedat a frequency of oneper minute. Rhythmicity could not be producedby increasing extracellular K+ concentration (20-120mM) in presenceof atropine (10m7M), but instead a tonic contraction occurred. These results suggest changesin excitation-contraction coupling mechanismwith agonists like acetylcholine, carbachol, or histamine during substrate deprivation. trachealis; histamine; glucose-freemedium
COUPLING is the sequence of events which, after an effective stimulus, results in shortening and/or tension development in the muscle. The detailed mechanism of excitation-contraction coupling in smooth muscle and how it is linked to cell metabolism remains to be determined. Bozler (6) has classified smooth muscle into multiunit nonpropagating types and single-unit, rhythmic muscles where cell-tocell propagation exists. The former respond with graded de$larization to agonists, while the latter exhibit spike electrogenesis. Somlyo and Somlyo (22, 23) have suggested the probable existence of a continuous spectrum of fibers with tonic and phasic activity. Rhythmic mechanical activity can be induced in certain multiunit muscles under special circumstances by agonists that stimulate the muscle directly (3,14), or by EXCITATION-CONTRACTION
Faculty
of Medicine,
University
of Manitoba,
treatment with pharmacological agents such as TEA (tetraethylammonium) (15, 17, 19), or by denervation (5). While st u d ying the biochemical changes associated with metabolic depletion of canine trachealis muscle, oscillatory behavior was observed (4). The present study was undertaken to establish if there was a link between the metabolic status and the single-unit type behavior of the multiunit dog trachealis. There are several reports of “peristaltic-like waves” in vivo, in trachea and bronchi of dogs (7,21,18). A cholinergic motor-control mechanism has been attributed to these (18). We report here the reversible induction of rhythmic contractions associated with electrical spikes on removal of glucose in a muscle which normally responds to exogenous chemical stimuli with graded depolarization and tonic contraction. METHODS
Trachea were obtained from 7- to 20.kg dogs which were anesthetized with 35 mg/kg pentobarbital. The tracheal m .uscle was di ssected out from the dorsal surface of the trachea and equilibrated in Krebs-Henseleit solution (in mM: NaCI-118, KC1 4.7, KH,PO, 1.4, NaHCO, 25, MgS04 1.2, CaCl, 2.5, glucose 11) at 37”C, during which period it was stimulated with a programmable stimulator which delivered 60-Hz sinusoidal pulses for 12 s at intervals of 5 min. The stimuli were delivered through platinum-plate electrodes placed parallel to the muscle strip. The muscle was stretched to optimum length (I+,, the length at which maximum active tension developed in response to electrical stimulation), and then exposed to 2 x low7 M carbachol in the presence or absence of glucose in the physiological solution. After 2-4 h in glucose-free solution, the carbacholtreated muscle strips exhibited stable regular rhythmic contractions which continued for several hours. Both continuous flow and fixed volume (10 ml) techniques have been used. In fixed volume experiments, bathing solution was changed after 60 min, if not earlier, or 0.5 mM glucose added every 90 min to ensure adequate supply of substrate. Paired strips were left in glucose containing Krebs-Henseleit solution for the same length of time. Isometric tension was recorded with a FT-03 transducer. Floating glass microelectrodes with a suspending con-
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ductor (courtesy of Drs. E. A. Kroeger and J. M. Marshall) were used for recording intracellular electrical activity. The microelectrodes were drawn from glass capillary tubes (1 mm OD, 0.5 mm ID) prefilled with glass fibers (Omegadot, Frederick Haer). The tip was filled with 2.7 M KC1 by capillary action and the rest back-filled with a syringe. Electrodes of 25-50 M resistance were used. Both electrical and mechanical activities were recorded on a Hewlett Packard 141 B oscilloscope as well as on a Brush Mark 280 recorder, through a Grass P-16 or Transidyne neuroprobe preamplifier. Sucrose-gap technique was also used for continuous recording of the membrane potential changes. Channels were made in a Perspex block so that a tracheal muscle strip was covered by free-flowing sucrose and Krebs solutions (1.8 x 20 mM). The solutions in the channel were left at 34OC. Muscle strips were equilibrated with and without glucose Krebs solution and tested with 2 x low7 M carbachol to see if a stable rhythmicity could be observed, at which time the muscle strips were quickly frozen in liquid nitrogen for ATP and CP estimations. Adenosine triphosphate (ATP) and creatine phosphate (CP) were measured by a coupled enzyme assay described previously (4). Data were analyzed by oneway classification analysis of variance. Multiple comparisons were made with Duncan’s new multiple-range test.
DEPLETION
c9
LKCL
4
0
80
mM
[J
OG
v
‘“ISTAMINE
OG
IO-‘M
+G L ‘ACETYLCHOLINE
lO+M
OG
FIG. 2. Isometric tension trace of a pair of TSM strips, one of which was in glucose-free medium (OG) up until stable rhythmicity appeared with carbachol and other remained in glucose-containing medium (+ G). After removing carbachol, various agonists (KCl, histamine, acetylcholine) were added separately, as indicated, to same pair of strips.
RESULTS
Effect of glucose removal on mechanical activity. Tracheal muscle, like other multiunit muscles, responds with a tonic contraction when exposed to 80 mM K+, acetylcholine, carbachol, or histamine, and this contraction is maintained as long as there is glucose and O2 in the medium (Fig. 1). However, when glucose was absent, the carbachol-stimulated strips went through a period of irregular contractions, which became regular at a frequency of 1.11 t 0.34 per minute (mean t SE) (n = 65) and lasted for l-5 h. At this stage the removal of carbachol and the addition of either histamine (low5 M) or acetylcholine (lo+ M) also produced rhythmic contractions. Addition of lo-120 mM K+ did not produce rhythmic contractions in presence of atropine (10B7 M). Instead, a tonic contraction was observed (Fig. 2). The rhythmically contracting trachea responded with a myogenic response when stretched by 1820% of I+, (Fig. 3).
FIG. 1. Isometric tension trace of a pair of tracheal smooth muscle (TSM) strips. After a l-h equilibration in normal Krebs-Henseleit solution at 37”C, carbachol (2 x 10m7M) was added as shown. Top trace in glucose-free medium (OG) and bottom trace in 11 mM glucose containing (+G) medium. Numbers between panels denote time intervals (hours). Both solutions were gassed with 95% oxygen and 5% carbon dioxide.
C
FIG. 3. Effect of quick stretch (18% of I+,) applied at (-) to a TSM strip which was A, rhythmically contracting in presence of 2 x 10B7 M carbachol in glucose-free medium; B , tonically contracted by 2 x low7 M carbachol in presence of glucose; C, relaxed in absence of glucose; and D , relaxed in presence of glucose. Myogenic response was seen only in A.
Myogenic response has been shown to occur only in single-unit muscles (8). In order to establish if these changes are due to alterations in the ATP concentration, addition of substrate and/or changing to conditions which are expected to alter the concentration of cellular ATP should change the nature of contraction. As shown in Fig. 4, a nonmetabolizable form of sugar, 3-O-methylglucose (10 mM), did not alter the contractions, whereas addition of phydroxybutyrate (10 mM), a substrate which would lead to the production of ATP, only through the mitochondria, abolished rhythmicity. Similarly, glucose (10 mM) rapidly increased tension and abolished phasic mechan-
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Cl0
R. BOSE AND 0. BOSE
ical activity. Anoxia produced with 95% Nz and 5% CO, after the addition of glucose was unable to maintain the tonic contraction, and oscillation reappeared. Reintroduction of oxygen again abolished rhythmicity. Since there are only two known sources of ATP in the cell, glycolysis and mitochondrial oxidative phosphorylation, and since P-hydroxybutyrate oxidation, but not glycolysis alone, was able to support the tonic contraction, it appears that mitochondrial ATP was required to sustain a tonic contraction produced by histamine or acetylcholine. Rhythmicity was also observed under anoxic or hypoxic (Paz < 100 mm Hg) conditions in muscles which had never been deprived of substrate, in presence of carbachol (lo+ M), but the oscillations were slower (0.53 per minute; n = 7) (Fig. 4, lower and last part of upper traces). Rhythmicity could not be observed in the absence of an agonist. The muscle strip could be repeatedly washed and exposed to carbachol (10e7 M), and stable rhythmicity could be observed each time. Atropine (10D7 M) abolished the rhythmicity induced by carbachol, but not that due to histamine. The possibility that a significant component of the effects of carbachol and other agonists, under condition of glucose deprivation, was by release of neurotransmitters from neural elements in the tissue seems unlikely since rhythmicity was not affected by the addition of either 3 x low6 M tetrodotoxin (which can abolish nerve action potential without affecting smooth muscle), 10m7M sotalol, a P-adrenergic receptor blocker, and 2 x 10m6M phentolamine, an a-adrenergic receptor blocker. Tissue ATP concentration and effect of extracellular ATP. In order to confirm that tissue levels of ATP and creatine phosphate (CP) decreased in the absence of glucose, we have measured the ATP and CP concentrations. As shown in Fig. 5, the ATP concentration decreased in the glucose-free medium from 1.1 to 0.5 mM, 2 min
4. Isometric tension trace of TSM strips in presence of 2 x M carbachol. Top trace: a nonmetabolizable sugar, 34-methylglucose (MeGI, 10 mM), added as indicated to glucose-free medium, and then 10 mM glucose was added at Gl. Asker a stable tonic response in presence of glucose, anoxia was produced with a 95% N, and 5% CO, mixture. Middle trace: 10 mM P-hydroxybutyrate (BOHB) was added to a tracheal strip which was rhythmically contracting with carbachol in absence of glucose. Bottom trace: anoxic TSM strip made to contract rhythmically with carbachol in presence of glucose. 95% OZ, 5% CO, introduced at 0,. FIG.
10e7
1.0 mM 0.5
0
+G Ic
OG Ic. ATP
OG
+G Ic
Ic CP
FIG. 5. ATP and creatine phosphate (CP) concentrations in tracheal strips in normal (+G) or glucose-free (OG) solution. One group from each contained 2 x low7 M carbachol (C). Vertical lines denote standard error values and horizontal bars denote a statistically significant difference (P < 0.05), as determined by Duncan’s new multiple-range test. n = 4-8 in each case.
and CP concentration decreased from 1.4 to 0.38 mM. In the presence of carbachol, the reduction in ATP concentration was greater (reduced from 1.2 to 0.29 mM). Since ATP has been shown to enter the soleus muscle (9), we decided to see if extracellular ATP had any effect on glucose-deprived muscles. Figure 6 shows the effects of various adenine nucleotides and nucleosides on the carbachol contractions. One interpretation of these results is that the return of tonic contraction is due to the ribose moiety in the nucleoside, which probably breaks down to form ribose l-phosphate, and with the help of hexose monophosphate shunt and glycolytic enzymes, provides fuel to Krebs cycle, and then produces ATP. Hence, ATP, ADP, AMP, adenine, inosine, but not adenine and sodium pyrophosphate, were able to produce tonic contractions (in 8 of 11 strips) following various degrees of relaxations. Iodoacetate (1 mM), known to inhibit glycolysis, blocked the tonic contraction with ATP and other nucleosides (seen in the absence of glucose) without affecting the relaxant action. Adenine group of the ATP appears to be responsible for the relaxant action at these concentrations (2 mM). The fact that K+ (80-120 mM) produced a sustained contraction suggested that ATP concentration was not varying in the vicinity of the contractile proteins. The possibility of K+ causing greater influx of Ca was ruled out by the use of a small amount of K+ (20 mM) in the presence of atropine (10e7 M), when a tonic contraction was also observed. These observations suggest that membrane properties were changed under conditions of metabolic depletion leading to alterations in excitationcontraction coupling. Effect of glucose removal on electrical activity. Intracellular electrical recording (Fig. 7A) showed a burst of spikes associated with each rhythmic contraction, which appears as partially fused tetanus (Fig. 8A) in glucose-free medium with carbachol. Oscillations in membrane potential, resulting in very slight oscillations of the tonic contraction, appeared long before the rhythmicity was observed (Fig. 7B). The maximum amplitude of spikes observed with rhythmic contractions were 20 mV and the average duration 0.75 t 0.29 s amplitude. On (mean t SE; n = 11) at half maximum returning glucose (10 mM), the membrane potential decreased (Fig. 9) and the membrane went through a
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RHYTHMICITY
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DEPLETION
Cl1
AMP
t ADENINE
INOSINE
4 IODOACETATE
10 0[
‘\
-
iINOSINE
4
Na Pi-Pi
,
4 min.
OL
#
b
2 min.
FIG. 6. Isometric tension trace with 2 x lo-’ M carbachol of a pair of TSM strips, one of which was in glucose-free medium (lower one of each pair) and other in glucose-containing medium; 2 mM of test
20
r
A
B
compounds (except iodoacetate, cated. A 10mV
1 mM) added to medium as indi-
I 10 sec.
FIG. 7. A: intracellular microelectrodes record (top) and tension (bottom) traces of a TSM strip made rhythmically active in glucosefree medium with lo-’ M carbachol present. B: intracellular microelectrode record showing oscillations with slight changes in tension of a tonically contracted (12 g) TSM strip after 30 min in glucose-free medium with carbachol present. Time scale five times faster than in A.
series of fast oscillations which decreased and disappeared. Sucrose-gap technique yielded similar results when a single gap was used (Fig. 8). On starting the second gap, the rhythmicity disappeared. As shown in Fig. 8A, each action potential resulted in a small contraction, many of which fused to form a big contraction. The membrane depolarized 7-15 mV before the oscillations in membrane potential were seen. In contrast, in presence of glucose, both membrane potential and tension were very stable following the initial graded depolarization [Fig. 8B and C) and oscillations, when observed, never exceeded 3 mV in amplitude. Effect of extracellular calcium (Ca). Since Ca2+ has a central role in excitation-contraction coupling, the effects of Ca addition, deprivation, and addition of D-600, a compound which has been shown to block the Ca current in cardiac muscles, were studied. Figure 10
C 1OmV
c mv&
+G
FIG. 8. A: electrical (top) and tension (bottom) traces obtained from a typical single sucrose-gap experiment with a TSM strip made to contract rhythmically in glucose-free medium (OG) with 10v7 M carbachol. B: electrical (top) and tension (bottom) traces obtained from a typical single sucrose-gap experiment. Sucrose solution (8%) had 10 mM glucose, and Krebs- Henseleit solution contained glucose and 10m7M carbachol. Initial graded depolarization and contraction with introduction of carbachol. C: stable membrane potential and tension seen after 2 h in carbachol.
shows that D-600 at lo-* M concentration blocked the rhythmicity due to carbachol of the strips in glucose-free medium without appreciably affecting the tonic contraction of the paired strip in glucose-containing medium. Higher concentrations (10-5 M) of D-600 relaxed the tonically contracted muscle. At this higher concentration, both rhythmicity and tone were abolished in tra-
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R. BOSE AND D. BOSE
the Ca2+ increased above 2.5 mM. This effect of high Ca may be due to membrane stabilization and needs to be investigated in greater detail. These observations also suggest differences in excitation-contraction coupling under conditions of metabolic deprivation. DISCUSSION FIG. 9. Intracellular electrical record (top) and isometric tension trace showing effect of adding 10 mM glucose (Gl) to a rhythmically contracting TSM strip. 4 D-600 i
5 0I &I
21clO-~M 2 mine
5 Z :
I
5 c
O
5 0
I
FIG. 10. Effect of D-600, added as indicated to TSM strips which were tonically contracted with carbachol (2 x 10s7 M) in presence of glucose (top and bottom trace) or in absence of glucose (middle trace).
A
5
6
9z if 10
5
I
II
VCa 1mM
2 min.
11. A: effect of cumulatively adding 0.5 mM EGTA (pH 7.0) at various points indicated (A) to TSM strips made to contract with carbachol (2 x 100~ M) in presence of glucose (+G) or in absence of glucose (OG). B: effect of cumulatively adding 1 mM Ca at various points indicated (v) to medium containing 2.5 mM Ca. FIG.
cheal strips in glucose-free solution. This further suggested that alterations in Ca movement had occurred under conditions of ATP depletion. Removing extracellular Ca in the medium completely abolished rhythmicity, whereas it decreased the tonic contraction only by 35%. Figure 1lA shows the effects of EGTA on tonic and rhythmic contractions and, here again, the rhythmic strips show greater sensitivity to Ca removal. Similarly, differences in the responses of the tonic and rhythmic contractions were observed when Ca was increased above 2.5 mM already present in the Krebs solution. Figure 1lB shows that increase in Ca, up to a concentration of 5 mM, increased the amplitude of contractions above which it did not have any effect. Very high concentration of Ca (9 mM) caused desynchronization of the contractions. In contrast, the tonically contracted tracheal strip in glucose relaxed as
In smooth muscle, the exact nature of excitationcontraction coupling and how it is affected by metabolic depletion is far from understood. Trachealis muscle is an electrically quiescent muscle, responding with graded and sustained depolarization in response to exogenous acetylcholine or histamine. Rabbit carotid artery and sheep carotid artery are also normally electrically quiescent but show action potentials when exposed to TEA (19) or cyanide (14), respectively. We have been able to produce rhythmicity in canine trachealis muscle by metabolic depletion as well as by parasympathetic denervation (unpublished observation) or TEA. The denervated trachealis also required carbachol for initiating rhythmic contractions. Whether a decreased level of ATP may be at least partly responsible for the production of rhythmicity in the latter cases (TEA and denervation) remains to be seen. Denervation of vas deferens leading to decreased level of ATP has been reported earlier (25). In a normally single-unit muscle (taenia coli), glucose was shown to function as a brake on the frequency of its spontaneous discharge, which persisted for hours after the mechanical response was abolished by glucose removal (1). Mechanisms by which tonic contraction is sustained only when ATP produced by mitochondria is available, but not with glycolysis alone, are not understood. This process of ATP production may be intimately coupled to other membrane processes. Why the mitochondria (or its ATP) are required for the tonic contraction with histamine, acetylcholine, and carbachol, and not to the same extent by K-induced tonic contraction, is not understood. Oscillation in heart mitochondria (at one per minute) has been demonstrated in vitro by measuring changes in transmittance, H+, K+, respiration rate, and morphology (11). ADP was found to have an important role in these oscillations. Gradmann and Slayman (12) have reported on an oscillating electrogenic pump in the poky mutants of Neurospora crassa, which have defective mitochondria. Rhythmicity in the presence of glucose and anoxia was much slower in frequency and amplitude and sometimes (6 of 13) not observed in fresh muscles. It is possible that, in these instances, the ATP concentration was insufficiently low to permit oscillation. However, even under these conditions, the tonic contraction was consistently reduced to 15.8 t 5% (n = S), whereas under similar conditions, the contraction with 80 mM KC1 decreased only to 63.9 t 6% (n = 8). ATP concentrations under hypoxic or anoxic conditions have not been determined in the tracheal muscle. NADH and NADPH concentrations under glucose-free normoxic conditions are expected to be low compared to anoxic conditions, and if the difference in rhythmicity under these two conditions is in any way related to this factor (ratio of reduced to oxidized form of nucleotides), it remains to be seen. Another factor for decreased
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rhythmicity in anoxic muscles could be the intracellular pH which is expected to decrease with lactate production. Acidosis has been shown to block Ca2+ influx (24). The nonmetabolizable analogue of glucose did not affect rhythmicity; therefore, structural requirement for glucose per se did not exist for the tonic contraction. This also rules out any osmotic effects due to glucose lack. Decreased ATP levels could alter the membrane characteristic by altering the Ca2+ bound to the membrane, thereby affecting Na conductance and directly increasing excitability (2). Histamine, under normoxic conditions with glucose present, did not produce slow waves or spikes but, on metabolically depleting the muscle, oscillations both in membrane potential and contractions were observed. Transmembrane flux of Ca2+ was found to be important in studies with rabbit aorta when the muscle was depolarized with K+ or when metabolically depleted (24). Our results also show similar changes in Ca sensitivity with metabolically depleted strips depending more on external Ca2+. It is possible that some internal store of Ca2+, which is normally used for maintaining a tonic contraction with carbachol, is depleted and unavailable for contraction. Whether this source is mitochondria, sarcoplasmic reticulum, or plasma membrane remains to be determined. Electron microscopic examination shows the presence of nexuses and also other types of junctions (R. Bose, unpublished observation) which may play a role in the conduction of impulses (10, 13, 23) and which may be affected by metabolic depletion. Demonstration of ac-
Cl3
DEPLETION
tion potentials across a single 8-mm sucrose gap shows that conduction is occurring in these muscles. However, since with double-sucrose gap rhythmicity disappeared, it is possible that enough pacemakers are not present, and carbachol produced the pacemakers which are masked under normal conditions and unmasked with glucose deprivation. It has been suggested that stable membranes may be labilized to produce action potentials (22). Our data support this view. The pulsatile mechanical activity during hypoxia may greatly aid the distribution of ventilation to the alveoli. To summarize our results, glucose deprivation leads to changes in electrical and mechanical properties of the tracheal muscle strips which normally show a multiunit behavior but, under conditions of metabolic deprivation, show a single-unit behavior. Sensitivity to extracellular Ca2+ is changed with decreased ATP levels and, hence, the study of mechanisms by which ATP alters the electrical properties and fluxes of ions becomes necessary; however, it is possible that processes associated with ATP synthesis, rather than the ATP molecule itself, may be responsible for the changes in excitation-contraction coupling. We extend sincere thanks to Dr. H. Yamaguchi for his design and generous help with the sucrose-gap apparatus. Expert technical assistance of Mrs. T. Chau is greatly appreciated. This research was supported by a grant from the Medical Research Council of Canada (MA-4339) to D. Bose and a fellowship to R. Bose from the Canadian Heart Foundation. Received for publication
13
July 1976.
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C. T. Excitation and contraction in bovine tracheal smooth muscle. J. Physiol., London 244: 263-281, 1975. KNULL, H. R., AND D. BOSE. Reversibility of mechanical and biochemical changes in smooth muscle due to anoxia and substrate depletion. Am. J. PhysioZ. 229: 329-333, 1975. KROEGER, E. A., AND N. L. STEPHENS. Effect of tetraethylammonium on tonic airway smooth muscle: initiation of phasic electrical activity. Am. J. PhysioZ. 228: 633-636, 1975. LOOFBOURROW, G. N., W. B. WOOD, AND I. L. BAIRD. Tracheal constriction in the dog. Am. J. Physiol. 191: 411-415, 1957. MEKATA, F. Electrophysiological studies of the smooth muscle cell membrane of the rabbit common carotid artery. J. Gen.
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