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Dexamethasone potentiates high-affinity beta -agonist binding and Gsalpha protein expression in airway smooth muscle

Karl Kalavantavanich and Craig M. Schramm

Pediatric Pulmonary Division, University of Connecticut School of Medicine, Farmington, Connecticut 06030


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Corticosteroids enhance beta -adrenergic responses by actions at both beta -adrenoceptor (beta -AR) and post-beta -AR sites. The present study investigated the effects of dexamethasone on beta -AR density, high-affinity beta -agonist binding, Gsalpha and Gialpha protein expression, and cAMP responses in bovine tracheal smooth muscle (bTSM). Dexamethasone treatment of cultured bTSM cells increased total beta -AR density 1.6- to 1.9-fold as assessed by the saturation binding of [3H]CGP-12177 and by displacement of radioligand binding with isoproterenol. Isoproterenol bound to the beta -AR at two sites, a high-affinity site with a density of 5.9 ± 1.2 fmol/mg protein and a low-affinity site with a density of 16.9 ± 1.0 fmol/mg protein. Dexamethasone increased both high- and low-affinity isoproterenol binding sites to 11.1 ± 2.2 and 25.9 ± 2.1 fmol/mg protein, respectively, without influencing agonist binding affinities. Dexamethasone also selectively increased Gsalpha protein levels from 0.99 ± 0.14 to 1.46 ± 0.17 µg/mg protein without affecting Gialpha levels. The net effect of these changes was a 1.8-fold increase in maximal isoproterenol-induced cAMP generation in dexamethasone-treated bTSM cells. These findings provide new insights into the corticosteroid regulation of beta -adrenergic signaling pathways in airway smooth muscle.

beta -adrenergic receptors; bovine; CGP-12177; corticosteroid; G proteins; isoproterenol


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE beta -ADRENERGIC RECEPTOR (beta -AR) is linked to its effector enzyme, adenylyl cyclase, via an intermediary guanine nucleotide regulatory binding protein termed Gs. The stimulatory effect of Gs on adenylyl cyclase activity is antagonized by a corresponding inhibitory G protein (Gi) that is activated by various other receptor systems. Agonist binding to Gs-coupled beta -ARs results in the activation and release of the alpha -subunit of Gs (Gsalpha ) that, in turn, activates adenylyl cyclase. In contrast, agonist binding to Gs-uncoupled beta -ARs does not result in adenylyl cyclase activation. To promote agonist-induced signal transduction, the Gs-coupled beta -AR has a much higher agonist binding affinity state than the uncoupled beta -AR. Thus beta -adrenergic agonists interact with the beta -AR at two affinity states, and the interaction between beta -AR and Gs proteins is reflected in the amount of high-affinity agonist-beta -AR binding.

There is considerable evidence that asthma is associated with dysfunction of the beta -AR-adenylyl cyclase pathway. Some inflammatory mediators have been shown to downregulate beta -AR expression (1, 6, 12); however, more recent studies (10, 12, 14) suggest that the beta -adrenergic defect in asthma results from impaired signal transduction, at least in part due to cytokine-induced overexpression of Gi proteins. As a result, the airway relaxant response to beta -adrenergic agonists is attenuated in asthmatic individuals. The administration of corticosteroids to asthmatic individuals reduces their chronic airway inflammation and helps restore beta -adrenergic responsiveness. Glucocorticoids directly influence many steps in the beta -adrenergic signaling pathway, including supersensitization through increased beta -agonist stimulation of adenylyl cyclase (3) and upregulation of beta -AR gene transcription and receptor expression in lung tissues and cells (7, 18-20, 22). Glucocorticoids have also been shown to increase high-affinity beta -agonist binding in human neutrophils (2) and in fetal rat lung tissue (17). Similar studies have not been done in airway smooth muscle, and the direct effects of corticosteroids on smooth muscle Gs protein expression have not been examined. Therefore, the present study was designed to investigate the effects of dexamethasone (Dex) on beta -AR density, high-affinity beta -agonist binding, and Gsalpha protein levels in airway smooth muscle cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture and cell membrane preparation. Bovine tracheal smooth muscle (bTSM) cells were generously provided by Dr. Mark Madison (Department of Medicine, University of Massachusetts Medical School, Worcester, MA) (5). The cells were maintained in culture in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 1% nonessential amino acids, 100 U/ml of penicillin, 0.1 mg/ml of streptomycin, and 0.5 µg/ml of amphotericin B. Under these culture conditions, bTSM cells grew with a population doubling every 1.5-3 days and reached confluence in 6-7 days. Cell passages 4 through 7 were used for the experiments. Confluent bTSM cells were placed in low-serum medium (3%) in the absence (control) and presence of Dex (1 µM). Our preliminary work with these cells demonstrated that a low concentration of serum was necessary for them to express beta -ARs at consistent levels and to demonstrate reproducible cAMP responses to isoproterenol. Additional studies with Dex concentrations of 0.01-10 µM showed that 1-10 µM elicited maximal increases in beta -AR expression and isoproterenol-induced generation of cAMP in airway smooth muscle cells (26).

After 24 h of incubation, the cells were homogenized in iced buffer consisting of 50 mM Tris · HCl (pH 8.3 at 25°C), 1 mM EDTA, and 1 mM dithiothreitol (DTT). Large cell fragments and debris were removed by an initial low-speed centrifugation (1,600 g), and the cell membrane was then pelleted by centrifugation at 35,000 g for 20 min at 4°C. The membrane pellet was resuspended in fresh homogenization buffer and centrifuged again at 35,000 g. The resulting washed membrane pellet was then suspended in binding buffer consisting of 50 mM Tris · HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA, and 1 mM DTT. Protein concentrations in the final suspensions were determined by the method of Lowry et al. (16) with bovine serum albumin as the standard.

beta -AR binding studies. The hydrophilic beta -AR ligand (-)-4- (3-t-butylamino-2-hydroxypropoxy)-[5,7-3H]benzimidazol-2-one ([3H]CGP-12177; specific activity 49.0 Ci/mmol) was chosen for radioligand studies because of its highly specific, single-site binding and its low nonspecific binding [< 20% of total binding around the binding affinity (Kd) concentration]. Initial studies assessed the saturation binding of [3H]CGP-12177 in control and Dex-treated bTSM cells to determine the influence of corticosteroids on [3H]CGP-12177 binding. Triplicate control and Dex samples containing ~100 µg of membrane protein were incubated with increasing concentrations of [3H]CGP-12177 (0.007-2.0 nM) for 45 min at 37°C. Specific binding was determined by the amount of ligand at each concentration displaced by 10 µM propranolol. Incubations were stopped with eight volumes of iced buffer. Bound [3H]CGP-12177 was extracted by rapid filtration through fiberglass filters (Whatman GF/C), rinsed two times with iced buffer containing 50 mM Tris · HCl, 1 mM EDTA, 1 mM DTT, and 0.1% bovine serum albumin. The filters were then counted for receptor-bound radioactivity in a liquid scintillation counter (EcoLite, ICN, Costa Mesa, CA). Maximum binding (Bmax) and Kd were determined for control and Dex-treated bTSM cell membranes with a computerized noniterative curve-fitting program (LIGAND).

To determine the effects of corticosteroids on beta -adrenergic agonist binding, competitive binding studies were performed with displacement of bound [3H]CGP-12177 by the beta -adrenergic agonist isoproterenol. In paired experiments, aliquots containing ~100 µg of control and Dex-treated membrane protein samples in binding buffer were exposed in triplicate to a constant concentration of [3H]CGP-12177 (2 nM) and 18 different concentrations of isoproterenol (0 and 1 × 10-12 to 3 × 10-4 M) for 45 min at 37°C. Nonspecific binding was determined by the presence of 20 µM propranolol. Bound [3H]CGP-12177 was extracted and counted as above. The data from all paired experiments (n = 5 for both control and Dex-treated bTSM) were analyzed with computerized noniterative curve fitting for one- and two-site displacement curves.

Western analysis of Gsalpha and Gialpha protein expression. Separate studies addressed the direct effects of corticosteroids on Gsalpha and Gialpha protein subunit expression as determined by quantitative Western analysis (11, 21). Washed membrane pellets (n = 9 each) were obtained from paired control and Dex-treated bTSM as in Cell culture and cell membrane preparation. Five serial dilutions ranging from 0.15 to 1.0 µg/µl of each control and Dex-treated membrane protein sample were made. One serial dilution of membrane protein was loaded to each of five lanes of a SDS-polyacrylamide gel (12%). Recombinant rat standard Gsalpha protein (47 kDa, 0.04 µg; Santa Cruz Biotechnology, Santa Cruz, CA) and standard Gialpha protein (42 kDa, 0.0107 µg; Santa Cruz Biotechnology) were loaded in separate lanes on each gel as positive controls and for density comparison of each protein sample band. Prestained high-range molecular-mass standards (Life Technologies) were also loaded in one lane as a transfer marker. The paired control and Dex-treated gels were run in parallel, and protein separation was performed on the Mini-PROTEAN II cell (Bio-Rad, Hercules, CA) at 200 V for 30-40 min. Separated proteins were transferred electrophoretically (200 V, 400 mA, 1 h) to nitrocellulose membranes (0.2 µm; Schleicher & Schuell) in transfer buffer containing 25 mM Tris base, 192 mM glycine and 20% (vol/vol) methanol (pH 8.2)

Thereafter, the nitrocellulose blots were incubated at room temperature for 1 h with blocking solution [0.2 M NaCl, 50 mM Tris · HCl, 0.2% (vol/vol) Tween, and 5% nonfat milk]. The blots were primed with rabbit polyclonal antibody raised to the Gsalpha subunit (1:1,000 dilution; Santa Cruz Biotechnology) for 1 h and then incubated with peroxidase-conjugated goat anti-rabbit IgG (1:10,000 dilution; Calbiochem, La Jolla, CA) for 1 h. In-between each incubation step, the blots were washed six times (5 min each time) with buffer containing 0.2 M NaCl, 50 mM Tris · HCl, and 0.2% (vol/vol) Tween. The blots were then exposed to enhanced chemiluminescence substrate (Pierce, Rockford IL) for 5 min and were immediately visualized on X-ray film (Kodak XAR-5). The visualized Gsalpha sample and standard bands were quantified with a densitometer.

The nitrocellulose membranes were then reprobed with a polyclonal antibody raised to the Gialpha subunit. Each nitrocellulose membrane was incubated for 30 min at 60°C in stripping buffer containing 2% (wt/vol) SDS, 62.5 mM Tris · HCl (pH 6.8), and 100 mM beta -mercaptoethanol and then rinsed three times with water. The membranes were reblotted with the same protocol and solutions as for anti-Gsalpha antibody above except that the primary antibody was changed to a polyclonal antibody recognizing all three subtypes of Gialpha (dilution 1:1,000; Santa Cruz Biotechnology). The sample and standard Gialpha bands were quantified with a densitometer. To ensure that there was no sequence effect to the blots, three of the nine paired blots were stained with anti-Gialpha first, then stripped and restained with anti-Gsalpha . The resulting blots were no different than the other six done in the opposite sequence.

The density units from each protein sample were compared with the density units of the known standard Gsalpha or Gialpha proteins. Thus the approximate amounts of Gsalpha and Gialpha proteins per membrane protein sample (in ng/µg) were obtained. Plots of the amounts of Gsalpha or Gialpha protein as a function of the corresponding amount of membrane protein were linear in all cases, and their slopes represent the average amount of Gsalpha or Gialpha protein (in ng) per microgram of membrane protein from the control or Dex-treated bTSM cells.

cAMP assay. To assess the physiological relevance of our observed changes in beta -adrenoceptor and Gsalpha densities, additional studies determined the influence of Dex on basal and maximal beta -agonist-stimulated levels of cAMP in the bTSM cells. Cells grown to confluence in T-25 plates were placed in low-serum medium for 24 h in the absence and presence of 1 µM Dex. After a 30-min incubation with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 10 µM), duplicate bTSM samples were exposed for 2 min to varying concentrations of isoproterenol (0 and 10-8 to 10-4 M) (27). The cells were quickly lysed in 6% perchloric acid and scraped from the flasks. Cellular cAMP levels were determined by displacement of [3H]cAMP from a commercial binding protein (Amersham International, Arlington Heights, IL). The cAMP values are expressed as a percentage of baseline, unstimulated levels in both control and Dex-treated cells.

Reagents. DMEM and nonessential amino acid solution were obtained from Life Technologies (Grand Island, NY). Fetal bovine serum, penicillin-streptomycin solution, and amphotericin B were obtained from Gemini Bio-Products (Calabasas, CA). [3H]CGP-12177 and the cAMP assay kit were purchased from Amersham Life Science (Cleveland, OH). Isoproterenol hydrochloride, dithiothreitol, EDTA, dexamethasone 21-acetate, Trizma hydrochloride, and Trizma base were obtained from Sigma (St. Louis, MO). Acrylamide, SDS, N,N'-methylene-bis-acrylamide, N,N,N',N'-tetramethylethylenediamine, and ammonium persulfate were obtained from Bio-Rad. Tween solution was obtained from Fisher Scientific (Pittsburgh, PA). Methanol was obtained from J. T. Baker (Phillipsburg, NJ), and glycine was from ICN (Aurora, OH). IBMX was purchased from Research Biochemicals International (Natick, MA).

Statistical analysis. All data are expressed as means ± SE. Slopes of the Galpha -membrane protein relationships were obtained by least-squares analysis. Statistical comparisons between control and Dex-treated results were performed by two-tailed, unpaired t-tests and by repeated-measures analysis of variance for isoproterenol-cAMP dose-response relationships (StatView 4.5, SAS Institute, Cary, NC). Cellular sensitivity to isoproterenol was characterized by the log concentration of isoproterenol associated with half-maximal responses (i.e., the pC2 values). P values < 0.05 were considered to be significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

beta -AR expression. The LIGAND analysis of beta -AR saturation binding with [3H]CGP-12177 demonstrated a single binding site in all control and Dex-treated bTSM membrane samples (n = 4 each). This finding was also reflected in linear Scatchard plots (correlation coefficients -0.791 to -0.984) and Hill coefficients near unity (range 0.847-1.03). Dex exposure did not affect the Kd of [3H]CGP-12177 for the beta -AR, with a Kd value of 0.164 ± 0.067 nM for Dex-treated bTSM cells vs. 0.213 ± 0.126 nM for control cells (P = 0.50). In contrast, maximal beta -AR binding was significantly increased with Dex, with Bmax equaling 30.2 ± 6.2 fmol/mg protein in Dex-treated cells and 15.8 ± 1.5 fmol/mg protein in control cells (P = 0.018). These findings suggested that Dex exposure increased total beta -AR numbers without changing the Kd for a beta -adrenergic-antagonist ligand.

beta -Agonist binding. In comparison to the ligand saturation binding experiments, LIGAND analyses of the isoproterenol displacement studies demonstrated two-site binding with biphasic curves and nonlinear Scatchard relationships. In each control and Dex experiment (n = 5 each), two-site binding resulted in a significantly better fit than one-site binding. Addition of the nonhydrolyzable GTP analog guanosine 5'-(beta ,gamma -imino)-triphosphate resulted in loss of high-affinity binding sites and a shift of the inhibition curves to the right (data not shown). The individual control and Dex displacement binding curves were combined in order for LIGAND to determine the average values for the high- and low-affinity agonist binding parameters (Fig. 1). The average high Kd was ~1,000 times higher than the average low Kd. Neither Kd value was changed by Dex treatment (Table 1). In contrast, Dex increased total isoproterenol binding (total Bmax) by 62% over control values (compared with the 91% increase obtained in the [3H]CGP-12177 saturation experiments). Low-affinity binding sites were increased by 53% above control levels, and high-affinity sites were increased by 88%. The ratio of high-affinity to total binding (i.e., high Bmax/total Bmax) was not affected by Dex exposure. The increase in high-affinity beta -agonist binding suggested that corticosteroid treatment either increased Gs protein expression or enhanced beta -AR-Gs coupling in bTSM (or both).


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Fig. 1.   Displacement analyses of [3H]CGP-12177 ([3H]CGP)-specific binding by cumulative administration of isoproterenol in control (A) and dexamethasone (Dex; B)-treated bovine tracheal smooth muscle membranes. Both displacement curves demonstrate biphasic shapes, suggesting presence of 2 binding sites with high- and low-binding affinities. Different symbols represent 5 different paired studies.


                              
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Table 1.   High- and low-affinity beta -agonist binding parameters in control and dexamethasone-treated bovine tracheal smooth muscle cells

Gsalpha and Gialpha protein expression. Paired Western analyses of Gsalpha protein expression in control and Dex-treated membrane protein samples (n = 9) demonstrated prominent bands at 45 kDa (Fig. 2A). The densities of the five bands in the five protein sample lanes in each blot were analyzed relative to the density of a known amount of Gsalpha protein standard (0.04 µg). Plots of the amount of Gsalpha protein present versus the amount of membrane protein sample loaded on the gel showed linear relationships (Fig. 2B), the slopes of which represented the average amount of Gsalpha present (in ng/µg or µg/mg membrane protein). Comparison of paired control and Dex-treated samples showed that the Dex-treated lines had consistently steeper slopes than the control lines. Statistical comparison between control and Dex-treated Gsalpha protein levels showed significantly increased Gsalpha expression in Dex-treated bTSM (1.46 ± 0.17 vs. 0.99 ± 0.14 µg/mg protein in control bTSM; P = 0.048).


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Fig. 2.   A: representative paired Western analyses of Gsalpha protein levels in control (CRL; top) and Dex-treated (bottom) membrane protein samples. Note a single prominent band at 45 kDa in each blot (slightly below the 47-kDa Gs standard) and decreasing band density with decreased protein loading. B: density analyses of each of the above bands relative to known Gsalpha standard were plotted against amount of membrane protein sample. , Dex-treated cells; open circle , control cells. Slope of this relationship represents an average amount of Gsalpha /µg membrane protein in this representative experiment. Significant increase in slope of Dex line demonstrated that Dex treatment increased Gsalpha protein level in treated cells.

A single prominent band was obtained at 40 kDa in each blot for Gialpha control and Dex-treated protein samples (Fig. 3A). However, the slopes of the relationship between Gialpha protein levels and the amount of membrane protein samples were not different between control and Dex-treated cells (Fig. 3B), suggesting that Dex treatment did not affect the amount of Gialpha in these cells. Indeed, average Gialpha levels were not changed with Dex treatment (0.421 ± 0.096 µg/mg protein) compared with levels in paired control cells (0.408 ± 0.072 µg/mg protein; P = 0.82). Thus Dex selectively increased Gsalpha protein levels without increasing Gialpha protein levels in cultured bTSM cells.


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Fig. 3.   A: representative paired Western analyses of Gialpha protein levels in control (top) and Dex-treated (bottom) membrane protein samples. Note a single prominent band at 40 kDa in each blot (slightly below the 42-kDa Gi standard) and decreasing band density with decreased protein loading. B: density analyses of each of the above bands relative to known Gialpha standard were plotted against amount of membrane protein sample. , Dex-treated cells; open circle , control cells. Slope of this relationship represents an average amount of Gialpha /µg membrane protein in this representative experiment. There was no increase in slope between control and Dex-treated cells, demonstrating that Dex treatment did not affect Gialpha protein levels.

cAMP responses. Treatment of control bTSM cells (n = 4 samples) with increasing doses of isoproterenol resulted in dose-dependent increases in cellular cAMP levels to a maximal stimulated level that was 309 ± 41% of the unstimulated baseline. Dex treatment potentiated the cAMP response (P = 0.046 vs. control value by ANOVA) and increased the maximal response to 541 ± 77% of baseline (P = 0.021 vs. control value). Unstimulated baseline levels of cAMP were not affected by Dex exposure (P = 0.30). There was also no difference in bTSM sensitivity to isoproterenol in Dex-treated (pC2 6.23 ± 0.28 - log M) and control cells (5.57 ± 0.24 - log M; P = 0.10).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study investigated the effects of corticosteroids on beta -AR density, beta -agonist binding, and Gsalpha and Gialpha protein levels in bTSM cells. Pretreatment of bTSM cells for 24 h with 1 µM Dex increased total beta -AR binding 1.9-fold over the control value in the saturation binding studies and 1.6-fold over the control value in the agonist displacement studies. These findings are consistent with observations in various other tissues and cell types and have been attributed to corticosteroid time- and concentration-dependent increases in beta 2-AR mRNA levels from enhanced gene transcription (18, 28). The increased beta -AR binding was accompanied by a 1.8-fold increase in maximal beta -agonist-induced cAMP generation in the cells. The parallel increases in beta -AR expression and beta -agonist-mediated cAMP generation have also been seen in rabbit tracheal smooth muscle after in vivo Dex administration (25). Thus there appears to be a direct relationship between beta -AR density and maximal beta -agonist cAMP responsiveness in airway smooth muscle.

The isoproterenol displacement studies revealed that both high- and low-affinity beta -agonist binding was enhanced by corticosteroid exposure due to increases in beta -agonist binding sites and not to changes in agonist Kd values. Isoproterenol bound to beta -ARs at two affinities in bTSM cells, with a proportion of high-to-low binding of ~1:2 and a difference in Kd values of ~1,000-fold. Both high- and low-affinity binding sites were increased by Dex treatment. The fraction of high-affinity beta -agonist binding was not affected by Dex in the bTSM cells, although cortisone treatment can enhance the percentage of high-affinity beta -agonist binding in human neutrophil membranes (2). Of interest, methylprednisolone has been shown to restore airway beta -adrenergic responsiveness in the Basenji-Greyhound dog model of asthma (24). At least one mechanism accounting for the impairment of beta -AR signaling in this model is a decrease in high-affinity beta -agonist binding sites in the lungs of these animals (4). Our studies suggest that corticosteroids could improve beta -adrenergic responsiveness in the Basenji-Greyhound dogs by increasing high-affinity beta -agonist binding sites in their airways. The increase in high-affinity beta -agonist binding in the bTSM cells implied either that Dex enhanced Gs expression in the cell membrane or that the corticosteroid somehow promoted the interaction between beta -ARs and the Gs proteins.

The mechanism of increased high-affinity binding was evaluated further by quantitative Western analysis of Gsalpha protein levels. These Western analyses demonstrated that Dex selectively increased Gsalpha protein levels in bTSM cells without affecting Gialpha levels. Similar observations have been reported in the rat cerebral cortex, wherein corticosterone increased Gsalpha levels 1.4-fold without influencing Gialpha levels (23). The unchanged Gialpha levels represent an important control for any potential loading inequalities between the samples and nonspecific steroid effects. It may be noted that the molecular masses of the recombinant rat Gsalpha and Gialpha protein standards were slightly higher than those of the bovine Gsalpha and Gialpha proteins, likely reflecting different posttranslational modifications of the recombinant alpha -protein standards. Corticosteroids can activate the transcription of many genes by binding as a ligand-receptor complex to certain DNA target sequences, termed glucocorticoid response elements, in the promoter region of steroid-sensitive genes. Dex could increase Gsalpha protein expression through this mechanism if the Gsalpha protein gene contains a glucocorticoid response element. To address this possibility, we reviewed the reported nucleotide sequence of the human Gsalpha gene (13) and found no classic sequences for glucocorticoid response elements in the 790 bases upstream from the initiation codon. Corticosteroids may act, therefore, through trans-activating factors or other indirect pathways to enhance Gsalpha protein expression in bTSM cells. Moreover, corticosteroid effects on Gsalpha protein expression may vary between cell types, with positive effects reported in the cerebral cortex (23), osteosarcoma cells (21), and pheochromocytoma cells (15) but negative effects seen in aortic endothelial membranes (9).

Agonist-induced GTPase stimulation is closely reflected in the affinity state of the receptor for the agonist (8). Thus although we did not measure GTPase activity directly, the finding that Dex increased high-affinity beta -agonist binding demonstrates that the steroid augments the function as well as the expression of Gsalpha . The enhancements were similar, with a 1.5-fold increase in Gsalpha protein expression and a 1.9-fold increase in Gsalpha function (i.e., high-affinity beta -agonist binding sites). These increases were reflected in the potentiated maximal cAMP response to isoproterenol in Dex-treated bTSM cells (175% of control value). Although these observations do not rule out any potential promoting effects of corticosteroids on beta -AR-Gs protein coupling, they suggest that the increased Gsalpha at least contributes to this steroid response.

In summary, our study showed that Dex potentiates beta -agonist cAMP responses and high-affinity binding in airway smooth muscle through parallel increases in both beta -AR levels and Gsalpha protein expression. This is the first direct demonstration that corticosteroids can influence Gsalpha protein levels in airway smooth muscle. The effect on Gsalpha is selective in that Gialpha protein levels were unaffected. Collectively, these findings provide new insights into the regulation of beta -adrenergic signaling pathways by corticosteroids and may partly explain the mechanisms of corticosteroid upregulation of beta 2-adrenergic responsiveness in asthmatic patients. Further studies are needed to fully understand the mechanisms underlying the potentiating effect of corticosteroids on the beta -adrenergic signaling pathway in airway smooth muscle cells.


    ACKNOWLEDGEMENTS

We thank Dr. Mitchell Kresch and his research technician Constance Christian for technical advice with the Western blot studies.


    FOOTNOTES

This research was sponsored by a Career Investigator Award cofunded by the American Lung Association and the American Lung Association of Connecticut (to C. M. Schramm).

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.

Address for reprint requests and other correspondence: C. M. Schramm, Pediatric Pulmonary Division, Connecticut Children's Medical Center, 282 Washington St., Hartford, CT 06106 (E-mail:cschram{at}ccmckids.org).

Received 14 June 1999; accepted in final form 11 February 2000.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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