Beclomethasone rapidly ablates allergen-induced beta 2- adrenoceptor pathway dysfunction in human isolated bronchi

Lorenzo Brichetto, Manlio Milanese, Pingfang Song, Mauro Patrone, Emanuele Crimi, Kai Rehder, and Vito Brusasco

Dipartimenti di Scienze Motorie e Riabilitative, di Medicina Interna, e di Medicina Sperimentale, Università di Genova, 16132 Genoa, Italy


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta 2-adrenoceptor (beta 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 beta 2-AR pathway dysfunction. Incubation with the Gsalpha 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. Gsalpha protein expression was not significantly altered by BDP, suggesting that the activity of Gsalpha protein was increased. Relaxation of challenged rings by forskolin was not significantly affected by BDP, suggesting that beta 2-AR pathway dysfunction was proximal to the adenylyl cyclase. In conclusion, short-term (3-h) treatment with BDP after allergen challenge ablated beta 2-AR pathway dysfunction by increasing the activity of the Gsalpha protein in human isolated bronchi.

asthma; cholera toxin; corticosteroids; Gsalpha protein; passive sensitization


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -adrenoceptor (beta -AR) function by promoting gene transcription and receptor expression in lung tissues and cells (5, 11, 12). In vitro, within 12-24 h, beta -AR downregulation induced by beta -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 beta -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 beta -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 Gsalpha 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 beta 2-adrenoceptor (beta 2-AR) pathway function in passively sensitized and allergen-challenged human isolated bronchi. The activity and expression of Gsalpha protein and the activity of adenylyl cyclase were determined to investigate possible effects of BDP at these levels of the beta 2-AR pathway.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue Preparation

Bronchi were obtained from 18 nonasthmatic male patients (50-70 yr) undergoing thoracotomy for lung cancer. None of the patients had received beta 2-agonists, theophylline, or anticholinergic drugs before or during surgery. After removal of tissue for pathological examination, bronchi were obtained from a site as far as possible from the malignancy. They were immersed in cold (4°C) and aerated (95% O2-5% CO2) physiological salt solution (PSS) of 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. Immediately after arrival in the laboratory, bronchial rings (3-5 mm ID, 4-5 mm long) were prepared, with care taken to avoid epithelial damage.

Passive 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 × 10-6 M BDP. Two other paired rings from the same six patients were incubated with PSS containing only the solvent for BDP, dimethyl sulfoxide (DMSO). In one patient, an unchallenged ring was not available. After 3 h of incubation, all rings were contracted with 10-6 M carbachol, and, after steady contractions had been achieved, salbutamol was added cumulatively (10-9-10-4 M, in half-log increments).

Gsalpha Protein Function and Expression

Two unchallenged and two challenged rings from each of six patients were incubated with 10-9 M carbachol. After a steady state had been achieved, the carbachol concentration was cumulatively increased to 10-4 M (in half-log increments). Thereafter, all rings were washed with PSS until resting force was reestablished. One challenged ring from each patient was incubated with 3 × 10-6 M BDP and 10 µg/ml cholera toxin (CTX) for 3 h. Two rings from each patient, one unchallenged and one challenged, were incubated for 3 h with 10 µg/ml CTX and no BDP. Second complete sets of carbachol concentration-response curves were then obtained for all rings. To correct for the effect of time, one unchallenged ring was incubated with PSS (30).

The expression of Gsalpha 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). Gsalpha -olf protein was identified by using the specific anti-Gsalpha -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 × 10-6 M BDP for 3 h, and another challenged ring was incubated with the solvent DMSO only. All rings were then contracted with 10-6 M carbachol. After the contractile responses stabilized, forskolin was added cumulatively (10-9-10-5 M, in half-log increments).

Gi 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 × 10-6 M BDP was added to the bath containing one of the rings incubated with PTX. At the end of the 4-h incubation, all rings were contracted with 10-6 M carbachol, and, after steady contractions had been achieved, salbutamol was added cumulatively (10-9-10-4 M, in half-log increments).

Data Analysis

Isometric forces developed by bronchial rings used for salbutamol and forskolin relaxation studies are expressed as percentage of contractile responses to 10-6 M carbachol. The time-corrected force developed from rings used for the CTX studies is expressed as percentage of contractile responses to 10-4 M carbachol. The concentrations of salbutamol inhibiting 50% of carbachol-induced contraction (IC50) were calculated by linear interpolation between the two adjacent points of the salbutamol relaxation curves.

Concentration-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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 10-6 M carbachol between the experimental groups.

Response to Salbutamol

Salbutamol relaxed all rings in a concentration-dependent manner (P < 0.001; Fig. 1). At 10-6-10-5 M salbutamol, the relaxation was significantly greater (P < 0.05) in BDP-treated challenged rings than in non-BDP-treated challenged rings. There was no significant difference in relaxation between non-BDP-treated unchallenged rings and BDP-treated challenged rings (P = 0.4).


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Fig. 1.   Relaxant effect of salbutamol in human isolated bronchial rings. Contraction is expressed as percentage of contraction with 10-6 M carbachol (CCh). Horizontal line, force before carbachol. Negative values represent relaxation of spontaneous active force. Values are means ± SD; n = 6 except for beclomethasone dipropionate (BDP)-treated unchallenged rings (n = 5). *P < 0.05, BDP-treated challenged rings vs. non-BDP-treated challenged rings. Reduction of relaxant effect of salbutamol by allergen challenge suggests beta 2-adrenoceptor (beta 2-AR) pathway dysfunction. Restoration of relaxant effect of salbutamol by BDP suggests reversal of beta 2-AR pathway function.

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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Table 1.   Salbutamol concentrations inhibiting 50% of active force

Gsalpha Protein Function and Expression

Before incubation with CTX, there were no significant differences in the maximal contractile forces (10-4 M carbachol) between the experimental groups. Incubation with CTX at 10 µg/ml displaced the mean carbachol concentration-response curves significantly downward (P < 0.01; Fig. 2). The displacement was significantly less in non-BDP-treated challenged rings than in the other rings (P < 0.001). In BDP-treated challenged rings, the mean concentration-response curve was displaced significantly more downward than in non-BDP-treated challenged rings, so there was no significant difference between mean concentration-response curves of BDP-treated challenged and unchallenged rings (P = 0.44).


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Fig. 2.   Effect of 3 × 10-6 M BDP on mean carbachol concentration-response curves in human isolated bronchial rings. Cholera toxin (CTX) displaced the mean carbachol concentration-response curves (P < 0.01) significantly downward and significantly reduced maximal forces (P < 0.01), presumably by the stimulatory effect of CTX on the Gsalpha subunit, which in turn activated adenylyl cyclase. There was a significant difference between mean concentration-response curves for non-BDP-treated unchallenged rings (A) and non-BDP-treated challenged rings (B; P < 0.001), suggesting an attenuated stimulation of the Gsalpha subunit by CTX in challenged rings. There was also a significant difference between the mean concentration-response curves of non-BDP-treated challenged rings (B) and BDP-treated challenged rings (C; P < 0.001), suggesting restoration of the Gsalpha subunit function by treatment with BDP. There was no significant difference between the mean concentration-contraction curves of BDP-treated challenged rings (C) and unchallenged rings (A; P = 0.44). Values are means ± SD; n = 6.

Expression of Gsalpha protein was not significantly different between non-BDP-treated challenged rings and BDP-treated challenged rings (Table 2).

                              
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Table 2.   Gsalpha subunit expression in human bronchial smooth muscle

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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Fig. 3.   Relaxant effect of forskolin on human isolated bronchial rings challenged with allergen and precontracted by 10-6 M carbachol. There was no significant difference between mean concentration-relaxation curves of BDP-treated challenged rings and non-BDP-treated challenged rings. Values are means ± SD; n = 4.

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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Fig. 4.   Effect of pertussis toxin (PTX, 1 µg/ml for 4 h) on concentration-relaxation curves to salbutamol in nonsensitized unchallenged bronchial rings from 2 patients. PTX consistently and similarly enhanced the relaxant effect of salbutamol in BDP-treated rings (3 × 10-6 M for 3 h) and non-BDP-treated rings, suggesting that BDP did not affect the function of Gi protein. CON, control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study confirms the attenuated responses to beta 2-AR and Gsalpha 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 beta 2-AR or Gsalpha protein stimulation. This finding suggests that BDP rapidly restored the beta 2-AR pathway function, and this effect involved Gsalpha 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 10-6 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 beta 2-AR agonist salbutamol and CTX activate the beta 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 beta -ARs are not present on parasympathetic nerve endings of human airways (4).

A 3-h incubation with BDP was chosen, because beta 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 Gsalpha protein function, definite proof of Gsalpha protein dysfunction in challenged rings and the effect of BDP on Gsalpha protein function requires direct measure(s) of Gsalpha 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 of beta 2-ARs can present a major problem in the treatment of acute asthma (1) and may be caused by high doses of beta 2-agonists (9) and/or severe airway inflammation (3, 20). Corticosteroids can reverse beta 2-AR downregulation by gene transcription within 17-24 h in vivo (5, 12) and in vitro (18). Corticosteroids can also antagonize beta 2-AR desensitization induced by beta 2-agonists; this effect occurs faster than the time required for gene transcription (6, 17, 31).

The results of this study suggest that BDP, a corticosteroid widely used in asthma therapy, rapidly antagonized beta 2-AR pathway dysfunction induced by allergen challenge of human isolated sensitized bronchial rings.

Response to salbutamol. Stimulation of beta 2-ARs by the selective beta 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 beta 2-AR pathway dysfunction (27-29). Dysfunction of the beta 2-AR pathway may be caused by an attenuated expression and/or function of the beta 2-AR, an altered intracellular signal transmission between receptor and effector.

Treatment of challenged rings with BDP for only 3 h restored the inhibitory effect of beta 2-AR stimulation in carbachol-precontracted rings, which suggests a restoration of the beta 2-AR pathway function. This conclusion seems justified, because BDP alone had no effect on relaxation by salbutamol in unchallenged rings (Fig. 1).

Gsalpha protein function and expression. CTX catalyzes ADP-ribosylation of the alpha -subunit of CTX-sensitive G proteins, which irreversibly activates the alpha -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 Gsalpha protein.

The attenuation of contractile response to carbachol by CTX was greater in BDP-treated than in non-BDP-treated challenged rings. Importantly, the contractile response to carbachol in these rings was not significantly different from that in unchallenged rings, which suggests that BDP treatment for only 3 h restored the inhibitory effect of CTX on airway smooth muscle contraction. Western blot analysis suggests that this was not due to an increased expression of Gsalpha protein. There are many beta -ARs on the epithelium that do not contribute to the relaxation response. To exclude the effects of these beta -ARs on the results of the Western blot analysis, the same analysis was carried out in two epithelium-denuded rings from an additional patient. The densitometric peaks were 621 mm2 for the non-BDP-treated ring and 520 mm2 for the BDP-treated ring, which supports the previous conclusion.

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 beta 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 beta 2-AR hyporesponsiveness (19) are consistent with this conclusion.

Possible underlying mechanisms. Corticosteroids may interfere with beta 2-AR pathway function by promoting gene transcription, which regulates expression of beta 2-AR and Gsalpha 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 Gsalpha protein expression. Second, interleukin-1beta (IL-1beta ) may interfere with coupling of Gsalpha protein to beta 2-ARs and, thus, activation of adenylyl cyclase (8, 26). Corticosteroids may ablate this effect by decreasing the stability of mRNA for IL-1beta (15). The effect of corticosteroids on IL-1beta seems to be mediated by inhibition of prostanoid formation (19). Blockade of prostanoid formation by inhibition of cyclooxygenase does not prevent beta 2-AR dysfunction (27), suggesting that this mechanism cannot explain our results. In addition, no detectable levels of IL-1beta 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 beta 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.

In this study the nonselective muscarinic agonist carbachol was used to contract airway smooth muscle. Stimulation of postjunctional M2 receptors inhibits adenylyl cyclase through the activity of the inhibitory Gi protein. Any change in Gi expression or function induced by challenge or BDP would have, therefore, affected the responses to beta 2-AR pathway stimulation. It seems unlikely that the results of the present study were determined by changes in Gi protein for several reasons: 1) allergen-induced dysfunction of the beta 2-AR pathway in this model was not associated with an increase in expression or function of Gi protein (28); 2) the relaxant effect of salbutamol was similar in unchallenged BDP-treated and non-BDP-treated rings (Fig. 1); 3) 24 h of incubation with dexamethasone did not increase Gi protein expression in bovine trachealis (13); and 4) the Gi protein-specific blocker PTX enhanced the relaxant effect of salbutamol similarly in BDP-treated and non-BDP-treated rings from two additional patients (Fig. 4).

Allergen-induced beta 2-AR dysfunction in human isolated sensitized bronchi can be prevented by leukotriene receptor blockade (27) and seems to be due to a dysfunction of the Gsalpha protein (28). Presumably, isoforms of protein kinase C (7, 22) are activated along the diacylglycerol pathway and cause phosphorylation of beta 2-AR and/or the coupled Gs protein. It is tempting to speculate that BDP may ablate this process.

Conclusions. The results of the present study in human isolated sensitized bronchi demonstrate that BDP rapidly reversed the dysfunction of the intracellular beta 2-adrenergic signal transmission pathway induced by allergen challenge. This effect does not appear to be dependent on gene transcription. Restoration of the intracellular beta 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 Gsalpha protein awaits further studies.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bai, TR. Abnormalities in airway smooth muscle in fatal asthma. Am Rev Respir Dis 141: 552-557, 1990[ISI][Medline].

2.   Barnes, PJ. Mechanism of action of glucocorticoids in asthma. Am J Respir Crit Care Med 154: S21-S27, 1996[ISI][Medline].

3.   Barnes, PJ. beta -Adrenergic receptors and their regulation. Am J Respir Crit Care Med 152: 838-860, 1995[ISI][Medline].

4.   Belvisi, MG, Patel HJ, Takahashi T, Barnes PJ, and Giembycz MA. Paradoxical facilitation of acetylcholine release from parasympathetic nerves innervating guinea-pig trachea by isoprenaline. Br J Pharmacol 117: 1413-1420, 1996[Abstract].

5.   Brodde, OE, Owe U, Egerzegi S, Konietzko N, and Michel MC. Effect of prednisolone and ketotifen on beta 2-adrenoceptors in asthmatic patients receiving beta 2-bronchodilators. Eur J Clin Pharmacol 34: 145-150, 1988[ISI][Medline].

6.   Ellul-Micallef, R, and Fenech FF. Effect of intravenous prednisolone in asthmatics with diminished adrenergic responsiveness. Lancet 2: 1269-1271, 1975[Medline].

7.   Grandordy, BM, Mak JWC, and Barnes PJ. Modulation of airway smooth muscle beta -adrenoceptor function by a muscarinic agonist. Life Sci 54: 185-191, 1994[ISI][Medline].

8.   Hakonarson, H, Herrick DJ, and Grunstein MM. Mechanisms of impaired beta -adrenoceptor responsiveness in atopic sensitized airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 269: L645-L652, 1995[Abstract/Free Full Text].

9.   Hayes, MJ, Qing F, Rhodes CG, Rahman SU, Ind PW, Sriskandan S, Jones T, and Hughes JMB In vivo quantification of human pulmonary beta -adrenoceptors: effect of beta -agonist therapy. Am J Respir Crit Care Med 154: 1277-1283, 1996[Abstract].

10.   Hirata, N, Kohrogi H, Iwagoe H, Goto E, Hamamoto J, Fujii K, Yamaguchi T, Kawano O, and Ando M. Allergen exposure induces the expression of endothelial adhesion molecules in passively sensitized human bronchus: time course and the role of cytokines. Am J Respir Cell Mol Biol 18: 12-20, 1998[Abstract/Free Full Text].

11.   Hirst, SJ, and Lee TH. Airway smooth muscle as a target of glucocorticoid action in the treatment of asthma. Am J Respir Crit Care Med 158: S201-S206, 1998[Abstract/Free Full Text].

12.   Hui, KKP, Connolly ME, and Taskin DP. Reversal of human lymphocyte beta -adrenoceptor desensitization by glucocorticoids. Clin Pharmacol Ther 32: 566-571, 1982[ISI][Medline].

13.   Kalavantavanich, K, and Schramm CM. Dexamethasone potentiates high-affinity beta -agonist binding and Gsalpha protein expression in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 278: L1101-L1106, 2000[Abstract/Free Full Text].

14.   Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[ISI][Medline].

15.   Lee, SW, Tsou AP, Chan H, Thomas J, Petrie K, Eugui EM, and Allison AC. Glucocorticoids selectively inhibit the transcription of the interleukin-1beta gene and decrease the stability of interleukin-1beta mRNA. Proc Natl Acad Sci USA 85: 1204-1208, 1988[Abstract].

16.   Lin, RY, Pesola GR, Bakalchuk L, Heyl GT, Dow AM, Tenenbaum C, Curry A, and Westfal RE. Rapid improvement of peak flow in asthmatic patients treated with parenteral methylprednisolone in the emergency department: a randomized controlled study. Ann Emerg Med 33: 487-494, 1999[ISI][Medline].

17.   Lipworth, BJ, and Aziz I. Bronchodilator response to albuterol after regular formoterol and effects of acute corticosteroid administration. Chest 117: 156-162, 2000[Abstract/Free Full Text].

18.   Mak, JCW, Nishikawa M, and Barnes PJ. Glucocorticosteroids increase beta 2-adrenergic receptor transcription in human lung. Am J Physiol Lung Cell Mol Physiol 268: L41-L46, 1995[Abstract/Free Full Text].

19.   Moore, PE, Laporte JD, Gonzalez S, Moller W, Heyder J, Panettieri RA, Jr, and Shore SA. Glucocorticoids ablate IL-1beta -induced beta -adrenergic hyporesponsiveness in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 277: L932-L942, 1999[Abstract/Free Full Text].

20.   Nijkamp, FP, Engels F, Henricks PAJ, and Van Oosterhout AJM Mechanism of beta -adrenergic receptor regulation in lungs and its implication for physiological responses. Physiol Rev 72: 323-367, 1992[Free Full Text].

21.   Palejwala, S, and Goldsmith LT. Ovarian expression of cellular ki-ras p21 varies with physiological status. Proc Natl Acad Sci USA 89: 4202-4206, 1992[Abstract].

22.   Pitcher, J, Lohse MJ, Codona J, Caron MG, and Lefkovitz RJ. The sensitization of the isolated beta -adrenergic receptor kinase, cAMP-dependent protein kinase, and protein kinase C occurs in distinct molecular mechanisms. Biochemistry 31: 3193-3197, 1992[ISI][Medline].

23.   Rodrigo, G, and Rodrigo C. Corticosteroids in the emergency department therapy of acute adult asthma: an evidence-based evaluation. Chest 116: 285-295, 1999[Abstract/Free Full Text].

24.   Schramm, CM, and Grunstein MM. Corticosteroid modulation of Na+-K+ pump-mediated relaxation in maturing airway smooth muscle. Br J Pharmacol 119: 807-812, 1996[Abstract].

25.   Seamon, KB, Padgett W, and Daly JW. Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc Natl Acad Sci USA 78: 3363-3367, 1981[Abstract].

26.   Shore, SA, Laporte J, Hall IP, Hardy E, and Panettieri RA, Jr. Effect of IL-1beta on responses of cultured human airway smooth muscle cells to bronchodilator agonists. Am J Respir Cell Mol Biol 16: 702-712, 1997[Abstract].

27.   Song, P, Crimi E, Milanese M, Duan J, Rehder K, and Brusasco V. Anti-inflammatory agents and allergen-induced beta 2-receptor dysfunction in isolated human bronchi. Am J Respir Crit Care Med 158: 1809-1814, 1998[Abstract/Free Full Text].

28.   Song, P, Milanese M, Crimi E, Bruzzone S, Zocchi E, Rehder K, and Brusasco V. Gs-protein dysfunction in allergen-challenged human isolated passively sensitized bronchi. Am J Physiol Lung Cell Mol Physiol 279: L209-L215, 2000[Abstract/Free Full Text].

29.   Song, P, Milanese M, Crimi E, Rehder K, and Brusasco V. Allergen challenge of passively sensitized human bronchi alters M2 and beta 2 receptor function. Am J Respir Crit Care Med 155: 1230-1234, 1997[Abstract].

30.   Song, P, Rocchi D, Lazzarotti M, Crimi E, Rehder K, and Brusasco V. Postjunctional effect of pinacidil on contractility of isolated bovine trachealis. Eur Respir J 9: 2057-2063, 1996[Abstract/Free Full Text].

31.   Tan, KS, Grove A, McLean A, Gnosspelius Y, Hall IP, and Lipworth BJ. Systemic corticosteroid rapidly reverses bronchodilator subsensitivity induced by formoterol in asthmatic patients. Am J Respir Crit Care Med 156: 28-35, 1997[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 284(1):L133-L139
1040-0605/03 $5.00 Copyright © 2003 the American Physiological Society




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