Impaired cAMP production in human airway smooth muscle cells
by bradykinin: role of cyclooxygenase products
Linhua
Pang,
Elaine
Holland, and
Alan J.
Knox
Division of Respiratory Medicine, City Hospital, University of
Nottingham, Nottingham NG5 1PB, United Kingdom
 |
ABSTRACT |
Interleukin
(IL)-1
impairs human airway smooth muscle (ASM) cell cAMP responses
to isoproterenol (Iso). We investigated if bradykinin (BK) could cause
a similar effect and the role of cyclooxygenase (COX) products in this
event, since we have recently reported that BK, like IL-1
, also
causes COX-2 induction and prostanoid release in human ASM cells. BK
pretreatment significantly attenuated Iso-induced cAMP generation in a
time- and concentration-dependent manner. cAMP generation by
prostaglandin (PG) E2 but not by
forskolin was also impaired. The COX inhibitor indomethacin completely
prevented the impairment, whereas the selective COX-2 inhibitors NS-398 and nimesulide, protein synthesis inhibitors cycloheximide and actinomycin D, and steroid dexamethasone were all partially effective. The impairment was mimicked by the
B2 agonist
[Tyr(Me)8]BK, the
Ca2+ ionophore A-23187, and
PGE2 and prevented by the
B2 antagonist HOE-140, but
anti-IL-1
serum was ineffective. The results indicate that BK
impairs human ASM cell responses to Iso, and the effect is largely
mediated by B2 receptor-related
COX product release via both COX isoforms and is independent of
IL-1
.
airway inflammation; prostaglandin
E2; asthma; cyclooxygenase
induction; isoproterenol; adenosine 3',5'-cyclic
monophosphate
 |
INTRODUCTION |
BRADYKININ (BK) is a naturally occurring inflammatory
peptide generated by the cleavage of kininogen. Asthmatic patients have elevated kinin concentrations in plasma and in nasal and
bronchoalveolar lavage fluid after allergen challenge (5, 7); BK
elicits many features of bronchial asthma such as bronchoconstriction (17), microvascular leakage (27), and recruitment of inflammatory cells
(28), and inhaled BK is a potent bronchoconstrictor in asthmatic
patients (26). BK may therefore play a role in the pathogenesis of
bronchial asthma.
Enhanced cytokine production, inflammation, and impaired responsiveness
of airway smooth muscle (ASM) to adrenoceptor agonists are
characteristic features in asthma. Increasing evidence suggests that
proinflammatory cytokines such as interleukin (IL)-1
and tumor
necrosis factor (TNF)-
play critical roles in the development of
inflammatory responses in the airway and in regulating ASM responsiveness to adrenoceptor agonists. IL-1
causes
-adrenergic hyporesponsiveness in human ASM cells (29) and guinea pig tracheas (34); TNF-
also causes similar impairment in guinea pig tracheas (34) and canine ASM cells (10). Although it has been suggested that
there is uncoupling of ASM
-receptors from adenylyl cyclase, the
precise mechanism(s) underlying this hyporesponsiveness has not been
fully explored, and there are no reports on whether BK can cause the
same hyporesponsiveness in ASM cells.
Cyclooxygenase [COX; prostaglandin (PG) endoperoxide synthase, EC
1.14.99.1] is the rate-limiting enzyme for the conversion of
arachidonic acid (AA) to prostanoids and exists in two isoforms, the
constitutive COX-1 and the inducible COX-2, which can be switched on by
cytokines and inflammatory mediators (3). 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. Enhanced expression of COX-2 in asthmatic airways has
recently been reported (30), suggesting that COX-2-derived products may
play an essential role in the inflammatory processes present in
asthmatic airways. We and others have shown that IL-1
(23, 33) or a
mixture of cytokines (6) induces COX-2 expression in cultured human ASM
cells and that the induction is accompanied by a marked increase in
PGE2 and
PGI2 production (23). BK has been
shown to stimulate AA release from cultured ASM cells via the rise in
cytosolic free Ca2+ and the
activation of the 85-kDa cytosolic phospholipase
A2 (24, 32). We have recently
reported that BK, like IL-1
, causes the induction of COX-2 and the
release of prostanoids from human ASM cells, and the effect is mediated
by the activation of the B2 receptors rather than the B1
receptors (24). Because PGE2 and PGI2 are both coupled to adenylyl
cyclase and increase intracellular cAMP, chronically elevated levels of
PGE2 and
PGI2 would be expected to cause
heterologous desensitization of adenylyl cyclase. We postulated that BK
may also cause impaired cAMP generation in response to
-adrenoceptor
agonists in BK-treated human ASM cells and BK-induced COX-2 expression,
and prostanoid release may be responsible for the impairment.
The present study was therefore aimed to investigate if BK impaired
cAMP production in human ASM cells in response to isoproterenol (Iso)
and if so to determine the mechanisms responsible for the impaired
response. We paid particular attention to whether COX-2 isoenzyme
induction and COX products were involved. The nonselective COX
inhibitor indomethacin (Indo), the selective COX-2 inhibitors N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide
(NS-398) and nimesulide (Nim), the protein synthesis inhibitors
cycloheximide and actinomycin D, and the anti-inflammatory steroid
dexamethasone were used to assess the role of COX-2 induction and COX
products in the process. We also characterized the receptors involved
by comparing the effect of BK with that of the selective
B1 and
B2 receptor agonists and by using
the selective B1 and
B2 receptor antagonists to prevent
the effect of BK. In addition, the ability of
Ca2+ ionophore A-23187, which has
a similar effect as BK in causing cytosolic free
Ca2+ increase and AA release, and
the exogenously applied COX product PGE2 to mimic the impaired
responses by BK was also investigated. Because BK has been shown to
stimulate IL-1 release from isolated lung strips (22), we used rabbit
anti-IL-1
(human) antiserum to examine if the effect of BK was
dependent on IL-1
release.
 |
MATERIALS AND METHODS |
Cell culture. Human tracheas were
obtained from two individuals postmortem (one male aged 44 and one
female aged 52, with no evidence of airway diseases) within 12 h of
death. Primary cultures of human ASM cells were prepared from explants
of ASM according to methods previously reported (16, 23, 24). Cells at
passages 3-4 were used for all experiments. We have
previously shown that the cells grown in this manner depict the
immunohistochemical and light-microscopic characteristics of typical
ASM cells (23).
Experiment protocol. The cells were
cultured to confluence in 10% fetal calf serum (Seralab, Crowly Down,
Sussex, UK)-Dulbecco's modified Eagle's medium (Sigma, Poole, Dorset,
UK) in humidified 5% CO2-95% air
at 37°C in 24-well culture plates and growth arrested in
serum-deprived medium for 24 h before experiments. Immediately before
each experiment, fresh serum-free medium containing BK (Sigma) was
added. In the time-course experiments, the cells were incubated with BK
(10 µM) for 1-24 h, whereas in the concentration-response experiments the cells were incubated for 24 h with 0.01-10 µM BK. In most experiments thereafter, the cells were incubated with 10 µM BK for 24 h. At the indicated times, the culture media were harvested and stored at
20°C until the radioimmunoassay of
PGE2 content (23) as a
representative of COX products. The
anti-PGE2 antiserum (Sigma) had
negligible cross-reactivity in our hands (23). To test the inhibition
by various drugs on the effect of BK, Indo, cycloheximide, actinomycin
D, dexamethasone, the B1 receptor
antagonist
des-Arg9,[Leu8]BK,
the rabbit anti-human IL-1
antiserum (Sigma), NS-398, and Nim
(Cayman Chemical, Ann Arbor, MI) and the
B2 receptor antagonist D-Arg[Hyp3,Thi5,Dtic7,Oic8]BK
(HOE-140, kind gift from Dr. R. N. Zahlten and Dr. B. A. Scholkens, Hoechst Aktiengesellschaft, Frankfurt, Germany) were added 30 min
before the addition of BK. Experiments with the selective BK
B1 receptor agonist
des-Arg9-BK, the
B2 receptor agonist
[Tyr(Me)8]BK,
Ca2+ ionophore A-23187, and
exogenous PGE2 (all from Sigma)
were conducted in the same way as BK. BK and its receptor agonists and
antagonists and the anti-human IL-1
antiserum were dissolved in
serum-free medium, and all other agents were dissolved in dimethyl
sulfoxide (Sigma; final concentration 1.0% vol/vol), except
PGE2, which was dissolved in
ethanol (Sigma; final concentration 1.0% vol/vol). In all of the
studies, a group of control cells was incubated with the vehicles used
to dissolve the agents applied in the experimental cells for the same
period of time.
cAMP assay. After the incubation with
BK, A-23187, or PGE2 and the
removal of culture media, the cells were washed three times with PBS
and incubated in 0.5 ml of fresh medium with 1.0 mM IBMX (Sigma) to
prevent cAMP degradation. The cAMP production reaction was initiated
with the addition of Iso (Calbiochem-Novabiochem, La Jolla, CA) and was
terminated 10 min later with 0.1 ml ice-cold trichloroacetic acid
(Sigma), which was then removed by amine-freon (Sigma) extraction (18),
and cAMP content in the extract was determined by a protein
binding assay (12). The protein kinase A-dependent cAMP
and cAMP used in the assay were from Sigma;
[8-3H]cAMP (specific
activity 962 GBq/mmol) was from Amersham Life Science
(Little Chalfont, Bucks, UK). The cAMP production in response to
forskolin and PGE2 (Sigma)
was conducted in the same way as Iso.
Cell viability. The toxicity of all
the chemicals used in this study and their vehicles dimethyl sulfoxide
and ethanol (final concentration 1.0% vol/vol; Sigma) to human ASM
cells was determined by thiazolyl blue
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide;
MTT; Sigma] assay (23, 24). After 24-h incubation with the
chemicals, 20 µl of 5 mg/ml 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 DMSO were added to solubilize the blue-colored
tetrazolium, the plates were shaken for 5 min, and values for the
optical density at 550 nm were read in a microplate reader. Viability
was set as 100% in control cells.
Statistical analysis. Data are
expressed as means ± SE from n
determinations. Statistical analysis was performed by using the
statistical software from SPSS (31). One-way analysis of variance
and/or unpaired two-tailed
t-test was used to determine the
significant differences between the means. The results were adjusted
for multiple testing by using Bonferroni's correction. P values of <0.05 were accepted as
statistically significant.
 |
RESULTS |
Effect of BK on cAMP formation in response to Iso,
PGE2, and forskolin.
The capacity of human ASM cells to synthesize cAMP in unstimulated
conditions and after BK pretreatment was examined first. As shown in
Fig. 1, basal cAMP levels were low in both
control cells and cells pretreated with BK (10 µM for 24 h). Iso
caused a concentration-dependent increase in cAMP synthesis. BK
markedly reduced cAMP formation in response to 1.0 and 10 µM Iso
(P < 0.01 and
P < 0.001 respectively). Forskolin,
a direct adenylyl cyclase activator, and
PGE2 also caused a
concentration-dependent increase in cAMP generation, and BK
significantly attenuated cAMP formation in response to 0.001 and 0.01 µM PGE2
(P < 0.01 and
P < 0.001, respectively, Fig.
2A).
However, unlike Iso and PGE2,
there was no significant change in cAMP between the control cells and
cells pretreated with BK (10 µM for 24 h) in response to forskolin
(Fig. 2B). Human ASM cells
pretreated with BK (10 µM) showed a time-dependent decrease in cAMP
formation in response to Iso in the time-course experiments (Fig.
3A), whereas cAMP formation in the
control cells over the time course remained unchanged, with cAMP
concentration around 12.5 pmol/well. The desensitization was
significant from after 8 h of incubation
(P < 0.001) and peaked after 24 h
(P < 0.001). Treatment of the cells
with various concentrations of BK for 24 h also produced a
concentration-dependent desensitization response of cAMP production
(Fig.
3B). The
effect was significant from 0.1 µM
(P < 0.001) and reached a maximum at
10 µM (P < 0.001).

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Fig. 1.
Concentration response of isoproterenol (Iso) on cAMP generation from
control human airway smooth muscle (ASM) cells and cells pretreated
with bradykinin (BK). After being pretreated with or without BK (10 µM) for 24 h, cells were washed with PBS and further incubated in
fresh medium with 1.0 mM IBMX plus various concentrations of Iso for 10 min. Each point represents the mean ± SE of 8 determinations from 2 independent experiments. ** P < 0.01 and *** P < 0.001 compared with control cells.
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Fig. 2.
Concentration response of prostaglandin (PG)
E2 and forskolin on cAMP
generation from control human ASM cells and cells pretreated with BK.
After being pretreated with or without BK (10 µM) for 24 h, cells
were washed with PBS and further incubated in fresh medium with 1.0 mM
IBMX plus various concentrations of either
PGE2
(A) or forskolin
(B) for 10 min. Each point is the
mean ± SE of 8 determinations from 2 independent experiments.
** P < 0.01 and
*** P < 0.001 compared with control cells.
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Fig. 3.
Time course and concentration response of BK on cAMP generation from
human ASM cells in response to Iso. After being pretreated with BK (10 µM) for the times indicated in the time- course experiment
(A) or for 24 h with increasing
concentrations of BK in the concentration-response experiment
(B), cells were washed with PBS and
further incubated in fresh medium with 10 µM Iso plus 1.0 mM IBMX for
10 min. Each point is the mean ± SE of 8 determinations from 2 independent experiments. *** P < 0.001 compared with control cells.
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Effect of various inhibitors on BK-induced changes in
cAMP formation. The effect of the nonselective COX
inhibitor Indo, selective COX-2 inhibitors NS-398 and Nim, protein
synthesis inhibitors cycloheximide and actinomycin D, and the steroid
dexamethasone was assessed on BK-induced desensitization in cAMP
production in response to Iso. As shown in Fig.
4, Iso (10 µM) caused a marked increase
in cAMP production; the response was strongly desensitized by BK
pretreatment (10 µM, 24 h, P < 0.001), and this desensitization was significantly attenuated by Indo,
NS-398, cycloheximide, actinomycin D, dexamethasone
(P < 0.001), and Nim
(P < 0.01). However, when the cells
were incubated with the inhibitors themselves, no significant change in
Iso-induced cAMP production was observed (data not shown), suggesting
that COX products after BK pretreatment, including those from the
inducible COX-2 isoform, are involved in BK-induced desensitization of
human ASM cell responses to Iso.

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Fig. 4.
Effect of various inhibitors on BK-induced changes in cAMP generation
in response to Iso. Cells were pretreated with or without 1.0 µM
indomethacin (Indo), NS-398, nimesulide (Nim), cycloheximide (CHX),
actinomycin D (Act), or steroid dexamethasone (Dex) for 30 min before
incubation with BK (10 µM) for 24 h. Cells were washed with PBS and
further incubated in fresh medium with 10 µM Iso plus 1.0 mM IBMX for
10 min. Each bar is the mean ± SE of 8 determinations from 2 independent experiments. ** P < 0.01 and *** P < 0.001 compared with the effect of BK.
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Effect of the selective BK receptor agonists on cAMP
formation in response to Iso. To
characterize the BK receptor(s) involved in BK-induced desensitization
of the cell responses to Iso, we examined the effect of the selective
BK B1 receptor agonist
des-Arg9-BK and the selective
B2 receptor agonist
[Tyr(Me)8]BK on the
cAMP production of human ASM cells in response to Iso. [Tyr(Me)8]BK mimicked
the effect of BK by reducing the cAMP production in a similar
concentration-dependent manner as that of BK, significant from 0.1 µM
(P < 0.001), and the maximum effect
was observed at 10 µM (P < 0.001;
Fig. 5). In contrast, pretreatment of the
cells with the B1 receptor agonist
des-Arg9-BK had no effect on cAMP
formation compared with the control cells (Fig. 5). The results suggest
that the B2 receptor is
responsible for the effect.

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Fig. 5.
Concentration response of BK receptor agonists on cAMP generation from
human ASM cells in response to Iso. After being pretreated for 24 h
with increasing concentrations of the selective
B1 receptor agonist
des-Arg9-BK or the selective
B2 receptor agonist
[Tyr(Me)8]BK, cells
were washed with PBS and further incubated in fresh medium with 10 µM
Iso plus 1.0 mM IBMX for 10 min. Each point is the mean ± SE of 8 determinations from 2 independent experiments.
*** P < 0.001 compared with
control cells.
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Effect of the selective BK-receptor antagonists on
BK-induced changes in cAMP formation. Pretreatment of
human ASM cells with the selective
B2 receptor antagonist HOE-140
(1-100 µM) strongly antagonized BK (10 µM)-induced attenuation
in cAMP production in a concentration-dependent fashion and abolished
the effect of BK at 100 µM (Fig. 6),
whereas pretreatment with the B1
receptor antagonist des-Arg9,
[Leu8]BK over the same
concentration range (1-100 µM) did not show any significant
effect (Fig. 6). The data therefore provide further evidence that
B2 receptors are largely
responsible for mediating BK-induced impairment of human ASM cell
responses to Iso.

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Fig. 6.
Effect of the selective BK receptor antagonists on BK-induced changes
in cAMP formation in response to Iso. Cells were pretreated with or
without increasing concentrations of the selective
B1 receptor antagonist
des-Arg9,[Leu8]BK
or the selective B2 receptor
antagonist HOE-140 for 30 min before incubation with 10 µM BK for 24 h and were then washed with PBS and further incubated in fresh medium
with 10 µM Iso plus 1.0 mM IBMX for 10 min. Each point is the
mean ± SE of 8 determinations from 2 independent
experiments. ** P < 0.01 and *** P < 0.001 compared with the effect of BK.
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Effect of the anti-human IL-1
serum on BK-induced
impaired changes in cAMP formation. Because reports
have shown that BK stimulates the release of IL-1 (22) and IL-1
causes impaired responses of human ASM cells to Iso (29), we examined
if BK-induced impaired responses to Iso were dependent on IL-1
by
using the anti-human IL-1
antiserum to block any effect of IL-1
during the 24-h incubation with BK. cAMP production by control cells in
response to Iso (10 µM, 10 min) was 11.0 ± 0.69 pmol/well. With
the pretreatment of BK (10 µM, 24 h), the cAMP production was reduced
to 1.64 ± 0.62 pmol/well. Coincubation of the cells with BK and a
series of dilutions of the antiserum (1:500, 1:250, 1:125, and 1:62.5)
did not significantly affect the impaired cAMP production induced by
BK, with the cAMP concentration ranging from 1.5 to 1.76 pmol/well.
However, the same range of dilution of the antiserum completely
abolished the impaired cAMP production induced by IL-1
(data not
shown), indicating that BK-induced impairment of human ASM cell
responses to Iso is independent of IL-1
.
Effect of the
Ca2+ ionophore
A-23187 on cAMP formation in response to Iso.
To further clarify the role of COX products in BK-induced
desensitization of the cell responses to Iso, we examined if
Ca2+ ionophore A-23187, which has
a similar effect as BK in causing free
Ca2+ increase and release of
endogenous AA, could result in similar impaired cAMP production.
A-23187 was found to cause a concentration-dependent generation of
prostanoids (measured as PGE2)
after 24 h of incubation with human ASM cells, significant from 0.1 µM (P < 0.001, Fig. 7A), and
the subsequent cAMP production in response to Iso (10 µM) was
strongly reduced by A-23187 in a concentration-dependent manner,
significant from 0.1 µM (P < 0.001; Fig. 7B). Coincubation of the
cells with A-23187 (1.0 µM) and the COX inhibitor Indo (0.01-1.0
µM) concentration dependently abolished the A-23187-induced PGE2 release (Fig.
8A) as
well as impairment of cAMP production (Fig.
8B), suggesting that it is the
prostanoids produced after A-23187 treatment that mediate the impaired
cAMP production.

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Fig. 7.
Effect of A-23187 on PGE2 release
(A) and cAMP formation in response
to Iso (B). After preincubation with
A-23187 (0.1-10 µM) for 24 h, the medium was removed for
PGE2 assay, and the cells were
washed with PBS and further incubated in fresh medium with 10 µM Iso
plus 1.0 mM IBMX for 10 min. Each point is the mean ± SE
of 8 determinations from 2 independent experiments.
*** P < 0.001 compared with
control cells.
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Fig. 8.
Effect of Indo on A-23187-induced
PGE2 release
(A) and changes in cAMP generation
in response to Iso (B). Cells were
pretreated with or without Indo (0.01-1.0 µM) for 30 min before
incubation with A-23187 (1.0 µM) for 24 h. Medium was removed for
PGE2 assay, and the cells were
washed with PBS and further incubated in fresh medium with 10 µM Iso
plus 1.0 mM IBMX for 10 min. Each point is the mean ± SE of 8 determinations from 2 independent experiments.
** P < 0.01 and
*** P < 0.001 compared with
the effect of A-23187.
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Effect of exogenous PGE2 on cAMP
formation in response to Iso and PGE2.
Pretreatment of the cells with the exogenously applied COX product
PGE2 also resulted in marked
attenuation of cAMP production in response to Iso (10 µM) in a
concentration-dependent fashion, significant from 0.01 µM
(P < 0.001, Fig.
9A).
When PGE2 was used as a cAMP
stimulant, it caused a concentration-dependent cAMP generation in
control cells, and PGE2 (1.0 µM)
pretreatment of the cells for 24 h markedly reduced cAMP
production in response to subsequent
PGE2 stimulation, significant for
all the subsequent PGE2
concentrations tested (P < 0.01 and
P < 0.001) compared with the control cells (Fig. 9B). The
results thus provide further evidence that the COX products can cause
heterologous desensitization of human ASM cells to various
receptor-lined cAMP stimulants and are mainly responsible for the
impaired responses caused by BK.

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Fig. 9.
Effect of exogenous PGE2 on cAMP
formation in response to Iso (A) or
PGE2
(B). After preincubation with
increasing concentrations of PGE2
(A) or without or with 1.0 µM
PGE2
(B) for 24 h, cells were washed with
PBS and further incubated in fresh medium with 10 µM Iso plus 1.0 mM
IBMX (A) or increasing
concentrations of PGE2 plus 1.0 mM
IBMX (B) for 10 min. Each point is
the mean ± SE of 8 determinations from 2 independent experiments.
** P < 0.01 and
*** P < 0.001 compared with 0 µM PGE2 (A) or with control cells
(B).
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Cell viability. Cell viability after
24 h of treatment with all the chemicals used in this study was
consistently >95% compared with cells treated with the vehicles.
 |
DISCUSSION |
Increasing evidence suggests that
-adrenergic relaxant mechanisms
may be dysfunctional in asthmatic airways (4). ASM relaxation to
-adrenoceptor agonists in vitro is impaired in airways taken from
patients who died of asthma exacerbation (2, 13), in surgical lobectomy
samples from patients with stable asthma (8), and in animal models of
asthma (9). Reports have accumulated to support the hypothesis that
cytokines, most notably IL-1
and to a lesser extent TNF-
,
contribute to the impaired airway relaxation in asthma. IL-1
and
TNF-
have been reported to cause
-adrenergic hyporesponsiveness
in various human and animal tissues and cells (10, 29, 34). Recently,
Hakonarson and associates (15) demonstrated that the altered
responsiveness of atopic/asthmatic sensitized rabbit ASM was largely
attributed to autologously induced expression and autocrine action of
IL-1
. However, very little is known about the role of other
proinflammatory mediators in the development of the
-adrenergic
hyporesponsiveness in the airway. Because asthmatic patients have
elevated levels of kinin concentrations in nasal and bronchoalveolar
lavage fluid (5, 7) and BK exerts similar effects as IL-1
in human
ASM cells such as causing COX-2 induction and prostanoid release (23, 24), we postulated that BK could also cause impaired responses of human
ASM cells to the
-adrenoceptor agonist Iso.
The results in our present study demonstrated that pretreatment of
human ASM cells with BK resulted in a time- and concentration-dependent impairment of the cell responses to Iso (Fig. 3). The impaired responses were completely reversed by the COX inhibitor Indo, which
blocked the generation of COX products, and were partially prevented by
the COX-2 selective inhibitors NS-398 and Nim, the protein synthesis
inhibitors cycloheximide and actinomycin D, and the steroid
dexamethasone (Fig. 4). We have recently reported the details of
BK-induced PGE2 release and COX-2
induction in human ASM cells and the inhibition of the induction by
some of the above inhibitors (24). In view of this, the Western blot results were not shown here. The fact that Indo produced a
greater effect than the COX-2 selective inhibitors, the protein
synthesis inhibitors, and dexamethasone suggests that prostanoids
produced by phospholipase A2
activation and constitutive COX-1 also play a role in BK-induced
impaired cAMP genera-tion. We have previously shown that phospholipase
A2 activation is responsible for
the early phase of prostanoid production (measured as
PGE2) in response to BK
and that COX-2 is responsible for the later phase (24). Our
findings therefore provide the first direct evidence that BK induces
impaired responses of human ASM cells to
-adrenoceptor agonists, and COX products from both COX-2 induction and constitutive COX-1 contribute largely to this impairment. The relevance of these
findings to human airway function could be made clear by further
investigations of the effect of BK on the responsiveness of intact
human airways.
Much effort has been made to understand the mechanism(s) by which
cytokines cause the
-adrenergic hyporesponsiveness. In ASM, Iso
binds to the
-adrenergic receptors that couple to the stimulatory G
protein Gs, the
-subunit of
which in turn activates the enzyme adenylyl cyclase to generate cAMP
(4). Increased cAMP activates protein kinase A and protein kinase G to
cause relaxation of ASM (4). The observations that IL-1
did not affect the ability of the direct adenylyl cyclase activator forskolin to cause cAMP accumulation (14, 29) or smooth muscle relaxation (14)
suggest that the decreased
-adrenergic responsiveness by IL-1
is
not due to any change in the activity or expression of adenylyl
cyclase. The fact that a phosphodiesterase inhibitor, IBMX, was used
together with Iso (29) also excludes the involvement of changes in
phosphodiesterase activity in the effects of IL-1
within the
experimental design. The effects, therefore, are likely to be mediated
upstream of the adenylyl cyclase enzyme. Studies looking at whether the
effect is at the level of G proteins have recently shown that, although
IL-1
had no effect on the expression of the stimulatory G protein
subunit Gs
(29),
IL-1
-attenuated relaxation of tracheal smooth muscle to Iso was
ablated by a muscarinic M2-receptor antagonist and was
associated with enhanced induction of the inhibitory G protein subunits
G
i-2 and
G
i-3 (14). This suggests that
the cytokine-induced impairment of airway responsiveness to
-adrenoceptor agonists is attributable to enhanced
M2
receptor/Gi protein-coupled
inhibition of adenylyl cyclase.
PGs (mainly PGE2 and
PGI2) activate the
PGEP2 and
PGEP4 receptors, which are also
coupled to Gs and adenylyl cyclase
(21), to increase cAMP production in human ASM cells. Thus PGs share a
similar receptor-mediated signal transduction system with
-adrenoceptor agonists, and the functional responses to
PGE2 receptor stimulation, like
that to
-adrenoceptor stimulation, are downregulated by the
activation of Gi protein (20). It
is therefore reasonable to speculate that elevated levels of PGs could
cause heterologous desensitization of adenylyl cyclase. In fact, BK not
only caused impaired responses to Iso but also caused impaired
responses to PGE2 (Fig.
2A), and exogenously applied
PGE2 also caused heterologous desensitization of cAMP production in response to both Iso and PGE2 (Fig. 9,
A and
B). BK caused impaired responses to
lower concentrations of PGE2
(0.001-0.01 µM) but not to higher concentrations (0.1-1
µM). The most likely explanation for this is that the
PGE2 concentration after 24 h of
incubation with BK was ~0.01-0.02 µM, which would only be
expected to impair cAMP accumulation to subsequent application of
similar concentration of PGE2. However, when the cells were
preincubated with 1 µM exogenously applied PGE2 for 24 h, impaired responses
to subsequent stimulation with PGE2 at concentrations up to 1 µM were observed (Fig. 9B).
Observations from our previous studies have demonstrated that IL-1
causes induction of COX-2 in human ASM cells and consequently results in a marked increase in prostanoid generation with
PGE2 and
PGI2 as the major products (23)
and that BK has a similar effect (24). BK attenuated the capacity of
human ASM cells to form cAMP in response to Iso, and this impairment
was prevented completely or partially by reagents that blocked either
the activity of COX or the induction of COX-2 (Fig. 4). The
Ca2+ ionophore A-23187, which has
a similar effect as BK in causing cytosolic free
Ca2+ increase and AA release,
induced PGE2 release and mimicked
the impaired responses by BK (Fig. 7,
A and
B), and when the
PGE2 release was blocked by Indo,
the impaired responses were also reversed (Fig. 8,
A and
B). The exogenously applied
PGE2 also mimicked the impaired
responses by BK (Fig. 9, A and
B). These findings are in agreement
with the report that pretreating human ASM cells with agents that
induce cAMP formation resulted in a marked decrease in the capacity of
the cells to produce cAMP after subsequent application of Iso (16). The
fact that the anti-human IL-1
antiserum did not affect BK-induced
impairment excludes the possibility that the effect of BK is mediated
by the release of IL-1
. It is therefore likely that BK-induced
impairment of responses of human ASM cells to
-adrenoceptor agonists
is largely mediated by the large quantity of COX products involving
both existing COX-1 and induced COX-2, possibly through the uncoupling of
-receptors from adenylyl cyclase activation. The precise
mechanism(s), however, remains to be further investigated.
Two BK receptor subtypes (B1 and
B2) have been identified (11).
B1 and
B2 receptor-mediated responses can
be distinguished pharmacologically on the basis of relative potencies
of agonists or by the use of receptor-selective antagonists. In the
present study, BK-induced impaired responses were mimicked by the
selective B2 receptor agonist
[Tyr(Me)8]BK but not
by the selective B1 receptor
agonist des-Arg9-BK (Fig. 5), and
the effect of BK was strongly reversed and abolished by the selective
B2 receptor antagonist HOE-140,
whereas the selective B1 receptor
antagonist
des-Arg9,[Leu8]BK
was ineffective (Fig. 6), suggesting that BK-induced impaired responses
to Iso are mediated by the B2
receptors. Our results are in agreement with previous findings that
B2 receptors are responsible for
various effects of BK in airway tissues and cells (17, 24, 27).
Our present findings also provide fresh insights into the argument
about whether the consequences of COX-2 induction and prostanoid production in human ASM would be detrimental or beneficial for airway
functions in asthma. PGE2 is an
important anti-inflammatory mediator that has bronchoprotective effects
in the airways (25). 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 the potent bronchoconstrictor of the lipoxygenase pathway toward the
synthesis of bronchodilators of the COX pathway, such as
PGE2 and
PGI2. However,
PGE2 at higher concentrations also
causes ASM contraction due to weak agonism at the thromboxane receptor
(1, 19), and other products of COX, such as
PGF2
, thromboxane
A2, and
PGD2, are potent proinflammatory modulators that cause bronchoconstriction via the activation of the
thromboxane prostanoid receptor (1, 19). In addition, we report here
that COX-2 induction forms an important part in BK-induced impairment
of human ASM cell responses to Iso, and we speculate that COX-2
induction may also be involved in IL-1
-induced impaired responses to
Iso. COX-2 induction and the consequent prostanoid production in human
ASM may therefore do more harm than good in respect to airway function
in inflammatory airway diseases such as asthma.
In summary, this study examined the role of COX products in BK-induced
impairment of human ASM cell cAMP responses to the
-adrenoceptor
agonist Iso. Our results demonstrated that
1) pretreatment of the cells with BK
resulted in a marked time- and concentration-dependent decrease in cAMP
accumulation after subsequent application of Iso;
2) reagents that inhibited or
blocked either the activity of COX or the induction of COX-2 also
completely or partially prevented BK-induced impairment in cAMP
formation; 3) BK-induced impaired
responses were mimicked and abolished by the selective B2 receptor agonist and
antagonist, respectively, but were not affected by the anti-IL-1
antiserum; and 4) pretreatment with A-23187 and exogenously applied
PGE2 also caused impairment on cAMP accumulation of the cells in a similar pattern as that of BK.
Collectively, these findings indicate that COX products (including those from COX-2 induction) are critical in the development of BK-induced impairment of human ASM cell responses to Iso, and this may
be helpful in the understanding of the pathogenesis of asthma.
 |
ACKNOWLEDGEMENTS |
We thank Colin Clelland for providing specimens of human trachea.
 |
FOOTNOTES |
This study was supported by grants from The National Asthma Campaign
(United Kingdom) and NHS Executive Trent.
Address for reprint requests: A. J. Knox, Div. of Respiratory Medicine,
City Hospital, University of Nottingham, Hucknall Rd., Nottingham NG5
1PB, UK.
Received 20 November 1997; accepted in final form 7 April
1998.
 |
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