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Extracellular Matrix Modulates ß₂-Adrenergic Receptor Signaling in Human Airway Smooth Muscle Cells

Division of Therapeutics and Molecular Biology, University of Nottingham, Queens Medical Centre, Nottingham, United Kingdom; Division of Critical Care, Pulmonary, Allergic and Immunologic Diseases, Thomas Jefferson University, Philadelphia, Pennsylvania; and Wake Forest University Health Science Center, Center for Human Genomics, Winston-Salem, North Carolina

Address correspondence to: Anette M. Freyer, Division of Therapeutics and Molecular Biology, University of Nottingham, D Floor, South Block, Queens Medical Centre, Nottingham NG7 2UH, UK. E-mail: anette.freyer{at}nottingham.ac.uk

	Abstract

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The airways of patients with chronic asthma commonly develop an element of fixed airway obstruction, which fails to reverse with inhaled ß₂-adrenoceptor agonists. Airway remodeling refers to the structural changes of the bronchi in longstanding asthma and is characterized by increased deposition and altered ratios of extracellular matrix (ECM) proteins. We therefore assessed whether ECM proteins alter ß₂-adrenoceptor signaling in human airway smooth muscle cells. We report that a fibronectin environment increases responses to ß₂-adrenoceptor stimulation, whereas exposure to collagen V or laminin decreases accumulation of the second messenger cyclic AMP when compared with collagens I or IV. These differences are likely to be physiologically significant as they translate into altered phosphorylation of the downstream target VASP. The altered cAMP levels are due to differences in adenylyl cyclase activity, although expression of the relevant isoforms of enzyme appears unaltered. However, inhibition of Gi abrogates the differences in ß₂-adrenoceptor–mediated cAMP accumulation in cells exposed to different matrix factors. The difference in Gi signaling is not due to altered Gi expression. We conclude therefore that ECM modulates Gi activity in human airway smooth muscle cells, and propose that these changes could contribute to the fixed airway obstruction seen in patients with chronic asthma.

Abbreviations: actinomycin D, ACD • airway smooth muscle, ASM • specific binding sites, Bmax • cyclic AMP, cAMP • cycloheximide, CHX • Dulbecco's modified Eagle's medium, DMEM • extracellular matrix, ECM • fibronectin, FN • forskolin, FSK • isoproterenol, ISO • laminin, LN • mitogen-activated protein kinase, MAPK • phosphate-buffered saline, PBS • phosphodiesterase, PDE • cAMP-dependent protein kinase, PKA • pertussis toxin, PTX • tris-buffered saline, TBS •

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Patients with chronic asthma often develop an element of fixed airway obstruction with lung function that progressively declines from predicted values. In these patients airways fail to fully bronchodilate in response to the most commonly used medication in asthma: inhaled ß₂-adrenoceptor agonists (1). A number of structural changes take place in the airways of patients with longstanding asthma including epithelial desquamation, mucous cell hyperplasia, and an increase in basement membrane thickness. In addition, increased deposition of extracellular matrix (ECM) (with increased proportions of collagens I and V, laminin [LN], and fibronectin) and enlargement of the airway smooth muscle (ASM) mass contribute significantly to the characteristic thickening of the remodeled airway wall (2). It is believed that this anatomical remodeling contributes to the fixed airflow obstruction and altered airway dynamics seen in patients with chronic asthma (3). However, it is also possible that the altered extracellular environment affects signaling through contractile and relaxant pathways in the ASM cells, thus contributing to airflow obstruction and a reduced ability to bronchodilate.

The major relaxant pathway in ASM cells is via ß₂-adrenoceptor–mediated stimulation of adenylyl cyclase activity, resulting in the formation of the intracellular second messenger cyclic AMP (cAMP). cAMP in turn activates the cAMP-dependent protein kinase (PKA). PKA phosphorylates numerous intracellular targets resulting in inhibition of inositol triphosphate (IP₃) production, increased Ca²⁺ reuptake, and downregulation of myosin light chain kinase, all of which promote ASM cell relaxation (4).

Over the last few years it has become increasingly apparent that signaling relays such as those governing relaxant signaling in ASM are not isolated unidirectional chains of events, but form part of a complex network of cross-regulated systems. The different components of the signaling system are present as complexes either in the cell membrane or anchored by the cytoskeleton. Further key components of these complexes are scaffold proteins and ECM receptors (integrins). Integrins activate several important signaling cascades, for example phosphatidylinositol-3-kinase, cAMP, and mitogen-activated protein kinase (MAPK) pathways (5), and are able to modulate G protein–mediated signal transduction (6, 7). We have profiled the expression of integrin subunits in primary cultures of human ASM cells and shown that they play a critical role in regulating smooth muscle cell homeostasis. The observation that ASM cells grown in different matrix environments demonstrated striking morphologic changes (8, 9) led us to investigate whether these matrix-induced phenotypic changes included alterations in the G protein–mediated signaling pathways controlling ASM relaxation.

Here we report that response to ß-agonists varies in human ASM cells in culture depending on their ECM environment, and demonstrate that this difference is due to alterations in Gi signaling.

	Materials and Methods

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Cell Culture
Primary cultures of human ASM cells were prepared from explants of trachealis muscle obtained from individuals without respiratory disease within 24 h of death as previously described (10). Briefly, a strip of trachealis muscle was dissected from just above the carina and washed in Dulbecco's modified Eagle's medium (DMEM) containing penicillin G (200 U/ml), streptomycin (200 µg/liter), and amphotericin B (0.5 µg/liter). Explants of airway muscle (0.2 x 0.2 cm) were excised, placed in 6-well plates, and allowed to adhere. DMEM containing the antibiotics, amphotericin B, 10% fetal calf serum, and glutamine (2 mM) was added to cover explants and replaced regularly until myocyte growth occurred (usually between Days 7 and 10), when culture medium was changed to exclude the antimicrobials. As cells approached confluence in some parts of the flask, explants were removed and the cells harvested by trypsinization 24 h later. All primary cell cultures exhibited > 95% cells staining for smooth muscle –actin antibody (11). Cells from passages 4–8 were used for the present study.

Coating of Culture Plates with Extracellular Matrix Factors
Experiments were conducted on uncoated tissue culture plastic or glass, or where indicated in wells coated with specific ECM proteins. Ten micrograms per milliliter solution of fibronectin (FN), collagens I, IV, V, or LN was allowed to adhere to culture plates overnight (12) before blocking with 1% albumin in phosphate-buffered saline (PBS) for 1 h. All plates were rinsed with PBS and sterilized by ultraviolet light before use.

³[H]-cAMP Assay
Cells were seeded in serum-free DMEM containing 2 mM glutamine into the matrix-coated wells or uncoated control wells of a 24-well plate. Under these culture conditions cell number does not differ between populations grown on different ECM factors after 24 h (9, 13). Protein synthesis inhibitors were added at this stage in the relevant experiments. After 16 h the cells were loaded with ³[H]-adenine (2 µCi/well) for 2 h before excess adenine was washed off. Antagonists were added for 20 min before the 15-min stimulation with isoproterenol (ISO) or forskolin (FSK). Reactions were terminated by the addition of 50 µl concentrated HCl followed by freezing at –20°C. cAMP was eluted by column chromatography and results corrected for adenine uptake and column efficiency with ¹⁴[C]-cAMP as described previously (14). Triplicate wells were counted per condition. The mean of these values is expressed as the % of the maximum mean within each experiment. Results are shown as means ± SEM of three separate experiments performed in cells of two different donors.

Receptor Ligand Studies and GTPS Assay
Human ASM cells were cultured under serum-free conditions in matrix-coated flasks for 18 h; 50 ng/ml pertussis toxin (PTX) was added to some flasks for the final 8 h where indicated. After harvesting with 5 mM ethylenediamine tetraacetic acid in PBS, the cells were resuspended in 50 mM Tris HCl, Complete protease inhibitor and 1 mM activated Na orthovanadate, pH 7.4, and manually homogenized. Homogenate was centrifuged and cell debris discarded. The membrane fractions were collected by high-speed centrifugation, rehomogenized, and stored at –80°C. For each sample 200 µg of membrane protein was exposed to a range of doses of the ß₂-receptor ligand ³[H]-CGP-12177 (55Ci/mmol) in the presence or absence of 100 nM of the ß₂-adrenoceptor antagonist ICI-18551 for the receptor ligand studies (15). Each condition was measured in triplicate in two separate experiments using cells from two different donors. The GTP-S assay was optimized for ASM cells and Gi activity: After 1 h incubation at 37°C (receptor ligand experiments) or room temperature (GTPS assays), samples were washed with ice-cold Tris HCl (pH 7.4) for receptor ligand experiments or harvesting buffer (NaCl 140 mM, KCl 3 mM, KH₂PO₄ 1.5 mM, NaH₂PO₄ 5 mM, pH7.5) for the GTPS activity assays and filtered through pre-soaked (0.3% Polyethylenimine in Tris HCl) Whatman GF/B filters using a Brandel cell harvester (SEMAT Technical Ltd, St. Albans, UK). Values for total and nonspecific binding were derived from tritium counts and receptor expression (Bmax) and affinity (Kd) calculated using Prism software without weighting, assuming one-site binding. For the GTPS assay we subtracted counts derived for the PTX-treated samples (nonspecific binding NSB) from the values gained in untreated samples (total) to give an indication of Gi-specific GTP binding.

Immunoblots
Cells were seeded in matrix-coated flasks under serum-free conditions for 18 h. After treatment with agonist for 15 min monolayers were washed with ice-cold PBS and cells harvested in tris-buffered saline (TBS) (0.9% NaCl, 20 mM Tris) containing 0.1% Triton-X 100, Complete protease inhibitor, and 1 mM activated Na orthovanadate. Samples were centrifuged and the supernatant retrieved for protein determination according to Bradford. Samples were boiled for 5 min in the presence of 2% sodium dodecyl sulfate sample buffer with 2.5% ß-mercaptoethanol and then equal amounts of protein were electrophoresed on a 10% sodium dodecyl sulfate–polyacrylamide gel. After electrophoresis, proteins were transferred to nitrocellulose membranes. Membranes were blocked for 1 h at room temperature in blocking buffer (TBS with 0.05% Tween-20 [TBST]) containing 5% (wt/vol) dried nonfat milk followed by an incubation overnight at 4°C in fresh blocking buffer containing anti-vasodilator–stimulated phosphoprotein (VASP) (1:2,500), anti–adenylyl cyclase V/VI (1:500), anti-Gi-2 (1:200), or anti-Gi-3/1 (1:200). The next day blots were washed three times with TBST then incubated for 1 h in TBST with 5% milk containing the required secondary antibody. Blots were then rinsed three times in TBST before visualization either with enhanced chemiluminescence or with the Odyssey Infrared Imaging System (LI-COR, Lincoln, NE).

Materials
All chemicals used were analytical grade or higher. Plastic culture ware was from Costar (High Wycombe, UK) and ECM factors except fibronectin were bought from Calbiochem (Nottingham, UK), as were cycloheximide and actinomycin D. ³[H]adenine and ¹⁴[C] cAMP were obtained from Amersham, ³[H]-CGP-12177 and ICI18551 were gifts from Jill Baker (University of Nottingham). Emulsifier Scintillation Plus was sourced from Packard Bioscience (Groningen, The Netherlands), Complete protease inhibitor cocktail from Roche (Welwyn Garden, UK). Anti-VASP Ab was bought from BD Biosciences (Oxford, UK), anti-adenylyl cyclase V/VI Ab (C-17, sc-590), anti–Gi-3/1 Ab (C-10, sc-262), and anti–Gi-2 Ab (L5, sc-13534) from Santa Cruz (Santa Cruz, CA and Heidelberg, Germany). The IRDye secondary Ab was from Rockland (Gilbertsville, PA), anti-mouse horseradish peroxidase–conjugated secondary was from Vector Laboratories (Burlingame, CA). CellTiter 96 MTS assay was bought from Promega (Southampton, UK) and used according to the manufacturer's instructions with eight replicate wells per condition in each experiment. All other chemicals and antibodies were bought form Sigma (Poole, UK).

Statistics
Results were analyzed using ANOVA with Bonferroni post-test or unpaired t test as appropriate. Prism software package (Graph-Pad software, San Diego, CA) was used for statistical analyses.

	Results

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ß₂-Adrenoceptor–Mediated cAMP Accumulation Differs between Cells on Different Matrix Factors
Initial experiments examined the effect of ECM environment on constitutive cAMP production (i.e., levels of cAMP accumulation in the absence of agonist). Basal cAMP levels did not differ significantly between cells grown on different matrix factors or those seeded onto plastic, except for slightly elevated levels in ASM cells grown on collagen V (1.83 ± 0.172-fold compared with cells grown on plastic, P < 0.05, n = 3, results not shown). However, when we stimulated cells with the ß-adrenoceptor agonist ISO, this resulted in increased cAMP accumulation in cells grown on FN and a decreased response in those plated on collagen V or LN relative to the responses seen in cells seeded onto the collagens I or IV (Figure 1A). ISO (10 µM ) maximally stimulated cAMP production on all ECM factors, with 15.2 ± 1.4-fold increase over plastic baseline on collagen I (range 7.4–18.7 for donor 1, 14.9–19.2 for donor 2), 16.9 ± 2.0 on collagen IV (range 12.3–19.2 donor 1, 17.5–20.2 donor 2), 8.3 ± 0.7 on collagen V (range 6.1–11.8 donor 1, 8.5–9.7 donor 2), 23.4 ± 2.4 on FN (range 15.5–21.5 donor 1, 29.7–35.1 donor 2), and 7.9 ± 0.9 on LN (range 5.1–10.4 donor 1, 7.1–12.4 donor 2), with EC₅₀ values that did not differ significantly between cells grown on different ECM proteins (131–279 nM). To explore these effects further we studied cells plated on FN and LN, as these conditions afforded the greatest difference in cAMP signaling. First we confirmed the biological significance of these results by investigating the ability of the downstream target PKA to phosphorylate the endogenous substrate VASP in cells grown on different matrix factors. In keeping with the cAMP data, low doses of ISO stimulated VASP phosphorylation in cells grown on FN but not on LN (Figure 1B).

View larger version (30K): [in this window] [in a new window]   Figure 1. ECM factors alter ß2-adrenoceptor-stimulated signaling. (A) cAMP accumulation in response to isoproterenol differs between cells grown on different matrix factors: filled squares, FN; open circles, collagen IV; triangles, collagen I; inverted triangles, collagen V, diamonds, LN. Means of three wells were expressed as % of the maximal value for each experiment. Results shown are means ± SEM of three separate experiments using cells derived from two different donors. Differences in cAMP levels reached statistical significance (P < 0.05 by ANOVA and Bonferroni) at 100 nM for FN compared with collagen V or LN and at 10 µM for collagens I or IV compared with FN, collagen V, or LN. (B) ECM modulates isoproterenol (ISO)–induced phosphorylation of the downstream target vasodilator-stimulated phosphoprotein (VASP) at the log(M) concentrations indicated. Densitometry confirms significant and maximal phosphorylation of VASP with 10–7 M ISO on FN but only with concentrations at or above 10–6 M ISO on LN when compared with baseline (P < 0.05 by ANOVA). A 50-kD region of a representative blot from one of four separate experiments is shown.

View larger version (16K): [in this window] [in a new window]   Figure 2. Differences in cAMP accumulation are not due to differences in breakdown. Addition of the phosphodiesterase inhibitor IBMX to unstimulated cells (hatched bars) nonsignificantly increased cAMP accumulation over baseline (solid bars) on both matrix factors studied. Before stimulation with isoproterenol human ASM cells grown on FN (filled squares) or LN (filled diamonds) were incubated with inhibitor IBMX (on fibronectin [open squares], on laminin [open diamonds]). Means of three wells were expressed as % of the maximal value for each experiment. Results shown are means ± SEM of three separate experiments. Differences in cAMP accumulation are statistically significant (P < 0.05 by unpaired t test) at isoproterenol concentrations 10 nM between cells grown on FN or LN. This observation holds true for both the naïve and the IBMX-pretreated cells.

This hypothesis was confirmed by using FSK to directly stimulate adenylyl cyclase. In these experiments differences in cAMP production were maintained between cells on different matrix factors (Figure 3). These data suggest that the locus of control lies at the postreceptor level, and possibly involves alteration in the expression or activity of adenylyl cyclase. Further support for this interpretation comes from the observation that stimulation with PGE₂ also led to similar differential cAMP responses in cells plated on FN and LN (data not shown).

View larger version (35K): [in this window] [in a new window]   Figure 3. Differences in cAMP accumulation are maintained when adenylyl cyclase is stimulated directly. Human ASM cells grown on FN, collagen I (Col1), collagen IV (Col4), collagen V (Col5), and LN were stimulated with 10 µM of the ß-adrenoceptor agonist ISO or 10 µM of the adenylyl cyclase agonist FSK. Means of three wells were expressed as % maximum of the ISO and % maximum of the FSK in each experiment. Results shown are means ± SEM of three separate experiments. cAMP accumulation differed significantly between cells grown on FN compared with those grown on LN or collagens I or V (P < 0.001), FN compared with collagen IV (P < 0.01), or collagen IV compared with collgen V or LN (P < 0.05, all by ANOVA and Bonferroni post-test).

To extend these observations we also used Western blot analysis to compare adenylyl cyclase expression between the FN- and LN-plated cells. Previous studies suggest adenylyl cyclase V and VI are the AC isoforms predominantly expressed in human ASM cells (17, 18). Anti–adenylyl cyclase V/VI antibodies failed to detect a difference in expression levels between cells grown on the two matrix factors (n = 3, results not shown). We therefore concluded that ECM-induced differences in cAMP accumulation occur via a mechanism that does not rely on changes in adenylyl cyclase expression but instead by a mechanism producing modulation of adenylyl cyclase activity.

ECM Affects Gi Activity
The adenylyl cyclase isoforms V and VI are negatively regulated by Gi (19). To investigate whether the difference in adenylyl cyclase function was due to altered control by Gi, we treated ASM cells with FSK in the presence of 50 ng/ml PTX to block Gi activity. PTX did not significantly affect basal levels of cAMP production, but increased responses to FSK on both FN and LN. In addition, PTX completely abrogated the matrix-induced differences in cAMP responses (Figure 4B). This effect by PTX was maintained when cells were stimulated with ISO instead of FSK (Figure 4A). This implies that the difference in ß₂-receptor–mediated cAMP production between cells grown on FN and LN is due to a difference in Gi signaling.

View larger version (44K): [in this window] [in a new window]   Figure 4. Differences in Gi signaling account for the differences in cAMP accumulation. Incubating human ASM cells grown on FN (solid bars) or LN (hatched bars) with pertussis toxin (PTX) to inhibit Gi abrogates differences in cAMP accumulation in response to ISO (A) or FSK (B) Means of three wells were expressed as % of the maximum for each experiment. Results shown are means ± SEM of three separate experiments (*P < 0.05).

Expression and Activation of Gi

The protein synthesis inhibition data (see above) suggest that the differences in Gi signaling are unlikely to be due to altered Gi expression. To confirm this we also examined Gi expression by Western blotting, but failed to see upregulation of Gi expression in the LN-plated cells (Figure 5).

View larger version (40K): [in this window] [in a new window]   Figure 5. Differences in Gi signaling are not due to altered expression. Gi expression is not increased in human ASM cells grown on LN even though these cells display greater Gi activity than those grown on FN, nor is it altered compared with cells plated on plastic (CON). Results show Gi-3/1 and Gi-2 and are representative of three separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The significance of airway remodeling in patients with asthma is increasingly being recognized. It is thought that the fixed airway obstruction and decreased bronchodilatory response to ß₂-adrenoceptor agonists that develops in some individuals with asthma with chronic disease is attributable to the physically narrowed lumen and altered airway mechanics afforded by the increased mass of ASM cells and ECM (2, 3). However, a recent presentation at the American Thoracic Society conference on the follow-up of the AMPUL cohort of patients with mild to moderate asthma revealed that decline of postbronchodilator lung function exceeds the decline in prebronchodilator lung function (20). Here we propose a potential mechanism for this functional contribution to the loss of lung function in subjects with asthma: the possibility that the altered extracellular environment of the remodeled airway modulates ß₂-adrenoceptor signaling and thus attenuates the bronchodilatory response. We demonstrate that ASM cells cultured on various matrix factors relevant to the airway environment display differing responses to ß₂-adrenoceptor stimulation. As ASM cell culture is a widely accepted model to study clinically relevant signaling mechanisms (4, 11), it is possible that these observations reflect a pathological process active in the airways of individuals with asthma.

The understanding that matrix factors can influence GPCR signaling is relatively recent. Short and colleagues reported that human venous endothelial cells stimulated with ATP and ECV30 cells stimulated with ATP or lysophosphatidic acid only activate MAPK when adherent to ECM (7), though the underlying mechanism was not apparent. Conversely angtiotensin-induced MAPK signaling is decreased by ß1-integrin activation in renal mesangial cells (21). It appears that the effect of ECM on GPCR signaling is dependent not only on the matrix factor and signaling cascade but also on the receptor and cell type.

In the current series of experiments basal levels of cAMP were similar between cells plated on the various matrix factors. However, cells grown in different matrix environments exhibited marked variation in their response to ß₂-adrenoceptor stimulation. We and others have previously shown that cell number does not differ between populations grown on different ECM factors after 24 h of serum-free culture (9, 13); in the current study there was no difference in adenine uptake between matrix conditions. These observations exclude a difference in cell number or adenine cycling as the cause for the observed differences in cAMP responses.

We believe that these findings are physiologically significant in these cells, as differences in cAMP accumulation were paralleled by differences in phosphorylation rates of the endogenous downstream target VASP. VASP phosphorylation in human ASM cells appears to be a sensitive and faithful indicator of intracellular cAMP accumulation and PKA activity (22). Even though there was no apparent difference in maximum VASP phosphorylation between cells grown on FN and LN, phosphorylation occurred with lower concentrations of ISO in the FN samples. This would be in keeping with the hypothesis that effective downstream signaling and physiologic response (such as smooth muscle relaxation) occurs with lower concentrations of agonist in cells in an FN environment versus a LN environment.

In an attempt to identify the level at which ECM exerts its effect on ß₂-receptor–mediated cAMP accumulation, we first examined possible effects on breakdown. PDE enzymes are important regulators of ASM cAMP content (23), and their activity can be regulated by compartmentalization (24). PDEs thus present an obvious target for possible regulation by matrix or integrins. However, the fact that differences in cAMP accumulation in cells grown on different matrix factors were preserved in the presence of the nonselective PDE inhibitor IBMX led us to conclude that the mechanism(s) resulting in different cAMP levels were related to altered cAMP production rather than degradation.

We therefore studied the determinants of isoproterenol-stimulated cAMP production in ASM cells in greater detail. Classical pharmacologic investigation into ß₂-receptor number and binding affinity, however, failed to identify any differences between cells grown on different matrix factors. These data suggest that the difference between cAMP production on different matrix factors is likely to be due to differences in adenylyl cyclase expression or activity. The latter could be explained in two ways. First, integrins could affect ß₂-receptor coupling to the relevant Gs protein and encourage "G protein switching." In this scenario, agonist binding to the ß₂-receptor would lead to activation of Gi rather than Gs, causing decreased adenylyl cyclase activity (both because of decreased Gs signaling and because of inhibition by Gi), which would translate into decreased cAMP accumulation. G protein switching is thought to be due to altered phosphorylation states of the ß₂-adrenoceptor (25) and activation of certain integrins is known to phosphorylate cell surface receptors (26). As FN and LN bind to and activate discrete integrins, this could explain the disparate cAMP accumulation results on these matrix factors. Second, integrins could regulate adenylyl cyclase activity by affecting other adenylyl cyclase modulators. The predominant adenylyl cyclases in human ASM cells are isoforms V and VI (17, 18); both isoforms are inhibited by calcium and Gi but stimulated by protein kinase C. Integrins are known to trigger calcium release (27) and influx (28), as well as cause PKC activation (29). ß₁-Integrins are essential for Gi function in certain cell types (30), and integrin associated protein (CD47) has been shown to have pertussis-sensitive effects (31). Conceivably ECM could therefore affect any of these known adenylyl cyclase modulators and thus regulate adenylyl cyclase function. These differences would not necessarily be apparent in unstimulated cells, as the basal activity of both adenylyl cyclases V and VI are very low (19). To explore these possibilities we therefore examined adenylyl cyclase function by direct stimulation with FSK: the differences in cAMP accumulation that we initially observed when we stimulated the system via the ß₂-adrenoceptor were maintained between cells grown on various matrix factors after stimulation with FSK. These results effectively exclude significant agonist-induced G protein switching as the underlying mechanism that could account for discrepancies in matrix-modulated ß₂-mediated cAMP responses. We therefore concluded that either the amount of adenylyl cyclase enzyme or its activity is altered by matrix factors.

To explore this, we used immunoblot to examine adenylyl cyclase expression in cells grown on different ECM factors, but could not detect a difference in the amount of adenylyl cyclase V or VI enzyme in cell lysates derived from cells grown on FN and LN. Also, inhibiting de novo protein synthesis in cells plated on the two matrix factors did not abrogate the difference in cAMP levels. However, adenylyl cyclase expression correlates poorly with observed function (19), and we therefore investigated the role of Gi in modulating adenylyl cyclase function. Preincubating the cells with PTX to inhibit Gi activity before stimulation with FSK completely obliterated the difference in cAMP accumulation between ASM cells grown on FN and LN. This implies that ASM cells in an LN environment have greater constitutive Gi activity than those in an FN environment, which translates into the observed difference in ß₂-agonist– and FSK-stimulated cAMP accumulation. Altered Gi activity could stem from changes in Gi expression or activation. Immunoblots failed to show differences in Gi expression between cells grown on different matrix factors, in keeping with the results from the experiments performed using protein inhibitors. Gi activity assays, however, point toward significantly higher baseline activity in cells grown on LN, as compared with those grown on FN. The fact that the differences in Gi activity were not apparent in the experiments measuring cAMP accumulation under basal conditions or in the presence of PTX may have several explanations: Gi may not significantly affect basal cAMP levels in ASM cells because basal cAMP production may not be dependent on adenylyl cyclase activity, because the major adenylyl cyclase isoenzymes contributing to basal cAMP levels are not Gi sensitive, or because the physical interaction between Gi and adenylyl cyclase under these conditions is unfavorable. Alternatively, the cAMP assay may not have the sensitivity to confidently detect the small differences in basal cAMP accumulation in the face of unstimulated adenylyl cyclase enzyme. We concluded that the ECM affects Gi activity and not expression.

Integrins could potentially link the ECM to Gi in several ways. Integrin clustering is a crucial step in focal adhesion formation, and the recruitment and regulation of scaffolding proteins in these cell signaling centers (32). Numerous studies demonstrate that GPCR signaling is highly compartmentalized and that experimentally disrupting this organization can dramatically affect function (33, 34). Integrins as well as G proteins localize to detergent insoluble glycoprotein domains, their association with the PTX-sensitive CD47 molcule dependent on cholesterol (35). Similarly caveolin-1, which binds integrin -subunits, is a requirement for integrin signaling (36) and can also regulate G protein–mediated signaling (37). We have previously shown that the ECM affects cytoskeletal morphology in ASM cells (8), which could form the basis of altered integrin–Gi or Gi–adenylyl cyclase interaction in these cells.

These findings offer a novel explanation for the diminished effect of ß₂-agonists in patients with chronic asthma. Disease duration and severity have been correlated with markers of airway remodeling, including airway wall thickness in patients with asthma (38). Collagen V and LN are increased in remodeled airways (2) and are also the matrix environments in which ß₂-adrenoceptor–mediated cAMP production was most depressed in our experiments. It is therefore conceivable that deposition of altered ECM in the airways of patients with asthma contributes not only to changes in airway mechanics, but also affects the response of bronchial smooth muscle to bronchodilator medication.

	Acknowledgments

 
This study has been funded by a grant from the National Asthma Campaign UK and HL58506 and HL65338. R.B.P. is the recipient of an American Lung Association Career Investigator Award.

Received in original form June 24, 2003

Received in final form May 25, 2004

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