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Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts
Submitted 21 November 2005 ; accepted in final form 30 January 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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laser scanning microscopy; pulmonary blood vessels; Rho kinase; protein kinase C; adenosine 3',5'-cyclic monophosphate
Phosphorylation of rMLC is primarily mediated by myosin light chain kinase (MLCK), which is activated by Ca2+-calmodulin. Dephosphorylation of rMLC is thought to be a Ca2+-independent process, mediated by myosin light chain phosphatase (MLCP) (44, 53). Therefore, an increase in intracellular Ca2+ concentration ([Ca2+]i) serves as the trigger for SMC contraction. However, this [Ca2+]i increase is not uniform or static but very dynamic in both the temporal and spatial domain (5). In airway SMCs agonists such as ACh, methacholine (MCh), 5-HT, ATP, and high-K+ solutions increase [Ca2+]i in the form of oscillatory waves to induced contraction. Most importantly, the extent of airway contraction correlates with the frequency but not the amplitude of the Ca2+ waves (3, 42, 43). However, it has been found in permeabilized SMCs, with the [Ca2+]i clamped at a constant concentration, that the relationship between [Ca2+]i and rMLC phosphorylation or contractile force varied depending on the excitatory stimulus. Generally, agonist-induced force development was higher than depolarization-induced force development at similar [Ca2+]i. This phenomenon is described as Ca2+ sensitivity of the contractile apparatus and is believed to occur by a Ca2+-independent increase in the amount of phosphorylated rMLC and cross-bridge activity. An abnormality in the Ca2+ sensitivity of SMCs may contribute to hyperreactive airways (34, 41). For example, repeated allergen challenge or inflammatory cytokines appear to increase Ca2+ sensitization via Rho-kinase (ROK) or CPI-17 (8, 9, 46, 50). Conversely, inhibitors of ROK (e.g., Y-27632) decreased airway responsiveness to various agonists in normal and antigen-sensitized animals (8, 24, 50).
Although a Ca2+-independent modulation of the phosphorylation of rMLC may occur by increased activity of a variety of kinases (i.e., zipper-interacting, integrin-linked, p21-activated protein kinase) (11, 38, 48), the most significant mechanism of Ca2+ sensitization appears to be mediated indirectly by agonists acting via G protein-coupled receptors (GPCRs). Activated GPCR subunits (G
q or G12,13) stimulate the RhoA/ROK pathway to phosphorylate the regulatory subunit (MYPT1) of MLCP to inhibit enzyme binding to myosin. Alternatively, active GPCRs stimulate protein kinase C (PKC), via production of diacylglycerol and arachidonic acid (AA), to phosphorylate CPI-17, which in turn, combines and inhibits the catalytic subunit of MLCP (PP1c) (53). Cross talk between the two pathways can occur via the activation of ROK by AA and phosphorylation of CPI-17 by ROK (44).
The majority of studies addressing Ca2+ sensitivity of airway SMCs have been performed with tracheal or large bronchial airways or with cultured airway SMCs. Although these results are important, it is not clear if they can be extrapolated to the SMCs of the intrapulmonary small airways. In addition, in most studies of Ca2+ sensitization, it was necessary to permeabilize the plasma membrane of the SMCs to gain control of intracellular events. The disadvantage of this approach is that membrane and intracellular components can be altered or lost and this may result in nonspecific responses (51). Consequently, our approach to investigate Ca2+ sensitivity of SMCs was to produce an "in situ SMC model" without the loss of cell components by exposing living lung slices to ryanodine and caffeine. A major advantage of the lung slice is that the SMCs of the intrapulmonary airways and arterioles can be simultaneously examined. With this new method, we examined the responses of both airway and arteriole SMCs to elevations of [Ca2+]i in the absence or presence of the GPCR agonist, MCh.
A major finding of these studies was that a sustained elevation of [Ca2+]i resulted in, after an initial contraction, the relaxation of the airways. By contrast, arterioles remained contracted. However, the contractile response of the airway SMCs was restored by MCh without altering the [Ca2+]i. This sensitivity to Ca2+ was influenced by ROK, PKC, and cAMP. One interpretation of these results is that in the airway, but not arteriole SMCs, MLCP activity is upregulated in a Ca2+-dependent manner. However, the mechanism coupling an increase in [Ca2+]i to an increase in MLCP activity remains to be established. The counterbalance to increased MLCP activity appears to be agonist-dependent inactivation of MLCP activity. Consequently, airway SMC contraction is controlled by the frequency of the Ca2+ oscillations that in turn appear to regulate both MLCK and MLCP activity, together with agonist-induced Ca2+ sensitization.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Lung slices.
The preparation of lung slices has been previously described in detail (43). In brief, male BALB/c mice (7–10 wk old; Charles River Breeding Labs, Needham, MA) were killed by intraperitoneal injection of pentobarbital sodium (Nembutal) as approved by the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School. After opening the chest cavity, we reinflated the lungs with 
1.3 ml of warm (37°C) 2% agarose (type VII-A: low gelling temperature) through a tracheal catheter (model 20G Intima, Becton Dickinson). Subsequently a 0.2-ml bolus of air was injected to flush the agarose out of the airways. To preserve the intrapulmonary blood vessels, 0.5 ml of warm (37°C) solution of gelatin (type A, porcine skin, 300 bloom, 6% in sHBSS) was perfused through a hypodermic infusion set (SVxS25BL, Terumo) inserted into the right ventricle of the heart to gain access to the pulmonary circulation. After gelling of the agarose and gelatin in the whole lung by cooling to 4°C, a single lobe was removed and cut into serial sections of 140 µm thickness with a vibratome (model EMS-4000, EMS) at 4°C. The slices were maintained in DMEM supplemented with antibiotics, antimycotics, and NaHCO3 (1 slice/1 ml) at 37°C and 10% CO2 for up to 3 days. No serum was used. Because gelatin dissolves at 37°C, the gelatin was absent from the vascular lumen when the slices were used.
Preparation of [Ca2+]i-"tunable" airway and arteriole SMCs in lung slices. The method used to manipulate or adjust the [Ca2+]i of the SMCs, without membrane permeabilization, is based on the principle of varying the amount of Ca2+ influx into a cell by altering the extracellular Ca2+ concentration ([Ca2+]o). However, this approach requires that a continuous Ca2+ influx current is activated and this was achieved by opening the ryanodine receptors (RyRs) with ryanodine to empty the sarcoplasmic reticulum (SR) of Ca2+. Because ryanodine is a use-dependent antagonist of the RyRs, caffeine was used to initially open RyRs in the presence of ryanodine.
Only lung slices containing viable airways or pairs of airways and arterioles, as judged by their morphology and active ciliated epithelium, were selected for model treatment. The lung slices were exposed twice to ryanodine (50 µM) and caffeine (20 mM) either sequentially or simultaneously for 3 min to activate and lock the RyR in the open state. Lung slices with either a low or high starting [Ca2+]i were obtained by performing the ryanodine-caffeine treatment in Ca2+-free sHBSS or normal sHBSS (containing 1.3 mM Ca2+), respectively. Before experimentation in most cases, the lung slices were washed for at least 5 min with either Ca2+-free or normal HBSS to remove the caffeine.
Measurement of the contractile response of airways and arterioles. Lung slices were transferred to a custom-made cover-glass perfusion chamber (43) and held in place with a small sheet of nylon mesh with a central hole to expose the selected airways and arterioles. Different experimental solutions were perfused through the chamber by gravity flow under electronic valve control. All experiments were performed at room temperature.
To measure airway lumen area, images of lung slices were recorded with phase-contrast microscopy on an inverted microscope in time lapse (0.5 Hz) with a charge-coupled device camera using a computer frame grabber (Road Runner, Bit Flow) and image acquisition software. The images were analyzed with National Institutes of Health Image/Scion software by initially selecting a gray-scale threshold to distinguish the airway or arteriole lumen from the surrounding tissue. The area of the lumen was calculated, with respect to time, by summing the number of pixels below the threshold gray level. Values were normalized to the prestimulation (initial) lumen area. Student’s or paired Student’s t-test was used to test for significant differences between means. All statistical values are expressed as means ± SE.
Measurement of intracellular Ca2+. The methods used to follow changes in [Ca2+]i in SMCs of lung slices have been previously described in detail (3, 43). In brief, lung slices were loaded with Oregon green AM (Molecular Probes) by immersion in 20 µM Oregon green AM in sHBSS containing 0.1% Pluronic F-127 (Molecular Probes) and 100 µM sulfobromophthalein for 45 min at 30°C and deesterified for 30 min at 30°C in sHBSS containing 100 µM sulfobromophthalein. In accordance with our previous studies, sulfobromophthalein, an anion-exchange inhibitor, was used to facilitate the loading of Oregon green. Cells were extensively washed before use and no detrimental effects have been observed in this or previous studies (3, 4, 42, 43).
Confocal microscopy with a x40 oil immersion [numerical aperture (NA) 1.3] objective was used to record the intracellular Ca2+ fluorescence intensity of SMCs. When required, two-photon microscopy with an Olympus x20 water immersion objective (NA 0.9) was used to record low-magnification fluorescence images of a paired intrapulmonary airway and arteriole simultaneously. Either a 488-nm beam from a diode laser or an 800-nm beam from a Ti-sapphire laser (Tsunami; Spectra-Physics, Mountain View, CA) pumped with a 5 W, 525 nm diode laser (Millennia, Spectra-Physics) was scanned across the specimen with two oscillating mirrors (for x- and y-scan) through the inverted microscope (Nikon DIAPHOT 200 for confocal microscopy; Olympus IX71FVSF-2 for two-photon microscopy). For confocal microscopy, the emitted fluorescence (>510 nm) was separated from the excitation light by a dichroic mirror, a long-pass filter and a confocal aperture (49). For two-photon microscopy, the emitted fluorescence was separated with a dichroic mirror (670uvdclp; Chroma Technology, Rockingham, VT) and a long-pass filter (E700SP, Chroma Technology) positioned immediately below the objective. Emitted fluorescence was detected by a photomultiplier tube (R5929; Hamamatsu USA, Bridgewater, NJ). Gray-scale images were recorded either at 15 or 30 Hz or at 1 Hz with a frame-grabber board (Raven, Bit Flow). Changes in the fluorescence intensity were obtained, frame-by-frame, from selected regions of interest (ROIs, 5 x 5 pixels). Only ROIs that represented the Ca2+ signaling occurring within the whole cell were used. Line-scan images were obtained by sequentially arranging frame-by-frame data from a line selected along or across an SMC. Lung slices were perfused as described above.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Responses of airways during model treatment with ryanodine and caffeine. Although airway contraction is modulated by the frequency of the Ca2+ oscillations, contraction also appears to be modulated by the sensitivity of the system to Ca2+. To explore this mechanism, it was necessary to experimentally manipulate the [Ca2+]i of the SMCs in a relatively nondisruptive way. This was achieved by locking RyRs of the SR in the open state with ryanodine (in the presence of caffeine) to empty internal Ca2+ stores. This, in turn, activated a Ca2+ influx current that was modulated by adjusting the [Ca2+]o.
Before treatment, and to confirm that airways displayed normal contraction and Ca2+ signaling, lung slices were exposed to MCh (200 nM) (Fig. 2). MCh induced the contraction of the airway (Fig. 2A) and induced Ca2+ oscillations within the SMCs (Fig. 2B and, as previously shown, Fig. 1). Washing the airways with sHBSS reversed these responses.
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Upon the first exposure to caffeine, in the presence of ryanodine, the [Ca2+]i increased with a transient spike followed by a sustained elevation [fluorescence ratio (F/F0) = 1.5–2.0, Fig. 2B]. These results are consistent with an initial emptying of internal Ca2+ stores and a sustained influx of Ca2+. Throughout these experiments, the fluorescence signal was constantly measured by confocal microscopy and showed a slow decline. When the fluorescence signal was measured over a similar time course, but with fewer sample points, the fluorescence was found not to significantly change (mean points, Fig. 2B). Consequently, the decline in the fluorescence signal was attributed to photobleaching and not to declines in [Ca2+]i.
The contractile response of the airway to model treatment consisted of a transient contraction that was quickly followed by relaxation (Fig. 2A), even through the [Ca2+]i remained elevated (Fig. 2B). The fact that the airway remained relaxed while the [Ca2+]i was high in the presence of caffeine also indicates that caffeine, in addition to emptying Ca2+ stores, served to desensitize the airway to Ca2+.
As expected, upon removal of caffeine by continuous washing with sHBSS, the [Ca2+]i of the SMCs remained at a sustained high level (Fig. 2B). However, the airways remained relaxed (Fig. 2A, airway 1) or responded with only a minimal contraction (Fig. 2A, airway 2; 4.83 ± 1.6% within 3 min, n = 8 airways from at least four different mice). On the other hand, the companion blood vessels fully recontracted (not shown). Because the effects of caffeine are rapidly reversed by washing, this weak airway contraction in the response to prolonged exposure to high [Ca2+]i implies that Ca2+ itself is also capable of inducing desensitization of contractile apparatus.
The second exposure to caffeine did not induce a further increase in [Ca2+]i. This result indicates that the internal Ca2+ stores remained empty. However, in the presence of high [Ca2+]i, caffeine again relaxed the small contraction of the airway and fully relaxed the accompanying blood vessel, results confirming that caffeine induced desensitization. When caffeine was removed a second time, no or only a small recontraction of the airway was observed, again implying Ca2+-dependent desensitization (Fig. 2A).
Upon reexposure of the model slice to MCh (200 nM), the airway showed a strong contraction similar to that induced by MCh in untreated lung slices although no Ca2+ oscillations or increases in [Ca2+]i were observed in the SMCs (Fig. 2B). This response indicates that agonist-dependent sensitization reversed Ca2+-dependent desensitization.
The absence of a caffeine-induced airway contraction and the ability of the airways to contract in response to MCh persisted at least 24 h after treatment. These responses are consistent with an irreversible opening of RyRs.
Response of model airway SMCs to sustained high [Ca2+]i. The implication of the previous results was that prolonged exposure to Ca2+ induced airway relaxation. However, by constructing model slices in the presence of extracellular Ca2+, the dynamic responses of the airway to increasing [Ca2+]i could not be observed. Consequently, we modified the experiment approach of making model SMCs by exposing the slices (twice) to ryanodine and caffeine in the absence of extracellular Ca2+. Under these conditions, model slices with low starting [Ca2+]i were obtained.
During the model treatment, the airways displayed a similar transient contraction to the initial exposure with caffeine, but this now correlated with only a transient increase in [Ca2+]i because Ca2+ influx was not available to sustain the [Ca2+]i (Fig. 3, A and B). The [Ca2+]i of the SMCs remained low and the airways remained relaxed, as expected, during the subsequent removal and addition of caffeine, confirming that model treatment was effective. The model slice was washed free of caffeine before exposure to external Ca2+.
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40% of lumen area, Fig. 3A), and this was associated with a rapid increase of [Ca2+]i in the SMCs (Fig. 3B). However, the contraction of the airway was not sustained even though the [Ca2+]i remained elevated. The uniformity of the increase in [Ca2+]i in the SMCs and the correlation with the changes in contractile state are clearly demonstrated in the line-scan image of the airway wall (Fig. 3C). The airway began to relax after 2 min of exposure to extracellular Ca2+ and was 50% relaxed after 5 min and >70% relaxed after 10 min (Fig. 3A). The airway completely relaxed when the extracellular Ca2+ was removed, and the [Ca2+]i was observed to decrease. After the slices were washed with Ca2+-free sHBSS for 15 min, a similar contractile and Ca2+ response of the airway SMCs was observed upon the readdition and removal of 10 mM Ca2+-sHBSS. These results indicate that while an initial exposure of airway SMCs to Ca2+ initiates contraction, the prolonged exposure to sustained high Ca2+ induces relaxation. Influence of airway epithelial cells on SMC responses. During the above exposure to high Ca2+, we also observed that the [Ca2+]i transiently increased in the epithelial cells (Fig. 4A). This raised the possibility that high [Ca2+]i within the epithelial cells may influence the SMCs by releasing some form of relaxing agent. In contrast to the SMCs, most epithelial cells reduced their [Ca2+]i to resting level within 5 min. If the release of a relaxing agent was coupled to the Ca2+ change, only a transient effect would be expected. However, airway relaxation continued for a further 5 min, and the accumulation of an extracellular messenger was unlikely because the lung slice was constantly perfused with fresh sHBSS.
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-monomethyl-L-arginine (L-NMMA), a nonspecific NO synthase (NOS) inhibitor, on the contractile response of airways to exposed high [Ca2+]i. Preincubation of lung slices with L-NMMA (100 µM) for at least 45 min did not block the relaxation response during exposure to 10 mM Ca2+-sHBSS (Fig. 3D). Similarly, incubation of model slices with indomethacin (100 µM for 30 min, n = 3) was found to have no effect on the relaxation responses of model slices to elevated [Ca2+]i. These results indicate that the SMC relaxation response was not due to a Ca2+-induced release of NO or prostaglandins from neighboring epithelium cells. The contraction of airway-arteriole pairs to sustained high [Ca2+]i. To confirm that relaxation of airways by high Ca2+ was an inherent property of airway SMCs and not a response resulting from the model treatment, we performed a comparative study of airways and arterioles. Because the airways and arterioles exist as paired structures in the same lung slice (Figs. 1 and 4), both SMC types were exposed to exactly the same treatment and experimental conditions.
As previously found, upon exposure of slices to 10 mM Ca2+, the airway SMCs displayed an initial contraction followed by gradual relaxation (Fig. 4, A and B) even though the [Ca2+]i was sustained at an elevated level (Fig. 4D). By comparison, the contractile response of the accompanying arteriole SMCs was very different. The onset of the Ca2+-induced contraction was slower but reached 80% of the maximal contraction after 2 min. More significantly, the arteriole showed no relaxation and continued to contract reaching a sustained contractile state within 10 min (40% of lumen area; Fig. 4, B and C). Despite these differences in the contractile response, the Ca2+ signaling of the arteriole SMCs was very similar to that of the airway SMCs (Fig. 4, D and E). Again, some variation in representative traces that document the [Ca2+]i with high-time resolution is observed. However, mean data from several experiments indicate there is little change in the [Ca2+]i during the course of these experiments (Fig. 4, D and E). These results indicate that relaxation to sustained high [Ca2+]i is an inherent property of airway SMCs and not arteriole SMCs.
Contraction of airway SMCs to MCh and sustained high [Ca2+]i.
To explore the antagonism of Ca2+ desensitization by MCh on the contraction of airway SMCs, we compared the contractile response of SMCs to sustained high [Ca2+]i in the absence or presence of MCh (200 nM). Lung slices were prepared in the absence of extracellular Ca2+ as described above. Upon exposure to Ca2+ (1.3 mM), the airway displayed contraction (15%) followed by relaxation (Fig. 5A). When the [Ca2+]o was increased to 10 mM, the airway responded with a second transient contraction that was followed by relaxation. However, relaxation was not complete, and a small contraction of 5% remained. When the Ca2+ concentration was reduced to 1.3 mM, the airway relaxed back to base line. However, the subsequent exposure to MCh (200 nM in 1.3 mM Ca2+) induced a large contraction (50%) even though this treatment did not further increase the [Ca2+]i (Fig. 5B). This MCh-induced contraction persisted with only a small change of lumen area (3%) for at least 60 min (Fig. 5C). These results indicate that MCh serves to counter Ca2+-induced Ca2+ desensitization of the SMC and that our model lung slice is viable for extended periods.
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-adrenergic agonists that elevate cAMP are commonly used as bronchodilators, we investigated the effect of compounds that elevate cAMP on the MCh-induced Ca2+-sensitization. Ryanodine caffeine-treated airways were contracted with MCh (200 nM) and subsequently exposed to forskolin (FSK; an adenylyl cyclase activator, 10 µM) or IBMX (a phosphodiesterase inhibitor, 10 µM). Although both agents induced an initial relaxation, the relaxation in response to IBMX occurred quickly (within 2 min) but was smaller (50% area change) compared with the relaxation induced by FSK, which took 3–5 min to induced a 70% area change (Fig. 9, A and D). The extent of FSK-induced relaxation increased in a concentration-dependent manner from 1 nM to 10 µM (Fig. 9C). In both cases, there was little change in the [Ca2+]i (Fig. 9, B and E). The decline in the F/F0 ratio that was independent of agonist application resulted from bleaching. There was no difference in the change of Ca2+ fluorescence intensity during these experiments in the absence or presence of antagonists. The mean fluorescence ratio after 5 min with MCh alone was 0.95 ± 0.03, compared with 0.92 ± 0.02, 0.93 ± 0.02, and 0.97 ± 0.04 in the presence of 10 µM FSK, 1 µM GF-109203X, and 10 µM Y-27623, respectively (each mean value from at least four airways of three different mice). Although higher concentrations were required, caffeine (acting as a phosphodiesterase inhibitor) induced a similar relaxation of MCh-contracted airways. At 10 µM, caffeine had a slight relaxant effect (20% area change) but, at 1 mM, induced prominent relaxation. In response to 20 mM caffeine, the airways showed a fast and full relaxation (Fig. 9F). An additional important point indicated by this result that applies to many of our preceding experiments is that washing efficiently reverses the effect of 20 mM caffeine. The results suggest that cAMP elevating agents relax airway SMCs by antagonizing the MCh-induced Ca2+ sensitization.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-toxin or with nonionic detergents such as saponin, -escin, or Triton X-100. Unfortunately, permeabilization of the cell membrane can also allow the uncontrollable loss of intracellular molecules. Although the extent of membrane permeability or disruption varies from mild (treatment with -toxin) to strong (treatment with Triton X-100), the loss of any key metabolic molecules is likely to preclude long-term maintenance of signaling and contractility (2, 45, 54). Consequently, to avoid the loss of cell constituents, we developed an alternative approach to control the [Ca2+]i of SMCs. Lung slices were exposed to ryanodine and caffeine to lock the RyR in a persistent "open" state. This treatment resulted in the sustained depletion of the intracellular Ca2+ store, which, in turn, led to an increased Ca2+ entry into the cell. Because this Ca2+ influx becomes the primary factor determining the [Ca2+]i, the [Ca2+]i could be adjusted by changing the [Ca2+]o. The viability of this method to make models of SMCs in lung slices was considered to be excellent, as judged by their ability to contract in response to MCh for more than a day after treatment. In addition, the MCh-induced contractile response of "model" SMCs was stable for at least 1 h.
With our model lung slices, we investigated the role of Ca2+ sensitivity in the regulation of contractility of airway SMCs. An initial finding, made during the process of ryanodine-caffeine treatment of lung slices, was that, even though the airway SMCs had a stable and high [Ca2+]i, these SMCs (and airways) were almost fully relaxed.
Although caffeine, in addition to activating the RyR, can induce Ca2+ desensitization, we are confident that caffeine was effectively washed from the preparation for several reasons. Firstly, under conditions where caffeine desensitizes airway contraction to MCh, we demonstrated a rapid recontraction of the airways during caffeine washout. Secondly, we show that a transient contraction in response to an elevation of [Ca2+]i is reproducible in the same model SMCs after 30 min of perfusion in the absence of caffeine. Thirdly, the blood vessels, which are also sensitive to caffeine, displayed contraction while the airways remained relaxed. And finally, previous studies with normal lung slices reveal a rapid reversal of the effects of caffeine upon washing (43).
To further test the idea that sustained high Ca2+ induced relaxation, we prepared lung slices in the absence of extracellular Ca2+. This produced airways and SMCs that were also fully relaxed but with low [Ca2+]i. Upon the addition of high [Ca2+]o, the SMCs initially contracted in response to the increasing [Ca2+]i but subsequently relaxed even though the [Ca2+]i remained high. However, upon exposure to MCh and without a significant change in [Ca2+]i, the SMCs recontracted. These results indicate that airway SMCs possess a mechanism of Ca2+-induced relaxation and that the antagonism of this response (i.e., by MCh) is essential to sustain Ca2+-induced contraction.
An alternative explanation for airway relaxation is a change in the external forces acting on the airway. However, because there is no agarose within the lumen and the airways are open ended and fluid filled, a relaxing pressure or surface tension force, which might oppose contraction or slowly drive relaxation, is not generated. Similarly, the tethering forces do not appear to substantially change with time because, in both normal and model lung slices, agonist-induced contraction is sustained for prolonged periods. Consequently, the contracted state of the airway stabilizes when the tension developed by the airway SMCs is equal to the recoil forces developed by tethering to the surrounding tissue. This infers that airway relaxation occurs by a reduction of tension within the SMCs.
We also examined the possibility that epithelial cells may release relaxing factors such as NO or prostaglandins to influence the SMC contractility (14). However, the inhibitor of NOS had no effect on airway relaxation. We also found that indomethacin had no effect on Ca2+-induced relaxation of the airway. This response is consistent with our previous findings that neither indomethacin nor PGE2 had any effects on agonist-induced contraction of SMCs (4). These results indicate that a Ca2+-dependent release of NO or prostaglandin does not contribute to airway relaxation. Endothelial cells could release other factors such as endothelin-1, 5-HT, or thromboxane (33), but these compounds stimulate airway contraction. Similarly, cytokines that would be expected to enhance airway contraction could be released by inflammatory cells. However, the lung slices appeared healthy and showed no signs of inflammation. It should also be pointed out that the lung slices were under constant perfusion, which would serve to quickly remove any soluble factors. In addition, the responses of the airway or arteriole SMCs to high [Ca2+]i were similar to those of ileum or pulmonary arterial muscle strips without epithelial or endothelial cells (20, 21, 29). These results all exclude the influence of the epithelium or endothelium on the effects of sustained high [Ca2+]i on SMCs.
In our experiments, F/F0 of Oregon green was used as an indicator of [Ca2+]i, and, in the presence of extracellular Ca2+, model lung slices displayed a high F/F0. In general, this ratio remained high during short-term experiments (<10 min). In longer experiments (>20 min), the slow decline in F/F0 was attributed to photobleaching by experiments of equal duration but with less light exposure due to lower sampling rates. The fact that the peak Ca2+ fluorescence ratio observed either during agonist-induced Ca2+ oscillations or during caffeine-induced transients was often higher than the steady-state ratio observed in model slices suggests that the dye was not saturated and still capable of recording Ca2+ changes. Although the [Ca2+]i is not linearly related to F/F0, the relatively stable high ratio implied that the [Ca2+]i remains elevated and does not decrease significantly to explain relaxation as a simple reversal of Ca2+ activated MLCK activity. In addition, treatment with ryanodine and caffeine induced a continuous leakage of Ca2+ from the SR that would eliminate the capacity of the SR to perform Ca2+ buffering while creating a conduit for Ca2+ delivery to the deeper regions of the cell. By abolishing the major stores or concentrations of Ca2+ within the cell, this treatment would also be expected to neutralize any microdomains of Ca2+ signaling. Although the uniformity of the bright fluorescence recorded by confocal imaging throughout the cell supports the view that, in model cells, the elevation of [Ca2+]i was homogenous, this result does not fully exclude the possibility that changes in [Ca2+]i can occur in microdomains beyond the resolution of our system. In view of a sustained homogenous increase in [Ca2+]i, which implies a constant activation of MLCK, we hypothesize that the variation in the airway contractile state reflects the regulation of MLCP activity.
Ca2+-induced relaxation has not been previously observed in airway SMCs, but a similar process has been reported in permeabilized SMCs of guinea pig ileum and rabbit portal vein where relaxation correlated with dephosphorylation of rMLC (36). The mechanism of rMLC dephosphorylation was not established although the participation of calcineurin (a Ca2+-regulated phosphatase) or Ca2+-dependent phosphorylation and inactivation of MLCK by calmodulin-dependent protein kinase II, PKA, and PKC were tested. In contrast to our studies, high [Ca2+]o induced a sustained isometric tension in permeabilized tracheal SMC strips (40, 56). The reasons for the discrepancy between these results are unclear but could be explained if SMCs at different locations within the respiratory tract have different contractile properties. A second explanation may relate to the extent of membrane permeabilization because SMC relaxation was only observed with mild permeabilization (36).
To explore the mechanism by which MCh reestablished Ca2+ sensitivity and contractility of model airways, we investigated the actions of ROK, PKC, and cAMP. We found that Y-27632, a specific ROK inhibitor and GF-109203X, a highly selective PKC inhibitor, induced a concentration-dependent relaxation of MCh-contracted airway SMCs, although neither compound altered the [Ca2+]i. These results are consistent with previous studies (15, 16, 52, 55) that suggest that agonist-induced Ca2+ sensitization of airway SMCs is mediated via ROK. Although our results suggest that PKC can also contribute to the Ca2+ sensitivity of intrapulmonary airways, the role of PKC is not as well established in tracheal or bronchial SMCs (1, 7, 19) as it is in vascular SMCs. A similar relaxation of model airways was also observed in response to the cAMP-elevating compounds, FSK and IBMX. This effect of cAMP may also act via the activation of MLCP because in previous studies, cAMP-dependent PKA (or cross-activated PKG) was reported to antagonize inhibitors of MLCP, including of RhoA, pre-RhoA signaling molecules (i.e., Gq or G12,13), and CPI-17 (12, 26, 37). Caffeine, presumably acting as a phosphodiesterase inhibitor to elevate cAMP, also served to relax the MCh-contracted airways but this effect required relatively high concentrations of caffeine compared with the effective concentrations of FSK or IBMX. This relaxing effect of caffeine was also observed in model or normal airways in the absence of MCh. These results indicate that the relaxing effect of high [Ca2+]i is reversed by GPCR agonists by the activation of ROK or PKC to inhibit MLCP activity. One implication of these results is that in airway SMCs, high [Ca2+]i may inhibit ROK or PKC to activate MLCP. It is currently unknown if [Ca2+]i can directly activate MLCP.
An enhancement of Ca2+-induced airway contraction in the presence of GPCR agonist such as ACh, carbachol and endothelin-1 has also been previously observed in permeabilized tracheal SMCs from pig, dog, rabbit, rat, and mouse as well as surgically resected human bronchial SMCs (6, 7, 10, 23, 25, 32). However, this vital role of GPCR agonists in establishing the sensitivity to Ca2+ for the maintenance of intrapulmonary airway contraction was not previously recognized.
A powerful experimental advantage of the lung slice preparation is the ability to examine the responses of two types of SMCs simultaneously under identical conditions. We found that while the airways relaxed, the arterioles remained contracted in response to a similar elevation of [Ca2+]i in the absence of agonist. In addition, the velocity of the initial contraction was slower in arteriole SMCs compared with airways and developed to a greater extent over a longer time. These differences strongly indicate that Ca2+-dependent modulation of the Ca2+ sensitivity of SMCs is specific to the type of SMC.
The mechanisms underlying these differences in Ca2+-modulated Ca2+ sensitivity are unknown, but a straightforward explanation would be a difference in the regulation of MLCP activity to alter the phosphorylation state of rMLC. For example, an increase in Ca2+ in arteriole SMCs appears to activate ROK that would lead to an inhibition of MLCP (22, 35, 47), but this mechanism does not appear to be in effect in airway SMCs. Similarly, in the absence of agonist, an activation of MLCP by cGMP was found to inhibit Ca2+-dependent force generation in vascular SMCs (30), but not in airway SMCs (27). However, as mentioned when GPCR agonists were present, we found that contracted airways relaxed in response to treatments that would be expected to activate MLCP (i.e., inhibition of ROK or PKC and elevations in cAMP). Similar findings were reported for the action of cGMP on ACh-contracted tracheal SMCs (27). A possible explanation for these results is that, in the absence of agonist, MLCP is activated in airway SMCs but inhibited in arteriole SMCs when the [Ca2+]i is elevated. However, in the presence of agonist, GPCR inactivation of MLCP serves to counterbalance the upregulation of MLCP in airway SMCs and enhances further contraction of arteriole SMCs.
A second mechanism that may contribute to sustained contraction of arteriole SMCs is the formation of "latch-like" actin-myosin cross bridges (39) that have slow cross-bridge cycling due to the dephosphorylated state of rMLC (18). Alternatively, slow cross-bridge cycling may result from stronger binding of ADP to phosphorylated rMLC (28, 31). A difference in cross-bridge cycling rates is consistent with the previously observed fast and slow relaxation rates of airway and arterioles (42, 43). An evaluation of the importance of this mechanism will require measurements of the phosphorylation state of rMLC, but this is beyond the scope of the present study.
In conclusion, intrapulmonary airway SMCs respond to sustained elevation of [Ca2+]i with only a transient contraction, and this implies a Ca2+-dependent activation of MLCP. MCh is essential for the airway SMCs to maintain their contraction and may upregulate the Ca2+ sensitivity by activating RhoA/ROK and/or PKC/CPI-17 pathways to decrease MLCP activity. Agents that elevated cAMP relaxed the airway SMCs by downregulating the Ca2+ sensitivity. These results emphasize that modulation of the Ca2+ sensitivity of airway SMCs is an important step in the control of their contraction or relaxation. One important implication of these results is that airway hyperreactivity could result from increased Ca2+ sensitivity to elevated [Ca2+]i.
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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.
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