Ionic mechanisms underlying electrical slow waves in canine airway smooth muscle

Luke J. Janssen, Chris Hague, and Roopung Nana

Asthma Research Group and Smooth Muscle Research Program, Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

In canine bronchial smooth muscle (BSM), spasmogens evoke oscillations in membrane potential ("slow waves"). The depolarizing phase of the slow waves is mediated by voltage-dependent Ca2+ channels; we examined the roles played by Cl- and K+ currents and Na+-K+-ATPase activity in mediating the repolarizing phase. Slow waves were evoked using tetraethylammonium (25 mM) in the presence or absence of niflumic acid (100 µM; Cl- channel blocker) or ouabain (10 µM; block Na+-K+-ATPase) or after elevating external K+ concentration ([K+]) to 36 mM (to block K+ currents); curve fitting was performed to quantitate the rates of rise/fall and frequency under these conditions. Slow waves were markedly slowed, and eventually abolished, by niflumic acid but were unaffected by ouabain or high [K+]. Electrically evoked slow waves were also blocked in similar fashion by niflumic acid. We conclude that the repolarization phase is mediated by Ca2+-dependent Cl- currents. This information, together with our earlier finding that the depolarizing phase is due to voltage-dependent Ca2+ current, suggests that slow waves in canine BSM involve alternating opening and closing of Ca2+ and Cl- channels.

voltage-dependent calcium currents; calcium-dependent chloride currents; calcium-dependent potassium currents; airway hyperresponsiveness

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

AIRWAY SMOOTH MUSCLE (ASM) exhibits several ionic currents that are activated during membrane depolarization. At least two different voltage-dependent Ca2+ currents (L type and T type) and two different K+ currents (delayed rectifier and Ca2+ dependent) are activated in direct response to the change in membrane potential (10, 15, 25, 26, 31). Opening of the Ca2+ channels results in Ca2+ influx, which in turn contributes further to Ca2+-dependent K+ channel activity and activates Ca2+-dependent Cl- currents (22). These membrane currents play key roles in many physiological responses. For example, voltage-dependent Ca2+ channels are involved in excitation-contraction coupling by mediating voltage-dependent Ca2+ influx (electromechanical coupling) as well as by refilling of the internal Ca2+ pool (15, 19, 25, 31). With the K+ equilibrium potential at -80 to -90 mV, the opening of K+ channels mediates membrane hyperpolarization. K+ channels are also important in setting the resting state of the cell and/or inhibiting excitation (14, 26, 31). Because the Cl- equilibrium potential (ECl) in smooth muscle is believed to range from -40 to -20 mV (2, 3), opening of Cl- channels leads to depolarization in resting cells (14, 18, 20) but hyperpolarization in cells that have already been depolarized to potentials more positive than ECl.

In the absence of stimulation by neurotransmitters or other pharmacological agents, canine ASM is mechanically and electrically quiescent (5). In addition to membrane depolarization and contraction, spasmogens such as cholinergic agonists (16), thromboxanes (17), leukotrienes (1), or K+ channel blockers such as tetraethylammonium (TEA) and 4-aminopyridine (16) evoke oscillations in membrane potential that are referred to as "slow waves"; these generally have a frequency of approx 1 Hz and amplitude of 10-25 mV. Similar oscillations occur spontaneously or in response to arachidonic acid metabolites in human (6, 11, 12), guinea pig (4, 27, 28, 33, 34), equine (8, 35), and bovine (34) ASM. In all cases, the slow waves are insensitive to neuronal blockers such as tetrodotoxin (6, 7, 27, 29). This suggests that a myogenic oscillatory mechanism is resident in all ASM tissues and is invoked by blockade of K+ channels or by excitatory stimulation by acetylcholine and histamine (canine and bovine ASM), by leukotrienes (human ASM), or by prostanoids (guinea pig and equine ASM).

The depolarizing phase of slow waves is mediated by voltage-dependent Ca2+ channels, since blocking Ca2+ influx (by removal of external Ca2+ or by addition of blockers of voltage-dependent Ca2+ channels, such as Cd2+, dihydropyridines, and verapamil) eliminates slow-wave activity and leaves the smooth muscle cell in a relatively hyperpolarized state (i.e., approximately equal to -45 mV; see Refs. 6, 7, 16, 27, 33). The Ca2+ channels are generally believed to be L type in nature, since the slow waves are sensitive to dihydropyridines (4, 7, 16, 33). The ionic conductance changes underlying the repolarizing phase of the slow waves in ASM, however, have not yet been resolved. In gastrointestinal smooth muscle, repolarization is attributed to opening of Ca2+-dependent K+ channels subsequent to influx of Ca2+ through voltage-dependent channels (9). In ASM, however, slow waves persist in the presence of 25 mM TEA or 5 mM 4-aminopyridine (16), arguing strongly against such a role for K+ currents. Instead, the repolarizing phase in ASM may be mediated by Ca2+-dependent Cl- currents, which are also triggered by voltage-dependent Ca2+ influx (22). Alternatively, Na+-K+-ATPase, which is also electrogenic, has been proposed to play a role, since slow waves in guinea pig ASM are reduced upon cooling or exposure to ouabain (28, 29, 33, 34).

In this study, we sought to characterize the roles played by Ca2+-dependent Cl- currents and Na+-K+-ATPase activity in slow waves in canine bronchial smooth muscle (BSM).

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Dissection. Adult mongrel dogs were euthanized with pentobarbital sodium (100 mg/kg); whole pulmonary lobes were excised and kept in oxygenated Krebs-Ringer solution (composition given in Solutions and chemicals) throughout the study. Parenchymal tissue and vasculature overlying the bronchi were dissected away (dissection carried out at 25°C), exposing the entire bronchiolar tree. The outer diameters of the tissues used in these studies ranged from 1 to 8 mm (3rd to 5th order). Ring segments 4-5 mm in length were excised from the lobe of lung and opened by cutting perpendicularly to the axis of the smooth muscle bundles; care was taken to not damage the epithelium.

Microelectrode studies. Tissues were carefully pinned out, epithelial face upward, in a chamber having a bath volume of 5 ml. Krebs-Ringer solution (composition given in Solutions and chemicals) was bubbled with 95% O2-5% CO2, heated to 37°C, and superfused over the tissues at a rate of 3 ml/min. Conventional microelectrodes (tip resistance of 30-80 MOmega when filled with 3 M KCl) were pulled from borosilicate capillary tubes. Smooth muscle cells were impaled from the epithelial surface of the tissue. Membrane potential changes were observed on a dual-beam oscilloscope (Tektronix D13; 5A22N differential amplifier; 5B12 dual-time base) and recorded on 0.25-in. magnetic tape with a Hewlett-Packard instrumentation recorder. Although we have found that impalements can be maintained for several hours provided no intervention is made that will induce a change in tension (causing the microelectrode to be pulled out of the cell), these generally last only 10-15 min in a typical experiment; frequently, impalement can be reestablished within seconds, although it is not clear whether this is the same cell or an adjacent one. However, ASM is highly coupled by gap junctions (23); as such, membrane potentials and electrical events in one cell are essentially identical to those in the surrounding cells. In general, the effects of only one drug or condition (i.e., niflumic acid, ouabain, high K+) were examined in a given tissue. Portions of these data were played back, digitized (Digidata 1200), and sampled using pCLAMP 6 software (Axon Instruments, La Jolla, CA) and then fitted using pCLAMP 6 and/or exported to SigmaPlot for graphical presentation.

Electrical field stimulation. Electrical field stimulation (EFS) was achieved through two silver plates on either side of the tissue (approx 1 cm apart). Electrical pulses (pulse width of 0.5 ms) were provided by a Grass S88 stimulator; both single pulses and pulse trains (frequencies ranging up to 20 pulses/s) were used. The voltage used was that which gave maximal responses (generally 50-100 V).

Solutions and chemicals. Tissues were studied using Krebs-Ringer buffer (KRB) containing (in mM) 116 NaCl, 4.2 KCl, 2.5 CaCl2, 1.6 NaH2PO4, 1.2 MgSO4, 22 NaHCO3, 11 D-glucose, and 0.01 indomethacin, which was bubbled continuously to maintain pH at 7.4. Isosmotic high-K+ KRB was prepared by substituting NaCl with KCl. Unless indicated otherwise, KRB solutions also contained TEA (25 mM). Chemicals were obtained from Sigma Chemical. Initially, niflumic acid was made up as a concentrated solution in 95% ethanol and was added to the bathing solution (final bath concentration of ethanol was 0.1%); later, however, we dissolved niflumic acid directly into KRB (10-4 M, without any ethanol) and superfused the tissues with this solution. Ouabain was prepared as an aqueous solution.

Data analysis. The rates of change for the various components (phases i-v; see legend of Fig. 1) of the slow waves were derived using Clampfit software (Axon Instruments). In Fig. 1, phase ii, phase iii, and phase iv could be well fit by linear functions, whereas a monoexponential function was used to fit phase v. The time course of phase i, on the other hand, was more complicated and varied; as a result, we were unable to obtain a satisfactory fit using the algorithms supplied by Clampfit. For each cell impaled, the values of phases ii-v were obtained from three different portions of a trace, and the average for that cell was calculated. Responses are reported as means ± SE; n refers to the number of tissues tested. Statistical comparisons were made using a paired Student's t-test, with P values <0.05 being considered significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Electrical slow waves are evoked by TEA in canine BSM. In canine BSM at rest, the membrane potential was -62 ± 4 mV (n = 21), and there was little or no spontaneous activity. Within 10 min after introduction of 25 mM TEA, however, the membrane depolarized and slow-wave activity was triggered. The slow waves were generally sinusoidal in appearance, with an exponential rise and fall ( phase i and phase v, respectively, in Fig. 1). In many cases (8 of 13 tested), superimposed upon the rising phase of the sinusoidal oscillations (i.e., phase i) were action potentials with a rapid spike-like depolarization ( phase ii in Fig. 1B, right) followed by a rapid decay ( phase iii) to a much more slowly decaying "plateau" or "shoulder" region (phase iv). The mean threshold potentials at which slow waves and action potentials were triggered were -43 ± 4 and -35 ± 6 mV, respectively. In the absence of any other experimental intervention, slow-wave activity persisted for at least 1 h, with no notable change in frequency or amplitude (Fig. 2).


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Fig. 1.   Slow waves in canine bronchial smooth muscle (BSM) depolarized using tetraethylammonium (TEA; 25 mM). A: slow waves were sinusoidal in appearance (left); in some cells, action potentials were superimposed on the rising phase of the sinusoid (right). B: individual electrical events indicated by * in A are displayed on a faster time scale; the rising and falling phases of the sinusoidal waves ( phases i and v, respectively) were best fit by monoexponential functions, whereas the rising and falling phases of the action potentials ( phases ii, iii, and iv) could be well fit by linear functions. t1/2, Time required for the slow wave per se, excluding the action potential, to decay to one-half of its peak value.


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Fig. 2.   Slow-wave time course is consistent over time. A: typical tracings obtained shortly after onset of TEA-induced slow waves (left) and 120 min later (right); there were no other pharmacological interventions during this period. B: although there was a slight decrease in slow-wave frequency over this period, the time course of the slow waves did not seem to change, as indicated by superimposition of the individual electrical events marked by * in A. t, Time.

Niflumic acid abolishes TEA-evoked slow waves. The depolarizing phase of the slow waves ( phase i) is mediated by dihydropyridine-sensitive voltage-dependent Ca2+ channels (16). The mechanism(s) underlying the repolarizing phases ( phases iv and v), however, are unclear but may involve Ca2+-dependent Cl- currents. We tested this hypothesis by examining what effect niflumic acid, a blocker of the Ca2+-dependent Cl- channels (22), might have on slow waves induced by TEA (25 mM). We used 100 µM niflumic acid because this was found to be sufficient to maximally block Ca2+-dependent Cl- current in this tissue (22).

Under control conditions, the decay of the shoulder region ( phase iii) was roughly linear, whereas the subsequent portion of the slow wave ( phase iv) decayed exponentially (Figs. 1-3 and Table 1). The time required for the membrane potential to drop midway between the "crest" or shoulder to the "trough" (t1/2) was 559 ± 37 ms, and the frequency of the slow waves was 0.54 ± 0.04 Hz. Within minutes after introduction of niflumic acid, however, t1/2 was markedly prolonged and slow frequency markedly reduced. These effects seemed to be secondary to a prolongation of the shoulder region of the slow waves (Fig. 3 and Table 1, phase iv) without any change in the other components of the slow waves. With more prolonged exposure to niflumic acid, slow waves appeared sporadically and eventually ceased altogether (Fig. 3). In some cases, these could be triggered again (albeit only temporarily) by EFS. Eventually, however, slow waves were abolished entirely. At this point, mean membrane potential was -46 ± 5 mV (not significantly different from the mean control value of -43 ± 4 mV, given in Electrical slow waves are evoked by TEA in canine BSM, at which slow waves are triggered).


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Fig. 3.   Slow waves are prolonged and eventually abolished by niflumic acid. A: representative traces showing slow-wave activity observed during perfusion with TEA-Krebs-Ringer buffer (KRB; left) and how this activity is suppressed (middle) and eventually abolished (right) upon perfusion with TEA-KRB containing niflumic acid (100 µM). B: individual electrical events indicated by * in A are superimposed to highlight the prolongation of the electrical event; this is primarily due to a prolongation of the plateau phase.

                              
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Table 1.   Pharmacological manipulation of TEA-evoked slow waves

Lack of effect of ouabain on TEA-evoked slow waves. The repolarization phase of the slow waves may also be mediated by Na+-K+-ATPase activity; to test this hypothesis, we examined the effect of 10-5 M ouabain, which is sufficient to maximally inhibit the Na+-K+ pump (34). After 30 min of exposure to ouabain, membrane potential was further decreased by approx 10 mV above the level existing in the presence of TEA. In addition, the mean frequency of the slow waves was increased and t1/2 somewhat decreased (Fig. 4A and Table 1), although these changes did not reach statistical significance (Table 1). More importantly, however, the rates of recovery ( phases iv and v) were not slowed; in fact, phase v was significantly faster in the presence of ouabain (Fig. 4 and Table 1).


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Fig. 4.   A: representative traces showing TEA-induced slow-wave activity before (left) and during (right) exposure to ouabain (10 µM). B: individual electrical events indicated by * in A were digitally made equivalent in size, while maintaining constant aspect ratio, and then superimposed to facilitate comparison; ouabain did not slow down the repolarization phase.

Lack of effect of high K+ on TEA-evoked slow waves. Although the persistence of slow waves in the presence of TEA argues against a causal role for large-conductance Ca2+-dependent K+ channels in mediating repolarization, it is possible that other types of K+ channels might be responsible, including delayed rectifier K+ channels (10, 31) and small-conductance K+ channels; in this light, it is interesting that niflumic acid has been reported to increase the activity of small-conductance K+ channels (32), which might account for its inhibitory effect on slow waves. We therefore raised external K+ concentration ([K+]o) isosmotically to 36 mM to elevate the K+ equilibrium potential to approximately -30 mV, thereby decreasing the outward driving force on K+ but not converting it to an inward driving force when the membrane potential was -30 mV (i.e., the peak of the oscillations). Upon introduction of high-K+ KRB, the troughs of the oscillations became progressively less negative (i.e., membrane depolarized), resulting in a significant decrease in slow-wave amplitude (Fig. 5 and Table 1); there was also an increase in slow-wave frequency (Fig. 5 and Table 1). Despite these changes, the rate of repolarization of the slow waves was not significantly decreased (Table 1). In one tissue during prolonged exposure to high-K+ medium, the membrane continued to depolarize until slow waves were barely discernable, began to become desynchronized, and were eventually lost; even seconds before they disappeared, however, the rate of repolarization was not slowed.


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Fig. 5.   A: slow waves in a tissue perfused with standard TEA-KRB (left) and after perfusion with high-K+ KRB containing TEA; elevating external K+ concentration ([K+]o) to 36 mM increased slow-wave frequency and decreased their magnitudes (by elevating the troughs to less negative potentials). B: individual slow waves indicated by * in A are superimposed here to show that elevation of [K+]o had no effect on the rate of repolarization of the slow waves (note: the slow wave obtained in the presence of high K+ was digitally increased in size, while maintaining constant aspect ratio).

Niflumic acid abolishes slow waves evoked by EFS. Usually, canine BSM exhibits a single spike-like excitatory junction potential in response to EFS (Fig. 6B); occasionally, however, the excitatory junction potential is followed by a series of slow waves (Fig. 6A). In this experiment, it was not our goal to ascertain the conditions that dictated whether or not slow waves would be evoked by EFS; instead, we investigated whether these slow waves were mediated by the same mechanisms as those seen during K+ channel blockade (see Niflumic acid abolishes TEA-evoked slow waves). In four cells that did in fact exhibit EFS-evoked slow waves, niflumic acid abolished the latter but not the excitatory junction potential evoked by EFS (Fig. 6).


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Fig. 6.   A: in this cell perfused with normal KRB (i.e., not containing TEA), electrical field stimulation (5 pulses at 20 pulses/s; indicated by bullet ) evoked an excitatory junction potential followed by a series of recurring oscillations or slow waves with varying amplitude. B: after exposure to niflumic acid (100 µM), excitatory junction potential could still be evoked, but the oscillations were completely abolished.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Slow waves have been recorded in the ASM of many species, including the dog (16), human (6, 11, 12), guinea pig (4, 27, 28, 33, 34), horse (8, 35), and cow (34). The depolarizing phase of slow waves involves opening of voltage-dependent Ca2+ channels. The ionic conductance changes underlying the repolarizing phase of the slow waves, however, have been unclear but may involve Ca2+-dependent Cl- currents, TEA-insensitive K+ channels, or electrogenic Na+-K+-ATPase. These mechanisms are easily distinguished using a variety of pharmacological approaches.

Contribution of K+ channels and Na+-K+-ATPase to TEA-induced slow waves. We were able to rule out any role for K+ channels or Na+-K+-ATPase in mediating the repolarization phase of the slow waves, since phase iv was not significantly slowed by elevating the [K+]o to 36 mM or by ouabain (10-5 M should maximally block Na+-K+-ATPase activity). Elevation of [K+]o should markedly decrease the outward driving force on K+ at a membrane potential of -30 mV (i.e., during the peaks of the slow waves) and thus minimize the ability of K+ channels to mediate hyperpolarization. In fact, to our surprise, we found that the t1/2 of the slow waves was significantly decreased, and slow-wave frequency increased, by high-K+ medium and that ouabain also seemed to accelerate slow-wave activity. This acceleration may be secondary to the depolarization that both interventions mediate; depolarization would influence the kinetics of the voltage-dependent Ca2+ channel activation and inactivation.

Contribution of Ca2+-dependent Cl- currents to TEA-induced slow waves. The Cl- channel blocker niflumic acid, on the other hand, markedly and significantly slowed phase iv and prolonged t1/2, indicating that this component of the slow waves is mediated by Cl- channels. Although this concentration of niflumic acid (100 µM) is sufficient to immediately block Cl- channels when applied by pressure ejection in the vicinity of an isolated canine tracheal smooth muscle cell (22), its effect was delayed in this study, presumably because of the time required for the drug to travel from the physiological saline solution reservoir and to diffuse into the tissue. Inevitably, however, slow waves were abolished after introduction of niflumic acid, consistent with the hypothesized role of Cl- channels in mediating slow waves.

The final phase of hyperpolarization ( phase v), however, was unaffected, suggesting that the final component of the repolarization phase is mediated by some other mechanism (possibly deactivation of the Ca2+ channels). Although niflumic acid may also enhance the activity of small-conductance K+ channels (32), we have already ruled out a role for K+ channels in general (see Lack of effect of high K+ on TEA-evoked slow waves). In addition, there have been numerous studies of the ion currents in canine ASM (26, 31), and none describe a small-conductance K+ current in this tissue. Previously, we have shown that niflumic acid does not affect the voltage-dependent Ca2+ current in this tissue (15, 22). Consistent with the conclusion that Ca2+-dependent Cl- currents play a central role in slow waves in canine ASM, Ba2+ is much less effective than Ca2+ in activating Ca2+-dependent Cl- currents, and we have previously shown that slow waves are greatly reduced or even abolished in the presence of Ba2+ (16).

Cl- current would not be expected to contribute greatly to the depolarizing phase ( phase i) of these slow waves, since it must first be triggered by Ca2+ influx (i.e., phase ii), and then, once triggered, it requires ~200 ms to be maximally activated (22), at which time the action potential has peaked and membrane potential is falling ( phase iii).

Slow waves in the absence of TEA. Slow waves can also be observed under conditions other than specific blockade of K+ currents. For example, they are occasionally evoked during neurogenic release of cholinergic agonists (Fig. 6) and are commonly seen in canine airway tissues during stimulation with exogenously added cholinergic agonist (16), with inflammatory mediators such as leukotrienes (1) or thromboxane A2 (17) or with aspirin (13); in addition, they are abolished by the cholinergic antagonist atropine (16). In this study, we found these to also be mediated by Ca2+-dependent Cl- currents, since they are blocked by niflumic acid (Fig. 6B). In single freshly dissociated canine ASM held under voltage clamp at -60 mV, recurring oscillations of membrane current are sometimes observed spontaneously (21) or after stimulation with acetylcholine (14, 18) or histamine (20). These membrane current oscillations were shown to be mediated by Ca2+-dependent Cl- current and are believed to indicate oscillations of cytosolic Ca2+ concentration ([Ca2+]i), possibly due to Ca2+-induced feedback (both positive and negative) on phospholipase C and/or the Ca2+ release sites on the sarcoplasmic reticulum. Thus the first component of each slow wave ( phase i) that triggers voltage-dependent Ca2+ influx ( phases ii and iii) and the subsequent activation of Ca2+-dependent Cl- current ( phase iv) may be an elevation of [Ca2+]i.

Contribution of voltage-dependent Ca2+ currents to slow waves. The Ca2+ channels that contribute to slow-wave activity are generally believed to be L type in nature, in part because slow waves are dihydropyridine sensitive, even though the threshold potentials for these channels and for slow waves are substantially different (approximately equal to -30 and -45 mV, respectively, in canine ASM). In addition, L-type currents are suppressed during agonist stimulation, whereas slow waves are not; consistent with this, oscillations in canine ASM depolarized using TEA have the appearance of action potentials, whereas those in canine ASM depolarized using cholinergic agonists are generally smaller and sinusoidal in appearance (this study and Ref. 16). T-type Ca2+ currents, however, may contribute to slow-wave activity, since 1) they are also dihydropyridine sensitive but unaffected by cholinergic agonists; 2) slow waves sweep the membrane continuously between -45 and -30 mV, which overlaps the threshold and peak potentials for T-type "window current"; and 3) recovery from inactivation of T-type currents occurs within 1 s, consistent with a role in oscillations that have a frequency of approx 1 Hz (recovery of L-type currents can take up to 30 s; see Refs. 15 and 30).

Sequence of molecular events underlying slow waves in canine ASM. We would therefore propose the following mechanism to account for the data presented in this and our previous studies (15, 16, 22) regarding the ionic mechanisms underlying slow-wave activity in canine ASM. An as yet poorly understood event serves as a pacemaker and triggers membrane depolarization ( phase i), which in turn activates T-type and L-type Ca2+ currents and a consequent action potential ( phase ii). The resultant Ca2+ influx leads to activation of the Ca2+-dependent Cl- current (22), which opposes the depolarizing influence of the Ca2+ currents and draws the membrane potential toward ECl ( phases iii and iv). As the membrane continues to hyperpolarize beyond approximately -30 to -40 mV, the L-type Ca2+ currents deactivate ( phase iv), leading to the fast repolarizing phase of the slow waves ( phase v). The T-type currents, which inactivate rapidly and nearly completely at -40 mV (15), would have already decreased to negligible levels at the peak of the slow wave. Deactivation of Ca2+ current is unaffected by Cl- channel blockers but is slowed by Ba2+, accounting for the insensitivity of the fast repolarizing phase to niflumic acid (this study) and the changes in slow-wave activity induced by Ba2+ (16). Finally, the subsequent deactivation of the Ca2+ channels leads to deactivation of the Cl- current, and the membrane is again primed for the next slow wave. Recovery of the T-type currents from inactivation occurs within 1 s (15, 30), consistent with their role in slow waves, which typically exhibit a frequency of 1 Hz.

This sequence of molecular events would account for the slow waves seen in isolated airway tissues (this study and Refs. 6, 7, 16, 27, 33) or in vivo electrophysiological recordings of ASM (24). In dissociated ASM cells studied under current-clamp conditions, however, irregular fluctuations in membrane potential are observed (18). It seems, then, that the regular, synchronized appearance of the slow waves is a product of the syncytial nature of this tissue and not of the individual cells themselves. That is, the high degree of coupling by gap junctions leads to electrotonic spread of the electrical changes, allowing synchronization of each event (since they are triggered by voltage-dependent Ca2+ influx) and "smoothing" of the subsequent membrane potential changes.

Conclusion and physiological significance. We conclude that the repolarization phase of slow waves in canine BSM is mediated by Ca2+-dependent Cl- currents. Slow waves are observed after stimulation with physiological agonists as well as under pathophysiological conditions such as aspirin-induced or allergen-induced airway hyperresponsiveness (1, 6, 11-13, 18, 20). Slow waves are associated with voltage-dependent Ca2+ influx and may maintain an excited state in the smooth muscle after stimulation with agonists or during inflammation; they may also play a role in the pathophysiology underlying airway hyperreactivity. Thus Cl- channel blockers, which abolish slow waves and reduce excitatory electrical events, may prove to be useful in the reversal of bronchoconstriction and treatment of airway disease.

    ACKNOWLEDGEMENTS

These studies were supported by grants from the Medical Research Council of Canada and the Ontario Thoracic Society and by a Career Award to L. J. Janssen from the Pharmaceutical Manufacturers Association of Canada and the Medical Research Council of Canada.

    FOOTNOTES

Address for reprint requests: L. J. Janssen, Dept. of Medicine, McMaster Univ., Hamilton, Ontario, Canada L8N 3Z5.

Received 29 December 1997; accepted in final form 19 May 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Lung Cell Mol Physiol 275(3):L516-L523
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