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Endotoxin stimulates endothelin release from cultured epithelial cells of guinea-pig trachea
European JwrnaI of P~armacoIo~, 203 (1991) 2?9-302 0 1991 Elsevier Science Publishers B-V. All rights reserved
0014.2999/91/$03.50
299
ADONIS 001429999100725V EJP 20932
Short comrn~~~ca~oa
Hiroki~inomiya, Yushi~~ Division
Uchida, Yukio Ishii, Akihiro Nomura, Masaaki Kameyama, Mikio Saotome, Takeo Endo and Shizuo Nasegawa
of Pulmonary Medicine, Institute of Clinical Medicine, University of Tsukubn, Tsukuba, Ibaraki 305, Japan Received 20 June 1991,revised MS received 13 August 1991,accepted 20 August 1991
We examined the release of endothelin from cultured eyithelial cells of guinea-pig trachea in response to treatment with endotoxin, using a sandwich-enzyme immunoassay. Cultured epithelial cells released endothelin in a time-dependent fashion (3, 6, 12, 24 h), and endotoxin (4-40 &ml) significantly increased endothelin release. Endotoxin (4-10 #g/ml) showed no cytotoxity against epithelial cells. These results suggest that guinea-pig airway epithelial cells are capable of producing endothelin and this peptide may be related to the pathophysiological effects of endotoxin. Endothelin; Endotoxin;Airwayepithelisl cells 1. Introduction
Endothelin-1 (ET-l) is a 21 amino acid peptide identified from cultured endothelial cells. ET-l has a potent contractile effect on both vascular and airway smooth muscles (Uchida et al., 1988), and is produced by airway epithelial cells as well as endothelial cells (Black et al., 1989). ET-l stimulates thromboxane A, (TXA,) r&ease from perfused guinea-pig lung (De Nucci et al., 1989), and ET-l induced airway constriction is suggested to be partly mediated by TXA, (Battistini et al., 1990). ET-l also stimulates airway epitheha1 cells to increase Cl- secretion (Piews et al., in press). These results indicate that ET-1 has a variety of effects in the lung. A dysfunction of epithelial cells is considered to play an important role in pathophysiological airway constriction. A recent study has shown that endotoxin exposure results in airway hyperreactivity to histamine and arecohne (a muscarinic receptcr agonists in the guinea-pig because of its cytotoxity to airway epithehum (Folkerts et al., 1989). In the present study, to elucidate the relationship between the airway epithehum and ET-l synthesis, we investigated whether endotoxin modulates ET-l release from cultured epitbeliai cells of the guinea-pig trachea. This is the first
Correspondence IO: S. Hasegawa, Division of Pulmonary Medicine, Institute of Clinical Medicine. University of Tsukuha, Tsukuba. fbaraki 305, Japan. Tel. 81.298.53 3361.
paper reporting the stimulant effect of endotoxin on ET-l production by airway epithelial cehs. 2. Materials and methods 2.1. Culture c$ trachea/ epitheliai cells
Tracheal epithelial cells were prepared by a modification of the method of Barnett et al. (1988); we used 24-well tissue culture plates instead of tissue culture flasks coated with human placenta collagen. Hartlcystrain guinea-pigs (body weight 300-400 g) were anesthetized with sodium pentobarbitai (50 mg/kg Lp.; Sigma., U.S.A.). Tracheas were remove: and trimmed of connective tissue. The tracheas were: shen incubated at 4°C overnight in 0.1% protease (Type XI!‘, Sigma, USA) dissolved in sterile 50% Dulbecco modified Eagle medium (DMEM) and 50% Ham nutrient F12 medium (GIBCO, USA) containing 5% fetal calf serum (Nakarai Chemical, Japan), penicillin (5 U/l; Sigma), streptomycin (100 mg/l; Sigma), gentamicin (50 mg/l; Sigma), and fungizone (2.5 mg/l; Sigma). This medium is referred to as DMEM/F12. Tracheal lumens were washed with 10 ml DMEM/Fl2 to detach the epitheha1 ceils, which were filtered with through 100 wrn Nitex mesh (Tetko, USA). The collected cells were centrifuged 100 x g at 4°C for 10 min and resuspended in DMEM/F12 twice. The total cell count was calculated using a standard hemocytometer, and the viability of the ceiis was examined by trypan blue dye exclusion
(Sigma). The \*iability of collected cells was 90-95%. al epithclial cells were plated at a density of 5 cells/dish on 15mm, 24-well cluster tissue plates (Corning, U.S.A.) and incubated at 37°C in 5% CO,-95% air. The medium was changed every day, and cells grew to confluent monolayers after 5-6 days. The study was performed with confluent cells. For identification, cells were stained with Giemsa as well as immu~ohistochemically with an antibody against cytokeratines (Cosmo Bio, Japan). 0
2.2. Experimental procedures Confluent primary epithelial cell cultures were washed three times with I ml DMEM/Fl2, and incubated with DMEM/FlZ containing 4, IO, and 40 pg/ml endotoxin (lipopo!ysaccharide B E. Coli 055: 85; DIFCO, U.S.A.) or vehicle control. Samples for the measurement of ET-I were obtained after 1 min, 6 h, 12 h and 24 h, and centrifuged 1200 Xg for 10 min. The concentration of ET-l in the supernatant was measured using a sandwich-enzyme immunoassay (EIA). After 24 h of stimulation, viability was tested by staining with trypan blue and morphological changes within the celis were examined by staining with Giemsa. 2.3. EL4 for ET-I The sandwich-EIA for ET-l has been described previously (Suzuki et al., 1989). In brief, we used immobilized mouse monoclonal antibody (AwETN 40), which recognizes the N-terminal portion of ET-l, and peroxidase-coupled rabbit anti-ET-l antibody against the C-terminal peptide (15-21). This assay does not cross-react with ET-3 or Big ET-l, but is able to detect ET-2 with a cross reactivity of approximately 160%. The sensitivity for ET-l was 0.2 pg/well, which corresponded to 0.4 pg/ml in this study.
2.4. Statistical analysis Data are expressed as means + S.E. The probability of differences between mean results was determined by using a one-way or two-way analysis of variance (ANOVA) as appropriate. If a significant variance ratio was indicated, individual group differences were determined with Dunnett’s test or Duncan’s multiple range test.
Confluent cultured cells formed a ‘cobble-stone’ appearance. Immunohistochemical studies of confluent cultured epithelial cells revealed cytoplasmic staining of cells with antibody to cytokeratines, a characteristic
0
6
12
10
24
TIME (h) Fig. 1. Release of immunoreactive ET-l from cultured epithelial cells as a function of time. Epithelial cells were incubated with endotoxin ), lOI.rgfO).4Opg( ) or vehicle control (0). Each point represents the mean of 6 wells; bars indicate SE. Significant differences were determined by using a two-way ANOVA and Duncan’s multiple range test. Asterisks show statistically significant differences between samples (* P < 0.01).
component of epithelial cells. The concentration of ET-1 in the medium increased in a time-dependent fashion, and endotoxin (4-40 pg) significantly increased the ET-l concentration in the supernatant from epithelial cells as compared to that of vehicle controls at 12 and 24 h (fig. 1). Supernatant obtained after i min of incubation and DMEM/F12 without cells exhibited no immunoreactive ET-l. Epithelial cell viability, based on trypan blue exclusion, was determined after incubation with endotoxin for 24 h. The number of cells in wells incubated with 40 pg endotoxin was significantly decreased compared to that of vehicle controls (21.5 of:0.6 x lo4 cells/well and 36.2 + 2.3 X lo4 cells/well, respectively). Endotoxin (4 pg. 10 pg), however, had no effect on epithelial cell viability (37.5 f 2.2 x lo4 cells/well and 35.0 f 1.8 x lo4 cells/well, respectively) (fig. 2). We could not detect obvious morphological changes in cultured epithelial cells incubated with endotoxin or vehicle control for 24 h. Figure 2 shows ET-l release per lo4 epithelial cells incubated with endotoxin for 24 h, and the dose-dependent increase in ET-1 release induced by endotoxin over this time period.
4. Discussion The result of the present study indicates that guinea-pig airway epithelial cells in culture are capable of releasing ET-l. Cultured canine epithelial cells also release ET-I (Black et al., 1989); however, because this previous study did not express ET-l release on a cell basis, we could not compare the extent of release between the two studies. In our study, the time course of ET-1 release showed that epithelial cells produced
301
v4llla cwnml a48 mlrnkn (10”44ll4/lv4H)
36.2+2s
Didoloxtn 44 @=e
37.5*2.2
Endotorln
Gleoloxln
~0~~)
(~~~
?&0*1Al
ndiaci*
Fig. 2. Release of immunoreactive ET-l per lo4 cells incubated with endotoxin for 24 h. Epithelial cells were incubated wirh endotoxin (4, 10,40 pg: n = 4) or vehicle control (n = 6). Each point represents the mean P SE. Numbers below each bar show the viable cell ~unt/well f SE. Si~ificant differences were determined by using a one-way ANOVA and Dunnett’s test. Asterisks show statistically significant differences between samples t* P < 0.01). N.S. means not significant.
ET-1 spontaneously and continuously. Several investigators have shown that endothelial cells in culture also produce ET-1 spontaneously and continuously. These cells are suggested to exhibit basal release of ET-l. In a previous study, we demonstrated that ET-1 was detectable in guinea-pig airway lavage and normal human bro~choalveolar Iavage fluids (Ninomiya et al., 19903. The present results suggest that epithelial cells in the normal airway release ET-i. In this study, ET-l release was significantly increased by treatment of epithelial cells with endotoxin. This increase was continuous and dose-related, it was even observed at levels of endotoxin that did not result in cell death. This continuous increase suggests that endotoxin may stimulate ET-1 mRNA production and increase basal release. The mechanism of endotoxin-induced ET-l release from airway epithelial cells remains obscure. Endot~xin stimulates ET-I release from endothelial cells in vivo and in vitro by an as yet unknown mechanism. Further investigation is needed to clarify the relationship between ET-l production and endotoxin in these cells. It should be noted that the antibody used in these studies is not specific for ET-l, but also reacts with ET-Z. Based upon the observation that ET-2 production by guinea-pig lung and trachea is not detected by high-performance liquid chromatography (HPLCI analysis (Ninomiya, unpublished data), we believe that the majority of activity released from the tissue in the current study can be attributed to ET-l, although the possibility that ET-2 contributed in part to the observed immunoreactivity cannot be ruled out. Airway epithelium plays an important role in host defense as the barrier against physical, pathological, and chemical stimuli, Currently, it is also considered as
an active metabolic and biosynthetic site for agents associated with the regulation of bronchomotor tone, for example, prostaglandin E, (PGE,) and epithelium-derived relaxing factor. A dysfunction of airway epithelium has been reported to cause airway hyperreactivity, and epithelial damage is associated with an increased severity of asthma (Leff, 1988).These investigations suggest that epithelial cells are related to pathophysiological bronchoconstricrion in asthma and airway iR~ammation. Endoto~n is known to cause airway epithelial damage and airway hyperreactivity. to bronchoconstrictive agents (Folkerts et al., 1989). In endotoxin shock, a reduction in dynamic compliance, an increase in resistance to airflow in the lung, and disturbances of gas exchange are observed, and gramnegative bacterial pulmona~ infection often leads to bro~~hoconstriction in asthmatic subjects. The increased release of ET-1 from epithelial cells may be associated with this airway hyperreactivity and other pathophysiological processes caused by endotoxin. In conclusion, cultured airway epithelial cells of guinea-pig trachea are capable of releasing ET-l, and endotoxin significantly stimulates this release. These results suggest that ET-1 from airway epithelium may represent an important regulatory mediator or*pathophysiologic states of the airways.
Acknowledgements This work would not have been possible without the guidance and assistance of N. Suzuki and H. Matsumoto (Takeda Chemical Industries, Japan). The writers thank Dr. E.W. Spannhake for his advice and assistance.
References Barnett, K., D.B. Jacoby, J.A. Nadel and SC. Lazarss, 1988, The effects of epithelial cell supernatant on contractions of isolated canine tracheal smooth muscle, Am. Rev. Respir. Dis. 138, 780. Battistini, B., J. Filep and P. Sirois. 1990, Potent thromboxane mediated in vitro bronchoconstrictor effect of endothelin in the guinea-pig, Eur. J. Pharmacol. 978, 141. Black, P.N., M.A. Ghatei, K. Takahashi, D. Bretherton-Watt, T. Krausz, CT. Dollery and S.R. Bloom, 1989, Formation of endothelin by cultured airway epithelial cells, FEBS Lett. 255, 129. De Nucci, G., R. Thomas, P. D’Orleans-Juste, E. Antunes, C. Walder, T.D. Warner and J.R. Vane, 1988, Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacycline snd endotheliumderived relaxing factor, Proc, Nat!. Acad. Sci. U.S.A. 85.9797. Folkerts, G., F. Engels and F.P. Nijkamp, 1989, Endotoxin-induced hy~rreactivi~ of the guinea-pig isolated trachea coincides with decreased prostaglandin Ez production by the epitheliai iayer, Br. J. Pharmacol. 96, 388. Leff. A.R., 1988. Endogeneous regulation of bronchomotor tone. Am. Rev. Respir. Dis. 137, 1198. Ninomiya, H., Y. Uchida, M. Kameyama, M. Saotome, A. Nomura, M. Ohtsuka, A. Watanabe and S. Hasegawa, 1990, Endothefin-re-
Icase into airway lumen in guinea-pig and man, Am. Rev. Respir. is. 141. Al04 (Abstract). PErwvs.P.L. Z.A. Abdel-Malek, C.A. Doupnik and G.D. Leikauf. Endothehn stimulates chloride secretion across canine tracheal epithelium. Am. J. Pbysiol. fin press).
Uchida, Y., H. Ninomiya, M. Saotome, A. Nomura, M. Ohtsuf.~, M. Yanagisawa, K. Goto, T. Masaki _.‘d S. Hasegawa, 1988, Endothelin, a novel potent vasoconstrictor. a, a potent bronchoconstrictor, Eur. J. Pharmacol. 1.54,227.
0014.2999/91/$03.50
299
ADONIS 001429999100725V EJP 20932
Short comrn~~~ca~oa
Hiroki~inomiya, Yushi~~ Division
Uchida, Yukio Ishii, Akihiro Nomura, Masaaki Kameyama, Mikio Saotome, Takeo Endo and Shizuo Nasegawa
of Pulmonary Medicine, Institute of Clinical Medicine, University of Tsukubn, Tsukuba, Ibaraki 305, Japan Received 20 June 1991,revised MS received 13 August 1991,accepted 20 August 1991
We examined the release of endothelin from cultured eyithelial cells of guinea-pig trachea in response to treatment with endotoxin, using a sandwich-enzyme immunoassay. Cultured epithelial cells released endothelin in a time-dependent fashion (3, 6, 12, 24 h), and endotoxin (4-40 &ml) significantly increased endothelin release. Endotoxin (4-10 #g/ml) showed no cytotoxity against epithelial cells. These results suggest that guinea-pig airway epithelial cells are capable of producing endothelin and this peptide may be related to the pathophysiological effects of endotoxin. Endothelin; Endotoxin;Airwayepithelisl cells 1. Introduction
Endothelin-1 (ET-l) is a 21 amino acid peptide identified from cultured endothelial cells. ET-l has a potent contractile effect on both vascular and airway smooth muscles (Uchida et al., 1988), and is produced by airway epithelial cells as well as endothelial cells (Black et al., 1989). ET-l stimulates thromboxane A, (TXA,) r&ease from perfused guinea-pig lung (De Nucci et al., 1989), and ET-l induced airway constriction is suggested to be partly mediated by TXA, (Battistini et al., 1990). ET-l also stimulates airway epitheha1 cells to increase Cl- secretion (Piews et al., in press). These results indicate that ET-1 has a variety of effects in the lung. A dysfunction of epithelial cells is considered to play an important role in pathophysiological airway constriction. A recent study has shown that endotoxin exposure results in airway hyperreactivity to histamine and arecohne (a muscarinic receptcr agonists in the guinea-pig because of its cytotoxity to airway epithehum (Folkerts et al., 1989). In the present study, to elucidate the relationship between the airway epithehum and ET-l synthesis, we investigated whether endotoxin modulates ET-l release from cultured epitbeliai cells of the guinea-pig trachea. This is the first
Correspondence IO: S. Hasegawa, Division of Pulmonary Medicine, Institute of Clinical Medicine. University of Tsukuha, Tsukuba. fbaraki 305, Japan. Tel. 81.298.53 3361.
paper reporting the stimulant effect of endotoxin on ET-l production by airway epithelial cehs. 2. Materials and methods 2.1. Culture c$ trachea/ epitheliai cells
Tracheal epithelial cells were prepared by a modification of the method of Barnett et al. (1988); we used 24-well tissue culture plates instead of tissue culture flasks coated with human placenta collagen. Hartlcystrain guinea-pigs (body weight 300-400 g) were anesthetized with sodium pentobarbitai (50 mg/kg Lp.; Sigma., U.S.A.). Tracheas were remove: and trimmed of connective tissue. The tracheas were: shen incubated at 4°C overnight in 0.1% protease (Type XI!‘, Sigma, USA) dissolved in sterile 50% Dulbecco modified Eagle medium (DMEM) and 50% Ham nutrient F12 medium (GIBCO, USA) containing 5% fetal calf serum (Nakarai Chemical, Japan), penicillin (5 U/l; Sigma), streptomycin (100 mg/l; Sigma), gentamicin (50 mg/l; Sigma), and fungizone (2.5 mg/l; Sigma). This medium is referred to as DMEM/F12. Tracheal lumens were washed with 10 ml DMEM/Fl2 to detach the epitheha1 ceils, which were filtered with through 100 wrn Nitex mesh (Tetko, USA). The collected cells were centrifuged 100 x g at 4°C for 10 min and resuspended in DMEM/F12 twice. The total cell count was calculated using a standard hemocytometer, and the viability of the ceiis was examined by trypan blue dye exclusion
(Sigma). The \*iability of collected cells was 90-95%. al epithclial cells were plated at a density of 5 cells/dish on 15mm, 24-well cluster tissue plates (Corning, U.S.A.) and incubated at 37°C in 5% CO,-95% air. The medium was changed every day, and cells grew to confluent monolayers after 5-6 days. The study was performed with confluent cells. For identification, cells were stained with Giemsa as well as immu~ohistochemically with an antibody against cytokeratines (Cosmo Bio, Japan). 0
2.2. Experimental procedures Confluent primary epithelial cell cultures were washed three times with I ml DMEM/Fl2, and incubated with DMEM/FlZ containing 4, IO, and 40 pg/ml endotoxin (lipopo!ysaccharide B E. Coli 055: 85; DIFCO, U.S.A.) or vehicle control. Samples for the measurement of ET-I were obtained after 1 min, 6 h, 12 h and 24 h, and centrifuged 1200 Xg for 10 min. The concentration of ET-l in the supernatant was measured using a sandwich-enzyme immunoassay (EIA). After 24 h of stimulation, viability was tested by staining with trypan blue and morphological changes within the celis were examined by staining with Giemsa. 2.3. EL4 for ET-I The sandwich-EIA for ET-l has been described previously (Suzuki et al., 1989). In brief, we used immobilized mouse monoclonal antibody (AwETN 40), which recognizes the N-terminal portion of ET-l, and peroxidase-coupled rabbit anti-ET-l antibody against the C-terminal peptide (15-21). This assay does not cross-react with ET-3 or Big ET-l, but is able to detect ET-2 with a cross reactivity of approximately 160%. The sensitivity for ET-l was 0.2 pg/well, which corresponded to 0.4 pg/ml in this study.
2.4. Statistical analysis Data are expressed as means + S.E. The probability of differences between mean results was determined by using a one-way or two-way analysis of variance (ANOVA) as appropriate. If a significant variance ratio was indicated, individual group differences were determined with Dunnett’s test or Duncan’s multiple range test.
Confluent cultured cells formed a ‘cobble-stone’ appearance. Immunohistochemical studies of confluent cultured epithelial cells revealed cytoplasmic staining of cells with antibody to cytokeratines, a characteristic
0
6
12
10
24
TIME (h) Fig. 1. Release of immunoreactive ET-l from cultured epithelial cells as a function of time. Epithelial cells were incubated with endotoxin ), lOI.rgfO).4Opg( ) or vehicle control (0). Each point represents the mean of 6 wells; bars indicate SE. Significant differences were determined by using a two-way ANOVA and Duncan’s multiple range test. Asterisks show statistically significant differences between samples (* P < 0.01).
component of epithelial cells. The concentration of ET-1 in the medium increased in a time-dependent fashion, and endotoxin (4-40 pg) significantly increased the ET-l concentration in the supernatant from epithelial cells as compared to that of vehicle controls at 12 and 24 h (fig. 1). Supernatant obtained after i min of incubation and DMEM/F12 without cells exhibited no immunoreactive ET-l. Epithelial cell viability, based on trypan blue exclusion, was determined after incubation with endotoxin for 24 h. The number of cells in wells incubated with 40 pg endotoxin was significantly decreased compared to that of vehicle controls (21.5 of:0.6 x lo4 cells/well and 36.2 + 2.3 X lo4 cells/well, respectively). Endotoxin (4 pg. 10 pg), however, had no effect on epithelial cell viability (37.5 f 2.2 x lo4 cells/well and 35.0 f 1.8 x lo4 cells/well, respectively) (fig. 2). We could not detect obvious morphological changes in cultured epithelial cells incubated with endotoxin or vehicle control for 24 h. Figure 2 shows ET-l release per lo4 epithelial cells incubated with endotoxin for 24 h, and the dose-dependent increase in ET-1 release induced by endotoxin over this time period.
4. Discussion The result of the present study indicates that guinea-pig airway epithelial cells in culture are capable of releasing ET-l. Cultured canine epithelial cells also release ET-I (Black et al., 1989); however, because this previous study did not express ET-l release on a cell basis, we could not compare the extent of release between the two studies. In our study, the time course of ET-1 release showed that epithelial cells produced
301
v4llla cwnml a48 mlrnkn (10”44ll4/lv4H)
36.2+2s
Didoloxtn 44 @=e
37.5*2.2
Endotorln
Gleoloxln
~0~~)
(~~~
?&0*1Al
ndiaci*
Fig. 2. Release of immunoreactive ET-l per lo4 cells incubated with endotoxin for 24 h. Epithelial cells were incubated wirh endotoxin (4, 10,40 pg: n = 4) or vehicle control (n = 6). Each point represents the mean P SE. Numbers below each bar show the viable cell ~unt/well f SE. Si~ificant differences were determined by using a one-way ANOVA and Dunnett’s test. Asterisks show statistically significant differences between samples t* P < 0.01). N.S. means not significant.
ET-1 spontaneously and continuously. Several investigators have shown that endothelial cells in culture also produce ET-1 spontaneously and continuously. These cells are suggested to exhibit basal release of ET-l. In a previous study, we demonstrated that ET-1 was detectable in guinea-pig airway lavage and normal human bro~choalveolar Iavage fluids (Ninomiya et al., 19903. The present results suggest that epithelial cells in the normal airway release ET-i. In this study, ET-l release was significantly increased by treatment of epithelial cells with endotoxin. This increase was continuous and dose-related, it was even observed at levels of endotoxin that did not result in cell death. This continuous increase suggests that endotoxin may stimulate ET-1 mRNA production and increase basal release. The mechanism of endotoxin-induced ET-l release from airway epithelial cells remains obscure. Endot~xin stimulates ET-I release from endothelial cells in vivo and in vitro by an as yet unknown mechanism. Further investigation is needed to clarify the relationship between ET-l production and endotoxin in these cells. It should be noted that the antibody used in these studies is not specific for ET-l, but also reacts with ET-Z. Based upon the observation that ET-2 production by guinea-pig lung and trachea is not detected by high-performance liquid chromatography (HPLCI analysis (Ninomiya, unpublished data), we believe that the majority of activity released from the tissue in the current study can be attributed to ET-l, although the possibility that ET-2 contributed in part to the observed immunoreactivity cannot be ruled out. Airway epithelium plays an important role in host defense as the barrier against physical, pathological, and chemical stimuli, Currently, it is also considered as
an active metabolic and biosynthetic site for agents associated with the regulation of bronchomotor tone, for example, prostaglandin E, (PGE,) and epithelium-derived relaxing factor. A dysfunction of airway epithelium has been reported to cause airway hyperreactivity, and epithelial damage is associated with an increased severity of asthma (Leff, 1988).These investigations suggest that epithelial cells are related to pathophysiological bronchoconstricrion in asthma and airway iR~ammation. Endoto~n is known to cause airway epithelial damage and airway hyperreactivity. to bronchoconstrictive agents (Folkerts et al., 1989). In endotoxin shock, a reduction in dynamic compliance, an increase in resistance to airflow in the lung, and disturbances of gas exchange are observed, and gramnegative bacterial pulmona~ infection often leads to bro~~hoconstriction in asthmatic subjects. The increased release of ET-1 from epithelial cells may be associated with this airway hyperreactivity and other pathophysiological processes caused by endotoxin. In conclusion, cultured airway epithelial cells of guinea-pig trachea are capable of releasing ET-l, and endotoxin significantly stimulates this release. These results suggest that ET-1 from airway epithelium may represent an important regulatory mediator or*pathophysiologic states of the airways.
Acknowledgements This work would not have been possible without the guidance and assistance of N. Suzuki and H. Matsumoto (Takeda Chemical Industries, Japan). The writers thank Dr. E.W. Spannhake for his advice and assistance.
References Barnett, K., D.B. Jacoby, J.A. Nadel and SC. Lazarss, 1988, The effects of epithelial cell supernatant on contractions of isolated canine tracheal smooth muscle, Am. Rev. Respir. Dis. 138, 780. Battistini, B., J. Filep and P. Sirois. 1990, Potent thromboxane mediated in vitro bronchoconstrictor effect of endothelin in the guinea-pig, Eur. J. Pharmacol. 978, 141. Black, P.N., M.A. Ghatei, K. Takahashi, D. Bretherton-Watt, T. Krausz, CT. Dollery and S.R. Bloom, 1989, Formation of endothelin by cultured airway epithelial cells, FEBS Lett. 255, 129. De Nucci, G., R. Thomas, P. D’Orleans-Juste, E. Antunes, C. Walder, T.D. Warner and J.R. Vane, 1988, Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacycline snd endotheliumderived relaxing factor, Proc, Nat!. Acad. Sci. U.S.A. 85.9797. Folkerts, G., F. Engels and F.P. Nijkamp, 1989, Endotoxin-induced hy~rreactivi~ of the guinea-pig isolated trachea coincides with decreased prostaglandin Ez production by the epitheliai iayer, Br. J. Pharmacol. 96, 388. Leff. A.R., 1988. Endogeneous regulation of bronchomotor tone. Am. Rev. Respir. Dis. 137, 1198. Ninomiya, H., Y. Uchida, M. Kameyama, M. Saotome, A. Nomura, M. Ohtsuka, A. Watanabe and S. Hasegawa, 1990, Endothefin-re-
Icase into airway lumen in guinea-pig and man, Am. Rev. Respir. is. 141. Al04 (Abstract). PErwvs.P.L. Z.A. Abdel-Malek, C.A. Doupnik and G.D. Leikauf. Endothehn stimulates chloride secretion across canine tracheal epithelium. Am. J. Pbysiol. fin press).
Uchida, Y., H. Ninomiya, M. Saotome, A. Nomura, M. Ohtsuf.~, M. Yanagisawa, K. Goto, T. Masaki _.‘d S. Hasegawa, 1988, Endothelin, a novel potent vasoconstrictor. a, a potent bronchoconstrictor, Eur. J. Pharmacol. 1.54,227.