Electrical behavior of guinea pig tracheal smooth muscle

Narelle J. Bramich

Department of Zoology, University of Melbourne, Parkville, Victoria 3052, Australia


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Intracellular recordings were taken from the smooth muscle of the guinea pig trachea, and the effects of intrinsic nerve stimulation were examined. Approximately 50% of the cells had stable resting membrane potentials of -50 ± 1 mV. The remaining cells displayed spontaneous oscillations in membrane potential, which were abolished either by blocking voltage-dependent Ca2+ channels with nifedipine or by depleting intracellular Ca2+ stores with ryanodine. In quiescent cells, stimulation with a single impulse evoked an excitatory junction potential (EJP). In 30% of these cells, trains of stimuli evoked an EJP that was followed by oscillations in membrane potential. Transmural nerve stimulation caused an increase in the frequency of spontaneous oscillations. All responses were abolished by the muscarinic-receptor antagonist hyoscine (1 µM). In quiescent cells, nifedipine (1 µM) reduced EJPs by 30%, whereas ryanodine (10 µM) reduced EJPs by 93%. These results suggest that both the release of Ca2+ from intracellular stores and the influx of Ca2+ through voltage-dependent Ca2+ channels are important determinants of spontaneous and nerve-evoked electrical activity of guinea pig tracheal smooth muscle.

excitatory junction potential


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MAMMALIAN AIRWAY SMOOTH MUSCLE receives a parasympathetic excitatory innervation originating from the vagus. In most species, stimulation of vagal nerve fibers evokes a contraction of tracheal smooth muscle that is abolished by the muscarinic-receptor antagonist atropine, indicating that acetylcholine (ACh) is released from postganglionic nerve fibers, causing the activation of muscarinic receptors (7). Contraction evoked by parasympathetic nerve stimulation is preceded by a membrane depolarization or excitatory junction potential (EJP). Studies examining the effect of dihydropyridine Ca2+ antagonists on responses evoked by parasympathetic nerve stimulation suggest that EJPs and contractions are partly mediated by the influx of Ca2+ through voltage-dependent L-type Ca2+ (CaL) channels (11, 21, 31). In contrast, most studies have shown that responses to exogenously applied ACh are little affected by either dihydropyridine Ca2+ antagonists or low external Ca2+ (1, 2, 13, 21, 26, 27). This has led to the suggestion that the influx of Ca2+ through CaL channels has little importance in the generation of tracheal smooth muscle contraction evoked by muscarinic-receptor stimulation. Instead, it has been proposed that muscarinic-receptor stimulation increases the intracellular concentration of Ca2+ after either the release of Ca2+ from intracellular stores via an inositol 1,4,5-trisphosphate-dependent process (see Ref. 4) or the entry of Ca2+ through receptor-operated Ca2+ channels (33).

The difference in effectiveness of Ca2+ antagonists to reduce responses to exogenously applied and neuronally released ACh in tracheal smooth muscle might suggest that the two sources of transmitter activate distinct receptors, bringing about different ionic changes. A difference in the effects of a neuronally released and exogenously applied transmitter has been observed at a number of neuroeffector junctions in the autonomic nervous system (see Ref. 16). For example, in guinea pig ileal smooth muscle different sources of Ca2+ are responsible for the membrane depolarizations and contractions evoked by exogenously applied and neuronally released ACh (8, 9). Although the ionic mechanisms underlying the responses to exogenously applied ACh in tracheal smooth muscle have been extensively studied, it is unclear whether the same mechanisms can account for the responses evoked by parasympathetic nerve stimulation (11, 31). The present study tested the hypothesis that nerve-evoked responses of tracheal smooth muscle cells depend on both release of Ca2+ from intracellular stores and the influx of Ca2+ through CaL channels.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The procedures described have been approved by the Animal Experimentation Ethics Committee at the University of Melbourne (Parkville, Australia). Guinea pigs of either sex, weighing 150-200 g, were killed by a blow to the head and exsanguination. A segment of trachea containing four to five cartilaginous rings was taken from just above the bifurcation of the two bronchi. A cut was made through the cartilage down the length of the trachea, and the epithelium along with any visible connective tissue was stripped from the muscle. Preparations were pinned in a shallow recording chamber (bath volume 2 ml) with pins cut from 100-µm tungsten wire. The base of the recording chamber consisted of a coverslip coated with Sylgard silicone resin (Dow Corning, Midland, MI). The preparations were placed over a platinum electrode, and a second platinum electrode was placed in the bath to allow stimulation of intrinsic nerve fibers (1-90 V, 0.1-1.0 ms). Preliminary experiments indicated that supramaximal responses could be obtained with pulse widths of 0.5 ms and amplitudes of 80-90 V. These stimulus parameters were used in all experiments. The preparations were continuously perfused with physiological saline (composition in mM: 119.8 NaCl, 5.0 KCl, 25 NaHCO3, 1.0 NaH2PO4, 2.5 CaCl2, 2.0 MgCl2, and 11 glucose, gassed with 95% O2-5% CO2) at a rate of 3 ml/min. Unless otherwise stated, experiments were performed in the presence of propranolol (1 µM) and indomethacin (10 µM). The drugs were added to the preparation by changing the inflow line from the control solution to one containing the appropriate concentration of drug. All experiments were performed at 35°C.

Intracellular recordings were made from the epithelial side of the tissue with the use of conventional techniques with fine glass microelectrodes (resistance 100-210 MOmega ) filled with 0.5 M KCl. All membrane potential records were low-pass filtered (cutoff frequency 1 kHz), digitized (100 Hz), and stored on disk for later analysis. At the start of each experiment, several successive recordings were made from each tissue, and the myogenic behavior of the tissue was characterized. Subsequently, the tissues were stimulated transmurally with a single impulse or trains of impulses at 2-min intervals. At least three consecutive EJPs with the same amplitude and time course were obtained before the addition of any drug. Control measurements were taken from the last EJP recorded before the addition of a drug. Measurements were taken from a single EJP after responses had reached a new steady state after drug treatment (after an equilibration time of at least 30 min). Due to variation in the amplitude of responses recorded from different cells, in any particular experiment all responses were recorded from the same cell. If an impalement was lost during the course of an experiment, these results were discarded. Latencies of EJPs were measured from the stimulus artifact to 10% of the peak amplitude. Rise times were measured from 10 to 90% of the peak amplitude. Half-widths were measured as the time between 50% of the peak amplitude on the rising and falling phases of the EJPs. All values are expressed as means ± SE. Each n value represents a measurement from a different animal. Where indicated, the significance of the difference between two means was determined with Student's t-test. To eliminate any possible effect of prostaglandins on nerve-evoked responses, experiments were performed in the presence of indomethacin (10 µM).

The drugs used in this study were nifedipine hydrochloride, hyoscine sulfate, tetrodotoxin, caffeine, isethionate, tetraethylammonium chloride, barium chloride, indomethacin, propranolol (all from Sigma, St. Louis, MO), and ryanodine (Calbiochem, Alexandria, Australia). All drugs were dissolved in distilled water except nifedipine and indomethacin, which were dissolved in absolute ethanol.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General observations. Intracellular recordings taken from guinea pig tracheal smooth muscle displayed three types of electrical activity. In all preparations, if sufficient different bundles of tracheal muscle were impaled, spontaneous oscillations in membrane potential were detected (Fig. 1, B and C). The oscillations had an amplitude of 25.6 ± 1.3 mV (n = 20) and a peak negative potential around -50 mV. The frequency of the membrane potential oscillations ranged between 11 and 44 cycles/min (mean 23.7 ± 1.9 cycles/min; n = 20). In some cells, an action potential spike was superimposed on the peak of each oscillation (Fig. 1C). In each preparation, the oscillations detected from successive bundles had similar properties. In 80% of the preparations, it was possible to record from a bundle of tracheal muscle in which the cells were quiescent; these had a stable resting membrane potential of -50 ± 1 mV (n = 26). The membrane potential of such cells was interrupted by brief membrane depolarizations with amplitudes up to 5 mV (Fig. 1A). In 35% of the preparations, a second type of rhythmic activity was detected in 20% of the tracheal bundles. These cells generated bursts of spontaneous activity that were interspersed with quiescent periods of 1-4 min in duration (Fig. 1D). In these cells, the resting membrane potential during periods of quiescence was -45 to -60 mV (mean -53.4 ± 4.0 mV; n = 5). A gradual depolarization of the resting membrane potential preceded each burst of activity.


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Fig. 1.   Different types of spontaneous activity recorded from cells of guinea pig tracheal smooth muscle. A: recording from a quiescent tracheal smooth muscle cell. Resting membrane potential of this cell was -48 mV. B and C: membrane potential recordings from 2 different cells that displayed spontaneous oscillations. Although oscillations had similar amplitudes, cell in C showed action potential spikes on rising phases. D: recording from a cell that displayed intermittent oscillations in membrane potential.

Indomethacin (1-10 µM) had no effect on the frequency or amplitude of membrane potential oscillations (n = 5; Fig. 2A). Oscillations ceased in the presence of nifedipine (10 µM), and the membrane potential settled near -50 mV (n = 10; Fig. 2B). Depletion of intracellular stores with ryanodine (10 µM) abolished the oscillations (Fig. 2C). Ryanodine initially depolarized the smooth muscle by ~30 mV before the depolarization settled at a value of -43 ± 4 mV (n = 5) after ~1 h.


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Fig. 2.   Effect of indomethacin, nifedipine, and ryanodine on spontaneous membrane potential oscillations recorded from tracheal smooth muscle of guinea pig. A: spontaneous membrane potential oscillations recorded before (a) and after (b) inhibition of prostaglandin synthesis with indomethacin. B: effect of nifedipine on generation of spontaneous oscillations. Membrane potential after inhibition of spontaneous activity was -53 mV. C: effect of depletion of intracellular stores with ryanodine on spontaneous membrane potential oscillations. Membrane potential recording in Cb was taken 60 min after addition of ryanodine, and membrane potential was -41 mV. Indomethacin (10 µM) was present in B and C.

Membrane potential changes evoked by intrinsic nerve stimulation. In quiescent bundles of tracheal muscle, stimulation of intrinsic nerve fibers with a single impulse evoked an EJP (Fig. 3, Aa and Ba). EJPs had amplitudes of 11.5-35.0 mV (mean 24.4 ± 1.4 mV), latency of 215 ± 12 ms, rise time of 246 ± 24 ms, and half-width of 525 ± 23 ms (n = 26). After a peak was reached, the membrane potential rapidly repolarized to a value close to the resting membrane potential and then gradually returned to the control level over the next 15-20 s (Fig. 3Ba). Usually, the slowly decaying phase of the EJP appeared as a separate component, with a peak amplitude of 2.7 ± 0.4 mV (n = 20; Fig. 3, Aa; see also Fig. 5A).


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Fig. 3.   Effect of increasing the number of transmural stimuli on responses recorded from quiescent cells of guinea pig tracheal smooth muscle. A: effects of stimulation with 1, 2, 3, and 4 impulses delivered at 10 Hz (a-d, respectively) in a cell in which increasing the number of stimuli caused a gradual increase in the 2nd component of excitatory junction potential (EJP) and eventually initiated membrane potential oscillations (d). Resting membrane potential of this cell was -54 mV. B: effects of stimulation with 1, 2, 5, and 10 impulses delivered 10 Hz (a-d, respectively) in a cell in which oscillations in membrane potential were unable to be evoked. Resting membrane potential of this cell was -48 mV. Indomethacin (10 µM) was present throughout.

Increasing the number of stimuli (1-10 impulses at 10 Hz) did not change the peak amplitude of the EJPs. However, in 30% of the cells recorded from, multiple stimuli triggered large fluctuations in membrane potential (Fig. 3A, b and c). A further increase in the number of stimuli resulted in the initiation of oscillations that lasted for some 20-60 s (Fig. 3Ad). In the other quiescent cells (70%), increased numbers of stimuli often initiated a second rapid phase and increased the amplitude of the slow secondary phase of the EJP (Fig. 3B, b and c). In these cells, stimulation with 10 impulses at 10 Hz evoked an initial depolarization of 26.2 ± 2.0 mV, a secondary depolarization of 19.3 ± 3.8 mV, and a slow membrane depolarization of 6.0 ± 0.8 mV (n = 11; Fig. 3Bd); further increases in the number of stimuli failed to initiate oscillations in membrane potential.

In spontaneously active cells, a single nerve stimulus had little effect on either the frequency or amplitude of the membrane potential oscillations (Fig. 4A). However, increasing the number of stimuli increased the frequency of the membrane potential oscillations for some 10 s after the period of stimulation. Thus 10 impulses at 10 Hz increased the frequency of the oscillations from 27 ± 4 to 48 ± 5 cycles/min (n = 7; Fig. 4B). During this time, the peak negativity of the membrane potential decreased by 6.3 ± 1.9 mV. This effect of nerve stimulation on rhythmic activity was more evident with lower frequencies of stimulation (2 Hz, 5 s; Fig. 4C).


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Fig. 4.   Effect of increasing the number of transmural stimuli on responses recorded from a spontaneously oscillating cell of guinea pig tracheal smooth muscle. A and B: effects of stimulation with 1 and 10 impulses, respectively, delivered at 10 Hz. Stimulation with a single impulse had little effect on either amplitude or frequency of membrane potential oscillations (A). Increasing the number of stimuli to 10 impulses caused an increase in frequency of oscillations (B). This was more evident with a train of low-frequency stimulation (10 impulses at 2 Hz; C). Peak negative potential in all records was around -60 mV. Indomethacin (10 µM) was present throughout.

The membrane potential changes evoked by transmural nerve stimulation resulted solely from the stimulation of parasympathetic nerve fibers and release of ACh because all responses were abolished after the addition of the muscarinic-receptor antagonist hyoscine (1 µM) to the physiological saline (n = 6). Both the spontaneous oscillations in membrane potential and the small spontaneous membrane depolarizations persisted with hyoscine.

The observation that, in any given preparation, three electrically different cell types could be recorded from suggests that either the behavior of the preparations changes with time or the cells within any given preparation may display quite different electrical properties. To distinguish between these two possibilities, pairs of recordings were made simultaneously from nearby smooth muscle cells in the same muscle bundle or from smooth muscle cells in neighboring muscle bundles. Pairs of cells impaled in the same muscle bundle, which originated from same cartilaginous ring, had similar electrical characteristics and responses to intrinsic nerve stimulation (n = 3; Fig. 5, A and B). However, if simultaneous recordings were made from muscle bundles that originated from different cartilaginous rings, the electrical responses were not synchronized (n = 3). For example, in a pair of quiescent cells, stimulation of the parasympathetic nerves with a single impulse evoked an EJP that had a similar amplitude and time course in both cells (Fig. 5C). However, short trains of stimuli evoked oscillations in one cell but not in the other (Fig. 5D).


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Fig. 5.   Simultaneous recordings of membrane potential taken from cells in the same and different muscle bundles of guinea pig trachea. A: recordings taken from 2 cells in the same muscle bundle. Stimulation with a single impulse evoked an EJP that was followed by initiation of membrane potential oscillations. Resting membrane potentials in Aa and Ab were -49 and -50 mV, respectively. B: 2 recordings made simultaneously from cells within the same muscle bundle. Spontaneous oscillations, which were recorded from both cells, were synchronous and of similar amplitude. Stimulation of intrinsic nerves with a single impulse caused a similar membrane potential change in both cells. Peak negative potential in B was around -49 mV. C and D: 2 simultaneous membrane potential recordings taken from cells in neighboring muscle bundles of trachea. Stimulation of intrinsic nerves evoked an EJP in both cells that had similar amplitudes and time courses. Resting membrane potentials in C, a and b, were -45 and -51 mV, respectively. When stimulus number was increased to 5 impulses delivered at 10 Hz, membrane potential oscillations were initiated in one cell (Da) but not in the 2nd cell (Db). Indomethacin (10 µM) was present throughout.

Sources of Ca2+ responsible for the generation of EJPs. Nifedipine (1-10 µM) reduced EJPs by ~30% (Fig. 6). In the control solution, the EJPs evoked by a single impulse had an amplitude of 23.1 ± 2.3 mV. With nifedipine (1 µM), the EJPs were reduced to 15.9 ± 2.6 mV (P < 0.05; n = 12). The slowly decaying component of the EJP was also inhibited by nifedipine (Figs. 6 and 7); with control physiological saline, the peak depolarization was 2.6 ± 0.6 mV, and after the addition of nifedipine, the depolarization was 0.8 ± 1.0 mV (P < 0.05; n = 12). Increasing the concentration of nifedipine (10 µM) did not further reduce the amplitude of EJPs (n = 3). Nifedipine abolished oscillations triggered by cholinergic nerve stimulation to reveal EJPs similar to those seen in quiescent cells (Fig. 6B, b and c).


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Fig. 6.   Effect of nifedipine on responses evoked by intrinsic nerve stimulation in guinea pig trachea. A: responses recorded in control physiological saline in response to 1 (a), 5 (b), and 10 (c) impulses delivered at 10 Hz. B: responses recorded from the same cell in presence of nifedipine (1 µM). Note presence of small spontaneous membrane depolarizations that persisted in presence of nifedipine. Resting membrane potential in all traces was -51 mV. Indomethacin (10 µM) was present throughout.



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Fig. 7.   Effect of nifedipine and ryanodine on responses evoked by intrinsic nerve stimulation in guinea pig trachea. A: effect of nifedipine followed by ryanodine on responses evoked by a single stimulus. Application of nifedipine (Ab) to physiological saline caused a reduction in initial component of response and abolished the 2nd slow depolarization. Subsequent depletion of intracellular stores with ryanodine (60 min; Ac) greatly reduced initial component of response. Response remaining in presence of nifedipine and ryanodine was abolished by muscarinic-receptor antagonist hyoscine (Ad). B: effects of adding ryanodine followed by nifedipine on EJPs evoked by intrinsic nerve stimulation. Application of ryanodine greatly reduced both initial and 2nd component of EJP evoked by a single impulse (Bb). Subsequent addition of nifedipine had no effect on small rapid depolarization but abolished slow secondary depolarization that persisted after store depletion (Bc). Hyoscine abolished rapid depolarization that persisted in presence of both ryanodine and nifedipine (Bd). Aa and Ba: responses in physiological saline. Resting membrane potential in A, a and b, was -49 mV. Resting membrane potential in Ba was -54 mV. In both experiments, ryanodine depolarized membrane potential by ~10 mV. Indomethacin (10 µM) was present throughout.

Ryanodine (10 µM) caused a 93% reduction in the peak amplitude (control, 26.5 ± 2.4 mV; ryanodine, 2.3 ± 1.2 mV; n = 5) and abolished the slowly decaying phase of EJPs evoked by single stimuli (Fig. 7B, a and b). Caffeine (3 mM) abolished the EJPs evoked by a single impulse (n = 3). Ryanodine or caffeine abolished the nifedipine-resistant small spontaneous depolarizations. In preparations pretreated with nifedipine (10 µM), ryanodine (10 µM, 60 min) reduced the amplitude of EJPs from 15.1 ± 2.3 to 4.0 ± 1.6 mV (P < 0.05; n = 7; Fig. 7Ac). However, if preparations were pretreated with ryanodine (10 µM), the subsequent addition of nifedipine had no effect on ryanodine-resistant EJPs (Fig. 7Bc). The EJPs had amplitudes of 29.0 ± 2.2 and 3.7 ± 1.3 mV in the control and ryanodine-containing solutions, respectively. After nifedipine was added, the EJPs had an amplitude of 3.8 ± 1.6 mV (n = 3).

The small nerve-evoked depolarizations that persisted in the presence of both nifedipine and ryanodine had time courses similar to the control EJPs. This resistant component increased in amplitude with an increasing number of stimuli and was invariably abolished by hyoscine (1 µM; n = 3; Fig. 7, Ad and Bd).

Ionic mechanisms underlying EJPs. If store-released Ca2+ contributed to the EJPs, the depolarizations would be expected to result from activation of sets of Ca2+-activated channels, presumably Ca2+-activated Cl- channels (5, 12, 29). The effect of reducing external Cl- concentration ([Cl-]o) was examined in preparations treated with nifedipine (10 µM) to block Ca2+ entry via CaL channels. Reducing [Cl-]o by substitution with isethionate ions did not change the resting membrane potential. In three of five cells, reducing [Cl-]o to 10% of the control value caused an immediate, but transient, 50% increase in the amplitude of EJPs. However, in all preparations, after 15 min in low [Cl-]o, both EJPs and small spontaneous depolarizations were abolished (Fig. 8).


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Fig. 8.   Effect of reducing external concentration of Cl- on EJPs recorded from tracheal smooth muscle of guinea pig. A: EJP recorded in control solution. Substitution of Cl- with isethionate ions abolished EJP evoked by the same stimulus (B). Perfusion with low-Cl- solution also abolished small spontaneous membrane depolarizations. Resting membrane potential in both traces was -53 mV. All recordings were made in presence of nifedipine (1 µM) and indomethacin (10 µM).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study has shown that the responses to cholinergic nerve stimulation in tracheal smooth muscle result both from the release of Ca2+ from intracellular stores and from the entry of Ca2+ via CaL channels. After release from the intracellular stores, Ca2+ activates sets of Ca2+-activated Cl- channels to trigger a depolarization, which, in turn, activates CaL channels. In addition, many preparations generate spontaneous myogenic activity that also appears to result from intracellular Ca2+ release and the influx of Ca2+ through CaL channels.

Few studies have indicated a role for Ca2+ entry through CaL channels in the generation of agonist-induced contractions (10, 15), with Ca2+ antagonists or a low external Ca2+ concentration ([Ca2+]) having little effect on the responses evoked by muscarinic-receptor agonists (1, 2, 13, 21, 26, 27). The insensitivity of agonist-induced contractions to Ca2+ antagonists has led to the suggestion that contraction of airway smooth muscle results solely from intracellular Ca2+ release after the production of inositol 1,4,5-trisphosphate (4). Membrane depolarization then results secondarily from an increase in intracellular [Ca2+] ([Ca2+]i) and the subsequent activation of Ca2+-dependent Cl- conductances (23, 24). Clearly, the role played by Ca2+ entering through CaL channels in the generation of tracheal smooth muscle contraction may vary depending on the source of ACh. In the present study, tracheal EJPs with characteristics similar to those previously described in guinea pig, dog, and ox airway smooth muscle (6, 13, 20, 30) were reduced but not abolished by nifedipine, suggesting that Ca2+ entry through CaL channels contributes to the membrane depolarization. EJPs were also greatly attenuated after the depletion of Ca2+ from the intracellular stores with either caffeine or ryanodine. Because nifedipine reduced the amplitude of EJPs before but not after Ca2+ store depletion, an initial membrane depolarization must be required to initiate Ca2+ entry through CaL channels. In the present study, there was no evidence for the activation of receptor-operated Ca2+ channels by parasympathetic nerve stimulation (see Ref. 33). Although a small membrane depolarization remained in the presence of ryanodine, this was abolished by a low [Cl-]o, suggesting that it results from the release of Ca2+ from inside the cells. Perhaps stores were not completely depleted by the addition of ryanodine or, alternatively, a second internal store may be involved in this component of the response. The latter suggestion is supported by the observation that caffeine, in contrast to ryanodine, was able to abolish EJPs.

Spontaneous electrical slow-wave activity has been observed in airway smooth muscle of the guinea pig (1, 17, 30, 32, 34), human (18), ox (28), and dog (21). Similar slow-wave activity may also be initiated by parasympathetic nerve stimulation (22), the bath application of ACh (6, 21), or blocking K channels with tetraethylammonium chloride (21). Both spontaneous and agonist-evoked slow waves were abolished after removal of external Ca2+ and after CaL channels were blocked with nifedipine (1, 3, 21, 27). It has therefore been suggested that Ca2+ entry through CaL channels is required for the generation of slow-wave activity in tracheal smooth muscle. In the present study, spontaneous and evoked membrane potential oscillations were abolished by nifedipine or after the depletion of intracellular Ca2+ stores with ryanodine. Thus an increase in [Ca2+]i resulting from either the influx of Ca2+ through CaL channels or the second messenger release of Ca2+ from intracellular stores may be required for the initiation of Ca2+-induced Ca2+ release from the intracellular stores and the maintenance of membrane potential oscillations. In isolated guinea pig tracheal smooth muscle cells, spontaneous transient inward currents (STICs), which result from activation of Ca2+-activated Cl- channels, display bursts of rhythmic activity that persist when the membrane potential is voltage clamped to -60 mV to inhibit the influx of Ca2+ through CaL channels (25). It has therefore been suggested that STICs, which result from the cyclic release of Ca2+ from intracellular stores, provide the basis for rhythmic activity (25). The oscillations in membrane potential and [Ca2+]i observed in tracheal smooth muscle might therefore result from the generation of STICs, causing membrane depolarization and the activation of CaL channels. The influx of Ca2+ through CaL channels would then result in further release and reuptake of Ca2+ from intracellular stores.

In the present experiments, it was apparent that different muscle bundles from within any given preparation often showed differing spontaneous activity and may respond differently to parasympathetic nerve stimulation. This suggests that smooth muscle bundles of the trachea are not well coupled. This has previously been implied in the guinea pig trachea where electrical and tension recordings taken simultaneously from two different regions of the trachea displayed quite different behaviors (14). This is despite the histological evidence that neighboring muscle bundles form cross-connections with one another (14, 19). Such poor coupling within muscle bundles of tracheal smooth muscle may explain the apparent discrepancies in observations in the actions of nifedipine on both spontaneous activity and the responses to cholinergic agonists in tracheal smooth muscle. For example, it has been suggested that membrane potential oscillations in tracheal smooth muscle are not directly representative of tone (1, 34). This is based on the observation that nifedipine abolishes oscillations in membrane potential but has no effect on overall tone of the tissue. If muscle bundles within the trachea act independently of one another, changes in membrane potential occurring in one region of the smooth muscle may not truly reflect the changes that are occurring in other regions of the smooth muscle. Therefore, although stimulation of the parasympathetic nerves appears to produce a synchronization of responses in all muscle bundles, the long-term effects of nerve stimulation may differ between muscle bundles. Presumably, this allows for both the local and overall regulation of tracheal smooth muscle tone.


    ACKNOWLEDGEMENTS

I thank Prof. David Hirst and Dr. Frank Edwards for helpful discussions and comments on the manuscript.


    FOOTNOTES

This work was supported by the National Health and Medical Research Council of Australia.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: N. J. Bramich, Dept. of Zoology, Univ. of Melbourne, Parkville, Victoria 3052, Australia (E-mail: n.bramich{at}zoology.unimelb.edu.au).

Received 28 December 1998; accepted in final form 10 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ahmed, F., R. W. Foster, and R. C. Small. Some effects of nifedipine in guinea-pig isolated trachealis. Br. J. Pharmacol. 84: 861-869, 1985[Abstract].

2.   Ahmed, F., R. W. Foster, R. C. Small, and A. H. Weston. Some features of the spasmogenic actions of acetylcholine and histamine in guinea-pig isolated trachealis. Br. J. Pharmacol. 83: 227-233, 1984[Abstract].

3.   Allen, S. L., R. W. Foster, R. C. Small, and R. Towart. The effects of the dihydropyridine Bay K 8644 in guinea-pig isolated trachealis. Br. J. Pharmacol. 86: 171-180, 1985[Abstract].

4.   Berridge, M. J. Inositol trisphosphate and calcium signalling. Nature 361: 315-325, 1993[ISI][Medline].

5.   Bramich, N. J., and G. D. S. Hirst. Sympathetic neuroeffector transmission in the rat anococcygeus muscle. J. Physiol. (Lond.) 516: 101-115, 1999[Abstract/Free Full Text].

6.   Cameron, A. R., and C. T. Kirkpatrick. A study of excitatory neuromuscular transmission in the bovine trachea. J. Physiol. (Lond.) 270: 733-745, 1977[ISI][Medline].

7.   Canning, B. J., and B. J. Undem. Parasympathetic innervation of airways smooth muscle. In: Airways Smooth Muscle: Structure, Innervation and Neurotransmission, edited by D. Raeburn, and M. A. Giembycz. Basel: Birkhauser Verlag, 1994, p. 43-78.

8.   Cousins, H. M., F. R. Edwards, and G. D. S. Hirst. Neuronally released and applied acetylcholine on the longitudinal muscle of guinea-pig ileum. Neuroscience 65: 193-207, 1995[ISI][Medline].

9.   Cousins, H. M., F. R. Edwards, G. D. S. Hirst, and I. R. Wendt. Cholinergic neuromuscular transmission in the longitudinal muscle of the guinea-pig ileum. J. Physiol. (Lond.) 471: 62-86, 1993.

10.   Croxton, T. L., C. Fleming, and C. A. Hirshman. Expression of dihydropyridine resistance differs in porcine bronchial and tracheal smooth muscle. Am. J. Physiol. Lung Cell. Mol. Physiol. 267: L106-L112, 1994[Abstract/Free Full Text].

11.   Cuss, F. M., J. D. Palmer, and P. J. Barnes. Effect of nifedipine on autonomic control of airway smooth muscle in vitro (Abstract). Am. Rev. Respir. Dis. 131: A283, 1985.

12.   Daniel, E. E., J. Jury, and J.-P. Bourreau. Chloride and depolarization by acetylcholine in canine airway smooth muscle. Can. J. Physiol. Pharmacol. 71: 284-292, 1993[ISI][Medline].

13.   Daniel, E. E., J. Jury, R. Serio, and L. P. Jager. Role of depolarization and calcium in contractions of canine trachealis from endogenous or exogenous acetylcholine. Can. J. Physiol. Pharmacol. 69: 518-525, 1991[ISI][Medline].

14.   Dixon, J. S., and R. C. Small. Evidence of poor conduction of muscle excitation in the longitudinal axis of guinea-pig isolated trachea. Br. J. Pharmacol. 79: 75-83, 1983[Abstract].

15.   Fanta, C. H., C. S. Venugopalan, P. G. Lacouture, and J. M. Drazen. Inhibition of bronchoconstriction in the guinea pig by a calcium channel blocker, nifedipine. Am. Rev. Respir. Dis. 125: 61-66, 1982[ISI][Medline].

16.   Hirst, G. D. S., J. K. Choate, H. M. Cousins, F. R. Edwards, and M. Klemm. Transmission by postganglionic axons of the autonomic nervous system: the importance of the specialized neuroeffector junction. Neuroscience 73: 7-23, 1996[ISI][Medline].

17.   Honda, K., T. Satake, K. Takagi, and T. Tomita. Effects of relaxants on electrical and mechanical activities in the guinea-pig tracheal muscle. Br. J. Pharmacol. 87: 665-671, 1986[Abstract].

18.   Honda, K., and T. Tomita. Electrical activity in isolated human tracheal muscle. Jpn. J. Physiol. 37: 333-336, 1987[ISI][Medline].

19.   Hoyes, A. D., and P. Barber. Innervation of the trachealis muscle in the guinea pig: a quantitative ultrastructural study. J. Anat. 130: 789-800, 1980[ISI][Medline].

20.   Ito, Y., and K. Tajima. Actions of indomethacin and prostaglandins on neuro-effector transmission in the dog trachea. J. Physiol. (Lond.) 319: 379-392, 1981[ISI][Medline].

21.   Janssen, L. J., and E. E. Daniel. Depolarizing agents induce oscillations in canine bronchial smooth muscle membrane potential: possible mechanisms. J. Pharmacol. Exp. Ther. 259: 110-117, 1991[Abstract].

22.   Janssen, L. J., C. Hague, and R. Nana. Ionic mechanisms underlying electrical slow waves in canine airway smooth muscle. Am. J. Physiol. Lung Cell. Mol. Physiol. 275: L516-L523, 1998[Abstract/Free Full Text].

23.   Janssen, L. J., and S. M. Sims. Acetylcholine activates nonselective cation and chloride conductances in canine and guinea-pig tracheal myocytes. J. Physiol. (Lond.) 453: 197-218, 1992[Abstract].

24.   Janssen, L. J., and S. M. Sims. Histamine activates Cl- and K+ currents in guinea-pig tracheal myocytes: convergence with muscarinic signalling pathway. J. Physiol. (Lond.) 465: 661-677, 1993[Abstract].

25.   Janssen, L. J., and S. M. Sims. Spontaneous transient inward currents and rhythmicity in canine and guinea-pig tracheal smooth muscle cells. Pflügers Arch. 427: 473-480, 1994[ISI][Medline].

26.   Kannan, M. S., and C. Davis. Mode of action of calcium antagonists on responses to spasmogens and antigen challenge in human airway smooth muscle. Respir. Physiol. 74: 15-24, 1988[ISI][Medline].

27.   Kannan, M. S., Y. S. Prakash, T. Brenner, J. R. Mickelson, and G. C. Sieck. Role of ryanodine receptor channels in Ca2+ oscillations of porcine tracheal smooth muscle. Am. J. Physiol. Lung Cell. Mol. Physiol. 272: L659-L664, 1997[Abstract/Free Full Text].

28.   Kirkpatrick, C. T. Tracheobronchial smooth muscle. In: Smooth Muscle: An Assessment of Current Knowledge, edited by E. Bülbring, A. F. Brading, A. W. Jones, and T. Tomita. London: Arnold, 1981, p. 385-395.

29.   Large, W. A., and Q. Wang. Characteristics and physiological role of the Ca2+-activated Cl- conductance in smooth muscle. Am. J. Physiol. Cell Physiol. 271: C435-C454, 1996[Abstract/Free Full Text].

30.   McCaig, D. J. Electrophysiology of neuroeffector transmission in the isolated, innervated trachea of the guinea-pig. Br. J. Pharmacol. 89: 793-801, 1986[Abstract].

31.   McCaig, D. J., and S. A. Aitken. Effects of calcium modulators on vagally-mediated constriction in the guinea-pig isolated trachea. Br. J. Pharmacol. 104: 343-348, 1991[Abstract].

32.   McCaig, D. J., and J. F. Souhrada. Alteration of electrophysiological properties of airway smooth muscle from sensitized guinea-pigs. Respir. Physiol. 41: 49-60, 1980[ISI][Medline].

33.   Murray, R. K., and M. I. Kotlikoff. Receptor-activated calcium influx in human airway smooth muscle cells. J. Physiol. (Lond.) 435: 123-144, 1991[Abstract].

34.   Small, R. C. Electrical slow waves and tone of guinea-pig isolated trachealis muscle: effects of drugs and temperature changes. Br. J. Pharmacol. 77: 45-54, 1982[ISI][Medline].


Am J Physiol Lung Cell Mol Physiol 278(2):L320-L328
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society




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