Vol. 278, Issue 2, L320-L328, February 2000
Electrical behavior of guinea pig tracheal smooth
muscle
Narelle J.
Bramich
Department of Zoology, University of Melbourne, Parkville, Victoria
3052, Australia
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ABSTRACT |
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
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INTRODUCTION |
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.
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METHODS |
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 M
) 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.
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RESULTS |
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.
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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.
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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.
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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.
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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.
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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.
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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).
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DISCUSSION |
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.
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ACKNOWLEDGEMENTS |
I thank Prof. David Hirst and Dr. Frank Edwards for helpful
discussions and comments on the manuscript.
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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.
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