Dipartimenti di Scienze Motorie e Riabilitative, di Medicina Interna, e di Medicina Sperimentale, Università di Genova, 16132 Genoa, Italy
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ABSTRACT |
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Bronchial rings from nonatopic humans were
passively sensitized with serum from allergic subjects. Allergen
challenge significantly reduced the relaxant effect of salbutamol on
carbachol-induced contractions, suggesting
2-adrenoceptor (
2-AR) pathway
dysfunction. Incubation of challenged rings for 3 h with 3 × 10
6 M beclomethasone dipropionate (BDP) restored the
relaxant effect, suggesting reversal of
2-AR pathway
dysfunction. Incubation with the Gs
protein-stimulating
cholera toxin attenuated contractile responses to carbachol
significantly less in challenged than in unchallenged rings. Treatment
of challenged rings with BDP resulted in an inhibitory effect of
cholera toxin that was similar to the effect in unchallenged rings.
Gs
protein expression was not significantly altered by
BDP, suggesting that the activity of Gs
protein was increased. Relaxation of challenged rings by forskolin was not significantly affected by BDP, suggesting that
2-AR
pathway dysfunction was proximal to the adenylyl cyclase. In
conclusion, short-term (3-h) treatment with BDP after allergen
challenge ablated
2-AR pathway dysfunction by increasing
the activity of the Gs
protein in human isolated bronchi.
asthma; cholera toxin; corticosteroids; Gs protein; passive sensitization
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INTRODUCTION |
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CORTICOSTEROIDS
ARE HIGHLY effective in the control of asthma, because they
inhibit expression of proinflammatory cytokines, thus blocking influx
and activation of inflammatory cells (2). In addition,
corticosteroids upregulate -adrenoceptor (
-AR) function by
promoting gene transcription and receptor expression in lung tissues
and cells (5, 11, 12). In vitro, within 12-24 h,
-AR downregulation induced by
-agonists is reversed by corticoids
(18). Administration of corticosteroids parenterally (16) or by inhalation (23) improves lung
function in patients with acute asthma and restores
-AR sensitivity
(6, 18, 31). Importantly, this occurs faster than the time
required for the gene transcriptional process.
Studies from this laboratory have shown that allergen challenge of
passively sensitized human isolated bronchi caused -AR pathway
dysfunction (29). This dysfunction could be prevented by a
leukotriene receptor antagonist (27) and was associated with a reduced activity, but not expression, of the receptor-coupled Gs
protein (stimulatory guanine nucleotide-binding
protein) (28), suggesting that mechanisms other than
downregulation were involved.
The aim of the present study was to investigate whether beclomethasone
dipropionate (BDP), a steroid widely used in the treatment of asthma,
can rapidly (within 3 h) restore 2-adrenoceptor
(
2-AR) pathway function in passively sensitized and
allergen-challenged human isolated bronchi. The activity and expression
of Gs
protein and the activity of adenylyl cyclase were
determined to investigate possible effects of BDP at these levels of
the
2-AR pathway.
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METHODS |
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Tissue Preparation
Bronchi were obtained from 18 nonasthmatic male patients (50-70 yr) undergoing thoracotomy for lung cancer. None of the patients had receivedPassive Sensitization
An atopic serum was obtained by pooling sera from asthmatic subjects with high concentrations of specific IgEs (>17.5 radio-allergo-sorbent test units/ml; Pharmacia, Uppsala, Sweden) against Dermatophagoides pteronyssinus and Dermatophagoides farinae and low total IgE concentrations (215 ± 34 IU/ml by paper radioimmunosorbent test). Bronchial rings were incubated overnight for 18 h with 1 ml of the atopic serum diluted in 9 ml of PSS, while they were continuously aerated at room temperature with 95% O2-5% CO2.Allergen Challenge
After repeated washes with PSS, sensitized rings were incubated for 60 min at 37°C with 5 ml of aerated PSS containing 200 arbitrary units of Dermatophagoides mix (challenged rings) or with PSS only (unchallenged rings).Isometric Force Measurements
Bronchial rings were placed in water-jacketed 25-ml tissue baths containing aerated PSS at 37°C. They were connected via a stirrup to a stationary hook at the bottom and via a silk string at the top of a force transducer (model FT03D, Grass Medical Instruments, Quincy, MA). The signal from the force transducer was continuously recorded (model TA 4000, Gould, Valley View, OH). The rings were allowed to equilibrate for 2 h, while they were washed every 20 min and progressively stretched to a resting force of 1 g (29). This length (i.e., the optimal length) was maintained throughout the studies.Verification of Passive Sensitization
At the end of experiments, one sensitized ring from each of 14 patients was incubated for 60 min with 25 ml of aerated PSS containing 1,000 arbitrary units of Dermatophagoides mix. Passive sensitization was considered successful if >0.5 g of contractile force was recorded.Response to Salbutamol
One unchallenged and one challenged ring from each of six patients was treated for 3 h with 3 × 10Gs Protein Function and Expression
The expression of Gs subunit was assayed by Western blot
analysis in eight challenged bronchial fragments from four separate patients. Paired muscles were incubated only with DMSO or with BDP for
3 h at 37°C in aerated (95% O2-5% CO2)
PSS. Aliquots of tissue derived from the bronchial fragments were
suspended in 20 mM Tris · HCl-buffered solution
(pH 7.4) of the following composition: 140 mM NaCl, 2.5 mM EDTA, 2.5 mM
EGTA, 1 mM phenylmethylsulfonyl fluoride, and 0.1 mg/ml leupeptin.
Tissues were homogenized in an ice bath, and cells were lysed by
sonication (6 bursts of 10-s duration each) at 0°C. After
centrifugation at 12,000 g for 5 min, supernatants of
samples were collected and suspended in SDS-PAGE loading buffer.
Samples were immediately heated to 95°C for 4 min and submitted to
SDS-PAGE on a 10% (wt/vol) slab gel (21) and then
electroblotted onto a pure nitrocellulose membrane (Amersham Pharmacia
Biotech, Milan, Italy). Gs
-olf protein was
identified by using the specific anti-Gs
-olf antibody
(C18) rabbit polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA).
Membranes were then probed with a peroxidase-conjugated secondary
antibody (Amersham Pharmacia Biotech) (14) and developed
with an enhanced chemiluminescence detection system (Amersham Pharmacia
Biotech). Blots were quantified by scanning analysis with a
densitometer (model CS-9000, Shimadzu, Kyoto, Japan). The relative
amount of each immunoreactive band was calculated by determining the
areas of the densitometric peaks in square millimeters.
Adenylyl Cyclase Activity
One challenged ring from each of four patients was incubated with 3 × 10Gi Protein Function
Three nonsensitized, unchallenged bronchial rings from each of two patients were used. Two rings were incubated with 1 µg/ml pertussis toxin (PTX) for 4 h and one with PSS but no PTX. After 1 h of incubation, 3 × 10Data Analysis
Isometric forces developed by bronchial rings used for salbutamol and forskolin relaxation studies are expressed as percentage of contractile responses 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 to be statistically significantly different at P < 0.05. Values are means ± SD.
Drugs
Salbutamol free base, carbachol (carbamylcholine chloride), CTX, forskolin, and PTX were purchased from Sigma-Aldrich (Milan, Italy). D. pteronyssinus and D. farinae were purchased from Laboratorio Farmaceutico Lofarma (Milan, Italy). BDP was generously provided by Chiesi Farmaceutici (Parma, Italy). BDP was dissolved in DMSO and forskolin in absolute ethanol. All other drugs were dissolved in distilled water. ![]() |
RESULTS |
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Successful sensitization was demonstrated in bronchial rings from 14 patients; in 2 patients used for the salbutamol study, verification was impossible because of insufficient amounts of tissue.
The mean weight of 55 bronchial rings from 18 patients (rings used for
verification of sensitization not included) was 101 ± 37 mg, and
the mean resting force was 0.9 ± 0.4 g. Mean weights and
resting forces were not significantly different between experimental groups (P = 0.4 and P = 0.4, respectively). There were no significant differences (P = 0.6) in contractile forces induced by 106 M carbachol
between the experimental groups.
Response to Salbutamol
Salbutamol relaxed all rings in a concentration-dependent manner (P < 0.001; Fig. 1). At 10
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The mean IC50 values were significantly (P < 0.05) greater for non-BDP-treated challenged rings than for
non-BDP-treated unchallenged, BDP-treated unchallenged, and BDP-treated
challenged rings (Table 1).
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Gs Protein Function and Expression
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Expression of Gs protein was not significantly different
between non-BDP-treated challenged rings and BDP-treated challenged rings (Table 2).
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Adenylyl Cyclase Activity
Forskolin relaxed non-BDP-treated and BDP-treated challenged rings in a concentration-related manner (P < 0.001). There was no significant difference (P = 0.3) between the two groups (Fig. 3).
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Gi Protein Function
In nonsensitized unchallenged rings, incubation with PTX at 1 µg/ml enhanced the relaxant effect of salbutamol, with no difference between BDP-treated and non-BDP-treated rings (Fig. 4).
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DISCUSSION |
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This study confirms the attenuated responses to
2-AR and Gs
protein stimulation in
passively sensitized and allergen-challenged human isolated bronchi
(28). The new major finding is that 3 h of incubation
with BDP increased the responses of challenged rings to
2-AR or Gs
protein stimulation. This
finding suggests that BDP rapidly restored the
2-AR
pathway function, and this effect involved Gs
protein.
Comments on Methodology
Isolated bronchi were passively sensitized by incubation with human serum containing low levels of total IgEs but high levels of specific IgEs (27-29). The method of sensitization used in this study does not alter contractile responses of human isolated airway smooth muscles, indicating that differences in contractions are not due to the force-generating capacity of the muscles. For this reason, unchallenged rings were used as control rings for the CTX studies.The relaxant effects of salbutamol and forskolin were studied by
precontracting bronchial rings with 106 M carbachol
(Figs. 1 and 3). Normalization to 10
6 M carbachol was
justified, because BDP, sensitization, and challenge with allergen did
not alter the force-generating capacity of the muscles.
The selective 2-AR agonist salbutamol and CTX activate
the
2-AR pathway in any cell type, including those in
prejunctional parasympathetic nerve endings. It is unlikely, however,
that the results of this study reflect prejunctional effects, because
it has been suggested that functional prejunctional
-ARs are not present on parasympathetic nerve endings of human airways
(4).
A 3-h incubation with BDP was chosen, because 2-AR
function after homologous desensitization is restored in vivo within
this time frame (6, 17, 31). The long-term effect of BDP
was not the subject of interest of this study. Although the restored effects of salbutamol and CTX suggest an effect of BDP on
Gs
protein function, definite proof of Gs
protein dysfunction in challenged rings and the effect of BDP on
Gs
protein function requires direct measure(s) of
Gs
protein activation. Finally, the results of this
study should be carefully extrapolated to in vivo conditions, inasmuch
as only cells in the bronchial wall are involved and the BDP
concentrations achievable by inhalation in the airways are uncertain.
These limitations do not invalidate the conclusions of the study.
Comments on Results
Hyporesponsiveness ofThe results of this study suggest that BDP, a corticosteroid widely
used in asthma therapy, rapidly antagonized 2-AR
pathway dysfunction induced by allergen challenge of human isolated
sensitized bronchial rings.
Response to salbutamol.
Stimulation of 2-ARs by the selective
2-AR agonist salbutamol relaxed all bronchial rings
precontracted with carbachol. However, the relaxation was significantly
attenuated in non-BDP-treated allergen-challenged rings, suggesting
2-AR pathway dysfunction (27-29).
Dysfunction of the
2-AR pathway may be caused by an
attenuated expression and/or function of the
2-AR, an
altered intracellular signal transmission between receptor and effector.
Gs protein function and expression.
CTX catalyzes ADP-ribosylation of the
-subunit of CTX-sensitive G
proteins, which irreversibly activates the
-subunit. This activation
results in increased intracellular cAMP concentration, reduced
intracellular calcium concentration, and reduced contractile response.
These effects are the underlying mechanisms for the attenuated
contractile responses to carbachol in the presence of CTX (Fig. 2). The
greater contractile response to carbachol of non-BDP-treated
CTX-incubated allergen-challenged rings (Fig. 2B) is
consistent with a dysfunction of Gs
protein.
Adenylyl cyclase activity.
Forskolin stimulates the activity of adenylyl cyclase, thus increasing
cAMP concentration and relaxing smooth muscle (25). BDP
did not alter the relaxant effect of forskolin in
carbachol-precontracted muscles (Fig. 3), suggesting that the effect of
BDP on the restoration of the 2-AR signal transmission
pathway was proximal to adenylyl cyclase. Consistent with this
conclusion is the report that the response to forskolin is not reduced
in allergen-challenged human isolated passively sensitized bronchi
(28). Also the results of studies using other models of
2-AR hyporesponsiveness (19) are consistent
with this conclusion.
Possible underlying mechanisms.
Corticosteroids may interfere with 2-AR pathway function
by promoting gene transcription, which regulates expression of
2-AR and Gs
protein. However, gene
transcription is unlikely the underlying mechanism for the results of
this study, because it has not been observed within 3 h
(13) and BDP did not increase Gs
protein expression. Second, interleukin-1
(IL-1
) may interfere with coupling of Gs
protein to
2-ARs and,
thus, activation of adenylyl cyclase (8, 26).
Corticosteroids may ablate this effect by decreasing the stability of
mRNA for IL-1
(15). The effect of corticosteroids on
IL-1
seems to be mediated by inhibition of prostanoid formation
(19). Blockade of prostanoid formation by inhibition of
cyclooxygenase does not prevent
2-AR dysfunction (27), suggesting that this mechanism cannot explain our
results. In addition, no detectable levels of IL-1
were found in the
majority of supernatants after 2 h of allergen challenge of
sensitized human airway tissues (10). Furthermore,
corticosteroids were administered before and not after
2-AR dysfunction (13, 19). Finally,
corticosteroids can modulate Na+-K+
pump-mediated relaxation (24). Again, these mechanisms
cannot explain the present data, because relaxation of
unchallenged rings was not affected by incubation with BDP.
Conclusions.
The results of the present study in human isolated sensitized bronchi
demonstrate that BDP rapidly reversed the dysfunction of the
intracellular 2-adrenergic signal transmission
pathway induced by allergen challenge. This effect does not appear
to be dependent on gene transcription. Restoration of the intracellular
2-AR effector pathway may contribute to the efficacy of
corticosteroids in the acute treatment of asthma. Elucidation of the
underlying mechanisms for the effect of BDP on Gs
protein awaits further studies.
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ACKNOWLEDGEMENTS |
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The authors are grateful to Dr. M. Chiaramondia and M. Zampini (Dept. of Pathology, S. Martino Hospital, Genoa, Italy), Prof. R. Fiocca and Dr. L. Mastracci (Dept. of Pathology, University of Genoa), Prof. G. Catrambone (Div. of Thoracic Surgery, S. Martino Hospital), and Prof. G. B. Ratto (Div. of Thoracic Surgery, S. Croce and Carle Hospital, Cuneo, Italy) for valuable assistance in selecting and providing tissues and S. Jemina for technical assistance.
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FOOTNOTES |
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This study was supported in part by a grant from Ministero dell' Università e della Ricerca Scientifica e Tecnologica (Rome, Italy). L. Brichetto was a Ph.D. student of Immunopathology at Università dell'Insubria (Varese, Italy). P. Song was supported by Chiesi Farmaceutici (Parma, Italy).
Address for reprint requests and other correspondence: V. Brusasco, Dipartimento di Medicina Interna, Università di Genova, Viale Benedetto XV, 6, 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. Section 1734 solely to indicate this fact.
August 16, 2002;10.1152/ajplung.00217.2002
Received 8 July 2002; accepted in final form 14 August 2002.
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