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Published ahead of print on December 14, 2006, doi:10.1165/rcmb.2006-0096OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 36, pp. 600-608, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0096OC

Acetylcholine-Induced Asynchronous Calcium Waves in Intact Human Bronchial Muscle Bundle

Jiazhen M. Dai, Kuo-Hsing Kuo, Joyce M. Leo, Peter D. Paré, Cornelis van Breemen* and Cheng-Han Lee*

The James Hogg iCAPTURE Center for Cardiovascular and Pulmonary Research, University of British Columbia, St. Paul's Hospital, Vancouver, British Columbia, Canada

Correspondence and requests for reprints should be addressed to Cornelis van Breemen, D.V.M., Ph.D., The James Hogg iCAPTURE Center for Cardiovascular and Pulmonary Research, St. Paul's Hospital, Room 166, 1081 Burrard Street, Vancouver, BC, V6Z 1Y6 Canada. E-mail: breemen{at}interchange.ubc.ca


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Calcium (Ca2+) is an important activator of the contractile machinery in airway smooth muscle (ASM). While agonist-induced Ca2+ signals are well characterized in animal ASM, little is known about what occurs in adult human ASM. In this study, we examined the Ca2+ signal elicited by acetylcholine (ACh) in smooth muscle cells of the intact human bronchial muscle strips obtained from fresh surgical specimens in relation to muscle contraction. We found that ACh induces repetitive Ca2+ waves that spread along the longitudinal axis of individual cells in the intact human bronchial smooth muscle strips. These Ca2+ waves display no apparent synchronization between neighboring cells, and their generation precedes force development. Comparison of the ACh concentration dependence of tissue contraction and selected parameters of the asynchronous Ca2+ waves (ACW) reveals that the graded force generation by ACh-stimulated human bronchial muscle strips is achieved by differential recruitment of cells to initiate Ca2+ waves and by enhancement of the frequency of ACW once the cells are recruited. Furthermore, pharmacologic characterization shows that the ACW are produced by repetitive cycles of SR Ca2+ release via ryanodine-sensitive channels followed by SR Ca2+ reuptake by sarco(endo)plasmic reticulum Ca2+ ATPase. Extracellular Ca2+ entry involving receptor-operated channels/store-operated channels, reverse-mode Na+/Ca2+ exchange, and to a lesser extent L-type voltage-gated Ca2+ channels is required to maintain the ACW. These findings for the first time demonstrate the occurrence and the role of ACW in excitation–contraction coupling in adult human ASM.

Key Words: simultaneous [Ca2+]i imaging and isometric force measurement • confocal microscopy • excitation-contraction coupling • human airway smooth muscle



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Our study provides insight into our understanding of Ca2+ regulation of airway smooth muscle contraction in health. This is important for the investigation of potential abnormalities in the Ca2+ signaling underlying airway hyperresponsiveness.

 
Airway smooth muscle cells (ASMC) are the basic contractile units of the tracheobronchial tree. Excessive airway smooth muscle (ASM) contraction can result in exaggerated airway narrowing, increased airway resistance, and during acute severe attacks of asthma or anaphylaxis may lead to respiratory failure. It is therefore important to understand the signaling molecules/pathways involved in the contractile regulation of ASM. In ASM, as in many other smooth muscle types, calcium (Ca2+) is the fundamental signaling molecule responsible for activating the contractile myofilament (1, 2). An increase in myoplasmic Ca2+ concentration ([Ca2+]) activates myosin light chain kinase and subsequent cross-bridge cycling. It is unknown at the present whether abnormal Ca2+ signaling contributes to the hyperresponsiveness of ASM, which is characteristic of obstructive airway diseases such as asthma. However, it is highly plausible that diseased ASM still uses Ca2+ to activate its myofilament and that inhibition of Ca2+-activating signals in ASMC could effectively reverse the acute airway obstruction produced by the hyperresponsive ASM. This logic was challenged when nifedipine—an effective blocker of the L-type voltage-gated Ca2+ channels (VGCC) was shown in clinical studies to be a relatively ineffective treatment of pharmacologically induced asthmatic attacks in human subjects (3, 4). These initial findings have resulted in diminished clinical interest in exploring therapeutic approaches that target Ca2+ signaling in ASM. However, recent advances in our understanding of the role of Ca2+ signaling in excitation–contraction (E-C) coupling of ASM have prompted us to reexamine this issue more carefully.

Traditionally, agonist-induced intracellular [Ca2+] ([Ca2+]i) elevation in ASM was thought to depend on Ca2+ influx through the plasma membrane with the L-type VGCC being implicated as the main Ca2+ channels. Ca2+ release from the sarcoplasmic reticulum (SR) was believed to contribute only in the initial generation of the Ca2+ signal, but was not to be involved in sustaining the Ca2+ signal. However, studies published around the late 1990s that employed fluorescence microscopy to visualize [Ca2+]i showed that acetylcholine (ACh) induced repetitive intracellular Ca2+ waves in the single ASMC enzymatically dissociated from the porcine trachea (5–10). The repetitive Ca2+ waves are produced by repetitive cycles of SR Ca2+ release followed by SR Ca2+ reuptake. The L-type VGCC were again implicated to be the main Ca2+ channels involved in providing the extracellular Ca2+ necessary to replenish the SR Ca2+ store and to support the ongoing Ca2+ waves (6). Most recently, instead of using the less physiologic enzymatically dissociated single ASMC, investigators have begun examining Ca2+ signals of ASMC of the intact airway tissue (11–15). These methods allowed us to examine [Ca2+]i regulation of the ASMC in their native milieu with little or no disruption of their intercellular adhesions or communication, while allowing for simultaneous measurement of their force generation. Bergner and Sanderson (11) were the first investigators to study agonist-induced Ca2+ signals in the in situ bronchiolar smooth muscle of the mouse lung slices. They found that ACh induced repetitive intracellular Ca2+ waves in individual ASMC, which were not synchronized between neighboring cells. Using the tracheal muscle strip preparation, we also observed a similar phenomenon of asynchronous Ca2+ waves in the in situ smooth muscle cells of the intact porcine tracheal muscle bundles. Furthermore, we and others have demonstrated that these asynchronous repetitive Ca2+ waves (ACW) are responsible for activating the myofilaments and thus producing tonic contraction in the ACh-stimulated ASM (12, 16). By altering the frequency and the inter-wave [Ca2+]i of the ACW with different degrees of ACh stimulation, the tissue is able to generate graded levels of tonic contraction. Most significantly, we found that high-dose nifedipine produced only partial attenuation of the ACW and tonic contraction in the ACh-stimulated porcine ASM. It is therefore likely that the poor bronchodilatory effect observed with nifedipine in the treatment of asthmatic attacks is due to the fact that nifedipine is unable to abolish the ACW responsible for ASM contractile activation. Furthermore, our recent characterizations of the mechanism of the ACW in porcine tracheal muscle strips also implicates several other Ca2+ transporting molecules in their generation (14).

Nonetheless, despite these recent advances in our understanding of ASM Ca2+ signaling in the animal airway, very little is known about contractile regulation and Ca2+ signaling in the intact human ASM. As we have previously demonstrated, the observation of ACW in animal tissues does not necessarily correlate with what occurs in the adult human tissues (17, 18). This likely reflects inter-specie differences or the effect of aging and relevant disease processes on smooth muscle physiology. It is therefore prudent to determine whether ACW are used by the adult human ASM for contractile activation. In this study we examined the mechanism of the ACh-induced Ca2+ signal in the intact human bronchial muscle bundle in relation to tissue contraction.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Tissue Preparation
Lung tissues were obtained from patients who required surgical resection of a lobe for bronchogenic carcinoma at St. Paul's Hospital (Vancouver, BC, Canada). Subjects were studied with the approval of the University of British Columbia-St. Paul's Hospital Ethics Committee and after obtaining informed consent from the subjects. The third- and fourth-branch bronchi were carefully dissected from a tumor-free part of the lobe, and immediately transferred to ice-cold sterile physiologic salt solution (PSS). From these bronchi, strips of smooth muscles (~ 5 x 1.5 x 0.3 mm in dimension) were isolated free of epithelium and cartilage. The strips were subsequently attached at both ends to aluminum foil clips designed for mounting onto the custom-built setup.

Solutions and Chemicals
Normal PSS containing (in mM): NaCl 140, KCl 5, CaCl2 1.5, MgCl2 1, glucose 10, HEPES 5, (pH 7.4 at 37°C) was used for all the studies. Zero Na+ PSS was identical in composition to normal PSS except Na+ was replaced with equimolar of N-methyl-D-glucamine (NMDG+). Fluo-4 AM and pluronic F-127 were purchased from Molecular Probes (Burlington, ON, Canada) and were dissolved in dimethyl sulfoxide (DMSO). Stocks of ryanodine, ACh, procaine, and tetracaine were prepared in normal PSS, and stocks of nifedipine, cyclopiazonic acid (CPA), KB-R7943, SKF96365 were prepared in 100% ethanol. These chemicals were purchased from Sigma-Aldrich (Oakville, ON, Canada).

Isometric Force Measurement
The bronchial muscle strips were attached to isometric force transducer with 1 mN baseline tension and equilibrated in PSS at 37oC for 1 h. Isometric tension was recorded on-line via a serial connection to a computer hard drive at a rate of 1 Hz. Myodaq 2.01/Myodata 2.02 (J. P. Trading, Copenhagen, Denmark) was employed for data acquisition and analysis.

Simultaneous Isometric Force Measurement and Confocal Microscopy of Ca2+-Induced Fluorescence
The clipped muscle strips were loaded with Fluo-4 AM (5 µM with 5 µM Pluronic F-127) for 120 min at 25°C and then left to equilibrate for 10 min in normal PSS. They were then immediately mounted onto the custom-made stiff force transducer setup for simultaneous isometric force and [Ca2+]i measurements. The employment of a stiff force transducer, the application of firm clipping to secure the tissue, and the use of small-sized muscle strips are all important for minimizing tissue movements during confocal Ca2+ imaging. Inside the organ bath, one end of the tissue was placed over a stiff metal bar mounted on a micromanipulator for adjustment of muscle strip length, and the other end was connected to the lever arm of a servo-controlled force transducer. Details of the force measurements have been described above. The details of confocal Ca2+ imaging have been described previously (12, 17, 19, 20). Briefly, once the muscle strips were isometrically mounted, the changes in [Ca2+]i were measured using a Leica TCS SP2 AOBS, laser scanning confocal microscope through a x10 air lens (n.a. 0.3). The muscle strip was excited with the 488 nm line of an argon-krypton laser and a high-gain photomultiplier tube collected the emission between the 505 nm and 550 nm line. The measured Fluo-4 fluorescence level indicates relative [Ca2+]i, and thus changes in [Ca2+]i are directly reflected by proportional changes in fluorescence level. All parameters (laser intensity, gain, etc.) were left unchanged during the experiment. The rate of image acquisition was 3 Hz.

Data Analysis
Confocal image analysis was performed with ImageProPlus software using a customized routine written in Visual Basic as described previously (12, 19). The muscle strips with recordings that showed significant horizontal and/or vertical movement artifacts were excluded from the study. To obtain data on recruitment of cells during ACh stimulation, a fixed field of view under the x10 lens was chosen, and the number of cells responding with Ca2+ wave(s) was recorded at each concentration of ACh. Recruitment of cells was calculated as a fraction of the number of cells that responded to the highest ACh concentration (100 µM). Further analysis of wave parameters was performed using a three-pixel-wide line along the longitudinal axis of a single cell. The resulting x-t plot revealed the point of origin and the progression of the apparent "Ca2+ wave." The frequency of the ACW was determined by counting the number of waves occurring during a period of 50 s. The amplitude of the ACW reflected the difference between the peak fluorescence level of individual Ca2+ spikes in the Ca2+ waves and the prestimulation baseline level. The baseline [Ca2+]i elevation is defined as the average difference between the trough fluorescence and the prestimulation resting fluorescence value. The representative fluorescence traces shown reflect the averaged fluorescence signals from a 3x3-pixel region (1.36 µm2) of a bronchial smooth muscle cell.

All numerical data are presented as mean ± SEM. The data were analyzed in Excel (Microsoft Corp., Bellevue, WA) or Prism (GraphPad Software, San Diego, CA). Paired Student's t tests were used, and a value of P < 0.05 was considered significant. The n-values indicated for the contraction studies represent the number of bronchial muscle strips used, and the n-values indicated for the Ca2+ studies represent the number of cells analyzed from the specified numbers of bronchial muscle strips. For Ca2+ studies ANOVA was performed to assess for inter-tissue variance within the same study group wherever appropriate and no significant variance was found.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Patient Background
Fresh bronchial tissues from eighteen patients with an average age of 66 ± 2 were used in this study. The demographic, clinical and physiologic data for the patients from whom the tissues were obtained are shown in Table 1. Fifteen of the twenty-two patients had a significant smoking history. Eight patients exhibited mild to moderate degree of airway obstruction on pulmonary function test with no significant reversible component. According to the Global Initiative for Chronic Obstructive Lung Disease (GOLD), patients nos. 5 and 20 were at GOLD stage 1, and patients nos. 3, 4, 6, 7, 9, 11, 13, and 18 were at GOLD stage 2 (21). Preoperative lung function test results were not available from three patients (Nos. 8, 12, and 22). Because of the small size of the specimens, we were only able to isolate two to four viable bronchial muscle strips from each specimen.


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  		TABLE 1. DEMOGRAPHIC INFORMATION OF THE PATIENTS

 
Characterization of ACh-Induced Ca2+ Signal in Human Bronchial Smooth Muscle
Confocal microscopy was performed on Fluo-4 loaded human bronchial muscle strip to visualize the changes in [Ca2+]i that occur in individual smooth muscle cells. As shown in Figure 1A, the ASMC of the intact bronchial smooth muscle strip appear as long ribbon-shaped cells. Application of 3 µM ACh to the intact muscle strip first induced a synchronized Ca2+ wave in the individual bronchial smooth muscle cells (n = 288 cells in 36 muscle strips from 19 patients) (Figure 1A). This Ca2+ wave gave rise to a transient [Ca2+]i elevation which subsided within 10 s. Subsequently, recurrent intracellular Ca2+ waves developed that traveled along the longitudinal axis of the ribbon-shaped cell. The x-t plot in Figure 1B displays the change in fluorescence level ({propto} [Ca2+]i) over a longitudinal section of the cell over time. As shown in this figure, a rapid rise in [Ca2+]i was first seen on the left side of this region of interest and subsequently propagated to the right side of the region in an apparent wave-like fashion. These apparent Ca2+ waves continue to recur in the same cell for as long as the agonist is present. According to Figure 1A, with the exception of the initial Ca2+ wave, the subsequent oscillatory Ca2+ signals did not occur in a synchronized fashion between neighboring cells. Thus ACh induces ACW in ASMC of the intact human bronchial muscle bundle. (Please visit http://www.mrl.ubc.ca/pare/ppare.html for a video clip of the ACW in ASMC.) Apart from the asynchronous nature, the ensuing repetitive Ca2+ waves were smaller in amplitude than the initial large Ca2+ transient. Interestingly, the ACW induced by ACh observed in the human bronchial smooth muscle cells are qualitatively indistinguishable from the ACh-induced ACW observed in the porcine tracheal smooth muscle cells.


Figure 1
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  		Figure 1. ACh-induced ACW in the intact ASMC of human bronchial muscle bundles. (A) These time series images depict the ACh-induced changes in [Ca2+]i over time (as revealed by the fluorescence level) in the intact ASMC within this selected field of view. At 1 s after ACh application, the intact ASMC responded with elevation in [Ca2+]i that was synchronized between different cells. At the 10-s time mark, when the initial [Ca2+]i elevation had subsided, the cells began to initiate oscillatory Ca2+ signals (recurring Ca2+ waves) in a nonsynchronized fashion since the rise in fluorescence level was not simultaneous in all cells in the latter four time series images. (B) The x-t plot is a 3-pixel (1.36-µm)–wide line scan that depicts the changes in fluorescence ([Ca2+]i) over time in this longitudinal line section of the ribbon-shaped ASMC stimulated with 3 µM ACh. The still frame image (left) delineates the placement of the line, and the x-t plot derived from the line is shown (right). The x-t plot shows recurring Ca2+ waves that are initiated on one end (X0) of the scanned cellular segment and subsequently propagated to the other end (X1). (C) Fluorescence changes in two 3x3-pixel intracellular regions (1.36 µm2) from two neighboring ASMC are depicted in the traces (right). The black and grey traces represent Ca2+ signals from the corresponding specified regions (indicated by white dots) in Cell 1 and Cell 2 shown in the still frame image (left). The scale bar indicates 10 µm.

 
Relationship between ACW and Force Development
We performed simultaneous measurements of both force and [Ca2+]i from the same muscle strip stimulated with ACh to study the relationship between the ACW and force generation. Figure 2 shows that the immediate response to ACh is a large [Ca2+]i transient, which precedes the onset of force development by approximately half a second. However, maximal force is not reached for another 20 s, at which time the ACW have been well established. For this reason we have examined the relationship between the steady state force development and the various parameters of the ACW. As shown in Figure 3A, increasing concentrations of ACh produces tonic contraction of increasing amplitude (n = 8 muscle strips from five patients, the concentration of ACh where 50% of its maximal effect is observed, EC50 = 4.99 ± 1.23 x 10–7 M). It is interesting to note that the ACh concentration–response relationship of the human bronchial smooth muscle is similar to that described earlier for the porcine tracheal smooth muscle (12). To examine how these Ca2+ waves modulate contraction, concentration dependences of selected quantitative parameters of the ACW were compared with the concentration dependence of force generation in ACh-stimulated tissues. As shown in the concentration–response curves in Figure 3B, increases in the concentration of ACh (over the lower concentration range from 0.01–1 µM) result in increasing recruitment of cells to initiate Ca2+ waves (EC50 = 8.58 ± 1.76 x 10–9 M), while increases in concentrations of ACh over the concentration range of 0.01–100 µM are correlated with rising frequency of ACW (Figure 3C, EC50 = 2.27 ± 0.27 x 10–7 M), reaching 0.47 ± 0.02 Hz at 100 µM ACh (n = 65 cells of 8 muscle strips from five patients). In contrast, baseline [Ca2+]i elevation (P = 0.4928) and amplitude of the ACW (P = 0.4958) show no significant concentration dependence (Figures 3D and 3E). These results indicate that cell recruitment and frequency of the ACW are two main parameters involved in determining the degree of force generation.


Figure 2
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  		Figure 2. Temporal association between ACh-induced force generation and Ca2+ signal. The representative experimental traces (n = 8 strips from eight patients) shown depict simultaneous measurement of the force generation (indicated by a black trace) and [Ca2+]i changes (indicated by a grey trace) that occur in the same muscle strip. After the application of 3 µM ACh, the appearance of ACW at the cellular level preceded the onset of force generation in the human bronchial muscle strip.

 

Figure 3
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  		Figure 3. Concentration–response relationships of ACh-induced force generation and ACW. These concentration–response curves are generated from simultaneous force and [Ca2+]i measurements of intact human bronchial muscle bundles (65 cells in eight muscle strips from five patients). (A) Concentration dependence of the magnitude of ACh-induced tonic contraction. (B) Concentration dependence of the percentage cell recruitment by ACh to initiate Ca2+ wave(s). (C) Concentration dependence of the frequency of ACh-induced ACW. (D) Concentration dependence of amplitude of ACh-induced ACW. (E) Concentration dependence of trough [Ca2+]i elevation (interspike baseline [Ca2+]i elevation).

 
Dependence of ACW on Plasmalemmal Ca2+ Influx
Various pharmacologic inhibitors were used to characterize the plasmalemmal Ca2+ entry pathway(s) important in ACh-mediated ACW and tonic contraction. Both the L-type VGCC and the receptor-operated channels/store-operated channels (ROC/SOC) have been implicated in the process of Ca2+ signaling in freshly isolated and cultured human bronchial smooth muscle cells (6, 22, 23). As shown in Figure 4, application of 10 µM nifedipine partially inhibited ACh-induced tonic contraction and attenuated frequency of the ACW, but did not abolish the ongoing ACW. The reduction in frequency of the ACW and tonic contraction induced by ACh was 35 ± 5% (n = 30 cells from four muscle strips from four patients, P < 0.001) and 35 ± 4% (n = 6 muscle strips from four patients, P = 0.0004), respectively. Additional application of SKF96365 (an inhibitor of the ROC/SOC) completely abolished the nifedipine-resistant component of ACh-induced ACW (n = 30 cells from four muscle strips from four patients) and reduced the nifedipine-resistant tonic contraction by 94 ± 1% (n = 6 muscle strips from four patients, P < 0.0001) of its peak level (Figure 4). These results suggest that ACh-induced tonic contraction in the human bronchial smooth muscle is maintained by plasmalemmal Ca2+ entry involving the L-type VGCC and the ROC/SOC. This is similar both qualitatively and quantitatively to our previous observations in porcine tracheal smooth muscle (12).


Figure 4
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  		Figure 4. Effect of 10 µM nifedipine and 50 µM SKF96365 on ACh-induced ACW and tonic contraction. (A) L-type VGCC blockade by nifedipine did not abolish ACh-induced ACW but reduced the frequency of the ACW. Additional application of SKF96365 abolished ACh-induced ACW completely. Experimental [Ca2+]i trace is representative of results in 30 cells in four muscle strips from four patients. (B) Application of nifedipine partially reduced the ACh-induced contraction, whereas SKF96365 nearly abolished the remaining contraction. Experimental tissue contraction trace is representative of results in six muscle strips from four patients. (C) Percentage reduction in the frequency of ACW and tonic contraction mediated by nifedipine and SKF96365 in ACh-stimulated human bronchial smooth muscle.

 
In vascular smooth muscle cells, reverse-mode Na+/Ca2+ exchange is implicated in the generation of agonist-induced ACW (17, 24). Although some studies reported lack of involvement of the Na+/Ca2+ exchanger (NCX) in ASM (25, 26), others have provided indirect and direct evidence that NCX may play a role in Ca2+ and contractile regulation of ASM (14, 27–30). We have previously proposed that an influx of Na+ through a nonselective cation permeable ROC/SOC across the plasma membrane can lead to a large subplasmalemmal rise in Na+ concentration near NCX, which then drives the NCX into its reverse mode of operation, bringing Ca2+ into the cell (14, 17). If Ca2+ entry via reverse-mode Na+/Ca2+ exchange contributes to the maintenance of ACh-mediated ACW, removal of extracellular Na+ should result in the inhibition of the ACW and the consequent force generation. Indeed, as shown in Figure 5, the removal of extracellular Na+ abolished the ongoing ACh-mediated ACW (n = 35 cells in five muscle strips from five patients) and reduced ongoing tonic contraction by 65 ± 11% (n = 5 muscle strips from four patients, P = 0.0039). In addition, we also employed a selective inhibitor of reverse-mode Na+/Ca2+ exchange, KB-R7943 (17, 24, 31). As shown in Figure 5, application of 10 µM KB-R7943 abolished nifedipine-resistant ACW (n = 32 cells of four muscle strips from four patients) and inhibited nifedipine-resistant tonic contraction by 78 ± 8% (n = 4 muscle strips from four patients, P = 0.0024). These results indicate that extracellular Ca2+ entry via reverse-mode Na+/Ca2+ exchange is required for maintaining ACh-induced ACW and tonic contraction.


Figure 5
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  		Figure 5. Effect of zero Na+ PSS and 10 µM KB-R7943 on ACh-induced ACW and tonic contraction. (A) Removal of extracellular Na+ resulted in the cessation of ACh-induced ACW. Experimental [Ca2+]i trace shown is representative of the result of 35 cells in five muscle strips from five patients. (B) Removal of extracellular Na+ inhibited ACh-induced tonic contraction. Experimental tissue contraction trace shown is representative of the result in 5 muscle strips from 4 patients. C. Application of 10 µM KB-R7943 inhibited nifedipine-resistant ACW. Experimental [Ca2+]i trace shown is representative of the results in 32 cells in four muscle strips from four patients. (D) Application of KB-R7943 resulted in the inhibition of ACh-induced nifedipine-resistant tonic contraction. Experimental tissue contraction trace shown is representative of the results in four muscle strips from four patients.

 
Dependence of ACW on SR Ca2+ Release
In intact porcine tracheal smooth muscle, ACh-induced ACW are the result of repetitive cycles of SR Ca2+ release and Ca2+ reuptake (14). As demonstrated by our group previously, the wave-like nature of the Ca2+ signal seen in ACh-induced ACW (Figure 1) indicates that they are the result of SR Ca2+ release rather than plasmalemmal Ca2+ entry (17). In addition, blockade of sarco(endo)plasmic reticulum ATPase (SERCA) with 10 µM CPA significantly reduced ACh-induced tonic contraction by 72 ± 10% (n = 4 muscle strips from four patients, P = 0.0073, Figure 6A) and rapidly abolished ongoing ACW (n = 28 cells from four muscle strips from three patients, Figure 6B). Furthermore, application of 200 µM ryanodine abolished the ongoing ACh-induced ACW (n = 50 cells in five muscle strips from four patients) (Figure 6). These observations show that, as in porcine ASM (14, 32), the ACW in human bronchial smooth muscle are dependent on periodic ryanodine-sensitive SR Ca2+ release channels (RyR)-mediated Ca2+ release from the SR. To confirm the involvement of RyR, we used two inhibitors of RyR, procaine and tetracaine (33–36). In the present study, the application of 2 mM procaine (n = 24 cells in three muscle strips from three patients) or 100 µM tetracaine (n = 24 cells in three muscle strips from three patients) caused a complete inhibition of ACh-induced ACW (Figure 7). Similarly, the application of the same dose of procaine (n = 5 muscle strips from four patients) and tetracaine (n = 5 muscle strips from four patients) resulted in 97 ± 1% (P < 0.0001) and 94 ± 4% (P < 0.0001) inhibition of ACh-induced tonic contraction, respectively (Figure 7).


Figure 6
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  		Figure 6. Effect of 10 µM CPA and 200 µM ryanodine on ACh-induced ACW and tonic contraction. (A) Application of CPA attenuated the ACh-induced tonic contraction. Experimental tissue contraction trace shown is representative of the results in four muscle strips from four patients. (B) Application of CPA to ACh-stimulated human bronchial smooth muscle cell completely abolished the ongoing ACW. Experimental [Ca2+]i trace shown is representative of the results in 28 cells in four muscle strips from three patients. (C) Application of ryanodine (200 µM) inhibited the ACh-induced ACW. Experimental [Ca2+]i trace shown is representative of the results in 50 cells in five muscle strips from four patients.

 

Figure 7
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  		Figure 7. Effects of 100 µM tetracaine and 2 mM procaine on the ACh-induced ACW and tonic contraction in human bronchial smooth muscle strips. (A) Application of tetracaine (100 µM) immediately abolished the ongoing ACh-induced ACW. Experimental [Ca2+]i trace shown is representative of 24 cells in three muscle strips from three patients. (B) Application of tetracaine (100 µM) nearly abolished the ongoing ACh-induced tonic contraction. Experimental tissue contraction trace shown is representative of the results in five muscle strips from four patients. (C) Application of procaine (2 mM) immediately abolished ongoing ACW mediated by ACh. Experimental [Ca2+]i trace shown is representative of the results in 24 cells in three muscle strips from three patients. (D) Application of procaine (2 mM) abolished ongoing tonic contraction mediated by ACh. Experimental tissue contraction trace shown is representative of the results in five muscle strips from four patients.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The present communication represents the first study of Ca2+-mediated E-C coupling in human bronchial smooth muscle and reveals several previously unexplored pharmacologic targets for the treatment of bronchospasm. In this study, ACh consistently and reproducibly induced ACW and tonic contraction in the human bronchial muscle strips from successive surgical specimens despite the potential variability that may exist between different patients such as age, disease status, and medication use. It has been shown previously by others and us that the ACW constitute the Ca2+ signals responsible for E-C coupling in ACh-stimulated porcine tracheal smooth muscle (11–13, 16). In this report, we show that the same applies to human bronchial smooth muscle as steady-state contraction is well correlatedwith the occurrence of ACW and their pharmacologic inhibition consistently leads to relaxation. Although our data indicate some variability, all cells were highly sensitive to ACh in that ACW were initiated at low ACh concentrations. The amplitude of the ACW was constant over the entire ACh concentration range, while the steady-state force development was best correlated with the frequency of the [Ca2+]i ACW.

ACW induced by ACh in the human ASM exhibit a similar spatiotemporal pattern as ACW observed in the animal ASM. There is, however, one noteworthy difference. Even though the dose–response relationships are similar between porcine tracheal smooth muscle and human bronchial smooth muscle, ACh-induced ACW at the highest concentration examined (100 µM) occurred at higher peak frequency of 0.47 Hz in human tissue than the frequency of 0.28 Hz observed in the porcine tissue (12). It is unclear at this time whether this difference in the frequency of ACW represents a physiologically significant interspecies difference or is the result of pathologic alterations in the human ASM related to the factors such as age and smoke exposure. With regard to the mechanism for the generation of ACW, the pharmacologic characterization in this study indicates that the ACW of human and animal ASM likely share similar mechanisms. Using the same maximally effective dose of nifedipine to inhibit the L-type VGCC, we observed a 35% reduction in ACh-induced tonic contraction in human ASM, a result that is similar to the 33% reduction in ACh-induced tonic contraction seen in porcine ASM. Correspondingly, nifedipine reduced the frequency of the ACW in the human and the porcine tissue by 35% and 29%, respectively. When SKF96365 is added in the presence of nifedipine, the nifedipine-resistant ACW were abolished in both human and porcine tissue, while the corresponding nifedipine-resistant tonic contraction was nearly completely inhibited as well in both human and porcine tissues. In addition, blockade of the reverse-mode Na+/Ca2+ exchange with either zero Na+ PSS or KB-R7943 resulted in complete abolition of the ACW and significant inhibition of tonic contraction induced by ACh in both human bronchial and porcine tracheal muscle. As for animal ASM (14), these human results indicate a role for ROC, reverse-mode NCX, and SERCA in refilling the SR in order to maintain the ACW. Consistent with the earlier observations by Sieck's group (5, 6, 9, 10) and ours (14), the present results of complete blockade of ACW by each of CPA, ryanodine, procaine, and tetracaine, indicate that SR Ca2+ release via RyR is crucial for the generation of ACW and contraction in human bronchial smooth muscle.

The new findings resulting from the present series of pharmacologic characterizations indicate the need to revise our knowledge of human ASM physiology. The concept that ASM contraction is brought on by stimulated Ca2+ influx via the L-type VGCC is oversimplified and no longer tenable. In contrast, the generation of ACW requires a coordinated sequence of Ca2+ fluxes inside the cell that occurs on a repetitive basis (17, 20). Multiple Ca2+ transporting molecules on the plasma membrane and the SR are required for this recurring sequence of Ca2+ fluxes. The L-type VGCC, the NCX, the SERCA, the RyR, and a putative nonselective cation-permeable ROC/SOC type channel have been implicated thus far in the generation of ACW in the human ASM. The signaling mechanism that initially activates RyR in human bronchial smooth muscle remains to be defined; however, an intracellular second messenger cyclic ADP ribose (cADPr) was found to activate RyR in airway as well as vascular smooth muscle (9, 37–40). In addition to the RyR, inositol trisphosphate–sensitive SR Ca2+ release channels (IP3R), although not essential, may contribute to the ACh-induced ACW in intact human bronchial smooth muscle, as IP3R have been implicated in Ca2+ waves found in smooth muscle cells of the murine lung slices and isolated porcine tracheal smooth muscle cells (5, 11). The ROC and SOC include a wide range of plasmalemmal channels that are activated secondary to receptor activation and SR Ca2+ depletion, respectively (41, 42). The exact molecular identities of the various types of ROC and SOC are not known at this time, but are likely related to transient receptor potential molecules (TRPs) (43). Identification of the various subtypes of TRPs and NCXs involved in the pathophysiology of human airway may lead to new therapeutic approaches.

Clinically, the appreciation of the importance of ACW in agonist-induced contractile activation of human ASM introduces many exciting therapeutic possibilities. Though it remains to be verified in future studies, it is plausible that aberrant control of ACW is involved in the hyperresponsive ASMC found in asthma. Further, it is possible that anomalies in ACW related to altered expression of TRPs, NCX, or RyR may actually be responsible for the altered ASM contractility (44). Therefore, pharmacologic agents that target the various Ca2+ translocators and effectively modulate or abolish the ACW may prove to be efficacious inhaled and/or systemic bronchodilators in the clinical setting. The development of novel inhalational therapeutic agents that target Ca2+ signaling in the ASM will certainly be a welcome addition to the beta-agonists that are the first-line bronchodilator used clinically at present. This is especially true in cases in which beta-agonists are poorly tolerated by the patients, and in cases in which other beneficial drugs such as beta-blockers used in the treatment of coronary vascular disease are withheld because of the potential adverse clinical interaction with the beta-agonists. Further, numerous recent studies have shown that inflammatory mediators and factors that are associated with pathogenesis of airway hyperresponsiveness alter agonist-induced Ca2+ signaling in ASM (45–49). However, future studies using asthmatic human airway tissue will be necessary to understand the importance of Ca2+ signaling in asthma and to determine if any change in Ca2+ signaling and ACW contribute to airway smooth muscle hyperresponsiveness in this condition.

In summary, we have demonstrated for the first time the presence of ACh-induced ACW in the smooth muscle cells of intact human bronchial muscle strips. The ACW are the primary mechanism by which the agonist used to signal for cellular contraction. Furthermore, we have identified a number of Ca2+ transport molecules that are involved in mediating ACh-induced ACW and tonic contraction, including the L-type VGCC, the NCX, the SERCA, the RyR, and a putative nonselective cation-permeable ROC/SOC type channel. Given that altered ASM contractility is known to contribute to the pathogenesis of obstructive airway diseases, an improved understanding of contractile regulation of human ASM in both health and disease states will undoubtedly provide better insight into these functionally debilitating and potentially lethal diseases.


   Footnotes
 
* This authors contributed equally to this article. Back

This study was supported by the James Hogg iCapture Centre for Cardiovascular and Pulmonary Research and by the St. Paul's Hospital Foundation.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0096OC on December 14, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form March 2, 2006

Accepted in final form December 7, 2006


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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