Introduction

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It is generally thought that smooth muscle tone is regulated by intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the sensitivity to Ca²⁺ of the contractile elements (1). These mechanisms play an important role in contraction by various agonists. Because hyperreactivity to muscarinic receptor agonists or histamine is a hallmark of bronchial asthma, an excessive augmentation in Ca²⁺ mobilization and Ca²⁺ sensitization by these agonists may be involved in the pathogenesis of this disease. Contraction induced by various receptor agonists such as acetylcholine and histamine is resistant to inhibitors of voltage-operated Ca²⁺ channels (VOCC) in airway smooth muscle (ASM), indicating that receptor-operated Ca²⁺ influx may be involved in agonist-induced contraction (2). Recently, the capacitative Ca²⁺ entry (CCE), which is activated by Ca²⁺ release or depletion of intracellular Ca²⁺ stores, has been proposed in numerous cell types (3). Ca²⁺ influx via CCE may be involved in receptor-operated Ca²⁺ influx, and contribute to tonic contraction by agonists in smooth muscle (4).

The sarcoplasmic reticulum (SR) Ca²⁺-ATPase inhibitors, such as thapsigargin and cyclopiazonic acid, cause sustained contraction in guinea pig tracheal smooth muscle (TSM) (7), and an increase in [Ca²⁺]_i in human bronchioles (8) and in bovine TSM (9). These results suggest the involvement of CCE in contraction of ASM. However, little is currently known about a causal relationship between [Ca²⁺]_i and contraction induced by these agents in detail. Moreover, recent reports have demonstrated that agonist-induced Ca²⁺ mobilization is mediated by another pathway for Ca²⁺ influx, noncapacitative receptor-operated Ca²⁺ entry (non-CCE), which is activated by the GTP-binding (G) protein-coupled receptors (10, 11). In ASM, the involvement of non-CCE in agonist-induced contraction is also still unclear.

A recent study shows that Rho-kinase plays a key role for smooth muscle contraction by enhancing Ca²⁺ sensitivity of the contractile apparatus through inactivation of myosin phosphatase (12). Rho-kinase is a target protein of a small G protein RhoA which is activated by trimeric G protein-coupled receptor agonists (12). Y-27632, a selective inhibitor of Rho-kinase (13), suppresses agonist-induced contraction via a reduction in sensitivity to Ca²⁺ in rabbit TSM and in human bronchioles (14). However, we reported that Y-27632 inhibits not only Ca²⁺ sensitivity but also Ca²⁺ mobilization by methacholine (MCh) in intact guinea pig TSM (15). Moreover, another report has indicated that the RhoA/Rho-kinase pathway contributes to agonist-induced Ca²⁺ mobilization in cultured cells (16). These results indicate the possibility of involvement of the RhoA/Rho-kinase pathway in Ca²⁺ influx by agonists. Recently, it has been demonstrated that novel gene families, transient receptor potential (TRP) and TRP-like (TRPL), encode Ca²⁺ entry channels for CCE and non-CCE (10, 11). However, the mechanism of CCE and non-CCE is currently unknown in ASM.

This study was designed to determine the role of signal transduction processes as the mechanisms underlying Ca²⁺ influx and contraction induced by agonists. We examined the involvement of the RhoA/Rho-kinase pathway in CCE and non-CCE by MCh or histamine in epithelium-denuded guinea pig TSM using simultaneous recording of [Ca²⁺]_i and tension.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Tissue Preparation and Solution

Male guinea pigs (250-350 g) were killed by stunning and bleeding, and tracheas were excised. The tracheal rings were opened by cutting longitudinally at the cartilaginous region, and the epithelium was dissected out. The normal bathing solution was composed of (in mM): NaCl 137, KHCO₃ 5.9, CaCl₂ 2.4, MgCl₂ 1.2, and glucose 11.8, bubbled with a mixture of 99% O₂ and 1% CO₂ (pH 7.4). Ca²⁺-free solution was prepared by replacing 2.4 mM CaCl₂ in the normal solution with 2.2 mM NaCl and 0.2 mM EGTA. The bathing solution was perfused in the organ bath at a constant flow of 3 ml/min. The temperature of the organ bath was maintained at 37°C.

Isometric Tension Recording and Measurement of Fura-2 Fluorescence

The methods are similar to those described previously (15, 17, 18). The tissues were placed horizontally in an organ bath (0.6 ml volume). Muscle strips containing three cartilaginous rings, one for isometric tension recording and two for [Ca²⁺]_i measurements, were prepared. One end of a cartilaginous ring was fixed to the chamber, and the other end was connected to a force-displacement transducer to monitor isometric tension. Both ends of two cartilaginous rings were fixed to the chamber for [Ca²⁺]_i measurements. To abolish the spontaneous tone, 2 µM indomethacin was present throughout the experiments. Muscle strips were treated with 10 µM acetoxymethyl ester of fura-2 (fura-2/AM) for 4 h at room temperature (22-24°C). The noncytotoxic detergent, pluronic F-127 (0.01% wt/vol), was added to increase the solubility of fura-2/AM. After the loading, the chamber was perfused with the normal solution at 37°C for 50 min to wash out the extracellular fura-2/AM before the measurements. Isometric tension and the fura-2 fluorescence of muscle strips were measured simultaneously, using a displacement transducer and a spectrofluorometer (CAF-110; Japan Spectroscopic, Tokyo, Japan). The mucosal side of the muscle strips was exposed to the excitation light, and the light emitted from the strip was collected into a photomultiplier through a 500-nm filter. The intensities of fluorescences due to excitation at 340 (F340) and 380 (F380) nm were measured after background subtraction. The absolute amount of [Ca²⁺]_i was not calculated because the dissociation constant of fura-2 for Ca²⁺ in smooth muscle cytoplasm is known to be different from that obtained in vitro (19). Therefore, the ratio of F340 to F380 (F340/F380) was used as a relative indicator of [Ca²⁺]_i. Measurements of tension and F340/F380 were performed when the muscle contraction had established a steady state.

Experimental Protocols

To assess the involvement of CCE in guinea pig TSM, 1 µM thapsigargin, an irreversible inhibitor of the SR Ca²⁺-ATPase (20), was applied under the condition that extracellular Ca²⁺ concentration ([Ca²⁺]_o) is nominally free (0 mM) to inhibit the Ca²⁺ uptake into the SR. Then, 10 µM MCh was applied for 3 min in the presence of 1 µM thapsigargin with 0 mM [Ca²⁺]_o to deplete the SR Ca²⁺ stores. After washing out MCh for 3 min, [Ca²⁺]_o was changed from 0 to 2.4 mM for 5 min at intervals of 5 min for 2-3 times in the presence of 1 µM thapsigargin to establish a control response to thapsigargin. Thapsigargin was present throughout the experiments. To assess the involvement of non-CCE, 1 µM MCh and 10 µM histamine was added for 5 min subsequent to the maximal Ca²⁺ influx and contraction by thapsigargin in the presence of 2.4 mM [Ca²⁺]_o. To assess the mechanism of Ca²⁺ influx in CCE, nifedipine, a VOCC inhibitor, or SKF-96365 (21) and Ni²⁺, nonselective Ca²⁺ channel inhibitors, were used. These inhibitors were applied 3 min before initiating CCE. To assess the role of the RhoA/Rho-kinase pathway on CCE and non-CCE, Y-27632 (10-100 µM), a specific Rho-kinase inhibitor (13), was applied. Y-27632 was added for 10 min to the strips contracted by CCE or by non-CCE. To assess the role of the actin cytoskeleton on [Ca²⁺]_i and contraction induced by MCh and thapsigargin, cytochalasin D was applied to disrupt the cytoskeletal structure of actin filaments (22). Cytochalasin D (10 µM) was subsequently added for 15 min to the strips stimulated by CCE for 5 min or by 1 µM MCh for 10 min in the normal solution. In the experimental conditions to assess CCE and non-CCE, the values of F340/F380 and tension by thapsigargin in response to the second Ca²⁺ application were taken as 100% (control). In the experimental conditions to assess the effects of cytochalasin D on MCh-induced contraction, those values in response to 1 µM MCh were taken as 100% (control). At the end of the experiments, [Ca²⁺]_o was decreased to 0 mM to obtain the basal levels of F340/ F380 and tension. Those values in response to 0 mM [Ca²⁺]_o were taken as 0% (the baseline).

Materials

Cytochalasin D, histamine, indomethacin, MCh, nifedipine, and pluronic F-127 were obtained from Sigma Chemical (St. Louis, MO). SKF-96365 was from Calbiochem (La Jolla, CA) and fura-2/AM was from Dojin Laboratories (Kumamoto, Japan). Y-27632 was a gift from Welfide Co. Ltd. (Osaka, Japan). Fura-2/AM was dissolved in dimethyl sulfoxide (DMSO), and the final DMSO concentration did not exceed 0.5%. Neither drug affected the fura-2 fluorescence ratio at the concentrations used.

Statistical Analysis

All data were expressed as means ± standard error; n is the number of preparations used. Student's paired t-test and analysis of variance was used to evaluate the significance of differences between means, with P < 0.05 as the level of significance.

	Results

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CCE in TSM

As shown in Figure 1A, when extracellular Ca²⁺ was removed, F340/F380 gradually decreased, reaching a new steady level (the baseline) with no change in tension. When [Ca²⁺]_o was returned to 2.4 mM, F340/F380 returned to the resting level, with no change in tension (data not shown). Thapsigargin alone did not evoke Ca²⁺ response nor contraction in Ca²⁺-free solution (Figure 1A). When 10 µM MCh was added for 5 min in the Ca²⁺-free solution to stimulate the Ca²⁺ release from the SR, sustained contraction with small or no increase in F340/F380 was observed. After washing out MCh for 3 min, [Ca²⁺]_o was increased from 0 to 2.4 mM for 5 min at intervals of 5 min. The first application of the extracellular Ca²⁺ (2.4 mM) caused a rapid increase in F340/F380 and tonic contraction. The value of F340/F380 was higher than that in the absence of thapsigargin (the resting level), indicating that this increase in F340/F380 was due to CCE. After removal of the extracellular Ca²⁺, both F340/F380 and tension rapidly returned to the baseline. The second and third Ca²⁺ application caused further increases in F340/F380 and contraction exceeding those of the first application, but there were no significant changes in F340/F380 and tension by thapsigargin between the second and the third application of Ca²⁺ (n = 12, Figures 1A and 1B). Those values of the first Ca²⁺ application were 84.8 ± 5.3% and 76.5 ± 5.1%, respectively (n = 12, P < 0.05; Figure 1B). Those values of the third Ca²⁺ application were 98.7 ± 3.4% and 99.8 ± 2.3%, respectively (n = 12, not significant; Figure 1B). When a concentration of thapsigargin was increased from 1 to 10 µM 5 min before the third Ca²⁺ application, an increase in F340/F380 and contraction were not affected (n = 6, Figure 1C). The values of percent F340/F380 and tension of the third Ca²⁺ application in the presence of 10 µM thapsigargin were 100.2 ± 1.6% and 99.7 ± 0.8%, respectively (n = 6, not significant).

View larger version (15K): [in this window] [in a new window]   View larger version (25K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   Figure 1.   (A) A representative trace showing CCE in guinea pig TSM. [Ca2+]o was changed three times from 0 to 2.4 mM in the presence of 1 µM thapsigargin, after pretreatment with 10 µM MCh. (B) Summarized data of percent F340/F380 and percent contraction in response to 2.4 mM [Ca2+]o in the presence of thapsigargin. (C ) A representative trace showing the effects of 10 µM thapsigargin on F340/F380 and tension. A concentration of thapsigargin was increased from 1 to 10 µM 5 min before the third Ca2+ application. F340/F380 and tension in response to 0 mM [Ca2+]o were taken as 0%, and percent F340/F380 and percent contraction were expressed by those in response to 2.4 mM [Ca2+]o in the second application as 100% (control). *P < 0.05, compared with the control values.

Effects of Ca²⁺ Channel Blockers on CCE

Nifedipine (1 µM) had few effects on an increase in F340/ F380 and tension induced by thapsigargin (Figure 2A), and the values of percent F340/F380 and tension were 96.2 ± 4.1% and 94.3 ± 4.8%, respectively (n = 6, not significant; Figure 2B). On the other hand, SKF-96365 (300 µM) caused roughly complete suppression (Figure 2C), and the values of percent F340/F380 and contraction with 300 µM SKF-96365 were 5.1 ± 1.9% and 3.8 ± 1.3%, respectively (n = 6, P < 0.05; Figure 2D). When SKF-96365 (10-300 µM) was applied, it caused a concentration-dependent inhibition of F340/F380 and tension induced by 1 µM thapsigargin (Figure 2D). The values of percent F340/F380 for thapsigargin with 10, 30, and 100 µM SKF-96365 were 97.5 ± 2.1%, 77.1 ± 4.2%, and 51.8 ± 6.2%, respectively (n = 6, Figure 2D). The values of percent contraction for thapsigargin with 10, 30, and 100 µM SKF-96365 were 91.6 ± 2.2%, 54.8 ± 4.3%, and 33.4 ± 5.9%, respectively (n = 6, Figure 2D). Ni²⁺ (0.1-1 mM) also caused a concentration-dependent inhibition of F340/F380 and tension induced by thapsigargin, and the values of percent F340/F380 and tension for 1 µM thapsigargin with Ni²⁺ (1 mM) were 29.4 ± 4.3% and 20.6 ± 5.2%, respectively (n = 6, P < 0.05; Figure 2E).

View larger version (17K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   View larger version (20K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   Figure 2.   A representative trace showing the effects of 1 µM nifedipine (Nif; A) or 300 µM SKF-96365 (SKF; C) on F340/F380 and tension induced by CCE. Summarized data of the effects of nifedipine (1 µM; B), SKF-96365 (10-300 µM; D) and Ni2+ (0.1- 1 mM; E) on F340/F380 and contraction. Values of percent F340/ F380 and percent tension were expressed as in Figure 1. Cont, control. *P < 0.05, compared with the control values.

Non-CCE Is Evoked by MCh and Histamine

We examined whether receptor activation would cause further Ca²⁺ influx under the condition of depletion of SR Ca²⁺ stores with thapsigargin. MCh (1 µM) and histamine (10 µM) were added for 5 min under the condition that CCE are fully activated by 1 µM thapsigargin. MCh (1 µM) caused a further increase in F340/F380 and contraction (Figure 3A). Histamine (10 µM) also caused a further increase in F340/F380 and contraction (Figure 3B). The values of percent F340/F380 and contraction for 1 µM MCh were 121.5 ± 4.2% and 165.6 ± 16.2%, respectively (n = 6, P < 0.05; Figure 3C). Those values for 10 µM histamine were 119.1 ± 6.8% and 183.7 ± 17.9%, respectively (n = 6, P < 0.05; Figure 3D).

View larger version (17K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   Figure 3.   A representative trace showing the effects of 100 µM Y-27632 on F340/F380 and tension induced by 1 µM MCh (A) or 10 µM histamine (B) in the presence of 1 µM thapsigargin. Summarized data of the effects of Y-27632 (10-100 µM) on F340/ F380 (open bars) and contraction (closed bars) induced by 1 µM MCh (C) or 10 µM histamine (D). Values of percent F340/F380 and percent tension were expressed as in Figure 1. *P < 0.05, compared with the control values. #P < 0.05, compared with the values induced by 1 µM MCh or 10 µM histamine.

Next, Y-27632 was applied to the strips contracted by 1 µM MCh and 10 µM histamine under the condition of depleted SR Ca²⁺ stores with 1 µM thapsigargin. As shown in Figures 3A and 3B, Y-27632 (100 µM) inhibited an increase in F340/F380 evoked by 1 µM MCh or 10 µM histamine, and values of F340/F380 returned to the level which was evoked by 1 µM thapsigargin alone. Y-27632 (10-100 µM) inhibited both an increase in F340/F380 and contraction induced by these agonists in a concentration-dependent manner (Figures 3C and 3D). The values of percent F340/ F380 for 1 µM MCh with 10, 30, and 100 µM Y-27632 were 123.3 ± 5.0%, 105.8 ± 5.0%, and 101.7 ± 4.1%, respectively (n = 6, Figure 3C). The values of percent contraction for 1 µM MCh with 10, 30, and 100 µM Y-27632 were 133.3 ± 8.3%, 112.5 ± 9.2%, and 65.0 ± 8.3%, respectively (n = 6, Figure 3C). The values of percent F340/F380 for 10 µM histamine with 10, 30, and 100 µM Y-27632 were 118.3 ± 5.8%, 103.3 ± 6.7%, and 100.1 ± 5.1%, respectively (n = 6, Figure 3D). The values of percent contraction for 10 µM histamine with 10, 30, and 100 µM Y-27632 were 145.0 ± 9.2%, 121.7 ± 9.1%, and 93.3 ± 9.8%, respectively (n = 6, Figure 3D).

Role of Rho-Kinase on Agonist-Induced Ca²⁺ Mobilization and CCE

The role of Rho-kinase on Ca²⁺ mobilization and contraction induced by MCh or histamine alone was investigated. Y-27632 (100 µM) inhibited both an increase in F340/F380 and contraction induced by 1 µM MCh (Figure 4A) and 10 µM histamine (Figure 4B). The values of percent F340/ F380 and tension for 1 µM MCh with Y-27632 (100 µM) were 83.2 ± 4.1% and 33.4 ± 2.8%, respectively (n = 6, P < 0.05), and those for 10 µM histamine were 84.1 ± 3.9% and 43.2 ± 3.6%, respectively (n = 6, P < 0.05).

View larger version (16K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   Figure 4.   A representative trace showing the effects of 100 µM Y-27632 on F340/F380 and tension induced by 1 µM MCh (A), 10 µM histamine (B), or 1 µM thapsigargin (C). (D) Summarized data of the effects of Y-27632 (10-100 µM) on F340/F380 and contraction induced by CCE. Values of percent F340/F380 and percent tension were expressed as in Figure 1. *P < 0.05, compared with the control values. (E ) A representative trace showing the effects of pretreatment with 100 µM Y-27632 on F340/F380 and tension induced by CCE.

Next, the role of Rho-kinase on Ca²⁺ mobilization and contraction induced by thapsigargin was investigated in the absence of receptor agonists. Y-27632 (100 µM) caused a marked inhibition of contraction induced by 1 µM thapsigargin (Figure 4C). The value of percent contraction for 1 µM thapsigargin with 100 µM Y-27632 was decreased to 17.6 ± 2.8% of the control values (n = 6, P < 0.05; Figure 4D). On the other hand, 100 µM Y-27632 did not affect F340/F380 increased by 1 µM thapsigargin (Figure 4C). The value of percent F340/F380 for 1 µM thapsigargin with 100 µM Y-27632 was 96.6 ± 3.0% (n = 6, not significant; Figure 4D). When Y-27632 (10-100 µM) was applied, it caused a concentration-dependent inhibition of 1 µM thapsigargin-induced contraction. The values of percent contraction for 1 µM thapsigargin with 10 and 30 µM Y-27632 were 55.6 ± 5.4% and 42.8 ± 5.6%, respectively (n = 6, P < 0.05; Figure 4D). The values of percent F340/ F380 for 1 µM thapsigargin were not affected by 10 and 30 µM Y-27632 (n = 6, Figure 4D). Even when 100 µM Y-27632 was applied 10 min before CCE activation, thapsigargin-induced contraction was blocked with no change in F340/ F380 (Figure 4E). The values of percent F340/F380 and tension were 98.8 ± 1.4% (n = 4, not significant) and 38.6 ± 3.7% (n = 4, P < 0.05), respectively.

Role of Actin Cytoskeleton on MCh-Induced Ca²⁺ Mobilization

Cytochalasin D (10 µM) was applied for 15 min to the strips precontracted by 1 µM MCh. Cytochalasin D (10 µM) inhibited MCh-induced contraction with no change in F340/ F380 (Figure 5A). The values of percent F340/F380 and contraction for 1 µM MCh with 10 µM cytochalasin D were 95.3 ± 3.8% (not significant) and 14.7 ± 3.3% (P < 0.05), respectively (n = 6, Figure 5B). Next, we investigated the effects of cytochalasin D on F340/F380 and tension induced by CCE. Cytochalasin D (10 µM) was applied for 15 min to the strips after extracellular Ca²⁺ was restored for 5 min in the presence of thapsigargin. Cytochalasin D inhibited thapsigargin-induced contraction with no change in F340/F380 (Figure 5C), similar to the results shown in Figure 5A. The values of percent F340/F380 and contraction for 1 µM thapsigargin with 10 µM cytochalasin D were 96.3 ± 2.4% (not significant) and 10.5 ± 1.2% (P < 0.05), respectively (n = 6, Figure 5D). As shown in Figure 5E, when cytochalasin D (10 µM) was applied 10 min before CCE activation, thapsigargin-induced contraction was also inhibited with no change in F340/F380. The values of percent F340/F380 and tension were 102.1 ± 3.4% (n = 4, not significant) and 9.6 ± 2.3% (n = 4, P < 0.05), respectively.

View larger version (16K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   Figure 5.   A representative trace showing the effects of 10 µM cytochalasin D (Cyt D) on F340/F380 and tension induced by 1 µM MCh (A) or 1 µM thapsigargin (C). Summarized data of the effects of 10 µM cytochalasin D on F340/F380 and contraction induced by 1 µM MCh (B) or CCE (D). F340/F380 and tension in response to 0 mM [Ca2+]i were taken as 0%. Values of percent F340/F380 and percent tension were expressed by taking those in response to 1 µM MCh (B) and the second application of 2.4 mM [Ca2 +]i (D) as 100% (control). *P < 0.05, compared with the control values. (E ) A representative trace showing the effects of pretreatment with 10 µM cytochalasin D on F340/F380 and tension induced by CCE.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the present study, we demonstrated for the first time that an increase in [Ca²⁺]_i via CCE evoked by thapsigargin causes tonic contraction in guinea pig TSM using simultaneous measurements of [Ca²⁺]_i and tension. We also showed the noncapacitative mechanisms for receptor-operated Ca²⁺ influx which are inhibited by Rho-kinase inhibition, not by the cytoskeletal disruption in TSM. As shown in Figure 1A, the second application of Ca²⁺ in the presence of thapsigargin was more potent in causing Ca²⁺ influx and contraction than the first application. The differences may depend on the degree of depletion in Ca²⁺ stores (23) or "overshoot" response for Ca²⁺ channels for CCE (24). An increase in [Ca²⁺]_i and tension by 10 µM thapsigargin was similar to those by 1 µM of this agent (Figure 1C), indicating that 1 µM thapsigargin is enough to inhibit the SR Ca²⁺-ATPase in guinea pig TSM. The increase in [Ca²⁺]_i and contraction by thapsigargin was not inhibited by nifedipine, but blocked by Ca²⁺-removal, SKF-96365, and Ni²⁺ (Figure 2), similar to observations in canine pulmonary artery (25) and in rat aorta (26). These results indicate that CCE is not mediated by VOCC but by other Ca²⁺ influx pathways. Because inositol, 1,4,5-trisphosphate (IP₃) produced by activation of the muscarinic receptor releases Ca²⁺ from the SR (27), CCE may be partially involved in Ca²⁺ influx and tonic contraction induced by receptor agonists in ASM.

Recent studies have revealed that there is another Ca²⁺ influx pathway via noncapacitative mechanisms in response to receptor activation (10, 11, 28). These mechanisms are considered to be regulated independently of store depletion, and may coexist with CCE in receptor activation. As shown in Figure 3, 1 µM MCh and 10 µM histamine caused a further increase in [Ca²⁺]_i and contraction after CCE has been fully activated by 1 µM thapsigargin. Our results also demonstrate for the first time that non-CCE exists in ASM, and that this pathway is involved in Ca²⁺ influx activated by MCh or histamine. The signal transduction cascade by second messengers such as IP₃ (29), arachidonic acid (30), protein kinase C (PKC) (28), or small G proteins (16) have been proposed for activation of non-CCE in different cell types. However, the mechanism of non-CCE in smooth muscle is still unclear. The G protein- coupled receptor agonists, such as MCh and histamine, activate the RhoA/Rho-kinase pathway in smooth muscle cells (1, 12). Moreover, Y-27632 attenuates an increase in [Ca²⁺]_i induced by MCh or histamine alone without thapsigargin treatment in TSM (15, see also Figure 4 above). Therefore, in the present study, we investigated whether the RhoA/ Rho-kinase pathway regulates the noncapacitative mechanisms in ASM. As shown in Figure 3, because Y-27632 inhibited an increase in [Ca²⁺]_i and contraction induced by MCh and histamine in a concentration-dependent manner after depletion of the SR Ca²⁺ stores, the RhoA/Rho-kinase pathway may be involved in activation of non-CCE by agonists. These results indicate that the RhoA/Rho-kinase pathway may affect agonist-induced Ca²⁺ mobilization, and that the coupling of receptors with the RhoA/Rho-kinase pathway is needed to initiate non-CCE in guinea pig TSM. Our findings are in agreement with those in C3H 10T1/2 cells (16).

CCE and non-CCE are generally considered to be involved in receptor-operated Ca²⁺ entry mechanisms. However, no specific inhibitors have been available. It may be difficult to completely distinguish these two pathways. In the present study, we sought to examine non-CCE by distinguishing it from CCE according to a previous report (28). Our results demonstrated that non-CCE can be distinguished from CCE because the former was sensitive to a Rho-kinase inhibitor but the latter was not (Figures 3 and 4). A recent report shows that human TRP3 stably expressed in human embryonic kidney 293 cells activates receptor-operated Ca²⁺ entry independently of store depletion (10). This report also supports our results.

An increase in [Ca²⁺]_i by CCE was not reduced by Y-27632 (Figure 4), different from that by non-CCE. The RhoA/Rho-kinase pathway may not contribute to CCE activation as described previously (31). In C3H 10T1/2 cells, direct inhibition of RhoA with Clostridium botulinum C3 toxin attenuates receptor-mediated Ca²⁺ mobilization by thrombin or platelet-derived growth factor, but does not affect thapsigargin-induced Ca²⁺ mobilization (16). These findings are consistent with those in the present study. In contrast, Y-27632 inhibited contraction evoked by CCE without affecting [Ca²⁺]_i (Figure 4). Our results suggest that Ca²⁺ sensitization via Rho-kinase is involved in CCE-induced contraction. However, thapsigargin is known to cause CCE without the confounding influence of G proteins (20). Y-27632 may inhibit, in part, CCE-induced contraction by reducing basal MLC phosphorylation levels in TSM.

The RhoA/Rho-kinase pathway is well known to control the organization of actin cytoskeleton in ASM (32). In NIH 3T3 cells, cytochalasin D, a membrane-permeable inhibitor of actin polymerization (22), blocks agonist-induced Ca²⁺ mobilization without altering CCE (33). However, as shown in Figure 5, cytochalasin D failed to inhibit MCh-induced Ca²⁺ mobilization in guinea pig TSM. Therefore, inhibitory effects of Y-27632 on non-CCE may not be mediated by cytoskeletal disruption. In contrast, recent reports have demonstrated the regulation of CCE by actin cytoskeleton as a conformational coupling model in endothelial cells (34) and in platelets (35). Because cytochalasin D did not affect Ca²⁺ influx by thapsigargin, the actin cytoskeleton is also unlikely to contribute to activation and maintenance of CCE in guinea pig TSM (Figure 5). However, cytochalasin D inhibited contraction induced by MCh or CCE with no change in [Ca²⁺]_i (Figure 5). The effects of Y-27632 on [Ca²⁺]_i and contraction induced by CCE were mimicked by those of cytochalasin D (Figures 4 and 5). Therefore, Y-27632 may cause an inhibition of CCE-induced contraction not only by attenuating Ca²⁺ sensitivity but also by disruption of the actin cytoskeleton. However, further studies are needed to clarify the mechanisms underlying this phenomenon.

In summary, both CCE and non-CCE are present and contribute to tonic contraction in guinea pig TSM. Ca²⁺ mobilization by Rho-kinase activation is involved in non-CCE, but not in CCE. Our results may provide the evidence that Ca²⁺ mobilization by CCE and non-CCE may be involved in the pathogenesis in bronchial hyperreactivity.