Cattedra di Fisiopatologia Respiratoria, Dipartimento di Scienze Motorie e Riabilitative, and Cattedra di Biochimica, Dipartimento di Medicina Sperimentale, Università di Genova, 16132 Genoa, Italy
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ABSTRACT |
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We studied the
intracellular mechanisms of allergen-induced
2-adrenoceptor dysfunction in human isolated passively
sensitized bronchi. Sensitization was obtained by overnight incubation
of bronchial rings with serum containing a high specific IgE level to
Dermatophagoides but a low total IgE level. Allergen
challenge was done by incubation with a Dermatophagoides
mix. The Gs protein stimulant cholera toxin (2 µg/ml) displaced the carbachol (CCh) concentration-response curves of
control and sensitized but not of challenged rings to the right.
Cholera toxin (10 µg/ml) displaced the concentration-response curves
to CCh of control, sensitized, and challenged rings to the right, but
this effect was less in challenged rings. The effects of the
Gi protein inhibitor pertussis toxin (250 ng/ml or 1 µg/ml) on salbutamol concentration-relaxation curves did not differ
significantly between challenged and sensitized rings. The adenylyl
cyclase activator forskolin and the Ca2+-activated
K+-channel opener NS-1619 relaxed CCh-contracted bronchial
rings without significant differences between control, sensitized, and challenged rings. Neither Gi nor Gs
-subunit
expression differed between control, sensitized, and challenged
tissues. We conclude that Gs protein dysfunction may be a
mechanism of allergen-induced
2-adrenoceptor dysfunction
in human isolated passively sensitized bronchi.
airway smooth muscle; calcium-activated potassium channels; adenylyl cyclase; Gi protein; -adrenergic receptors
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INTRODUCTION |
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DECREASED
AIRWAY RELAXATION to -adrenoceptor stimulation has been
proposed as a potential mechanism of airway hyperresponsiveness in
asthma (6, 19). This hypothesis is supported
by the demonstration of impaired
-adrenoceptor function in isolated
bronchi from patients with fatal asthma (1) or from
antigen-sensitized animals (20, 22).
Recently, it has been shown that
2-adrenoceptor
dysfunction can also be induced by allergen challenge of human
passively sensitized isolated bronchi (18). The
dysfunction seems to be related to the release of peptido-leukotrienes
(17), but the intracellular mechanisms involved have not
been elucidated.
The relaxant effect of -adrenoceptor stimulation is mediated through
a series of intracellular events, including receptor-effector coupling
by activation of the stimulating guanine nucleotide regulating protein
(Gs protein), increase in cAMP by activation of adenylyl cyclase, and cell membrane hyperpolarization by opening of
Ca2+-activated K+
(KCa) channels. Moreover, the response to
-adrenoceptor stimulation is negatively affected by M2
receptor or Gi protein signaling.
The aim of the study was to investigate which mechanisms are affected by allergen challenge of human isolated passively sensitized bronchial rings. The functions of Gs protein, Gi protein, KCa channel, and adenylyl cyclase were compared between control, sensitized, and allergen-challenged rings. The expression of Gs protein and Gi protein in the three conditions was determined by Western blot analysis.
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METHODS |
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Tissue Preparation
Materials. Bronchi were obtained from 35 patients who underwent lobectomies for resection of lung cancer. After tissue for microscopic examination had been removed, the bronchi were immersed in chilled (4°C) and aerated (95% O2-5% CO2) physiological salt solution (PSS) and transported to the laboratory. The PSS had the following composition (in mM): 110.5 NaCl, 3.4 KCl, 2.4 CaCl2, 0.8 MgSO4, 1.2 KH2PO4, 25.7 NaHCO3, and 5.6 dextrose. Within 4 h after resection, bronchi (3- to 5-mm ID) were dissected from the surrounding tissue and cut into rings. Sera from asthmatic patients with concentrations of specific IgE > 17.5 Phadebas radioallergosorbent text (RAST) units/ml (4th RAST class; Pharmacia, Uppsala, Sweden) to Dermatophagoides pteronyssinus and D. farinae were obtained; the concentration of total IgE in the serum was 220 IU/ml. The serum was diluted 1:10 with PSS before use as a sensitizing serum.
Passive sensitization. Aerated bronchial rings were incubated with the diluted sensitizing serum for 18 h at room temperature. These rings are referred to as sensitized rings.
Paired rings from each patient were treated identically with sera from nonallergic subjects (paper radioimmunosorbent test < 20 IU/ml and negative RAST for D. pteronyssinus and D. farinae). These rings are referred to as control rings.Allergen challenge. Sensitized rings from each patient were washed for 15 min with PSS and then incubated for 60 min with 5 ml of aerated (37°C) PSS containing 200 arbitrary units/ml of Dermatophagoides mix (Laboratorio Farmaceutico Lofarma, Milan, Italy). These rings are referred to as challenged rings.
Verification of passive sensitization and exclusion of natural sensitization. Passive sensitization and absence of natural sensitization was verified by incubating sensitized and control rings with 1,000 arbitrary units of Dermatophagoides mix in 25 ml of the tissue bath for 1 h. Successful passive sensitization was assumed if the allergen challenge resulted in a contractile response > 0.5 g. Absence of natural sensitization was assumed if control rings did not contract after allergen challenge.
Functional Studies
Bronchial rings were suspended between two stirrups in water-jacketed 25-ml tissue baths containing aerated PSS at 37°C. The lower stirrup was connected via a silk string to a fixed hook at the bottom of the tissue bath. The upper stirrup was connected via a silk string to a force transducer (model FT 03D, Grass Medical Instruments, Quincy, MA) mounted on a micromanipulator. Isometric forces were continuously recorded (model TA 4000, Gould, Valley View, OH). All rings were equilibrated for 2 h with PSS and washed every 20 min with PSS. During this time, the rings were gradually stretched to a resting force of 1 g. The length of the rings was not altered during the experiments.Effect of cholera toxin on carbachol concentration-response
curves.
One control, one sensitized, and one challenged ring from each of nine
patients were incubated with 109 M carbachol (CCh), and
the contractile responses were recorded. After a steady contractile
response was achieved, the CCh concentration was cumulatively increased
in half-log increments to 10
4 M, and the contractile
responses were recorded. After completion of the CCh
concentration-response curves, the rings were washed with PSS until the
resting forces had been reestablished. The rings were then incubated
with 2 (n = 6 patients) or 10 (n = 3 patients) µg/ml of cholera toxin (CTX) for
6 h and then washed with PSS. Complete sets of CCh
concentration-response curves were then obtained.
Effect of pertussis toxin on relaxant salbutamol
concentration-relaxation curves.
Two sensitized and two challenged rings from each of 10 patients were
incubated with 250 ng/ml (n = 4 patients) or 1 µg/ml (n = 6 patients) of pertussis toxin (PTX) for 4 h.
The rings were then half-maximally contracted with 106 M
CCh (18). After the contractile responses had become
stable, salbutamol was added cumulatively (10
9 to
10
4 M in half-log increments), and the relaxant responses
were recorded.
Relaxant effect of forskolin.
One control, one sensitized, and one challenged ring from each of six
patients were incubated with 106 M CCh. After the
contractile responses had become stable, forskolin was added
cumulatively (10
9 to 10
5 M in half-log
increments), and the relaxant responses were recorded.
Relaxant effect of NS-1619.
One control, one sensitized, and one challenged ring from each of six
patients were incubated with 106 M CCh. After the
contractile responses had become stable, NS-1619 was added cumulatively
(10
9 to 10
4 M in half-log increments), and
the relaxant responses were recorded.
G Protein Expression
Expression of Gi and GsDrugs
CCh (carbamylcholine chloride), CTX, PTX, salbutamol free base, forskolin, and NS-1619 {1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one} were purchased from Sigma-Aldrich (Milan, Italy). Stock solutions of forskolin and NS-1619 were prepared in absolute ethanol; other drugs were dissolved in distilled water.Data Analysis
The force developed by muscles used for relaxation studies is expressed as a percentage of the contractile response to 10Concentration-response curves were analyzed by two- or three-factor repeated-measures ANOVA with Newman-Keuls post hoc test. Bronchial ring characteristics were compared by a between-within groups mixed ANOVA. Differences were considered significant if P values were <0.05. Data are presented as means ± SD.
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RESULTS |
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Passive sensitization was demonstrated in all patients and the absence of natural sensitization in 30 of the 35 patients.
Functional Studies
Effect of CTX on CCh concentration-response curves.
The mean weight of the 27 rings was 107 ± 53 mg, the mean resting
force was 1.1 ± 0.4 g, and the maximal response to
104 M CCh was 28 ± 15 g/g (force/wet weight of
tissue). Mean weight, resting force, and maximal response were not
significantly different between control, sensitized, and challenged rings.
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Effect of PTX on salbutamol concentration-relaxation curves.
The mean weight of the 38 rings used (a pair of sensitized rings was
not included because one of the two rings did not contract in response
to CCh) was 136 ± 49 mg, the mean resting force was 1.0 ± 0.3 g, and the response to 106 M CCh was 13 ± 7 g/g (force/wet weight of tissue). Mean weight and resting force were
not significantly different between sensitized and challenged rings.
Salbutamol relaxed CCh-contracted sensitized and challenged rings in a
concentration-related manner (P < 0.001; Fig.
3). The presence of 250 ng/ml of PTX did
not alter the salbutamol concentration-relaxation curves of either
sensitized (P > 0.95) or challenged (P > 0.8) rings (Fig. 3, A and B, respectively). PTX (1 µg/ml) significantly displaced the salbutamol
concentration-relaxation curves of both sensitized (Fig. 3C)
and challenged (Fig. 3D) rings to the left
(P < 0.001), without a significant difference between the two conditions (P > 0.95).
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Relaxant effect of forskolin.
The mean weight of the 18 rings was 110 ± 36 mg, the mean resting
force was 0.9 ± 0.3 g, and the response to 106
M CCh was 17 ± 5 g/g (force/wet weight of tissue). Mean weight, resting force, and response to CCh were not significantly different between control, sensitized, and challenged rings.
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Relaxant effect of NS-1619.
The mean weight of the 18 rings was 78 ± 28 mg, the mean
resting force was 0.9 ± 0.1 g, and the response to
106 M CCh was 15 ± 5 g/g (force/wet weight of
tissue). Mean weight, resting force, and response to CCh were not
significantly different between control, sensitized, and challenged rings.
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G Protein Expression
Western blot analysis showed comparable expression of either Gi
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DISCUSSION |
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The major findings of this study are that allergen challenge of human isolated passively sensitized bronchi did not reduce the relaxant effects of either the KCa channel opener NS-1619 or the adenylyl cyclase activator forskolin. An inhibitory effect of the Gs protein stimulator CTX on CCh-induced contractile response was observed in control and sensitized rings, but this inhibitory effect was reduced in allergen-challenged rings.
Before the underlying mechanisms for 2-adrenoceptor
dysfunction are considered, the current understanding of
2-adrenoceptor activation and signal transduction is
briefly discussed. Agonist binding to
2-adrenoceptors
causes conformational changes of the receptor, allowing activation of
the
-subunit of the Gs protein. The activated
Gs
stimulates adenylyl cyclase, which converts ATP into
cAMP. The latter, in turn, activates protein kinase (PK) A and PKG,
which induce several intracellular events, resulting in the relaxation
of the smooth muscle. The activated Gs
can also directly
activate the KCa channel independent of an increase in cAMP (8). Dysfunction of the
2-adrenoceptor can occur at any level in this cascade of
events. In addition "cross talk" between muscarinic or inflammatory
mediator receptors and
2-adrenoceptors may result in
phosphorylation of both the
2-adrenoceptor itself and
the Gs protein, thus inducing
2-adrenoceptor
dysfunction (5, 9, 15,
16).
In the current study, dysfunction of 2-adrenoceptors in
airway smooth muscle was induced by allergen challenge of human
passively sensitized isolated bronchial rings (17,
18). Control, sensitized, and challenged rings were then
incubated with compounds with specific actions on the function of
either the Gs protein, the KCa channel, or adenylyl cyclase. The effects of these compounds on muscle contractions and relaxation were compared in the three conditions.
The function of the KCa channel was assessed by incubating the muscles with the KCa channel opener NS-1619 (13) and comparing the relaxation before and after incubation with NS-1619. The relaxant effect of NS-1619 on the control, sensitized, and allergen-challenged rings was not significantly different, suggesting that K+ transport through the KCa channel was not affected by allergen challenge of human isolated bronchial rings.
The effect of allergen challenge on adenylyl cyclase activity was inferred from its effect on the relaxant effect of forskolin in control, sensitized, and allergen challenged rings. If the activity of adenylyl cyclase were reduced by allergen challenge, muscle relaxation should be less after incubation with forskolin. Allergen challenge did not reduce the relaxation of the bronchial rings by forskolin, suggesting that the activity of the adenylyl cyclase and the function of the relaxation machinery were not affected. This conclusion is similar to that by Kume and Tagaki (10). These authors reported that homologous desensitization of guinea pig trachealis did not alter adenylyl cyclase activity.
Incubation of control and sensitized (but not of challenged) rings with
CTX resulted in a rightward displacement of the CCh concentration-response curves and a reduction in the maximal force. Incubation with CTX irreversibly stimulates the -subunit of the Gs protein, increasing the intracellular concentration of
cAMP and thereby inhibiting smooth muscle contraction. Allergen
challenge greatly reduced the inhibitory effects of CTX on CCh-induced
contractions, suggesting that the Gs protein was
dysfunctional. Western blot analysis showed no difference in
Gs protein expression between control, sensitized, and
challenged tissues. Therefore, the reduced response to CTX seems to be
the result of reduced Gs protein activity rather than
expression. Homologous
-adrenoceptor desensitization of isolated
guinea pig trachealis can also be prevented by incubation with CTX,
suggesting that the
-subunit of the Gs protein is
dysfunctional (10).
The present study cannot rule out that desensitization through
phosphorylation of -adrenoceptors may contribute to the reduced response to
2-agonists. Iralukast, a peptido-leukotriene
receptor antagonist prevents
2-adrenoceptor dysfunction
induced by allergen challenge (17). But the underlying
mechanisms for this effect are still unclear. Allergen challenge may
phosphorylate
2-adrenoceptors, Gs protein,
or both (5, 16). A cross talk between
2-adrenoceptors and inflammatory mediator receptors via
PKC may occur (3, 7). The PKC may
phosphorylate both
2-adrenoceptors and Gs protein.
-Adrenoceptor hyporesponsiveness may also be induced by treatment of
human isolated smooth muscle cells with interleukin-1
(12). This effect has been suggested to be mediated by the
release of prostanoids from the smooth muscle cells as a result of
cyclooxygenase-2 activation because it is prevented by indomethacin. In
human passively sensitized bronchi, indomethacin does not prevent
2-adrenoceptor dysfunction induced by allergen
challenge (17).
As in a previous study (18) on human isolated
bronchi passively sensitized with sera containing a low level of IgE,
neither sensitization nor allergen challenge altered the contractile
responses to CCh, suggesting that the capacity to generate force was
similar between the two conditions and the control condition.
Therefore, differences in response to CTX between allergen-challenged
and control or sensitized rings are not due to a difference in force generation capacity. Sensitization with sera containing high levels of
total IgE caused -adrenoceptor dysfunction (6) and
variable effects on airway smooth muscle response to contractile
agonists as the response to histamine was increased (2),
whereas the response to cholinergic agonists was either decreased
(2) or increased (6). Whether Gs
protein, adenylyl cyclase, or KCa channel function can be
altered by passive sensitization with high total IgE levels without
allergen challenge has not been determined.
The nonselective muscarinic agonist CCh was used to contract airway
smooth muscle in functional studies. Activation of M2 receptors or Gi protein signaling pathway may inhibit
adenylyl cyclase, thus causing a reduced response to -adrenoceptor
or Gs protein stimulation. Increased expression or
increased activity of Gi protein would, therefore, have
negatively affected the response to CTX. In rabbit bronchi sensitized
with human serum containing a high level of total IgE, Hakonarson et
al. (6) found an increased expression of Gi
protein and an enhanced relaxant response to isoproterenol with PTX,
suggesting an increased Gi protein function. In the present
study, with human bronchi sensitized with human serum containing a
relatively low level of total IgE, neither sensitization nor allergen
challenge increased Gi protein expression. Furthermore, the
effect of PTX on salbutamol concentration-relaxation curves was not
different between sensitized and challenged rings. It is therefore
unlikely that the present data are accounted for by increased
M2 receptor or Gi protein signaling. The
difference between the present findings and those of Hakonarson et al.
may be either species related or sensitizing serum related.
In conclusion, exposure to sensitizing allergens may cause
Gs protein dysfunction in human isolated passively
sensitized bronchi, an effect that may be due to phosphorylation of the
Gs protein by PKC. The Gs protein dysfunction
may be a contributing mechanism underlying
2-adrenoceptor dysfunction in bronchial asthma.
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ACKNOWLEDGEMENTS |
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We thank Dr. Maurizio Chiaramondia and Mauro Zampini (Department of Pathology, S. Martino Hospital, Genoa, Italy), Prof. Roberto Fiocca and Dr. Luca Mastracci (Department of Pathology, University of Genoa, Genoa, Italy), Prof. G. Catrambone (Division of Thoracic Surgery, S. Martino Hospital), and Prof. G. B. Ratto (Department of Surgery, University of Genoa) for the valuable assistance in selecting and providing tissues.
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FOOTNOTES |
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This work was supported in part by a grant from Ministero dell'Universitá della Ricerca Scientifica e Tecnologica (Rome, Italy) and the University of Genoa.
P. Song was supported by a grant from Rhöne-Poulenc Rorer (Milan, Italy).
Address for reprint requests and other correspondence: V. Brusasco, Dipartimento di Scienze Motorie e Riabilitative, Università di Genova, Largo R. Benzi, 10, 16132 Genoa, Italy (E-mail: brusasco{at}dism.unige.it).
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. §1734 solely to indicate this fact.
Received 23 November 1998; accepted in final form 8 March 2000.
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