In vitro sensitization of human bronchus by
2-adrenergic agonists
Christophe
Faisy1,2,
Emmanuel
Naline1,
Jean-Luc
Diehl2,
Xavier
Emonds-Alt3,
Thierry
Chinet1, and
Charles
Advenier1
1 Unité Propre de Recherche de l'Enseignement
Supérior Equipe d'Accueil 220, Faculté de Médecine
Paris-Ouest, Unité de Formation et de Recherche Biomédicale
des Saint-Pères, 75006 Paris; 2 Service de
Réanimation Médicale, Hôpital Européen Georges
Pompidou, 75908 Paris Cedex 15; and 3 Sanofi
Synthelabo Recherche, 34000 Montpellier, France
 |
ABSTRACT |
Incubation of human distal bronchi from 48 patients for 15 h with 10
7 M fenoterol induced
sensitization characterized by an increase in maximal contraction to
endothelin-1 (ET-1) and acetylcholine (ACh). Incubation of human
bronchi with 10
6, 3 × 10
6, and
10
5 M forskolin (an adenyl cyclase activator) reproduced
sensitization to ET-1 and ACh. The sensitizing effect of fenoterol was
inhibited by coincubation with gliotoxine (a nuclear factor-
B
inhibitor), dexamethasone, indomethacin (a cyclooxygenase inhibitor),
GR-32191 (a TP prostanoid receptor antagonist), MK-476 (a cysteinyl
leukotriene type 1 receptor antagonist), SR-140333 + SR-48968 + SR-142801 (neurokinin types 1, 2, and 3 tachykinin receptor
antagonists) with or without HOE-140 (a bradykinin B2
receptor antagonist), SB-203580 (an inhibitor of the 38-kDa
mitogen-activated protein kinase, p38MAPK), or calphostin C
(a protein kinase C blocker). Our results suggest that chronic exposure
to fenoterol induces proinflammatory effects mediated by nuclear
factor-
B and pathways involving leukotrienes, prostanoids,
bradykinin, tachykinins, protein kinase C, and p38MAPK,
leading to the regulation of smooth muscle contraction to ET-1 and ACh.
2-agonists; airway sensitization; airway smooth
muscle; endothelin-1; asthma
 |
INTRODUCTION |
THE FATAL ACUTE ASTHMA
CASES attributable to fenoterol abuse in the 1980s in New Zealand
started a controversy concerning the potential worsening of the
bronchial hyperresponsiveness by the
2-adrenoceptor
agonists (7, 14, 42). Studies in animals and humans showed
that chronic exposure to fenoterol or salbutamol induces a nonspecific
bronchial sensitization, whereas the relaxant effects of these
2-agonists on the airway smooth muscle are not decreased
(11, 59, 60). The bronchial sensitization induced by
fenoterol is similar to the sensitization provoked by ovalbumin in
sensitized guinea pigs (60). Chronic administration of
salbutamol at low doses to guinea pigs increases airway reactivity to
histamine and methacholine (11). In humans, long-term use
of salbutamol increases the bronchial hyperresponsiveness to histamine
but does not cause subsensitization of
2-adrenoceptors
to salbutamol (59). In a bovine tracheal model, Katsunuma
and colleagues (36) showed that prolonged incubation with
fenoterol induced an increased contractile responsiveness to neurokinin
A (NKA). In 1995, Peters and colleagues (47) suggested
that the continuous activation of the intracellular signal transduction
caused by the
2-adrenoceptor stimulation could induce a
proinflammatory process mediated by nuclear transcription factors in
rat lung. A recent study in our institution showed that the
transcription factor nuclear factor-
B (NF-
B) is involved in
fenoterol-induced hyperresponsiveness to NKA in guinea pig isolated
trachea (52).
Endothelin-1 (ET-1) is a 21-amino acid peptide recently implicated in
chronic inflammatory airway diseases such as asthma and chronic
obstructive pulmonary disease (25, 26, 41, 46). ET-1 is
synthesized and metabolized in lung, and ET-1 receptors (ETA and ETB) are widely distributed in airway
cells (21, 26, 41). ET-1 is one of the most potent
contractile agents of human airway smooth muscle and can induce airway
inflammation, airway hyperresponsiveness, and airway remodeling in
animals and humans (25, 26, 27, 41), suggesting that ET-1
could be a major component of asthma pathophysiology (10, 22, 26,
27). The purpose of this study was to determine the sensitizing
effect of fenoterol on the contraction to ET-1 of human bronchi and to investigate the role of inflammatory mediators and signal transduction pathways involved in airway sensitization to ET-1 induced by
2-adrenoceptor agonists.
 |
METHODS |
Human bronchial tissue preparations.
Bronchial tissues were surgically removed from 54 patients with lung
cancer (45 men and 9 women, 62 ± 10 yr of age); all patients were
smokers or ex-smokers. Just after resection, segments of human bronchi
(1-3 mm ID) were taken as far as possible from the malignant
lesion. They were placed in oxygenated Krebs-Henseleit solution
composed of (in mM) 119 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 25 NaHCO3, and 11.7 glucose. After removal of adhering lung parenchyma and connective
tissues, rings of the same bronchus were prepared (5-7 mm long,
1-3 mm ID) and divided into two paired groups: one (control) group
was placed in Krebs-Henseleit solution at room temperature (21°C) for
15 h; the other (pretreated) group was treated with
10
7 and 10
6 M fenoterol, 10
7
and 10
6 M formoterol, 10
6 M salbutamol,
10
6 M salmeterol, or a cAMP activator, i.e., forskolin
(10
6, 3 × 10
6, and 10
5
M), for 15 h at room temperature. Incubation time and temperature were chosen in agreement with the work of Katsunuma and colleagues (36) and our previous work (52). Fenoterol,
formoterol, salbutamol, salmeterol, and forskolin concentrations were
chosen according to Wang and colleagues (60), Katsunuma
and colleagues, and our previous work (45, 52).
Experiments were performed in parallel (control and pretreated groups)
for 15 h at room temperature in the absence or presence of
1) an NF-
B inhibitor, gliotoxine (10
6 M),
2) a glucocorticoid, dexamethasone (10
6 M),
3) a cyclooxygenase (COX) inhibitor, indomethacin
(10
6 M), 4) a prostanoid and prostaglandin TP
receptor antagonist, GR-32191 (10
7 and 10
6
M), 5) a cysteinyl leukotriene (Cys-LT1)
receptor antagonist, MK-476 (10
8 and 10
7
M), 6) a nitric oxide (NO) synthase (NOS) inhibitor,
nitro-L-arginine methyl ester (L-NAME,
10
3 M), 7) a mixture of the tachykinin
NK1, NK2, and NK3 receptor antagonists, SR-140333 (10
7 M), SR-48968
(10
7 M), and SR-142801 (10
7 M),
respectively, 8) a bradykinin B2 receptor
antagonist, HOE-140 (10
7 M), 9) an inhibitor
of the 38-kDa mitogen-activated protein kinase (MAPK), SB-203580,
10) a protein kinase C (PKC) blocker, calphostin C
(10
7 M), and 11) SB-203580 + calphostin
C. Because SB-203580 has been shown to inhibit COX-1 and COX-2, on the
one hand, and thromboxane synthase, on the other hand, with
IC50 for both enzymes of 2 × 10
6 M in
human platelets (9) and 1.8 × 10
6 M in
human airway smooth muscle (24), we studied in preliminary experiments the potential inhibitory effect of SB-203580, calphostin C,
and SB-203580 + calphostin C on the contraction of human bronchus induced by
[Sar9,Met(O2)11]substance P
(10
7 M), which acts in part through the COX and
thromboxane synthase pathway (4). Moreover, we used
10
7 M calphostin C according to Barman (5).
To investigate the mechanisms of change in airway contractility after
sensitization by fenoterol, we conducted a second protocol, where drugs
(10
6 M indomethacin, 10
6 M GR-32191,
10
7 M MK-476, 10
3 M L-NAME,
10
7 M SR-140333, 10
7 M SR-48968,
10
7 M SR-142801, and 10
7 M HOE-140) were
added, after 15 h of incubation at room temperature with
10
7 M fenoterol, to the organ bath at 37°C 45 min
before addition of ET-1 and ACh.
Experimental procedure.
After incubation with drugs, the bronchial rings were suspended on
hooks in a 5-ml organ bath containing Krebs-Henseleit solution, gassed
with 95% O2-5% CO2 and maintained at 37°C.
Each preparation was connected to a force displacement transducer
(model UF1, Statham), and isometric tension changes were recorded on a
polygraph. Preparations were suspended with an initial tension of
1.5 g in organ baths and equilibrated for 60 min, with changes in
fresh Krebs-Henseleit solution every 10 min during the first 30 min of
the equilibrium period before the start of data acquisition. A load of
1-1.5 g was maintained throughout the first 30 min of the
equilibrium period. Concentration-response curves to ET-1
(10
10-10
7 M with logarithmic
increments) were then obtained by applying increasing concentrations at
10- to 15-min intervals. Only one concentration-response curve was
recorded for each ring. Then human bronchi were maximally contracted
with 3 × 10
3 M ACh. Previous exposure of human
bronchus to ET-1 has no effect on contraction induced by exogenous ACh
(17).
Drugs.
ET-1 was obtained from Novobiochem (Laüfelfingen, Switzerland),
ACh hydrochloride from Pharmacie Centrale des Hôpitaux (Paris, France), forskolin, gliotoxine, dexamethasone, indomethacin,
L-NAME, and calphostin C from Sigma (St. Louis, MO),
GR-32191 from Glaxo (Greenford, UK), MK-476 from Merck (Paris, France),
SR-140333, SR-48968, and SR-142801 from Sanofi Research Center
(Montpellier, France), HOE-140 from Peninsula (Merceyside, UK),
SB-203580 from Calbiochem (San Diego, CA), and
[Sar9,Met(O2)11]substance P from
Bachem (Bubendorf, Switzerland). All drugs except indomethacin and
SB-203580 were dissolved in distilled water; indomethacin was dissolved
in pure ethanol and then diluted in Krebs solution, and SB-203580 was
dissolved in pure ethanol and DMSO and then diluted in Krebs solution.
The final amount of ethanol (0.03%) did not alter ACh reactivity
(4). ET-1 was dissolved in water at 2.5 × 10
4 M and kept in small aliquots (200 µl) at
20°C
until used. A fresh aliquot was used for each experiment.
Expression and analysis of data.
Contractile responses were expressed in tension (g) compared with the
basal tone recorded before the start of the concentration-response curve. Values are means ± SE. The data are expressed in terms of
Emax for efficacy and
log EC50
(pD2) for potency. Emax represents the maximal
contraction induced by ET-1 and ACh and is expressed in tension
compared with the basal tone.
Emax represents the difference between Emax obtained with the pretreated
bronchi and Emax obtained with the paired control human
bronchi.
Emax was expressed in grams compared with basal
tone.
log EC50 values were derived graphically from the
logarithmic concentration-effect curves and defined as the negative
logarithm of the drug concentration that caused 50% of maximal effect
of ET-1 (10
7 M).
(
log EC50) represents
the difference between
log EC50 obtained with the
pretreated bronchi and
log EC50 obtained with the paired
control human bronchi. Bronchi with Emax of ET-1 <0.7 g
were excluded from analysis, because we considered that a low level of
Emax is a reliable marker of dysfunction of contractility. Statistical analysis of the results was performed using Student's t-test (2-tailed, for paired samples). P < 0.05 was considered significant.
 |
RESULTS |
Sample.
Bronchi of 48 from 54 patients (89%) yielded an Emax of
ET-1
0.7 g. Incubation of these 48 bronchi for 15 h at 21°C
with fenoterol (10
7 M) significantly increased their
maximal contraction to ET-1 (Fig. 1) and
ACh (Emax of ACh = 2.66 ± 0.18 and 1.99 ± 0.15 g in the presence and absence of fenoterol, respectively,
n = 48, P < 0.01). Incubation of human
bronchi with fenoterol did not change significantly the potency of ET-1
(
log EC50 = 8.52 ± 0.05 and 8.41 ± 0.06 in the presence and absence of fenoterol, respectively, n = 48, not significant; Fig. 1). Because sensitization
of human bronchi by fenoterol was characterized by an increase in
maximal contraction to ET-1, we investigated the human bronchi with
Emax of ET-1 >0 after 15 h of fenoterol exposure.
Among the 48 bronchi, 38 (79%) presented a
Emax of ET-1
>0. Analysis of data was performed from this sample of 38 human
bronchi.

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Fig. 1.
Concentration-response curves for endothelin-1 (ET-1) in
human bronchi after incubation for 15 h at 21°C with
10 7 M fenoterol. Emax, difference between
maximal contraction (maximal efficacy) to ET-1 in pretreated bronchi
and maximal contraction to ET-1 in paired control bronchi. Values are
means ± SE (n = 48).
P < 0.01;
 P < 0.001 vs. control.
|
|
Effect of incubation with
2-adrenoceptor agonists or
forskolin on potency of ET-1 and on maximal efficacy of ET-1 and ACh.
Emax was statistically significant when fenoterol
(10
7 and 10
6 M), formoterol
(10
8 and 10
7 M), salbutamol
(10
6 M), or salmeterol (10
6 M) was added to
the incubation medium of bronchi (Table
1). Potency of ET-1 (
log
EC50) was not significantly modified by incubation with
these drugs (Table 1). Forskolin, a cAMP activator, increased the
maximal contraction to ET-1 and ACh in a concentration-dependent manner
(Table 1). Incubation with forskolin (10
6, 3 × 10
6, and 10
5 M) had no significant effect
on potency of ET-1 (Table 1).
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Table 1.
Effect of incubation of human bronchi with 2
-adrenoceptor agonists or forkolin on potency of ET-1 and maximal
efficacy of ET-1 and ACh
|
|
Effect of incubation with anti-inflammatory drugs, proinflammatory
mediator receptor antagonists, or NOS inhibitor on maximal contraction
to ET-1 and ACh in the absence of fenoterol.
In the absence of fenoterol, incubation of human bronchi for 15 h
at 21°C with gliotoxine (10
6 M), dexamethasone
(10
6 M), indomethacin (10
6 M), GR-32191
(10
7 and 10
6 M), MK-476 (10
8
and 10
7 M), L-NAME (10
3 M),
SR-142801 (10
7 M), and SR-140333 + SR-48968 + SR-142801 ± HOE-140 (10
7 M) had no significant
effect on the Emax of ET-1 and ACh (Table 2).
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Table 2.
Effect of incubation of human bronchi with anti-inflammatory drugs,
proinflammatory mediator receptor antagonists, or NO synthase inhibitor
on maximal contraction to ET-1 and ACh in absence of fenoterol
|
|
Effect of coincubation with anti-inflammatory drugs,
proinflammatory mediator receptor antagonists, or NOS inhibitor on
fenoterol-induced sensitization.
When the same paired human bronchi were incubated with fenoterol
(10
7 M) for 15 h at 21°C, coincubation with
gliotoxine, dexamethasone, indomethacin, GR-32191 (10
6
M), MK-476 (10
7 M for ACh, 10
8 and
10
7 M for ET-1), and combinations of SR-140333 + SR-48968 + SR-142801 or SR-140333 + SR-48968 + SR-142801 + HOE-140 significantly decreased the rise of maximal
response to ET-1 and ACh elicited by fenoterol (control bars, Figs.
2 and 3).
Incubation with L-NAME or SR-142801 had no significant
effect on the sensitization induced by fenoterol (Fig. 2). Addition of
HOE-140 to SR-140333 + SR-48968 + SR-142801 did not
significantly increase the inhibition of SR-140333 + SR-48968 + SR-142801 on the fenoterol-induced sensitizing effect (Fig. 3).

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Fig. 2.
Effect of coincubation for 15 h at 21°C with
gliotoxine (a nuclear factor- B inhibitor), dexamethasone,
indomethacin (a cyclooxygenase inhibitor), GR-32191 (a prostanoid and
prostaglandin TP receptor antagonist), MK-476 (a cysteinyl leukotriene
receptor antagonist), nitro-L-arginine methyl ester
(L-NAME, a nitric oxide synthase inhibitor), and SR-142801
(a tachykinin NK3 receptor antagonist) on Emax
of ET-1 and ACh in the presence of 10 7 M fenoterol.
Values are means ± SE (n = 12).
*P < 0.05; **P < 0.01;
***P < 0.001 vs. control.
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Fig. 3.
Effect of coincubation for 15 h at 21°C with
SR-140333 + SR-48968 + SR-142801 (tachykinin NK1,
NK2, and NK3 receptor antagonists) and
SR-140333 + SR-48968 + SR-142801 + HOE-140 (a bradykinin
B2 receptor antagonist) on Emax of ET-1 and ACh
in the presence of 10 7 M fenoterol. Values are means ± SE (n = 8). *P < 0.05;
**P < 0.01 vs. control.
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|
Effect of coincubation with MAPK inhibitor, PKC blocker, and MAPK
inhibitor + PKC blocker on fenoterol-induced
sensitization.
In preliminary experiments, we found that 10
6 M, but not
10
7 and 3 × 10
7 M, SB-203580
inhibited the contraction of nine human bronchi induced by
[Sar9,Met(O2)11]substance P,
which acts through the COX and thromboxane synthase pathway (Table
3). Therefore, we used
<10
6 M SB-203580 (i.e., 10
7 and 3 × 10
7 M). In control experiments (absence of fenoterol),
15 h of incubation at 21°C with the inhibitor of
p38MAPK SB-203580 (10
7 M) alone and the PKC
blocker calphostin C (10
7 M) had no significant effect on
the Emax of ET-1 and ACh (Table 2). Addition of SB-203580
(3 × 10
7 M) to calphostin C (10
7 M)
did not modify significantly the Emax of ET-1 and ACh
(Table 2). When the same paired bronchi were sensitized to ET-1 and ACh
by fenoterol for 15 h at 21°C (control bars, Fig.
4), coincubation with SB-203580
(10
7 and 3 × 10
7 M), calphostin C
(10
7 M), or calphostin C + SB-203580 significantly
decreased the rise of maximal response induced by fenoterol (Fig. 4).
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Table 3.
Effects of indomethacin, SB-203580, and calphostin C on contraction of
human bronchi induced by [Sar9,
Met(O2)11]substance P
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Fig. 4.
Effect of coincubation for 15 h at 21°C with
calphostin C (a protein kinase C blocker), SB-203580 (a 38-kDa
mitogen-activated protein kinase inhibitor), and calphostin C + SB-203580 on Emax of ET-1 and ACh in the presence of
10 7 M fenoterol. Values are means ± SE
(n = 8). *P < 0.05;
**P < 0.01 vs. control.
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|
Effects of the anti-inflammatory drugs, the proinflammatory
mediator receptor antagonists, or an NOS inhibitor on maximal
contraction to ET-1 and ACh after sensitization by fenoterol.
In control experiments (absence of fenoterol), 45 min of incubation at
37°C with L-NAME (10
3 M) increased
significantly the maximal contraction to ET-1 and ACh (Table
4). Incubation for 45 min at 37°C with
indomethacin, GR-32191, MK-476, or combinations of the tachykinin
NK1 + NK2 + NK3 receptor
antagonists did not alter the maximal contraction of the human bronchi
(Table 4). Addition of HOE-140, a bradykinin B2 receptor
antagonist, to the tachykinin NK1 + NK2 + NK3 receptor antagonists did not
modify the Emax of ET-1 and ACh of the bronchi (Table 4).
When the same paired bronchi were sensitized to ET-1 and ACh by
fenoterol for 15 h at 21°C (control bars, Fig.
5), incubation for 45 min at 37°C with
indomethacin, GR-32191, MK-476, and L-NAME did not affect
the sensitizing effect induced by fenoterol (Fig. 5). In contrast,
incubation for 45 min at 37°C with SR-140333 + SR-48968 + SR-142801 or SR-140333 + SR-48968 + SR-142801 + HOE-140 significantly decreased the rise of the
maximal response elicited by fenoterol (Fig.
6). Addition of HOE-140 to SR-140333 + SR-48968 + SR-142801 did not significantly increase the
inhibition of SR-140333 + SR-48968 + SR-142801 on the
sensitizing effect induced by fenoterol.
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Table 4.
Effect of incubation of human bronchi with anti-inflammatory drugs,
proinflammatory mediator receptor antagonists, or NO synthase inhibitor
on maximal contraction to ET-1 and ACh in absence of fenoterol
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Fig. 5.
Effect of incubation for 45 min at 37°C with
indomethacin, GR-32191, MK-476, and L-NAME on
Emax of ET-1 and ACh after sensitization of human bronchi
by 10 7 M fenoterol for 15 h at 21°C (control
bars). Values are means ± SE (n = 8). Difference
between pretreated groups and paired control group was not
statistically significant.
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Fig. 6.
Effect of incubation for 45 min at 37°C with
SR-140333 + SR-48968 + SR-142801 and SR-140333 + SR-48968 + SR-142801 + HOE-140 on Emax of ET-1
and ACh after sensitization of human bronchi by 10 7 M
fenoterol for 15 at 21°C (control). Values are means ± SE
(n = 9). *P < 0.05;
**P < 0.01 vs. control.
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 |
DISCUSSION |
In this study, we observed an in vitro sensitization to ET-1 and
ACh of human bronchi by fenoterol. This sensitization is not specific
to fenoterol, inasmuch as we found the same phenomenon with formoterol,
salbutamol, and salmeterol. We then investigated the transduction
pathways involved in sensitization of human bronchi by
2-adrenoceptor agonists and showed that the nuclear
transcription factor NF-
B and p38MAPK play a pivotal
role in this event. Furthermore, several inflammatory processes appear
to be involved in the sensitization of human bronchi by fenoterol.
Sensitization of human bronchi is not limited to fenoterol but is also
observed with several
2-adrenoceptor agonists in a range
of concentrations known to cause submaximal relaxation
(45). We found that prolonged exposure of human bronchi to
fenoterol affects maximal efficacy of ET-1 but not its potency. Our
results are in agreement with the work reported by Wang and colleagues (60), who showed that chronic fenoterol exposure increased
maximal airway response to ACh but not ACh EC50 in guinea
pigs. Potency of an agonist depends in part on the affinity of
receptors for binding the agonist and in part on the efficiency with
which agonist-receptor interaction is coupled to response. Maximal
efficacy of an agonist is determined by the characteristics of the
receptor-effector system involved. In this way, our results suggest
that chronic exposure to
2-adrenoceptor agonists
involves changes in the contractile proteins of human bronchi but does
not alter affinity of the receptors for ET-1. In addition, forskolin, a
cAMP activator, increased the maximal response to ET-1 and ACh of human
bronchi in a concentration-dependent manner. This suggests that
prolonged activation of the cAMP-protein kinase A (PKA) system could
cause sensitization. Indeed, prolonged activation of cAMP-PKA may
induce stimulation of proinflammatory nuclear transcription factors,
such as NF-
B or AP-1 (1, 33), and may enhance the
expression of several types of receptors, such as bradykinin
B1 and B2 and NK1 and
NK2 receptors implied in nonspecific airway
hyperresponsiveness in animals and humans (23, 32, 36,
52). However, short-term activation of the cAMP-PKA system may
decrease the activity of proinflammatory enzymes such as constitutive
phospholipase A2 (cPLA2), COX-2,
5-lipoxygenase, and MAPK (38, 54, 57). Eickelberg and
colleagues (16) showed that incubation with salmeterol or
salbutamol induced, probably via calmodulin stimulation, a
ligand-independent activation of the glucocorticoid receptor in
cultured human lung fibroblasts and vascular smooth muscle cells. Our
results conflict with these findings, but it is not well known whether
inflammatory processes may regulate activation of the glucocorticoid
receptor in human bronchus. In asthma, various studies underlined that
long-acting
2-adrenoceptor agonists exhibit very small,
if any, anti-inflammatory effects when given alone (37).
Katsunuma and colleagues (36) showed that dexamethasone
and cycloheximide (a protein synthesis blocker) inhibited the increased bovine tracheal smooth muscle contractile response to NKA induced by
fenoterol. Saulnier and colleagues (52) abolished the
fenoterol-induced tracheal sensitization in guinea pigs with two
transcription factor NF-
B inhibitors (gliotoxine and pyrrolidine
dithiocarbamate). In agreement with these authors, we found that
gliotoxine (an NF-
B inhibitor) and dexamethasone (an NF-
B and
AP-1 inhibitor) abolished the sensitization induced by fenoterol in
human bronchi. These results underline the pivotal role played by
NF-
B in the process of sensitization. NF-
B is involved in the
expression of proinflammatory molecules and mediators
(cPLA2, COX-2, prostanoids, and leukotrienes) implicated in
cellular events in asthma (2, 4, 6, 13). Effects of
2-adrenoceptor agonists on NF-
B pathways are not well
known in humans. Korn and colleagues (39) recently showed
that expression of interleukin-8, a proinflammatory cytokine stimulated
in part by NF-
B, was markedly increased by formoterol
(10
10 M) in cultured human bronchial epithelial cells. In
contrast, Wilson and colleagues (62) observed a reduction
of NF-
B expression in mucosal eosinophils and epithelial cells in
bronchial biopsies from 10 atopic asthmatic patients after 8 wk of
treatment with formoterol. In this study, formoterol did not reduce the
immunoreactivity for adhesion molecules and proinflammatory cytokines
stimulated by NF-
B, in contrast to glucocorticosteroid treatment. We
found that indomethacin, GR-32191, and MK-476 significantly decreased or abolished the sensitization induced by fenoterol, suggesting that
prolonged activation of the cAMP-PKA system by fenoterol may induce an
enzymatic inflammatory process (cPLA2 and COX-2) mediated
by NF-
B.
We established that a mixture of tachykinin NK1,
NK2, and NK3 receptor antagonists decreased the
sensitization elicited by fenoterol. In contrast to results obtained
previously in the guinea pig trachea by Saulnier and colleagues
(52), we found that the NK3 receptor
antagonist SR-142801, when used alone, did not significantly reduce the
fenoterol-induced sensitization. NK3 receptors seem to be
involved in airway sensitization in guinea pigs (43), but
its role is not well known in humans. However, SR-142801 has been shown
to inhibit interleukin-1
-induced hyperresponsiveness to
[Sar9,Met(O2)11]substance P
(4) and nerve growth factor in human bronchi
(19). Also, our results suggest that the tachykinin
NK1 and NK2 receptor agonists are involved in
the mechanisms of sensitization of human bronchi by fenoterol. This is
in agreement with the recent works reported by Katsunuma and colleagues
(35, 36), who found an increase of NK2
receptor expression in bovine tracheal smooth muscle after treatment
with fenoterol.
Leukotrienes could amplify neurogenic inflammation by increasing
release of the tachykinins from the C-fibers of the nonadrenergic noncholinergic system in asthma (28). We found that the
bradykinin B2 receptor antagonist HOE-140 did not
significantly enhance the inhibition of the fenoterol-induced
sensitization by the tachykinin NK1, NK2, and
NK3 receptor antagonists. Ricciardolo and colleagues (49) showed that a combination of the NK1 and
NK2 tachykinin receptor antagonists abolished the increased
bronchoconstriction produced by NKA and inhibited partially the
contractile response induced by bradykinin in ovalbumin-sensitized
guinea pigs, whereas HOE-140 had no effect on the increase in
bronchoconstriction produced by NKA, suggesting that bradykinin induces
the release of tachykinins from sensory nerves in guinea pig airways.
Moreover, bradykinin may stimulate the MAPK pathways via the activation
of the protein Rho GTPase, PKC, and NF-
B in human lung
(29). Also, our study highlights the role of NF-
B,
leukotrienes, prostanoids, tachykinin NK1 and
NK2 receptor agonists, and bradykinin in the
mechanisms of sensitization of human bronchi by fenoterol (Fig.
7).

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|
Fig. 7.
Proposed mechanisms underlying sensitization of human airway smooth
muscle induced by fenoterol. NF- B, nuclear factor- B;
cPLA2, constitutive phospholipase A2; COX,
cyclooxygenase; 5-LPO, 5-lipoxygenase; TxA2, thromboxane
A2; MAPK, mitogen-activated protein kinase; PKA, protein
kinase A.
|
|
Our results also show that SB-203580, a p38MAPK inhibitor,
at a concentration that did not inhibit COX-1 or COX-2 activity and thromboxane synthesis (Table 3), abolishes the sensitization elicited
by fenoterol. Recent studies underline the role of the MAPK in the
intracellular processes of airway smooth muscle proliferation and
sensitization (2, 30, 32, 46, 58, 61). Three distinct MAPK
pathways have been identified in mammals: 1)
p42/44MAPK, 2) stress-activated protein kinase
(SAPK) or c-Jun NH2-terminal kinase (JNK), and
3) p38MAPK (15). MAPKs are involved
in multiple proinflammatory mechanisms, implying humoral and neurogenic
mediators. Expression of NF-
B, cPLA2, COX-2,
tachykinins, bradykinin, ETB, and muscarinic M3 receptors is upregulated by MAPK in airway smooth muscle cells (12, 23, 32, 53). Moreover, MAPK increases the
Gi protein activity, which results in a functional
uncoupling between Gs protein and
2-adrenoceptor (2, 32, 37). In addition,
MAPK enhances the myosin light chain kinase activity and the heavy chain of myosin expression (32) and may increase smooth
muscle contraction probably via h-caldesmon phosphorylation and actin-F remodeling (30). Interestingly, prostanoids such as
thromboxane A2 stimulate the MAPK by coupling the TP
receptor with G
q (activation of PKC) or
Gi
proteins (2, 32, 34), and
leukotrienes increase the MAPK expression in humans (48,
49). These data suggest that MAPK pathways could amplify the
inflammatory processes induced by NF-
B and could sensitize the
airway smooth muscle after prolonged exposure to fenoterol (Fig. 7).
PKC is a cyclic nucleotide-independent protein kinase implicated in
regulation of airway smooth muscle tone (61). PKC-
enhances the activity of the protein Raf-1 and NF-
B, which activate the p38MAPK and SAPK/JNK pathways (12). Recent
publications showed that bradykinin and thromboxane A2
activate MAPK pathways via PKC-dependent Gi
protein
coupling in human cells (20, 29). In addition, PKC may
increase the contractility of the airway smooth muscle by inhibiting
caldesmon (via the MAPK pathways) and calponin (directly), which are
involved in modulation of the actin-myosin interaction (30,
61). We show here that blockage of PKC by calphostin C
effectively inhibits fenoterol-induced sensitization. Thus our results
suggest that PKC plays a major role in the intracellular mechanisms
leading to fenoterol-induced sensitization of human airway smooth
muscle (Fig. 7).
We also investigated the mechanisms involved in the increase of the
contractility to ET-1 and ACh of human bronchi after sensitization by
fenoterol in a protocol where drugs were added after incubation with
fenoterol but 45 min before addition of ET-1 or ACh at 37°C for
contraction. Our results showed that neither prostanoids nor leukotrienes were involved in this mechanism. In contrast, the NK1, NK2, and NK3 receptors
appeared to be implicated in the increase of contractility
after sensitization by fenoterol as well as bradykinin, which tended to
potentiate, but not significantly, inhibition of the fenoterol-induced
sensitization by the NK1, NK2, and
NK3 receptor antagonists. In the absence of incubation with
fenoterol, we found that tachykinins and bradykinin were not involved
in the process of contraction to ET-1 and ACh. Additional studies are
needed to clarify the role of tachykinins and bradykinin in the
contraction mechanisms after sensitization by fenoterol in human bronchi.
Studies in animal and human airways have shown that the epithelial
ETA receptor may mediate NO production (via the
constitutive NOS) and prostaglandin E2 production (via the
epithelial COX-2) (3, 8, 31, 44). Epithelial NO and
prostaglandin E2 are relaxant for the airway smooth muscle.
In a recent study, Naline and colleagues (44) found that
NO is the major determinant of the epithelial regulation of the human
airway smooth muscle contraction to ET-1. Our results are in agreement
with these authors, because we showed that L-NAME, but not
indomethacin and GR-32191, enhanced the contractility to ET-1. The
epithelial regulation of the contractility to ACh was also mediated by
NO. On the contrary, after sensitization by fenoterol,
L-NAME failed to enhance the maximal contractility to ET-1
and ACh. We suggest that chronic exposure to fenoterol induces a
disruption of the epithelial regulation of the airway smooth muscle
contraction to ET-1 and ACh. Further investigations are needed to
confirm and elucidate this mechanism.
Our study has several limitations. First, we studied bronchi obtained
from nonhealthy subjects, who were all previous smokers.
Emax was increased by fenoterol exposure in only 38 of
48 bronchi.
2-Adrenoceptor polymorphisms inducing
variable response to
2-agonists may constitute a
possible explanation of this fickle sensitizing effect
(55). For instance, the Gly16
2-adrenoceptor polymorphism could be associated with
asthma severity (37). However, in patients treated
chronically with salmeterol, exacerbations are not correlated with the
Gly16 polymorphism (56). The
2-agonist concentrations that we used to sensitize the
bronchi may not be consistent with the in vivo concentrations obtained
by actual treatment using
2-agonists. In a randomized
study comparing clinical efficacy of nebulized vs. intravenous
salbutamol in severe acute asthma, Salmeron and colleagues
(51) found plasma concentrations of salbutamol on the
order of 10
5 M. In healthy volunteers, plasma formoterol
concentration reached 10
9 M after inhalation of a single
dose of 120 µg of formoterol fumarate (40). Therapeutic
plasma concentration of fenoterol of ~10
8 M was
recommended (18), but plasma concentration associated with
serious toxicity is not known.
In summary, our study demonstrates the proinflammatory effects of
chronic exposure to
2-agonists in human bronchus. Our
results suggest that these proinflammatory effects are mediated by
NF-
B and lead to sensitization of airway smooth muscle. MAPK, PKC, and tachykinins seem to play a major role in the sensitization of the
human bronchus after chronic exposure to
2-adrenergic agents.
 |
ACKNOWLEDGEMENTS |
This study was supported by grants from Assistance
Publique-Hôpitaux de Paris and Société de Pneumologie
de Langue Française.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: C. Advenier, UPRES EA220, UFR Biomédicale des Saint-Pères, 45 rue des Saint-Pères 75006 Paris (E-mail:
charles.advenier{at}wanadoo.fr).
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.
June 10, 2002;10.1152/ajplung.00063.2002
Received 19 February 2002; accepted in final form 5 June 2002.
 |
REFERENCES |
1.
Adcock, IM.
Role of transcription factors in mediating cytokine-induced inflammation.
Eur Respir J
10:
289-293,
2000.
2.
Anderson, GP.
Interactions between corticosteroids and
-adrenergic agonists in asthma disease induction, progression, and exacerbation.
Am J Respir Crit Care Med
161:
S188-S196,
2000[Free Full Text].
3.
Baraniuk, JN,
Molet S,
Mullol J,
and
Naranch K.
Endothelins and the airway mucosa.
Pulm Pharmacol Ther
11:
113-123,
1998[ISI][Medline].
4.
Barchasz, E,
Naline E,
Molimard M,
Moreau J,
Georges O,
Edmonds-Alt X,
and
Advenier C.
Interleukin-1
-induced hyperresponsiveness to [Sar9,Met(O2)11]substance P in isolated human bronchi.
Eur J Pharmacol
379:
87-95,
1999[ISI][Medline].
5.
Barman, SA.
Effect of protein kinase C on hypoxic pulmonary vasoconstriction.
Am J Physiol Lung Cell Mol Physiol
280:
L888-L895,
2001[Abstract/Free Full Text].
6.
Barnes, PJ.
Pharmacology of airway smooth muscle.
Am J Respir Crit Care Med
158:
S123-S132,
1998[Abstract/Free Full Text].
7.
Barnes, PJ,
and
Chung KF.
Questions about inhaled
2-agonists in asthma.
Trends Pharmacol Sci
13:
20-23,
1992[ISI][Medline].
8.
Bertrand, C,
Naline E,
and
Advenier C.
In vitro effects of the endothelins on airway and vascular smooth muscle tone.
In: Pulmonary Actions of the Endothelins. Basel: Birkhäuser Verlag, 1999, p. 107-123.
9.
Börch-Haubold, AG,
Pasquet S,
and
Watson SP.
Direct inhibition of cylooxygenase-1 and -2 by the kinase inhibitors SB 203580 and PD 98059.
J Biol Chem
273:
28766-28772,
1998[Abstract/Free Full Text].
10.
Bousquet, J,
Jeffery PK,
Busse WW,
Jonhson M,
and
Vignola AM.
Asthma. From bronchoconstriction to airways inflammation and remodeling.
Am J Respir Crit Care Med
161:
1720-1745,
2000[Free Full Text].
11.
Buchheit, KH,
Hofman A,
and
Fozard JR.
Salbutamol-induced airway hyperreactivity in guinea pigs is not due to a loss of its bronchodilator effects.
Eur J Pharmacol
287:
85-88,
1995[ISI][Medline].
12.
Carlin, S,
Poronnik P,
Coock DI,
Carpenter L,
Biden TJ,
Johnson PRA,
and
Black JL.
An antisense of protein kinase C-
inhibits proliferation of human airway smooth muscle cells.
Am J Respir Cell Mol Biol
23:
555-559,
2000[Abstract/Free Full Text].
13.
Christman, JW,
Sadikot RT,
and
Blackwell TS.
The role of nuclear factor-
B in pulmonary diseases.
Chest
117:
1482-1487,
2000[Abstract/Free Full Text].
14.
Crane, J,
Pearce N,
Flatt A,
Burgess C,
Jackson R,
Kwong T,
Ball M,
and
Beasley R.
Prescribed fenoterol and death from asthma in New Zealand, 1981-83: case control study.
Lancet
8644:
917-922,
1989.
15.
Davis, RJ.
MAPKs: new JNK expands the group.
Trends Biochem Sci
19:
470-473,
1994[ISI][Medline].
16.
Eickelberg, O,
Roth M,
Lörx R,
Bruce V,
Rüdiger J,
Johnson M,
and
Block LH.
Ligand-independent activation of the glucocorticoid receptor by
2-adrenergic receptor agonists in primary human lung fibroblasts and vascular smooth muscle cells.
J Biol Chem
274:
1005-1010,
1999[Abstract/Free Full Text].
17.
Fernandes, LB,
Henry PJ,
and
Goldie RG.
Endothelin-1 potentiates cholinergic nerve-mediated contraction in human isolated bronchus.
Eur Respir J
14:
439-442,
1999[Abstract/Free Full Text].
18.
Flanagan, RJ.
Guidelines for the interpretation of analytical toxicology results and unit of measurement conversion factor.
Ann Clin Biochem
35:
261-267,
1998[ISI][Medline].
19.
Frossard, N,
Naline E,
Olgart C,
Mathieu E,
Edmonds-Alt X,
and
Advenier C.
Increased production of NGF by IL-1
in the human bronchus in vitro is related to bronchial hyperresponsiveness. Effects of SR 142801 (Abstract).
Am J Respir Crit Care Med
163:
A55,
2001.
20.
Gao, Y,
Tang S,
Zhou S,
and
Ware JA.
The thromboxane A2 receptor activates mitogen-activated protein kinase via protein kinase C-dependent Gi coupling and Src-dependent phosphorylation of the epidermal growth factor receptor.
J Pharmacol Exp Ther
296:
426-433,
2001[Abstract/Free Full Text].
21.
Goldie, RG.
Endothelin receptor subtypes: distribution and function in the lung.
Pulm Pharmacol Ther
11:
89-95,
1998[ISI][Medline].
22.
Goldie, RG.
Endothelins in health and disease: an overview.
Clin Exp Pharmacol Physiol
26:
145-148,
1999[ISI][Medline].
23.
Haddad, EB.
Regulation of airway bradykinin B1 and B2 receptor expression and function by inflammatory insults.
Eur Respir J
10:
276-279,
2000.
24.
Hallsworth, MP,
Moir LM,
Lai D,
and
Hirst SJ.
Inhibitors of mitogen-activated protein kinases differentially regulate eosinophil-activating cytokine release from human airway smooth muscle.
Am J Respir Crit Care Med
164:
688-697,
2001[Abstract/Free Full Text].
25.
Hay, DW.
Endothelins.
In: Airways Smooth Muscle: Peptides Receptors, Ions Channels and Signal Transduction. Basel: Birkhäuser Verlag, 1995, p. 1-50.
26.
Hay, DW.
Endothelin-1: an interesting peptide or an important mediator in pulmonary diseases?
Pulm Pharmacol Ther
11:
141-146,
1998[ISI][Medline].
27.
Hay, DW.
Putative mediator role of endothelin-1 in asthma and other lung diseases.
Clin Exp Pharmacol Physiol
26:
168-171,
1999[ISI][Medline].
28.
Hay, DW,
Thorphy TJ,
and
Undem BJ.
Cysteinyl leukotrienes in asthma: old mediators up to new tricks.
Trends Pharmacol Sci
16:
304-309,
1995[ISI][Medline].
29.
Hayashi, R,
Yamashita N,
Matsui S,
Fujita T,
Araya J,
Kassa K,
Arai N,
Yoshida Y,
Kashii T,
Maruyama M,
Sugiyama E,
and
Kobayashi M.
Bradykinin stimulates IL-6 and IL-8 production by human lung fibroblasts through ERK- and p38 MAPK-dependent mechanisms.
Eur Respir J
16:
452-458,
2000[Abstract/Free Full Text].
30.
Hedges, JC,
Yamboliev IA,
Ngo M,
Horowitz B,
Adam LP,
and
Gerthoffer WT.
p38 Mitogen-activated protein kinase expression and activation in smooth muscle.
Am J Physiol Cell Physiol
275:
C527-C534,
1998[Abstract/Free Full Text].
31.
Henry, PJ.
Endothelin receptor distribution and function in the airways.
Clin Exp Pharmacol Physiol
26:
162-167,
1999[ISI][Medline].
32.
Hirst, SJ,
Walker TR,
and
Chilvers ER.
Phenotypic diversity and molecular mechanisms of airway smooth muscle proliferation in asthma.
Eur Respir J
16:
159-177,
2000[Abstract/Free Full Text].
33.
Jaffuel, D,
Demoly P,
Gougat C,
Balaguer P,
Mautino G,
Godard P,
Bousquet J,
and
Mathieu M.
Transcriptional potencies of inhaled glucocorticoids.
Am J Respir Crit Care Med
162:
57-63,
2000[Abstract/Free Full Text].
34.
Jarpe, MB,
Knall C,
Mitchell FM,
Buhl AM,
Duzic E,
and
Johnson GL.
[D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P acts as a biased agonist toward neuropeptide and chemokine receptors.
J Biol Chem
273:
3097-3104,
1998[Abstract/Free Full Text].
35.
Katsunuma, T,
Fujita K,
Mak JC,
Barnes PJ,
Ueno K,
and
Likura Y.
-Adrenergic agonists and bronchial hyperreactivity: role of
2-adrenergic and tachykinin neurokinin-2 receptors.
J Allergy Clin Immunol
106:
S104-S108,
2000[ISI][Medline].
36.
Katsunuma, T,
Roffel AF,
Elzinga CR,
Zaagsma J,
Barnes PJ,
and
Mak JC.
2-Adrenoceptor agonist-induced upregulation of tachykinin NK2 receptor expression and function in airway smooth muscle.
Am J Respir Cell Mol Biol
21:
409-417,
1999[Abstract/Free Full Text].
37.
Kips, JC,
and
Pauwels RA.
Long-acting inhaled
2-agonist therapy in asthma.
Am J Respir Crit Care Med
164:
923-932,
2001[Free Full Text].
38.
Koch, A,
Lindsay MA,
Barnes PJ,
and
Giembycz MA.
2-Adrenoreceptor agonist induced dephosphorylation of extracellular signal regulated kinases (p44ERK1 and p42ERK2) in airway smooth muscle (Abstract).
Am J Respir Crit Care Med
161:
A450,
2000.
39.
Korn, HS,
Jerre A,
and
Brattsand R.
Effect of formoterol and budesonid on GM-CSF and IL-8 secretion by triggered human bronchial epithelial cells.
Eur Respir J
17:
1070-1077,
2001[Abstract/Free Full Text].
40.
Lecaillon, JB,
Kaiser G,
Palmisano M,
Morgan J,
and
Della Cioppa G.
Pharmacokinetics and tolerability of formoterol in healthy volunteers after a single high dose of Foradil dry powder inhalation via aerosolizer.
Eur J Clin Pharmacol
55:
131-138,
1999[ISI][Medline].
41.
Michaël, JR,
and
Markewitz BA.
Endothelins and the lung.
Am J Respir Crit Care Med
154:
555-581,
1996[ISI][Medline].
42.
Morley, L,
Sanjar S,
and
Newth C.
Viewpoint: untoward effects of
-adrenoreceptor agonists in asthma.
Eur Respir J
3:
226-233,
1990.
43.
Myers, A,
and
Undem BJ.
Electrophysiological effects of tachykinins and capsaicin on guinea-pig parasympathetic ganglion neurons.
J Physiol
470:
665-679,
1993[Abstract].
44.
Naline, E,
Bertrand C,
Biyah K,
Fujitani Y,
Okada T,
Bisson A,
and
Advenier C.
Modulation of ET-1-induced contraction of human bronchi by airway epithelium-dependent nitric oxide release via ETA receptor activation.
Br J Pharmacol
126:
529-535,
1999[Abstract/Free Full Text].
45.
Naline, E,
Zhang Y,
Qian Y,
Mairon N,
Anderson GP,
Grandordy B,
and
Advenier C.
Relaxant effects and duration of action of formoterol and salmeterol on the isolated bronchus.
Eur Respir J
7:
914-920,
1994[Abstract/Free Full Text].
46.
Panettieri, RA.
Cellular and molecular mechanisms regulating airway smooth muscle proliferation and cell adhesion molecule expression.
Am J Respir Crit Care Med
158:
S133-S140,
1998[Abstract/Free Full Text].
47.
Peters, MJ,
Adcock IM,
Brown CR,
and
Barnes PJ.
-Adrenoceptor agonists interfere with glucocorticoid receptor DNA binding in rat lung.
Eur J Pharmacol
289:
275-281,
1995[Medline].
48.
Peters-Golden, M,
and
Brock TG.
Intracellular compartmentalization of leukotriene biosynthesis.
Am J Respir Crit Care Med
161:
S36-S40,
2000[Free Full Text].
49.
Ricciardolo, FL,
Nadel JA,
Graf PD,
Bertrand C,
Yoshihara S,
and
Geppetti P.
Role of kinins in anaphylactic-induced bronchoconstriction mediated by tachykinins in guinea-pigs.
Br J Pharmacol
113:
508-512,
1994[Abstract].
50.
Roland, M,
Bhowmick A,
Sapsford RJ,
Seemungal TA,
Jeffries DJ,
Warner TD,
and
Wedzicha JA.
Sputum and plasma endothelin-1 levels in exacerbations of chronic obstructive pulmonary disease.
Thorax
56:
30-35,
2001[Abstract/Free Full Text].
51.
Salmeron, S,
Brochard L,
Mal H,
Tenaillon A,
Henry-Amar M,
Renon D,
Duroux P,
and
Simonneau G.
Nebulized vs. intravenous albuterol in hypercapnic acute asthma. A multicenter, double-blind, randomized study.
Am J Respir Crit Care Med
149:
1466-1470,
1994[Abstract].
52.
Saulnier, JP,
Naline E,
Edmonds-Alt X,
and
Advenier C.
Transcription factor NF-
B is involved in fenoterol-induced hyperresponsiveness to neurokinin A on guinea pig trachea. Effects of tachykinin NK1, NK2 and NK3 receptor antagonists (Abstract).
Am J Respir Crit Care Med
161:
A437,
2000.
53.
Schmidlin, F,
Landry Y,
and
Gies JP.
Regulation of bradykinin B2 receptor expression.
Eur Respir Rev
10:
274-275,
2000.
54.
Schmidt, D,
and
Rabe KL.
The role of leukotrienes in the regulation of tone and responsiveness in isolated human airways.
Am J Respir Crit Care Med
161:
S62-S67,
2000[Free Full Text].
55.
Sears, MR.
Deleterious effects of inhaled
-agonists.
Chest
119:
1297-1299,
2001[Free Full Text].
56.
Taylor, DR,
Drazen JM,
Herbison GP,
Yandava CN,
Hancox RJ,
and
Town GI.
Asthma exacerbations during long-term
-agonist use: influence of
2-adrenoceptor polymorphism.
Thorax
55:
762-776,
2000[Abstract/Free Full Text].
57.
Touqui, L.
Regulation of pulmonary synthesis of type II secretory phospholipase A2 and potential role in acute lung inflammation.
Eur Respir Rev
10:
286-288,
2000.
58.
Underwood, DC,
Osborn RR,
Kotzer CJ,
Adams JL,
Lee JC,
Webb EF,
Carpenter DC,
Bochnowicz S,
Thomas HC,
Hay DW,
and
Griswold DE.
SB 239063, a potent p38 MAP kinase inhibitor, reduces inflammatory cytokine production, airways eosinophil infiltration, and persistence.
J Pharmacol Exp Ther
293:
281-288,
2000[Abstract/Free Full Text].
59.
Van Schayck, CP,
Graasma SJ,
Visch MB,
Dompelling E,
Van Weel C,
and
Van Herwaarden CL.
Increased bronchial hyperresponsiveness after inhaling salbutamol during one year is not caused by subsensitization to salbutamol.
J Allergy Clin Immunol
86:
783-790,
1990.
60.
Wang, ZL,
Bramley AM,
McNamara A,
Paré PD,
and
Bai TR.
Chronic fenoterol exposure increases in vivo and in vitro airway responses in guinea pigs.
Am J Respir Crit Care Med
149:
960-965,
1994[Abstract].
61.
Webb, BLJ,
Hirst SJ,
and
Giembycz MA.
Protein kinase C isoenzymes: a review of their structure, regulation and role in airways smooth muscle tone and mitogenesis.
Br J Pharmacol
130:
1433-1452,
2000[Abstract/Free Full Text].
62.
Wilson, SJ,
Wallin A,
Della-Cioppa G,
Sandströn T,
and
Holgate ST.
Effects of budesonide and formoterol on NF-
B, adhesion molecules, and cytokines in asthma.
Am J Respir Crit Care Med
164:
1047-1052,
2001[Abstract/Free Full Text].
Am J Physiol Lung Cell Mol Physiol 283(5):L1033-L1042
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