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Am J Physiol Lung Cell Mol Physiol 290: L459-L469, 2006. First published October 7, 2005; doi:10.1152/ajplung.00092.2005
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Mechanism of ACh-induced asynchronous calcium waves and tonic contraction in porcine tracheal muscle bundle

Jiazhen M. Dai,* Kuo-Hsing Kuo,* Joyce M. Leo, 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

Submitted 1 March 2005 ; accepted in final form 3 October 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Stimulation of the tracheal muscle bundle by acetylcholine (ACh) results in the generation of asynchronous repetitive Ca2+ waves (ACW) in intact tracheal smooth muscle (TSM) cells. We showed previously that ACW underlie cholinergic excitation-contraction coupling in porcine TSM and that Ca2+ entry through the L-type voltage-gated Ca2+ channel (VGCC) contributes partially to maintenance of the ACW. However, the mechanism of the ACW remains undefined. In this study, we pharmacologically characterized the mechanism of ACh-induced ACW in the intact porcine tracheal muscle bundle. We found that inhibition of receptor-operated channels/store-operated channels (ROC/SOC) by SKF-96365 completely abolished the nifedipine-insensitive component of ACh-mediated ACW and tonic contraction. Blockade of Na+/Ca2+ exchange with KB-R7943 or 2',4'-dichlorobenzamil or removal of extracellular Na+ resulted in nearly complete inhibition of the nifedipine-insensitive component of ACh-mediated ACW and tonic contraction. Inhibition of the sarco(endo)plasmic reticulum Ca2+-ATPase by cyclopiazonic acid abolished the ongoing ACW. Application of 2-aminoethoxydiphenyl borate (2-APB) or xestospongin C to inhibit the inositol 1,4,5-trisphosphate-sensitive sarcoplasmic reticulum (SR) Ca2+ release channels produced no effect on ACh-mediated ACW and tonic contraction. However, pretreatment with caffeine or ryanodine inhibited ACh-induced ACW. Furthermore, application of procaine or tetracaine prevented the generation and abolished the ongoing ACh-mediated ACW and tonic contraction. Collectively, these results indicate that the ACh-stimulated ACW in porcine TSM are produced by repetitive cycles of Ca2+ release from SR through 2-APB- and xestospongin C-insensitive Ca2+ release channels, and plasmalemmal Ca2+ entry involving reverse-mode Na+/Ca2+ exchange, ROC/SOC, and L-type VGCC is required to refill the SR via SERCA to support the ongoing ACW.

confocal calcium imaging; excitation-contraction coupling; intact airway smooth muscle

ACTIVATION OF THE MUSCARINIC RECEPTOR by acetylcholine (ACh) or other cholinergic agonists is known to result in tonic contraction of airway smooth muscle. Recent findings by Bergner and Sanderson (4) and by our laboratory (19) have revealed that smooth muscle cells of the intact tracheal and bronchial muscle bundle respond to cholinergic stimulation with repetitive Ca2+ waves that are not synchronized between neighboring cells. Functionally, these asynchronous Ca2+ waves (ACW) were found to be responsible for the development of force in the tracheal smooth muscle cells (TSMC) (19). In the case of ACh stimulation, the regulation of force generation by the TSMC is achieved initially by differential recruitment of cells to initiate ACW and subsequently by the enhancement of the frequency of the ACW and the elevation of interspike intracellular Ca2+ concentration ([Ca2+]i) once the cells are recruited (19). Mechanistically, we found that the blockade of the L-type voltage-gated Ca2+ channel (VGCC) with high-dose nifedipine resulted in only partial reduction in frequency of the ACW and partial inhibition of the tonic contraction induced by ACh (19). However, the detailed mechanism for the generation of ACh-induced ACW and tonic contraction has not been elucidated in the smooth muscle cells of the intact tracheal muscle bundle.

In enzymatically isolated porcine TSMC, ACh-induced repetitive Ca2+ waves have been described, and their mechanism has been extensively studied. It was found that these agonist-induced repetitive Ca2+ waves are produced by sarcoplasmic reticulum (SR)-mediated Ca2+ release (9, 18, 23, 36, 37, 42). More specifically, Ca2+ release through the inositol-1,4,5-trisphosphate-sensitive SR Ca2+ release channels (IP3R) is important in initiating the Ca2+ waves, whereas Ca2+ release through the ryanodine-sensitive SR Ca2+ release channels (RyR) is responsible for the recurrent wave generation (9, 18, 23). The repetitive Ca2+ waves in the isolated TSMC can be abolished with 100 nM nifedipine (36), indicating that Ca2+ entry through the L-type VGCC is the main pathway utilized for refilling the SR Ca2+ store and the maintenance of the repetitive cycles of SR Ca2+ release in these cells. However, our initial investigation into the mechanism of the ACW of the intact porcine TSMC revealed a crucial difference from the isolated cell preparation. Blockade of the L-type VGCC with high-dose nifedipine attenuated the frequency of the ACW observed in TSMC of the intact tissue but did not abolish the ongoing ACW or the tonic contraction stimulated with ACh. This finding suggests that, unlike the enzymatically isolated TSMC, Ca2+ influx through the L-type VGCC is not obligatory for maintaining the ACW of the TSMC of the intact tissue. Such a significant difference in the phenotypic characteristics between freshly dissociated cells and the intact tissue may be the result of the disruption of crucial intercellular communication or may reflect damage of important surface proteins on the TSMC due to nonspecific enzymatic digestion (16). Because of the observed discrepancy between the isolated TSMC and the TSMC of the intact tissue, the data deduced from studies that examined either the cultured or the enzymatically isolated TSMC may not accurately depict the physiology of the intact airway smooth muscle. Therefore, the detailed mechanism for the generation of ACW in the intact tracheal muscle bundle requires further investigation.

In this study, we examine the mechanism for the generation of ACh-induced ACW in the TSMC of the intact porcine tracheal muscle bundle. The focus of this study was to identify the Ca2+ transport molecules involved in the generation and maintenance of ACh-mediated ACW.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue preparations. Porcine trachea obtained from a local abattoir was placed in physiological saline solution (PSS) at 4°C. Tracheal smooth muscle strips (~6 x 1.5 x 0.3 mm in dimension) free of epithelium and connective tissue were isolated from the trachea. Each tracheal muscle strip contains multiple muscle bundles (19). The tracheal muscle strips were subsequently attached at both ends to aluminum foil clips designed for mounting onto the custom-built setup.

Cell permeabilization. Tracheal muscle strips were permeabilized using 10 µM digitonin in an intracellular substitution solution (see Solutions and chemicals) for 10 min. The experiments were performed in intracellular substitution solution for permeabilized tracheal muscle strips. Successful permeabilization of tracheal muscle bundle was verified by the observation of force generation following either the application of inositol 1,4,5-trisphosphate (IP3) or increased extracellular [Ca2+] at the end of each experiment.

Isometric force measurement. The porcine tracheal muscle strips were attached to an isometric force transducer and equilibrated in PSS at 37°C for 1 h. During this time, the resting tension was maintained at 0.3 g. Exchange of bathing solutions was accomplished by simultaneously draining and refilling the tissue bath. The muscle strips were stimulated twice with 80 mM K+ PSS for 5 min each time. The experiment protocols were applied after complete relaxation of the tissue from the second dose of 80 mM K+ stimulation. Chart v3.4.5 software (ADIinstruments) was employed for data acquisition and analysis.

Confocal imaging. The details of the confocal Ca2+ imaging method have been described previously by investigators in our laboratory (19). Briefly, 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 isometrically mounted onto the custom-made stiff force transducer setup for [Ca2+]i measurements. The changes in [Ca2+]i were measured using an inverted Leica TCS SP2 AOBS laser scanning confocal microscope with an air x10 (numerical aperture 0.3) lens. The tissue was illuminated using the 488-nm line of an argon-krypton laser, and a high-gain photomultiplier tube collected the emission at wavelengths between 505 and 550 nm. The acquisition rate was 3 frame/s. The measured changes in fluo-4 fluorescence level are proportional to the relative changes in [Ca2+]i. All parameters (laser intensity, gain) were maintained during the experiment.

Data analysis. All confocal image analysis was performed with ImageProPlus software using customized routines written in Visual Basic. Analysis of frequency of the ACW 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 as well as 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 reflects the difference between the peak fluorescence of individual Ca2+ spikes in the ACW and the prestimulation baseline level. The fluorescence level derived in each region is linearly proportional to the [Ca2+]i in that region in such a fashion that any fluctuation in [Ca2+]i would be proportionally reflected in the changes in fluorescence.

All summarized data are presented as means ± SE. For numerical analysis, all data were analyzed with Excel or SigmaPlot software using the appropriate statistical tests. Paired Student's t-test was used for comparisons. A value of P < 0.05 was considered significant. The n values indicated for contraction experiments represent the number of animals studied, and the n values indicated for the Ca2+ studies represent the number of TSMC studied from the specified numbers of animals. For each study protocol, only one muscle strip from each animal was used, and the number of tissues indicated therefore reflects the number of animals. For the analysis of the Ca2+ signals, when the data obtained from individual TSMC were pooled together from different animals, ANOVA was performed and variance is reported when significant. In our current study, no significant variance was found.

Solutions and chemicals. Normal PSS containing (in mM) 140 NaCl, 5 KCl, 1.5 CaCl2, 1 MgCl2, 10 glucose, and 5 HEPES (pH 7.4 at 37°C) was used for all of the studies. High-K+ (80 mM extracellular K+) PSS was identical in composition to normal PSS with the exception of (in mM) 65 NaCl and 80 KCl. Zero-Ca2+ PSS and zero-Na+ PSS were prepared in the same way as normal PSS, but CaCl2 was replaced with 1 mM EGTA and NaCl was replaced with equimolar N-methyl-D-glucamine. The intracellular substitution solution used contained (in mM) 130 KCl, 10 NaCl, 5 K2HPO4, 5.6 glucose, 1 MgSO4, 5 Tris-succinate, 1 ATP, 75 µM EGTA, and 20 HEPES (pH 7.0 at 37°C). Fluo-4 AM, Pluronic F-127, and 2',4'-dichlorobenzamil (2',4'-DCB) were purchased from Molecular Probes and were dissolved in dimethyl sulfoxide (DMSO). Stocks of ACh, caffeine, digitonin, and IP3 (Sigma) were prepared in normal PSS, and stocks of 2-aminoethoxydiphenyl borate (2-APB), xestospongin C, nifedipine, cyclopiazonic acid (CPA), KB-R7943, SKF-96365, procaine, and tetracaine (Sigma) were prepared in 100% ethanol.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Dependence of ACW on plasmalemmal Ca2+ influx. There are two potential sources of Ca2+ that can contribute to the generation of ACh-mediated ACW: Ca2+ release from intracellular store and Ca2+ influx from the extracellular space. To determine the significance of plasmalemmal Ca2+ entry in ACh-mediated ACW, we studied the effect of extracellular Ca2+ removal on the ACh-induced ACW. As shown in Fig. 1, ACh-mediated ACW were completely abolished in zero-Ca2+ PSS within 5 min of treatment (n = 30 cells from 5 animals). This finding indicates that Ca2+ entry from the extracellular space is required for maintaining ACh-induced ACW.


Figure 1
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  		Fig. 1. Effect of removal of extracellular Ca2+ on acetylcholine (ACh)-induced asynchronous Ca2+ waves (ACW). Application of 3 µM ACh elicited ACW in porcine tracheal smooth muscle cells (TSMC) placed in normal physiological saline solution (PSS) with 1.5 mM extracellular Ca2+. Replacement of the normal PSS with 0-Ca2+ PSS resulted in the cessation of the ACW. Experimental intracellular Ca2+ concentration ([Ca2+]i) trace is representative of results in 30 cells from 5 different animals.

 
To further define the Ca2+ entry pathways that are responsible for maintaining the ACW, we used SKF-96365, an inhibitor of receptor-operated channels (ROC) and store-operated channels (SOC), together with nifedipine, a selective inhibitor of L-type VGCC. Results from a previous study in our laboratory (19) showed that high-dose nifedipine (10 µM) reduced the frequency of ACh-mediated ACW by 29% and partially inhibited ACh-mediated tonic contraction by 32.8%. It is important to note that at 10 µM, nifedipine was able to completely abolish the contraction induced by 80 mM K+-PSS in the tracheal muscle bundle. These findings indicate that Ca2+ influx via L-type VGCC plays only a minor role in the maintenance of ACh-induced ACW and tonic contraction in the porcine TSMC. Therefore, an additional pathway(s) for Ca2+ entry must be responsible for supporting the nifedipine-resistant component of the ACh-mediated ACW and tonic contraction. In the isolated airway smooth muscle cells, both SOC and ROC have been implicated in Ca2+ signaling (3, 24, 28, 29). As shown in Fig. 2, 50 µM SKF-96365 abolished the nifedipine-resistant ACW within 2 min of its application (n = 30 cells from 4 animals). A corresponding time control trace of the ACh-mediated ACW in Fig. 2C shows the persistent generation of ACW up to 45 min after ACh stimulation, and the frequency of the ACW at 30 s and 45 min poststimulation was 0.36 ± 0.02 and 0.33 ± 0.02 Hz, respectively (P = 0.168, n = 40 cells from 5 animals). In parallel contraction studies (Fig. 2), a combination of 10 µM nifedipine and 50 µM SKF-96365 abolished 93.7 ± 1% of ACh-induced tonic contraction compared with the 32.8 ± 2.9% reduction in ACh-induced tonic contraction seen with nifedipine alone (P < 0.0001, n = 5 animals). SKF-96365 was used after the addition of nifedipine but not in the reverse sequence, because it is known to inhibit the L-type VGCC as well. To examine the possibility that the high-dose nifedipine may produce nonspecific inhibition beyond the blockade of the L-type VGCC, we used 100 nM nifedipine, and it produced a degree of inhibition (33.0 ± 3.3%, P = 0.0096, n = 3 animals) of ACh-induced tonic contraction similar to that produced by 10 µM nifedipine. These findings suggest that Ca2+ entry via the ROC/SOC and the L-type VGCC are important in maintaining ACh-induced ACW as well as tonic contraction in the tracheal muscle bundle.


Figure 2
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  		Fig. 2. Effect of 10 µM nifedipine and 50 µM SKF-96365 on ACh-induced ACW and tonic contraction. A: L-type voltage-gated Ca2+ channel (VGCC) blockade by nifedipine did not abolish ACh-induced ACW but reduced the frequency of the ACW. Additional application of SK-F96365 abolished ACh-induced ACW completely. Experimental [Ca2+]i trace is representative of results in 30 cells from 4 different animals. B: application of nifedipine partially reduced the ACh-induced contraction, whereas SKF-96365 nearly abolished the remaining contraction. Experimental tissue contraction trace is representative of results from 5 different animals. C: time-control traces of ACh-mediated ACW at 30 s and 45 min poststimulation.

 
In addition to the conventional plasmalemmal Ca2+-permeable channels, the Na+/Ca2+ exchanger (NCX) operating in the reverse mode can be an important pathway for Ca2+ entry into smooth muscle cells (1, 6). The role of NCX in airway smooth muscle is controversial. Although some studies have reported little contribution by NCX (13, 17), others have implicated NCX in the Ca2+ and contractile regulation of airway smooth muscle (8, 11, 12, 30, 35, 38). To examine whether the reverse-mode Na+/Ca2+ exchange is involved in supporting the nifedipine-resistant ACW in the porcine TSMC, we used an inhibitor of both the forward- and the reverse-mode Na+/Ca2+ exchange, 2',4'-DCB, and a selective reverse-mode inhibitor of NCX, KB-R7943 (6, 21, 41, 43, 45, 48). As shown in Fig. 3, the application of 10 µM 2',4'-DCB abolished nifedipine-resistant ACW induced by ACh (n = 45 cells from 7 animals) and inhibited nifedipine-resistant tonic contraction by 91.0 ± 1.4% (P < 0.001, n = 8 animals). Similarly, the application of 20 µM KB-R7943 abolished nifedipine-resistant ACW induced by ACh (n = 45 cells from 7 animals) and inhibited the corresponding tonic contraction by 86.9 ± 4.3% (P < 0.0001, n = 8 animals). In addition to the use of the above-mentioned pharmacological inhibitors, we also examined the effect of removal of extracellular Na+ on ACh-mediated ACW and tonic contraction. As shown in Fig. 3C, the removal of extracellular Na+ with the use of zero-Na+ PSS abolished ACh-mediated ACW within 10 min (n = 40 cells from 5 animals). In a parallel contraction study (Fig. 3C), the removal of extracellular Na+ reduced ACh-mediated tonic contraction by 72.7 ± 2.2% (P < 0.001, n = 5 animals). These data uniformly indicate that reverse-mode Na+/Ca2+ exchange is involved in maintaining ACh-induced ACW and tonic contraction. The sensitivity of the nidefipine-resistant ACW and tonic contraction to SKF-96365, 2',4'-DCB, and KB-R7943 suggest that the ROC/SOC are likely operating in series with the NCX, as proposed previously (1, 21).


Figure 3
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  		Fig. 3. Effect of 10 µM 2',4'-dichlorobenzamil (2',4'-DCB), 20 µM KB-R7943, and 0-Na+ PSS on the nifedipine-resistant component of ACh-induced ACW and tonic contraction. A: application of 10 µM nifedipine reduced the frequency of ACh-induced ACW, whereas additional application of 2',4'-DCB resulted in inhibition of the nifedipine-resistant component of the ACh-induced ACW and tonic contraction. Experimental [Ca2+]i trace is representative of results in 45 cells from 7 different animals, and tissue contraction trace is representative of results from 8 different animals. B: the nifedipine-insensitive portion of the ACh-induced ACW and tonic contraction were inhibited by KB-R7943. Experimental [Ca2+]i trace is representative of results in 45 cells from 7 different animals, and tissue contraction trace is representative of results from 8 different animals. C: replacement of the bathing solution with 0-Na+ PSS resulted in cessation of ACh-induced ACW and significant inhibition of the tonic contraction. Experimental [Ca2+]i trace is representative of results in 40 cells in 5 different animals, and tissue contraction trace is representative of results from 5 different animals.

 
Dependence of ACW on SR Ca2+ release. As described previously (19), ACh induces ACW in the TSMC of the intact porcine tracheal muscle bundle. The wavelike nature of the Ca2+ signal implies that SR Ca2+ release is most likely responsible for raising the [Ca2+]i, because the Ca2+ signal produced by extracellular Ca2+ entry would typically result in a spatially more uniform elevation of [Ca2+]i (39). To examine the role of plasmalemmal Ca2+ entry in the generation of the ACW, we pretreated the tracheal muscle bundle with 10 µM nifedipine and 50 µM SKF-96365 for 5 min before ACh stimulation to block all the Ca2+ entry pathways that were found to be important in supporting the ACW (Fig. 2). As indicated in Fig. 4, blockade of the L-type VGCC and ROC/SOC did not prevent the induction of ACW, because ACh was able to induce Ca2+ waves initially (n = 20 cells from 4 animals). However, the ACW did not persist as they did in the absence of nifedipine and SKF-96365 (Fig. 4). More interestingly, caffeine (12.5 mM) induced no significant rise in [Ca2+]i after the cessation of the ACW, because the peak [Ca2+]i levels before and immediately after the addition of caffeine were 100.9 ± 0.8 and 100.6 ± 1.0% of baseline level pre-ACh stimulation, respectively (P = 0.862, n = 20 cells from 4 animals), indicating that the SR Ca2+ stores had been emptied. It is important to note that the pretreatment of the muscle bundle with only nifedipine and SKF-96365 at the same concentration did not affect the amplitude of the caffeine (12.5 mM)-induced Ca2+ transient compared with the control amplitude before the addition of nifedipine and SKF-96365 (P = 0.896, n = 28 cells from 4 animals). This finding suggests that the ACW are the result of SR Ca2+ release and that the SR Ca2+ release can be initiated in the absence of extracellular Ca2+ entry, thereby excluding plasmalemmal Ca2+ entry-induced SR Ca2+ release by the process of Ca2+-induced Ca2+ release as the primary activation mechanism. Furthermore, if SR Ca2+ release is responsible for the generation of the ACW, blockade of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) should completely inhibit the ACW, because the SR Ca2+ store can no longer be replenished. As shown in Fig. 5, the application of CPA (10 µM), a selective inhibitor of SERCA, resulted in a brief broadening followed by complete abolition of the ACh-mediated ACW within 10 s, leaving behind a small but significant elevation in baseline [Ca2+] (P < 0.0001, n = 30 cells from 4 animals) that corresponds to 26 ± 2% of the peak [Ca2+] of the ACW. In a parallel contraction study, the application of 10 µM CPA produced a 83.4 ± 2.8% inhibition of the tonic contraction induced by ACh (P < 0.001, n = 4 animals,). These findings collectively indicate that ACW are produced by repetitive cycles of SR Ca2+ release followed by Ca2+ reuptake and that Ca2+ entry through the L-type VGCC, ROC/SOC, and NCX pathway is necessary to ensure the continual proper refilling of the SR Ca2+ store to sustain the ongoing ACW.


Figure 4
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  		Fig. 4. Effect of 10 µM nifedipine and 50 µM SKF-96365 on the initiation of ACh-induced ACW. Application of ACh elicited transient repetitive Ca2+ waves in tissues pretreated with nifedipine (L-type VGCC blocker) and SKF-96365 [receptor-operated channels/store-operated channels (ROC/SOC) blocker] in contrast to the sustained repetitive Ca2+ waves in the control tissue. Experimental [Ca2+]i trace is representative of results in 20 cells from 4 different animals.

 

Figure 5
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  		Fig. 5. Effect of 10 µM cyclopiazonic acid (CPA) on ACh-induced ACW. Application of CPA to ACh-stimulated porcine TSMC completely abolished the ongoing ACW. Experimental [Ca2+]i trace is representative of results in 30 cells from 4 different animals.

 
Given that SR Ca2+ release produces the ACW, we proceeded to identify the type(s) of SR Ca2+ release channels involved in the generation of ACh-induced ACW. As mentioned earlier, in isolated TSMC, SR Ca2+ release through the IP3R is required for the initiation of ACh-induced repetitive Ca2+ waves (18). To determine whether SR Ca2+ release through the IP3R is responsible for producing ACh-induced ACW in the intact porcine tracheal muscle bundle, we used 2-APB and xestospongin C, both well-established cell-permeable inhibitors of IP3R that have been found to block IP3R-mediated Ca2+ release in a variety of tissues, including the airway smooth muscle as well as non-smooth muscle cell types (2, 14, 25, 26, 27, 32, 33, 34, 46). As shown in Fig. 6, after pretreatment of the tissue with 75 µM 2-APB for 30 min, ACh (3 µM) was still able to induce ACW and tonic contraction in a manner similar to that observed in the absence of 2-APB in the same tissue. Furthermore, the addition of 2-APB to ongoing ACh-mediated ACW and tonic contraction produced no measurable effect on the ACW and the tonic contraction (Fig. 6). It is important to note that the 30-min pretreatment of the tissue with the same batch of 2-APB at 75 µM was able to inhibit phenylephrine (5 µM)-induced tonic contraction of the porcine aorta by 94.4 ± 5.4% (P < 0.0001, n = 4 animals). More importantly, in a separate experiment in which we permeabilized the tracheal muscle bundle with 10 µM digitonin (31, 40, 44, 47), the addition of IP3 (10 µM) produced a transient contraction that was completely prevented with 2-APB (75 µM) pretreatment (n = 4 animals) (Fig. 6E). In contrast, as shown in Fig. 6F, 2-APB pretreatment did not prevent or attenuate caffeine-induced contraction (n = 4 animals). These positive control studies show that 75 µM 2-APB is able to effectively inhibit porcine airway smooth muscle IP3R and porcine vascular smooth muscle IP3R, because the vascular smooth muscle in the aorta is known to exhibit ACW, and the phenylephrine-induced ACW in the vascular smooth muscle depend on SR Ca2+ release through the IP3R (22). Similar to the results with 2-APB, pretreatment of the tissue for 45 min with 10 µM xestospongin C produced no significant effect on ACh-induced ACW and tonic contraction in the intact porcine muscle bundle, and the addition of 10 µM xestospongin C to tissues already stimulated with ACh produced no measurable effect on the ongoing ACW and tonic contraction (Fig. 7). The lack of effect seen with these two structurally distinct inhibitors of the IP3R suggests that Ca2+ release via the IP3R is not required for the generation or maintenance of ACh-induced ACW and tonic contraction.


Figure 6
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  		Fig. 6. Effect of 75 µM 2-aminoethoxydiphenyl borate (2-APB) on ACh-induced ACW and tonic contraction and on inositol 1,4,5-trisphosphate (IP3)-induced contraction in intact smooth muscle cell from porcine trachea. A: applications of ACh initiated comparable ACW in cells from the control group and the 2-APB-pretreated (30 min) group. Experimental [Ca2+]i traces are representative of results in 20 cells from 4 different animals. B: application of 2-APB did not affect the ongoing ACW mediated by ACh. Experimental [Ca2+]i trace is representative of results in 20 cells from 4 different animals. C: ACh stimulated tension development in both the control smooth muscle bundle and the 2-APB-pretreated (30 min) muscle bundle. Experimental tissue contraction traces are representative of results from 5 different animals. D: application of 2-APB did not affect the ongoing tonic contraction mediated by ACh. Experimental tissue contraction trace is representative of results from 5 different animals. E: pretreatment with 75 µM 2-APB prevented the generation of force transient induced by 10 µM IP3 shown in control trace in digitonin (10 µM)-permeabilized tracheal muscle bundle. Experimental tissue contraction traces are representative of results in 4 animals. F: pretreatment with 75 mM 2-APB did not affect the generation of force transient induced by 12.5 mM caffeine in permeabilized tracheal muscle bundle. Experimental tissue contraction traces are representative of results in 4 animals.

 

Figure 7
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  		Fig. 7. Effect of 10 µM xestospongin C (Xe-C) on ACh-induced ACW and tonic contraction. A: preexposure of porcine tracheal muscle strip with Xe-C (10 µM) for 45 min did not prevent the initiation of ACh-mediated ACW. Experimental [Ca2+]i traces are representative of results in 30 cells from 5 different animals. B: application of Xe-C did not affect the ongoing ACW induced by ACh. Experimental [Ca2+]i trace is representative of results in 30 cells from 5 different animals. C: preexposure of porcine tracheal muscle strip with Xe-C for 45 min did not prevent the generation of ACh-induced tissue contraction. Experimental tissue contraction traces are representative of results in 5 different animals. D: application of Xe-C did not affect the ACh-induced tonic contraction. Experimental tissue contraction trace is representative of results in 5 different animals.

 
In addition to the IP3R, another type of SR Ca2+ release channel that is known to be functionally important in the TSMC is the RyR. As shown in Fig. 8, pretreatment of the tracheal muscle bundle for <2 min with either 12.5 mM caffeine or 25 µM ryanodine to empty Ca2+ from the RyR-sensitive SR Ca2+ store completely prevented the generation of ACh-induced ACW. These findings support the notion that SR Ca2+ release from the RyR-dependent SR store is responsible for the generation of ACW but nonetheless do not prove the involvement of RyR in the Ca2+ release. We therefore employed procaine and tetracaine, both known membrane-permeable inhibitors of RyR-mediated SR Ca2+ release, to block the RyR channels (7, 10, 15). As demonstrated in Fig. 9, pretreatment of tracheal muscle bundle with 2 mM procaine for 30 min completely prevented ACh-induced ACW and tonic contraction. When 2 mM procaine was applied to ACh-stimulated tracheal muscle bundles exhibiting ongoing ACW and tonic contraction, it immediately abolished ongoing ACW and reduced the ongoing tonic contraction to 5.3 ± 0.7% of the original level (P < 0.0001, n = 6 animals). Similarly, as shown in Fig. 10, pretreatment of the tracheal muscle bundle with 100 µM tetracaine for 30 min completely prevented ACh-induced ACW and tonic contraction as well. The application of 100 µM tetracaine to ACh-stimulated tissues resulted in the immediate cessation of the ongoing ACW and near-complete inhibition of the ongoing tonic contraction to 13.3 ± 5.3% of the original level (P = 0.0038, n = 5 animals). These findings together suggest that SR Ca2+ release via the RyR channel is responsible for both the initiation and maintenance of ACh-mediated ACW and tonic contraction in the porcine tracheal smooth muscle.


Figure 8
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  		Fig. 8. Effect of 12.5 mM caffeine and 25 µM ryanodine on ACh-induced ACW in smooth muscle cells from intact porcine trachea. A: application of ACh initiated ACW in cells from the control group, whereas it resulted in no measurable Ca2+ signal in cells from the caffeine-pretreated group. Experimental [Ca2+]i traces are representative of results in 20 cells from 4 different animals. B: application of ACh stimulated ACW in control smooth muscle bundle but produced no response in ryanodine-pretreated muscle bundles. Experimental [Ca2+]i traces are representative of results in 20 cells from 4 different animals.

 

Figure 9
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  		Fig. 9. Effect of 2 mM procaine on ACh-induced ACW and tonic contraction in intact smooth muscle cells from porcine trachea. A: applications of ACh initiated ACW in cells from the control group, whereas it resulted in no measurable Ca2+ signals in cells from the procaine-pretreated (30 min) group. Experimental [Ca2+]i traces are representative of results in 20 cells from 4 different animals. B: application of procaine immediately abolished the ongoing ACW mediated by ACh. Experimental [Ca2+]i trace is representative of results in 20 cells from 4 different animals. C: application of ACh stimulated tension development in control smooth muscle bundle but elicited no response in procaine-pretreated (30 min) muscle bundle. Experimental tissue contraction traces are representative of results from 6 different animals. D: application of procaine abolished the ongoing tonic contraction mediated by ACh. Experimental tissue contraction trace is representative of results from 6 different animals.

 

Figure 10
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  		Fig. 10. Effect of 100 µM tetracaine on ACh-mediated ACW and tonic contraction in porcine tracheal smooth muscle cells. A: ACh-mediated ACW were observed in control porcine tracheal muscle cells but were absent in porcine tracheal muscle strip preincubated with tetracaine for 30 min. Experimental [Ca2+]i traces are representative of results in 30 cells from 5 different animals. B: application of tetracaine immediately abolished the ongoing ACh-induced ACW. Experimental [Ca2+]i trace is representative of results in 30 cells from 5 animals. C: ACh induced tonic contraction in control muscle bundle, whereas it produced no response in tetracaine-pretreated (30 min) muscle bundle. Experimental tissue contraction traces are representative of results in 5 different animals. D: application of tetracaine immediately attenuated the ongoing ACh-induced tonic contraction. Experimental tissue contraction trace is representative of results in 5 different animals.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The aim of this study was to examine the mechanism of the ACW in the porcine tracheal smooth muscle. Our findings show that extracellular Ca2+ entry is not directly responsible for the generation of the Ca2+ waves (Fig. 4) and that the ACW are produced by recurring cycles of SR Ca2+ release and SR Ca2+ reuptake (Fig. 5). We also characterized the type of SR Ca2+ release channels involved. As shown in Figs. 6 and 7, the application of IP3R inhibitors 2-APB and xestospongin C produced no measurable effect on the initial generation and the maintenance of ACh-induced ACW and tonic contraction. It is unlikely that two structurally unrelated inhibitors of IP3R with well-demonstrated efficacy in various smooth muscle and non-smooth muscle cell types would be completely ineffective in inhibiting the IP3R in the porcine tracheal smooth muscle (2, 14, 25–27, 32–34, 46). In addition, our control studies demonstrate that 2-APB is an effective inhibitor of porcine airway IP3R, because 2-APB pretreatment completely inhibited IP3-induced force transient in permeabilized tracheal muscle bundle. 2-APB also produced significant inhibition of phenylephrine-induced tonic contraction of the nonpermeabilized porcine aorta, a process that is driven by IP3R-mediated ACW in the vascular smooth muscle (20). Furthermore, a similar concentration of xestospongin C has been shown to be effective in preventing ACh and ATP-induced ACW in the mouse bronchiolar smooth muscle cells (5, 34), suggesting that xestospongin C is able to inhibit IP3R in the airway smooth muscle as well. The observed difference in the sensitivity to xestospongin C between ACh-induced ACW in the porcine tracheal smooth muscle and ACh-induced ACW in the mouse bronchial smooth muscle may reflect interspecies or inter-airway segment heterogeneity in the mechanism of ACW. The lack of effect with 2-APB and xestospongin C observed in our study therefore suggests that Ca2+ release through the IP3R is not involved in the generation of the ACW in ACh-stimulated intact porcine tracheal smooth muscle. In contrast, pretreatment of the tracheal muscle bundle with caffeine or ryanodine prevented the generation of the ACW by ACh. More importantly, application of procaine or tetracaine prevented the generation of the ACW and produced immediate and complete inhibition of ongoing ACW induced by ACh. These findings show that ACh-induced ACW in the intact porcine smooth muscle are the result of repetitive waves of SR Ca2+ release through the RyR. Even though extracellular Ca2+ entry is not immediately required for the generation of the ACW, it is necessary to support ongoing ACW over time, as shown in Fig. 4. This is likely due to the fact that a proportion of the Ca2+ released by the SR to produce the Ca2+ waves is inevitably extruded to the extracellular space, possibly via the plasma membrane Ca2+-ATPase. In the absence of sufficient extracellular Ca2+ entry, the SR is unable to continually replenish itself after repetitive waves of Ca2+ release, and the ACW cease after a period of time because the SR Ca2+ store is depleted (Fig. 4). Therefore, to maintain the ongoing ACW, it is important to replenish the SR Ca2+ store with additional Ca2+ from the extracellular space. Our pharmacological characterizations have implicated the L-type VGCC, the NCX operating in the reverse mode, and the ROC/SOC-type channel. The role of the L-type VGCC is ascertained when both low-dose (100 nM) and high-dose (10 µM) nifedipine produce comparable partial inhibitory effects on ACh-induced ACW and tonic contraction (Fig. 2). The exact nature of the ROC/SOC-type channel is not known at this point. However, our findings have revealed a few characteristics of this ROC/SOC-type channel. First, it is sensitive to SKF-96365 but resistant to nifedipine, 2-APB, and xestospongin C. Second, this channel is likely a nonselective cation channel, given the involvement of the reverse-mode NCX in supporting the ACW. For the NCX to contribute to Ca2+ entry into the cell, it would need to operate in its reverse mode. To activate reverse-mode Na+/Ca2+ exchange, ACh-stimulated TSMC must allow for an influx of Na+ into the cell, and such influx of Na+ in smooth muscle cells typically occurs through a nonselective cation channel (1). Na+ entry from the nonselective cation channel then accumulates in the subplasmalemmal space and drives the NCX into the reverse mode of operation. In this model, the nonselective cation ROC/SOC-type channel is coupled in series to the NCX operating in the reverse mode. This would explain our findings that the nifedipine-resistant ACW and tonic contraction are similarly sensitive to both SKF-96365, an inhibitor of the ROC/SOC, and 2',4'-DCB and KB-R7943, inhibitors of the NCX. It is important to note that although the phenomenon of reverse-mode Na+/Ca2+ exchange coupled with the nonselective cation channel has never been reported previously in airway smooth muscle, it has been well described in vascular smooth muscle (1, 21). In both animal and human airway smooth muscle, it is known that NCX is present and is capable of operating in the reverse mode (8, 35, 38).

Our study of the mechanism of ACW in ACh-stimulated TSMC of the intact tissue has revealed some crucial differences from the enzymatically dissociated porcine TSMC. First, in isolated porcine TSMC challenged with ACh, it was shown previously that Ca2+ release through IP3R is important in initiating the repetitive Ca2+ waves (18). However, in the porcine TSMC of the intact tissue, two structurally unrelated inhibitors of IP3R did not affect either the initial generation or the maintenance of ACh-induced ACW and tonic contraction. Second, Prakash et al. (36) showed that the repetitive Ca2+ waves could be abolished by 100 nM nifedipine, which suggested that Ca2+ entry through the L-type VGCC is the main pathway utilized for refilling the SR Ca2+ store and maintenance of the repetitive cycles of SR Ca2+ release. Nonetheless, our previous study showed that the inhibition of the L-type VGCC by high-dose nifedipine attenuated the frequency of the ACW but did not abolish the ongoing ACW or the tonic contraction stimulated with ACh (19). This finding indicates that Ca2+ entry through the L-type VGCC is not obligatory for maintaining the ACW in intact tracheal smooth muscle, and as shown in this report, Ca2+ entry via the ROC/SOC and the NCX operating in the reverse mode are able to sustain the ACW in the absence of Ca2+ entry through the L-type VGCC. Third, results from earlier single-cell studies also showed that NCX plays only a minor role in the regulation of Ca2+ signaling in the tracheal smooth muscle (17). However, the significant inhibitory effects produced by KB-R7943 and 2',4'-DCB, as well as extracellular Na+ removal, on ACh-induced ACW and tonic contraction indicate that the NCX plays an important role in agonist-induced Ca2+ signaling in the porcine TSMC. These discrepancies in the characteristics between single-cell preparations and intact tissue show that enzymatically isolated TSMC are phenotypically different from the intact TSMC of intact tissue. It would appear that the process of enzymatic dissociation may have altered the phenotype of these TSMC, possibly as a result of the nonspecific proteolysis and the disruption of intercellular communication as suggested previously (16).

In summary, the data presented in this article show that multiple Ca2+ translocating proteins are involved in the generation of the ACW observed in ACh-stimulated smooth muscle cells of the intact porcine tracheal muscle bundle. The ACW appear to be produced by repetitive cycles of RyR-mediated SR Ca2+ release followed by SERCA-mediated SR Ca2+ reuptake. Because a proportion of the released Ca2+ is lost to the extracellular space, extracellular Ca2+ entry involving the L-type VGCC, ROC/SOC, and reverse-mode Na+/Ca2+ exchange is important to supply additional Ca2+ to refill the SR Ca2+ and to sustain the repetitive Ca2+ waves. In the future, more detailed characterization of the molecules important in ACh-mediated excitation-contraction coupling of the airway smooth muscle may help to identify a novel therapeutic target for the treatment of airway diseases characterized by excessive muscle contraction.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
J. Dai is the recipient of a Canadian Institutes of Health Research Doctoral Research Award. C.-H. Lee is a Post Graduate Year 2 resident in Anatomical Pathology (University of British Columbia, Vancouver, BC, Canada).


   ACKNOWLEDGMENTS
 
We thank the St. Paul's Hospital Foundation for generous support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C.-H. Lee, The iCAPTURE Center, Univ. of British Columbia, St. Paul's Hospital, Rm. 292, 1081 Burrard St., Vancouver, BC, Canada V6Z 1Y6 (e-mail: breemen{at}interchange.ubc.ca)

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.

* J. M. Dai and K.-H. Kuo contributed equally to this work. C. van Breemen and C.-H. Lee contributed equally to this work. Back


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