Introduction

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In conditions characterized by airflow obstruction, such as asthma and chronic obstructive pulmonary disease (COPD), there may be structural alterations in the airway wall in addition to alterations in airway tone. The thickening of the bronchial smooth muscle in these conditions contributes to increased tone in the asthmatic airway and ultimately leads to fixed airflow obstruction in COPD (1). Proliferation and hypertrophy of airway smooth-muscle (ASM) cells (ASMCs) are implicit in this process of airway remodeling (2).

Many endogenous mediators have been implicated in the control of airway tone and structure. Of these, endothelin (ET)-1 has an important role in the human airway, being a potent constrictor of ASM (3, 4), and a promoter of ASMC growth (5). ET is expressed and secreted by airway epithelial cells (6, 7) and vascular endothelial cells, as well as inflammatory cells such as macrophages (7). Evidence supporting a role for ET-1 in the pathogenesis of airway disease comes from studies demonstrating increased levels of ET-1 in bronchoalveolar lavage fluid from patients with asthma (8), and increased expression of ET-1 in biopsies of asthmatic airways (6). ET_A and ET_B receptor subtypes have been identified in human ASM (9) and cultured ASMCs (5, 10). Both ET_A and ET_B receptors have been reported to mediate bronchoconstriction in humans (4, 11), but only the ET_A receptor appears to be involved in ASMC proliferation (5).

Another peptide with a potentially important role in the human airway is adrenomedullin (ADM). This recently discovered peptide, originally isolated from human phaeochromocytoma (12), has been intensively studied in the circulation, where it acts as vasodilator (13, 14). It is expressed at a higher level in the lung than in other tissues, except the adrenal glands (15, 16), and is a potent bronchodilator in guinea pigs (17). In human lung, immunohistochemical staining has demonstrated that ADM is expressed in ASM (18), and a high density of ADM receptors has also been demonstrated in rat lung (19). Distinct cross talk has been demonstrated between ADM and ET in vascular cells, where ADM inhibits ET release (20) and ET stimulates ADM release (21). Recently it was shown that ADM secretion by canine aortic endothelial cells is enhanced by activation of the ET_B receptor (22).

Despite the potential importance of ADM in the control of airway tone and remodeling, there is no information regarding the regulation of ADM secretion from ASMCs. We hypothesized that ADM is released by human ASM cells and that its secretion may be regulated by the ET system. Our results demonstrate that ADM is expressed and released by human ASM. Further, the rate of ADM release from individual isolates was heterogeneous, higher rates of release being associated with the possession of ET_B receptors. Stimulation of ET_B receptors also lead to increased ADM secretion. Our findings support the paracrine/autocrine regulation of ADM release by ET in the bronchial wall.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Reagents

Medium 199 (M199), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), antibiotic/antimycotic solution, trypsin- ethylenediaminetetraacetic acid (EDTA), and Trizol reagent were purchased from Life Technologies (Paisley, Renfrewshire, UK). Sarafotoxin 6c (S6c) and ET-3 were from Peninsula Laboratories (St. Helens, UK). ET-1 and BQ123 were from Calbiochem-Novabiochem Corp. (Beeston, Nottingham, UK). Na¹²⁵I, ¹²⁵I-ET-1 (specific activity = 74 Bq/fmol) ligand, [-³²P]deoxycytidine triphosphate (dCTP), [methyl-³H]-thymidine (25 Ci/mmol), and the Megaprime DNA labeling system were from Amersham Pharmacia Biotech (Little Chalfont, Bucks, UK). Human ADM(1-52) and human ADM(22-52) were synthesized by Dr. P. Byfield (CRC, Hammersmith Hospital, London, UK). Cy3-conjugated antirabbit immunoglobulin (Ig) G was from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Platelet-derived growth factor (PDGF)- BB, the monoclonal antibody to -smooth-muscle actin (IA4) and fluorescein isothiocyanate (FITC)-conjugated antimouse IgG were purchased from Sigma (Poole, Dorset, UK) and all other reagents were from Sigma or Merck (Leicestershire, UK).

Isolation of Human ASMC

Proximal segments of lobar bronchi (luminal diameter, approximately 5 to 7 mm) were obtained from resected lung specimens from patients undergoing lung or heart-lung transplantation for fibrosing alveolitis (n = 1), pulmonary hypertension (PH) secondary to Eisenmenger's syndrome (n = 1), emphysema (n = 1), or COPD (n = 1); or from lobectomy for bronchial carcinoma (n = 3). Further specimens were available from donor tissues at transplantation (n = 1). Ethical approval was obtained from the Ethics Committees of Harefield and Hammersmith Hospitals. Samples of bronchus were placed in M199 for transport to the laboratory and kept at 4°C for up to 12 h. Airways were opened to expose the epithelial surface, which was removed by gentle scraping with a scalpel; the tissue was rinsed with phosphate-buffered saline (PBS); and the smooth-muscle layer was cut into approximately 2-mm³ cubes. In initial studies, pooled cell populations were obtained from multiple explants from the same airway. Explants were placed at regular intervals in 25-cm² flasks coated with collagen (Type I rat tail; Becton Dickinson, Franklin Lakes, NJ) and left to adhere overnight. Fresh M199/20% FBS was then added to the flasks. Cells were generally seen growing from explants after 7 to 10 d in culture, and reached confluence by 3 to 4 wk. All cells were grown at 37°C in an atmosphere of 5% CO₂ in air in a humidified incubator (ICN Model 160; ICN Biomedicals Ltd., Thame, Oxon., UK). At confluence, cells were trypsinized and plated into 75-cm² flasks in M199/10% FBS. Subsequent passages were carried out at confluence, dividing one flask into three. Cells were used for experiments between passages 3 and 7.

For isolation of cells from individual explants, a cloning ring approach was adopted, based on the method described by Frid and colleagues (23). Explants from the same bronchial segment were placed at least 20 mm apart on a 90-mm petri dish coated with collagen, allowed to adhere overnight, and then incubated in M199/ 20% FBS. When cells were observed growing out of individual explants to cover a diameter of 6 to 7 mm, the medium was removed and small polypropylene cloning rings (internal diameter, 6 mm) were placed over each explant and its surrounding cells. A watertight seal with the petri dish was achieved with silicone grease on the underside of each ring. The cells inside the polypropylene ring were trypsinized as described earlier and the cells from each individual explant were plated into one well of a 12-well plate. On subsequent passages, the cells were transferred into a 25-cm² flask and, on attaining confluence, into a 75-cm² flask. Subsequently, cells were grown and passaged as described earlier.

Phenotypic Characterization of Cells

The smooth-muscle phenotype of isolated cells was investigated with IA4 and a polyclonal antibody to smooth muscle-specific myosin (kindly provided by Dr. Maria Frid, University of Colorado Health Sciences Center, Denver, CO) (23). For immunostaining, cells were seeded at a density of 2 × 10³ cells/well in M199/10% FBS in eight-well chamber slides (Life Technologies) coated with collagen and grown for a further 2 d. Cells were fixed in methanol at 20°C for 10 min, followed by three brief washes in PBS at room temperature. Cells were incubated with primary antibody for 1 h at room temperature and washed three times with PBS. FITC-conjugated antimouse IgG or Cy3-conjugated antirabbit IgG secondary antibody were added, as appropriate, for 1 h at room temperature. Cells were counterstained with the nuclear stain 4,6-diamidino-2-phenylindole (0.5 µg/ml in PBS) for 2 min, rinsed with PBS, and mounted in PBS/glycerol (1:1). Staining was visualized by fluorescence microscopy.

Serum-Induced Proliferation of ASMCs

To assess the rate of serum-induced proliferation of isolates, cells were seeded at a density of 1.5 × 10⁴ cells/well in M199/10% FBS in 24-well plates. The day of plating was designated Day 0. Cells were replenished with fresh M199/10% FBS every 48 h. On Days 1, 2, 5, 7, 9, 13, and 15, three wells were washed with PBS and trypsinized with 0.25% trypsin-EDTA in PBS, and cells were counted by hemocytometer. Viability was assessed by trypan blue exclusion.

¹²⁵I-ET-1 Binding Studies on ASMCs

Ligand binding studies in cultured cells were performed as previously described (24). Briefly, ASMCs were seeded at a density of 5 × 10³ cells/well in 48-well plates and grown to confluence. For studies of ¹²⁵I-ET-1 binding, cells were incubated for 120 min at 37°C in 0.5 ml DMEM containing 0.3% bovine serum albumin (BSA) and 1,000 Bq (50 pM) ¹²⁵I-ET-1. Nonspecific binding was determined in the presence of excess (1 µM) unlabeled human ET-1. The proportion of ET_A/ET_B receptor subtypes present was initially estimated by incubating cells with ¹²⁵I-ET-1 in the presence or absence of BQ123 (10 nM), a specific ET_A receptor antagonist. This concentration of BQ123 was previously determined in our laboratory to completely compete for ¹²⁵I-ET-1 binding at ET_A sites but not at ET_B sites. Binding sites were further characterized by competition experiments whereby cells were incubated with ¹²⁵I-ET-1 (50 pM) in the presence of increasing concentrations (0 to 10 µM) of either BQ123 or ET-3.

After incubation, cells were washed twice with 0.5 ml ice-cold assay buffer and lyzed with 0.2 M NaOH containing 0.1% (wt/ vol) sodium dodecyl sulfate (SDS) for counting bound ¹²⁵I-peptide. Protein concentration was assayed with the Bio-Rad DC protein assay (Bio-Rad, North Yorkshire, UK) according to the manufacturer's instructions. Binding data were analyzed by nonlinear regression using GraphPad Prism version 2.01 (GraphPad Software, Inc., San Diego, CA).

Quantitative In Vitro Autoradiographic Analysis of ¹²⁵I-ET-1 Binding in Human Bronchi

ET binding sites in human bronchi (5 to 7 mm luminal diameter; n = 8) were localized and characterized using in vitro autoradiography as previously described (25). Briefly, 10-µm-thick cryostat sections were cut at 20°C and thaw-mounted onto Vectabond-coated microscope slides. Sections were preincubated for 10 min at 20 to 22°C in binding buffer (50 mM Tris-HCL [pH 7.4], 100 mM NaCl, 5 mM MgCl₂, and 28 µM bacitracin), followed by incubation for 120 min in binding buffer containing 0.5% BSA and 100 pM ¹²⁵I-ET-1. Sections were then washed three times for 5 min each time in binding buffer at 4°C, and briefly washed in cold distilled water followed by rapid drying under a stream of cold air. Nonspecific binding was defined as that remaining in adjacent sections coincubated with 100 nM unlabeled human ET-1. The proportions of ET_A and ET_B receptor subtypes were characterized by coincubation of consecutive sections with either BQ123 or S6c. Sections were apposed to Hyperfilm-³H together with radiolabeled standards for 3 d at 4°C. The resulting macroautoradiographic images were quantified using a KS300 Imaging System (Image Associates, Thame, Oxon., UK). Standard curves were calculated, converting relative grey levels to ligand-bound (amol/ mm²) for each film.

ADM and ET Release from ASMCs

To determine whether ASMCs release ADM or ET, cells were seeded at a density of 5 × 10⁴ cells/well in six-well plates and grown to 80 to 90% confluence. The cells were serum-starved by washing once with serum-free M199 (SFM) followed by incubation in SFM for 2 h. For time-course studies, serum-starved cells were then incubated in 2 ml M199/0.1% FBS for 6, 10, 24, and 48 h. At the designated time point, cells were counted and aliquots (1 ml) of culture media were collected, frozen immediately, dried by rotary evaporation, and stored at 20°C. The dried samples were resuspended in assay buffer and submitted to radioimmunoassay (RIA) for ADM or ET.

In parallel experiments, we determined the effects of different concentrations of serum on ADM and ET immunoreactivity in ASMC conditioned medium. Cells were seeded in 24-well plates at a density of 1.5 × 10⁴ cells/well and grown to confluence. Cells were serum-starved for 2 h, followed by incubation for 24 h in 400 µl SFM alone or with 0.01, 0.03, 0.1, 0.3, 1, 3, or 10% FBS. At the end of this period, medium was collected for RIA. The cells treated with SFM and M199/10% FBS were trypsinized and counted by hemocytometer. Viability was assessed by trypan blue exclusion.

ADM RIA

Samples were assayed for ADM immunoreactivity using a previously reported specific human RIA (26). Briefly, assays were performed in a final volume of 700 µl assay buffer comprising 0.06 M sodium phosphate (pH 7.2) containing 0.3% (wt/vol) BSA, 10 mM EDTA, and 7 mM sodium azide. The antibody (designated FB8) was used at a final dilution of 1:10,000. The tracer was prepared by iodination of synthetic human ADM(22-52) by the Iodogen method and purification of the ¹²⁵I-ADM(22-52) tracer by reversed-phase high-performance liquid chromatography (HPLC). Assays were incubated at 4°C for 3 d. Bound and free tracer were separated using dextran-coated charcoal. The detection limit of the assay was 2 fmol/tube and the intra- and interassay coefficients of variation were 3.2 and 12.2%, respectively.

Characterization of ADM Immunoreactivity in ASMC Conditioned Medium

Characterization of immunoreactive ADM detected in conditioned medium was performed using Fast Protein Liquid Chromatography (FPLC) (Pharmacia, Herts., UK). For conditioning of medium, cells were grown to 80 to 90% confluence in T75 flasks. The cells were serum-starved for 2 h, followed by incubation in 12 ml of M199/0.1% FBS for 24 h. The conditioned medium was acidified with glacial acetic acid to a concentration of 0.5 M and extracted using Sep-Pak C₁₈ cartridges (Waters, Herts., UK) as previously described (24). FPLC of extracted medium was performed according to a previously published protocol (27). Dried samples were resuspended in water containing 0.1% trifluoroacetic acid, loaded onto a Pep-RPC C₂/C₁₈ reversed-phase column, and eluted with a linear gradient of 10 to 50% acetonitrile over 60 min. Fractions collected over 1-min periods were dried by rotary evaporation and submitted to RIA for ADM.

Northern Blot Analysis of ADM Messenger RNA

To extract total RNA, 10⁶ to 10⁷ cells were trypsinized, followed by centrifugation at 200 × g for 5 min. The cell pellets were lyzed in Trizol Reagent (Life Technologies) and total RNA was extracted according to the manufacturer's instructions. Total RNA (50 µg) was fractionated, using denaturing 3-(N-Morpholino)propanesulfonic acid/formaldehyde/1% agarose gels, and transferred to Hybond-N (Amersham Pharmacia Biotech) nylon membranes, followed by ultraviolet crosslinking.

Membranes were hybridized with a 900-base pair complementary DNA (cDNA) probe that included the coding sequence for human ADM (16). The probe was labeled by the random primer method using the Megaprime DNA labeling kit. Briefly, 10 ng of the cDNA probe was mixed with the primers, boiled for 5 min and cooled on ice. The reaction buffer containing deoxyadenosine triphosphate, deoxythymidine triphosphate, and deoxyguanidine triphosphate was added together with [-³²P]dCTP and the Klenow fragment of DNA polymerase. The reaction was incubated at 37°C for 1 h and terminated by addition of 3 vol of TES buffer (10 mM Tris-HCl [pH 7.5], containing 1 mM EDTA [pH 8.0] and 0.1% [wt/vol] SDS). The membranes were prehybridized in 30 ml of 5× saline sodium citrate (SSC) (1× = 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0), 5× Denhardt's (1× = 0.025% polyvinylpyrrolidine, 0.02% Ficoll, and 0.02% BSA), 1% SDS, and 5 ng/ml salmon-sperm DNA (Life Technologies) for 2 h at 60°C. Hybridization was performed overnight at 60°C in 15 ml of prehybridization buffer plus 10% (wt/vol) dextran sulfate. Membranes were washed once with 2× SSC/0.2% SDS at 22°C for 10 min, followed by 0.2× SSC/0.1% SDS at 60°C for 60 min. Washed membranes were exposed to Kodak Biomax MR-1 film (Sigma) at 70°C for 1 to 3 d. Membranes were then stripped of radioactive probe by incubation in 10 mM Tris-HCl (pH 7.5) containing 1 mM EDTA (pH 8.0) containing 0.5% (wt/vol) SDS for 15 min at 80°C. Membranes were then probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to determine the RNA loading in each lane.

Effect of ADM on Intracellular Cyclic Adenosine Monophosphate Accumulation

The effect of exogenous ADM on intracellular cyclic adenosine monophosphate (cAMP) accumulation was measured as an indicator of a functional role for ADM. ASMCs were seeded at a density of 1.5 × 10⁴ cells/well in 24-well plates and grown to confluence. On the day of the experiment, cells were serum-starved for 3 h, then treated in the absence or presence of 100 nM ADM in SFM containing 50 µM 3-isobutyl-1-methylxanthine for 15 min as previously described (24). After treatment, cells were extracted overnight at 20°C in 250 µl acid ethanol (75% ethanol and 16 mM HCl). Extracts were dried and assayed for cAMP content using a commercially available RIA kit (DuPont, Stevenage, Herts., UK).

ET RIA

Samples of conditioned media were assayed for ET immunoreactivity using a previously reported specific RIA (28). Briefly, assays were performed in a final volume of 700 µl assay buffer comprising 0.06 M sodium phosphate (pH 7.2) containing 0.3% (wt/ vol) BSA, 10 mM EDTA, and 7 mM sodium azide. The antibody (designated BP6) was used at a final dilution of 1:5,000. The ¹²⁵I- ET-1 tracer was prepared by iodination of synthetic human ET-1 using the Iodogen method and purified by reversed-phase HPLC. Assays were incubated at 4°C for 3 d. Bound and free tracer were separated using dextran-coated charcoal. The detection limit of the assay was 0.05 fmol/tube and the intra- and interassay coefficients of variation were 3.2 and 11.4%, respectively. This assay cross-reacts fully with ET-1 and displays 60 and 40% cross-reactivity with ET-2 and ET-3, respectively.

Effect of ET on ADM Release from ASMCs

To investigate whether there was cross talk between the ET and ADM systems, the effects of different concentrations of ET-1 and ET-3 on basal ADM release by ASMCs were examined. For these experiments, cells were seeded in 48-well plates at a density of 5 × 10³ cells/well and grown to confluence. Cells were serum-starved for 2 h, followed by incubation for 24 h in 400 µl M199/ 0.1% FBS containing 0.01 to 100 nM ET-1 or ET-3 with 0.1% FBS alone as a control. At the end of this period, 300 µl of the medium was frozen immediately, dried by rotary evaporation, and stored at 20°C until RIA for ADM. Cells treated with M199/0.1% FBS alone, M199/0.1 %FBS containing 100 nM ET-1, or M199/0.1% FBS containing 100 nM ET-3 were all counted by hemocytometer. Viability was assessed by trypan blue exclusion.

Effect of ET on DNA Synthesis

The growth response of human ASM cells was determined by measurement of [³H]thymidine incorporation, representing DNA synthesis. Cells were seeded in 48-well plates at a density of 5 × 10³ cells/well, grown to 80 to 90% confluence, and then quiesced by serum-starvation for 2 h, followed by incubation in M199/ 0.1% FBS for 72 h. To determine the effect of treatments upon DNA synthesis, quiescent cells were incubated in the presence of 0.25 µCi/well [³H]thymidine for 24 h. To examine the effect of ET-1 or ET-3 on basal or PDGF-stimulated [³H]thymidine incorporation, quiescent cells were incubated in M199/0.1% FBS with or without 10 ng/ml PDGF-BB for 24 h in the presence or absence of 100 nM of each peptide. At the end of this period, the cells were rapidly washed three times with PBS at 4°C, followed by addition of 1 ml of 10% trichloroacetic acid (TCA) at 4°C, to each well. The plate was left at 4°C for 30 min, the TCA was discarded, and the TCA insoluble fraction was dissolved in 0.2 M NaOH. The plate was stored at 4°C overnight, after which the [³H]thymidine content of the lysates was determined by scintillation counting.

To assess whether ET-1 or ET-3 potentiated the PDGF-induced proliferation of ASMCs, cells were seeded in 24-well plates at a density of 1.5 × 10⁴ cells, grown to approximately 30 to 40% confluence, and quiesced for 72 h as described earlier. Quiescent cells were then incubated in M199/0.1% FBS with or without 10 ng/ml PDGF-BB in the presence or absence of 100 nM of either ET-1 or ET-3, which were replenished every 2 d. Cells were counted at Days 2, 4, and 7 by hemocytometer. Viability was assessed by trypan blue exclusion.

Statistical Analyses

To compare responses between isolates and concentration-response data within isolates, data were compared by one-way analysis of variance with post hoc Tukey's test using GraphPad Prism version 2.01 (GraphPad Software).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Heterogeneity between ASMC Isolates

For our initial studies, we used isolates from six separate subjects (one secondary PH, one emphysema, one fibrosing alveolitis, and three carcinoma), each comprising a pooled population of cells from multiple explants from the same individual. All populations demonstrated > 90% positive immunofluorescent staining for -smooth-muscle actin and smooth muscle-specific myosin (not shown). However, there were evident phenotypic differences as the cells of four populations (one secondary PH, one emphysema, one fibrosing alveolitis, and one carcinoma) grew to confluence more rapidly and appeared smaller and more densely packed than the other two populations (two carcinoma). BQ123, an ET_A-selective antagonist, completely inhibited ¹²⁵I-ET-1 binding in the faster-growing populations (designated ET_A cells). In the slower-growing populations (designated ET_A/ET_B cells), BQ123 only partially inhibited binding, implying that a proportion of the sites were likely to be of the ET_B subtype (Figure 1A). In addition, we demonstrated that all the ASMC populations secreted immunoreactive ADM into the culture medium (Figure 1B). However, the rate of release differed markedly between the two cell populations, secretion from ET_A/ET_B cells being 3- to 5-fold higher than that from ET_A cells.

View larger version (24K): [in this window] [in a new window]   Figure 1.   ET binding and ADM release by ASMC isolates. (A) Competition for 50 pM 125I-ET-1 was assessed in the absence (Total) or presence of either 1 µM ET-1 (NSB; nonspecific binding) or 100 nM BQ123. Representative of two separate ASMC isolates measured in triplicate. (B) ADM release by ETA and ETA/ETB ASMC isolates from individual subjects (1) over 24 h. Each point represents an individual observation measured in triplicate (***P < 0.001, *P < 0.05).

Subsequently, cloning rings were used to isolate these two ASMC phenotypes from the same individuals by isolating cells growing from individual explants. Using this technique, two explant isolates were obtained from one individual (donor) and six from a second individual (COPD). Initial characterization of these cells using competition with 10 nM BQ123 to distinguish between ¹²⁵I-ET-1 binding to receptor subtypes indicated that 50% of the isolates from each individual possessed ET_A receptors and 50% possessed a mixed population of ET_A/ET_B receptors. All isolates demonstrated > 90% positive immunofluorescent staining for -smooth-muscle actin and smooth muscle- specific myosin, confirming their smooth-muscle phenotype (data not shown).

Characterization of ASMCs: Morphology and Serum-Induced Growth

When ET_A and ET_A/ET_B ASMCs were grown to a confluent monolayer, differences were noted in their morphology under phase-contrast illumination. The ET_A isolates (Figure 2A) appeared smaller and more densely packed than the ET_A/ET_B isolates (Figure 2B). In addition, the ET_A ASMCs demonstrated a higher doubling rate (mean ± standard error of the mean [SEM]; 1.73 ± 0.12 d) than ET_A/ET_B cells (mean ± SEM; 3.16 ± 0.68 d) in the presence of 10% FBS (Figure 2C). Further, the plateau density of the ET_A ASMCs (71.9 ± 8.3 cells/mm²) was significantly greater (P < 0.01) than that of the ET_A/ET_B ASMCs (25.3 ± 4.0 cells/mm²).

View larger version (53K): [in this window] [in a new window]   Figure 2.   Morphology and growth characteristics of ETA and ETA/ETB ASMCs. Representative photographs of ETA (A) and ETA/ETB ASMC isolates (B) grown to confluence and photographed using phase contrast at ×40 magnification. (C) Proliferation of ETA ASMCs (squares) and ETA/ETB ASMCs (triangles) in the presence of 10% FBS. Medium was replenished every 2 d and the cells in three wells were trypsinized and counted at each time point. The graph represents pooled data of all growth curves (n = 3).

Analysis of ¹²⁵I-ET-1 Binding Sites on ASMCs

To confirm the ET receptor profiles of the isolated ASMCs, we examined competition for ¹²⁵I-ET-1 binding using the ET_A-selective antagonist BQ123 and the ET_B-selective agonist ET-3. Specific binding of ¹²⁵I-ET-1 to ET_A ASMCs (Figure 3A) demonstrated concentration-dependent competition by both BQ123 and ET-3 with nonlinear regression analysis of the competition data, indicating that binding curves were best explained by a single-site model. The affinity of this site was higher for BQ123 (concentration for half-maximal inhibition [IC₅₀] = 5.21 ± 2.53 nM) than ET-3 (IC₅₀ = 327.7 ± 94.1 nM) (n = 3 per group), indicating these cells possess ET_A receptors alone. In contrast, analysis of the competition curves for ET_A/ET_B cells revealed that the binding was best explained by a two-site model (Figure 3B). The ET_A sites were defined according to their high affinity for BQ123 (IC₅₀ = 3.75 ± 2.12 nM) and low affinity for ET-3 (IC₅₀ = 622.7 ± 89 nM), whereas the ET_B sites displayed high affinity for ET-3 (IC₅₀ = 0.25 ± 0.07 nM) and low affinity for BQ123 (> 1 µM). The relative proportions of ET_A and ET_B sites were 31 ± 3% and 69 ± 3%, respectively. In addition, the density of equilibrium ¹²⁵I-ET-1 binding (50 pM) was significantly greater (P < 0.05) in ET_A/ET_B ASMCs (84.6 ± 15.1 fmol/mg protein; n = 4) than in ET_A ASMCs (25.8 ± 7.6 fmol/mg protein; n = 4).

View larger version (19K): [in this window] [in a new window]   Figure 3.   Characterization of ET binding sites in ASMC isolates from individual explants. Equilibrium competition binding for 125I-ET-1 with BQ123 (squares) or ET-3 (triangles). Data are expressed as a percentage of the maximum specific binding for two representative isolates. Data points represent means ± SEM of triplicate measurements.

ET Receptor Localization in Human Bronchi

To determine the ET receptor profile in human bronchial smooth muscle in vitro, we performed macroautoradiographic analysis of ¹²⁵I-ET-1 binding sites to bronchi in cryostat sections (Figure 4). ET binding was localized to the bronchial smooth muscle and epithelium as well as the lung parenchyma and pulmonary vasculature. Competition for ET binding in serial sections using BQ123 and S6c to eliminate ET_A and ET_B binding, respectively, demonstrated ET_A binding in the airway epithelium and bronchial vessels and a relatively low level of ET_A binding to bronchial smooth muscle. In contrast, binding to ET_B sites predominated in bronchial smooth muscle. Equilibrium ¹²⁵I-ET-1 binding to bronchial smooth muscle was determined as 271.9 ± 50.8 amol/mm², whereas the nonspecific binding was 7.3 ± 1.0 amol/mm² (n = 8). The density of ET_A binding sites was 50.4 ± 12.3 amol/mm² and that of ET_B binding sites was 213 ± 34.5 amol/mm². The proportion of total binding sites was 19.2 ± 2.9% ET_A and 80.7 ± 5.0% ET_B.

View larger version (100K): [in this window] [in a new window]   Figure 4.   Macroautoradiographic images of 125I-ET-1 binding in serial sections of human bronchus. (A) Hematoxylin and eosin-stained section. Serial sections incubated for 120 min at 20 to 22°C in binding buffer containing 100 pM 125I-ET-1, in the absence (B; total binding) or presence of either BQ123 (C; ETB binding) or S6c (D; ETA binding). The bronchial epithelium (arrows) and smooth muscle (asterisks) are indicated.

ADM and ET Release from ASMCs

All ASMC isolates secreted immunoreactive ADM into the culture medium at a linear rate, but the amount released differed markedly between the ET_A and ET_A/ET_B isolates (Figure 5A). The rate of immunoreactive ADM release by ET_A/ET_B ASMCs (mean 261 ± 16 fmol/10⁵ cells/24 h; n = 4) was significantly greater (P < 0.001) than by ET_A ASMCs (mean = 57 ± 12 fmol/10⁵ cells/24h; n = 4) (Figure 5B). ADM release was modulated by the serum concentration in both cell types, release in the presence of 0.1 to 0.3% FBS being significantly greater (140 to 150%; P < 0.05) than in the absence of serum. In contrast, serum concentrations of 3 and 10% significantly inhibited 24-h ADM release in both cell types to approximately 80 and 35% (P < 0.01), respectively, of that measured in the absence of serum. FPLC analysis of conditioned medium from both ET_A and ET_A/ET_B confirmed that the majority of the immunoreactive ADM co-eluted with synthetic human ADM (not shown).

View larger version (27K): [in this window] [in a new window]   Figure 5.   Release of immunoreactive ADM from ETA and ETA/ ETB ASMCs. (A) Time course of ADM release from ETA (squares) and ETA/ETB ASMCs (triangles) seeded in six-well plates and incubated in M199/0.1% FBS for up to 48 h. Data are expressed as means ± SEM (n = 4). (B) Results of 24-h ADM release by separate ETA and ETA/ETB ASMC isolates. Bars represent the means ± SEM for four separate experiments measured in triplicate (**P < 0.01, *P < 0.05 with respect to ETA isolates).

In contrast, there was no detectable immunoreactive ET in the conditioned medium from any ASMC isolate of either phenotype over a 48-h period in M199/0.1% FBS or over 24 h in any of the serum concentrations tested.

Expression of ADM Messenger RNA in ASMCs

Northern blot analysis of total RNA from each isolate probed for ADM messenger RNA (mRNA) demonstrated a band of the expected 1.6 kb (16) in all eight explant isolate ASMCs (Figure 6A). Steady-state mRNA levels for ADM were consistently higher in the ET_A/ET_B ASMCs than in the ET_A ASMCs.

View larger version (58K): [in this window] [in a new window]   Figure 6.   Northern blot analysis of ADM mRNA from ETA and ETA/ETB ASMCs hybridized with a 32P-labeled ADM cDNA (upper panel). A 1.6-kb band was detected in ASMCs, as expected for human ADM. Lanes A-H correspond to explant isolates A-H defined in Figure 5B. Blots were stripped of radioactive probe and RNA loading checked by probing for GAPDH mRNA (lower panel).

Effect of ADM on Intracellular cAMP in ASMCs

To establish whether ET_A and ET_A/ET_B ASMCs demonstrate functional responses to ADM, the effect of ADM on intracellular cAMP accumulation was assessed. In ET_A cells treated with ADM, cAMP was elevated to approximately 250% of the SFM control (5.67 ± 0.61 pmol/10⁵ cells versus 2.41 ± 0.24 pmol/10⁵ cells; P < 0.05). In contrast, there was no effect of ADM on cAMP levels in ET_A/ET_B ASMCs (3.65 ± 0.18 pmol/10⁵ cells versus 3.28 ± 0.11 pmol/10⁵ cells).

Effect of ETs on ADM Release by ASMCs

To determine whether ADM release by ET_A and ET_A/ ET_B ASMCs was modulated via ET receptors, we studied the effects of ET-1 (nonselective ET_A and ET_B) or ET-3 (ET_B-selective) on 24-h ADM release. At a dose of 100 nM, ET-1 elicited a modest dose-dependent stimulation of ADM release by ET_A ASMCs (Figure 7A). A stimulatory effect on ADM release from ET_A ASMCs was observed only with a dose of 100 nM ET-3, whereas lower concentrations had no effect. In contrast, both ET-1 and ET-3 elicited concentration-dependent increases (> 150% of basal) in ADM release by ET_A/ET_B ASMCs (Figure 7B).

View larger version (20K): [in this window] [in a new window]   Figure 7.   Effect of ET-1 and ET-3 on ADM release by ETA (squares) and ETA/ETB ASMCs (triangles). Cells were incubated with SFM (Basal) or medium containing 0.01 to 100 nM ET-1 or ET-3 for 24 h. Conditioned medium was submitted to RIA for human ADM and cells were counted by hemocytometer. Data represent the means of four separate experiments with different isolates. Data are expressed as means ± SEM (n = 4) (**P < 0.01; *P < 0.05 compared with basal ADM release).

Effect of ETs on [³H]Thymidine Uptake by ASMCs

To determine whether ET-1 or ET-3 modulates DNA synthesis in ET_A and ET_A/ET_B ASMCs, the effects of these peptides were studied on either basal (Figure 8A) or PDGF-BB-stimulated [³H]thymidine uptake over 24 h (Figure 8B). In ET_A ASMCs, no effects of 100 nM ET-1 or ET-3 were observed upon either basal or PDGF-BB (10 ng/ml) stimulated DNA synthesis. In addition, 100 nM ET-1 or ET-3 did not significantly affect basal DNA synthesis in ET_A/ET_B ASMCs. In contrast, 100 nM ET-1, but not ET-3, significantly (P < 0.05) potentiated PDGF-stimulated DNA synthesis in ET_A/ET_B cells to 140% of the response observed with PDGF-BB alone.

View larger version (36K): [in this window] [in a new window]   Figure 8.   Effects of ET-1 and ET-3 on basal and PDGF-BB- stimulated [3H]thymidine uptake by quiescent ETA ASMCs and ETA/ETB ASMCs at 80 to 90% confluence in 24-well plates. (A) Effect on basal [3H]thymidine uptake by ETA and ETA/ETB ASMCs. Cells were incubated with M199/0.1% FBS alone (0.1%) or with 100 nM of either ET-1 or ET-3. (B) Effects of ET-1 and ET-3 on PDGF-BB-stimulated [3H]thymidine uptake in ETA and ETA/ETB ASMCs. Cells were incubated with M199/ 0.1% FBS alone (0.1%) and with 10 ng/ml PDGF-BB in the absence and presence of 100 nM of either ET-1 or ET-3. Data represent the means of four separate experiments with different isolates. Data are expressed as means ± SEM (*P < 0.05 compared with PDGF alone).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The main finding of this study is that two subpopulations of smooth-muscle cells derived from the human airway are distinct with regard to their expression of ET receptors and ADM release. These subpopulations were also distinct with regard to cell morphology and serum-stimulated proliferation.

Explant isolates with mixed ET_A/ET_B receptors grew more slowly and released more ADM than ET_A ASMCs. In addition, ET_A/ET_B ASMCs demonstrated enhanced secretion of ADM, predominantly in response to the ET_B receptor agonist ET-3. Further, exogenous ET-1 potentiated PDGF-BB-stimulated DNA synthesis in ET_A/ET_B ASMCs, but not in ET_A ASMCs. At first sight this implicates the ET_B receptor in the observed responses. However, ET-3, an ET_B agonist, had no effect on [³H]thymidine incorporation. Thus our favored explanation is an ET_A mediated effect limited to ET_A/ET_B cells. This is plausible because differences in intracellular signaling pathways determining growth and differentiation between our populations are more likely to determine the outcome of a given mitogenic stimulus rather than simply the expression of cell-surface receptors.

In initial experiments studying isolates comprising pooled cells from individual subjects, we determined that there were phenotypic differences between populations on the basis of their ET-binding characteristics and rates of ADM release. In subsequent experiments, we successfully isolated distinct subpopulations of ASMC from individual explants derived from the same segment of airway wall, suggesting that these subpopulations exist in vivo. Using the cloning ring technique, we were able to isolate ET_A and ET_A/ET_B populations from both patients studied. From our observations, the reason for isolating ET_A cells more frequently using standard explant techniques is that ET_A cells tend to proliferate faster from the explants than do the ET_A/ET_B cells and are thus over-represented in mixed culture.

Studies of isolated animal ASMCs have consistently shown that they possess a mixed ET_A/ET_B receptor population (29). In culture, human ASMCs possessing only ET_A receptors were isolated from the bronchus (30), whereas tracheal cells with a mixture of ET_A and ET_B receptors have been demonstrated (5). In the present study, each ASMC isolate exhibited either ET_A receptors alone or ET_A and ET_B receptors, and the existence of these two cell phenotypes may explain the apparent discrepancies between previous reports. Our study has not determined whether the ET_A/ ET_B cultures comprise cells that express both receptor subtypes or instead a mixed population of cells that are exclusively ET_A or ET_B. This will require further evaluation, but cell morphology by light microscopy indicated a homogeneous population of cells. Our results further demonstrate that human ASMCs secrete immunoreactive ADM but at different rates, according to their ET receptor profile. The concept of cellular heterogeneity in the human airway is well known, with groups reporting morphologically and pharmacologically distinct ASMC subtypes (30, 32). Heterogeneity is recognized between cells isolated from proximal and distal bronchi. In addition, distinct subpopulations of cell types are found at the same anatomical site in the bovine pulmonary artery (23). Evidence for a similar heterogeneity of airway smooth muscle is now emerging with evidence for biochemical, morphologic, and functional differences in cells isolated from the same airway segment (33). In addition, ASM demonstrates considerable plasticity of phenotype in culture, which makes comparison between phenotypes of cultured and in vivo cells difficult. However, our results indicate that stable differences in cell phenotype can be maintained in cell culture systems and suggest important functional differences between cells in the airway wall.

To characterize ET receptor profiles, full competition curves for ¹²⁵I-ET-1 were derived using the specific ET_A antagonist BQ123 and the selective ET_B agonist ET-3. In each case, half of the isolates displayed typical high-affinity ET_A receptors (ET_A ASMCs) and half displayed a mixed population of ET_A and ET_B receptors (ET_A/ET_B ASMCs). The relative proportions of ET_A and ET_B receptors (31 versus 69%) in the ET_A/ET_B cells were similar to previously reported ratios in rat ASMCs (30) and human tracheal ASMCs (5). In addition, the density of ET receptors in ET_A/ET_B ASMCs was approximately 3-fold higher than ET_A cells, which suggests that the density of ET_A receptors was equivalent in both populations. We did not observe any change in the relative proportions of ET_A and ET_B receptors on ET_A/ET_B cells, a phenomenon reported in ovine ASMCs (31). The consistency of the ratio of ET_A to ET_B sites implies that the ET_A/ET_B isolates are a homogeneous population, inasmuch as cells that express only ET_A receptors proliferate faster. Our data regarding localization of ¹²⁵I-ET-1 binding sites in the ASM, airway epithelium, and lung parenchyma by in vitro macroautoradiography are consistent with previously reported studies (7, 9). We determined the relative proportions of ET_A and ET_B sites to be 19.2 and 80.7%, respectively, in bronchial smooth muscle, which is similar to a previous report (9).

Immunostaining for ADM has been demonstrated previously in human ASM (18), although secretion from isolated ASMCs has not been reported. We have now shown that ASMCs secrete authentic ADM at a linear rate, with the ET_A/ET_B ASMCs releasing ADM at a significantly faster rate than the ET_A isolates. This difference in release rates was consistent with the relative levels of ADM mRNA expression by individual isolates. ADM release was stimulated in low (0.1 to 0.3%) concentrations of serum but inhibited at concentrations > 1%. Because serum controls were used in the RIA, it is unlikely this observation is an artifact due to serum cross-reactivity. A similar profile of serum responsiveness was previously reported in rat vascular endothelial cells (34). The implications of this effect have yet to be established regarding whether proliferation affects serum-stimulated proliferation or vice versa.

We examined the effect of ADM on cAMP elevation in the ASMC isolates as an indicator of receptors demonstrating a functional response to this peptide (20, 24). ADM stimulated cAMP accumulation in ET_A ASMCs, whereas no response was observed in ET_A/ET_B ASMCs. This does not exclude the possibility of functional receptors on the ET_A/ ET_B ASMCs, inasmuch as their high rate of ADM release could mask a cAMP response. However, our observation of cAMP elevation in response to ADM in ET_A ASMCs implies a functional role for ADM in ASM. ADM may act as a bronchodilator (17), but can also inhibit cell proliferation (35) and inhibit ET-1 release (20). Therefore the contribution of ADM as a regulator of muscle tone or modulator of remodelling, or its involvement in other aspects of airway physiology, have yet to be established.

In contrast to the high-level ADM release, we did not observe ET release from any of the ASMC isolates. It has been shown that ovine tracheal smooth-muscle cells express prepro-ET-1 and secrete ET-1 (36). Failure to detect ET-1 may be due to very low levels of release or an absence of ET-1 production, and indicates that there may be species differences in ET expression in ASMCs. In addition, our observation is consistent with immunohistochemical studies indicating a lack of ET immunoreactivity in human ASM (6, 7).

We investigated whether stimulation of ET receptor subtypes is involved in the regulation of ADM secretion in ASMCs. ET-1 is a weak stimulator of ADM release by rat vascular smooth-muscle cells (21) and enhances ADM secretion by human coronary artery cells by ET_B receptor activation (22). In ET_A cells, ET-1 elicited a weak concentration-dependent increase in ADM release, whereas ET-3 stimulated release only at a concentration of 100 nM, which is known to stimulate the ET_A receptor (37). In contrast, both ET-1 and ET-3 elicited a concentration-dependent stimulation of ADM secretion by ET_A/ET_B cells. The effects of both peptides were comparable in ET_A/ET_B cells, implying that the ET_B receptor was mediating this response. The functional significance of these findings remains to be determined. However, it is conceivable that ET_B-stimulated ADM release is involved in homeostatic regulation of ASM tone, and partly attenuates the ET_B-mediated bronchoconstriction reported in human airways (11).

We investigated whether ET-1 or ET-3 stimulates mitogenesis in human ASMCs. ET-1 is a potent mitogen to ASMCs isolated from experimental animals (29, 38), although the involvement of the ET_A and ET_B receptors in this response differs between reports (29, 38). ET-1 did not affect basal DNA synthesis in ET_A or ET_A/ET_B ASMCs in the present study, and did not alter PDGF-BB-stimulated DNA synthesis in ET_A cells. However, in ET_A/ET_B ASMCs, ET-1 potentiated the response to PDGF-BB by approximately 40%, whereas ET-3 had no effect, indicating that the response to ET-1 was mediated via the ET_A receptor. Previous studies have demonstrated mitogenic (39) or comitogenic (5) effects of ET-1 on human ASMCs, ET-1 acting via the ET_A receptor. As in the current study, no mitogenic effect was observed after ET_B stimulation with ET-3 or S6c. However, our finding that ET_A-mediated [³H]thymidine uptake is restricted to isolates with a mixed population of ET_A and ET_B receptors suggests that the ET_B receptor may be a marker for a cell type possessing ET_A-mediated signaling pathways coupled to growth and/or differentiation.

In summary, two phenotypically distinct smooth-muscle cell populations were isolated from explants of human ASM. These two populations differed both morphologically and in their rates of proliferation to serum, and could be differentiated by their expression of ET receptor subtypes and rates of ADM release. Interactions between the ET and ADM systems were established in ASMCs and demonstrated a stimulatory effect of ET on ADM release mainly via ET_B receptors and a comitogenic effect of ET via ET_A receptors. These observations provide further support for heterogeneity of human ASM and provide functional evidence for paracrine interactions between the ADM and ET systems within the human airway.