Introduction

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The ETs are a family of potent vasoactive peptides which include ET-1, -2 and -3 and exert their effects by binding to specific G protein-coupled receptor subtypes, ETA and ETB. The two subtypes can be distinguished pharmacologically by different rank orders of affinity toward the three ET isopeptides; ETA is ET-1-selective, showing an affinity rank order of ET-1  ET-2  ET-3, whereas ETB exhibits similar affinities to all the three isopeptides (Masaki et al., 1992).

Since the original identification of ET-1 in the culture medium of vascular endothelial cells (Yanagisawa et al., 1988), this peptide has been produced by a variety of cell types including airway epithelial cells. Cultured tracheobronchial epithelial cells from various species, including guinea pig, rabbit, dog and human, secret ET-1 into the medium (Black et al., 1989; Mattoli et al., 1990; Ninomiya et al., 1991; Rennick et al., 1993; Noguchi et al., 1995). As in vascular endothelial cells, the secretion of ET-1 by the tracheobronchial epithelial cells can be modulated by various agents such as interleukins, tumor necrosis factor, transforming growth factor, thrombin or bacterial endotoxins (Ninomiya et al., 1991; Endo et al., 1992; Rennick et al., 1993). Airway epithelium is composed of heterogeneous cells, and in trachea and large bronchi, it includes at least three distinct cell types, basal, ciliated columnar and nonciliated secretory cells (Nettesheim et al., 1990; Jetten, 1991). Immunohistochemical studies with anti-ET-1 antibodies indicated that, among the three cell types, nonciliated secretory cells are the major site of ET-1 secretion in the tracheobronchial epithelium of rat and mouse (Rozengurt et al., 1990).

Several lines of evidence indicated that, in addition to the ligand, the tracheobronchial epithelial cells also express ET receptors. Ligand binding studies indicated the presence of ET-1 binding sites in cultured feline and canine tracheal epithelial cells (Wu et al., 1993; Ninomiya et al., 1995), and autoradiographic studies with guinea pig tracheal sections indicated the presence of both subtypes of ET receptors in the epithelium (Tschirhart et al., 1991). Besides, functional studies have described various effects of ET-1 on the physiological functions of the epithelial cells. When applied to isolated tissues or cultured tracheal epithelial cells from ferret, dog, rabbit or human, ET-1 stimulates chloride secretion (Plews et al., 1991; Satoh et al., 1992; Webber et al., 1992), ciliary motility (Tamaoki et al., 1991; Amble et al., 1993) and fibronectin secretion (Marini et al., 1996). ET-1 also stimulates the release of prostanoids from feline tracheal epithelial cells (Wu et al., 1993) and that of NO from guinea pig tracheal epithelium (Filep et al., 1993). Furthermore, ET-1 works as a mitogen on cultured porcine tracheal epithelial cells (Murlas et al., 1995). Although these studies indicated the autocrine/paracrine role of ET-1 in the tracheal epithelium, localization of ET receptors in the epithelium has not been examined and the receptor subtype involved in the individual effect of ET-1 remains unclear, except for a few cases in which the roles of ETA in the stimulation of fibronectin secretion (Marini et al., 1996) and cell proliferation (Murlas et al., 1995) have been described.

Among the various effects of ET-1 on the tracheal epithelial cells, the mitogenic effect of ET-1 may be particularly relevant to the possible role of this peptide in the normal turnover of epithelium and/or abnormal turnover of it under pathological conditions such as asthma or airway inflammation. Given the heterogeneity of the tracheal epithelial cells, we wished to identify the target cell type of the mitogenic effect of ET-1 and the receptor subtype responsible for it. For this purpose, we first examined the localization of ET receptor subtypes by immunofluorescent staining and tested the effects of receptor antagonist/agonist on the proliferation of cultured epithelial cells. The results obtained in the present study indicated the presence of paracrine ET-1 signaling pathways in the control of the basal cell proliferation that involved all three major cell types in the tracheal epithelium and both subtypes of ET receptors.

	Methods

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Materials. Synthetic human ET-1, big ET-1 and ET-3 were obtained from Peptide Institute Inc. (Osaka, Japan). BQ123 (an ETA-selective antagonist), BQ788 (an ETB-selective antagonist) and IRL1620 (an ETB-selective agonist) were from Peninsula Laboratories, Inc. (Belmont, CA). [¹²⁵I]ET-1 (74 TBq/mmol) was from Amersham International (Buckinghamshire, UK). The serum-free BEGM for the tracheal epithelial cell culture was from Kurabo (Osaka, Japan). BEGM was supplemented for use with the following: insulin (5 µg/ml), epidermal growth factor (0.5 ng/ml), hydrocortisone (0.5 µg/ml), transferrin (10 µg/ml), epinephrine (0.5 µg/ml), triiodothyronine (6.5 ng/ml), gentamycin (50 µg/ml), amphotericin B (50 µg/ml), retinoic acid (0.1 ng/ml) and bovine pituitary extract (0.4% v/v). Vitrogen 100 collagen gel was from Collagen Corp. (Fremont, CA). Rabbit polyclonal anti-ET-1 and anti-bigET-1 antibodies were from Peninsula Laboratories, Inc. (Belmont, CA). Rabbit polyclonal anti-eNOS antibody was from Transduction Laboratories (Lexington, KY). Goat polyclonal anti-p53 antibody (M-19) was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Affinity-purified rabbit polyclonal antibodies against the synthetic peptides that correspond to the carboxyl-terminal 12 amino acids of human ETA and ETB were kindly provided by Dr. S. Hori and Dr. M. Takimoto at the Takarazuka Research Institute, Novartis Pharma K. K. (Takarazuka Japan). The cell proliferation assay kit with MTT was from Promega (Madison, WI). SNP and L-NMMA were from Sigma Chemical Co. (St. Louis, MO). All other reagents used were of the purest grade available.

Immunofluorescent staining. Hartley-strain male guinea pigs (250-300 g) were anesthetized with sodium pentobarbital (50 mg/kg i.p.; Sigma) and the tracheae were removed and trimmed of the connective tissue. After fixation at 4°C overnight in 4% paraformaldehyde/0.1 M phosphate buffer (pH 7.4) followed by serial incubations each at 4°C for 24 h in 10% sucrose/PBS and in 20% sucrose/PBS, they were embedded in OCT compound (Miles Inc., Elkhart, IN) and 10-µm sections were cut with a cryostat and stored at 4°C. The sections were immunostained with anti-ETA, ETB and eNOS antibodies by the indirect fluorescent technique. After blocking in Block Ace (Dainippon Pharmaceutical Co., Suita, Japan), the sections were incubated with the antibodies at 4°C for 24 h in a humidity chamber. The working concentrations of the antibodies were anti-ETA, 2 µg/ml; anti-ETB, 2 µg/ml; and anti-eNOS, 1 µg/ml. The sections were rinsed with PBS and sequentially incubated with biotinylated anti-rabbit IgG (1:1000, Amersham) and with Cy2-avidin (1:500, Amersham). They were mounted in fluoromount G (Southern Biotechnology Associates, Inc., Birmingham, AL) and fluorescent images were obtained with a MRC1024 confocal microscope (BioRad Laboratories Inc., Osaka, Japan). Staining with anti-ET-1 and anti-bigET-1 antibodies was done exactly in the same way except for the additional incubation of the sections in 100% methanol at the room temperature for 1 h before the blocking (Yoshizawa et al., 1990). Controls were performed by processing the slides lacking the primary antibody and also by preincubating the primary antibody with excess antigen peptides (anti-ETA, ETB), synthetic ET-1 or bigET-1 (anti-ET-1, bigET-1). HE and PAS stains were done with Meyer's hematoxylin, eosin Y and PAS staining kit (Muto chemicals, Tokyo, Japan).

Primary culture of tracheal epithelial cells. Tracheal epithelial cells were isolated by the method of Yu et al. (1992) with slight modifications. The tracheae were isolated, rinsed with HBSS (Flow Labs, MacLean, VA) and trimmed of the connective tissue. They were incubated at 4°C for 24 h in HBSS containing pronase (1 mg/ml; Sigma), rinsed with HBSS and opened with a longitudinal incision. The tracheal lumen was washed with HBSS to remove the epithelial cells that then were filtered through a 70-µm pore nylon mesh. The cells were centrifuged at 100 × g for 5 min at 4°C and resuspended in BEGM. The number of cells was counted with a Coulter cell counter (Coulter Electronics Inc., Hialeah, FL). Approximately 2.5 × 10⁶ cells were recovered from one trachea. The cells were used for immersion or air-interphase cultures. Both the cell culture dishes (immersion culture) and the membrane inserts of Costar Transwells (air-interphase culture) were coated with vitrogen 100 (Collagen Corp.) according to the manufacture's instructions. For immersion cultures, basal cell- and non-basal cell-enriched cultures were obtained by differential plating (Chang et al., 1985). The resuspended cells first were seeded on noncoated culture dishes (~2.5 × 10⁶ cells from one trachea to a 75-cm² 100-mm dish) and incubated for 12 h in a CO₂ incubator. Loosely attached cells and firmly attached cells were collected in succession by washing the culture bed and then scraping the remaining cells. The cells were transferred to the vitrogen-coated dishes to give basal- and non-basal-enriched cultures. All of the cells were recovered by scraping without washing and seeded on the vitrogen-coated dishes to give a mixed cell culture. The identity of he basal cells was verified by immunostaining with antiepithelial keratin-AE1/AE3 mixture (ICN Flow, Costa Mesa, CA). Air-interphase culture was obtained as described by Yamaya et al. (1992) with some modifications. The recovered cells (without differential plating) were seeded directly on the vitrogen-coated membrane inserts of Costar Transwells (6.5-mm diameter, 0.1-µm pore; Costar, Cambridge, MA) at the density of 10⁵/well. Twenty-four hours later, the upper medium was removed to expose the cells to the air. Thereafter, the cells were fed by replacing the lower medium with fresh BEGM and maintained up to 10 days.

Western blotting. Crude membrane preparations from dissociated epithelial cells were used for Western blotting with anti-ETA and anti-ETB antibodies. The cells suspended in the lysis buffer (10 mM Tris-HCl, pH 7.8, 1 mM ethyleneglycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1 mM ethylenediaminetetraacetic acid) were lysed by sonication followed by centrifugation at 100,000 × g for 30 min. The pellet was suspended in the lysis buffer and protein concentration was determined by a BCA micoprotein assay kit (Pierce, Rockford, IL). The samples were mixed with Laemmli's sample buffer, electrophoresed on a polyacrylamide gel and transferred to a poly(vinylidene difluoride) membrane. The membrane was probed with the antibody, and the bound antibody was detected with ¹²⁵I-labeled protein A (Amersham). Autoradiographs were developed with a BAS2000 image analyzer (Fujitsu, Tokyo, Japan). The specificity of anti-ETA/ETB antibodies was verified by Western blotting of the membrane proteins obtained from Chinese hamster ovary cells expressing human ETA or ETB (data not shown). Whole cell lysates from the cultured epithelial cells were used for Western blotting with an anti-p53 antibody. The cells were harvested by scraping, centrifuged and then incubated for 30 min at 4°C in the lysis buffer (20 mM Tris-HCl, pH 7.8, 137 mM NaCl, 5 mM ethyleneglycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1 mM ethylenediaminetetraacetic acid, 2 mM dithiothreitol, 1 mM 4-(2-aminoethyl)-benzenesulfonylfluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml pepstatin A, 0.1% Triton X-100). After brief centrifugation, the soluble fraction was subjected to SDS-PAGE, Western transfer and immunoblotting. The blots were developed with an ABC immunodetection kit (Vector Laboratories, Inc., Burlingame, CA) and Konica immunostain HRP-1000 (Konica, Tokyo, Japan). Densitometric analysis was performed with NIH Image software.

Enzyme-linked sandwich immunoassay for ET-1. The conditioned medium from cultured epithelial cells was filtrated through a 0.2-µm pore filter and the concentration of irET-1 was determined as described (Suzuki et al., 1989).

Displacement of [¹²⁵I]ET-1 binding. To minimize the effects of endogenous ETs, membrane preparations were prepared from the cultured cells that had been exposed to 0.1 mM phosphoramidon for 24 h (Takimoto et al., 1996). The cells were harvested by scraping and suspended in PBS. They were lysed by sonication followed by centrifugation at 100,000 × g for 30 min. The pellets were resuspended in PBS. The membrane preparations (20 µg protein/assay) were incubated with 50 pM [¹²⁵I]ET-1 and increasing concentrations (1 pM-1 µM) of either ET-1 or ET-3 in 0.2 ml of PBS/0.2% bovine serum albumin. After incubation for 60 min at 25°C, bound ligand was separated from free ligand by filtration through Whatman GF/C glass fiber filters preimmersed in the ice-cold binding medium. The filters were washed with ice-cold PBS and counted for radioactivity in a -counter. Nonspecific binding was defined as the binding in the presence of 100 nM unlabeled ET-1 and was always less than 10% of the total binding capacity.

Cell proliferation assay. Cells were grown on vitrogen-coated 96-well plates and the number of viable cells in each well was estimated by the measurement of the rate of mitochondrial metabolism of MTT with a cell proliferation assay kit (Promega). The cells were exposed for 4 h to MTT (1 mg/ml) and then lysed with acidic lysis buffer containing SDS. The optical densities of the lysate at 562 and 630 nm were measured with an EL340 microtiter plate reader (BIO-TEK Instruments, Inc., Tokyo, Japan).

cGMP measurement. Cells grown on vitrogen-coated 6-well plates for 48 h were used for the assay. The medium was gently replaced (so as not to detach the loosely attached nonbasal cells) with 1.8 ml of HBSS containing a phosphodiesterase inhibitor 3-isobuthyl-1-methylxanthine (1 mM). After 5 min incubation, 0.2 ml of HBSS containing increasing concentrations of IRL1620 was added to start the reaction. Because IRL1620 caused a linear increase of cGMP content at least for 5 min in the mixed cell culture (data not shown), the reaction time was set to 5 min. The reaction was halted by adding ice-cold ethanol at the final concentration of 65% v/v. The solvent was evaporated by centrifugation under reduced pressure and the cGMP content in the resultant pellet was measured with an enzyme-linked immunoassay kit (Amersham).

Statistical analysis. Where necessary, statistical analysis was performed by analysis of variance.

	Results

Top Abstract Introduction Methods Results Discussion References

Cellular/subcellular localization of ET receptors, ET-1 and eNOS in guinea pig tracheal epithelium. Western blotting with anti-ETA/ETB antibodies indicated the expression of both subtypes of ET receptors in guinea pig tracheal epithelial cells (fig. 1a). In the membrane proteins from dissociated cells, a single band at 49 kDa was detected with the anti-ETA antibody, whereas a major band at 49 kDa and a minor band at 32 kDa were detected with the anti-ETB antibody. The 32 kDa band corresponds to a proteolytic derivative with amino-terminal truncation (Kozuka et al., 1991).

View larger version (0K): [in this window] [in a new window]   View larger version (0K): [in this window] [in a new window]   Fig. 1.   A-C.  Cellular/subcellular localization of ET receptors, ET-1, bigET-1 and eNOS in guinea pig tracheal epithelium. (a) Western blotting of membrane proteins from dissociated tracheal epithelial cells with anti-ET receptor antibodies. Membrane preparations were prepared from dissociated tracheal epithelial cells as described under "Methods." Proteins were separated on SDS 12%-PAGE and transferred to a poly(vinylidene difluoride) membrane. The membrane was probed with affinity-purified anti-ETA antibody (lanes 1, 2) or anti-ETB antibody (lanes 3, 4). Excess antigen peptide for each antibody was included in the blotting in lanes 2 and 4. Molecular weights are indicated on the left (kDa). (b-g) Immunofluorescent staining of tracheal epithelial sections. Ten-micrometer horizontal sections of trachea were stained with antibodies against ETA (b), ETB (c, d), ET-1 (e), big ET-1 (f) and eNOS (g) (bar, 10 µm). The bound antibodies were detected with biotinylated secondary antibody and Cy2-avidin, and fluorescent images were obtained with confocal microscopy. Shown in panel d is the fluorescent image of anti-ETB staining (green) merged with a DIC image of the same field.

Differential plating and culture of tracheal epithelial cells. To examine the effects of ET-1 on the individual cell type, we attempted to obtain primary cultures enriched in basal and other types of cells (the latter including the ciliated columnar and nonciliated secretory cells) by differential plating. Figure 2a shows the morphological appearance of the cells 24 h after seeding on the vitrogen-coated dishes. The cells that were attached firmly to the noncoated dishes appeared rather homogeneous with an epithelioid shape. In contrast, the loosely attached cells showed heterogeneous morphology and contained large oval cells with translucent cytoplasm, small cells with dense cytoplasm and an amorphous assembly of large cells that apparently were attached to the substratum. The mixed cell culture apparently contained all types of cells.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Differential plating of tracheal epithelial cells. (a) DIC images of the cells in culture. Dissociated epithelial cells first were seeded on noncoated culture dishes and incubated for 12 h in a CO2 incubator. Loosely attached and firmly attached cells were collected in succession by washing the culture bed and then scraping the remaining cells. The cells were seeded on vitrogen-coated dishes to give basal- and non-basal-enriched cultures. All the cells were recovered without washing and seeded on the vitrogen-coated dishes to give a mixed cell culture. The cell density at seeding was 5 × 104/cm2 in every culture condition. DIC images of the cells were obtained 24 h later by confocal microscopy. (b) Secretion of ET-1 by cultured epithelial cells. Cells were seeded on vitrogen-coated 6-well plates at the density of 5 × 104/cm2. Conditioned medium from each cell culture was collected, and the concentration of irET-1 was determined by enzyme-linked sandwich immunoassay. Each bar represents the mean ± S.E.M. of three determinations, each done in duplicate. Unconditioned medium from cell-free incubations contained undetectable levels of ET-1. (c) Displacement of [125I]ET-1 binding to membrane preparations from cultured epithelial cells. Membrane preparations were prepared from the cells that had been exposed for 24 h to 0.1 mM phosphoramidon. Binding assays were performed as described under "Methods" with 20 µg protein/assay, 50 pM [125I]ET-1 and increasing concentrations of ET-1 () or ET-3 (). Shown are the representative results from three independent determinations.

Effects of exogenous ET-1 and ET receptor antagonists on the growth of cultured tracheal epithelial cells. Figure 3a shows the growth kinetics of the cells under the control conditions. On day 0, the cells were seeded on vitrogen-coated 96-well plates at a low density (10⁴/cm²) to allow proliferation, and on days 1, 3, 5 and 7, they were fed by replacing half of the medium with fresh BEGM. The rate of MTT metabolism increased steadily up to 9 days both in the basal-enriched and mixed cell cultures, whereas there was little increase in the non-basal-enriched culture. Microscopic observation of the culture bed revealed an obvious increase of the basal cell number after day 3 both in the basal-enriched and mixed cell cultures (not shown). Few ciliated cells were found in the non-basal-enriched culture after day 3 when it was occupied largely by an amorphous assembly of large cells (not shown). These results agree with the previous observation of the proliferative capacity of basal cells (Nettesheim et al., 1990) and also with the loss of differentiated characters of ciliated cells under the immersion cultures (Chang et al., 1985; Ostrowski and Nettesheim, 1995). With the culture conditions, we tested the effects of exogenous ET-1 and ET receptor antagonists on the cell proliferation. Because time-course analysis showed the maximum effect of ET-1 (applied on days 1, 3 and 5) on the basal cell proliferation was observed after day 5 (data not shown), the incubation time was set to 7 days.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Effects of ET-1 and ET receptor antagonists on the proliferation of cultured tracheal epithelial cells. (a) Kinetics of the cell proliferation. On day 0, differentially plated (basal- and non-basal-enriched) or mixed cells were seeded on vitrogen-coated 96-well plates at the density of 104/cm2. The cells were fed by replacing half of the medium with fresh BEGM every other day. At the indicated time, the number of viable cells in each well was estimated by MTT assay. Shown are the means ± S.E.M. of triplicate determinations obtained in a single experiment. (b) Effects of exogenous ET-1 on the cell proliferation. Cells were seeded as described above and on days 1, 3 and 5, half of the medium was replaced with fresh BEGM containing 2 times the final concentrations of ET-1. (c) Modulation of the effect of exogenous ET-1 by ET receptor antagonists. ET-1 (1 nM) was applied either alone (hatched bars) or together with BQ123 (1 µM; open bars) or BQ788 (1 µM; closed bars). *P < .01; significantly different from the values in the presence of ET-1 alone. (d) Effects of ET receptor antagonists on the cell proliferation. On days 1, 3 and 5, half of the medium was replaced with fresh BEGM containing 2 times the final concentrations of BQ123 () or BQ788 (). *P < .01; significantly different from the values in the absence of the drug. In panels b, c and d, the number of viable cells in each well was estimated by MTT assay on day 7 and the results were expressed as relative to the values in the absence of the drug (100%). Shown are the mean ± S.E.M. of three determinations, each done in triplicate.

Growth inhibitory effect of IRL1620 mediated by NO. IRL1620 caused dose-dependent inhibition of the cell growth in the mixed cell culture conditions with the maximum effect of ~60% decrease at 10 nM, whereas it caused no effect either in the basal-enriched or non-basal-enriched cultures (fig. 4a). The growth inhibitory effect of IRL1620 (10 nM) was blocked by BQ788 (1 µM), as expected from the selectivity of the drugs (data not shown). Furthermore, BQ788 (1 µM) still caused an enhancement of the cell growth in the presence of IRL1620 (10 nM), but the effect was not as evident as that shown in figure 3d (data not shown). These results gave strong support for the second hypothesis, i.e., the presence of an intercellular diffusible molecule that is released by the ciliated cells in response to ETB activation and suppresses the basal cell growth.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Growth inhibitory effect of IRL1620 mediated by NO. (a) Effects of IRL1620 on the cell proliferation. Basal-enriched (), non-basal-enriched () and mixed () cell cultures were maintained as described in the legend to figure 2, and on days 1, 3 and 5, half of the medium was replaced with fresh BEGM containing 2 times the final concentrations of IRL1620. The number of viable cells in each well was determined by MTT assay on day 7 and the results were expressed as relative to the values in the absence of the drug (100%). Shown are the means ± S.E.M. of three determinations, each done in triplicate. * P < .01; significantly different from the values in the absence of IRL1620. (b) Attenuation of the growth inhibitory effect of IRL1620 by L-NMMA and hemoglobin. The mixed cell culture was exposed on days 1, 3 and 5 to IRL1620 in the absence or presence of the indicated drugs. Each bar represents the means ± S.E.M. of three determinations, each done in triplicate. *P < .01; significantly different from the values in the presence of IRL1620 alone. (c) IRL1620-induced cGMP formation. Basal-enriched (), non-basal-enriched () and mixed () cells were seeded on 6-well plates at the density of 5 × 104/cm2. Twenty-four hours later, they were stimulated with increasing concentrations of IRL1620 for 5 min, and the cGMP content was determined with an immunoassay kit (Amersham). Shown are the means ± S.E.M. of three determinations, each done in duplicate. (d) Effects of SNP on the cell proliferation. As in panel a, basal-enriched (), non-basal-enriched () and mixed () cell cultures were supplemented on days 1, 3, 5 with fresh BEGM containing 2 times the final concentrations of SNP. The number of viable cells in each well was determined by MTT assay on day 7, and the results were expressed as relative to the values in the absence of the drug (100%). Shown are the means ± S.E.M. of three determinations, each done in triplicate. *P < .01; significantly different from the values in the absence of SNP.

SNP- and IRL1620-induced expression of p53. In many types of cells including epithelial cells, NO-induced growth inhibition was linked to the induction of the tumor suppresser protein p53 (Messmer et al., 1994; Muhl et al., 1996). Therefore, we tested the effects of SNP and IRL1620 on the p53 expression level in the cultured tracheal epithelial cells (fig. 5). In the basal-enriched and mixed cultures 24 h after seeding, there was a trace level of p53 and a single application of SNP (0.1 mM) caused a significant increase in the expression that lasted at least 2 days. IRL1620 (100 nM) caused a similar increase but only in the mixed cell culture, and this effect was attenuated by a concomitant application of L-NMMA (100 µM). Cells in the non-basal-enriched culture expressed undetectable levels of p53 and neither SNP nor IRL1620 caused any change. Thus, SNP- and IRL1620-induced growth inhibition of cultured tracheal epithelial cells was paralleled by an increase of the p53 expression level.

View larger version (45K): [in this window] [in a new window]   Fig. 5.   Induction of p53 by SNP or IRL1620. Cells were seeded in 6-well plates at the density of 5 × 104/cm2. Twenty-four hours later, they were stimulated with the indicated drugs and maintained up to 3 days. Because of the loose attachment of nonbasal cells, the cells were harvested, without washing, by scraping and the call lysate was processed for Western blotting with anti-p53 antibody as described under "Methods." Ten micrograms of protein was loaded on each lane. The representative blots from three independent experiments are shown in panel a. The data from densitometric analysis on the day 1 blots (means ± S.E.M. of three independent experiments) are shown in panel b.

Effects of ET receptor antagonists and IRL1620 on the epithelial layer formation under the air-interphase culture. To examine whether the ET signaling may operate during epithelial remodeling, we tested the effects of BQ123, BQ788 and IRL1620 on the epithelial cell layer formation under the air-interphase culture. When the dissociated cells were allowed to adhere to the vitrogen-coated membrane for 24 h under immersion and then exposed to the air (day 1), they secreted ET-1 into the lower medium and the content of ET-1 in the conditioned medium gradually decreased to reach a steady level after day 4 (fig. 6a). In agreement with the previous observation on the guinea pig tracheal epithelial cells cultured on a human amniotic membrane (Noguchi et al., 1995), the cells also secreted ET-1 to the upper medium but in smaller amounts (fig. 6a). Under the control conditions in which the cells were fed by replacing the lower medium with fresh BEGM on days 2, 4 and 6, morphological examinations on day 7 revealed a continuous cell layer formed by a single column of cuboidal cells, and many of them had fine cilia (fig. 6b). When the drugs were applied to the lower medium, to ensure the presence of the drug on the apical side of the cells, we also applied a tiny volume (20 µl) of the drug-containing BEGM in the upper chamber. This procedure did not inhibit the cell differentiation under the air-interphase culture as reported previously (Ostrowski and Nettesheim, 1995). With these procedures, we found completely different effects of BQ123 and BQ788 on the epithelial cell layer formation (fig. 6b). In the presence of BQ123, the cells failed to cover the substratum and remained poorly organized. In contrast, in the presence of BQ788, the cells did cover the substratum, but they were multilayered and failed to differentiate into the ciliated columnar cells. The outcome of IRL1620 application was quite similar to that of BQ123, the cells failed to cover the substratum and retained the oval, undifferentiated shape. When L-NMMA was applied with IRL1620, the cells did cover the substratum but remained oval and failed to differentiate to the cuboidal ciliated cells, which suggests the partial blockade of the effect of IRL1620 by L-NMMA.

View larger version (41K): [in this window] [in a new window]   Fig. 6.   Effects of ET receptor blockade or ETB stimulation on epithelial cell layer formation under the air-interphase culture. (a) Secretion of ET-1 by the epithelial cells under the air-interphase culture. Dissociated epithelial cells were seeded on vitrogen-coated Costar Transwells (6.5 mm) at the density of 105/well. Twenty-four hours later, the cells were exposed to the air by removing the medium in the upper chamber (day 0). Thereafter, the medium in the lower chamber was replaced everyday with fresh BEGM, and the content of ET-1 in the conditioned medium was determined by enzyme-linked sandwich immunoassay (). In separate sets of cultures, the cells were immersed for 24 h in 200 µl of BEGM in the upper chamber, and the content of ET-1 was determined (). Shown are the means ± S.E.M. of triplicate determinations obtained in a single experiment. Similar results were obtained in two other occasions. (b) Morphology of the epithelial cell layer. The air-interphase culture was obtained as in panel a, and on days 0, 2, 4 and 6, the lower medium was replaced with fresh BEGM containing the indicated drugs. To ensure the presence of the drug on the apical side of the cells, a tiny volume (20 µl) of the drug-containing BEGM also was applied to the upper chamber. On day 7, they were subjected to morphological examinations. Shown in the left panel is the microscopic appearance of the culture bed seen from above (×200, HE stain). Shown in the right panel are the 10-µm vertical sections of the cell layer (bar; 10 µm, HE stain). Presented are the representative results of three independent experiments.

	Discussion

Top Abstract Introduction Methods Results Discussion References

The normal turnover of the tracheal epithelium depends on a delicate balance between the cell loss and the cell renewal. The loss of the terminally differentiated cells including the ciliated and secretory cells is compensated by proliferation and differentiation of the stem cells that include the basal cells and, possibly, a part of the secretory cells (Nettesheim et al., 1990; Jetten, 1991). The maintenance of such a balance requires intercellular interactions between the various cell types in the epithelium that fall into two main categories. One involves direct cell-cell interactions through cell surface components such as tight junctions, gap junctions, desmosomes and cell adhesion molecules such as integrins. Another type of intercellular communication occurs via paracrine factors that are synthesized and released by one cell type and influence the growth and differentiation of another cell type. The paracrine factors so far described include molecules such as the epidermal growth factor, transforming growth factor- and -, insulin-like growth factor, keratinocyte growth factor and retinoids that activate cell surface receptors with tyrosine kinase or serine/threonine kinase activities or a nuclear receptor with a transcription factor activity (Jetten, 1991). The present study described the first example of a ligand for the G protein-coupled receptors, ET-1, that is involved in the intercellular communication as a paracrine factor. The ET-1 paracrine signaling pathways apparently include all three major cell types in the epithelium, each with a specialized function.

Immunofluorescent staining with anti-ET-1 and bigET-1 antibodies (fig. 1, e and f) indicated that the nonciliated secretory cells, but not the ciliated columnar or basal cells, are the major site of ET-1 production in guinea pig tracheal epithelium. These results agree with the previous observation of the scattered distribution of anti-ET-1-positive cells in rabbit tracheal epithelium (Rennick et al., 1992; Amble et al., 1993) and also with that of anti-ET-1 immunostaining of the nonciliated secretory cells in rat and mouse tracheobronchial epithelium (Rozengurt et al., 1990). The high concentration of ET-1 in the non-basal-enriched cultures further substantiates this notion, given the recovery of the majority of the secretory cells in this fraction (Chang et al., 1985). A definite amount of ET-1 ~1/10 of that in the non-basal-enriched culture was detected in the basal-enriched cultures, in accordance with the previous observations of ET-1 secretion by the cultured tracheal epithelial cells that were allowed to attach the substratum and washed (Black et al., 1989; Mattoli et al., 1990; Ninomiya et al., 1991; Rennick et al., 1992). The source of ET-1 under the basal-enriched culture can be caused by the presence of secretory cells recovered in this fraction and/or by the acquisition of ET-1-producing capacity by the basal cells in the culture conditions. Therefore, these results do not contradict the conclusion that the secretory cells are the major source of ET-1.

Apart from the source of ET-1, previous observations by Noguchi et al. (1995) and us (fig. 6a) on the polarized cells under the air-interphase culture indicated the ability of the tracheal epithelial cells to secrete ET-1 both to apical and basolateral sides of the cells, the two compartments separated by the tight junctions that do not allow free diffusion of the peptides such as ET-1. Our immunofluorescent analyses indicated the different receptor subtypes localized in each of the two compartments.

Both anti-ETA immunostaining (fig. 1b) and [¹²⁵I]ET-1 binding assay (fig. 2c) indicated almost exclusive expression of ETA by the basal cells that is located on the basolateral side of the epithelial layer. ET-1, by activating ETA, stimulated the proliferation of the basal cells (fig. 3b). This ETA-mediated mitogenic effect is consistent with the previous observations on the cultured porcine tracheal epithelial cells (Murlas et al., 1995) as well as many other cell types that express ETA (Masaki et al., 1992; Rubanyi and Polokoff, 1994). The inhibition of the basal cell proliferation by BQ123 under the mixed cell culture conditions (fig. 3d) indicated the dependence of the basal cell proliferation on the ETA-mediated mitogenic signaling activated by endogenous ET-1. The importance of this endogenous mitogenic signal on the basal proliferation in epithelial remodeling was underscored by the total failure of the cells to form the epithelial cell layer under the air-interphase culture in the presence of BQ123 (fig. 6).

Both anti-ETB immunostaining (fig. 1, c and d) and [¹²⁵I]ET-1 binding assay (fig. 2c) indicated almost exclusive expression of ETB by the ciliated columnar cells, and within the cells, the receptors are localized in the apical side of the cells. Nonbasal cells in culture (including ciliated columnar and nonciliated secretory cells) did not undergo significant proliferation under the immersion cultures, and neither ETB stimulation by exogenous ET-1 nor blockade by BQ788 affected the growth rate (fig. 3, b and d). These results suggested the independence of the growth/survival of the nonbasal cells from ETB-mediated signaling. Under the mixed cell cultures, however, activation of ETB caused a significant effect on the basal cell proliferation. ETB blockade by BQ788 (fig. 3d) and stimulation by IRL1620 (fig. 4a) caused increase and decrease of the proliferation, respectively, which indicates the presence of cell-cell communication that is activated by ETB and inhibit the basal cell proliferation. Blockade of this inhibitory cell-cell communication by L-NMMA and hemoglobin (fig. 4b) indicated the role of NO as a diffusible messenger. IRL1620-induced generation of cGMP (fig. 4c) gave supportive evidence for this notion. Besides, NOS, an essential component for this transduction, was expressed by the ciliated columnar cells (fig. 1g). Collectively, these results indicated the role of ETB/NO-paracrine signaling pathway in the negative control of the basal cell proliferation. In the signaling pathway, the ciliated columnar cells are supposed to play a role of a signal transducer in which ET-1 signal is converted to NO signal.

In accordance with this notion, an NO donor SNP caused obvious suppression of the basal cell proliferation (fig. 4d). NO was reported to cause growth arrest and/or apoptosis in various types of cells including epithelial cells (Hirano, 1996; Schobersberger et al., 1996). The signaling mechanism underlying the effect of NO remains to be elucidated; however, studies on several cell lines indicated the role of the tumor suppresser protein p53 (Messmer et al., 1994; Muhl et al., 1996). The SNP- and IRL1620-induced increase of the p53 expression levels (fig. 5) suggested that this factor also was involved in the NO-mediated growth inhibition of the basal cells in tracheal epithelium.

The importance of this inhibitory signaling in the remodeling of the epithelial cell layer was underscored by the compromised epithelial layer formation under the air-interphase culture in the presence of ETB-selective agonist/antagonist. Blockade of ETB with BQ788 apparently inhibited the differentiation of the cells to the ciliated cells, whereas overstimulation of it with IRL1620 apparently inhibited the cell proliferation (fig. 6), which suggests that the tuning of the ETB/NO-mediated negative signal was as essential in epithelial remodeling as the tuning of the ETA-mediated positive signal.

The proposed paracrine ET signaling system in the control of the basal cell proliferation is illustrated in figure 7. This model predicts that the loss of the terminally differentiated cells that continuously slough off into the lumen turns off the negative signal on the basal cell proliferation. Therefore, the signaling might be involved in the automatic adjustment of the total cell number in the epithelial cell layer (Jetten, 1991). Given the role of the basal cells as the stem cell in tracheal epithelium, this model also predicts that the ET receptor subtype expressed by the cells is switched from ETA to ETB during differentiation. The ET receptor subtype expressed by bovine corneal epithelial cells apparently switches from ETA to ETB during their proliferation and differentiation within the epithelial cell layer (Tao et al., 1995). According to the model, the balance between the positive and negative signals can be controlled by the local concentrations of ET-1 between the apical and basolateral sides of the epithelial cell layer. The intracellular sorting mechanism to determine the side of ET-1 secretion presently is totally unknown and should be the subject for a future study. Also unaddressed in the present study was a possible role of ET-3 which also is secreted by cultured tracheal epithelial cells (Black et al., 1989). ET-3, with its affinity for ETB higher than that for ETA, may work as a negative regulator of the epithelial cell growth.

View larger version (138K): [in this window] [in a new window]   Fig. 7.   Schematic representation of the paracrine ET signaling pathways in the control of the basal cell proliferation in guinea pig tracheal epithelium. The paracrine loop consists of three types of cells; secretory cells that produce ET-1, basal cells that express ETA and ciliated cells that express ETB and produce NO in response to ET-1. Secretory cells can secrete ET-1 both to apical and basolateral sides of the cells. ET-1 in the basolateral side activates ETA on the basal cells to stimulate their proliferation. ET-1 in the luminal side binds ETB on the ciliated cells to stimulate the production of NO, which in turn acts on the basal cells to inhibit their proliferation. See text for details.

Despite the numerous studies on the biological effects of ET-1, the anatomical localization and functional co-operation of ET receptor subtypes so far have been clarified only in the vascular wall, where ET-1 secreted by the endothelial cells activates ETA expressed on the smooth muscle cells to induce their contraction, whereas it also binds, in an autocrine manner, ETB expressed on the endothelial cells to stimulate the production of NO, which in turn causes relaxation of the smooth muscle cells. This autocrine/paracrine signaling system enables the single ligand ET-1 to exert countervailing effects on the smooth muscle tension. We have shown in the present study that the same set of signaling molecules is used in the epithelial paracrine signaling pathways that enable ET-1 to exert countervailing effects on the basal cell proliferation: ETA on the target cells to transmit the positive signal and ETB coupled with NOS to transmit the negative signal. Whether a similar paracrine system operates in other tissues must be the subject for a future study.

In conclusion, we have demonstrated the paracrine ET signaling pathways in the control of the basal cell proliferation in guinea pig tracheal epithelium. The signaling pathways involve all three major cell types of the epithelial cells and depend on the selective expression and distinct localization of the ET receptor subtypes. They can convey both positive and negative signals on the basal cell proliferation and the normal epithelial remodeling appears to be critically dependent on a balance between the positive and negative signals. The physiological role of the system in vivo will be clarified in a future study.