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Published ahead of print on November 1, 2007, doi:10.1165/rcmb.2007-0259OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 38, pp. 407-413, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0259OC

A Novel Ca2+ Influx Pathway Activated by Mechanical Stretch in Human Airway Smooth Muscle Cells

Satoru Ito1, Hiroaki Kume1, Keiji Naruse2,3, Masashi Kondo1, Naoya Takeda1, Susumu Iwata1, Yoshinori Hasegawa1 and Masahiro Sokabe3–5,

1 Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan; 2 Department of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Okayama, Japan; 3 ICORP/SORST Cell Mechanosensing, JST, Nagoya, Japan; 4 Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya, Japan; and 5 Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki, Japan

Correspondence and requests for reprints should be addressed to Hiroaki Kume, M.D., Ph.D., Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: hkume{at}med.nagoya-u.ac.jp


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
In response to mechanical stretch, airway smooth muscle exhibits various cellular functions such as contraction, proliferation, and cytoskeletal remodeling, all of which are implicated in the pathophysiology of asthma. We tested the hypothesis that mechanical stretch of airway smooth muscle cells increases intracellular Ca2+ concentration ([Ca2+]i) by activating stretch-activated (SA) nonselective cation channels. A single uniaxial stretch (3 s) was given to human bronchial smooth muscle cells cultured on an elastic silicone membrane. After the mechanical stretch, a transient increase in [Ca2+]i was observed. The [Ca2+]i increase was significantly dependent on stretch amplitude. The augmented [Ca2+]i due to stretch was completely abolished by removal of extracellular Ca2+ and was markedly attenuated by an application of Gd3+, an inhibitor of SA channels, or ruthenium red, a transient receptor potential vanilloid (TRPV) inhibitor. In contrast, the stretch-induced rises of [Ca2+]i were not altered by other Ca2+ channel inhibitors such as nifedipine, BTP-2, and SKF-96365. Moreover, the [Ca2+]i increases were not affected by indomethacin, a cyclooxygenase inhibitor, U-73122, a phospholipase C inhibitor, or xestospongin C, an inhibitor of the inositol-trisphosphate receptor. These findings demonstrate that a novel Ca2+ influx pathway activated by mechanical stretch, possibly through the Ca2+-permeable SA channel activated directly by stretch rather than by indirect mechanisms via intracellular messenger production, is involved in human airway smooth muscle cells. A molecular candidate for the putative SA channel may be one of the members of the TRPV channel family. Thus, abnormal Ca2+ homeostasis in response to excessive mechanical strain would contribute to the pathogenesis of asthma.

Key Words: Ca2+ channels • mechanotransduction • mechanical stress • transient receptor potential



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Mechanical stretch alters airway smooth muscle (ASM) functions implicated in the pathophysiology of asthma. We demonstrate a novel Ca2+ influx pathway activated by stretch in ASM cells that could be a novel target molecule for the treatment of asthma.

 
It is well established that intracellular free Ca2+ plays a central role in a variety of cellular functions. In airway smooth muscle (ASM) cells, intracellular Ca2+ concentration ([Ca2+]i) determines contractile force, cell proliferation, migration, and cytokine release (1–4), all of which are significantly associated with the pathophysiology of asthma. Therefore, abnormality in Ca2+ homeostasis of ASM cells may contribute to development of airway hyperresponsiveness in patients with asthma (5, 6). [Ca2+]i is regulated by Ca2+ release from internal stores and Ca2+ influx from the extracellular side through the plasma membrane in smooth muscle cells (7). As in other cells, different Ca2+ influx pathways, voltage-dependent Ca2+ channels, receptor-operated Ca2+ entry, and store-operated Ca2+ entry have been observed in ASM cells (3, 8, 9). Moreover, another Ca2+ influx pathway, Ca2+-permeable cation channels activated by mechanical stretch, called stretch-activated (SA) cation channels, has been found in various cell types, including smooth muscle cells (10–15). Recent evidence has indicated that some SA channels are encoded by mammalian homologs of the Drosophila transient receptor potential (TRP) genes, specifically the TRP vanilloid (TRPV) subfamily (16).

During tidal breathing, the ASM within airway walls is continuously exposed to mechanical strain. In particular, the airway walls in patients with asthma are exposed to excessive mechanical strain during bronchoconstriction (17). Thus, the cellular properties of ASM such as contraction, stiffness, reorganization of the actin cytoskeleton, and cytokine release are influenced by the mechanical strain in both normal and asthmatic conditions (18–27). Therefore, it is important to characterize how mechanical stress triggers signal transduction in the lung cells to fully understand the pathogenesis of asthma. However, the fundamental mechanisms converting mechanical force into intracellular signaling which is referred to as mechanotransduction (28) remain unclear in these cells (26, 29). The Ca2+ influx through SA channels is considered to be involved in this mechanotransduction process (28). Indeed, contractile response and activation of intracellular signaling are sensitive to Gd3+, an inhibitor of SA cation channels, in ASM cells (4, 24). Nevertheless, there is no direct evidence for Ca2+ influx in response to mechanical stretch in human ASM cells yet.

This study was designed to determine whether mechanical strain affects ASM cell properties. We focused on intracellular Ca2+ dynamics and investigated the mechanisms underlying regulation of [Ca2+]i in response to mechanical stretch in cultured human ASM cells.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Reagents
Indomethacin, nifedipine, and capsazepine were obtained from Sigma (St. Louis, MO). BTP-2, U-73122, xestospongin C, and SKF-96365 were from Calbiochem (La Jolla, CA). GdCl3 was from Wako (Osaka, Japan). Fura-2/AM was from Dojin (Kumamoto, Japan). Ruthenium red was from Latoxan (Valence, France).

Human Bronchial Smooth Muscle Cell Culture
Primary cultures of normal human bronchial smooth muscle cells from multiple donors were obtained from Cambrex (Walkersville, MD). The cells were maintained in culture medium containing 5% fetal bovine serum, human recombinant epidermal growth factor (1 ng/ml), insulin (10 µg/ml), human recombinant fibroblast growth factor (2 ng/ml), gentamicin (50 µg/ml), and amphotericin B (50 ng/ml, SmGM-2 BulletKit; Cambrex) in an atmosphere of 5% CO2 and 95% air at 37°C (30, 31). The cells retain protein expression of smooth muscle markers ({alpha}-smooth muscle actin, calponin, and smooth muscle myosin heavy chain) and exhibit the morphologic characteristic of smooth muscle. Cell viability was determined by morphology and trypan blue exclusion.

For detection of mRNA for TRPVs, primary cultures of normal human bronchial epithelial cells (Cambrex) were maintained in culture medium supplemented with bovine pituitary extract (52 µg/ml), hydrocortisone (0.5 µg/ml), human recombinant epidermal growth factor (0.5 ng/ml), epinephrine (0.5 µg/ml), transferrin (10 µg/ml), insulin (5 µg/ml), retinoic acid (0.1 µg/ml), triiodothyronine (6.5 µg/ml), gentamicin (50 µg/ml), and amphotericin B (50 ng/ml) (BEGM; Cambrex) (32).

Application of Uniaxial Stretch
Uniaxial mechanical stretch was applied to the human bronchial smooth muscle cells at the sixth to eighth passage using a stretch apparatus (ST-150; Strex, Osaka, Japan) (33, 34). The cells were removed from the dish with 0.01% EDTA-0.02% trypsin and transferred to a silicon chamber (10 mm long, 10 mm wide, and 5 mm deep) coated with 50 µg/ml human fibronectin (BD Biosciences, Bedford, MA) at a density of 2.0 x 103 cells/cm2. One end of the chamber was clamped in a fixed metal frame, and the other end in a movable frame that was connected to a shaft driven by a computer-controlled stepping motor. This apparatus was able to control the amplitude and the rate of stretch. In this study, the chamber was uniaxially stretched by 10 to 40% of the initial length at a rate of 0.1%/millisecond. Then the chamber was held for 1 second in the stretched position, and returned to the initial unstretched state at the same rate as the stretching phase. We confirmed that the bottom of the chamber was stretched uniformly over the area of interest.

Measurement of Intracellular Ca2+ Concentrations
The cells (50% confluence) in the silicon chamber were treated with fura-2/AM (10 µM) for 30 minutes at 37°C in HEPES-buffered saline (HBS) containing (in mM): NaCl 145, KCl 5, CaCl2 2, MgCl2 1, glucose 10, and HEPES 10 (pH 7.40). After the cells were washed with HBS, [Ca2+]i was assessed by the fluorescence of fura-2 using a fluorescence microscope with a x20 objective (Fluor 20; Nikon, Tokyo, Japan) (33, 34). Data were analyzed using a digital fluorescence imaging system (Aquacosmos, Hamamatsu Photonics, Hamamatsu, Japan). The excitation wavelengths were set at 340 and 380 nm, and the emission was detected at 510 nm by a photomultiplier. The intensity of the fura-2 fluorescence due to excitation at 340 nm (F340) and at 380 nm (F380) was measured after subtraction of the background fluorescence. The absolute amount of [Ca2+]i was not calculated because the dissociation constant of fura-2 for Ca2+ in smooth muscle cytoplasm is different from that in vitro (35). Thus, the ratio of F340 to F380 (F340/F380 ratio) was used as an indicator of the relative level of [Ca2+]i (3). The experiments were performed at room temperature (25–27°C). The cells were preincubated with each pharmacologic agent for 15 to 20 minutes.

RNA Isolation and RT-PCR
Total cellular RNA was extracted from human bronchial smooth muscle cells and normal human bronchial epithelial cells by using a commercial kit (Takara, Otsu, Japan). RNA was reverse transcribed to cDNA using a Superscript III kit (Invitrogen, Carlsbad, CA). PCR amplification was performed with 35 cycles of 30 seconds at 94°C, 30 seconds at 60°C, and 60 seconds at 72°C. The sequences of the forward and reverse specific primers, respectively, were designed as follows: TRPV1, 5'-GCGGCCTGGATTCTACTTC-3' and 5'-ACTCGGTGAACTTCCTGGAC-3'; TRPV2, 5'-CTTCTTCCAGAAGGGCCAAG-3' and 5'-AGGTGGCTCAGTCCTGAAAA-3'; TRPV4, 5'-GCCCCACATTGTCAACTACC-3' and 5'-TCCAGGGAGGAGAGGTCATA-3'; and GAPDH, 5'-AACGGATTTGGTCGTATTGG-3' and 5'-TGAGTCCTTCCACGATACCA-3'. The product sizes for TRPV-1, -2, -4, and GAPDH were 392, 384, 376, and 498 bp, respectively.

Statistical Analysis
All data are expressed as means ± SD. ANOVA followed by the Bonferroni test for post hoc analysis was used to evaluate the statistical significance. P < 0.05 was considered statistically significant.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Mechanical Stretch Increases Intracellular Ca2+ Concentration
Human bronchial smooth muscle cells cultured on an elastic silicon membrane were stretched equivalent to 10%, 20%, and 40% of loading in terms of length for 3 seconds and were returned to the initial unstretched state. During stretching, the cells were out of focus as shown in the supplemental movie. Therefore, the change in [Ca2+]i of the cell was not detected until the cells were returned to the initial position. Representative fluorescent images and time courses of [Ca2+]i changes of the cells in response to different stretch amplitudes are shown in Figures 1A and 1B, respectively. Following the application of a uniaxial stretch and subsequent unloading, the F340/F380 ratio, a measure of [Ca2+]i, quickly increased transiently, and then the increase in the F340/F380 ratio declined to the initial basal level within 100 seconds (Figure 1B). The increases in the F340/F380 ratio were significantly strain amplitude–dependent (P < 0.001) (Figure 1C). The augmentation of the F340/F380 ratio was not observed in several cells at the lowest (10%) strain amplitude (Figure 1A; see movie in the online supplement). Transient increases in the F340/F380 ratio were observed even when the stretch duration was shorter than 1 second (data not shown). The cell viability was not affected by mechanical stretch under these experimental conditions (10–40% strain).


Figure 1
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  		Figure 1. (A) Representative images of changes in fura-2 fluorescence signals (F340/F380 ratio), an index of intracellular Ca2+ concentrations, in the same position after a uniaxial mechanical stretch equivalent to 10%, 20%, or 40% of strain amplitude in HEPES-buffered saline (HBS) containing 2 mM of Ca2+ are shown. The bright (red, yellow, and green) and dark (black and blue) colors represent higher and lower F340/F380 levels, respectively. (B) Representative time course and strain amplitude-dependent effects on the changes in the F340/F380 ratio. (C) The F340/F380 ratios without stretch and in response to 10%, 20%, or 40% mechanical stretch. Bars represent means ± SD (n = 7). *Significantly different from the values of the unstretched condition (P < 0.05).

 
Ca2+ Source of Stretch-Induced [Ca2+]i Increase
The contribution of the Ca2+ influx from the extracellular side to the stretch-induced [Ca2+]i increases was examined. When mechanical stretch (10–40% strain) was applied to the ASM cells in a Ca2+-free solution, few changes in the F340/F380 ratio other than the artifact signals during stretching were detected (Figure 2A). The F340/F380 ratio elicited by stretch in the Ca2+-free solution was significantly lower than that in the control condition in HBS (P < 0.001) (Figure 2B).


Figure 2
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  		Figure 2. Roles of Ca2+ influx from the extracellular side in the F340/F380 signals after a mechanical stretch equivalent to 10%, 20%, or 40% of strain amplitude. (A) A representative trace of the changes in the F340/F380 ratio in a Ca2+-free solution. (B) Values of F340/F380 ratio in response to mechanical stretch. Extracellular medium contains either 2 mM (Control, solid bars) or 0 mM (Ca2+-free, open bars) of Ca2+. Bars represent the means ± SD (n = 6). *Significantly different from the control values (P < 0.05).

 
Involvement of SA Channels in Stretch-Induced Ca2+ Influx
When mechanical stretch (10–40%) was applied to the cells in the presence of 10 µM Gd3+ (a potent inhibitor of SA channels), the augmentation of the F340/F380 ratio was almost completely abolished (Figure 3A). The increases in the F340/F380 ratio elicited by stretch were significantly inhibited by application of Gd3+ (1 and 10 µM) at 20% and 40% strain amplitude (P < 0.001) (Figure 3B).


Figure 3
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  		Figure 3. Inhibitory effects of Gd3+, a stretch-activated cation channel inhibitor, on the levels of F340/F380 ratio after a mechanical stretch equivalent to 10%, 20%, or 40% of strain amplitude. (A) A representative trace of the changes in the F340/F380 ratio after mechanical stretch in the presence of 10 µM Gd3+. (B) The F340/F380 ratios in response to stretching in the absence (control) or presence of Gd3+ (1 and 10 µM). Bars represent means ± SD (n = 5). *Significantly different from the control values (P < 0.05).

 
Involvement of TRPV Family Ion Channels in Stretch-Induced Ca2+ Influx
Involvement of TRPV in the stretch-induced [Ca2+]i increases was examined. A representative trace of the changes in the F340/F380 ratio due to stretch in the presence of 1 µM ruthenium red, an inhibitor of TRPV subfamily channels, is shown in Figure 4A. The increases of the F340/F380 ratio induced by stretch were significantly inhibited by 1 and 10 µM ruthenium red at 20% and 40% strain amplitude (P < 0.001) (Figure 4B). We next determined the TRPV isoforms present in human bronchial smooth muscle cells and human bronchial epithelial cells using RT-PCR. Expression of TRPV-1, -2, and -4 mRNA was detected in human bronchial smooth muscle cells (Figure 4C). TRPV-1 and -4 expression was detected, but TRPV-2 was not expressed in human bronchial epithelial cells (Figure 4C).


Figure 4
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  		Figure 4. Inhibitory effects of ruthenium red (a TRPV subfamily channel inhibitor) on the F340/F380 ratio after a mechanical stretch equivalent to 10%, 20%, or 40% of strain amplitude. (A) A representative trace of the changes in the F340/F380 ratio in the presence of 1 µM ruthenium red. (B) The F340/F380 ratios in response to stretching in the absence (control) or presence of ruthenium red (RuR, 1 and 10 µM). Bars represent means ± SD (n = 5). *Significantly different from the control values (P < 0.05). (C) TRPV family (TRPV-1, -2, and -4) and GAPDH mRNA expression detected by RT-PCR in human bronchial smooth muscle cells (SM) and human bronchial epithelial cells (EP) are shown. The product sizes for TRPV-1, -2, -4, and GAPDH were 392 bp, 384 bp, 376 bp, and 498 bp, respectively. M and N indicate DNA marker and negative control, respectively.

 
Effects of Ca2+ Channel Inhibitors on Stretch-Induced Ca2+ Influx
To determine whether the rise in [Ca2+]i was via voltage-gated Ca2+ channels due to membrane depolarization, the effect of nifedipine, an inhibitor of the L-type Ca2+ channel, was examined. The increases of the F340/F380 ratio due to stretch were not inhibited by 10 µM nifedipine (Figure 5). Moreover, the elevation of the F340/F380 ratio was not inhibited by an application of either 100 µM SKF-96365, a nonselective inhibitor of Ca2+ channels; 10 µM BTP-2, a potent inhibitor of classic TRP (TRPC) channels; or capsazepine, a TRPV-1 inhibitor (Figure 5).


Figure 5
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  		Figure 5. Effects of the Ca2+ channel inhibitors, 10 µM nifedipine (Nif) (n = 4), 10 µM BTP-2 (BTP) (n = 4), 100 µM SKF-96365 (SKF) (n = 4), and 10 µM capsazepine (n = 4) on the F340/F380 ratios after a mechanical stretch equivalent to 40% of strain amplitude. Bars represent the means ± SD.

 
Intracellular Second Messengers Are Not Involved in Stretch-Induced Ca2+ Influx
We further tested whether the increase of [Ca2+]i induced by mechanical stretch was regulated by intracellular second messengers, specifically phospholipase C (PLC), inositol trisphosphate (IP3), and cyclooxygenase (COX). Stretch-induced increases in the F340/F380 ratio were not affected either by 10 µM U-73122, a PLC inhibitor; 50 µM xestospongin C, an inhibitor of IP3 receptors; or 10 µM indomethacin, a COX inhibitor (Figure 6).


Figure 6
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  		Figure 6. Effects of 50 µM xestospongin C, an inhibitor of the inositol trisphosphate receptor (Xest) (n = 4); 10 µM U-73122, a phospholipase C inhibitor (PLC) (n = 4); and 10 µM of indomethacin, an inhibitor of cyclooxygenase (Ind) (n = 4), on the F340/F380 ratios after a mechanical stretch equivalent to 40% of strain amplitude. Bars represent the means ± SD.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
We demonstrate the novel Ca2+ influx pathway activated by mechanical stretch for the first time in human bronchial smooth muscle cells. This stretch-induced Ca2+ response was sensitive both to Gd3+, a potent inhibitor of SA channels, and ruthenium red, an inhibitor of TRPVs. On the other hand, the Ca2+ signal induced by stretch was not mediated by intracellular second messengers. These findings suggest a pivotal role for SA channels and possible involvement of TRPVs in the stretch-induced Ca2+ response.

The Ca2+-permeable, nonselective SA cation channels have been considered one of the pivotal mechanosensory molecules responsible for triggering mechanotransduction in smooth muscle cells (7, 12). In this study, we found that single uniaxial mechanical stretch elevated [Ca2+]i in human ASM cells. This intracellular Ca2+ mobilization subsequent to stretch was completely suppressed by removal of extracellular Ca2+ (Figure 2). These results demonstrate that the increases in [Ca2+]i elicited by mechanical stretch are mediated by Ca2+ influx from the extracellular side rather than via Ca2+ release from internal Ca2+ stores in human ASM cells. Moreover, the stretch-evoked increases in [Ca2+]i were also blocked by Gd3+ (Figure 3). Therefore, the present findings strongly suggest that mechanical stretch causes Ca2+ influx through SA channels in human ASM cells.

Both direct and indirect mechanisms for activating Ca2+ influx after cell stretching have been reported (7, 28). Activation of PLC has been proposed as a mechanism responsible for mechanically activated Ca2+ mobilization in endothelial cells and vascular smooth muscle cells (34, 36). It is possible that PLC activation stimulates receptor-operated Ca2+ entry as well as store-operated Ca2+ entry via Ca2+ release from IP3 receptors on the sarcoplasmic reticulum (7, 28). However, the present results revealed that the stretch-induced Ca2+ influx was not inhibited either by U-73122, a PLC inhibitor, or by xestospongin C, an inhibitor of the IP3 receptor, in human bronchial smooth muscle cells (Figure 6). Although it is known that the COX activation is involved in the mechanotransductory processes in ASM cells (20, 24), the stretch-induced Ca2+ influx was not affected by a nonselective COX inhibitor indomethacin (Figure 6). Thus, it is unlikely that the stretch-induced Ca2+ influx is mediated indirectly by the activation of intracellular signaling molecules such as PLC, IP3, and COX in response to mechanical stretch in human ASM cells. It was reported that mechanical stretch causes ATP release, which may in turn stimulate neighboring cells and propagate intracellular Ca2+ waves (28, 37). However, Ca2+ waves were not observed in human bronchial smooth muscle cells under the present experimental condition in which a mechanical stretch was applied to the cells at a relatively low (< 50%) confluence to minimize the effects of cell–cell interaction. In addition, it is possible that Ca2+ influx via SA channels indirectly activates voltage-gated Ca2+ channels by causing membrane depolarization (28). Nevertheless, the stretch-induced Ca2+ influx was unaffected by nifedipine (Figure 5), demonstrating that voltage-gated L-type Ca2+ channels are not involved in the present mechanisms. Taken together, our findings suggest that the Ca2+ influx appears to be directly mediated by mechanical stretch rather than indirect mechanisms in human ASM cells.

There is increasing evidence that the mammalian TRP superfamily cation channels transduce a remarkable spectrum of signals ranging from chemical to physical stimuli including temperature, osmotic pressure, shear stress, and mechanical stretch (7, 38–40). Moreover, defects and dysfunctions in TRP channels are found in the pathogenesis of renal and neuronal diseases (41), and TRP channels have been considered as potential drug targets for respiratory diseases such as asthma and chronic obstructive pulmonary disease (41–43). TRPV subfamily channels, specifically TRPV-2 and -4, have been proposed as mechanical stretch–activated channels (16). As shown in Figure 4, the increases in [Ca2+]i induced by mechanical stretch were significantly inhibited by ruthenium red, an inhibitor of TRPVs (38, 40). Indeed, TRPV-1, -2, and -4 genes are expressed in human ASM cells (Figure 4C). Although ruthenium red and Gd3+ might not always be specific for TRPVs and SA channels, respectively (38), these findings suggest the possible involvement of TRPVs in the stretch-induced Ca2+ influx in human ASM cells.

It is well established that TRPV-2 and -4 are widely expressed in various cell types, including smooth muscle cells (7, 16, 36, 38). In contrast, TRPV-1 has been observed in neurons and epithelial cell (38, 40). Jia and coworkers (39) have reported that human ASM cells express TRPV-2 and -4, but not TRPV-1. However, recent studies have demonstrated the TRPV-1 expression in rat pulmonary arterial smooth muscle, rat aorta (44), and human bronchial smooth muscle cells (45). In this study, expression of TRPV-1 mRNA was also observed both in human bronchial smooth muscle cells and epithelial cells (Figure 4C), consistent with these previous findings. Although TRPV-2 and -4 are candidates for SA channels, there are no previous reports that suggest contribution of TRPV-1 to SA Ca2+ influx in smooth muscle cells. The Ca2+ mobilization elicited by mechanical stretch was not affected by capsazepine, an inhibitor of TRPV-1, in human ASM cells (Figure 5). Thus, TRPV-1 is unlikely to be involved in the stretch-activated Ca2+ influx pathways in spite of its expression in ASM cells.

In this study, application of SKF-96365 and BTP-2 did not affect the Ca2+ influx in response to mechanical stretch (Figure 5). Both SKF-96365 and BTP-2 have been reported to inhibit TRPCs, specifically TRPC-3 and -6, when these channels are overexpressed in HEK293 cells (38, 40). While TRPC-3 and -6 are functionally expressed in human ASM cells as candidates for the receptor-operated Ca2+ entry (43), our results suggest that TRPC-3 and -6 are not likely involved in the stretch-activated Ca2+ influx. Moreover, it is known that SKF-96365 inhibits both receptor- or store-operated Ca2+ entry pathways in smooth muscle cells (3, 7). Thus, the characteristics of the Ca2+ influx activated by stretch are different from those of receptor- or store-operated Ca2+ entry. Further studies would be beneficial to fully elucidate the role of TRPV subfamilies in sensing mechanical stretch and to determine which TRPs are responsible for the putative SA channels in ASM cells.

The lung and airway cells, including ASM cells, are exposed to a mechanically dynamic environment. Hence, the cellular functions and development of the respiratory system are influenced by mechanical stimuli (4, 18–25, 46–51). It has been considered that abnormal Ca2+ mobilization affects ASM cell functions (5, 6). Thus, mechanical stretch may up-regulate contractile force, proliferation, and cytokine production in ASM cells via altered Ca2+ homeostasis by activating SA channels. In the present study, we applied 10 to 40% of uniaxial stretch in terms of length to the silicone chamber on which ASM cells were cultured using a cell stretch apparatus. Under physiologic conditions, ASM is exposed to 4 to 5% of strain during tidal breathing (47, 52). When lung volume is doubled by a deep inspiration, ASM is expected to be stretched by as much as 25 to 30% (47, 52). Thus, stretch amplitude employed in our study appears to represent both the physiologic and maximum strain levels in vivo.

Primary cultures of ASM cells have widely been used to assess cell functions because of its great advantage for the availability of molecular biology (43, 53, 54). In contrast, the cells may lose some features of the differentiated ASM cell when they are cultured with serum and/or growth factors (53, 54). Our preliminary study demonstrated that a significant [Ca2+]i elevation was still observed in response to mechanical stretch even when human ASM cells were cultured under the fetal bovine serum–free condition for 6 to 24 hours (data not shown). These findings suggest that the stretch-activated Ca2+ influx is involved in ASM cells which retain characteristics of the contractile phenotype.

In summary, we found a novel Ca2+ influx pathway, presumably an SA channel, which is activated by mechanical stretch in human ASM cells. The Ca2+ mobilization via the SA channel in response to mechanical stretch would enhance the contractile force, proliferation, and synthesis ability of ASM cells. Taken together, excessive mechanical strain during acute exacerbation of asthma may lead to further bronchial hyperresponsiveness. Moreover, one or more SA Ca2+ channels, possibly members of the TRPV subfamily, could be a novel target molecule for the treatment of asthma.


   Acknowledgments
 
The authors thank to Dr. N. Sakaguchi (JST) for her technical assistance in [Ca2+]i measurements.


   Footnotes
 
This work was supported by a Grant-in Aid from the Ministry of Education, Science, Sports, and Culture of Japan (17790531, 19689017 to S.I. and 19590891 to H.K.), Grants for Scientific Research on Priority Areas (15086270 to M.S.), and Creative Scientific Research (16GS0308 to M.S.) from MEXT, Japan.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org.

Originally Published in Press as DOI: 10.1165/rcmb.2007-0259OC on November 1, 2007

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 July 6, 2007

Accepted in final form October 8, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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