Introduction

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Endothelins (ETs) were first identified as vasoconstrictors (1) and consist of at least 3 isopeptides (ET-1, -2, or -3) (2). In mammalian tissues, ETs exhibit various biological actions through at least two distinct receptors, namely ET_A-receptors (ET_A-Rs) and ET_B-Rs (3). ET_A-Rs are selective for ET-1 and ET-2 (4), whereas ET_B-Rs are highly sensitive to all ET isopeptides (5). The distinct distributions of ET_A- and ET_B-Rs and the distinct production of both ET-1 and ET-3 have been demonstrated in various types of tissue and cells (6). We are interested in the ET systems in the airway, because it has been suggested that ETs may play an important role in the pathophysiology of bronchial asthma (10), pulmonary hypertension (11), pulmonary fibrosis (12) and hypoxic pulmonary vasoconstriction (13, 14).

Concerning the mode of action of ETs in the airway, it is plausible that ETs act not only as a circulating hormone but also as a local hormone in an autocrine and/or paracrine manner, because it has been reported that airway epithelial cells (EpCs) (6) and endocrine cells (15) produce and secrete ET-1 and ET-3, while in addition the airway smooth muscle cells (SMCs) express prepro ET-1 (ppET-1) mRNA (16). To assess the autocrine and/or paracrine role of ETs in the airway, we thus considered it to be important to investigate the expression of ppETs, ET-Rs and endothelin converting enzyme (ECE) in airway SMCs and EpCs. However, the expression and function of ppETs, ET-Rs and ECE in airway systems are not fully documented.

The objectives of the present study were to test the hypothesis that ETs exert actions as autocrine and/or paracrine transmitters in the porcine airway systems, as well as to investigate the mechanism of contraction of tracheal SMCs induced by ETs. For this, we characterized the ET-R subtypes of both tracheal SMCs and EpCs, using both pharmacological experiments and reverse transcription polymerase chain reaction (RT-PCR). We also examined the expression of the ppET-1, ppET-3, and ECE-1 mRNAs in the porcine tracheal SMCs and EpCs. We obtained evidence that mRNAs for ETs-Rs, ppETs, and ECE-1 were expressed in porcine tracheal SMCs and EpCs.

	Materials and Methods

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Tissue Preparation for the Fura-2 Fluorometry

Tracheas were dissected from adult pigs at a local slaughterhouse using a protocol approved by the Animal Research Committee of Research Institute of Angiocardiology, Faculty of Medicine, Kyushu University. The tracheas were placed in ice-cold physiological salt solution (PSS) (NaCl 123, KCl 4.7, NaHCO₃ 15.5, KH₂PO₄ 1.2, CaCl₂ 1.25 and D-glucose 11.5, in mM) and brought to our laboratory. The lower end of the trachea, just above the first bronchus branching and three tracheal rings in length, was used for the experiments. After removing the cartilage, the mucosa and adventitial tissue were removed under microscopic observation. The muscle sheets were transversely cut into rectangular strips measuring approximately 3 mm in length and 1 mm in width.

Fura-2 Loading

Muscle strips were loaded with the Ca²⁺ indicator dye, fura-2, by incubating in oxygenated (95% O₂/5% CO₂) Dulbecco's modified Eagle's medium (DMEM) containing 50 µM fura-2/AM (an acetoxymethyl ester form) and 5% fetal bovine serum for 3-4 h at 37°C. The strips were then rinsed with PSS for 1 h prior to the start of measurements.

Measurement of Tension Development and Fura-2 Fluorescence

The measurements of tension development and fura-2 fluorescence were done as previously described (17). During the 1 h fura-2 equilibration period, the strips were stimulated with 40 mM K⁺ PSS, which was made by an equimolar substitution of KCl for NaCl, at 5-10 min intervals, and the muscle length was increased stepwise after each stimulation until the developed tension reached a maximum. The responsiveness of each strip to 40 mM K⁺ PSS was then recorded before starting the experimental protocol. The developed tension was expressed as a percentage, assigning the values in normal (5.9 mM K⁺) PSS and steady state of 40 mM K⁺ PSS to be 0% and 100%, respectively.

The fluorescence of the fura-2-Ca²⁺ complex was monitored with front-surface fluorometry (18), using equipment specifically designed for fura-2 fluorometry (CAM-OF; Japan Spectroscopic Co., Tokyo, Japan). The 500 nm ratio of the fluorescence intensities at 340 nm excitation to those at 380 nm excitation was monitored with the sampling rate of 400 Hz and expressed as a percentage, while the values were assigned in normal PSS (at rest: 5.9 mM K⁺) and the steady state of 40 mM K⁺ PSS (at high K⁺- depolarization) recorded at the beginning of each measurement to be 0% and 100%, respectively. The absolute values of [Ca²⁺]_i were determined in separate measurements, using the method described by Grynkiewicz and associates (19). In brief, after recording the 0% and 100% levels of the fluorescence ratio, the minimum and the maximum fluorescence ratios were determined by the addition of 25 µM ionomycin to Ca²⁺-free PSS containing 2 mM ethyleneglycol-bis-(-amino-ethylether)-N,N,N',N'-tetraacetic acid (EGTA), followed by replacement with normal PSS (1.25 mM Ca²⁺), respectively. In each measurement, the levels of [Ca²⁺]_i were expressed as percentage levels of the fluorescence ratio. The absolute values of [Ca²⁺]_i in normal PSS (0%) and the steady state of 40 mM K⁺ PSS (100%) were calculated and they were determined to be 90 ± 14 nM and 499 ± 54 nM (n = 8), respectively (17). The responsiveness of each strip to 40 mM K⁺ PSS was then recorded before starting the experimental protocol.

Experimental Protocol for the Determination of Ca²⁺ Sensitivity of the Myofilaments

To examine the effect of ET-1, ET-3, or sarafotoxin S6c (S6c) on Ca²⁺ sensitivity of the contractile apparatus, we determined the [Ca²⁺]_i-tension relationships of the contractions induced by the stepwise increases in the extracellular Ca²⁺ concentration (0.0125-2.5 or 5 mM) during 40 mM K⁺-induced depolarization in the presence or absence of these peptides. After a 5 min incubation in Ca²⁺-free PSS containing 2 mM EGTA, and then a 5 min incubation in Ca²⁺-free PSS without EGTA, the strips were immersed in Ca²⁺-free 40 mM K⁺ solution. Next, the extracellular Ca²⁺ concentration was increased by the cumulative addition of CaCl₂. In addition, either ET-1, ET-3 or S6c was applied when the Ca²⁺-free PSS containing 2 mM EGTA was replaced with the Ca²⁺-free PSS without 2 mM EGTA.

Preparation of Total RNA from Tracheal SMCs, EpCs and Aortic ECs

Total RNA was isolated from the porcine tracheal SMCs, EpCs, and aortic ECs according to the method described by Chomczynski and Sacchi (20). During the tissue trimming of the tracheal SMCs, care was taken not to include any mucosal or adventitial tissue, as described above under the heading "Tissue Preparation for the fura-2 Fluorometry." For the preparation of total RNA from the tracheal EpCs, the posterior portion of the trachea was opened longitudinally and the mucosal surface was scraped by a rubber policeman to collect the EpCs. To obtain the ECs, the inner surface of the aorta was scraped by a rubber policeman. The total RNA preparation was further digested by RNase free DNase to rule out the possibility of contamination by genomic DNA. The amount of total RNA was then determined by a spectrophotometer.

Primers for RT-PCR

The oligonucleotide sequences of the primers for RT-PCR of ppET-1, ppET-3, ECE-1 and coagulation factor VIII (Factor VIII) are shown in Table 1. Regarding the primers for pig ET_A-Rs, ET_B-Rs, and -actin, we used the same primers as previously reported (21, 22). The primers for ppET-1 were designed according to the published sequence for pig ppET-1 by Yanagisawa and colleagues (1). Since the sequence of the pig ppET-3 mRNA has not been described, we chose the primers, based on the rat sequence, from the conserved regions between the rat (23) and human (24) ppET-3 sequences. The primers for ECE-1 were designed based on the conserved regions among the bovine (25), rat (26), and human (25) ECE-1 sequences. The primers for Factor VIII were designed from the conserved regions between mouse (27) and human (28) Factor VIII sequence. For the control, we also performed RT-PCR for -actin. The expected size of the PCR product for ET_A-R is 255 base pair (bp) and should be digested into 198 bp and 57 bp fragments by Hae III. The expected size for ET_B-R is 304 bp and should be digested into 175 bp and 129 bp fragments by Eco RI. The expected size for ppET-1 is 389 bp and should be digested into 286 bp and 103 bp fragments by Hind III. The expected size for ppET-3 is 122 bp and should be digested into 67 bp and 55 bp fragments by Sau 3A I, according to the sequence for both rat and human ppET-3. It is thus expected that the PCR product of pig ppET-3 would also be digested in the same manner. The expected sizes for ECE-1 and Factor VIII are 235 and 371 bp, respectively. The PCR product for pig Factor VIII should be digested into 284 bp and 87 bp fragments by Eco RI, according to the sequence for both mouse and human Factor VIII.

Detection of Pig ET_A-R, ET_B-R, ppET-1, ppET-3, ECE-1 and Factor VIII mRNA in Porcine Tracheal SMCs and EpCs by RT-PCR

The RT-PCR was done as previously reported (21, 22). In brief, total RNA (1 µg) was used for the RT reaction in a total volume of 20 µl. An aliquot (1 µl) of RT product was applied for PCR amplification in a total volume of 11 µl. The thermal cycle profiles used for ET_A-R, ppET-1, ppET-3, and ECE-1 were (1) denaturing for 30 s at 94°C, (2) annealing primers for 60 s at 55°C, (3) extending the primers for 30 s at 72°C for 35 cycles. The PCR amplifications for Factor VIII were done in the same manner except that the annealing was done for 90 s at 55°C. In the case of ET_B-R, the annealing was done for 60 s at 60°C and the amplification was repeated for 32 cycles. For -actin, the annealing was done for 60 s at 50°C and the amplification was repeated for 25 cycles. A portion (10 µl) of the PCR mixture was electrophoresed in 3% agarose gel in a TAE buffer. The gel was stained with ethidium bromide and then photographed.

Chemicals

Synthetic ET-1 and ET-3 were obtained from the Peptide Institute Co. Ltd. (Osaka, Japan), fura-2/AM was purchased from DOJINDO (Kumamoto, Japan). BQ-123 (cyclo [-D-Trp-D-Asp-Pro-D-Val-Leu-]) was kindly donated by the Banyu Pharmaceutical Co., Ltd. (Tokyo, Japan). M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase was purchased from BRL (Gaithersburg, MD). NuSieve^TM 3:1 agarose was from TaKaRa (Kyoto, Japan). RNase inhibitor and X174/Hinc II digest were purchased from TOYOBO (Osaka, Japan). Taq DNA polymerase was from Wako (Osaka, Japan). The oligonucleotides for primers were synthesized by Sawady Technology Inc. (Tokyo, Japan). All other chemicals were of the highest grade commercially available. Fura-2/AM was dissolved in dimethyl sulfoxide (DMSO) as a stock solution and then diluted in the medium just before loading the dye. The final concentration of DMSO was 5%.

Data Analysis

The values were expressed as the mean ± standard error (SE). Student's t test was used to determine statistical difference between two mean values. One-way analysis of variance for repeated measurements was used to determine the concentration-dependent effects of the drugs. P values less than 0.05 were considered to be statistically significant. An analysis of covariance was used to determine the statistical significance of the shift of the [Ca²⁺]_i-tension relationship curves.

	Results

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Tension Development and [Ca²⁺]_i Transients Induced by ET-1, ET-3, and S6c, and the Effects of BQ-123 in Tracheal Smooth Muscle Strips

Figure 1a shows the representative recordings of the responses of [Ca²⁺]_i and tension to the depolarization with 40 mM K⁺ PSS and the elevations of [Ca²⁺]_i and tension induced by the 10⁷ M ET-1 in the porcine tracheal smooth muscle strips. The maximal increases in [Ca²⁺]_i and tension induced by 10⁷ M ET-1 were 65.01 ± 4.65% and 129.82 ± 14.88% of those induced by 40 mM K⁺ PSS, respectively (n = 5). When 10⁶ M BQ-123 was applied 10 min before and during the stimulation, 10⁷ M ET-1- induced increases in [Ca²⁺]_i and tension were inhibited to 47.02 ± 5.57% and 46.61 ± 15.89% (n = 5), respectively (Figure 1b). The concentration-dependent increases in [Ca²⁺]_i and tension induced by ET-1, ET-3 or S6c in the absence or presence of 10⁶ M BQ-123 in the porcine tracheal smooth muscle strips are shown in Figure 2. As shown in Figures 2a and b, BQ-123 shifted the concentration-response curve of the [Ca²⁺]_i and tension to the right (P < 0.05 by analysis of variance, n = 5 for each concentration). On the other hand, 10⁶ M BQ-123 had no significant effect on the increases in [Ca²⁺]_i and tension induced by 10¹⁰ M to 3 × 10⁷ M ET-3 (Figures 2c and 2d; P < 0.05 by analysis of variance, n = 5 for each concentration) or by S6c (Figures 2e and 2f; P < 0.05 by analysis of variance, n = 5 for each concentration).

View larger version (18K): [in this window] [in a new window]   Figure 1.   Representative recordings showing changes in [Ca2+]i (upper trace) and tension (lower trace) induced by 107 M ET-1 in the absence (a) or presence (b) of BQ-123, an ETA-R antagonist. In each protocol, 40 mM K+ solution was applied before the application of ET-1. The fluorescence ratio and tension were expressed as a percentage by assigning the values in normal physiological salt solution (5.9 mM K+) and at the steady state during 40 mM K+ depolarization to be 0 and 100%, respectively. BQ-123 (106 M) was applied 10 min before and was present during the application of ET-1.

View larger version (25K): [in this window] [in a new window]   Figure 2.   Effects of various concentrations of ET-1 (a and b), ET-3 (c and d) and S6c (e and f  ) on tension (a, c, and e) and [Ca2+]i (b, d and f  ), in the absence (open symbols) or presence (closed symbols) of 106 M BQ-123. To construct the dose-response curves of tension and [Ca2+]i for ET-1 (a and b), the same protocols as shown in Figure 1 were used. Only one dose of ET-1 was applied to each strip, and the changes in [Ca2+]i and tension were measured at the time point of 20 min after the application. In the case of ET-3 (c and d) and S6c (e and f  ), the cumulative applications were used to construct dose-response curves of tension and [Ca2+]i. The fluorescence ratio and tension were expressed as percentages by assigning the values at normal PSS (5.9 mM K+) and the steady state during 40 mM K+ depolarization to be 0 and 100%, respectively. The plots represent the means of five preparations, with S.E. shown by vertical bars.

Effects of ET-1 on the Increases in [Ca²⁺]_i and Tension Induced by Increases in Extracellular Ca²⁺ Concentration during High K⁺ Depolarization

Figure 3a shows a representative recording of changes in [Ca²⁺]_i and tension induced by the cumulative application of CaCl₂ during depolarization with 40 mM K⁺. When the strips were immersed in Ca²⁺-free PSS containing 2 mM EGTA, and then, in Ca²⁺-free PSS without EGTA, [Ca²⁺]_i decreased to reach a new steady state level, whereas, the tension was not affected. In response to the stepwise increment of extracellular Ca²⁺ concentration (0.0125-2.5 mM), [Ca²⁺]_i and tension increased in a concentration-dependent manner. When the extracellular Ca²⁺ was 2.5 mM, [Ca²⁺]_i and tension were 105.6 ± 7.2% and 85.3 ± 10.5%, respectively (n = 10). Treatment with 10⁷ M ET-1 (Figure 3b) for 5 min before and during the cumulative application of extracellular Ca²⁺ (0.0125-5 mM) significantly increased both [Ca²⁺]_i and tension (P < 0.01 for both, by two way analysis of variance). When the extracellular Ca²⁺ was 5 mM with 10⁷ M ET-1, [Ca²⁺]_i and tension were 99.28 ± 7.85% and 209.49 ± 9.85%, respectively (n = 6).

View larger version (24K): [in this window] [in a new window]   Figure 3.   Representative recordings of changes in [Ca2+]i and tension induced by the cumulative application of extracellular Ca2+ (0.0125-2.5 or 5 mM) during 40 mM K+ depolarization, in the absence (a) or presence (b) of 107 M ET-1. After 5 min incubation in Ca2+-free PSS containing 2 mM EGTA, and then 5 min incubation in Ca2+-free PSS without EGTA, strips were immersed in Ca2+-free 40 mM K+ solution and then the extracellular Ca2+ concentration was increased by the cumulative addition of CaCl2.

Effect of ET-1, ET-3 and S6c on the [Ca²⁺]_i-tension Relationship

Figure 4 represents the summary of the measurements of [Ca²⁺]_i (abscissa)-tension (ordinate) relationships obtained at the steady state during the contractions induced by cumulative applications of the extracellular Ca²⁺ during high K⁺ depolarization in the absence or presence of ET-1, ET-3 or S6c. The [Ca²⁺]_i-tension relationship of the contractions observed in the presence of ET-1, ET-3, or S6c significantly (P < 0.05 by analysis of covariance) shifted upward and left from that observed in the absence of these peptides. The shift of the [Ca²⁺]_i-tension relationship induced by ET-1 was significantly (P < 0.05 by analysis of covariance) greater than that induced by ET-3 or S6c.

View larger version (18K): [in this window] [in a new window]   Figure 4.   Summary of the effect of ET-1, ET-3 and S6c on the [Ca2+]i-tension relationship. The [Ca2+]i-tension relationship was obtained from the similar experiments shown in Figure 3. Each symbol indicates value obtained from the steady state of the contractions induced by changes in extracellular Ca2+ during 40 mM K+ depolarization, without (open circles) (n = 10) or with 107 M ET-1 (closed squares) (n = 6), 107 M ET-3 (closed triangles) (n = 6) and 107 M S6c (closed circles) (n = 6) treatment.

Detection of ET_A- and ET_B-R mRNAs in Porcine Tracheal SMCs and EpCs by RT-PCR

As shown in Figure 5a, using the total RNA prepared from the porcine tracheal SMCs and the specific primers for ET_A- and ET_B-Rs, RT-PCR produced the bands of 255 bp and 304 bp, respectively, in agarose gel electrophoresis, only when the cDNA was added. Possible amplifications of the genomic ET_A- and ET_B-R sequences were excluded since such bands were only detected when reverse transcriptase was added (data not shown). These PCR products could be digested into the predicted size of the fragments (Figures 7a and 7b) by the selected restriction enzymes, thus indicating that the PCR products obtained by these primers derived from the pig ET_A- and ET_B-R cDNA. In the porcine tracheal EpCs, ET_A-R mRNA was detected predominantly, while ET_B-R mRNA was only slightly detected if at all (Figure 5b). These results indicated that porcine tracheal SMCs express both ET_A- and ET_B-R mRNA, while EpCs predominantly express ET_A-R mRNA.

View larger version (44K): [in this window] [in a new window]   Figure 5.   The expression of ETA- and ETB-R mRNA in porcine tracheal SMCs (a) and EpCs (b) detected by RT-PCR. The PCR amplifications for ETA-receptor (ETA-R) (lanes 1- 3), ETB-receptor (ETB-R) (lanes 6-8) and -actin (Actin) (lanes 11-13) were performed for 35, 32 and 25 cycles, respectively. The predicted sizes of these PCR products are 255 bp for ETA-R, 304 bp for ETB-R and 241 bp for -actin, which are shown on the left side of the photograph. We used three different pigs for this experiment (lanes 1, 6 and 11 from pig 1; lanes 2, 7 and 12 from pig 2; lanes 3, 8 and 13 from pig 3). Lanes 4, 9 and 14 represent the negative control where the cDNA template had been omitted. Lanes 5 and 10 represent the DNA size marker (M; øX174/Hinc II digest). The size of each band was, from top to bottom, 1057, 770, 612, 495, 392, 345+341+335, 297+291, 210 and 162 bp.

View larger version (53K): [in this window] [in a new window]   Figure 7.   The PCR products for ETA-R (a), ETB-R (b), ppET-1 (c) and ppET-3 (d) mRNA in tracheal EpCs identified by the digestion with the restriction enzyme. (a) ETA-R: Lane 1; the PCR product without digestion by Hae III. Lane 2; the PCR product digested by Hae III. Lane 3; DNA size marker (øX174/Hinf I digest; the size of each band was, from top to bottom, 726+713, 553, 500, 427+417+413, 311, 249, 200, 151, 140, 118, 100, 82, 66 and 48 bp). The size of each band in lanes 1 and 2 is shown on the left side of the photograph. The PCR product for ETA-R (255 bp) was digested into 198 bp and 57 bp fragments as predicted. (b) ETB-R: Lane 1; the PCR product without digestion by Eco RI. Lane 2; the PCR product digested by Eco RI. Lane 3; DNA size marker (øX174/Hinf I digest). The size of each band in lanes 1 and 2 is shown on the left side of the photograph. The PCR product for ETB-R (304 bp) was digested into 175 bp and 129 bp fragments as predicted. (c) ppET-1: Lane 1; the PCR product digested by Hind III. Lane 2; the PCR product without digestion by Hind III. Lane 3; DNA size marker (øX174/Hinc II digest). The size of each band in lanes 1 and 2 is shown on the left side of the photograph. The PCR product for ppET-1 (389 bp) was digested into 286 bp and 103 bp fragments as predicted. (d) ppET-3: Lane 1; DNA size marker (øX174/Hinc II digest). Lane 2; the PCR product digested by Sau 3A I. Lane 3; the PCR product without digestion by Sau 3A I. The size of each band in lanes 2 and 3 is shown on the left side of the photograph. The PCR product for ppET-3 (122 bp) was digested into 67 bp and 55 bp fragments as predicted.

Detection of ppET-1 and ppET-3 mRNA in the Porcine Tracheal SMCs and EpCs by RT-PCR

In RT-PCR, using total RNA prepared from the porcine tracheal SMCs, EpCs and aortic ECs and the specific primers for ppET-1 (Figure 6a) and ppET-3 (Figure 6b), the expected size of the bands (namely, 389 bp and 122 bp, respectively) were detected. These specific bands could be detected only when reverse transcriptase was added (data not shown). These specific bands could be digested into the predicted size of fragments by the selected restriction enzymes (Figures 7c and 7d), thus indicating that the PCR products obtained by these primers derived from the pig ppET-1 and ppET-3 cDNA. These results therefore indicated that porcine tracheal SMCs and EpCs express both ppET-1 and ppET-3 mRNAs.

View larger version (58K): [in this window] [in a new window]   Figure 6.   RT-PCR for ppET-1 (a), ppET-3 (b), ECE-1 (c), Factor VIII (d) and -actin (e) mRNA in porcine tracheal SMCs, EpCs and aortic ECs. PCR amplifications for ppET-1, ppET-3, ECE-1, Factor VIII and -actin were performed for 35 (ppET-1, ppET-3 and ECE-1), 30 (Factor VIII) and 25 cycles (-actin). The predicted sizes of these PCR products are 389 bp for ppET-1, 122 bp for ppET-3, 235 bp for ECE-1, 371 bp for Factor VIII and 241 bp for -actin, which are shown on the right side of the photograph. Lanes 1-3 are tracheal EpCs, lanes 5-7 are tracheal SMCs and lanes 9-11 are aortic ECs. We used three different pigs for this experiment (lanes 1, 5 and 9 from pig 1; lanes 2, 6 and 10 from pig 2; lanes 3, 7 and 11 from pig 3). Lanes 4 and 8 represent the DNA size marker (M; øX174/Hinc II digest).

Detection of ECE-1 mRNA in the Porcine Tracheal SMCs, EpCs and Aortic ECs by RT-PCR

RT-PCR using the specific primers for ECE-1 and the total RNA prepared from the porcine tracheal SMCs, EpCs, and aortic ECs gave the expected size of bands (235 bp), as shown in Figure 6c. The level of ECE-1 expression in tracheal EpCs appeared to be even higher than that in aortic ECs, although ppET-1 mRNA was most abundantly expressed in aortic ECs (Figure 6a). We sequenced the PCR product for pig ECE-1 and found that the homology of the amino acids of this region was 96.8% to that of the corresponding region of the human ECE-1 (data not shown), which indicated that the PCR products obtained by these primers derived from the pig ECE-1 cDNA. Therefore, it was indicated that porcine tracheal SMCs, EpCs, and aortic ECs thus express ECE-1 mRNA.

Detection of Factor VIII mRNA in the Porcine Tracheal SMCs, EpCs and Aortic ECs by RT-PCR

In RT-PCR, using the specific primers for Factor VIII and total RNA prepared from the porcine tracheal SMCs, EpCs and aortic ECs, the expected size of bands (371 bp) could be detected in the aortic ECs but not in tracheal SMCs and EpCs, under the condition described in the legends. The PCR product for Factor VIII could be digested by Eco RI into the fragments of the expected size (data not shown).

	Discussion

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The working hypothesis of the present study is that ETs may act as autocrine and/or paracrine mediators in the airway. To test this hypothesis, we attempted to characterize the function and distribution of ET_A-, ET_B-Rs, ppET-1, ppET-3, and ECE-1 in porcine tracheal SMCs and EpCs, using functional and molecular biological technique. The results obtained in the present study are as follows: (1) porcine tracheal SMCs were found to have both functioning ET_A- and ET_B-Rs, (2) ETs increase the Ca²⁺ sensitivity of myofilaments of the porcine tracheal SMCs, (3) tracheal SMCs expressed both ET_A- and ET_B-R mRNAs, (4) tracheal EpCs predominantly express ET_A-R mRNAs, (5) tracheal SMCs and EpCs express both ppET-1 and ppET-3 mRNAs, (6) tracheal SMCs and EpCs express ECE-1 mRNA.

The presence of both functioning ET_A- and ET_B-Rs in porcine tracheal SMCs was assessed as follows: A functional study indicated that ETs increased [Ca²⁺]_i and tension in tracheal SM, in a concentration-dependent manner. These results are consistent with the findings in the previous reports (29). As for the mechanism, Lee and colleagues (29) suggested that ET modulates the airway smooth-muscle tone by direct activation and/or depolarization-induced activation of sarcolemmal calcium channels. As shown in Figure 1, both ET-1 and ET-3 induced increases in [Ca²⁺]_i and tension in a similar concentration range, although the maximal increases in [Ca²⁺]_i and tension induced by ET-1 were much greater than those induced by ET-3. This results contrasts with our previous data obtained in the porcine coronary artery (32), in which the concentration range for ET-1-induced tension was much lower than that for ET-3-induced tension. We thus considered that these differences may be due to differences in the distribution of ET-Rs between coronary arterial SMCs and tracheal SMCs, namely, the predominant distribution of ET_A-Rs in coronary artery (32) versus a fairly similar distribution of ET_A- and ET_B-Rs in tracheal SMCs. This speculation was supported by the use of BQ-123, an ET_A-R antagonist, and S6c, an ET_B-R agonist (Figure 2). BQ-123 partially inhibited the ET-1-induced contraction and had little effect on ET-3- and S6c-induced contraction. These results thus indicated that the porcine tracheal SMCs do possess functioning ET_A- and ET_B-Rs. This conclusion is consistent with the results of the previous tension studies (33) and a binding study of cultured guinea pig tracheal SMCs (31).

As shown in Figure 1a, ET-1-induced contraction was much greater than the 40 mM K⁺-induced tension, although the increase in [Ca²⁺]_i induced by ET-1 was much smaller than that induced by 40 mM K⁺. Based on this observation, we further explored the effect of ET ligands on the Ca²⁺ sensitivity of the contractile apparatus in tracheal SMCs. In the presence of ET-1, ET-3 or S6c, the [Ca²⁺]_i (abscissa)-tension (ordinate) relation-curve of the contraction induced by the increases in the extracellular Ca²⁺ concentration during high K⁺-depolarization shifted significantly upward and left from that obtained in the absence of these peptides (Figure 4). In other words, at a given [Ca²⁺]_i level, the tension development in the presence of ET ligands or S6c were much greater than that in the absence of these peptides, thus indicating that the Ca²⁺ sensitivity of the contractile apparatus is increased by ET ligands and S6c. The leftward shift of the [Ca²⁺]_i-tension relationship induced by ET-1 was much greater than those induced by ET-3 and S6c (Figure 4). This could be explained by the difference in the potency to increase Ca²⁺ sensitivity of the contractile apparatus by ETs, which is thought to be one of the mechanisms for the smooth muscle contraction induced by ET-1 (34, 35). Since, to our knowledge, there has been no report on the simultaneous measurements of [Ca²⁺]_i and tension induced by ETs in tracheal smooth-muscle strips, this is the first report regarding an increase in Ca²⁺ sensitivity by ETs in tracheal SMCs. A similar phenomenon has also been reported in the pulmonary vein (36).

Because this pharmacological study indicated the presence of both ET_A- and ET_B-Rs in the porcine tracheal SMCs, we next attempted to confirm this by determining the presence of ET_A- and ET_B-R mRNAs by using RT-PCR. To avoid or minimize the possible contamination of ECs into tracheal SMCs and EpCs, in the present study, special care was given in the preparation of these cells, including the removal of the mucosa and adventitial tissues under microscopy. In addition, the extent of the possible contamination with ECs was assessed by use of RT-PCR. In the present study, the mRNA expression of Factor VIII, one of the oldest markers of ECs (37, 38), was determined in tracheal SMCs and EpCs, and compared with that in the aortic ECs. If there is a significant contamination of endothelium in tracheal smooth muscle tissue and EpCs used for RT-PCR study, there will be a significant expression of Factor VIII in these samples as much as aortic ECs. However, the level of the expression of Factor VIII mRNA (371 bp) of tracheal SMCs and EpCs was very much lower than that in the aortic ECs (Figure 6d), indicating that the contamination by the vascular ECs into the tracheal and epithelial preparation would be negligible. Figure 5 clearly shows the expression of both ET_A- and ET_B-R mRNAs in porcine tracheal SMCs. The specificity of the PCR primers used in the present study for ET_A- and ET_B-R mRNAs has already been shown in our previous study (21). Inui and associates (31) reported that ET_A- and ET_B-Rs are present in a primary culture of tracheal SMCs of the guinea pig, based on the findings of both binding studies and functional studies. In the present study, we documented for the first time the expression of ET_A- and ET_B-R mRNAs in the intact porcine tracheal SMCs.

ET-Rs are classified by their selectivity to ET isopeptides (3). They are classified into ET_A- and ET_B-Rs. Subclassifications of ET_A- and ET_B-Rs into ET_A1-, ET_A2-, ET_B1-, and ET_B2-subtypes have recently been proposed, based on their selectivity to various newly synthesized ET-R antagonists (39, 40). Although these subclassifications have not yet been generally accepted, Shyamala and colleagues (41) recently reported that ET_B1- and ET_B2-Rs are produced by the alternative splicing in humans. They reported only a small difference between these two subtypes (only 30 bp was longer in one form than the other). Even if a similar alternative splicing was present in the pig, these differences may not be detected by RT-PCR if special attention is not paid to the alternative splicing site. Thus, it is speculated that the ET_B-R mRNA detected in the present study might involve net ET_B-Rs (ET_B1- and ET_B2-). Similarly, it would thus be speculated that ET_A1- and ET_A2-Rs also derived from alternative splicing from the same gene. Thus, we considered that we are detecting net ET_A- or ET_B-R mRNAs in the RT-PCR experiment.

In the present study, we found that the tracheal EpCs express predominantly ET_A-R mRNA. Consistent with this finding, Ninomiya and associates (42) reported that canine tracheal EpCs possess specific binding sites for ET-1 with characteristics similar to those of the ET_A-R subtype, using a radioligand binding study and cultured tracheal EpCs. The function of the ET_A-R of the tracheal EpCs was not, however, examined in the present study. In contrast, previous studies have indicated that ET-1 causes an increase in chloride secretion, ciliary beat frequency, and the release of arachidonic acid and/or its metabolites in the tracheal EpCs (43, 44). Using tracheal strips with an intact epithelium, evidence has been provided for an epithelium-dependent relaxation induced by ET-1 in guinea pig (45) and rabbit (30). This has been proposed to be due to ET_A-R activation (47) and the release of nitric oxide (46). Thus, there is a possibility that ET-1 might be involved in the autocrine control mechanisms in the airway EpC function, since the EpCs express ppET-1 and ECE-1 mRNAs as shown in the present study.

In addition to the determination of the distribution of ET_A- and ET_B-Rs (mRNA) in the porcine tracheal SMCs and EpCs, in the present study, the distribution of the production of ppET-1 and ppET-3 was characterized in the porcine tracheal SMCs and EpCs, to show the autocrine and/or paracrine regulation of ET system in the airway. As shown in Figure 6, we could clearly show the expression of both ppET-1 and ppET-3 mRNAs both in porcine tracheal SMCs and EpCs. Concerning the production of ET-1 and/or ET-3 in the airway, Black and associates (6) first reported that the immunoreactivity for ET-1 and ET-3 could be detected in the conditioned medium of the cultured canine and porcine airway EpCs. Similar results have also been reported in human bronchial EpCs (48). Subsequently, Giaid and colleagues (15) further characterized the cellular localization of ET-1 and ET-3 immunoreactivity and mRNA by immunocytochemistry and in situ hybridization. They also reported that the ET-1 and ET-3 immunoreactivity and mRNA are localized in the endocrine cells of the human airway. The present study also confirmed these previous reports. In the case of tracheal SMCs, Ergul and associates (16) reported that the airway SMCs express ET-1 mRNA based on the findings of a Northern analysis. However, there have apparently been no reports which described the expression of ppET-3 mRNA in tracheal SMCs.

Although it is well known that proETs need to be converted into mature ETs catalyzed by ECE to show their biological activities, the expression of ECE in the tracheal SMCs or EpCs has not been documented. Thus, we performed RT-PCR for ECE-1 mRNA in the porcine tracheal SMCs and EpCs. As shown in Figure 6c, ECE-1 mRNA was abundantly expressed in tracheal SMCs and EpCs. The expression of ECE-1 mRNA in these cells further supported the hypothesis that ETs may act as autocrine and/or paracrine transmitters in the porcine airway. In summary, the present study strongly indicates that ET-1 and ET-3 could potentially be autocrine and/or paracrine transmitters in tracheal SMCs and EpCs, which mediate the cell-to-cell signaling between either the same kinds of cells or different kinds of cells.