Altered ETB- but not ETA-receptor density and function in sheep airway smooth muscle cells in culture

Michael J. Maxwell, Roy G. Goldie, and Peter J. Henry

Department of Pharmacology, University of Western Australia, Nedlands, Western Australia 6907, Australia

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

The possibility that significant changes in endothelin (ET)A- and ETB-receptor density and function occur in airway smooth muscle cells (ASMCs) during cell growth and extended cell culture was investigated in sheep tracheal ASMCs. As in intact tracheal smooth muscle tissue from this species, early-passage sheep ASMCs contained a homogeneous population of ETA receptors. However, growth of ASMCs from seeding to postconfluence and repeated passage of ASMCs (6th to 14th passages) was associated with a substantial increase in ETB-receptor density, with no change in ETA-receptor density. ET-1-induced stimulation of ETB receptors increased the intracellular Ca2+ concentration in single ASMCs. Interestingly, a 2-day period of serum deprivation completely eliminated the increase in ETB-receptor density and the ETB receptor-mediated change in intracellular Ca2+ concentration. In summary, growth and repeated passage of sheep ASMCs were associated with a profound and selective increase in the density and function of the ETB receptor, a receptor subtype not present in early-passage ASMCs and not detected in intact sheep tracheal airway smooth muscle.

endothelin-1; endothelin receptors; calcium; trachea

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE ENDOTHELINS (ETs) induce potent contraction in intact preparations of tracheal and bronchial airway smooth muscle from the human and many animal species, although the relative densities of the ETA and ETB receptors and their contribution to ET-1-induced contraction differ markedly among species (9). A recent autoradiographic study (8) revealed that airway smooth muscle from an isolated human bronchus contains a greater proportion of ETB than ETA receptors. Consistent with this, ET-1-induced contraction in isolated human bronchial preparations was mediated predominantly via the activation of ETB receptors (8), although the less abundant ETA receptors also contributed to the contractile response (6). In stark contrast, autoradiographic and isometric tension recording studies of intact tracheal smooth muscle from sheep have identified a homogeneous population of ETA receptors that were linked to ET-1-induced contraction (7). The inability to detect autoradiographically the existence of a significant population of ETB receptors over airway smooth muscle in sheep isolated tracheal preparations was entirely consistent with the finding that the ETB receptor-selective agonist sarafotoxin S6c did not induce a significant contractile response in this tissue (7). However, recent studies by Ergul et al. and Carratu et al. using cell cultures derived from sheep trachea suggest that although ETA receptors predominated in these cells (85% of total ET receptors), there existed a significant population of ETB receptors (5) and both subtypes mediated ET-1-induced proliferation of these cells (3). One possible explanation for this apparent discrepancy is that ETB-receptor expression was enhanced during the culturing process. In support of this explanation, recent studies in human omental arteries have demonstrated that organ culture is associated with an increase in ETB-receptor mRNA content and the appearance of a substantial ETB receptor-mediated contraction (1). These findings suggest a considerable plasticity in smooth muscle expression of contractile (and perhaps mitogenic) ETB receptors.

The utilization of ET receptor subtype-selective ligands in combination with radioligand binding techniques provides an invaluable means for the investigation and characterization of ET receptors in cultured cells. Furthermore, the fluorescent Ca2+ indicator dye fura 2 and Ca2+-imaging technology can be employed to examine coupling of these receptors to intracellular Ca2+ (Ca2+i) mobilization. The aim of the present study was to systematically investigate the influence of cell growth and extended cell culture on ETA- and ETB-receptor density and function in sheep tracheal airway smooth muscle cells (ASMCs).

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

ASMC culture. Enzymatic digestion of the tracheal smooth muscle band obtained from 4- to 9-mo-old sheep was performed with a modified version of the methods of Hirst (11). Briefly, smooth muscle bundles were digested in two steps in "low-Ca2+" Krebs containing BSA (0.25%), collagenase I (2, then 1 mg/ml), and elastase IV (5, then 10 U/ml) and incubated at 37°C for 30-45 and 45-60 min, respectively. After the second digestion, DMEM-F-12 containing 10% FCS and antibiotic-antimycotic (100 U/ml of penicillin, 0.1 mg/ml of streptomycin, and 0.25 µg/ml of amphotericin B) was added. The digestion mix was centrifuged (10 min at 1,000 g), and the medium was aspirated and replaced with fresh DMEM-F-12. This wash process was repeated, and then loosely attached ASMCs were gently aspirated with a heat-smoothed Pasteur pipette to liberate single cells. ASMCs were then seeded onto Falcon Primaria plates (33 × 10 mm) (Becton Dickinson) in a 2-ml volume of DMEM-F-12 (final seeding density of 5 × 104 viable cells/cm2) and maintained in a humidified atmosphere of 95% O2-5% CO2 at 37°C. ASMCs were subcultured by gentle enzymatic dissociation (0.2% trypsin) coupled with minimal mechanical manipulation with a heat-smoothed Pasteur pipette. Subcultured cells were plated onto Falcon Primaria plates (33 × 10 mm) in a 2-ml volume of DMEM-F-12 at an estimated final seeding density of 5 × 104 viable cells/cm2 and maintained as described above. In some experiments, cultured ASMCs were deprived of serum by replacement of DMEM-F-12 with a serum-free medium (SFM) composed of DMEM-F-12 basal medium (no added FCS), 5 µg/ml of transferrin, 0.01 U/ml of insulin, and 0.25% (wt/vol) BSA.

Immunocytochemistry. The identity of cultured cells was determined after incubation with a fluorescently labeled monoclonal antibody to alpha -smooth muscle actin (15). ASMCs were subcultured onto glass coverslips overnight; washed thoroughly with PBS containing (in mM) 145 NaCl, 1.8 KH2PO4, and 8.2 Na2HPO4; fixed in 3% formaldehyde (5 min); and dehydrated in acetone (-20°C) for 2 min. A 1-h incubation with alpha -smooth muscle actin antibody (diluted 1:400) at 22°C was followed by an additional 1-h incubation with goat anti-mouse IgG conjugated with FITC. Nonspecific binding of the FITC conjugate was determined by substituting mouse ascites fluid (diluted 1:400) for alpha -smooth muscle actin antibody. ASMCs were washed in PBS, mounted in 90% glycerin, and viewed with a confocal microscope (Bio-Rad MRC1024; excitation wavelength of 494 nm, with viewing of the resultant FITC fluorescence at 520 nm).

Radioligand binding in ASMCs in culture. Radioligand binding experiments were performed on ASMCs seeded onto 24-well culture dishes at various stages of culture and, where indicated, deprived of serum for 2 days before the experiment. ASMCs were repeatedly washed in PBS containing 0.25% BSA and 10 µM phenylmethylsulfonyl fluoride (binding medium) and left to air-dry for 3 h. Air-dried cells were incubated in binding medium (0.5 ml) containing 0.2 nM 125I-labeled ET-1 for 30-180 min at 22°C either alone, in the presence of 1 µM BQ-123 (ETA receptor-selective ligand) (12) or 200 nM sarafotoxin S6c (ETB receptor-selective ligand) (18), or in the combined presence of 1 µM BQ-123 and 200 nM sarafotoxin S6c (to assess nonspecific binding). At the end of the appropriate incubation period, the cells were washed (2 × 10 min) in cold binding medium and solubilized during a 15-min incubation in 200 µl of NaOH (1 M). Cellular debris was removed by wiping with glass microfiber filter paper (Whatman GF/A), and the associated radioactivity in each well was determined in a Packard auto-gamma counter (model 5650). To relate these levels to individual cells (in binding sites/cell), the average cell number per well was determined by a hemocytometer in wells that were set aside earlier.

Data obtained from association time-course experiments were employed to calculate the maximal binding capacity (Bmax) values for specific 125I-ET-1 binding via nonlinear least squares regression analysis according to the equation RLt = Bmax (1 - e-k1t), where RLt is the bound receptor concentration at time t and k1 is the association rate constant (16). ET-receptor subtype proportions were assessed by comparing the specific binding of 125I-ET-1 in the absence and presence of subtype-selective ligands. The degree to which BQ-123 or sarafotoxin S6c attenuated 125I-ET-1-specific binding was employed as a measure of 125I-ET-1-specific binding to ETA and ETB receptors, respectively. ET-receptor subtype proportions were calculated by division of this subtype-specific 125I-ET-1 binding density by the total 125I-ET-1-specific binding density.

Ca2+ imaging in ASMCs in culture. Unless otherwise indicated, sheep tracheal ASMCs were seeded overnight onto glass coverslips and incubated in SFM for an additional 2 days before experimentation. The cells were incubated in SFM containing 2.5 µM fura 2-AM for 45 min, and the cells were washed three times with 1.5 ml of imaging medium containing (in mM) 137 NaCl, 5.4 KCl, 2.5 CaCl2, 1.47 MgSO4, 11 glucose, 1.47 KH2PO4, 2.8 Na2HPO4, and 1.4 NaHCO3 and 0.25% BSA.

Digital-imaging fluorescence microscopy system. Excitation light from a 75-W high-pressure xenon-vapor lamp (Osram, Berlin, Germany) was passed through a computer-controlled, single-shutter, dual-filter wheel system (MAC 2000, LUDL Electronics Products, Hawthorne, NY), which consisted of a neutral density and narrow band-pass filter wheel assembly to select for intensity and wavelength, respectively. The fluorescence signal from single ASMCs was imaged with a ×40 dry objective (Olympus D Plan Apo 40X UV; numerical aperture 0.85) and detected by a low-light, intensified-silicon intensifier target (ISIT) camera (ISIT-66 series, Dage-MTI, Michigan City, IN). The video signal from the ISIT camera was fed to a Comdek 80386-DX computer and digitized by an FG100-1024 imaging card (Imaging Technology, Woburn, MA), which consisted of an analog-to-digital converter, frame memory, image-processing hardware, and a digital-to-analog converter for a pseudocolor display of images on an NEC Multisync 3D color monitor. Each pixel element was digitized as an eight-bit number and stored on the computer to form a pixel map of the image.

Estimation of free Ca2+i concentration in single cells. Free Ca2+i concentration ([Ca2+]i) was estimated by ratiometric analysis of fura 2 fluorescence images elicited by excitation at 340 and 380 nm captured ~700 ms apart. The potential influence of background fluorescence was assessed by capturing a background image for both excitation wavelengths at the commencement of each experiment. The resultant average pixel values were subtracted from subsequently captured images at each respective excitation wavelength. After background subtraction, each 340-nm fluorescence image was divided by its corresponding 380-nm fluorescence image on a pixel-by-pixel basis, and the individual pixel ratios were averaged to provide a 340- to 380-nm fluorescence ratio (F340/F380) value for that time point. Calculation of [Ca2+]i from the fura 2 F340/F380 was according to Grynkiewicz et al. (10).

Fura 2-loaded ASMCs bathed in imaging medium were allowed to equilibrate on the heated microscope stage (37°C) for 15 min to ensure complete fura 2-AM hydrolysis, after which time 340- and 380-nm fluorescence intensity image pairs were recorded at 30-s intervals (baseline fluorescence ratio). Acquisition of fluorescence images was interrupted, the cells were exposed to prewarmed imaging medium (37°C) containing the required concentration of ET-receptor agonist, and image acquisition was resumed at 3-s intervals. In experiments with ET-receptor antagonists, BQ-123 and BQ-788 were initially introduced to the cells for 20 min before agonist challenge and were included in subsequent medium changes.

Drugs. Drugs and chemicals utilized were ET-1, 125I-ET-1, sarafotoxin S6c, BQ-123 (all from Auspep, Melbourne, Australia), BQ-788 (Banyu Pharmaceutical, Tsukuba, Japan), DMEM-F-12 (Life Technologies), pentobarbital sodium (Nembutal, Boehringer Ingelheim), phenylmethylsulfonyl fluoride (Calbiochem, La Jolla, CA), BSA, antibiotic-antimycotic, DMSO, trypsin, transferrin, alpha -actin smooth muscle antibody, mouse ascites fluid, goat anti-mouse IgG-FITC conjugate (all from Sigma, St. Louis, MO), FCS, insulin (both from Commonwealth Serum Laboratories, Melbourne, Australia), rat IgG (generously donated by Dr. Max Cake, School of Biological and Environmental Sciences, Murdoch University, Perth, Australia), fura 2-AM, and Pluronic F-127 (both from Molecular Probes, Eugene, OR). Stock solutions of ET-1 and sarafotoxin S6c were prepared in 0.1 M acetic acid, whereas BQ-123 was prepared in 100 mM Na2CO3 and BQ-788 in DMSO. Fura 2-AM (1 mg) was dissolved in 1 ml of dry DMSO containing 15% (wt/vol) Pluronic F-127, a detergent that facilitates cellular uptake of fura 2-AM, to a final concentration of 1 mM and was stored desiccated in 5-µl aliquots at -20°C. All other drugs were prepared in normal saline. All drugs were kept on ice throughout an experiment, and subsequent dilutions were performed in saline.

Data analysis. Numerical data are presented as means ± SE, and unless otherwise stated, differences between treatment means were assessed by one-way analysis of variance followed by a modified t-statistic (17). P values < 0.05 were considered to be statistically significant.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Sheep tracheal ASMC cultures. Typically, 80-90% of cells liberated by the enzymatic and mechanical dispersion of sheep tracheal smooth muscle were viable. The vast majority of cells maintained in culture for up to at least 14 passages stained positively for immunoreactive alpha -actin, which was arranged in long fibers traversing the length of the cells. Within 7-9 days, cells formed confluent monolayers, which if left undisturbed for an additional 3-5 days, formed multilayered ridges or "hills" randomly distributed across the surface, separated by monolayers of cells or "valleys." A 2-day incubation in SFM inhibited cell growth at all time points examined such that cell numbers in SFM were consistently lower than the corresponding density of the cells exposed to 10% serum (Fig. 1).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Time course of sheep airway smooth muscle cell (ASMC) growth in medium containing 10% fetal calf serum (open circle ) and after a 2-day period in serum-free medium (bullet ). Data are means ± SE and are representative of 3 separate experiments.

Radioligand binding studies. In time-course studies, total specific 125I-ET-1 binding to ASMCs (sixth passage; day 7) increased rapidly in a time-dependent manner and plateaued after 120 min at a Bmax of ~20,000 binding sites/cell (Fig. 2A). Nonspecific binding determined in the presence of BQ-123 (1 µM) and sarafotoxin S6c (200 nM) increased in a linear fashion over time but represented substantially <10% of the specific binding at any given time point. In subsequent competition binding studies, coincubation of 125I-ET-1 with the ETB receptor-selective ligand sarafotoxin S6c (200 nM) did not reduce 125I-ET-1 binding, whereas the ETA receptor-selective ligand BQ-123 (1 µM) almost abolished specific 125I-ET-1 binding (Fig. 2B).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   A: time course of 125I-endothelin (ET)-1 binding in confluent monolayer cultures of sheep ASMCs (6th passage) depicting total specific binding (open circle ) and nonspecific binding (bullet ). Data are means ± SE and are representative of 2 separate experiments; n = 3 cells. B: relative proportion of ET-receptor subtypes in sheep ASMCs (day 9; 6th passage). Specific 125I-ET-1 binding maxima were assessed in confluent cells to provide estimates of total, nonspecific (NS; in presence of 1 µM BQ-123 and 200 nM sarafotoxin S6c), ETA receptor-specific (sites occluded by 1 µM BQ-123), and ETB receptor-specific (sites occluded by 200 nM sarafotoxin S6c) binding. Data are means ± SE and are representative of 3 separate experiments; n = 3 cells.

Throughout the growth period of sixth-passage ASMCs, ranging from freshly subcultured cells to confluent monolayers, the number of ETA-specific binding sites remained relatively constant at ~20,000-25,000 sites/cell for all time points examined (Fig. 3A). However, the ETB-specific binding site number increased from negligible levels on days 1 and 3 to ~10,000 sites/cell as the cells neared confluence on day 9. An additional 6 days in culture had no further effect on the number of ETB-specific binding sites per cell (Fig. 3A).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3.   Changes in ETA- and ETB-receptor density from seeding to confluence and during extended culture of sheep ASMCs. A: density of ETA (circles) and ETB (triangles) receptor-specific binding sites/cell determined in 7th-passage sheep ASMCs (open symbols) and effect of 2 days of incubation in serum-free medium (solid symbols). n = 3 cells. B: density of ETA (circles) and ETB (triangles) receptor-specific binding/cell determined on day 7 from passages 6-14 (open symbols) and effect of serum deprivation (solid symbols). Data are means ± SE from 3 separate experiments.

Serum deprivation had a negligible effect on ETA-specific binding site density throughout the culture period of 15 days (Fig. 3A) but abolished the increase in ETB-specific binding site numbers at all time points examined (Fig. 3A). Thus serum deprivation of sheep tracheal ASMCs in culture promoted either the retention (at early stages of cell growth) or the conversion (at later stages of cell growth) of the ET-receptor population to an essentially homogeneous ETA-receptor population at all time points examined (Fig. 3A).

During serial passage of ASMCs (6th to 14th passages), the number of ETA-specific binding sites on day 7 of culture remained relatively constant, whereas the number of ETB-specific binding sites increased from negligible levels in passages 6 and 7 to levels that were comparable with those for ETA sites by passage 14 (Fig. 3B). In concurrently performed experiments, a 2-day period of serum deprivation did not affect the number of ETA-specific binding sites but completely abolished the increase in ETB-specific binding site numbers previously seen in the 6th- to 14th-passage ASMCs exposed to 10% serum (Fig. 3B). Thus serum deprivation of sheep tracheal ASMCs in culture maintained an essentially homogeneous ETA-receptor population in all passages examined.

Ca2+ mobilization studies. ET-1 induced a concentration-dependent increase in [Ca2+]i in ASMCs, which typically involved an early transient [Ca2+]i peak followed by a smaller, sustained increase in [Ca2+]i (Figs. 4A and 5A). Both components of the ET-evoked [Ca2+]i increase were blocked by BQ-123 (Fig. 5B) but were unaffected by BQ-788 (Fig. 5C). Consistent with this, sarafotoxin S6c failed to induce any significant [Ca2+]i increase in these cells (Fig. 5D). Removal of extracellular Ca2+ by placement of the cells in a Ca2+-free medium did not affect the peak [Ca2+]i increase induced by ET-1 but abolished the smaller, sustained [Ca2+]i increase (Fig. 5E).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 4.   ET-1-induced intracellular Ca2+ concentration ([Ca2+]i) increase in individual sheep ASMCs in culture. A: representative curves illustrating effects of increasing concentrations of ET-1 ([ET-1]; 2.5-100 nM) on [Ca2+]i. B: dose-response relationship between [ET-1] and [Ca2+]i [difference (Delta ) between baseline and peak [Ca2+]i levels]. Data are means ± SE; n = 38-45 cells for each [ET-1].


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5.   Representative curves of effects of selective ET-receptor subtype activation on [Ca2+]i in individual sheep tracheal ASMCs in culture. A: ET-1 (50 nM). B: sarafotoxin S6c (10 nM). C: ET-1 (50 nM) + BQ-788 (1 µM). D: ET-1 (50 nM) + BQ-123 (3 µM). E: ET-1 (50 nM) in Ca2+-free medium.

Given that an increase in ETB-receptor number was observed as sheep tracheal ASMCs exposed to 10% FCS approached confluence and that these changes were completely reversed by serum deprivation (Fig. 3), the coupling of these receptors to Ca2+ mobilization was assessed. The number of cells that responded to ETA-receptor stimulation was unaffected by either time in culture or serum deprivation and remained stable at 100% (Fig. 6C), in keeping with the consistency of receptor expression under these conditions (Fig. 6B). Moreover, the magnitude of the ETA receptor-induced [Ca2+]i response was unaffected, with a maximal increase of between 400 and 450 nM under all conditions examined (Fig. 6D). In contrast, the appearance of ETB receptors on day 15 of culture (Fig. 6B) was associated with a significant increase in the number of cells responding to sarafotoxin S6c (to ~45% of cells; P < 0.05; Fig. 6C). However, although a substantial increase in responsive cells was seen, only a modest [Ca2+]i increase (~70 nM) linked to ETB-receptor activation was recorded (P < 0.05; Fig. 6D). Furthermore, the increase in cell numbers responding to ETB-receptor activation and the resulting Ca2+ mobilization were abolished after 2 days of serum deprivation (Fig. 6, G and H), in keeping with the ability of SFM to reverse the appearance of ETB receptors (Fig. 6F).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 6.   Linkage of ETA and ETB receptors to Ca2+ mobilization in sheep tracheal ASMCs in culture (7th passage) from seeding to confluence (A-D) and effect of serum deprivation (E-H). Solid bars, ETA receptors; open bars. ETB receptors. A and E: time course for growth of 7th-passage sheep tracheal ASMCs. A: open circle , cells obtained after different periods in 10% FCS; bullet , cells used for further studies (B-D) after 3 and 15 days in 10% FCS. E: open circle , cells obtained after different periods in 10% FCS before 2-day period in serum-free medium; bullet , cells obtained after 2-day period in serum-free medium; shaded circle, cells used for further studies (F-H) after 2-day period in serum-free medium. n = 3 experiments. B and F: ET-receptor subtype numbers on days 3 and 15. n = 3 experiments. C and G: percentage of cells responding to ET-receptor subtype challenge. n = 46-50 cells. D and H: peak increase in [Ca2+]i in response to ET-receptor subtype activation. n = 45-49 cells. Data are means ± SE. * P < 0.05 compared with day 3 response.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

This paper describes the differential changes that occurred in the density and function of ETA and ETB receptors on sheep tracheal ASMCs during cell growth and extended culture and how these changes were modified by various culture conditions such as a serum-free period. In early cultures, radioligand binding studies identified a homogeneous population of ETA receptors on sheep tracheal ASMCs, consistent with a previously published autoradiographic study (7) that used tissue sections of intact sheep tracheal smooth muscle. However, cellular growth was found to substantially modify the proportions of ETA and ETB receptors, as did extended culture of ASMCs. In both instances, a marked increase in ETB-receptor density was observed, whereas ETA-receptor density remained unchanged. Furthermore, selective stimulation of ETB receptors in these sheep tracheal ASMCs was associated with a modest increase in [Ca2+]i, suggesting a functional role for these newly revealed ETB receptors. Under all conditions examined, a 2-day period of serum deprivation dramatically reduced ETB-receptor density and function and promoted the maintenance of ET-receptor subtype proportions in cell culture that were similar to those receptor subtype proportions previously observed in intact tissues.

In the present study, specific binding of 125I-ET-1 to sheep tracheal ASMCs in early culture was fully inhibited by an ETA receptor-selective ligand, BQ-123, but was insensitive to blockade by an ETB receptor-selective ligand, sarafotoxin S6c. These findings indicate the presence of an essentially homogeneous population of ETA receptors in early-culture ASMCs and are entirely consistent with a recent quantitative autoradiographic study (7) that estimated that the ratio of ETA to ETB receptors was 100:0 in intact sheep tracheal smooth muscle. The present study has also demonstrated that ET-1, via the stimulation of ETA receptors, increased [Ca2+]i in sheep tracheal ASMCs, an effect that may well be linked to the production of a contractile response because BQ-123-sensitive contractions induced by ET-1 in intact sheep tracheal smooth muscle preparations were strictly dependent on increases in [Ca2+]i (7). In addition to contraction, ETA receptors might also be linked to other cellular functions, including mitogenesis, in cultured ASMCs from sheep trachea (3).

As sheep tracheal ASMCs approached confluence, there was a marked increase in the density of ETB receptors, which represented the appearance of a receptor subtype that is not expressed in significant numbers in intact tracheal smooth muscle in this species. Despite the marked increase in overall ETB-receptor density, ETB-receptor stimulation with ET-1 or sarafotoxin S6c evoked a modest increase in [Ca2+]i in only 50% of ASMCs. This may indicate that ETB receptors are not expressed in all ASMCs and/or that not all ETB receptors are necessarily linked to increases in [Ca2+]i. The precise role for these emergent ETB receptors is not entirely clear, but Carratu et al. (3) have recently demonstrated that ET-1-induced mitogenesis in cultured sheep tracheal ASMCs is, at least in part, mediated by stimulation of a small population of ETB receptors. Whether the ETB receptors identified in the present study contribute to ASMC growth through Ca2+-dependent or Ca2+-independent mechanisms remains to be established. In addition to promoting cell division, one hitherto untested possibility is that these ETB receptors, like ETA receptors, are also linked to contraction. Evidence supporting this possibility comes from Adner et al. (1) and Moller et al. (14), who demonstrated that organ culture of segments of human omental arteries, which are normally devoid of ETB receptors, resulted in the development of a substantial ETB receptor-mediated contractile response.

Despite the marked increase in expression of functional ETB receptors, the density of ETA receptors on these cells remained relatively stable during ASMC growth. The selective alteration of ETB-receptor number is consistent with recent functional reports of the plasticity of contractile ETB receptors in human and animal blood vessels when subjected to short-term organ culture (1, 2, 14). Although these workers suggested that organ culture induces transcription and subsequent translation of contractile ETB receptors, the possibility of such a mechanism operating in cell cultures of ASMCs remains to be tested.

Similar to the results obtained during cellular growth, repeated passage of sheep tracheal ASMCs elicited a selective and substantial increase in ETB-receptor number. This increase in ETB-receptor number showed no signs of abatement by the 14th passage, at which stage the ETB receptors comprised 50% of the total ET-receptor pool. In line with these findings, Eguchi et al. (4) also reported a significant increase in the relative proportion of ETB receptors during the extended culture of rat vascular smooth muscle cells. However, in contrast, Inui et al. (13) have reported that repetitive subculture of guinea pig tracheal ASMCs resulted in the complete disappearance of ETB-receptor binding sites and in Ca2+ responses to 10 nM ET-3, suggesting that culture conditions may induce species-dependent changes in ET-receptor subtype proportions.

Exposure of sheep tracheal ASMCs in culture to SFM completely abolished both the increase in ETB-receptor number and the ETB receptor-evoked increase in [Ca2+]i that occurred during cellular growth and extended culture. Despite this, serum deprivation was without effect on ETA-receptor density or ETA receptor-mediated Ca2+ signaling at any time during the growth cycle. Regardless of the passage number or ET-receptor subtype proportions before exposure to SFM, serum deprivation completely reversed any changes in ETB-receptor number and restored an essentially homologous ETA-receptor population. Although the ability of serum deprivation to modify receptor expression is not without some precedent because it has been shown to selectively alter receptor subunit mRNA levels and the density and proportion of N-methyl-D-aspartate-receptor subtypes (19), this has not been directly demonstrated before in relation to ET receptors either in non-smooth muscle or smooth muscle tissues.

The stimulus for change in ET-receptor expression and also possibly in functional linkages may be a product of cell cycle phase-dependent processes, differing cell densities in culture, the lack of cofactors released by neighboring cell types in vivo, or perhaps serum factors to which many cells are not normally exposed. At present, there are few firm indicators as to the underlying cause of the observed changes in ET-receptor density and function; however, the possibility of altered receptor systems occurring in all cultures should not be overlooked and must be investigated before the validity of a cell culture model can be assessed.

    ACKNOWLEDGEMENTS

We thank Glenn Self for developing the calcium-imaging software.

    FOOTNOTES

We acknowledge the financial support of the National Health and Medical Research Council (Australia) and the Asthma Foundation of Western Australia.

Address for reprint requests: P. J. Henry, Dept. of Pharmacology, Univ. of Western Australia, Nedlands, Western Australia 6907, Australia.

Received 1 December 1997; accepted in final form 18 February 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Adner, M., L. Cantera, F. Ehlert, L. Nilsson, and L. Edvinsson. Plasticity of contractile endothelin-B receptors in human arteries after organ culture. Br. J. Pharmacol. 119: 1159-1166, 1996[Abstract].

2.  Adner, M., D. Erlinge, L. Nilsson, and L. Edvinsson. Upregulation of a non-ETA receptor in human arteries in vitro. J. Cardiovasc. Pharmacol. 26, Suppl. 3: S314-S316, 1995.

3.   Carratu, P., M. Scuri, J. L. Styblo, A. Wanner, and M. K. Glassberg. ET-1 induces mitogenesis in ovine airway smooth muscle cells via ETA and ETB receptors. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L1021-L1024, 1997[Abstract/Free Full Text].

4.   Eguchi, S., Y. Hirata, T. Imai, K. Kanno, and F. Marumo. Phenotypic change of endothelin receptor subtype in cultured rat vascular smooth muscle cells. Endocrinology 134: 222-228, 1994[Abstract].

5.   Ergul, A., M. K. Glassberg, A. Wanner, and D. Puett. Characterization of endothelin receptor subtypes on airway smooth muscle cells. Exp. Lung Res. 21: 453-468, 1995[Medline].

6.   Fukuroda, T., S. Ozaki, M. Ihara, K. Ishikawa, M. Yano, T. Miyauchi, S. Ishikawa, M. Onizuka, K. Goto, and M. Nishikibe. Necessity of dual blockade of endothelin ET(A) and ET(B) receptor subtypes for antagonism of endothelin-1-induced contraction of human bronchi. Br. J. Pharmacol. 117: 995-999, 1996[Abstract].

7.   Goldie, R. G., P. S. Grayson, P. G. Knott, G. J. Self, and P. J. Henry. Predominance of endothelinA (ETA) receptors in ovine airway smooth muscle and their mediation of ET-1-induced contraction. Br. J. Pharmacol. 112: 749-756, 1994[Abstract].

8.   Goldie, R. G., P. J. Henry, P. G. Knott, G. J. Self, M. A. Luttmann, and D. W. P. Hay. Endothelin-1 receptor density, distribution, and function in human isolated asthmatic airways. Am. J. Respir. Crit. Care Med. 152: 1653-1658, 1995[Abstract].

9.   Goldie, R. G., P. G. Knott, M. J. Carr, D. W. P. Hay, and P. J. Henry. The endothelins in the pulmonary system. Pulm. Pharmacol. 9: 69-93, 1996[Medline].

10.   Grynkiewicz, G., M. Poenie, and R. Y. Tsien. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260: 3440-3450, 1985[Abstract].

11.   Hirst, S. J. Airway smooth muscle cell culture: application to studies of airway wall remodelling and phenotype plasticity in asthma. Eur. Respir. J. 9: 808-820, 1996[Abstract/Free Full Text].

12.   Ihara, M., K. Noguchi, T. Saeki, T. Fukuroda, S. Tschida, S. Kimura, and M. Yano. Biological profiles of highly potent novel endothelin antagonists selective for the ETA receptor. Life Sci. 50: 247-255, 1992[Medline].

13.   Inui, T., A. F. James, Y. Fujitana, M. Takimoto, T. Okada, T. Yamamura, and Y. Urade. ETA and ETB receptors on single smooth muscle cells cooperate in mediating guinea pig tracheal contraction. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L113-L124, 1994[Abstract/Free Full Text].

14.   Moller, S., L. Edvinsson, and M. Adner. Transcriptional regulated plasticity of vascular contractile endothelin ETB receptors after organ culture. Eur. J. Pharmacol. 329: 69-77, 1997[Medline].

15.   Skalli, O., P. Ropraz, A. Trzeciak, G. Benzonana, D. Gillessen, and G. Gabbiani. A monoclonal antibody against alpha -smooth muscle actin: a new probe for smooth muscle differentiation. J. Cell Biol. 103: 2787-2796, 1986[Abstract].

16.   Waggoner, W. G., S. L. Genova, and V. A. Rash. Kinetic analyses demonstrate that the equilibrium assumption does not apply to [125I]-endothelin-1 binding data. Life Sci. 51: 1869-1876, 1992[Medline].

17.   Wallenstein, S., C. L. Zucker, and J. L. Fleiss. Some statistical methods useful in circulation research. Circ. Res. 47: 1-9, 1980[Abstract].

18.   Williams, D. L., Jr., K. L. Jones, D. J. Pettibone, E. V. Lis, and B. V. Clinesschidt. Sarafotoxin S6c: an agonist which distinguishes between endothelin receptor subtypes. Biochem. Biophys. Res. Commun. 175: 556-561, 1991[Medline].

19.   Zhong, J., S. L. Russell, D. B. Pritchett, P. B. Molinoff, and K. Williams. Expression of mRNAs encoding subunits of the N-methyl-D-aspartate receptor in cultured cortical neurons. Mol. Pharmacol. 45: 846-853, 1994[Abstract].


Am J Physiol Lung Cell Mol Physiol 274(6):L951-L957
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society