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
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ABSTRACT |
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
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INTRODUCTION |
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 |
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 |
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.
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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.
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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.
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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.
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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.
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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.
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 |
DISCUSSION |
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.
 |
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Am J Physiol Lung Cell Mol Physiol 284(6):L1045-L1054
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