Division of Respiratory Medicine, City Hospital, University of Nottingham, Nottingham NG5 1PB, United Kingdom
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ABSTRACT |
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Prostanoids may be involved in bradykinin (BK)-induced bronchoconstriction in asthma. We investigated whether cyclooxygenase (COX)-2 induction was involved in prostaglandin (PG) E2 release by BK in cultured human airway smooth muscle (ASM) cells and analyzed the BK receptor subtypes responsible. BK stimulated PGE2 release, COX activity, and COX-2 induction in a concentration- and time-dependent manner. It also time dependently enhanced arachidonic acid release. In short-term (15-min) experiments, BK stimulated PGE2 generation but did not increase COX activity or induce COX-2. In long-term (4-h) experiments, BK enhanced PGE2 release and COX activity and induced COX-2. The long-term responses were inhibited by the protein synthesis inhibitors cycloheximide and actinomycin D and the steroid dexamethasone. The effects of BK were mimicked by the B2-receptor agonist [Tyr(Me)8]BK, whereas the B1 agonist des-Arg9-BK was weakly effective at high concentrations. The B2 antagonist HOE-140 potently inhibited all the effects, but the B1 antagonist des-Arg9,(Leu8)-BK was inactive. This study is the first to demonstrate that BK can induce COX-2. Conversion of increased arachidonic acid release to PGE2 by COX-1 is mainly involved in the short-term effect, whereas B2 receptor-related COX-2 induction is important in the long-term PGE2 release.
prostaglandin E2; airway inflammation; asthma; bradykinin-receptor agonists; bradykinin-receptor antagonists
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INTRODUCTION |
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BRADYKININ (BK) is a nine-amino acid peptide that is formed locally in body fluids and tissues from the plasma precursor kininogen during inflammatory processes. It has been reported that asthmatic patients have elevated kinin concentrations in plasma and in nasal and bronchoalveolar lavage fluid (BALF) after allergen challenge (5, 9); BK elicits many features of bronchial asthma such as bronchoconstriction (16), microvascular leakage (27), hypersecretion of mucus (3), and recruitment of inflammatory cells (28), and inhaled BK is a potent bronchoconstrictor in asthmatic patients but has no effect even in high concentrations in normal individuals (26). It has therefore been considered to play a part in the pathogenesis of asthma (4).
Two subtypes of the BK receptor, B1 and B2, have been cloned (15, 20). The B1 receptor has a higher affinity for the BK metabolite des-Arg9-BK than for BK itself and is blocked by the selective antagonist des-Arg9,(Leu8)-BK (11). The B2 receptor has a higher affinity for [Tyr(Me)8]BK and BK than for des-Arg9-BK and is blocked by the potent and selective antagonist HOE-140 (11). The B1 receptor does not appear to be present to any significant extent in most normal tissues but becomes evident only under pathological conditions, whereas the B2 receptor is normally expressed in a large number of tissues (11) and is believed to mediate the BK-induced contractile effect on airway smooth muscle (ASM) (16, 21).
The release of arachidonic acid (AA) is an important event preceding
the production of prostanoids. Cyclooxygenase [COX; prostaglandin (PG) endoperoxide synthase, EC 1.14.99.1] is the enzyme at the rate-limiting step for the conversion of AA to PGs and thromboxane (Tx)
A2. BK has been reported to cause
AA release via the rise in cytosolic free
Ca2+ and the activation of the
85-kDa cytosolic phospholipase A2
(cPLA2) (30) and consequently
increases prostanoid production in the airways (16, 21). Reports have
shown that prostanoids may be involved in BK-induced
bronchoconstriction because COX inhibitors such as indomethacin
(Indo) significantly attenuated BK-induced bronchoconstriction (16, 21). It is now known that two isoforms of COX
mediate prostanoid production (32). COX-1 produces physiological levels
of prostanoids and is constitutively expressed under normal conditions
in most tissues (31). COX-2, the inducible isoform of the enzyme, is
induced in many cells under the stimulation of inflammatory mediators
such as lipopolysaccharides and cytokines (23, 24). Accumulating
evidence suggests that the induction and regulation of COX-2 may be key
elements in the pathophysiological process of a number of inflammatory
disorders such as asthma. Studies in bovine (10) and guinea pig (13)
ASM cells have shown that BK causes
PGE2 release, but the same effect
has not been demonstrated in cultured human ASM cells and the
underlying mechanisms have not been fully investigated. Our group and
others have recently shown that interleukin-1 (IL-1
) (24) or a
mixture of cytokines (6) induces COX-2 in cultured human ASM cells and
that this induction is mainly responsible for IL-1
-induced prostanoid release (24). Although BK has been reported to cause COX-2
mRNA expression in cultured fibroblasts (29), direct evidence for COX-2
protein induction in response to BK has not been shown in any cell type
and the role of COX induction in BK-induced prostanoid generation in
ASM cells has not been explored. The aim of this study was, therefore,
to investigate the effect of BK on prostanoid (PGE2) production in human ASM
cells and to determine the mechanisms responsible, particularly whether
COX isoenzyme induction is involved. In addition, we characterized the
receptors involved in this BK-induced action by comparing the effect of
BK with selective B1- and
B2-receptor agonists and by using
selective B1- and
B2-receptor antagonists to inhibit
the responses. We also assessed the effect of the COX inhibitor Indo,
the protein synthesis inhibitors cycloheximide (CHX) and actinomycin D
(Act), and the anti-inflammatory steroid dexamethasone (Dex) on
PGE2 release, COX activity, and
the induction of COX-2 isoenzyme after stimulation by BK.
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MATERIALS AND METHODS |
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Cell Culture
Primary cultures of adult human ASM cells were prepared from explants of ASM according to methods previously reported (14, 24). Briefly, human tracheae were obtained from two postmortem individuals (one man aged 44 yr and one woman aged 52 yr with no evidence of airway diseases) within 12 h of death. The trachealis muscle was then dissected free of epithelium and connective tissue under sterile conditions. Small (2 × 2-mm) explants of ASM were then excised, and ~10 explants were placed in one small petri dish. The explants were incubated in 10% fetal calf serum (FCS)-Dulbecco's modified Eagle's medium (DMEM) in humidified 5% CO2-95% air at 37°C, and the medium was changed every 3 days. Smooth muscle cells were usually seen ~7 days later. Once confluent, the cells were trypsinized and subpassed into 175-cm2 tissue culture flasks, grown to confluence again, trypsinized, and cryopreserved at 1 × 106 cells/ml. ASM cells seeded out of cryopreservation were plated at a density of 2 × 104 cells/well in 12-well culture plates, cultured to confluence in 10% FCS-DMEM, and growth arrested in FCS-free DMEM for 24 h before the experiment. Cells at passage 3 were used for all experiments. We (24) have previously shown that cells grown in this manner show the immunohistochemical and light-microscopic characteristics of pure ASM cells.PGE2 Release
Immediately before each experiment, the cells were treated with fresh serum-free medium containing the reagent to be tested. To evaluate the possible role of COX-1 and COX-2 induction in BK-induced PGE2 release, the study was carried out in short-term (15-min) and long-term (4-h) incubations, respectively, according to the time-course response of BK. At the indicated time intervals, the culture medium was transferred to separate microcentrifuge tubes and stored at[3H]AA Release
Confluent human ASM cells in 12-well plates were growth arrested for 24 h in serum-free medium and then incubated with [5,6,8,9,11,12,14,15-3H]AA (specific activity 7.40 TBq/mmol, 18.5 kBq/well; Amersham Life Science, Little Chalfont, UK) for 16 h in medium containing 0.1% FCS. After incorporation, the cells were washed three times and fresh medium was added before the experiment, and the cells were then incubated with BK (10 µM) for the times indicated. Radioactivity in the medium and inside the cells was counted separately, and [3H]AA release is expressed as the percentage of total radioactivity.COX Activity
For experiments designed to measure COX activity functionally, the cells were washed three times with phosphate-buffered saline (PBS) after they were treated differently for a certain period of time with BK, the B1-receptor agonist des-Arg9-BK (Sigma Chemical), or the B2-receptor agonist [Tyr(Me)8]BK (Calbiochem-Novabiochem, Beeston, UK) and were then incubated with exogenous AA (final concentration 5 µM; Sigma Chemical) for a further 30-min incubation in serum-free medium. The samples generated were subjected to radioimmunoassay for PGE2, and the resulting PGE2 production from AA was taken as an index of COX activity.Cell Viability
The toxicity of all the chemicals used in this study and of the drug vehicle dimethyl sulfoxide (final concentration 1.0%; Sigma Chemical) to human ASM cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [thiazolyl blue (MTT); Sigma Chemical] assay (23, 24). After a 24-h incubation with the chemicals, 20 µl of 5 mg/ml of MTT were added to the culture medium in 96-well plates and incubated for 1 h at 37°C. After the medium was removed, 200 µl of dimethyl sulfoxide were added to solubilize the blue-colored tetrazolium, the plates were shaken for 5 min, and the optical density values at 550 nm were read in a microplate reader. Viability was set as 100% in control cells.Western Blot Analysis
Identification of COX-2 by Western blotting was performed by culturing human ASM cells in 12-well plates. After being treated differently, the cells were washed with PBS and incubated for 5 min with an extraction buffer [0.9% NaCl, 20 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, pH 7.6, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 0.01% leupeptin (Sigma Chemical)] with gentle shaking. The cell extract was centrifuged (4,000 g at 4°C for 10 min), and the protein concentration in the supernatant was determined with the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hemel Hempstead, UK). Fifty to sixty micrograms of protein were harvested from each well. Sufficient aliquots of the sample (30 µg protein/track) were mixed 1:1 with sample buffer [20 mM Tris · HCl, pH 6.8, 20% glycerol, 2% sodium dodecyl sulfate (SDS), 5% 2-mercaptoethanol, and 0.025% bromphenol blue] and boiled for 5 min before electrophoresis in 20 × 20-cm 7.5% SDS-polyacrylamide gel (45 mA, 5 h). The separated proteins were electroblotted (150 V, 3 h) to pure nitrocellulose membranes (Gelman Sciences, Northampton, UK), and the blot was blocked for 2 h at 4°C in blocking reagent [8% fat-free dried milk powder in PBS, pH 7.4, with 0.3% Tween 20 (PBS-T)]. The blot was then incubated with primary monoclonal anti-human COX-2 antibody (1:2,000 in blocking reagent; Cayman Chemical, Ann Arbor, MI) for 2 h at room temperature. The blot was subsequently washed with PBS-T and incubated with polyclonal anti-mouse immunoglobulin G coupled with horseradish peroxidase (1:2,000 in blocking reagent; Sigma Chemical) for 1 h at room temperature. Semiquantitative staining was achieved by using enhanced chemiluminescence (ECL) detection. The blot was washed with PBS-T, then incubated with the SuperSignal CL-HRP substrate system (Pierce, Rockford, IL) for 1 min, and finally exposed to hyperfilm-ECL (Amersham Life Science). The position and molecular weight of COX-2 and COX-1 were validated by reference to rainbow-colored molecular-weight markers (Amersham Life Science). Reprobing of COX-1 was carried out by incubating the membrane in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris · HCl) at 50°C for 30 min with occasional agitation, washing the membrane in a large volume of PBS-T, blocking the membrane for 2 h in blocking reagent at 4°C, and then following the above steps to detect COX-1 with monoclonal anti-ovine COX-1 antibody (with cross-reactivity to human COX-1, 1:2,000 in blocking reagent; Cayman Chemical).Statistical Analysis
Data are expressed as means and SE from n determinations. Statistical analysis was performed with the statistical software SPSS (22). Unpaired two-tailed t-test or one-way analysis of variance was used to determine the significant differences between means. The results were adjusted for multiple testing with Bonferroni's correction. P values < 0.05 were accepted as statistically significant. ![]() |
RESULTS |
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Time Course of PGE2 Release, AA Mobilization, COX Activity, and COX Isoform Induction in Response to BK
Treatment of human ASM cells with BK (10 µM) caused a time-dependent accumulation of PGE2 (Fig. 1A). The increase was significant after only 5 min of incubation (P < 0.001) and was sustained for 16 h of treatment. BK also stimulated [3H]AA release in a time-dependent manner that was significant after 5 min of incubation (P < 0.001; Fig. 1B). The time-dependent enhancement of PGE2 generation was associated with a time-dependent increase in COX activity (Fig. 1C). The increase was significant as early as 2 h after stimulation (P < 0.01), reached a peak at 4-8 h, and declined gradually at 16 and 24 h. No changes were seen in the time-matched control cells. Direct evidence for a time-dependent induction of COX-2 enzyme protein in human ASM cells after exposure to BK was obtained by Western blotting of cell extracts with a specific antibody that resolves COX-2 from COX-1. As shown in Fig. 2, COX-2 was undetectable in untreated human ASM cells. After treatment, however, COX-2 protein bands started to appear at 2 h, reached a maximum at 4 h, and gradually reduced from 8 to 24 h. Reprobing of the same blot with an antibody specific to COX-1 showed that COX-1 enzyme protein bands existed in both treated and untreated cells and remained virtually unchanged (Fig. 2). Because there was a parallel increase in COX-2 protein and COX activity after a 2- to 4-h incubation with BK, different mechanisms were considered to be involved in the short- and long-term PGE2 production. Some of the following experiments aimed at investigating the BK receptors responsible for the BK response were therefore conducted in the short term (15 min) and long term (4 h), respectively.
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Pharmacological Characterization of BK Receptors Involved Using Selective Agonists
Concentration response of PGE2 release,
COX activity, and COX-2 induction in response to BK and BK-receptor
agonists.
Treatment of human ASM cells with BK for 15 min (short term) produced a
concentration-dependent release of
PGE2. The effect was significant
from the lowest concentration tested
(108 M;
P < 0.001) and reached a peak at
10
5 M (Fig.
3). Treatment of the cells with the
B2-receptor agonist [Tyr(Me)8]BK for 15 min also produced a concentration-dependent release of
PGE2 in a pattern similar to that
of BK, but the maximum effect was reached at
10
6 M (Fig. 3). The
concentrations of PGE2
released into the medium were also similar after stimulation with BK
and [Tyr(Me)8]BK. In
contrast, PGE2 production after
stimulation with the B1-receptor agonist des-Arg9-BK was much lower
than that with BK and
[Tyr(Me)8]BK. The
small effect seen was only significant from
10
6 M
(P < 0.01), and even at a
very high concentration
(10
4 M),
PGE2 release was still much
smaller (Fig. 3). The order of potency of
[Tyr(Me)8]BK
BK > des-Arg9-BK suggested that the
response was mediated by B2
receptors.
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Full time course of PGE2 release, COX activity, and COX isoform induction in response to BK-receptor agonists. After the concentration-response relationship in short-term and long-term experiments was characterized, the full time course of the effect of the selective agonists was studied. The B2-receptor agonist [Tyr(Me)8]BK (1 µM) and the B1-receptor agonist des-Arg9-BK (100 µM) both caused time-dependent enhancement in PGE2 release. The enhancement was significant for both agonists after only 5 min of stimulation (63.0 ng/mg protein, P < 0.001 for [Tyr(Me)8]BK and 9.8 ng/mg protein, P < 0.01 for des-Arg9-BK compared with 2.0 and 3.3 ng/mg protein, respectively, for the control cells) and was sustained for 16 h of treatment. The enhancement was accompanied by a time-dependent increase in COX activity that became significant after a 2-h incubation (102.3 ng/mg protein, P < 0.01 for [Tyr(Me)8]BK and 94.2 ng/mg protein, P < 0.01 for des-Arg9-BK compared with 76.0 and 69.3 ng/mg protein, respectively, for the control cells), reached the peak at 8 h (253.0 and 96.1 ng/mg protein, respectively), and declined gradually at 16 and 24 h. COX-2 induction became visible at 1 h and peaked at 4 h for both agonists and gradually reduced from 8 h for [Tyr(Me)8]BK or diminished at 16 h for des-Arg9-BK (blot not shown). No change in the constitutive COX-1 isoform was observed. The time-course pattern of the B2-receptor agonist [Tyr(Me)8]BK was very similar to that of BK in terms of PGE2 release, COX activity, and COX-2 induction. The B1-receptor agonist des-Arg9-BK was much less potent than BK and [Tyr(Me)8]BK despite being used at a much higher concentration. The results therefore are consistent with activation of B2 receptors rather than B1 receptors being involved in BK-induced PGE2 release, COX activity, and COX-2 induction in human ASM cells.
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Pharmacological Characterization of BK Receptors Involved Using Selective Antagonists
Effect of BK-receptor antagonists on PGE2 release, COX activity, and COX-2 induction in response to BK and BK-receptor agonists. In the short-term experiments, pretreatment of human ASM cells with the B2-receptor antagonist HOE-140 (1-100 µM) strongly inhibited BK-induced PGE2 release in a concentration-dependent manner and abolished the effect of BK (10 µM) at 100 µM (Fig. 6), whereas pretreatment with the B1-receptor antagonist des-Arg9,(Leu8)-BK (1-100 µM) did not show any significant inhibition of PGE2 release caused by BK (Fig. 6).
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Effect of Various Inhibitors on BK-Stimulated PGE2 Synthesis, COX Activity, and COX-2 Induction
The effects of the nonsteroidal anti-inflammatory drug Indo, the protein synthesis inhibitors CHX (a translation inhibitor) and Act (a transcription inhibitor), and the steroid Dex were also assessed on BK-induced short-term and long-term PGE2 release and COX activity. In the short-term experiments, Indo (1 µM) completely blocked the BK (10 µM)-induced increase in PGE2 synthesis (P < 0.001), whereas CHX, Act, and Dex (all 1 µM) were without effect (Fig. 9A). Although BK did not significantly increase short-term COX activity, the basic COX activity (COX-1) was still strongly inhibited by Indo (P < 0.001) but not by CHX, Act, or Dex (Fig. 9A), suggesting that the de novo synthesis of protein and mRNA was not involved in BK-induced short-term PGE2 production. In the long-term experiments (Fig. 9B), BK-induced enhancement in PGE2 accumulation was also abolished by Indo (P < 0.001) and markedly inhibited by CHX (P < 0.05), Act (P < 0.001), and Dex (P < 0.001). As observed before, BK caused a significant enhancement in COX activity, and the increase was not only blocked by Indo (P < 0.001) but also strongly suppressed by CHX, Act, and Dex (P < 0.001; Fig. 9B). The effect of the protein synthesis inhibitors and Dex on COX-2 induction was further examined. As detected by Western blotting analysis, CHX strongly inhibited and Act and Dex abolished BK-induced COX-2 induction (blot not shown). These results provide further and direct evidence that COX-1 is mainly responsible for the accumulation of PGE2 after short-term stimulation and that the exaggerated PGE2 release after long-term incubation with BK in human ASM cells is at least partly composed of de novo synthesis of COX-2 protein that is solely responsible for the enhancement in COX activity.Cytotoxicity of the Reagents Used in the Study
Cytotoxicity to human ASM cells after 24 h of treatment with all the reagents used in the study was measured by MTT assay, and cell viability was consistently >95% compared with the untreated cells (data not shown). Furthermore, no change in the protein level was observed after any of the treatments (data not shown). ![]() |
DISCUSSION |
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Our results showed that BK induced PGE2 production in cultured human ASM cells and that the PGE2 production had a rapid and a sustained phase. We studied the early production at 15 min, and we chose 4 h to study the long-term effect of BK because 4 h was the time of maximum COX-2 induction as determined by Western blotting. In short-term experiments, BK induced a concentration-related increase in PGE2 release but had no effect on COX activity or COX isoenzyme induction. The exaggerated PGE2 release was abolished by the COX inhibitor Indo but was not affected by the protein synthesis inhibitors CHX and Act and the steroid Dex. The results, therefore, indicate that the constitutive COX-1 is responsible for PGE2 release in the short-term experiments. Because BK has been reported to cause AA release via the rise in cytosolic free Ca2+ and activation of the 85-kDa cPLA2 in a number of cultured cells, including ASM cells (30), and because it was found to cause AA release in a time-dependent manner in this study, the short-term increase in PGE2 production by BK in human ASM cells is likely to be due to the increased mobilization of AA and subsequent conversion to PGE2 by the existing COX-1. Increased release of AA is also likely to have contributed to the late increase in PGE2 seen in our long-term experiments. We considered whether the induction of cPLA2 might have contributed to the late increase in PGE2 but found no evidence of cPLA2 induction (data not shown).
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In our long-term experiments, we also found, however, that BK
stimulated COX activity, and this was accompanied by a corresponding increase in COX-2 enzyme induction that was first apparent at 2 h. In
contrast, COX-1 expression was not affected by BK. The late enhancement
of PGE2 production and COX
activity were abolished by Indo and markedly inhibited by CHX, Act, and
Dex. The protein synthesis inhibitors and Dex also strongly suppressed
COX-2 induction in response to BK stimulation. The results suggest that
the enhanced PGE2 generation in
the long-term experiments after BK treatment is largely attributable to
the induction of COX-2, consistent with the hypothesis that COX-1 is a
constitutive isoform, whereas COX-2 is an inducible isoform responsible
for the generation of prostanoids under inflammatory conditions (31).
Although BK has been reported to cause COX-2 mRNA expression in
cultured fibroblasts (29), the present study is the first that we are
aware of to demonstrate directly that BK causes the induction of COX-2
protein in any cell type and that the induction forms part of the
BK-induced PGE2 generation.
Previous studies of airway cells have shown that cytokines (mainly
IL-1) can increase inflammatory gene expression. Our results suggest
that kinins can also regulate inflammatory gene expression. Because
asthmatic patients have elevated levels of kinin concentrations (5, 9)
in nasal fluid and BALF, our results may be of importance to the
understanding of the airway inflammation in asthma.
Two BK receptor subtypes (B1 and
B2) have been identified (15,
20). B1 and
B2 receptor-mediated responses can
be distinguished pharmacologically on the basis of the
relative potencies of agonists or by the use of receptor-selective
antagonists. For the B2 receptor, the order of potency of agonists is
[Tyr(Me)8]BK BK > des-Arg9-BK, whereas for the
B1 receptor, the order is
reversed: des-Arg9-BK > BK > [Tyr(Me)8]BK (11).
In our experiments with selective agonists, the
B2-receptor agonist
[Tyr(Me)8]BK
reproduced all the effects of BK in a very similar time- and concentration-dependent pattern, whereas the
B1-receptor agonist des-Arg9-BK was effective only at
much higher concentrations compared with BK. The
PGE2 concentration and the COX
activity in response to
des-Arg9-BK stimulation were also
considerably lower than those of BK. The order of potency, therefore,
was [Tyr(Me)8]BK
BK > des-Arg9-BK,
suggesting that the BK response is mediated by
B2 receptors. The results with
selective agonists were consistent with those obtained with selective
antagonists. The selective
B2-receptor antagonist HOE-140
concentration dependently and potently suppressed all the effects of
BK, whereas the selective
B1-receptor antagonist des-Arg9,(Leu8)-BK
was without effect. These results suggest that all of the actions of BK
are mediated by B2 receptors. It
is unlikely that the B1 receptors
were also involved because the B1
antagonist des-Arg9,(Leu8)-BK
was only slightly effective and the weak effect of the
B1-receptor agonist
des-Arg9-BK could be explained by
the lack of receptor selectivity at high concentrations. Our results
are in agreement with the findings that
B2 receptors are responsible for
BK-induced bronchoconstriction observed in isolated human airways (16,
21) and PGE2 production in
cultured guinea pig ASM cells (13).
On the basis of the observations that B1- and B2-receptor antagonists have very weak inhibitory effects on BK-induced bronchoconstriction in guinea pigs, on [3H]BK binding in guinea pig and sheep airway tissues, and on the BK-induced efflux of Ca2+ in cultured guinea pig tracheal smooth muscle cells, a novel BK receptor subtype, B3, has been proposed (12, 13). All of the effects of BK in our present study were inhibited by the selective B2-receptor antagonist HOE-140, which does not provide any evidence to support the existence of a novel B3 receptor in cultured human ASM cells, at least in terms of PG production.
It is not clear whether the consequences of COX-2 induction and
PGE2 production by BK in human ASM
would be detrimental or beneficial for airway function in inflammatory
airway diseases. Prostanoids are important mediators known to influence
many aspects of airway function such as airway tone, mucus secretion,
and cell proliferation. PGE2 is an
important anti-inflammatory mediator and has considerable
bronchoprotective effects in the airways (25), and because
PGI2, like
PGE2, is also coupled to adenosine 3',5'-cyclic monophosphate elevation, it could have a
similar protective effect (17). It is possible, therefore, that the exaggerated PGE2 production as a
result of COX-2 induction is part of a negative feedback mechanism that
is exerting a braking effect on the inflammatory response. The
induction of COX-2 itself may also shunt the released AA away from the
generation of a potent bronchoconstrictor of the lipoxygenase pathway
toward the synthesis of bronchodilators such as
PGE2 and
PGI2 of the COX pathway. However, PGE2 at higher concentrations also
causes ASM contraction (2) due to weak agonism at the Tx receptor (18),
and other products of COX, such as
PGF2,
TxA2, and
PGD2, are potent proinflammatory modulators that cause bronchoconstriction via the activation of the Tx
prostanoid receptor (2, 18). BK has been shown to generate prostanoids
in various airway tissues, and
PGE2 and
PGI2 have been shown to be the
dominant COX products (7, 16). In our previous study (24), we also
found that after stimulation with IL-1
human ASM cells released
large quantities of PGE2 and PGI2 and the release of
PGF2
,
TxA2, and
PGD2 was much lower. Although we
did not measure the spectrum of prostanoids produced after COX-2
induction in the present study, it is likely that it would be similar.
Further studies are required to determine whether BK alters the
activity of specific PG synthases. Accumulating reports have shown that
BK-induced bronchoconstriction is significantly attenuated by COX
inhibitors such as Indo (16, 21) and by TxA2 receptor/synthetase
inhibitors (1, 16). BK-induced airway responses may therefore be
largely mediated by the release of prostanoids that contract airways
via the activation of the TxA2 receptor. However, the overall impact of prostanoids on the airway may
then depend on the cell type, the balance of the prostanoids released,
and the prostanoid receptor subtype activated.
The fact that BK causes bronchoconstriction in asthmatic subjects but not normal subjects is intriguing. This may be a dose-related phenomenon, and if enough BK was given to normal subjects, they would develop bronchoconstriction. Alternatively, it could be speculated that the spectrum of prostanoids produced may differ between asthmatic and normal subjects or that because COX-2 is already partially induced by cytokines in asthma, BK may be having an additive or synergistic effect. Immunohistochemical studies of asthmatic airways would be useful to determine the relative COX-1 and COX-2 distribution in ASM cells.
In conclusion, our studies have demonstrated that human ASM cells
release large quantities of PGE2
in response to BK stimulation. The underlying mechanisms are different
for the short-term and long-term responses. Although both are mediated
by B2 receptors, short-term
increases are due to the conversion by existing COX-1 of increased AA
release to PGE2, whereas the
long-term increases are mainly due to the induction of COX-2. Because
elevated levels of kinin (9) and proinflammatory cytokines (8, 19) have been found in BALF in asthmatic subjects and BK (this study) and IL-1 (24) have been shown to cause prostanoid generation and COX-2
induction in human ASM cells, it is tempting to speculate that the
coexistence of proinflammatory mediators and cytokines in the airway,
and the consequential induction of COX-2 may play an important role in
the pathogenesis of airway inflammation in asthma.
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ACKNOWLEDGEMENTS |
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We thank Colin Clelland for providing the specimens of human trachea.
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FOOTNOTES |
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This study was supported by a grant from The National Asthma Campaign (UK).
Address for reprint requests: A. J. Knox, Division of Respiratory Medicine, City Hospital, Univ. of Nottingham, Hucknall Rd., Nottingham NG5 1PB, UK.
Received 2 June 1997; accepted in final form 29 August 1997.
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