1 Research Group in Mechanisms of Diseases, Department of Physiology, Faculty of Medicine, The University of Hong Kong, and 2 Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China SAR
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ABSTRACT |
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Full muscarinic stimulation in bovine tracheal smooth muscle caused a sustained contraction and increase in intracellular Ca2+ concentration ([Ca2+]i) that was largely resistant to inhibition by nifedipine. Depletion of internal Ca2+ stores with cyclopiazonic acid resulted in an increased efficacy of nifedipine to inhibit this contraction and the associated increase in [Ca2+]i. Thus internal Ca2+ store depletion promoted electromechanical coupling between full muscarinic stimulation and muscle contraction to the detriment of pharmacomechanical coupling. A similar change in coupling mode was induced by ryanodine even when it did not significantly modify the initial transient increase in [Ca2+]i induced by this stimulation, indicating that depletion of internal stores was not necessary to induce the change in excitation-contraction coupling mode. Blockade of the Ca2+-activated K+ channel by tetraethylammonium, charybdotoxin, and iberiotoxin all induced the change in excitation-contraction coupling mode. These results suggest that in this preparation, Ca2+ released from the ryanodine-sensitive Ca2+ store, by activating Ca2+-activated K+ channels, plays a central role in determining the expression of the pharmacomechanical coupling mode between muscarinic excitation and the Ca2+ influx necessary for the maintenance of tone.
calcium release; calcium influx; potassium channels; tracheal smooth muscle
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INTRODUCTION |
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AIRWAY SMOOTH MUSCLES contract tonically in response to muscarinic stimulation. This contraction is associated with a biphasic change in intracellular free Ca2+ concentration ([Ca2+]i) and a graded membrane depolarization that often oscillates (4, 11, 17). Activation of muscarinic receptors stimulates phospholipase C, resulting in the formation of inositol trisphosphate [Ins(1,4,5)P3] that acts as second messenger to release Ca2+ from internal stores (7, 8). Release of Ca2+ from Ins(1,4,5)P3-sensitive internal stores is transient and occurs immediately after muscle stimulation, leading to a transient contraction. An influx of extracellular Ca2+ is required to maintain the sustained tension observed during prolonged muscarinic stimulation (5, 6).
The route of Ca2+ entry into the cytosol in airway smooth
muscle cells during muscarinic stimulation is multiple. Two types (L
and T) of voltage-operated Ca2+ channels (VOCCs) have been
identified in airway muscles (13). The L-type VOCC is
activated at 30 to
15 mV, and maximum current is seen at positive
potentials in tracheal smooth muscle (20). Because ACh
depolarizes the plasma membrane from about
60 to
40 or
30 mV
(4, 9, 11), the channel is
little activated, and voltage-dependent Ca2+ entry into
cells through the VOCCs is not the main contributor for maintaining the
tone induced by muscarinic stimulation in nearly all animal airway
preparations (3). The Ca2+ influx that is the
main contributor to the maintenance of tone does not occur through
voltage-dependent channels and is not sensitive to inhibition by
Ca2+ channel blockers (4-6,
23). Thus airway smooth muscle uses a mode of coupling
between muscarinic stimulation and Ca2+ entry that is
independent of membrane potential changes and was labeled
pharmacomechanical coupling by Somlyo and Somlyo (30). This fact may explain the lack of beneficial clinical effects of
Ca2+ channel blockers in the management of most obstructive
lung diseases (3).
Amaoko et al. (1), Baba et al. (2), and Bourreau (4) have reported previously that the coupling between the stimulation of muscarinic receptors and airway muscle contraction can switch from the pharmacomechanical mode to a mode dependent on membrane potential, labeled electromechanical coupling (30). This switch in excitation-contraction (E-C) coupling occurs after inhibition of the sarcoplasmic reticulum (SR) Ca2+ pumps with cyclopiazonic acid (CPA), and this renders Ca2+ entry blockers and K+ channel openers very efficient in inhibiting contractions induced by muscarinic agonists (1, 4). Because CPA eventually depletes internal Ca2+ stores and eliminates the Ca2+ release from these stores induced by muscarinic stimulation (4, 6, 10, 16), this observation suggests that the coupling mode between muscarinic receptor stimulation and muscle contraction in airway muscle may be under the influence of Ca2+ release from internal Ca2+ stores. However, the exact mechanism(s) responsible for this switch are not known.
The membrane of airway smooth muscle strongly rectifies due to the activity of K+ channels (12, 18, 19, 22). This profound rectification limits depolarization during muscarinic stimulation, which contributes to the observation that Ca2+ influx through the VOCCs is not the main direct contributor to the maintenance of tone. The activation of a large-conductance Ca2+-activated (KCa) channel by Ca2+ released from the SR (24, 25, 28) could be an essential element in controlling the amount of depolarization during muscarinic stimulation and, perhaps, Ca2+ entry through the VOCCs. It has been shown in other smooth muscles that the local increase in [Ca2+]i (Ca2+ spark) resulting from activation of ryanodine-sensitive channels in the SR limited muscle contraction by activating the KCa channel (24, 38). A similar mechanism could explain why the E-C coupling mode in bovine tracheal smooth muscle switches from a pharmacomechanical to an electromechanical mode after depletion of internal Ca2+ stores by CPA. Indeed, if muscarinic stimulation is no longer able to release Ca2+ from internal stores, one could expect that the stimulation of Ca2+-activated conductances would decrease (12, 14, 15, 19). Ca2+ entry through the VOCCs could thus be promoted, whereas Ca2+ entry through the non-VOCCs (23) would be decreased because the driving force for Ca2+ decreases when the membrane depolarizes. If this is true, compounds that block the KCa channel should have similar effects as compounds, such as CPA or ryanodine, that affect Ca2+ release from the SR on the E-C coupling mode in our preparation (10, 37). The aim of this study was to test this hypothesis, and we report in this manuscript supporting data from contraction experiments on bovine tracheal smooth muscle strips and studies with the fura 2 fluorescence technique in single bovine tracheal myocytes.
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MATERIALS AND METHODS |
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Tissue Preparation and Organ Bath Experiment
Fresh bovine tracheae were obtained from a local abattoir and transported to the laboratory in ice-cold, HEPES-buffered solution containing (in mM) 154 NaCl, 4.6 KCl, 1.25 CaCl2, 1.5 MgCl2, 5.95 NaHCO3, 10 HEPES, and 10 glucose and 3.5 mg/l of indomethacin, pH 7.4. The smooth muscle layer of the trachealis was dissected free of cartilage, mucosa, connective tissue, and fat and cut into thin strips (1 mm wide and 1 cm long) with scissors. The strips were mounted for isometric tension recording (Grass FT.03) under a load of 2 g in organ baths containing Krebs solution at 37°C and gassed with 5% CO2-95% O2 (pH 7.4). Preparations were then repetitively challenged with 60 mM KCl until stable contractile responses were obtained. After this equilibration procedure, the preparations were incubated for 1 h with vehicle or drugs that affect Ca2+ release from internal stores or K+ channel blockers. Preparations were then challenged with a maximally effective concentration of bethanechol (100 µM), and stable tone was allowed to develop. When the tonic contraction induced by bethanechol had stabilized, 1 µM nifedipine was added to the medium and the tension was allowed to relax. Atropine (10 µM) was used to induce relaxation of the bethanechol-contracted strips, and the amplitude of the atropine-sensitive tonic contraction induced by bethanechol in the various conditions was normalized to the contraction induced by 60 mM KCl in the control solution.Preparation of Isolated Bovine Tracheal Smooth Muscle Cells
After dissection as described in Tissue Preparation and Organ Bath Experiment, the smooth muscle strips (0.5 mm wide and 1 mm long) were transferred to a digestion solution (in mM: 136.9 NaCl, 6.2 KCl, 0.2 CaCl2, 0.5 MgCl2, 10 HEPES, and 10 glucose and 1.0 g/l of bovine serum albumin, pH 7.4) containing 200 U/ml of collagenase (type I), 10 U/ml of elastase (porcine pancreas), and 0.2 mg/ml of trypsin inhibitor (type 1-S). The tissues were incubated with gentle shaking for 40 min at 37°C. After this incubation, the digestion solution containing the enzymes was removed, and the preparations were washed three times in digestion solution without enzyme. Digested tissue was then gently pushed and sucked through a pipette for six steps of 5 min each to liberate individual myocytes. At the end of each step, isolated myocytes in suspension were collected (1-ml samples). The samples were pooled together, and Ca2+ concentration in the solution was increased gradually from 0.2 to 1.5 mM in 20 min. The solution was then centrifuged at 1,000 rpm for 5 min, and the cells were resuspended at a density of ~1 × 105 cells/ml in medium 199 containing 10% fetal bovine serum. The cells were then incubated for 24 h on glass coverslips coated with poly-L-lysine in 24-well tissue culture clusters in a 95% air-5% CO2 atmosphere at 37°C.Measurement of [Ca2+]i
After incubation, the coverslips were removed from the culture well and washed in an incubation well with a physiological salt solution (PSS) containing (in mM) 130 NaCl, 5.6 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 11 D-glucose and 1 mg/ml of bovine serum albumin, pH 7.4. The cells attached to the coverslips were then incubated with fura 2-AM at a concentration of 3 µM in PSS for 40 min. The unincorporated dye was then washed out twice, and the cells loaded on the coverslips were stored at room temperature (20-22°C) in the dark until used 30-240 min after the end of the loading period. The coverslips were then fitted at the bottom of an incubation chamber mounted on the stage of an inverted microscope (Nikon). The PSS solution containing 1% bovine serum albumin in the incubation chamber (1 ml) could be drained by vacuum suction and replaced with test solution within 5 s. The inverted microscope was coupled to a dual-excitation spectrofluorometer system (PTI), and the emitted light was filtered at 510 nm before being recorded. Fluorescent signals induced by excitation at 340 and 380 nm were stored for subsequent data processing and analysis. The changes in the ratio of the signals obtained at 340 and 380 nm were used as an index of the fluctuations in [Ca2+]i induced by drugs.Drugs and Chemicals
Bethanechol chloride, ryanodine, nifedipine, CPA, charybdotoxin (ChTX), and iberiotoxin (IbTX) were purchased from RBI. Type I collagenase was from Worthington Biochemical; elastase was from Boehringer Mannheim; and fura 2-AM was from Molecular Probes. Trypsin inhibitor, tetraethylammonium (TEA), medium 199, and bovine serum albumin were obtained from Sigma. All the chemicals were dissolved in distilled water except fura 2-AM, CPA, and ryanodine, which were dissolved in dimethyl sulfoxide, and nifedipine, which was dissolved in 95% ethyl alcohol. The final concentration of the solvents was <0.1%.Statistical Analysis
For contractility data, the number of experiments stated refers to the number of tracheae obtained from different animals. Tests were performed in triplicate on strips dissected from each trachea. For Ca2+ measurements in isolated cells, sample cells in each batch prepared from tissue from one animal were tested for their ability to respond to bethanechol stimulation, and the batch was used for experimentation if bethanechol induced a biphasic change in [Ca2+]i in these cells similar to the biphasic change in [Ca2+]i induced in freshly isolated cells and muscle strips from bovine trachealis muscle. Experiments were carried out in separate batches, and the number of cells tested from these batches is given.All data were analyzed with GraphPad PRISM software (GraphPad, San Diego, CA.) and are presented as means ± SE. Unpaired Student's t-test was used to determine the effects of drugs. Probability values < 0.05 were considered to be significant.
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RESULTS |
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Contractile Responses
Effect of nifedipine and removal of extracellular Ca2+
on atropine-sensitive tonic contractions induced by bethanechol.
In Ca2+-containing medium, 100 µM bethanechol induced
sustained contractions of bovine tracheal smooth muscle strips (Fig.
1A), whereas in a
Ca2+-free solution (plus 100 µM EGTA), the
contractile response to bethanechol stimulation was only
transient (Fig. 1B).
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Effect of nifedipine on atropine-sensitive tonic contractions
induced by bethanechol in the presence of compounds that affect
intracellular Ca2+ stores.
CPA. After equilibration, the addition of 30 µM CPA to
the medium induced a biphasic increase in tone, which often oscillated (Fig. 2A). The addition of 100 µM
bethanechol further increased the tone, and there was no difference
between the maximum amplitudes of the tonic contraction induced by
bethanechol in control and CPA-containing media (P > 0.05; n = 9 experiments; Fig. 2B). In the
presence of CPA, when the tension induced by bethanechol had stabilized, 1 µM nifedipine almost completely relaxed the
preparations (Fig. 2, A and B). The steady-state
contraction induced by bethanechol was similar in a Ca2+
medium containing CPA and nifedipine and in a Ca2+-free
medium (Figs. 1C and 2B). The increase in the
ability of nifedipine to relax tonic contractions induced by
bethanechol was significant (P < 0.05;
n = 4-9 experiments) when CPA was present at
concentrations between 1 and 30 µM, and the effect of CPA was apparently concentration dependent, reaching a maximum at 10 µM (Fig. 3A).
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Effect of nifedipine on atropine-sensitive tonic contractions
induced by bethanechol in the presence of TEA.
Ten millimolar TEA itself induced a large increase in tone. Bethanechol
(100 µM) could further increase the muscle tension, and under these
experimental conditions, nifedipine almost completely relaxed the tone
induced by TEA and bethanechol (Fig. 5,
A and B). In the presence of TEA, the
maximum amplitude of the tonic contraction induced by bethanechol was
smaller than that in the control medium (P < 0.05;
Fig. 5B). The inhibition by nifedipine of the
atropine-sensitive contraction induced by bethanechol was larger
(P < 0.05) in the presence of TEA at concentrations
between 0.5 and 10 mM (Fig. 3C).
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Depolarizing effect of CPA and ryanodine. Figure 4 shows that the contractions of bovine tracheal smooth muscle strips induced by 30 µM CPA and 30 µM ryanodine were sensitive to inhibition by the K+ channel opener cromakalim and by the L-type channel blocker nifedipine. Although contractions induced by CPA and ryanodine shared a similar sensitivity to inhibition by cromakalim and nifedipine, the amplitude of the contraction induced by CPA was larger than the contraction induced by ryanodine in this preparation (P < 0.05; Fig. 4D).
Caffeine (10 mM) induced a contraction similar in amplitude to the contraction induced by ryanodine (Fig. 4D). However, this contraction was not sensitive to inhibition by cromakalim or nifedipine (Fig. 4, C and D).Ca2+ Signals
Nifedipine sensitivity of the Ca2+ influx
evoked by full muscarinic stimulation: effect of CPA and ryanodine.
In Ca2+-containing medium, single cells responded to
bethanechol stimulation with a rapid increase in
[Ca2+]i, which decayed and stabilized above
the resting level. This response was not affected by 1 µM nifedipine,
whereas in a Ca2+-free solution, the steady-state increase
in [Ca2+]i induced by bethanechol was absent
(Fig. 6).
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Effects of selective blockade of KCa channels
on the efficacy of nifedipine to inhibit
Ca2+ influx induced by muscarinic
stimulation.
Two K+ channels have been described in bovine tracheal
smooth muscle cells: the delayed rectifier K+ channel,
which is blocked preferentially by 4-aminopyridine, and a
large-conductance KCa channel, which is inhibited
preferentially by TEA at millimolar concentrations and more potently
and more selectively by ChTX and IbTX (19,
21, 22). In single bovine tracheal smooth
muscle cells, in the presence of ChTX (100 nM) or IbTX (100 nM), full
muscarinic stimulation with 100 µM bethanechol still induced a
biphasic increase in [Ca2+]i (Fig.
9, A and C), which
was not significantly different from the control value (Fig.
9E). However, in the presence of ChTX (100 nM) or IbTX (100 nM), nifedipine inhibited the sustained increase in
[Ca2+]i induced by bethanechol stimulation
(Fig. 9, B, D, and F).
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DISCUSSION |
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In this study, we observed that full activation of muscarinic receptors in bovine tracheal smooth muscle by large concentrations of bethanechol in PSS induced sustained contractions of muscle strips and a biphasic change in [Ca2+]i in isolated myocytes. This biphasic change in [Ca2+]i resulted from the recruitment of two Ca2+ pools, i.e., the intracellular CPA-sensitive Ca2+ stores and the extracellular space. The initial transient increase in [Ca2+]i was completely abolished after prolonged incubation of cells with CPA, which results in depletion of internal agonist-recruitable Ca2+ stores as previously described (5, 6, 10, 16). Thus this component of the Ca2+ signal was due to Ca2+ release from intracellular Ca2+ stores. The sustained increase in [Ca2+]i was absent in Ca2+-free medium, indicating the requirement of the extracellular Ca2+ pool for this component of the Ca2+ signal, but was not very sensitive to nifedipine, suggesting that the Ca2+ influx induced by muscarinic stimulation and contributing to the maintenance of tone and the elevation in [Ca2+]i occurs mainly via non-VOCCs. These data are in agreement with previous reports (5, 9, 11, 23) showing that airway smooth muscle uses a coupling mode between full muscarinic stimulation and contraction that is independent of membrane potential under normal conditions, labeled pharmacomechanical coupling by Somlyo and Somlyo (30).
The efficacy of nifedipine to inhibit the contractile response of
strips to bethanechol stimulation was significantly increased after
depletion of intracellular Ca2+ stores with CPA as
previously reported (1, 4). In single cells,
pretreatment with CPA abolished the initial transient increase in
[Ca2+]i but largely increased the sustained
increase in [Ca2+]i induced by bethanechol
stimulation (see also Ref. 16). This larger influx of Ca2+
induced by bethanechol stimulation in the presence of CPA was also very
sensitive to inhibition by nifedipine. These results agree with
previous data by Bourreau (4) suggesting that depletion of
Ca2+ stores can switch the coupling mode between muscarinic
stimulation and contraction from pharmacomechanical to
electromechanical and that the coupling mode between muscarinic
stimulation and Ca2+ influx is regulated by intracellular
Ca2+ stores. A simplified model to explain these
observations would be that Ca2+ released from internal
stores immediately after muscarinic stimulation activates
Ca2+-dependent Cl and K+
conductances (15, 34). The balance between
activation of these conductances, together with activation of
nonspecific cation conductances (15), would then dictate
the amount of depolarization induced by agonist stimulation and the
rate of Ca2+ influx through VOCCs. Because the rate of
Ca2+ influx determines the amount of Ca2+ that
can escape the superficial buffer barrier and reach the contractile
machinery (14, 32, 33), the
amount of depolarization induced by agonist stimulation should affect
the sensitivity of the tonic contraction to inhibition by VOCC
blockers. Inhibition of Ca2+ release from internal stores
would reduce activation of KCa channels (27,
38), which might result in an increased depolarization induced by muscarinic stimulation, increased activation of VOCCs, and
an increased sensitivity of the tonic contraction to inhibition by VOCC
blockers (20). In our preparations, CPA could, by itself, induce a large contraction that was associated with an increase in
[Ca2+]i. Most of the response to CPA can be
inhibited if preparations are hyperpolarized with the K+
channel opener cromakalim or if L-type Ca2+ channels are
blocked with nifedipine, suggesting that the response of bovine
trachealis muscle to CPA stimulation resulted from activation of L-type
Ca2+ channels after membrane depolarization, perhaps due to
activation of depolarizing currents by CPA (36). In the
presence of CPA, i.e., when internal Ca2+ stores are
depleted and the membrane is depolarized, both the tonic contraction
and Ca2+ influx induced by bethanechol become sensitive to
inhibition by nifedipine. These data support the model described above.
The model predicts that blockade of K+ channels (especially
KCa channels) should promote depolarization in our
preparations and should result in an increased sensitivity of the tonic
contraction induced by muscarinic stimulation to inhibition by
nifedipine. Data obtained in the presence of TEA, ChTX, or IbTX fit
this prediction and support the model.
The data obtained in the presence of ryanodine suggest that the Ins(1,4,5)P3-mediated Ca2+ released from agonist-recruitable stores did not control the mode of E-C coupling in our preparations. Indeed, a 10-min incubation with ryanodine was sufficient to induce the switch in coupling mode without inducing depletion of internal stores that significantly modified Ins(1,4,5)P3-mediated Ca2+ release induced by bethanechol stimulation. This is suggested in these experiments by the ability of bethanechol to still induce a CPA-sensitive transient increase in [Ca2+]i (see Fig. 8). On average, prolonged stimulation with ryanodine was able by itself to induce a contraction that was nifedipine sensitive. This contraction induced by ryanodine was significantly smaller than the contraction induced by CPA under similar experimental conditions. This probably reflects the ability of CPA, but not of ryanodine, to inhibit SR Ca2+ pumps and to disrupt the superficial buffer barrier (10, 14). Consequently, in the presence of CPA, influx via VOCCs is directly available for contraction (33). Ryanodine, on the other hand, does not inhibit SR Ca2+ pumps but stabilizes Ca2+ release channels in a subconductance state, which prevents their full opening (37). Thus in the presence of ryanodine, the superficial SR should still buffer part of the increased Ca2+ influx via VOCCs. This is supported by data in Figs. 7 and 8 showing that the increase in [Ca2+]i induced by ryanodine alone or by bethanechol in the presence of ryanodine is significantly smaller than the increase in [Ca2+]i induced by CPA alone or by bethanechol in the presence of CPA.
In smooth muscle cells, it has been shown that two distinct Ca2+-release channels exist in the membrane of the SR, Ins(1,4,5)P3-sensitive channels and ryanodine-sensitive channels (8, 25, 26). Stimulation of muscarinic receptors induces Ca2+ release from both channels, and Ca2+ release from ryanodine-sensitive channels has been implicated in the generation of Ca2+ waves and sparks in smooth muscle preparations (25, 26, 28, 29). Recently, Janssen et al. (14) have shown that in airway smooth muscle, Ca2+ release from ryanodine-sensitive channels was targeted toward the plasma membrane and that Ca2+ released from these channels was not a major contributor to the average [Ca2+]i but was to activated Ca2+-dependent conductances. Our data are compatible with these findings and suggest a mechanism that could be responsible for the dominant pharmacomechanical mode of muscarinic E-C coupling in airway smooth muscle.
Muscarinic stimulation causes only a limited, graded membrane
depolarization from a resting value of about 60 to
30 mV
(4, 11), and the strong rectification of the
plasmalemma due to the activity of K+ channels
(12, 18, 19, 22,
34) is probably responsible for limiting depolarization.
Although there is little activation of L-type VOCCs within this voltage
window (20), the channels are not fully inactivated,
thereby allowing a substantial amount of Ca2+ to enter the
cells through VOCCs to reload internal Ca2+ stores
(5). Two K+ channels described in airway
smooth muscle cells confer on the plasma membrane its outward
rectifying property, the delayed rectifier and KCa channels
(19, 21); and muscarinic stimulation of
tracheal smooth muscle cells by acetylcholine causes activation of
KCa channels (34; see, however, Ref. 35). Activation of
KCa channels by ryanodine-sensitive Ca2+
release has been well described in vascular smooth muscle cells, where
local transient increases in [Ca2+]i
(Ca2+ sparks) resulting from Ca2+ release via
ryanodine-sensitive Ca2+ channels in the SR just under the
cell membrane can stimulate KCa channels while having
little contribution to a global increase in
[Ca2+]i (24). In airway smooth
muscle, ryanodine-sensitive Ca2+ release (Ca2+
sparks) can also activate K+ channels (27,
29, 39), and our data with caffeine support this. In our experiment, caffeine had an effect compatible with its
action on ryanodine-sensitive Ca2+ release channels (i.e.,
release of internal Ca2+ via these channels). However,
contractions induced by caffeine were not due to membrane
depolarization because they were not blocked by cromakalim or
nifedipine. This suggests that unlike ryanodine, caffeine did not
inhibit KCa channels but rather, perhaps, activated these
channels as already proposed (34). Thus unlike ryanodine,
caffeine should not modify the coupling between muscarinic stimulation
and muscle contraction from pharmacomechanical to electromechanical
because it should not suppress Ca2+ release through
ryanodine-sensitive channels. Data in Fig. 3E, showing that
at all concentrations tested caffeine did not promote electromechanical
coupling, support this assumption.
Based on these findings, we propose that in our experimental model, blockade or removal of activation of KCa channels with TEA, ChTX, IbTX, or CPA [which also blocks KCa channels (31)] rendered bethanechol-induced contraction very sensitive to inhibition by nifedipine, probably by increasing the rate of Ca2+ influx via VOCCs. The increase in the rate of Ca2+ influx via VOCCs was probably due to a decrease in membrane rectification (22) allowing greater membrane depolarization during muscarinic stimulation. This increased rate of Ca2+ influx would allow Ca2+ to reach the inner cytosol, bypassing the superficial buffer barrier (33), to contribute to contraction.
Thus in bovine tracheal smooth muscle cells, activation of KCa channels by ryanodine-sensitive Ca2+ release may play a central role in limiting the Ca2+ influx through VOCCs after full muscarinic stimulation. Because the Ca2+ influx through VOCCs is limited, the superficial SR can efficiently prevent its access to the inner cytosol (32, 33), limiting its contribution to tonic contraction (4, 5, 9) but making it a significant component of internal stores reloading (5).
What triggers ryanodine-sensitive Ca2+ release during muscarinic stimulation in our preparation remains to be established. Ca2+-induced Ca2+ release and cyclic ADP-ribose-induced Ca2+ release have both been described in airway smooth muscle cells (26, 35), and Ca2+-induced Ca2+ release can activate KCa channels (35). Also, our data show that in the presence of ryanodine or the selective KCa channel inhibitors ChTX and IbTX, the Ca2+ influx during muscarinic stimulation is not different from the Ca2+ influx observed in the absence of these compounds. This implies that only the route of Ca2+ entry to the inner cytosol has changed. Also, when the membrane is depolarized in the presence of TEA and Ca2+ entry via VOCCs is blocked by nifedipine, the tonic contraction induced by full muscarinic stimulation is not different from the contraction induced by the same stimulation in Ca2+-free medium. This suggests that Ca2+ entry is absent in these experimental conditions. Whether Ca2+ entry through non-VOCCs is decreased, perhaps due to a decrease in the driving force for Ca2+ entry, or whether this influx is buffered remains to be established.
In conclusion, our study suggests that ryanodine-sensitive Ca2+ release plays a central role in determining the expression of pharmacomechanical coupling mode between stimulation of muscarinic receptors and Ca2+ influx by activating KCa channels under physiological conditions.
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ACKNOWLEDGEMENTS |
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The contribution of W. Y. Wong in performing some of the experiments presented is greatly appreciated.
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FOOTNOTES |
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This work was supported by a grant from the Committee on Research and Conference (University of Hong Kong, Hong Kong, China) and by Research Grant Council of Hong Kong Grants HKU 7216/97M and HKU 7196/99M.
Address for reprint requests and other correspondence: J.-P. Bourreau, Dept. of Physiology, The Univ. of Hong Kong, 5 Sassoon Rd., Hong Kong, China SAR (E-mail: bourreau{at}hkucc.hku.hk).
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. Section 1734 solely to indicate this fact.
Received 25 October 1999; accepted in final form 13 April 2000.
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