Department of Pharmacology, University of Western Australia, Nedlands, Western Australia 6907, Australia
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ABSTRACT |
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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
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INTRODUCTION |
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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).
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METHODS |
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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 -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
-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
-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, ![]() |
RESULTS |
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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 -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).
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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).
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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).
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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).
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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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We thank Glenn Self for developing the calcium-imaging software.
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FOOTNOTES |
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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.
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