Introduction

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Phospholipase (PL) A₂ is a membrane-bound lipolytic enzyme that regulates the conversion of glycerolphospholipids to lysophospholipids, a precursor of platelet-activating factor (PAF), and free fatty acids such as arachidonic acid (AA), the substrate for synthesis of biologically active eicosanoids. Therefore, activation of PLA₂ is generally considered to be implicated in the pathophysiology of bronchial asthma. On the other hand, lysophosphatidylcholine (Lyso-PC) is another type of lysophospholipid which is synthesized from phosphatidylcholine by PLA₂ in the cell membrane; however, the role(s) of Lyso-PC in the pathogenesis of this disease remains unclear. Although Lyso-PC is not a precursor of PAF and eicosanoids, this lysophospholipid has been demonstrated to correlate with hyperreactivity to histamine in guinea pigs (1) and humans (2). Previous clinical trials also have demonstrated that antigen inhalation causes a marked augmentation in values of not only PLA₂ and AA but also Lyso-PC in bronchoalveolar lavage in patients with atopic asthma (3, 4). Moreover, inasmuch as it has been revealed that extracellular application of Lyso-PC induces expression of adhesion molecules such as intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 on the surface of endothelial cells (5), this lysophospholipid may be involved in the infiltration of leukocytes, including eosinophils, in airways. These results indicate that the extracellular generation of Lyso-PC by secretory PLA₂ may promote tissue inflammation, and, moreover, that chronic exposure to Lyso-PC may be responsible for some of the pathophysiology of bronchial asthma.

Reduced responsiveness to -adrenergic receptor agonists (-agonists) is well known to be a characteristic feature of bronchial asthma. This undesirable dysfunction of -adrenergic receptors may be mediated by proinflammatory cytokines that participate in the airway inflammation of this disease (6, 7). Previous reports have demonstrated that preincubation with PLA₂ leads to a reduction in sensitivity to -agonists in lung membrane (8), and that preincubation with AA also causes a reduction in subsequent relaxation by -agonists in airway smooth muscle (9, 10). Because the reduced responsiveness to -agonists by AA is prevented in the presence of indomethacin, a cyclooxygenase inhibitor, synthesis of eicosanoids is directly involved in causing desensitization of the -adrenergic receptors. PLA₂/AA processes are generally considered to play an important role in not only airway inflammation but also desensitization to -agonists, similar to these cytokines. However, little is currently known about the involvement of exogenous Lyso-PC in the pathophysiology of bronchial asthma.

This study was designed to determine causal relationships between chronic exposure to lysophospholipids and -adrenergic desensitization in airway smooth muscle. We examined subsequent response to -agonists after continuous exposure to Lyso-PC in isolated guinea-pig tracheal smooth muscle. Moreover, we examined the involvement of signal transduction processes as the mechanisms underlying the reduced responsiveness to -agonists by Lyso-PC.

	Materials and Methods

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Tissue Preparation and Tension Records

The methods were essentially similar to those described previously (11, 12). Male guinea pigs (350 to 450 g) were killed by stunning and bleeding, and tracheas were excised from the animals. The tracheal ring was opened by cutting longitudinally through the cartilaginous region and the epithelium was dissected away. The muscle strips containing one cartilaginous ring were removed. Muscle strips were placed vertically in a 1-ml organ bath to measure tension isometrically, and were perfused with solution at a constant flow rate 2 ml/min throughout the experiments. The normal bathing solution had the following composition (in mM): 137 NaCl, 5.9 KHCO₃, 2.4 CaCl₂, 1.2 MgCl₂, and 11.8 glucose, bubbled with a gas mixture of 99% O₂/1% CO₂. For the Ca²⁺-free solution, 2.4 mM CaCl₂ was replaced with 2.2 mM NaCl and 0.2 mM ethyleneglycol-bis-(-aminoethyl ether)-N,N',-tetraacetic acid. The passive tension was adjusted to 0.5 g after equilibrating the preparation in the normal bathing solution for 60 min. The quantity of 1 µM methacholine (MCh) was applied to the strips for 10 min at intervals of 20 min until the control response to 1 µM MCh was established, then the experiments were started. Indomethacin (2 µM) was perfused throughout the experiments to abolish the resting tone. The relaxation induced by exposure to the Ca²⁺-free solution was defined as complete relaxation (0% contraction). The Ca²⁺-free solution was applied to the tissues at the end of each experiment to determine the level of 0% contraction. All experiments were carried out at 37°C.

Measurement of Fura-2 Fluorescence

Segments containing two cartilaginous rings were placed horizontally in a chamber (0.6 ml volume). One end of the segment was fixed to the chamber and the other end was connected to a force-displacement transducer. The muscle strips of guinea-pig tracheas were treated with 10 µM acetoxymethyl ester of fura-2 (fura-2/AM) for 4 h at room temperature (22 to 24°C). The noncytotoxic detergent, pluronic F-127 (0.01% wt/vol), was added to increase the solubility of fura-2/AM. After loading, the chamber was perfused with the normal bathing solution at 37°C for 50 min to wash out the extracellular fura-2/AM before the measurements were taken. According to the method described previously (13), 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 intensity of fluorescence due to excitation at 340 nm (F340) and that at 380 nm (F380) were measured after background subtraction. The absolute amount of intracellular Ca²⁺ concentration ([Ca²⁺]_i) was not calculated because the dissociation constant of fura-2 for Ca²⁺ in smooth muscle cytoplasm may be different from that obtained in vitro (14). Therefore, the ratio of F340 to F380 (F340/ F380 ratio) was used as a relative indicator of [Ca²⁺]_i. To assess the contamination of Ca²⁺-independent fluorescence mediated by intermediate metabolites of fura-2/AM, photobleached fura-2, and changes in the cell geometry, we checked that F340 and F380 move in opposite directions when [Ca²⁺]_i is changed. Muscle tension and F340/F380 ratio in the resting state were defined as 0%. Percent contraction and percent F340/F380 ratio were expressed by taking response to 1 µM MCh as 100%.

Experimental Protocols

To determine the effects of Lyso-PC on -adrenergic receptors in airway smooth muscle, response to MCh in the presence of isoproterenol (ISO) was examined before and after continuous exposure of the tissues to Lyso-PC. MCh (1 µM) was applied to the tissues for 10 min and the normal bathing solution was perfused for 15 min to wash out this agent. The quantity of 1 µM MCh was again applied in the presence of 0.3 µM ISO for an equivalent time, and the normal bathing solution was perfused for 15 min to wash out these agents. Then 10 µM Lyso-PC was perfused for 15, 60, and 120 min. After continuous exposure to Lyso-PC, the normal bathing solution was perfused for 15 min to wash out Lyso-PC. Next, MCh and MCh with ISO were applied to the identical tissues in the same way. To obtain concentration-inhibition curves for ISO on MCh, ISO was cumulatively applied to the tissues precontracted by 1 µM MCh at intervals of 10 min at each concentration before and after exposure to the normal bathing solution and 10 µM Lyso-PC for 120 min. The curves for ISO on MCh were constructed after exposure to the normal bathing solution and Lyso-PC. To determine the role of intracellular cyclic adenosine monophosphate (cAMP) in the effects of exposure to Lyso-PC, we examined the effects of exposure to Lyso-PC on the relaxant effects of agents that increase concentration of cAMP, bypassing -adrenergic receptors such as forskolin (a direct activator of adenylyl cyclase), theophylline (a nonselective phosphodiesterase inhibitor), and db-cAMP (the stable analog of cAMP). Concentration-inhibition curves for these agents on MCh were constructed in the same way. To determine the effects of Lyso-PC on MCh-induced contraction, MCh was cumulatively applied for 10 min at each concentration before and after exposure to Lyso-PC. Concentration-response curves for MCh were constructed in the same way. To determine the involvement of rho, a small monomeric G protein, in the effects of exposure to Lyso-PC, Y-27632, a selective inhibitor of rho-kinase, was perfused throughout the experiments to exclude the effects of Y-27632 on muscarinic and -adrenergic action. To determine the involvement of protein kinase (PK) C in this phenomenon, 5 µM bisindolylmaleimide (BIS), a membrane-permeable inhibitor of PKC, was applied 30 min before exposure to 10 µM Lyso-PC and then the tissues were incubated with Lyso-PC and BIS for 120 min. To determine the involvement of a pertussis toxin (PTX)-sensitive G protein (G_i) in this phenomenon, the tissues were incubated with 1 µg/ml PTX for 6 h; then after the normal bathing solution was perfused for 15 min to wash them out, the tissues were exposed to 10 µM Lyso-PC for 120 min. To determine the involvement of the stimulatory G protein of adenylyl cyclase, G_s, the tissues were incubated with 2 µg/ml cholera toxin (CTX), and after washout, Lyso-PC was perfused in the same way. Moreover, concentration-inhibition curves for prostaglandin (PG) E₂, an activator of G_s, on MCh were constructed in the same way as ISO and other agents. In the fura-2 experiments, because the experiments must be carrried out under the condition that the loaded fluorescence is steady, the experimental protocol described earlier was modified as follows: (1) the periods for application of MCh and MCh with ISO were shortened to 7 min, (2) the periods for washing out were shortened to 5 min, and (3) the control response to MCh after exposure to Lyso-PC was not examined. Time-matched control tissues were treated similarly to the test tissues but exposed continuously to the normal bathing solution (sham incubation) instead of Lyso-PC, CTX, PTX, and BIS. The subsequent relaxation by ISO after exposure to these agents was compared with time-matched control tissues.

Materials

Lyso-PC (palmitoyl), MCh, ISO, CTX, PTX, BIS, forskolin, theophylline, db-cAMP, PGE₂, and indomethacin were obtained from Sigma Chemical Co. (St. Louis, MO). Y-27632 was kindly provided by Welfide Corp. (Osaka, Japan).

Analysis of Results

All data are expressed as means ± standard deviation. The responses to an agent under each condition are described as percentages of the control response. Values of concentration of relaxant agents that produce 50% inhibition (EC₅₀) of contraction induced by 1 µM MCh were determined using linear regression analysis applied to the linear portion of each concentration-response curve. Parameters were compared using the unpaired Student's t test, and P values of < 0.05 were considered statistically significant.

	Results

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Effects of Continuous Exposure to Lyso-PC on Subsequent Response to -Agonists

Application of 0.3 µM ISO caused a marked inhibition of 1 µM MCh-induced contraction; however, after exposure of the tissues to Lyso-PC (10 µM) for 15 min, inhibitory effects of ISO markedly attenuated (Figure 1A). Values of percent contraction for MCh with ISO inhibition after exposure to the normal bathing solution (control) and 10 µM Lyso-PC for 15 min were 9.5 ± 5.6% (n = 12) and 34.6 ± 9.4% (n = 12), respectively (P < 0.001). Pre-exposure to Lyso-PC caused a reduction in subsequent relaxation by ISO against MCh-induced contraction in a time-dependent manner (Figure 1B). When the tissues were exposed to an equimolar Lyso-PC for 60 min, values of percent contraction for MCh with ISO significantly increased to 67.9 ± 6.9 % (n = 12; P < 0.001), whereas those values after incubation with the normal bathing solution for an equivalent time were 10.6 ± 5.6% (n = 12). Moreover, when the period of exposure to Lyso-PC was prolonged to 120 min, subsequent response to ISO almost disappeared (Figures 1A and 1B) and values of percent contraction for MCh with ISO increased to 97.2 ± 2.9% (n = 12). Concentration-inhibition curves for ISO (0.0003 to 10 µM) on MCh were not affected after exposure to the normal bathing solution for 120 min, whereas these curves were markedly shifted to the right after exposure to 10 µM Lyso-PC for an equivalent time (Figure 1C). EC₅₀ values for the curves for ISO after exposure to the normal bathing solution and 10 µM Lyso-PC for 120 min were 0.032 ± 0.009 (n = 8) and 6.1 ± 2.3 µM (n = 8), respectively (P < 0.0001). When MCh was cumulatively applied, 30 µM MCh produced the maximal contraction. Concentration-response curves for MCh (0.003 to 30 µM) were not affected after exposure to 10 µM Lyso-PC for 120 min (Figure 1D). EC₅₀ values for the curves for MCh after exposure to the normal bathing solution and Lyso-PC for an equivalent time were 0.18 ± 0.08 (n = 6) and 0.21 ± 0.11 µM (n = 6), respectively (not significant).

View larger version (16K): [in this window] [in a new window]   View larger version (11K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Figure 1.   A reduction in subsequent response to ISO after continuous exposure to Lyso-PC. (A) A typical example of the inhibitory effects of 0.3 µM ISO on contraction induced by 1 µM MCh before and after exposure to 10 µM Lyso-PC for 15 (upper trace) and 120 (lower trace) min. (B) Values of percent contraction for MCh with ISO inhibition after exposure to the normal bathing solution (control, open columns) and Lyso-PC (filled columns) for 15, 60, and 120 min. These values were expressed by taking the previous response to 1 µM MCh for each experimental condition as 100%. (C) Concentration-inhibition curves for ISO on precontraction by 1 µM MCh after exposure to the normal bathing solution (open circles) and 10 µM Lyso-PC (filled circles) for 120 min. The curves were generated by taking the response to 1 µM MCh before incubation under each experimental condition as 100%. (D) Concentration- response curves for MCh after exposure to the normal bathing solution (open circles) and 10 µM Lyso-PC (filled circles) for 120 min. The curves were generated by taking the response to 30 µM MCh before incubation under each experimental condition as 100%. The abscissa expresses molar concentration on a log scale.

Inhibitory Effects of G_s on the Reduced Responsiveness to -Agonists by Lyso-PC

Preincubation with CTX caused a marked inhibition of reduction in subsequent relaxation by ISO after continuous exposure to Lyso-PC (Figure 2A). Values of percent contraction for MCh with ISO after exposure to 10 µM Lyso-PC for 120 min subsequent to incubation with the normal bathing solution and 2 µg/ml CTX for 6 h were 97.9 ± 1.5 (n = 8) and 14.9 ± 7.1% (n = 8), respectively (P < 0.001). In contrast, preincubation with PTX did not inhibit reduction in the relaxant effects of ISO induced by Lyso-PC (Figure 2A). Values of percent contraction for MCh with ISO after exposure to 10 µM Lyso-PC for 120 min subsequent to incubation with the normal bathing solution and 1 µg/ml PTX for 6 h were 96.9 ± 2.2 (n = 8) and 96.4 ± 2.8% (n = 8), respectively (not significant). Moreover, incubation with BIS also did not inhibit reduced responsiveness to ISO induced by Lyso-PC (Figure 2A). Values of percent contraction for MCh with ISO after exposure to 10 µM Lyso-PC in the absence and the presence of 5 µM BIS for 120 min were 97.1 ± 1.9 (n = 8) and 95.9 ± 3.8% (n = 8), respectively (not significant). PGE₂ (0.1 to 300 nM) was cumulatively applied to the tissues precontracted by 1 µM MCh before and after exposure to 10 µM Lyso-PC for 120 min. PGE₂ caused an inhibition of MCh-induced contraction in a concentration-dependent manner, however concentration-inhibition curves for PGE₂ on MCh were not affected after exposure to Lyso-PC (Figure 2B). EC₅₀ values for the curves for PGE₂ after exposure to the normal bathing solution and Lyso-PC were 121.9 ± 38.6 (n = 8) and 172.3 ± 44.7 nM (n = 8), respectively (not significant).

View larger version (13K): [in this window] [in a new window]   View larger version (12K): [in this window] [in a new window]   Figure 2.   Involvement of Gs in the prevention of -adrenergic desensitization induced by Lyso-PC. (A) Relaxant effects of 0.3 µM ISO on 1 µM MCh-induced contraction after exposure to 10 µM Lyso-PC for 120 min after treatment with PTX, BIS, CTX (filled columns), and the normal bathing solution (control, open columns) under each experimental condition. Values of percent contraction for MCh with ISO were expressed as in Figure 1B. (B) Concentration-inhibition curves for PGE2 on contraction by 1 µM MCh after exposure to the normal bathing solution (open circles) and 10 µM Lyso-PC (filled circles) for 120 min. The curves for PGE2 were generated as in Figure 1C. The abscissa expresses molar concentration on a log scale.

Effects of Pre-exposure to Lyso-PC on Subsequent Response to Agents that Bypass -Adrenergic Receptors

We examined subsequent relaxation induced by agents that bypass -adrenergic receptors after exposure to Lyso-PC, similar to Figures 1C and 2B. Forskolin (0.001 to 10 µM) was cumulatively applied to the tissues precontracted by 1 µM MCh before and after exposure to 10 µM Lyso-PC for 120 min. Concentration-inhibition curves for forskolin on MCh were not affected after exposure to Lyso-PC (Figure 3A). EC₅₀ values for the curves for forskolin after exposure to the normal bathing solution and Lyso-PC were 0.19 ± 0.09 (n = 8) and 0.22 ± 0.12 µM (n = 8), respectively (not significant). Theophylline (0.1 to 300 µM) was cumulatively applied to the tissues precontracted by 1 µM MCh before and after exposure to 10 µM Lyso-PC for an equivalent time. Concentration-inhibition curves for theophylline on MCh were also not affected after exposure to Lyso-PC (Figure 3B). EC₅₀ values for the curves for theophylline after exposure to the normal bathing solution and Lyso-PC were 136.8 ± 41.2 (n = 8) and 184.9 ± 55.3 µM (n = 8), respectively (not significant). Further, db-cAMP (0.1 to 1,000 µM) was cumulatively applied to the tissues precontracted by 1 µM MCh before and after exposure to equimolar Lyso-PC for an equivalent time. Concentration- inhibition curves for db-cAMP on MCh were not affected after exposure to Lyso-PC (Figure 3C). EC₅₀ values for the curves for db-cAMP after exposure to the normal bathing solution and Lyso-PC were 196.3 ± 25.4 (n = 8) and 210.6 ± 30.8 µM (n = 8), respectively (not significant).

View larger version (13K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Figure 3.   The effects of exposure to Lyso-PC on subsequent relaxation by agents that bypass -adrenergic receptors. Concentration-inhibition curves for forskolin (A), theophylline (B), and db-cAMP (C) on 1 µM MCh-induced contraction after exposure to the normal bathing solution (open circles) and 10 µM Lyso-PC (filled circles) for 120 min. The curves for these relaxant agents were generated as in Figure 1C. The abscissa expresses molar concentration on a log scale.

Involvement of Ca²⁺ Sensitization in the Reduced Responsiveness to -Agonists Induced by Lyso-PC

To determine the effects of [Ca²⁺]_i on subsequent reduction in relaxation by ISO after exposure to Lyso-PC, we continuously measured relationships between isometric tension and F340/F380 ratio. In the presence of ISO (0.3 µM), MCh (1 µM) was applied to the fura-2-loaded tissues for 7 min before and after exposure to 10 µM Lyso-PC for 15 min. Application of Lyso-PC did not affect basal tone or F340/F380 ratio (Figure 4A, lower trace). However, the relaxant effects of ISO on MCh-induced contraction were significantly reduced after exposure to Lyso-PC for 15 min, similar to the results in Figure 1 (Figure 4A, upper trace). Values of percent contraction for MCh with ISO after exposure to the normal bathing solution (n = 4) and Lyso-PC were 10.8 ± 4.1 and 37.9 ± 9.1% (n = 4), respectively (P < 0.001; Figure 4B). On the other hand, values of F340/F380 ratio did not increase after exposure to Lyso-PC (Figure 4A, lower trace). Values of percent F340/ F380 ratio for MCh with ISO after exposure to the normal bathing solution and Lyso-PC were 34.2 ± 8.2 (n = 4) and 35.1 ± 9.9% (n = 4), respectively (not significant; Figure 4C).

View larger version (17K): [in this window] [in a new window]   View larger version (9K): [in this window] [in a new window]   View larger version (8K): [in this window] [in a new window]   Figure 4.   The effects of sensitivity to Ca2+ on -adrenergic desensitization by Lyso-PC. A typical example of a simultaneous record of tension (A, upper trace) and F340/F380 ratio (A, lower trace) induced by 1 µM MCh with 0.3 µM ISO inhibition before and after exposure to 10 µM Lyso-PC for 15 min. Values of percent contraction (B) and percent F340/F380 ratio (C) for MCh with ISO after exposure to the normal bathing solution (control) and Lyso-PC. These values were expressed by taking the response to 1 µM MCh before exposure to the normal bathing solution and Lyso-PC for each experimental condition as 100%.

Involvement of Rho-Kinase in the Reduced Responsiveness to -Agonists Induced by Lyso-PC

To determine the mechanisms of reduced responsiveness to -agonists without any change in [Ca²⁺]_i after exposure to Lyso-PC, the relaxant action of -agonists on MCh- induced contraction was examined before and after exposure to Lyso-PC in the presence of Y-27632. The quantity of 1 µM MCh was initially applied for 10 min (data not shown), and after washing out for 15 min, 10 µM Y-27632 was applied. Then the same experiments as shown in Figure 1A were done in the presence of 10 µM Y-27632 throughout the experiments. The contraction induced by 1 µM MCh was attenuated in the presence of 10 µM Y-27632 and the value of percent contraction for MCh was decreased to 63.2 ± 7.8% (n = 16; not shown). Although 0.001 µM Y-27632 did not change subsequent relaxation by ISO after exposure to 10 µM Lyso-PC for 120 min, Y-27632 caused an inhibition of reduced responsiveness to ISO induced by Lyso-PC in a concentration-dependent manner (Figure 5A). Values of percent contraction for MCh with ISO after exposure to 10 µM Lyso-PC for 120 min in the presence of 0.001, 0.1, and 10 µM Y-27632 were 96.2 ± 3.1 (n = 8; not significant), 70.2 ± 5.6 (n = 8; P < 0.001), and 38.4 ± 6.9% (n = 8; P < 0.001), respectively (Figure 5B). The values of percent contraction for MCh with ISO after exposure to 10 µM Lyso-PC for 15 min in the presence of these equivalent concentrations of Y-27632 were 32.9 ± 5.9 (n = 8; not significant), 18.7 ± 4.8 (n = 8; P < 0.001), and 4.9 ± 3.1% (n = 8; P < 0.001), respectively (Figure 5B). Moreover, the values of percent contraction for MCh with ISO after exposure to 10 µM Lyso-PC for 60 min in the presence of the equivalent concentrations of Y-27632 were 63.1 ± 8.1 (n = 8; not significant), 42.8 ± 8.4 (n = 8; P < 0.001), and 22.8 ± 5.4% (n = 8; P < 0.001), respectively (Figure 5B).

View larger version (16K): [in this window] [in a new window]   View larger version (22K): [in this window] [in a new window]   Figure 5.   Involvement of rho-kinase on -adrenergic desensitization by Lyso-PC. (A) A typical example of the inhibitory action of 0.3 µM ISO on 1 µM MCh-induced contraction before and after exposure to 10 µM Lyso-PC for 15 (upper trace) and 120 (lower trace) min in the presence of 10 µM Y-27632 throughout the experiments. (B) Values of percent contraction for MCh with ISO after exposure to Lyso-PC for 15, 60, and 120 min in the presence of 0.001 (open columns), 0.1 (shaded columns), and 10 (filled columns) µM Y-27632. These values were expressed as in Figure 1B.

	Discussion

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In this study we examined the effects of continuous exposure to Lyso-PC on subsequent response to -agonists in airway smooth muscle. Extracellular exposure to Lyso-PC caused a reduction in subsequent relaxation by -agonists in guinea-pig tracheal smooth muscle in a time-dependent manner (Figure 1). However, the reduced responsiveness to -agonists by Lyso-PC occurred even though indomethacin was present throughout the experiments, different from that induced by AA. Although AA plays an important role in the subsequent reduction in response to -agonists, our results indicate that desensitization of -adrenergic receptors induced by PLA₂ may be mediated by Lyso-PC, independent of eicosanoid synthesis by AA. A reduction in subsequent relaxation by ISO does not occur after exposure to 30 nM PGE₂ in human tracheal smooth muscle (15). Further, pre-exposure to 30 nM leukotriene D₄ for 120 min did not cause a reduction in subsequent response to ISO in guinea-pig tracheal smooth muscle (our unpublished observation). Chronic exposure to eicosanoids does not always cause a reduction in subsequent responsiveness to -agonists in airway smooth muscle.

In vessels, Lyso-PC is well known to inhibit endothelium-dependent relaxation by acethylcholine (16), and to increase [Ca²⁺]_i mediated by influx through a verapamil-sensitive channel (17). The effects of Lyso-PC are considered to be mediated by PKC (18) and G_i (19). However, in guinea-pig tracheal smooth muscle, pre-exposure to 10 µM Lyso-PC attenuated subsequent relaxation induced by ISO via the independent pathways of PKC and G_i in the absence of epithelium (Figure 2), and moreover, did not affect subsequent contraction induced by MCh (Figure 1D). These effects induced by continuous exposure to Lyso-PC were mimicked when epithelium was present (our unpublished observation). These results indicate that the reduced responsiveness to -agonists by Lyso-PC is mediated by nonepithelial processes in airway smooth muscle, and that Lyso-PC does not affect muscarinic contraction in both the presence and the absence of epithelium. In vascular smooth muscle, a muscarinic receptor agonist leads to relaxation induced by endothelium-derived relaxation factors in the presence of endothelium, whereas this agent leads to contraction in the absence of endothelium. In contrast, in airway smooth muscle, a muscarinic receptor agonist causes contraction under the condition of both the presence and the absence of epithelium. Mechanical response to muscarinic receptor agonists via epithelium in airways is not consistent with the response via endothelium in vessels. These differences in the mechanisms of exposure to Lyso-PC between airways and vessels may be due to the differences between responses mediated by epithelium and endothelium.

Relaxant effects of agents that increase concentrations of intracellular cAMP bypassing -adrenergic receptors were not affected after exposure to Lyso-PC in guinea-pig tracheal smooth muscle (Figure 3). The inhibitory effects of PGE₂, which activates G_s via PG receptors, were also not affected after exposure to Lyso-PC (Figure 2B). Although it is not currently known whether PLA₂- and AA-induced desensitization to -agonists are mediated by homologous or heterologous desensitization, our results indicate that a reduction in subsequent relaxation by -agonists after exposure to Lyso-PC is mediated by homologous desensitization, and that after continuous exposure to Lyso-PC, adenylyl cyclase/cAMP processes are still intact. Further, as shown in Figure 2A, because irreversible activation of G_s by CTX reversed the reduced mechanical response to -agonists after continuous exposure to Lyso-PC, similar to continuous exposure to -agonists (12, 15), pre-exposure to Lyso-PC may cause damage to -adrenergic receptor/G protein processes, and not to cAMP/cAMP-dependent PKA processes. Recently it has been revealed that G_s protein dysfunction is involved in allergen-induced desensitization of -adrenergic receptors in isolated passively sensitized human bronchi (20). In transgenic mice in which the -subunit of G_s is overexpressed in myocardium, desensitization to -agonists does not occur after chronic exposure to these agents (21). These results indicate that activation of G_s may lead to the prevention of -adrenergic desensitization. Moreover, in human platelets, extracellular application of Lyso-PC results in an augmentation of membrane guanidine triphosphatase (GTPase) activity and preincubation with CTX reduces the Lyso-PC-stimulated increase in membrane GTPase by adenosine diphosphate ribosylation of the -subunit of G_s (22). The membrane GTPase may be one of the affected proteins in the inhibitory effects of CTX on the reduced responsiveness to -agonists induced by pre-exposure to Lyso-PC.

Previous reports have demonstrated that the number of -adrenergic receptors markedly reduces after exposure to PLA₂ and AA in lung parenchyma (8, 23). Exposure to Lyso-PC may lead to a reduction in the number of -adrenergic receptors in airway smooth muscle, although the assay of the receptors was not examined in this study. However, a reduction in the mechanical response to -agonists after exposure to the agonists is not always associated with the number of -adrenergic receptors in airway smooth muscle (24) because of the uncoupling of the receptors with G proteins (25), and of the existence of spare receptors for -adrenaline (26).

In vascular smooth muscle, Lyso-PC enhances contraction mediated by an increase in Ca²⁺ influx (27) or by Ca²⁺ sensitization (28), however little is currently known about the involvement of [Ca²⁺]_i in a reduction in subsequent response to -agonists in airway smooth muscle. In this study, to determine which is involved in the -adrenergic desensitization by Lyso-PC, Ca²⁺ mobilization or Ca²⁺ sensitization, we simultaneously measured tension and [Ca²⁺]_i for MCh with ISO before and after exposure to Lyso-PC. As shown in Figure 4, Lyso-PC has no direct effects on tension and [Ca²⁺]_i in airway smooth muscle, different from vascular smooth muscle. However, after continuous exposure to Lyso-PC the inhibitory effects of ISO markedly attenuated without an increase in F340/F380 ratio. Our results indicate that in airway smooth muscle the reduced responsiveness to -agonists by Lyso-PC is involved in an augmentation in Ca²⁺ sensitivity, not affecting Ca²⁺ mobilization, although the mechanisms of -adrenergic desensitization induced by PLA₂ and AA remain unclear. Fura-2 fluorescence may be interfered with by several endogenous fluorescent substances that are insensitive to Ca²⁺, leading to an error when the results are quantitatively compared, as shown in Figure 4. However, in preliminary experiments, the relationship between tension and F340/F380 ratio by 1 µM MCh was not affected during the observation period (data not shown). Moreover, F340 and F380 always moved in opposite directions throughout the experiments. Therefore, comparison of relative Ca²⁺ level may not be significantly affected by these substances in this study.

Recent reports have demonstrated that rho enhances sensitivity to Ca²⁺ mediated by myosin light chain processes in airway smooth muscle (29, 30), and that AA is considered to contribute an ancillary pathway of rho-mediated Ca²⁺ sensitization in the permeabilized vascular smooth muscle (31). We examined the involvement of rho in an augmentation in Ca²⁺ sensitivity induced by continuous exposure to Lyso-PC. Rho-kinase, which is a target protein of rho (32), causes an augmentation in sensitivity to Ca²⁺ via a reduction in myosin light chain phosphatase activity. The inhibition constant (K_i) value of Y-27632 for inhibiting rho-kinase is generally considered to be 0.14 µM in vitro (33). However, concentrations higher than 30 µM of Y-27632 cause an inhibition in PKA and PKC because K_i values of Y-27632 for inhibiting these kinases are 25 and 26 µM, respectively (33). As shown in Figure 5, a reduction in subsequent relaxation by ISO after continuous exposure to Lyso-PC was suppressed by 0.001 to 10 µM Y-27632, which is close to the K_i value for inhibiting rho-kinase, in a concentration-dependent manner. The quantity of 10 µM Y-27632 caused an approximate 40% inhibition in the contraction induced by 1 µM MCh in guinea-pig tracheal smooth muscle (our unpublished observation). Contraction induced by 1 µM MCh with 10 µM Y-27632 inhibition is roughly equivalent to contraction induced by 0.2 µM MCh. When the concentration of MCh was lowered to 0.2 µM, the inhibitory effects of 0.3 µM ISO on 0.2 µM MCh were reduced after exposure to Lyso-PC, similar to the results shown in Figure 1 (our unpublished observation). These results indicate that prevention of the Lyso-PC-induced desensitization by Y-27632 may not be due to an inhibition in response to MCh by Y-27632. Therefore, pre-exposure to Lyso-PC may lead to -adrenergic desensitization precipitated by Ca²⁺ sensitization via rho/rho-kinase processes, and a reduction in sensitivity to Ca²⁺ by inhibition of rho-kinase may prevent the -adrenergic desensitization by Lyso-PC. It is generally considered that Ca²⁺ sensitization is involved in contraction by various agonists, including MCh (29, 30). In this study, Lyso-PC induced Ca²⁺ sensitization by rho-kinase; however, MCh-induced contraction was not augmented after exposure to Lyso-PC. Although our results are inconsistent with some previous reports, other previous reports have demonstrated that cAMP-PKA processes inhibit rho activity (34, 35). Rho activation induced by Lyso-PC may be more potent in a reduction in relaxation by ISO than an augmentation in contraction by MCh in guinea-pig tracheal smooth muscle. As shown in Figure 3, cAMP-PKA processes are not involved in the Lyso-PC-mediated -adrenergic desensitization. There is no evidence for the regulation of rho-dependent pathways through G_s yet; however, our results suggest that a reduction in G_s activity leads to an augmentation in rho activity. Rho GTPase may also be one of the affected proteins in the -adrenergic desensitization induced by Lyso-PC. Although interaction between "large" and "small" G proteins remains unclear, pre-exposure to Lyso-PC may act on both the membrane GTPase and rho GTPase.

In conclusion, continuous exposure of airway smooth muscle to Lyso-PC causes homologous desensitization of -adrenergic receptors mediated by rho-induced Ca²⁺ sensitization. Preactivation of G_s prevents the reduced responsiveness to -agonists induced by Lyso-PC. Our results may provide the evidence that sensitivity to -agonists is attenuated by PLA₂/Lyso-PC processes in patients with chronic asthma.