Airway smooth muscle (ASM) cells express voltage-dependent Ca2+ channels, primarily of the L-subtype. These may play a role in excitation-contraction coupling of ASM, although other signaling pathways may also contribute: one of these includes Rho and its downstream effector molecule Rho-associated kinase (ROCK). Although voltage-dependent Ca2+ influx and Rho/ROCK signaling have traditionally been viewed as entirely separate pathways, recent evidence in vascular smooth muscle suggest differently. In this study, we monitored contractile activity (muscle baths) in bronchial and/or tracheal preparations from the pig, cow, and human, and further examined Rho and ROCK activities (Western blots and kinase assays) and cytosolic levels of Ca2+ (fluo 4-based fluorimetry) in porcine tracheal myocytes. KCl evoked substantial contractions that were suppressed in tracheal preparations by removal of external Ca2+ or using the selective L-type Ca2+ channel blocker nifedipine; porcine bronchial preparations were much less sensitive, and bovine bronchi were essentially unaffected by 1 μM nifedipine. Surprisingly, KCl-evoked contractions were also highly sensitive to two structurally different ROCK inhibitors: Y-27632 and HA-1077. Furthermore, the inhibitory effects of nifedipine and of the ROCK inhibitors were not additive. KCl also caused marked stimulation of Rho and ROCK activities, and both these changes were suppressed by nifedipine or by removal of external Ca2+. KCl-induced elevation of [Ca2+]i was not affected by Y-27632 but was reversed by NiCl2 or by BAPTA-AM. We conclude that KCl acts in part through stimulation of Rho and ROCK, possibly secondary to voltage-dependent Ca2+ influx.
- myosin light chain phosphatase
- Rho-associated kinase
- voltage-dependent calcium channels
excitation-contraction coupling in smooth muscle is exceedingly complex, involving several different signaling pathways. One of these is entirely dependent on membrane depolarization, which regulates the amount of Ca2+ entering across the membrane through voltage-dependent Ca2+ channels. Another pathway involves changes in intracellular Ca2+ concentration ([Ca2+]i) mediated by an internal Ca2+ pool: the sarcoplasmic reticulum. Both of these mechanisms have been studied extensively in airway smooth muscle (ASM), although the importance of these pathways in excitation-contraction coupling is still not fully understood (8).
Recently, a third pathway has been found to operate in smooth muscle. Instead of influencing tone through changes in [Ca2+]i, this pathway involves changes in the Ca2+ sensitivity of the contractile apparatus (14, 24). In particular, many constrictor agonists activate the monomeric G protein Rho, which in turn activates its downstream effector molecule Rho-associated kinase (ROCK). ROCK phosphorylates myosin light chain phosphatase (MLCP), decreasing its activity. As a result, there is a greater accumulation of phosphorylated myosin for any given change in [Ca2+]i and thus a change in the Ca2+ sensitivity. In numerous studies using vascular smooth muscle and other cell types, activation of Rho has been shown to involve the heterotrimeric G proteins G12/13 (23, 25). The recent development of highly selective inhibitors of ROCK such as Y-27632 and HA-1077 (2) has greatly facilitated the study of this signaling pathway.
The Rho/ROCK signaling pathway is also employed in ASM in the contractile responses to cholinergic stimulation (6, 13), leukotrienes (21), sphingosine-1-phosphate (19), or to mechanical stress (22), in airway hyperresponsiveness (4, 5), and in the migratory responses to platelet-derived growth factor or leukotrienes (1, 18). However, very little is known about the interactions between this pathway and other signaling pathways (particularly those involving changes in [Ca2+]i) in ASM. Also, it should be emphasized that numerous studies have documented important differences between tracheal and bronchial smooth muscles (TSM and BSM, respectively) as well as between airways from different species, with respect to excitation-contraction coupling (13).
The role of voltage-dependent Ca2+ channels in ASM physiology is not well understood. Although it has long been held that they play the same role in ASM as they do in vascular or gastrointestinal smooth muscles, that is, in electromechanical coupling, the full body of literature is not consistent with this view (8, 11). The robust contractions that can be elicited by high-millimolar potassium chloride (KCl) could suggest that voltage-dependent Ca2+ influx alone is sufficient for contraction. However, this interpretation is based on the assumption that KCl-evoked contractions are mediated solely by voltage-dependent Ca2+ influx. Recent investigations using vascular smooth muscle have revealed a novel mechanism whereby ROCK activation can be triggered by depolarization and/or elevation of [Ca2+]i (16, 20).
In this study, we sought to examine the relative contributions of voltage-dependent Ca2+ influx and Rho/ROCK signaling in the responses to high-millimolar KCl, making comparisons between TSM and BSM as well as among porcine, bovine, and human airways.
Preparation of isolated tissues and single cells.
All experimental procedures were approved by the McMaster University Animal Care Committee, the McMaster University Biosafety Committee, and the St. Joseph's Healthcare Research Ethics Board and conform to the guidelines set by the Canadian Council on Animal Care.
Lobes of lung and tracheae were obtained from pigs (20–90 kg) and cows (136–454 kg) killed at a local abattoir and were immediately put in ice-cold physiological solution for transport to the laboratory. TSM was isolated by removing connective tissue, vasculature, and epithelium and was then cut into strips parallel to the muscle fibers (∼1 mm wide). Lobes of lung were pinned out, the overlying parenchyma and pulmonary vasculature were removed, and ring segments (∼4–5 mm long) of fifth- to sixth-order bronchi (outer diameter 1.5–3 mm) were excised.
Segments of donor (i.e., nondiseased) human main stem bronchi were provided by the Lung Transplant Program (Toronto, Ontario, Canada). The overlying connective tissue, vasculature, and thicker portions of the epithelium were removed, and the smooth muscle was then cut into strips (∼1 mm wide) parallel to the muscle fibers.
Muscle bath technique.
Ring segments were mounted into 2.5-ml muscle baths using stainless steel hooks inserted into the lumen. One hook was tied with 5-0 silk suture to a Grass FT.03 force transducer; the other was attached to a Plexiglas rod that served as an anchor. Tracheal strips were likewise tied to the anchoring rod and the force transducer using silk thread. Tissues were bathed in Krebs-Ringer buffer (see below for composition) containing indomethacin (10 μM), Nω-nitro-l-arginine (l-NNA; 10−4 M), and atropine (1 μM; to block the effects of KCl-induced release of acetylcholine), bubbled with 95% O2/5% CO2, and maintained at 37°C; tissues were passively stretched to impose a preload tension of ∼1 g (determined to allow maximal responses). Isometric changes in tension were amplified, digitized (2 samples/s), and recorded online (DigiMed System Integrator; MicroMed, Louisville, KY) for plotting on the computer. Tissues were equilibrated for 1 h before the experiments commenced, during which time the tissues were challenged with 60 mM KCl three times to assess the functional state of each tissue. In one set of experiments, tissues were pretreated for 20 min (e.g., Y-27632, HA-1077, or nominally Ca2+-free Krebs supplemented with EGTA) before being challenged with increasing concentrations of KCl (0–60 mM) added in cumulative fashion. In a second set of experiments, tissues were pretreated with Y-27632 or vehicle for 20 min and then preconstricted with 60 mM KCl, after which the nifedipine concentration-response relationship was examined, also in cumulative fashion.
Assay for Rho activity.
Tissues that had been flash-frozen were homogenized in ice-cold buffer [50 mM Tris·HCl, pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 750 mM NaCl, 5% Igepal CA-630, 50 mM MgCl2, 10% glycerol, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM PMSF, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 2 mM sodium orthovanadate] with total protein content determined (Bradford method) and adjusted (by addition of media) to make uniform. Tissue homogenates were incubated (60 min at 4°C) with rhotekin-coated cellulose beads (rhotekin specifically binds activated Rho and not inactive Rho). The sample was then lightly centrifuged (14,000 g for 5 s at 4°C) to “pull down” the beads, and the supernate (unbound material) was discarded, after which Rho was dissociated from the beads by incubating with Laemmli sample buffer (62.5 mM Tris·HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.1% 2-mercaptoethanol, 0.01% bromphenol blue). Samples were boiled for 5 min in loading buffer containing SDS, mercaptoethanol, and DTT, subjected to electrophoresis, and then transferred to nitrocellulose membrane (blocked with 3% BSA). Rho was visualized using a rabbit anti-Rho polyclonal antibody preparation (Upstate Biotechnology, Waltham, MA).
ROCK assay: Western blot.
Tissues were homogenized, and protein content was adjusted as outlined above. Tissue homogenates were incubated (10 min at 30°C) with 0.5 ng of MYPT (the myosin-targeting subunit of MLCP), after which the reaction was terminated by addition of Laemmli sample buffer. Samples were then subjected to Western blot analysis as outlined above. Phospho-MYPT was visualized using a rabbit anti-phospho-MYPT1 polyclonal antibody preparation (Upstate Biotechnology).
ROCK assay: 32P assay.
Tissue homogenates were centrifuged (10,000 g for 10 min at 4°C). The pellet was resuspended, and an aliquot was used to quantify the total protein content (Bradford method). The remainder was resuspended (1 mg/ml) in kinase assay buffer (20 mM 3-[N-morpholino]propane sulfonic, 25 mM β-glycerophosphate, 15 mM MgCl2, 1 mM EGTA, 0.1 mM NaF, 1 mM Na3VO4, 1 mM DTT, pH 7.2) containing 50 μM MYPT as a substrate. The kinase reaction was started by adding 100 μM ATP (containing 10 μCi/ml [γ-32P]ATP) and incubated for 10 min at 30°C with agitation. Aliquots of reaction mixture were spotted on P81 paper and washed five times with ice-cold 0.75% H3PO4 and then with acetone. Paper squares were dried, and radioactivity was counted (Cerenkov method).
Porcine tracheal strips were digested in Hanks' buffered saline solution using collagenase (type II; 0.9 U/ml) and elastase (type IV; 12.5 U/ml), and single cells were dissociated by gentle trituration. The tissue digest was centrifuged (200 rpm for 1 min at room temperature), the pellet was resuspended in Ringer buffer, and cells were incubated with fluo 4-AM (2 μM, dissolved in DMSO containing 0.1% Pluronic F-127, 37°C, 30 min). Cells were placed in the recording chamber of an Olympus microscope equipped with a charge-coupled device camera and imaging software (ImageMaster for Windows 2.0; Photon Technology, Lawrenceville, NJ). Cells studied were chosen on the basis of morphology (spindle-shaped; relaxed; absence of blebs; phase-bright) and responsiveness to 10 mM caffeine (applied by micropipette brought into vicinity of the cell using a hydraulic micromanipulator). Dye was excited using light from a mercury arc lamp (75 W, 494-nm excitation), and emitted light images (516 nm) were acquired at 1 Hz. Fluorescence intensities from regions of interest (central, nonnuclear regions of the cell) were saved and plotted against time.
Solutions and chemicals.
Tissues were studied using Krebs-Ringer buffer containing (in mM): 116 NaCl, 4.2 KCl, 2.5 CaCl2, 1.6 NaH2PO4, 1.2 MgSO4, 22 NaHCO3, and 11 d-glucose, bubbled to maintain pH at 7.4. l-NNA (10−4 M) and indomethacin (10 μM) were also added to prevent generation of nitric oxide and of cyclooxygenase metabolites of arachidonic acid, respectively.
All chemicals were obtained from Sigma Chemical. Pharmacological tools were prepared as 10 mM stock solutions, either in distilled water or in absolute ethanol (nifedipine, Y-27632). Aliquots were then added to the muscle baths; the final bath concentration of ethanol did not exceed 0.1%, which we have found elsewhere to have little or no effect on mechanical activity.
Constrictor responses were generally expressed as a percent of the response to 60 mM KCl delivered during the equilibration period, unless noted otherwise. IC50 for nifedipine was calculated by linear interpolation of the concentration-response relationship obtained in each individual tissue, as we have described previously (10). All responses are reported as means ± SE; n refers to the number of animals. Statistical comparisons were made using Student's t-test (for single pairwise comparisons) or one-way ANOVA (for multiple comparisons of mean values). P < 0.05 was considered statistically significant.
Effect of ROCK inhibitors and a Ca2+ channel blocker on KCl-evoked contractions.
We first examined the effect of two structurally different ROCK inhibitors on KCl-evoked contractions in a variety of ASM preparations (porcine and bovine tracheal strips and bronchial rings as well as human main stem bronchi). Tissues were pretreated with either Y-27632 (10−5 M) or HA-1077 (10−5 or 3 × 10−5 M) or neither for 20 min before the concentration-response relationship for KCl (0–60 mM) was examined. Responses were standardized as a percent of the third control response to 60 mM KCl obtained during the equilibration period (∼30 min before treatment with the ROCK inhibitors or with vehicle). After we examined the KCl concentration-response relationship, all tissues were additionally challenged with nifedipine (10−6 M) to block voltage-dependent Ca2+ influx.
Y-27632 (10−5 M) and HA-1077 (3 × 10−5 M) had no statistically significant effect on basal tone in porcine or bovine airway preparations but significantly decreased in human main stem bronchi (Table 1). Both ROCK inhibitors also markedly (and significantly) suppressed contractile responses to KCl (30–60 mM) in all preparations except bovine bronchial rings (Fig. 1).
As expected, nifedipine reversed the active tone exerted by 60 mM KCl in all control tissues except the bovine bronchial rings. In the former group, active KCl tone was reversed 60–80%, whereas tone in the bovine bronchial rings was not significantly affected at all (Fig. 1). Likewise, in tissues pretreated with ROCK inhibitors before examination of the KCl concentration-response relationship, subsequent addition of nifedipine reversed active tone in all preparations except the bovine bronchi (Fig. 1). Interestingly, in all types of tissues, the mean tone remaining in the presence of ROCK inhibitors plus nifedipine was not significantly different from that remaining in the presence of nifedipine alone (Fig. 1).
In a separate set of experiments, we examined the concentration dependence of nifedipine inhibition of KCl contractions in the presence vs. the absence of Y-27632. Porcine trachea and bronchi and bovine trachea were pretreated with Y-27632 (10−5 M) or its vehicle for 20 min and were then preconstricted with 60 mM KCl. Tone stabilized within 20 min after addition of KCl, at which point we added nifedipine at various concentrations (10−9 to 10−5 M) in cumulative fashion, making comparisons with other tissues not treated with nifedipine (time controls). Nifedipine caused a concentration-dependent reversal of KCl-induced tone (Fig. 2), which was maximal (80–100% reversal) at 10−6 M nifedipine in all three preparations. However, there were interesting regional differences with respect to the potency of nifedipine. In particular, the mean nifedipine log IC50 was −8.0 ± 0.4 and −8.1 ± 0.3 in the porcine and bovine tracheal preparations, respectively, but ∼10-fold higher (−6.8 ± 0.4) in the porcine bronchi (Fig. 2); bovine bronchi were not tested in this set of experiments, but it can be seen from Fig. 1 that the log IC50 is clearly in excess of −6. Pretreatment of the tissues with Y-27632 had no statistically significant effect on the nifedipine concentration-response relationship (Fig. 2). The log IC50 values for the same three sets of tissues were −8.2 ± 0.4, −7.9 ± 0.5, and −7.8 ± 0.8, respectively.
Effect of KCl on Rho and ROCK activities.
Collectively, the data above suggest that KCl evokes contractions in part through activation of ROCK. We probed this more directly using the Western blot technique (Rho and ROCK) as well as a radiometric kinase assay (ROCK). Porcine tracheal strips were mounted and equilibrated in muscle baths, pretreated with atropine (1 μM), and then challenged for 20 min with 60 mM KCl in the presence or absence of nifedipine (10−6 M, added 20 min before KCl challenge). In parallel, other tissues were challenged with nifedipine or vehicle for 40 min but were not challenged with KCl. In all four cases, tissues were flash-frozen (liquid nitrogen) and then assayed for Rho and ROCK activities. KCl (60 mM) more than doubled the activities of Rho and ROCK over and above those activities measured in tissues not exposed to KCl, and this increase was largely reversed when the tissues were pretreated with nifedipine (Fig. 3). Nifedipine alone had no statistically significant effect on Rho or ROCK activities (Fig. 3).
Effect of Ca2+ removal on KCl-induced responses.
Rho translocates to the membrane upon activation. It may be that its translocation and/or activity is sensitive to either the membrane potential or to [Ca2+]i, which might account for the effects of high-millimolar KCl described here. To distinguish between whether membrane depolarization per se and/or voltage-dependent Ca2+ influx is involved in the ROCK-mediated contractions, we examined the effect of removing external Ca2+ on KCl-induced changes in ASM function.
One set of porcine TSM tissues was bathed in either standard (i.e., Ca2+-containing) Krebs buffer or Ca2+-free Krebs buffer containing 1 or 10 mM EGTA (to chelate all trace amounts of Ca2+) before we examined the KCl concentration-response relationship. As expected, removal of external Ca2+ markedly suppressed KCl-evoked contractions. These were markedly reduced in the presence of 1 mM EGTA and essentially abolished with 10 mM EGTA (Fig. 4A).
Another set of porcine TSM tissues (n = 4) was bathed in standard Krebs buffer or Ca2+-free Krebs buffer containing 10 mM EGTA before being challenged with 60 mM KCl or vehicle for 20 min, flash-frozen, and processed for Rho and ROCK activities, as described above. Removal of external Ca2+ had no significant effect on basal Rho or ROCK activities but completely abrogated the KCl-induced elevation of both enzymatic activities (Fig. 4B).
Effect of KCl on [Ca2+]i.
[Ca2+]i was examined in porcine tracheal myocytes using the fluo 4 fluorimetric technique. As shown elsewhere (9, 12), caffeine (10 mM in application pipette; 10-s duration) evoked a spike-like increase in fluorescence, reaching a peak within a few seconds of applying caffeine and then falling back to baseline within 15 s (Fig. 5A). After caffeine was washed out and allowed 10 min for recovery, KCl (60 mM) was introduced via the bathing medium, leading to a marked increase in fluorescence (indicative of elevated [Ca2+]i; Fig. 5A). On average, the magnitude of this response was 90 ± 24% of that evoked by 10 mM caffeine. Y-27632 did not significantly reverse the KCl-induced elevation of fluorescence (n = 5; Fig. 5B). It was, however, completely reversed by addition of NiCl2 (1 mM; n = 6; Fig. 5, A and B) or by loading of the cells with the Ca2+-chelating agent BAPTA (10−4 M BAPTA-AM for 20 min; Fig. 5B).
In this study, we examined the relative contributions of voltage-dependent Ca2+ influx and Rho/ROCK signaling in the responses to high-millimolar KCl, making comparisons between tracheal and bronchial preparations as well as among various species (porcine, bovine, and human).
We found the contractile responses to high-millimolar KCl to be markedly attenuated by two structurally different selective inhibitors of ROCK (Y-27632 and HA-1077) (2). One interpretation of this finding is that, in the absence of these blockers, there is a certain degree of ongoing ROCK activity that augments the small contractile responses triggered by voltage-dependent Ca2+ influx. However, if this were the case, one would expect these ROCK inhibitors to drop basal tone, an observation that is not true for the porcine or bovine airways (although it is true for human airway smooth muscle; Table 1).
A second interpretation is that these two different ROCK inhibitors inhibit KCl-evoked contractions through some nonspecific side effect [e.g., blockade of the Ca2+ channels or direct inhibition of other enzymes such as myosin light chain kinase (MLCK)], a hypothesis that is not consistent with the existing literature for these compounds (2). We also ruled out the possibility that KCl-induced stimulation of Rho/ROCK activities was secondary to depolarization of nerve endings and release of acetylcholine by pretreating the tissues with atropine.
Another interpretation is that not only does KCl trigger voltage-dependent Ca2+ influx and subsequent activation of MLCK but it also somehow increases ROCK activity, as has been shown to occur in vascular smooth muscle (17, 20). We tested this hypothesis directly using Western blot techniques and found stimulation with KCl to indeed enhance the activities of both Rho and ROCK.
The means by which high KCl increases Rho/ROCK signaling is not entirely clear. There is evidence that depolarization and/or Ca2+ influx increases the activity of Rho GTPase-activating protein (24), which in turn would result in activation of ROCK. Membrane depolarization per se may affect these activities by triggering/increasing Rho activation, accelerating its recruitment to the membrane, prolonging its lifetime in the plasma membrane, and/or enhancing ROCK activity itself. Many groups have shown ion channels to form large complexes with a wide variety of kinases and that formation of these complexes and interactions between the channels and enzymes is a prerequisite for signaling via those enzymes (26). It is possible that the ion channels sense the change in membrane potential and signal to the associated kinases through some conformational change. Our findings that KCl-induced changes in tone and Rho/ROCK activities were largely suppressed by nifedipine or removal of external Ca2+ suggest that membrane depolarization alone is not sufficient for enhancement of Rho/ROCK activities, since those interventions should not affect the change in membrane potential per se. Alternatively, it may be that Rho/ROCK activities are modulated by changes in [Ca2+]i such as those secondary to any voltage-dependent Ca2+ influx triggered by KCl, which could explain the effects of nifedipine and of removing external Ca2+. We are now examining whether other stimuli that elevate [Ca2+]i mimic these effects on Rho/ROCK signaling. Collectively, our data suggest that KCl-evoked contractions in ASM (except bovine bronchi) are mediated to a large extent by activation of ROCK and that this appears to be dependent on voltage-dependent Ca2+ influx.
We also noted intriguing species and regional differences in the sensitivity to the pharmacological agents used in this study. For example, whereas KCl-evoked contractions in the central airways (trachea of the pig and cow as well as main stem bronchi of the human) all exhibited a marked sensitivity to Y-27632 and/or nifedipine (Fig. 1), the peripheral airways (4th- to 6th-order bronchi) of the cow showed essentially no sensitivity to these agents (Fig. 1), and those of the pig also showed reduced sensitivity to nifedipine (Fig. 2). The underlying basis of these differences is unclear but may be related to the finding that trachea and main stem bronchi express L-type Ca2+ currents almost exclusively, whereas T-type Ca2+ currents can also be found in bronchial preparations from the same species (7, 27). The KCl-induced tone remaining in the presence of both Y-27632 and nifedipine, although clearly not involving Ca2+ influx through L-type channels nor Rho/ROCK signaling, nonetheless appears to involve some Ca2+ influx pathway (T-type Ca2+ channels?), since removal of external Ca2+ completely abolishes all KCl-induced tone (Fig. 4).
Furthermore, with respect to the issue of the sensitivity of the responses to nifedipine and the relative distribution of L-type vs. T-type Ca2+ currents in ASM, we found nifedipine to be highly potent in the porcine and bovine tracheal preparations, both of which exhibit L-type Ca2+ currents almost exclusively (3, 27). In both cases, the IC50 for nifedipine against KCl-induced contraction was ∼10 nM, which compares quite favorably with the published IC50 for this agent as a selective blocker of L-type Ca2+ channels (15). Many groups studying ASM physiology, including our own, often use Ca2+ channel blockers at micromolar concentrations and conclude an involvement of L-type Ca2+ channels upon finding positive results (i.e., inhibition of the particular response being studied). However, at this concentration, the blockers may be exerting nonselective actions. Our group and others have shown previously that this can include partial inhibition of T-type Ca2+ currents (7, 27). The data presented in this study clearly show that submicromolar concentrations are wholly sufficient to deal with the L-type Ca2+ currents.
In conclusion, we found high-millimolar KCl to stimulate Rho and ROCK activities and ROCK inhibitors to markedly suppress KCl-induced contraction in ASM of the pig, cow, and human. Collectively, the data suggest that Rho is somehow stimulated by voltage-dependent Ca2+ influx. Further investigation will be required to elucidate the mechanism(s) underlying this voltage- and/or [Ca2+]-dependent activation of the Rho/ROCK signaling pathway.
These studies were supported by an Investigator Career Award from the Canadian Institutes of Health Research as well as operating grants from the Canadian Institutes of Health Research, the Ontario Thoracic Society, and Astra-Zeneca Canada.
The authors wish to thank Dr. A. Yoshimura (Welfide Corp., Osaka, Japan) for the kind gift of Y-27632.
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.
- Copyright © 2004 the American Physiological Society