ChTX induces oscillatory contraction in guinea pig trachea: role of cyclooxygenase-2 and PGE2

Yukihiro Yagi, Masayoshi Kuwahara, and Hirokazu Tsubone

Department of Comparative Pathophysiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the possible role of cyclooxygenase (COX) in charybdotoxin (ChTX)-induced oscillatory contraction in guinea pig trachea. Involvement of prostaglandin E2 (PGE2) in ChTX-induced oscillatory contraction was also investigated. ChTX (100 nM) induced oscillatory contraction in guinea pig trachea. The mean oscillatory frequency induced by ChTX was 10.7 ± 0.8 counts/h. Maximum and minimum tensions within ChTX-induced oscillatory contractions were 68.4 ± 1.8 and 14.3 ± 1.7% compared with K+ (72.7 mM) contractions. ChTX-induced oscillatory contraction was completely inhibited by indomethacin, a nonselective COX inhibitor. Valeryl salicylate, a selective COX-1 inhibitor, did not significantly inhibit this contraction, whereas N-(2-cyclohexyloxy-4-nitro-phenyl)-methanesulfonamide, a selective COX-2 inhibitor, abolished this contraction. Exogenously applied arachidonic acid enhanced ChTX-induced oscillatory contraction. SC-51322, a selective PGE receptor subtype EP1 antagonist, significantly inhibited ChTX-induced oscillatory contraction. Exogenously applied PGE2 induced only a slight phasic contraction in guinea pig trachea, but PGE2 induced strong oscillatory contraction after pretreatment with indomethacin and ChTX. Moreover, ChTX time-dependently stimulated PGE2 generation. These results suggest that ChTX specifically activates COX-2 and stimulates PGE2 production and that ChTX-induced oscillatory contraction in guinea pig trachea is mediated by activation of EP1 receptor.

airway smooth muscle; calcium-activated potassium channel; prostaglandin E2; charybdotoxin


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CA2+-ACTIVATED K+ CHANNELS (KCa) are located on the surface of a variety of cells, including airway smooth muscle cells (14), where they are involved in the regulation of membrane polarization and, in turn, muscle tone (12). Actually there is some evidence that inhibitors such as charybdotoxin (ChTX), iberiotoxin (IbTX), and tetraethylammonium ions (TEA) affect the sensitivity and responsiveness of smooth muscle to contractile and relaxant drugs. Furthermore, we recently showed that ChTX and IbTX induce oscillatory contractions in guinea pig trachea (32) and that ChTX had a lesser effect on salbutamol relaxation response when the trachea was pretreated with indomethacin (7). ChTX does not evoke a significant contraction in the presence of indomethacin in guinea pig airway smooth muscle (29). Although there are no reports on the effects of ChTX on cyclooxygenase (COX) in the airway smooth muscle, COX may play an important role in ChTX-induced responses.

COX is the enzyme that converts arachidonic acid (AA) to prostaglandin H2, which can then be further metabolized to prostanoids, including prostaglandin E2 (PGE2), prostacyclin, and thromboxane A2 (28). There are two distinct isoforms of COX (COX-1 and COX-2) that have differences in structural and kinetic properties (8, 20, 21, 27). COX-1 is generally thought to be involved in the production of prostanoids that serve to maintain cellular homeostasis and is known to be expressed constitutively in many cell types. COX-2, the inducible isoform of the enzyme, is the major isoenzyme associated with inflammation. COX-2 is induced by stimuli such as lipopolysaccharide (LPS) (10) and proinflammatory cytokines (2, 26) in cells in vitro and at the site of inflammation in vivo (4). Moreover, prostanoids are produced by COX under physiological and pathophysiological conditions by many cell types present in the lung and are known to modify various airway functions. It has recently been shown that the predominant product of COX in airway smooth muscle cells is PGE2 (16). PGE2 has been demonstrated to cause both contraction and relaxation of airway smooth muscle (30). Furthermore, four different prostanoid receptors have been identified for PGE2 and are known as EP1, EP2, EP3, and EP4 (22). Contraction is mediated through EP1 and EP3 receptor activation, and relaxation is mediated through EP2 and EP4 receptor activation (23).

We hypothesized that activation of COX was involved in ChTX-induced oscillatory contraction. Therefore, the purpose of this study was to investigate the possible role of COX in ChTX-induced oscillatory contraction in guinea pig trachea. In addition, involvement of PGE2 in ChTX-induced oscillatory contraction was investigated by the application of PGE2 exogenously and blockage of EP1 with the selective EP1 blocker SC-51322.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of guinea pig tracheal smooth muscle strips. This study was performed in accordance with the ethical guidelines, which meet generally accepted international criteria, for animal care, handling, and euthanasia of our institution. Forty male Hartley guinea pigs weighing 450-520 g (Saitama Experimental Animals Supply) were used. The guinea pigs were killed by stunning and exsanguination after pentobarbital sodium (50 mg/kg ip) anesthesia. The trachea was rapidly excised and placed in physiological solution. Fat and connective tissue were removed. Then the trachea was opened by cutting longitudinally opposite the tracheal muscle and was cut into small strips (~3 mm wide and 7 mm long). The luminal surface was scraped with a cotton wool swab to remove the epithelium.

Each strip was mounted in a 20-ml organ bath in physiological solution, placed under an initial resting tension of 10 mN, and allowed to equilibrate for 1 h before the experiment was begun. First, the maximum contractile response to high-K+ (72.7 mM) solution was determined. The contraction in absolute force induced by this solution was 9.85 ± 0.6 mN. Data were normalized to contraction induced by the high-K+ solution as the reference (100%). Muscle contractions were measured continuously with an isometric force transducer (TB-612T, Nihon Kohden, Tokyo, Japan).

RT-PCR in mouse smooth muscle cells. Fresh mice tracheas were obtained from 5-wk-old male C57BL/6 mice and were immediately transferred to Hanks' balanced salt solution. The smooth muscle layer was isolated from the posterior membranous portion of the trachea by removing surrounding connective tissue and epithelium, and the isolated smooth muscle strips were cut into small pieces. These small pieces were then dispersed enzymatically into single cells with collagenase (0.1%) and elastase (0.2%) for 40 min at 37°C. The solution containing dissociated smooth muscle cells was centrifuged (5 min at 500 g), and the pellet was resuspended in Clonetics modified molecular cellular developmental biology 131 medium containing 0.5 µg/l epidermal growth factor, 5 mg/l insulin, 2 µg/l fibroblast growth factor, 50 mg/l gentamicin, 50 mg/l amphotericin, and 5% fetal bovine serum. These cells were cultured in 25-cm2 flasks at 37°C in an atmosphere of 5% CO2.

Total RNA from mice tracheal smooth muscle cells, which were treated with and without 100 nM ChTX for 1 h, was isolated with a Catrimox-14 RNA isolation kit, version 2.11 (Takara Biomedicals, Shiga, Japan). RT-PCR was performed with an RNA PCR kit, version 2.1 (Takara Biomedicals). RNA was RT by adding 1 µg of RNA per sample to a reaction mixture that contained 2 µl of 10× RNA PCR buffer, 8.5 µl of RNase-free dH2O, 0.125 µM oligo dT-primer, 5 mM MgCl2, 1 mM of all four dNTPs, 1 U/µl RNase inhibitor, and 0.25 U/µl AMV RT, in a 20-µl total reaction volume. Samples were then incubated at 42°C for 30 min, at 95°C for 5 min, and at 5°C for 5 min in a thermal cycler (PCR Thermal Cycler PERSONAL, Takara Biomedicals).

For PCR, a volume of 20 µl of the RT products was amplified in the presence of 0.2 µM each of forward and reverse primers and 2.5 units of Taq polymerase (Takara Taq) in a final volume of 100 µl. Taq polymerase was added with a hot start to reduce nonspecific binding. The COX-1-specific primers were 5'-CTGCATGTGGCTGTGGATGTCATC-3' and 5'-GGTCTTGGTGTTGAGGCAGACCAG-3'. The COX-2-specific primers were 5'-GTCTGATGATGTATGCCACAATCTG-3' and 5'-GATGCCAGTGATAGAGGGTGTTGAA-3'. The GAPDH-specific primers were 5'-CGGAGTCAACGGATTTGGTCGTAT-3' and 5'-AGCCTTCTCCATGGTGGTGAAGAC-3'. The reactions were carried out for 40 cycles (94°C for 30 s; 61°C for 30 s; 72°C for 90 s) for COX-1 and COX-2 mRNA measurements, and 30 cycles (94°C for 30 s; 55°C for 30 s; 72°C for 90 s) for GAPDH mRNA measurements. After electrophoresis (5 µl/lane, 3% agarose), PCR products were visualized by staining with ethidium bromide. The sizes of the PCR products were 398 bp for COX-1, 276 bp for COX-2, and 307 bp for GAPDH. Digitized images of the gels were obtained and analyzed with a charge-coupled device image analyzer (FULA-3000, Fujifilm, Tokyo, Japan). Amounts of COX-1 and COX-2 mRNA were standardized with GAPDH that was used as an internal control.

Measurement of PGE2 generation. Guinea pig tracheal strips were placed for 30 min, 1 h, and 2 h in normal physiological solution added to ChTX (100 nM). The weight of the tracheal strips was 30.8 ± 1.0 mg. The solution was collected and used for enzyme immunoassay of PGE2. To measure the concentration of PGE2, a PGE2 enzyme immunoassay kit-monoclonal (Cayman Chemical, Ann Arbor, MI) was used. The results were calculated in terms of percent B/B0, where B and B0 represent absorbance measurements of the bound fraction in the presence and absence of PGE2, respectively. The standard curve and the results of quantitative determination of PGE2 in biological samples were analyzed with a linear log-logit transformation.

Drugs and solutions. The normal bath physiological solution used was (in mM) 136.9 NaCl, 5.4 KCl, 1.5 CaCl2, 1.0 MgCl2, 23.8 NaHCO3, 5.5 glucose, and 0.01 EDTA. A high-K+ (72.7 mM) solution was made by replacing NaCl with equimolar KCl. These solutions were saturated with a 95%O2-5% CO2 mixture at 37°C and pH 7.4. ChTX, indomethacin, AA, and ethidium bromide were obtained from Sigma Chemical (St. Louis, MO). PGE2, valeryl salicylate (VSA), and N-(2-cyclohexyloxy-4-nitro-phenyl)-methanesulfonamide (NS-398) were obtained from Cayman Chemical. SC-51322 was obtained from BioMol Research Laboratories (Plymouth Meeting, PA). Cycloheximide was obtained from Nacalai Tesque (Kyoto, Japan).

Statistical analysis. All values are expressed as means ± SE, and statistical significance was assessed by one-way ANOVA. P < 0.05 was considered to be significant.


    RESULTS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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ChTX (100 nM) and TEA (1 and 3 mM) produced oscillatory contractions in guinea pig trachea (Fig. 1, A and B). At a concentration of <100 nM, ChTX induced slight sustained contraction, and no oscillatory contraction occurred. The mean oscillatory frequency induced by ChTX was 10.7 ± 0.8 counts/h. Maximum tension (Tmax) and minimum tension (Tmin) within ChTX-induced oscillatory contractions were 68.4 ± 1.8 and 14.3 ± 1.7%. After treatment with indomethacin, a nonselective COX inhibitor, ChTX and TEA did not induce contraction (Fig. 1, C and D). Exposure to indomethacin inhibited ChTX-induced oscillatory contraction in a dose-dependent manner (Fig. 2). Indomethacin (100 nM) significantly decreased Tmin and Tmax values and the frequency of ChTX-induced oscillatory contractions. A high concentration of indomethacin (1 µM) completely abolished ChTX-induced oscillatory contractions. IbTX, a more specific blocker of large-conductance KCa (BKCa), also produced oscillatory contractions in guinea pig trachea (Fig. 3A). This IbTX-induced oscillatory contraction occurred without prior treatment with high-K+ solution. Furthermore, indomethacin (1 µM) completely blocked IbTX-induced oscillatory contractions. Clotrimazole, a calmodulin-sensitive KCa blocker, did not produce oscillatory contractions (Fig. 3B).


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Fig. 1.   Charybdotoxin (ChTX; A)- and tetraethylammonium ions (TEA; B)-induced oscillatory contraction in guinea pig tracheal smooth muscle. Effects of pretreatment with indomethacin, a nonselective cyclooxygenase (COX) inhibitor, on ChTX- and TEA-induced oscillatory contraction were measured. Tmax and Tmin, maximum and minimum tension, respectively, within ChTX-induced oscillatory contraction. ChTX (C) and TEA (D) induced no contraction after pretreatment with indomethacin. Each trace is representative of 7 independent experiments.



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Fig. 2.   Effects of indomethacin on ChTX-induced oscillatory contraction were measured. A: original trace shows that indomethacin (1 µM) abolished ChTX-induced oscillatory contraction. Bar graphs summarize the data on amplitude (B) and frequency (C) of oscillatory contractions from 6-7 separate experiments. +indo, With indomethacin. *P < 0.05 compared with control.



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Fig. 3.   A: iberiotoxin (IbTX)-induced oscillatory contraction in guinea pig tracheal smooth muscle. This IbTX-induced oscillatory contraction occurred without prior treatment with high-K+ solution. Indomethacin (1 µM) blocked this contraction. Calbachol (CCh; 1 µM) was applied at the end of experiment. B: clotrimazole, a calmodulin-sensitive Ca2+-activated K+ channel blocker, did not produce oscillatory contractions. Each trace is representative of 5 independent experiments.

VSA, a selective COX-1 inhibitor, did not significantly inhibit ChTX-induced oscillatory contraction (Fig. 4). Although contraction was slightly inhibited for a few contractions, the oscillatory contraction was restored to the same as before VSA administration. Tmin and Tmax values and the frequency of ChTX-induced oscillatory contraction were not significantly decreased by VSA. NS-398, a selective COX-2 inhibitor, significantly inhibited ChTX-induced oscillatory contraction (Fig. 5). Similar to indomethacin, NS-398 (100 nM) significantly decreased the Tmin and Tmax values and the frequency, and NS-398 (1 µM) abolished ChTX-induced oscillatory contractions.


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Fig. 4.   Effects of valeryl salicylate (VSA), a selective COX-1 inhibitor, on ChTX-induced oscillatory contraction were measured. A: original trace shows that VSA (10 mM) did not inhibit ChTX-induced oscillatory contraction. Bar graphs summarize the data on amplitude (B) and frequency (C) of oscillatory contractions from 6 to 7 separate experiments. +VSA, with VSA. *P < 0.05 compared with control.



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Fig. 5.   Effects of N-(2-cyclohexyloxy-4-nitro-phenyl)-methanesulfo-amide (NS-398), a selective COX-2 inhibitor, on ChTX-induced oscillatory contractions were measured. A: original trace shows that NS-398 (1 µM) abolished ChTX-induced oscillatory contraction. Bar graphs summarize the data on amplitude (B) and frequency (C) of oscillatory contraction from 6 to 7 separate experiments. +NS-398, with NS-398. *P < 0.05 compared with control.

Furthermore, effects of ChTX on mRNA expression of COX-1 and COX-2 in mice bronchial smooth muscle cells were evaluated by RT-PCR analysis. As shown in Fig. 6, although ChTX did not increase the expression of COX-1 mRNA, ChTX significantly increased the expression of COX-2 mRNA. These observations provide further evidence that ChTX-induced oscillatory contractions are greatly related to COX-2 activity.


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Fig. 6.   Effects of ChTX on mRNA expression of COX-1 and COX-2 in tracheal smooth muscle cells. A: total RNA was prepared with and without 100 nM ChTX for 1 h, and RT-PCR was performed. Lane 1, control; lane 2, treatment with ChTX. B: intensities of the bands were densitometrically quantified, and the results were normalized to the internal standard band GAPDH from 5 separate experiments. *P < 0.05 compared with control.

A selective EP1 blocker, SC-51322, significantly inhibited ChTX-induced oscillatory contraction (Fig. 7). SC-51322 (1 µM) abolished ChTX-induced oscillatory contractions. SC-51322 (100 nM) significantly decreased Tmin and Tmax values and frequency, and SC-51322 (1 µM) completely abolished ChTX-induced oscillatory contractions.


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Fig. 7.   Effects of SC-51322, a selective EP1 blocker, on ChTX-induced oscillatory contraction were measured. A: original trace shows that SC-51322 (1 µM) abolished ChTX-induced oscillatory contraction. Bar graphs summarize the data of amplitude (B) and frequency (C) of oscillatory contraction from 6-7 separate experiments. *P < 0.05 compared with control.

The tissue was pretreated with cycloheximide, a protein synthesis inhibitor, for 25 min before administration of ChTX. In all experiments, pretreatment with cycloheximide (10 µM) completely suppressed ChTX-induced contraction (Fig. 8). Application of AA (10 µM) induced oscillatory contraction, and this AA-induced oscillatory contraction was significantly inhibited by 1 µM SC-51322 or 1 µM indomethacin. But NS-398 (1 µM) did not significantly inhibit this AA-induced oscillatory contraction. Exogenously applied AA (1 and 10 µM) enhanced ChTX-induced oscillatory contraction (Fig. 9). AA significantly increased Tmin value and the frequency of ChTX-induced oscillatory contraction, but AA did not influence the Tmax value.


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Fig. 8.   Effects of cycloheximide on ChTX-induced oscillatory contraction were measured. Cycloheximide inhibited ChTX-induced oscillatory contraction, but additional application of arachidonic acid (AA) induced oscillatory contraction. This AA-induced oscillatory contraction was inhibited by SC-51322 (A) or indomethacin (B). Each trace is representative of 6 independent experiments.



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Fig. 9.   Effects of exogenously applied AA on ChTX-induced oscillatory contraction were measured. A: original trace shows that exogenous AA (1 and 10 µM) enhanced ChTX-induced oscillatory contraction. Bar graphs summarize the data on amplitude (B) and frequency (C) of oscillatory contraction from 6-7 separate experiments. +AA, with AA. *P < 0.05 compared with control.

Exogenously applied PGE2 (1 µM) induced slight phasic contraction in guinea pig trachea (Fig. 10A). This contraction was very small (<0.5 mN). On the other hand, after pretreatment with indomethacin and ChTX, PGE2 (1 µM) induced strong oscillatory contraction (Fig. 10B). Under this condition, PGE2 could mimic the response of only ChTX application. This PGE2-induced oscillatory contraction was abolished by exposure to 1 µM SC-51322. Furthermore, ChTX (100 nM) time-dependently increased PGE2 generation in tracheal smooth muscle (Fig. 11).


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Fig. 10.   Effect of exogenously applied prostaglandin E2 (PGE2) application in guinea pig trachea. A: original trace shows that PGE2 (1 µM) induced slight phasic contraction in guinea pig trachea. B: after pretreatment with indomethacin (1 µM) and ChTX (100 nM), PGE2 (1 µM) induced strong oscillatory contraction. The PGE2-induced oscillatory contraction was abolished by SC-51322 (1 µM). Each trace is representative of 6 independent experiments.



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Fig. 11.   Generation of PGE2 was measured in ChTX-treated guinea pig tracheal smooth muscle. Generation of PGE2 was significantly increased in guinea pig tracheal smooth muscle treated with ChTX for 30 min, 1 h, and 2 h. These results were obtained from 4 separate experiments. *P < 0.05 compared with control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study has shown that COX-2 and PGE2 may play important roles in ChTX-induced oscillatory contraction in guinea pig trachea. ChTX-induced oscillatory contraction was abolished by the nonselective COX inhibitor indomethacin and by the selective COX-2 inhibitor NS-398, but it was not inhibited by the selective COX-1 inhibitor VSA. After treatment with cycloheximide, ChTX did not induce contraction. ChTX specifically enhanced COX-2 mRNA expression. These results support the hypothesis that COX activation, particularly COX-2 activation, is involved in ChTX-induced oscillatory contraction. Furthermore, ChTX increased generation of PGE2, and EP1 receptor antagonist SC-51322 blocked ChTX-induced oscillatory contraction. Therefore, it seems that PGE2 is the main product of ChTX-induced COX-2-mediated action.

KCa are distributed abundantly in the surface of airway smooth muscle cells (15, 17), where they are involved in the regulation of membrane polarization and, in turn, muscle tone (12). In this study, we have shown that ChTX, IbTX, and TEA induced oscillatory contraction. The oscillatory contractions induced by these agents seem to depend on the block of BKCa because IbTX is a specific blocker of BKCa. However, ChTX and TEA inhibit not only BKCa but also calmodulin-sensitive KCa. The possibility that inhibition of calmodulin-sensitive KCa produced oscillatory contractions was considered unlikely because clotorimazole, a calmodulin-sensitive KCa blocker (31), did not produce oscillatory contractions. These results reveal that BKCa play an important role in regulating muscle tone, and blockage of BKCa induces contraction in isolated guinea pig trachea. However, ChTX-induced oscillatory contraction was abolished by COX inhibitor indomethacin. This result suggests that COX activation may be relevant to ChTX-induced contraction in guinea pig trachea.

There are two distinct isoforms of the COX enzyme: COX-1 and COX-2 (8). It is generally thought that COX-1 is responsible for physiological or "housekeeping" functions, whereas COX-2 is an inducible enzyme involved in inflammation, mitogenesis, and specialized signal transduction. Actually, COX-1 is thought to be constitutively expressed under physiological conditions in healthy cells in which it maintains cellular homeostasis by producing physiological levels of prostaglandins (1), and COX-2 has been shown to be expressed in response to many proinflammatory stimuli, including cytokines (1, 26) and proinflammatory mediators such as bradykinin in human tracheal smooth muscle (25). Although it was thought that COX-2 was present only in cells that are irritated by inflammatory factors, low basal concentrations of the enzyme have been detected recently in the brain, kidney, and gravid uterus (13). It has also been reported that prostanoid contributing muscle tone is dependent on COX-2 activity rather than COX-1 activity in isolated guinea pig trachea (3); so in guinea pig trachea, COX-2 may play an important role in the physiological as well as the pathophysiological condition.

In this study, ChTX-induced oscillatory contraction was inhibited by NS-398 but not by VSA. These results suggest that ChTX induces oscillatory contraction resulting from selective activation of COX-2 in guinea pig trachea. In addition, ChTX enhanced the expression of COX-2 mRNA, but no increase in COX-1 mRNA was observed. Thus ChTX seems to specifically activate COX-2 and induce expression of COX-2 mRNA as do LPS and cytokines (2, 10, 26). Because cycloheximide inhibits ChTX-induced oscillatory contraction, it appears that COX-2 synthesis is necessary for ChTX-induced oscillatory contraction. However, an additional application of AA-induced oscillatory contraction. This AA-induced oscillatory contraction was inhibited by indomethacin and SC-51322 but not by NS-398. These results indicate that cycloheximide inhibits COX-2 synthesis and, additionally, that applied AA might be converted to PGE2 by COX-1, because cycloheximide suppresses induction of COX-2 but does not suppress PGE2 generation by COX-1 (24). Therefore, even if induction of COX-2 is suppressed, PGE2 that is converted from exogenously applied AA by COX-1 can induce oscillatory contraction in ChTX-treated tracheal smooth muscles.

We normalized ChTX-induced oscillatory contraction to maximal contraction evoked by high-K+ solution at the beginning of the experiment; thus high-K+ treatment might have affected the responses of KCa in the subsequent studies. However, IbTX induced oscillatory contraction without pretreatment with high-K+ solution. Moreover, COX-2 mRNA expression was enhanced only by ChTX treatment. Although it has been reported that high K+ (3 M) can induce COX-2 (18), this K+ concentration was much higher than in our experiment (72.7 mM).

The main substrate for COX is AA, which is converted from phospholipids by phospholipase A2. An exogenously applied low concentration of AA enhanced ChTX-induced oscillatory contraction. It seems that this exogenously applied AA is mainly utilized as the substrate for COX-2 when COX-2 exists, because a recent publication (27) reported that a released low concentration of AA was only converted by COX-2 in NIH3T3 fibroblasts. Moreover, it is generally thought that the predominant product of COX in airway smooth muscle cells is PGE2 (16). In this study, ChTX increased PGE2 generation and ChTX-induced oscillatory contraction was abolished by EP1 receptor blocker SC-51322. Although exogenously applied PGE2 induced only slight phasic contraction in guinea pig trachea, after pretreatment with indomethacin and ChTX, oscillatory contraction was induced by exogenously applied PGE2. These results suggest that ChTX stimulates PGE2 generation and that PGE2 induces oscillatory contraction through activation of EP1 receptor.

Four different prostanoid receptors have been identified for PGE2, and they are known as the EP1, EP2, EP3, and EP4 receptors (22). In guinea pig trachea, activation of EP1 and EP3 receptors on the smooth muscle causes contraction (5, 23), whereas activation of EP2 and EP4 receptors mediates relaxation (6, 23). It seems that EP2 and EP4 receptor-mediated relaxation is dependent on cAMP increase, because EP2 is coupled to adenylate cyclase and increases intracellular cAMP (9, 22). Indeed, PGE2 increased generation of cAMP and relaxed the contraction due to electric field stimulation in tracheal smooth muscle (16, 19). Moreover, most relaxants increase intracellular cAMP or cGMP and open some types of K+ channels. ChTX inhibited relaxation induced by these relaxants in guinea pig trachea (11, 12). It is therefore reasonable to speculate that a higher level of PGE2 might cause oscillatory contraction, because PGE2-induced relaxation is already inhibited by ChTX treatment in guinea pig trachea.

In conclusion, this study first demonstrated that ChTX activates COX-2 specifically and stimulates PGE2 generation in guinea pig trachea and that ChTX-induced oscillatory contraction is mediated by activation of EP1 receptor. These results suggest that COX-2 activation and prostanoids generation may play important roles in airway pathogenesis.


    FOOTNOTES

Address for reprint requests and other correspondence: M. Kuwahara, Dept. of Comparative Pathophysiology, Graduate School of Agricultural and Life Sciences, The Univ. of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan (E-mail: akuwam{at}mail.ecc.u-tokyo.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 31, 2003;10.1152/ajplung.00054.2002

Received 6 February 2002; accepted in final form 24 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 284(6):L1045-L1054
1040-0605/03 $5.00 Copyright © 2003 the American Physiological Society




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