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Am J Physiol Lung Cell Mol Physiol 291: L923-L931, 2006. First published July 7, 2006; doi:10.1152/ajplung.00185.2006
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Functional expression of the GABAB receptor in human airway smooth muscle

Yoko Osawa,1 Dingbang Xu,1 David Sternberg,2 Joshua R. Sonett,2 Jeanine D’Armiento,3 Reynold A. Panettieri,4 and Charles W. Emala1

Departments of 1Anesthesiology, 2Surgery, and 3Medicine, College of Physicians and Surgeons of Columbia University, New York, New York, and 4Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

Submitted 22 May 2006 ; accepted in final form 6 July 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
{gamma}-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system and exerts its actions via both ionotropic (GABAA/GABAC) and metabotropic (GABAB) receptors (R). In addition to their location on neurons, GABA and functional GABAB receptors have been detected in nonneuronal cells in peripheral tissue. Although the GABABR has been shown to function as a prejunctional inhibitory receptor on parasympathetic nerves in the lung, the expression and functional coupling of GABAB receptors to Gi in airway smooth muscle itself have never been described. We detected the mRNA encoding multiple-splice variants of the GABABR1 and GABABR2 in total RNA isolated from native human and guinea pig airway smooth muscle and from RNA isolated from cultured human airway smooth muscle (HASM) cells. Immunoblots identified the GABABR1 and GABABR2 proteins in human native and cultured airway smooth muscle. The GABABR1 protein was immunohistochemically localized to airway smooth muscle in guinea pig tracheal rings. Baclofen, a GABABR agonist, elicited a concentration-dependent stimulation of [35S]GTP{gamma}S binding in HASM homogenates that was abrogated by the GABABR antagonist CGP-35348. Baclofen also inhibited adenylyl cyclase activity and induced ERK phosphorylation in HASM. Another GABABR agonist, SKF-97541, mimicked while pertussis toxin blocked baclofen’s effect on ERK phosphorylation, implicating Gi protein coupling. Functional GABAB receptors are expressed in HASM. GABA may modulate an uncharacterized signaling cascade via GABAB receptors coupled to the Gi protein in airway smooth muscle.

G protein; adenylyl cyclase; mitogen-activated protein kinase; [35S]GTP{gamma}S binding; guinea pig; trachea

GAMMA-AMINOBUTYRIC ACID (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system (CNS). GABA acts at two distinct types of receptors, ligand-gated ionotropic GABAA and GABAC receptors, and G protein-linked metabotropic (GABAB) receptors (R). The GABAB receptor is composed of two subunits (GABABR1 and GABABR2), and to date four splice variants of the human GABABR1 have been described (2, 15, 17, 26). The GABAB receptor typically functions as a Gi protein-coupled receptor and is fully functional only when both subunits are expressed and are linked in a heterodimeric assembly (2). In the CNS, presynaptically located GABABRs suppress neurotransmitter release by inhibiting voltage-sensitive Ca2+ channels (14). Postsynaptically, GABABRs stimulation causes inhibition of adenylyl cyclase via the Gi protein as well as activation of the Kir3 type potassium channels by liberated Gbeta{gamma}-subunits (15). GABABRs are also capable of directly interacting with transcription factors and can regulate gene transcription regulation upon stimulation (29). In addition to their presence on neurons, GABA and functional GABAB receptors have been detected in peripheral tissues such as adrenal medulla, islets of Langerhans, placenta, and smooth muscle cells of the urinary bladder and uterus. However, the expression and functional coupling of GABAB receptors to Gi in airway smooth muscle has never been described.

It is known that GABAB-specific agonists decrease airway responsiveness to various bronchoconstricting agents by modulating presynaptic acetylcholine release from parasympathetic nerves (6, 28). On the other hand, a GABAB receptor agonist, baclofen, can worsen airway responses following the administration of methacholine to asthmatic patients (7). This paradoxical enhancement by baclofen of airway responsiveness led us to hypothesize that there may be postganglionic (i.e., smooth muscle) GABAB functional receptors that couple to the Gi protein, known to impair relaxation of airway smooth muscle (3, 23).

In the present study, we investigated the expression of GABAB receptors in native guinea pig, human airway smooth muscle (HASM), and cultured HASM cells, and assessed the functional coupling of the GABAB receptor to the Gi protein by demonstrating adenylyl cyclase inhibition and ERK activation.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cells were cultured in SmGM-2 smooth muscle medium (Cambrex, Walkersville, MD). [35S]GTP{gamma}S (1,250 Ci/mmol) was obtained from Perkin Elmer (Boston, MA). [{alpha}-32P]ATP (800 Ci/mmol) and [3H]cAMP (32 Ci/mmol) were obtained from MP Biomedicals (Irvine, CA). Human brain protein was obtained from BD Biosciences (Palo Alto, CA). All other chemicals were obtained from Sigma (St. Louis, MO) unless otherwise stated.

Cell culture. Primary cultures of HASM cells were obtained from lung transplant donors in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings as previously described (20). The cells were grown to confluence on 24- (adenylyl cyclase) or 6-well (immunoblotting) plates in culture medium (SmGM-2 supplemented with 5% FBS, 5 µg/ml insulin, 1 ng/ml human fibroblast growth factor, 500 pg/ml human epidermal growth factor, 30 µg/ml gentamicin, and 15 ng/ml amphotericin B, Cambrex) at 37°C in 5% CO2-95% air. In all studies, cell culture media was not changed for 72 h (conditioned media) before the beginning of treatment with GABAB agonists.

For analysis of ERK phosphorylation, cells were treated with GABAB agonists (baclofen or SKF-97541) for 5 min in 72-h conditioned culture medium. Pertussis toxin (100 ng/ml) was preincubated for 4 h prior to the addition of GABAB agonists. After treatment, cells were rinsed with cold phosphate-buffered saline (PBS), and ice-cold lysis buffer [50 mM Tris·HCl, pH 8.0, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1:200 dilution of protease inhibitor cocktail III (Calbiochem, San Diego, CA), 1 mM Na3VO4, 1 mM NaF] was added. Thereafter, the whole cell lysates were sonicated four times on ice for 15 s, and the protein concentration was determined. Cell lysates were solubilized by heating at 95°C for 5 min in sample buffer (final concentrations: 50 mM Tris·HCl, pH 6.8, 2.5% SDS, 6% glycerol, 2.5% 2-mercaptoethanol, bromophenol blue) and were stored at –20°C.

Isolation of smooth muscle from human trachea and guinea pig trachea. Studies were approved by Columbia University’s Institutional Review Board and deemed not human subjects research under 45 CFR 46. Human trachea came from two sources. Snap-frozen tracheas obtained at autopsy from nonasthmatic adults within 8 h of death were obtained from the National Disease Research Exchange (Philadelphia, PA). Additional tracheas were obtained from discarded regions of healthy donor lungs harvested for lung transplantation at Columbia University. Lung transplant excess tissue was transported to the laboratory in cold (4°C) Krebs-Henseleit buffer (in mM: 118 NaCl, 5.6 KCl, 0.5 CaCl2, 0.24 MgSO4, 1.3 NaH2PO4, 25 NaHCO3, 5.6 glucose, pH 7.4). Frozen tracheas were thawed in cold Krebs-Henseleit buffer.

Adult male guinea pigs were deeply anesthetized by intraperitoneal pentobarbital, the chest cavity was opened, and the animal was exsanguinated before the dissection of the trachea. The trachea was surgically removed and placed in cold (4°C) PBS.

The exterior of either guinea pig or human trachea was dissected free of adhering connective tissue under a dissecting microscope. Tracheas were then cut open longitudinally along the anterior border. The tracheal epithelium was removed, and the airway smooth muscle between the noncontiguous ends of the cartilaginous tracheal rings was dissected free and homogenized in cold (4°C) buffer (10 mM HEPES, pH 7.4, 1 mM EDTA with a 1:200 dilution of protease inhibitor cocktail III) using a Tekmar Ultra Turrax T25 high-speed homogenizer set at top speed for 30 s. The homogenate was filtered through 125-µm Nitex mesh and centrifuged at 1,000 g for 10 min at 4°C. The supernatant was transferred into new tubes and centrifuged at 50,000 g for 15 min at 4°C. The pellet was resuspended in the same buffer and centrifuged at 50,000 g for 15 min at 4°C. The final pellet was resuspended in the same buffer and stored at –80°C.

RNA isolation and RT-PCR. Total RNA was extracted from freshly dissected native guinea pig or HASM, cultured HASM cells, and guinea pig whole brain using TRI Reagent (Ambion, Austin, TX) according to the manufacturer’s recommendations. Total RNA from whole human brain was purchased from Clontech and used as a positive control. Using the Advantage RT-for-PCR Kit (Clontech, Mountain View, CA), 1 µg of total RNA was reverse transcribed at 42°C for 1 h in 20 µl including 200 units of Moloney murine leukemia virus reverse transcriptase, 20 units of RNase inhibitor, 20 pmol oligo(dT) primer, and 0.5 mM each of dNTP mix in reaction buffer (50 mM Tris·HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2).

PCR was performed by adding 5 µl of newly synthesized cDNA to a 45-µl reaction mixture yielding final concentrations of 0.2 mM of each dNTP, 1x Advantage 2 Polymerase Mix, PCR buffer (Clontech, Mountain View, CA), and 0.4 µM of both sense and antisense primers (Sigma) for corresponding GABABR subunits including four splice variants of human GABABR1 (Table 1, Fig. 1). For the detection of the GABABR1b splice variant, 20% glycerol was added in the PCR reaction mixture because of a GC-rich sequence of GABABR1b. Two-step PCR (annealing and extension at same temperature) was performed with a PTC-200 Peltier thermal cycler (MJ Research, Waltham, MA). PCR conditions for all reactions included an initial denaturation step at 94°C for 1 min, 40 cycles of a denaturation step at 94°C for 10 s, and an annealing/extension step at 72°C for 1 min except for PCR amplification of human GABABR1e, which used an annealing/extension step at 70°C for 1 min, and for amplification of human GABABR1a/c, which used an annealing/extension time of 2 min. PCR products were electrophoresed on 5% nondenaturing polyacrylamide gel in 1x Tris, acetate, EDTA buffer. The gel was stained with ethidium bromide (Molecular Probes, Eugene, OR), visualized using ultraviolet illumination, and analyzed using Quantity One software (BioRad, Hercules, CA).


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  		Table 1. GABABR1 and GABABR2 primer sequences

 

Figure 1
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  		Fig. 1. Schematic summary of the currently known 4 isoforms of the human GABABR1 subunit and primer design used in the present study. Human GABABR1a contains 22 exons indicated by numbered white boxes 1-22. An alternatively spliced exon of GABABR1b is indicated by a gray box. Gaps linked by a "V" represent omitted exons in GABABR1c and GABABR1e. The locations of sense and antisense primers that were used in RT-PCR analysis for each GABABR1 subunit are indicated by arrows.

 
Immunoblotting. Whole cell or tissue lysates were electrophoresed (8–10% SDS-PAGE) and immunoblotted using antibodies directed against GABABR1 (rabbit 1:1,000, sc-14006; Santa Cruz Biotechnology, Santa Cruz, CA), GABABR2 (rabbit 1:1,000, AB5848; Chemicon, Temecula, CA), and total or phospho-ERK (both rabbit 1:1,000, nos. 9102 and 9101, respectively; Cell Signaling Technology, Danvers, MA). Epitopes were visualized with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, 1:5,000, Santa Cruz Biotechnology) using ECL Plus (Amersham Biosciences) and developed on film (Kodak BioMax light film; Kodak, Rochester, NY). Film was developed such that band intensities were within the linear range of film responses, and band intensities were quantified using Quantity One software (Bio-Rad). Data presented are means ± SE.

Immunohistochemistry. Guinea pig tracheal rings were fixed using 10% formalin for 24 h at room temperature for GABABR1 immunostaining and using 4% paraformaldehyde/1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 4 h at 4°C for GABA immunostaining. Tracheal rings were paraffin embedded, sectioned (5 µm), dewaxed in xylene, and rehydrated in a graded alcohol series to water. Endogenous peroxidase was blocked in 0.3% hydrogen peroxide. Heat-mediated antigen retrieval was performed with 10 mM sodium citrate buffer, pH 6.0 for 30 min. An avidin biotin blocking kit (Vector Laboratories, Peterborough, UK) was used (in 10% serum in PBS) to block endogenous biotin. Slides were rinsed with PBS and incubated overnight at 4°C in primary antibody against GABABR1 (rabbit, sc-14006, Santa Cruz Biotechnology) or GABA (mouse, MAB316, Chemicon) at a concentration of 1:250 or 1:50 in 2% serum in PBS, respectively. A tracheal ring section was incubated with the appropriate isotype IgG antibody as a negative control. A tracheal ring section was also incubated with a primary antibody directed against human {gamma}- and {alpha}-smooth muscle actin (1:10,000, mouse, MAB1522, Chemicon) to identify smooth muscle in trachea. Following overnight incubation at 4°C, slides were washed with PBS, and primary antibodies were detected using biotinylated anti-mouse or anti-rabbit antibodies (Vector Laboratories) at a concentration of 1:100. The antigen antibody complex was then visualized by the enzymatic reduction of 3,3-diaminobenzidine tetrahydrochloride. Sections were counterstained with hematoxylin and dried, and cover slides were mounted using Poly-mount (Polysciences, Warrington, PA).

GTP{gamma}S binding assays. The binding of [35S]GTP{gamma}S was assayed as follows. Smooth muscle homogenates from human trachea (50 µg of protein) were resuspended in reaction buffer (final volume 200 µl) containing 10 mM HEPES-NaOH, 100 mM NaCl, 10 mM MgCl2 (pH 7.4), and 10 µM GDP in the absence or presence of the GABAB receptor agonist R(+)-baclofen HCl (10–1,000 µM) or with 300 µM baclofen in the presence of the GABAB receptor antagonist CGP-35348 [(3-aminopropyl)(diethoxymethyl)phosphinic acid, 1 mM]. The reaction was initiated by the addition of [35S]GTP{gamma}S (final concentration 0.3 nM) and incubated for 30 min at 30°C. The reaction was stopped by adding 3 ml of ice-cold buffer containing 10 mM HEPES, 100 mM NaCl, and 5 mM MgCl2 (pH 7.4), immediately followed by rapid filtration on glass fiber filters presoaked in the same buffer. The filter was washed five times with 3 ml of the same buffer, and the radioactivity trapped was determined by liquid scintillation spectrometry. Nonspecific binding was determined in the presence of 200 µM GTP and corresponded to <10% of total binding. Assays were performed in triplicate.

Adenylyl cyclase assays. Adenylyl cyclase activity was measured as previously described (24). Briefly, confluent cultured HASM cells in 24-well plates were washed once with warm PBS (37°C). One hundred microliters of warm PBS were added to each well. Subsequently, 50 µl of 3x adenylyl cyclase buffer (13) with or without 100 µM baclofen were added directly to the wells [to achieve final concentrations of 50 mM HEPES, pH 8.0, 50 mM NaCl, 0.4 mM EGTA, 1 mM cAMP, 7 mM MgCl2, 0.1 mM ATP (20 µCi/ml [{alpha}-32P]ATP), 0.1 mg/ml BSA, 50 U/ml creatine phosphokinase, and 7 mM phosphocreatine] in the presence of 10 µM forskolin, and plates were incubated at 37°C for 15 min. The reactions were terminated by the addition of 100 µl of stop buffer [50 mM HEPES, pH 7.5, 2 mM ATP, 0.5 mM cAMP (0.5 µCi/ml [3H]cAMP), 2% SDS], and newly synthesized [32P]cAMP was separated by sequential column chromatography over Dowex and alumina (24). Recovery of [3H]cAMP was used to correct for individual column recoveries, and radioactivity was quantitated by scintillation counting.

Statistics. Statistical analysis was performed using repeated measures of ANOVA, followed by Bonferroni posttest comparison using Prism 4.0 software (GraphPad, San Diego, CA). Data are presented as means ± SE; P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
RT-PCR analysis of GABABR isoforms in airway smooth muscle. RT-PCR analysis demonstrated mRNA encoding four splice variants of the human GABABR1 protein (Fig. 2, A–C) and the GABABR2 protein (Fig. 2D) in both native human tracheal smooth muscle and cultured HASM cells. Several splice variants of the GABABR1 protein are expressed in human tissues, and we used primer sets designed to distinguish between each of these GABABR1 variants. The expression of mRNA encoding the GABABR1a and 1c variants was examined using a common primer set and was distinguishable by virtue of the omitted exons in GABABR1c, resulting in a smaller PCR product for GABABR1c (1,343 bp) compared to GABABR1a (1,529 bp; Fig. 2A). We identified PCR products corresponding to the expected size of GABABR1a and GABABR1c in both native human tracheal airway smooth muscle and cultured HASM cells (Fig. 2A). Additionally, mRNA encoding the splice variant GABABR1b was detected in both native HASM and cultured HASM cells (Fig. 2B). We then confirmed the existence of GABABR1e using primers designed to flank the omitted exons of GABABR1e and therefore could distinguish 1e from other isoforms (i.e., 1a, b, and c). We observed two PCR products of predicted sizes corresponding to splice variants GABABR1a, b, and c (302 bp) and GABABR1e (150 bp) in human trachea and cultured HASM cells (Fig. 2C). In human brain, used as a positive control, GABABR1e was detected as a faint band. mRNA encoding GABABR2 was also detected in native and cultured HASM (Fig. 2D). Guinea pig airway smooth muscle also expressed mRNA encoding both the GABABR1 and GABABR2 protein (Fig. 2E).


Figure 2
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  		Fig. 2. Representative RT-PCR analysis of total RNA using primers that specially recognize mRNA for each GABABR subtype. Expression of GABABR1a (expected size: 1,529 bp) and 1c (1,343 bp) subunits (A); GABABR1b (272 bp; B); GABABR1a, b, c (302 bp), and GABABR1e (150 bp; C); and GABABR2 (239 bp; D) in human tracheal airway smooth muscle (ASM) and cultured human ASM (HASM) cells. Human brain cDNA was used as a positive control. E: expression of GABABR1 and R2 in guinea pig (GP) tracheal ASM. Expected product sizes were 446 bp for the R1 and 348 bp for the R2. GP brain was used as a positive control.

 
Immunoblot analysis of GABABR isoforms in airway smooth muscle. The antibody used for immunoblot analysis of GABABR1 recognizes the common COOH terminus region of three of the four known human isoforms of GABABR1 (i.e., GABABR1a, b, and c, but not GABABR1e). Two immunoreactive bands of ~100 and 130 kDa were identified in freshly isolated human and guinea pig tracheal smooth muscle or whole brain from both species (Fig. 3A). Whereas the larger 130-kDa band corresponds to the expected molecular mass of GABABR1a, the 100-kDa band corresponds to the molecular mass of either GABABR1b or GABABR1c. In contrast, only the larger immunoreactive band corresponding to the GABABR1a protein was identified in cultured HASM cells (Fig. 3A). An antibody raised against the GABABR2 protein identified a single immunoreactive band of ~110 kDa in protein samples from whole human brain and both native and cultured HASM (Fig. 3B), consistent with the predicted size for GABABR2. This antibody was weakly reactive using a protein sample from whole guinea pig brain (data not shown), and thus this antibody was considered not sufficiently reactive with guinea pig protein samples to analyze guinea pig airway smooth muscle expression.


Figure 3
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  		Fig. 3. Representative immunoblot analyses using protein prepared from GP brain, GP tracheal ASM, whole human brain, human tracheal ASM, and cultured HASM cells. A: immunoblot with antibody directed against GABABR1a (130 kDa) and GABABR1b and c (100 kDa). Whole human brain (25 µg), smooth muscle isolated from human trachea (100 µg), cultured HASM cells (100 µg), GP brain (25 µg), and smooth muscle isolated from GP trachea (50 µg) were analyzed. M.W., molecular weight. B: immunoblot with antibody against GABABR2, whole human brain (25 µg), smooth muscle isolated from human trachea (100 µg), and cultured HASM cells (100 µg).

 
Immunohistochemical analysis of GABABR1 and GABA expression in guinea pig trachea. To confirm the protein localization of GABABR1 to airway smooth muscle in guinea pig airways, immunohistochemistry was performed. GABABR1 immunoreactivity in guinea pig trachea was detected in airway smooth muscle, airway epithelium, and tracheal cartilage chondrocytes (Fig. 4A) with no staining in the negative control studies with rabbit IgG (Fig. 4B). The identity of the airway smooth muscle layer (and vascular smooth muscle) was confirmed using an anti-{alpha}-smooth muscle actin antibody (Fig. 4C), and no staining was seen with control mouse IgG2a (Fig. 4D).


Figure 4
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  		Fig. 4. Representative immunohistochemical staining of GABABR1 (A) and {alpha}-smooth muscle (SM) actin (C) in formalin-fixed GP trachea. The GABABR1 was localized in tracheal epithelium (Epi), tracheal SM, and cartilage (CL), whereas {alpha}-SM actin was localized in tracheal and vascular smooth muscle. B: isotype controls for GABABR1. D: isotype control for {alpha}-SM actin. All sections were counterstained with hematoxylin (original magnification, x100).

 
Next, we examined whether the endogenous ligand for the GABAB receptor, GABA, was expressed in guinea pig airways. GABA was localized to epithelium, chondrocytes, and an area immediately adjacent to airway smooth muscle with limited staining in small punctuate areas within the airway smooth muscle itself (Fig. 5, A and C). No specific staining was apparent within these structures using a negative control, mouse IgG1 (Fig. 5, B and D).


Figure 5
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  		Fig. 5. Representative immunohistochemical staining of GABA (A and C) and isotype control (B and D) in paraformaldehyde-glutaraldehyde-fixed GP trachea. GABA was localized in Epi, chondrocytes (CL), and connective tissue near the SM. Limited positive staining was also found in ASM [original magnification, x100 (A and B) and x200 (C and D)].

 
Functional coupling of the GABAB receptor to Gi signaling pathways in HASM. Demonstration of GABAB receptor mRNA and protein in airway smooth muscle led us to determine whether the receptor demonstrated classical coupling to the Gi protein by evaluating 1) agonist-induced enhanced [35S]GTP{gamma}S binding, 2) agonist-induced inhibition of adenylyl cyclase activity, and 3) pertussis toxin-sensitive, agonist-induced activation of mitogen-activated protein kinase (MAPK ERK).

Agonist-enhanced [35S]GTP{gamma}S binding in native HASM. Baclofen, a specific GABAB agonist, elicited a concentration-dependent stimulation of [35S]GTP{gamma}S binding with a 152 ± 6% increase above basal values (P < 0.001, n = 3) at 1 mM baclofen (Fig. 6A). A GABAB antagonist, CGP-34358 (1 mM), blocked baclofen (300 µM)-induced increases (P < 0.001, n = 3) in [35S]GTP{gamma}S binding (Fig. 6B). These results suggest that GABAB receptors are functionally coupled to a G protein in HASM.


Figure 6
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  		Fig. 6. A: concentration-dependent stimulation of [35S]GTP{gamma}S binding by baclofen in native human tracheal smooth muscle. Tissue membranes (50 µg of protein) were incubated with the indicated concentrations of baclofen for 30 min. *P < 0.05, ***P < 0.001 compared with control. B: effect of CGP-35348 (1 mM) on the stimulation of [35S]GTP{gamma}S binding by baclofen. Tissue membranes were incubated with 300 µM baclofen in the presence or absence of 1 mM CGP-35348 for 30 min. Data are expressed as percentage of basal [35S]GTP{gamma}S binding and are the means ± SE of 3 experiments. Each experimental value was determined in triplicate. ***P < 0.001 compared with control. ###P < 0.001 compared with baclofen. Bacl, baclofen; cont, control.

 
Agonist-induced inhibition of adenylyl cyclase activity in human cultured airway smooth muscle cells. GABAB receptor-mediated inhibition of adenylyl cyclase activity via coupling to the Gi protein is well known in neurons (12). To examine whether baclofen inhibits the adenylyl cyclase activity in cultured HASM cells, we measured 10 µM forskolin-stimulated adenylyl cyclase activity in the presence or absence of 100 µM baclofen. Baclofen significantly inhibited forskolin-stimulated adenylyl cyclase activity (P < 0.01, n = 8; Fig. 7).


Figure 7
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  		Fig. 7. Ten micromolar forskolin-stimulated adenylyl cyclase activity in cultured HASM cells in the presence or absence of baclofen (100 µM). N = 8 experiments; within each experiment, values were determined in triplicate. Data represent means ± SE. **P < 0.01 compared with control.

 
Agonist-induced activation of ERK in cultured HASM cells. Many Gi-coupled receptors are known to activate the ERK pathway via G protein beta{gamma}-subunits (11). ERK activation was identified by increased phospho-ERK on immunoblots and expressed as a ratio of phospho/total ERK for each sample. Baclofen (100 µM, 5 min) significantly increased phosphorylation of ERK (Fig. 8, left; n = 3, P < 0.05). In separate experiments, a second selective GABAB agonist, SKF-97541, also increased ERK phosphorylation (Fig. 8, right; P < 0.01, n = 3). Four-hour pretreatment of cells with pertussis toxin (100 ng/ml) abrogated increases in ERK phosphorylation by either GABAB agonist (P < 0.05 and P < 0.001 for pertussis toxin + agonist vs. baclofen or SKF-97541 alone, respectively, n = 3) confirming the role of Gi in GABAB receptor activation of ERK.


Figure 8
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  		Fig. 8. A: Western blot analysis of phospho-ERK or total ERK expression in cultured HASM cells. Cells were treated with 100 µM baclofen (left) or 100 µM SKF-97541 (right) for 5 min in the presence or absence of pertussis toxin (100 ng/ml for 4 h pretreatment prior to baclofen or SKF-97541 treatment). A representative of 3 experiments is shown. B: relative band intensities of phospho-/total ERK from 4 separate immunoblots. The effect of baclofen (left) and SKF-97541 (right) on ERK phosphorylation is given as percentage of control. Means ± SE. *P < 0.05, **P < 0.01 compared with control. #P < 0.05, ###P < 0.001 compared with baclofen or SKF. PTX, pertussis toxin.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study is the first to demonstrate that functional GABAB receptors are expressed in both native human and guinea pig airway smooth muscle and cultured HASM cells. Both GABABR1 and GABABR2 subunits were detected at the level of mRNA (by RT-PCR) and protein (by immunoblot) at appropriate molecular weights. Immunocytochemistry localized the GABABR1 subunit to airway smooth muscle in guinea pig tracheal rings. The GABAB receptor agonist baclofen significantly increased GTP{gamma}S binding (implying G protein coupling) in a manner sensitive to the GABAB receptor antagonist CGP-35348. Baclofen also inhibited adenylyl cyclase activity and induced ERK phosphorylation, which was abrogated by pretreatment of cultured HASM cells with pertussis toxin, confirming classical coupling of the GABAB receptor to Gi protein signaling pathways in these cells.

Several studies indicate the possible expression of functional GABAB receptors in peripheral and nonneuronal cells such as pancreatic beta cells (4), adrenocortical cells (18), cardiomyocytes (16), chondrocytes (27), and osteoblasts (9). Furthermore, GABABR1 mRNA expression was detectable using RT-PCR analysis in many peripheral organs including heart, spleen, lung, liver, intestine, kidney, stomach, adrenal gland, testis, ovary, and urinary bladder (5). Although mRNAs encoding GABABR1a and 1b have been reported in RNA isolated from whole lung (5), the present study is the first to demonstrate the expression of both the GABABR1 and GABABR2 localized to airway smooth muscle.

Recent evidence has shown that GABAB receptors must exist as a heterodimer to form a functional Gi protein-coupled receptor in the plasma membrane (2). The presence of both subunits in airway smooth muscle cells suggests that they can assemble functional GABAB receptor heterodimers. We demonstrated that baclofen was effective in activation of G protein as well as inhibition of adenylyl cyclase, thus showing that functional GABAB receptors are expressed in HASM. To date, four different splice variants (GABABR1a, b, c, and e) of GABABR1 have been identified in the human. Indeed, we identified not only the expression of major splice variants (GABABR1a and 1b) but also minor variants 1c and 1e in HASM. The GABABR1e splice variant encodes a truncated protein lacking the transmembrane and intracellular domains and is known to be present in a variety of peripheral human tissues. At the present time, there are no confirmed GABABR2 splice variants (2). The GABABR1e is suggested to compete for heterodimerization with the GABABR2 subunit and affect the formation of functional GABAB receptors in a dominant negative manner (26). In the present study, a high concentration of baclofen was needed to achieve significant stimulation of [35S]GTP{gamma}S binding. This may be due to the low expression of GABAB receptors in airway smooth muscle relative to brain. However, the expression of the GABABR1e subunit in airway smooth muscle may decrease the number of functional GABAB receptors.

Although we identified mRNA encoding all four known splice variants of the GABABR1 subunit and the GABABR2 subunit in RNA isolated from freshly dissected human and guinea pig airway smooth muscle, RNA isolated from freshly dissected tissues invariably will contain some RNA from nonmuscle cells despite careful dissection. Therefore, to confirm the smooth muscle cell-specific expression of the GABABR1 and GABABR2 subunits, we analyzed RNA isolated from homogenous cultures of HASM cells and confirmed the expression of the same splice variants of GABABR1 and GABABR2 specifically in airway smooth muscle cells. Furthermore, immunohistochemistry in guinea pig tracheal rings localized the GABABR1 protein to the airway smooth muscle layer. In HASM, the GABABR1 antibody we used for immunoblot analysis reacted strongly and apparently nonspecifically with a 50-kDa protein making its use for immunocytochemistry in HASM unreliable. Interestingly, the GABABR1 protein was also abundantly expressed in tracheal epithelial cells and chondrocytes. Whereas the identification of the GABAB receptor on chondrocytes has been previously described (9, 27), the identification of the GABAB receptor on airway epithelium is novel and suggests that multiple cell types in the airway may be responsive to endogenous GABA.

The endogenous ligand for GABAB receptors, GABA, has also been identified in many peripheral tissues, especially in endocrine organs such as the pituitary, pancreas, testis, gastrointestinal tract, ovary, placenta, uterus, and adrenal medulla (10). Peripheral GABA has been suggested to act not only as a neurotransmitter or neuromodulator in the autonomic nervous system but also as a hormone or trophic factor in nonneuronal tissue (19). To identify the existence and localization of GABA in airway, we performed immunohistochemical analysis using a specific antibody against GABA in guinea pig trachea. In the present study, we detected GABA immunoreactivity in the connective tissue near the smooth muscle as well as tracheal epithelium and cartilage chondrocytes. This result suggests that GABA may bind to GABAB receptors in airway smooth muscle, although the origin of GABA in airway is still unclear.

After demonstrating the mRNA and protein expression of GABAB receptors in airway smooth muscle, we sought to confirm its coupling to G proteins in general and its specific classical coupling to the Gi protein. Activation of GTP binding is a standard measure of receptor coupling to heterotrimeric G proteins, and indeed the GABAB receptor agonist baclofen enhanced GTP binding in HASM. Two Gi-specific coupling pathways were investigated using the GABAB agonist baclofen. Inhibition of adenylyl cyclase is a well-known effect of Gi protein activation and is known to occur in airway smooth muscle in response to activation of several Gi-coupled receptors (e.g., M2 muscarinic receptor). Activation by phosphorylation of ERK is a ubiquitous signaling pathway following Gi activation, and pertussis toxin is a widely used tool to inactivate and implicate Gi proteins in signaling events. Indeed, baclofen inhibited adenylyl cyclase and activated ERK phosphorylation in a pertussis toxin-sensitive manner in HASM cells, confirming the coupling of GABAB receptors to Gi proteins in these cells. The specificity of baclofen’s effect at the GABAB receptor in the present study is supported by the finding that the antagonist CGP-35348 blocked the effect of baclofen in GTP{gamma}S binding and by the finding that another GABAB agonist, SKF-97541, could mimic the baclofen’s effect on ERK activation.

The physiological role of GABAB receptors in airway smooth muscle and of GABA-ergic modulation of intercellular cAMP or ERK activation is at present unclear. Because cAMP is known to induce relaxation of airway smooth muscle, an inhibitory effect of baclofen on adenylyl cyclase suggests that GABAB receptor activation in airway smooth muscle could inhibit cAMP-mediated relaxation. A well known example of a Gi-coupled receptor that modulates airway smooth muscle relaxation is the M2 muscarinic receptor. The M2 muscarinic receptor couples to Gi, inhibits adenylyl cyclase, and is known to inhibit beta2-adrenoceptor-induced smooth muscle relaxation (25), whereas M2 muscarinic receptor antagonists are known to facilitate isoprenaline- and forskolin-mediated relaxation of acetylcholine-induced contraction of airway smooth muscle (8). These findings support the idea that the GABAB receptor could modulate airway smooth muscle tone via activation of the Gi protein. Consistent with this, GABAB receptors have been implicated in modulation of contractility in the rabbit uterus (22), where the receptors appear to be nonneuronal, and are most likely expressed in smooth muscle cells.

Baclofen can increase cell proliferation in a GABAB receptor antagonist-sensitive manner in rat growth plate chondrocytes (27). In the present study, we found that baclofen induced ERK phosphorylation. This result was consistent with observations in HEK-293 cells transfected with GABABR1 and R2 subunits (1). The requirement for ERK activation in HASM mitogenic signaling pathways has been well established (21). Since hyperplasia and hypertrophy of smooth muscle is considered to contribute to airway hyperresponsiveness in asthma (30), stimulation of GABAB receptors coupling to ERK activation would theoretically favor HASM cell proliferation and be associated with airway hyperresponsiveness. Further investigations are required to identify the physiological and possibly pathophysiological role of GABAB receptors in airway smooth muscle cells.

Although the source of the endogenous ligand GABA for GABAB receptors in airway is unclear at present, GABA may modulate an uncharacterized signaling cascade via GABAB receptors expressed in airway smooth muscle. This signaling cascade could be a target for new therapeutic interventions in controlling airway tone.


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This work was supported by National Heart, Lung, and Blood Institute Grant HL-58519.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. W. Emala, Dept. of Anesthesiology, College of Physicians and Surgeons of Columbia Univ., 630 W. 168th St., P&S Box 46, New York, NY 10032 (e-mail: cwe5{at}columbia.edu)

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.


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 REFERENCES
 

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