Epithelial Modulation of Airway Smooth Muscle Response to Endothelin-1 1- 4
STEVEN R. WHITE,5 DARREN ~ HATHAWAY,6 JASON G. UMANS,7 JULIO TALLET, CYRIL ABRAHAMS, and ALAN R. LEFF
I t has been proposed that bronchomotor tone is regulated in part by localized secretion from the adjacent epithelium (1-3). However, the mechanisms by which this occurs remain controversial. Airway reactivity is increased during viral respiratory infection and is associated with inflammation and damage to the airway epithelium (4). Chronic inflammatory infiltration of the epithelium and lamina propria is a common finding in asthmatic patients (5, 6). Prior investigations have demonstrated that removal of the epithelium from trachea or bronchial airways augments contraction of the underlying airway smooth muscle after stimulation with contractile agonists (1-3). In those studies, it was postulated that epithelial ablation caused nonspecific augmentation of airway contractile responses by removal of a tonically secreted epithelialderived inhibitory factor. Subsequent studies have not demonstrated the putative epithelial-derived inhibitory factor (2, 3), and recent investigations of epithelial function in situ suggest that epithelial inflammation leads to synthesis and local secretion of substances that augment airway smooth muscle contraction (7, 8). The 21-amino acid polypeptide endothelin-l (ET-l), isolated from the supernatant of cultured vascular endothelial cells (9), is a potent constrictor ofvascular smooth muscle (9, 10). A family of three similar human endothelin peptides has been recognized, each encoded by a different gene (11). Kimura and coworkers (12) have demonstrated that the carboxy-terminus of the molecule is important in eliciting the effects ofET-l in porcine coronary smooth muscle. Prior investigations indicate that endothelin can elicit contraction in lowerairways (13, 14); this response may be altered by blockade of J}-adrenergic receptors or inhibition of cyclooxygenase. However, the mechanisms by which endothelin alters
SUMMARY We Investigated the role of epithelial modulation of contraction caused by endothelln-1 In airway smooth muscle In guinea pigs In situ. Airway responses were assessed Isometrlcallyas tracheal force and simultaneously as change In lung resistance. Intravenous administration of 10-8 mol/kg endothelln-1 caused a blphaslc response In tracheal active tension: Initial relaxation (-0.82 ± 0.22 g/cm after 30 s, p < 0.05 versus baseline) followed by contraction (1.65 ± 0.28 g/cm after 7 min, p < 0.05 versus baseline). Endothelln-1 also elicited Immediate bronchoconstrlctlonj lung resistance Increased from 0.148 ± 0.030 to 0.992 ± 0.274 em H20/Us (p < 0.005) after 10-8 mol/kg endothelln-1 given Intravenously. Active tension elicited by 10-8 mol/kg endothelln-1 after removal of the epithelium from the tracheal segment (0.59 ± 0.16 glcm) was less than In segments with an Intact epithelium (1.65 ± 0.28 glcm, p < 0.01).Both tracheal contraction and bronchoconstrlctlon were attenuated by pretreatment with Indomethacin orally, BW 755C Intravenously, or substitution of endothelln-C-termlnal hexapeptlde for endothelln-1. However, the Initial tracheal relaxation response was similar after each Intervention. These data suggest actions of endothelln-1 that have not been demonstrated previously: (1) endothelln-1 elicits a blphaslc response In tracheal smooth muscle (an Initial relaxation response elicited by the carboxy-terminal residues and a later contractile response that requires synthesis of a cyclooxygenase mediator) and (2) epithelium adjacent to the airway smooth muscle modulates contraction elicited by endothelln-1. AM REV RESPIR DIS 1991; 144:373-378
bronchomotor response have not been defined completely. The aims of this study were (1) to assessthe effect of ET-l on tracheal smooth muscle, (2) compare these effects with the effect of ET-l in lower airways, (3) determine the mechanisms of action of ET-l, and (4) determine the dependence of the effects of ET-l on epithelial activation. We used a newly developed epithelium-smooth muscle preparation, which preserves in situ relationships, to assess the role of ET-l in airway smooth muscle contraction. Wedemonstrate that endothelin causes a biphasic response in airway smooth muscle in vivo: (1) a short, initial relaxation response that is not mediated by eicosanoids followed by (2) a sustained, eicosanoid-mediated airway contraction that can be measured independently of airwayedemagenesis.Unlike many inflammatory mediators, endothelin does not augment airway responsiveness to muscarinic stimulation. Finally, we find that the effects of endothelin depend substantially upon the presence of an intact epithelium. These studies demonstrate both direct and epithelium-mediated effects of endothelin and suggest that contraction caused
by ET-l is mediated through eicosanoid synthesis either by airway smooth muscle or by the overlying epithelium. (Receivedin originalform November 26, 1990and in revised form February 19, 1991) 1 From the Section of Pulmonary and Critical Care Medicine and Section of Nephrology, Department of Medicine, the Committee on Clinical Pharmacology and the Department of Pathology, Divi. sion of the Biological Sciences, The University of Chicago, Chicago, Illinois. 2 Supported by Grants HL-02484, HL-32495, and HL-35718 from the National Heart, Lung, and Blood Institute and by grants from the American Lung Association, the Chicago Heart Association, the Pharmaceutical Manufacturers Association, the Schweppe Foundation, and the Mid-States Science and Mathematics Consortium. 3 Presented in part at the joint Meeting of the American Physiological Society and the American Thoracic Society, Rochester, MN, October 18, 1989. 4 Correspondence and requests for reprints should be addressed to Steven R. White, M.D., University of Chicago, Section of Pulmonary and Critical Care Medicine, 5841S. Maryland Ave.,Box 98, Chicago, IL 60637. 5 Recipient of a Clinical Investigator Award from the National Heart, Lung, and Blood Institute, and is an Edward Livingston Trudeau Fellow of the American Lung Association and a Fellow of the Schweppe Foundation. 6 Undergraduate Scholar of the Pew Charitable Trust. 7 Fellow of the Schweppe Foundation.
WHITE, HATHAWAY, UMANS, TALLET, ABRAHAMS, AND LEFF
Preparation of Animals All experiments were approved by the UnivesityCommittee on Animal Care. Male Hartley guinea pigs weighing 600 to 900 g were anesthetized with pentobarbital (50 mg/kg intraperitoneally), and a lower cervical tracheostomy was performed. Animals wereventilated continuously with pure oxygen using a constant-volume ventilator (Model 681; Harvard Apparatus Co., South Natik, MA) so that Pao] was> 300 mm Hg. Tidal volume was set at 6 ml/kg; this tidal volume was demonstrated in preliminary studies to maintain Paco] between 35 and 45 mm Hg and arterial blood pH between 7.35 and 7.45 (8). Respiratory rate was 60 breaths/min in all animals. Rate, tidal volume and Flo] remained constant for all animals during the study. Full anesthesia was verified periodically by checking withdrawal reflexes; additional anesthesia was administered when required. A l.o-cm segment of cervical trachea proximal to the tracheostomy site was prepared for measurement of isometric tension (8).This is a modification of a preparation that wehave described previously (7, 15)that preserves the epithelium-smooth muscle relationship in situ. The tracheal segment was attached by sutures to a parallel bar on one side and a Grass model FT.03 force-displacement transducer (Grass Instruments, Quincy, MA) on the other side.Active force of contraction was determined by subtracting initial resting force of the tracheal segment from total force achieved after administration of an agonist. Active force was normalized by dividing by the longitudinal length of the fixed segment (8, 15) and expressed as active tension (AT) in grams force per centimeter longitudinal length (g/cm) of the segment (8). Resting tension was set to 10 g/cm; this corresponds to the optimal resting length of the tracheal segment from which maximal, reproducible contraction occurs (8). This resting tension was necessary to overcome the elastance of the split cartilage and attachments of the trachea in this in situ preparation. Resting tension was stable to 0.02 g/cm in these studies; the sensitivity of active tension did not vary during these studies. Airway pressure during ventilation was measured through a side port in the tracheostomy apparatus by a Honeywell model 140PC differential air pressure transducer (Honeywell, Denver, CO) referenced to atmospheric pressure. Airflow was measured with a Fleisch pneumotachograph connected to a Honeywell 140PC differential air pressure transducer. For calculation of lung resistance, pleural pressure was made equal to atmosphere by bilateral thoracotomy. Lung resistance was determined by an adaptation of the method of von Neergaard and Wirz (16). This method permitted continuous monitoring of lung resistance. All physiologic measurements wererecorded continuously on a Gould 3800 chart recorder (Gould Instruments, Cleveland, OH).
A catheter was placed in the external jugular vein for administration of agonists. All agonists for intravenous injection were dissolved in normal saline. Bilateral cervical vagotomy was performed at the levelofthe third tracheal cartilage. At the end of each experiment, guinea pigs were killed with an overdose of pentobarbital followed by 2 ml saturated potassium chloride given intravenously.
Effect of Endothelin-I In six guinea pigs prepared for simultaneous measurement of isometric force of contraction and lung resistance, the effect of intravenously administered ET-l was determined. A single dose of 3 x 10-7 mol/kg ACh was administered intravenously initially to demonstrate viability of the preparation (8). Twenty minutes after tracheal tension and lung resistance returned to baseline, a cumulative dose-response curve was generated with 10-10 to 10-8 mol/kg ET-l given intravenously in half-log increments. Each dose was given at the plateau of the maximal contractile response (over 7 min) of the tracheal segment to the previous dose. Endothelin doseresponse curves were preceded by 0.5 ml phosphate-buffered normal saline given intravenously (the solvent for ET-l). Ten minutes after completion of the ET-l dose-response curve, 3 x 10-7 mol/kg ACh was administered intravenously again to verify viability of the preparation (8). Preliminary experiments in two animals demonstrated that the response to 10-8 mol/kg ET-l given as a single dose was equivalent to the response to the same dose given in a cumulative dose-response curve. To determine if the initial relaxation response (see RESULTS) resulted from sympathetic activation caused by hypotension elicited by intravenously administered ET-l (17),doseresponse curves from 10-10 to 10-8 mol/kg ET-l were generated in six additional guinea pigs receiving prior ~-adrenergic blockade with propranolol, 1 mg/kg givenintravenously and 2 mg/kg given intraperitoneally (18). In six other guinea pigs, the effect of the epithelium on the response to ET-l was assessed. A dose-response curve was generated with 10-10 to 10-8 mol/kg ET-l given intravenously 30 min after the epithelium of the tracheal segment was removed with a cotton applicator. Viability of the preparation was confirmed by intravenous administration of 3 x 10-7 mol/kg ACh as above. Effect of Cyclooxygenase Inhibition on the Response to Endothelin-I In five guinea pigs, the response to ET-l was assessed after administration of indomethacin. Each animal was fed 25 mg/kg indomethacin daily for 3 days; the last dose was administered 45 min prior to the start of the protocol. After preparation, a dose-response curve was generated with 10-10 to 10-8 mol/kg ET-l administered intravenously. To demonstrate viability of the preparation, the response to 3 x 10-7 mol/kg ACh given intravenously was assessed as above 20 min before and 10 min after ET-l.
In four additional guinea pigs, 10 mg/kg 3-amino-l-(3-trifluoromethylphenyl)-2-pyrazoline hydrochloride (BW755C) was administered intravenously 30 min before the first dose-response study. This dose has been demonstrated to cause inhibition of both cyclooxygenase and 5-lipoxygenase in guinea pig tracheal smooth muscle (19). Intravenously administered ACh and ET-l dose-response curves were generated identically as for' animals receiving indomethacin. In four other guinea pigs, the specificity of the response to ET-l was assessed using endothelin-Csterrninal-hexapeptide (ECH). This fragment contains the C-terminal tryptophan residue demonstrated to be necessary for contraction of porcine coronary arterial smooth muscle (12)but not the double disulfide bridges in the endothelin molecule. After preparation, a cumulative dose-response curve was generated with 10-10 to 10-8 mol/kg ECH given intravenously. Dose-response curveswerepreceded by 0.5 ml saline as above. To demonstrate viability of the preparation, the response to 3 X 10-7 mol/kg ACh was assessed as above 20 min before and 10 min after ECH.
Drugs and Suppliers Endothelin-l and ECH were obtained from Peninsula Laboratories (Belmont, CA). Sodium pentobarbital was obtained from Abbott Laboratories (North Chicago, IL). Acetylcholine hydrochloride, propranolol bitartrate, and indomethacin were obtained from Sigma Chemical (St. Louis, MO). 3-amino-l(3-trifluoromethylphenyl)-2-pyrazoline hydrochloride (BW 755C) was a gift of Wellcome Research Laboratories (Beckingham, Kent, UK). All reagents were laboratory grade or better. All agonists for intravenous administration were dissolved in phosphate-buffered normal saline immediately prior to use.
Analysis of Data Data are expressed as the mean ± SEM. Comparisons between groups of active tension or changes in lung resistance (RL) elicited by a specific dose of ET-l were made using Student's t test. Paired comparisons were made within groups when appropriate. Bonferroni's correction was applied as required for multiple comparisons. Comparisons of active tension elicited by ET-l or ECH to baseline (passive) tension weremade with the 95070 confidence interval (CI). Comparisons between groups for the maximal relaxation or contraction response in the tracheal segment was made by analysis of variance; when significant differences were found, further comparisons were made using Dunnett's t test. Statistical significance was claimed at p < 0.05.
Validation and Control Studies In all studies, the preparation remained viable for the duration of the experiment. Injection of normal saline prior to either ET-l or ECH dose-response curves did
EPITHELIAL-DEPENDENT TRACHEAL SMOOTH MUSCLE CONTRACTION BY ENDOTHELlN-1
not alter either tracheal tension or lung resistance. There was no difference in the response to 3 X 10-7 mol/kg ACh given intravenously initially and after completion of ET-1 dose-response studies. In control animals, active tension generated by ACh was 0.74 ± 0.09 g/cm before and 0.65 ± 0.15 g/cm after ET-1 (p = NS). Tracheal activetension generated by ACh in other groups was comparable to that in control animals, both before and after ET-1, and was not altered by administration of eicosanoid synthetase inhibitors, removal of the epithelium, or administration of ECH. Complete removal of the epithelium was verified by histologic examination in all animals in all protocols requiring epithelial removal. Epithelial integrity was demonstrated in randomly selected animals in all protocols requiring an intact epithelium.
Effects of Endothelin-l In all six guinea pigs, ET-1 administered intravenously caused a biphasic response of tracheal tension; initial relaxation, which was maximal in < 30 s, was followed by contraction that was maximal for each dose in rv 3 min (figure 1).Each dose of ET-1 > 10-10 mol/kg elicited both relaxation and contraction phases in the tracheal segment. Tracheal contraction was maximal after 10-8 mol/kg (figure 2), whereas relaxation reached a plateau after 10-9 mol/kg (figure 3). The tracheal contractile response to ET-l was attenuated by epithelial removal. After 10-8 mol/kg ET-l, active tension was 0.59 ± 0.16 g/cm (95070 CI, 0.18 to
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Fig. 1. A representative tracing of responseof trachea and lower airwaysto intravenouslyadministered endothelin-1. Endothelin-1 eliciteda biphasic responsein the isolated tracheal segment of each animal studied: initial relaxation of tracheal smooth muscle, which was maximal in < 30 s, followed by contraction, which reachedstableplateauin < 3 min.Transpulmonary pressure (Ptp) increased substantiallyduring this time, but it did not demonstratea biphasicresponse.Lung resistance was calculated from Ptp and ventilatory flow (not shown); (AT = active tension).
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-1.20 - l - - - - , - - - - . - - - - - - . - - - , . . . - - - - - , -8.5 -9.5 -9.0 -8.0 -10.0
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(log mol/kg iv)
Fig. 2. Effect of epithelium removalon tracheal smooth muscle responseelicited by intravenouslyadministered endothelin-1. Left panel. Relaxation phase of the response. Tracheal smooth muscle relaxation was not altered significantly by removalof the epitheliumof the isolated trachealsegment (closedcircles) comparedwith animals with an intact epithelium (opencircles); AT = active tension. Rightpanel. Contractile phase of the response. Tracheal smooth muscle contractionwas attenuatedSUbstantially after removal of the epithelium of the isolatedtracheal segment (n = 6) (closed circles) compared with animals with an intact epithelium (n = 6) (opencircles).
Fig. 3. Maximalrelaxation andcontraction elicited by endothelin-1. Relaxation wasnotdifferent in anygroupfrom maximalrelaxation elicitedin controlanimals (closedsquares). Forcontraction, differencesare as shown (opensquaies). For BW 755C, n = 4. Ep- = epitheliumremoved(n = 6); Indo = indomethacin (n = 5); ECH = endothelin-C-terminal hexapeptide(n = 4); Prop = proprano101 (n = 6). (*, P < 0.02; **, P < 0.005; ***, P < 0.002 from control by analysis of variancefollowedby Dunnett's t-test).
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0.99 g/cm) for animals in which the epithelium had been removed versus 1.65 ± 0.28 g/cm (95070 CI, 0.94 to 2.37 g/cm) for animals having an intact epithelium (p < 0.01) (figures 2 and 3). In contrast, the initial relaxation response to ET-l was not affected by epithelial removal. Maximal relaxation after intravenously administered ET-1 was - 0.50 ± 0.25 g/cm (95070 CI, -1.14 to 0.13 g/cm) for animals in which the epithelium had been removed versus - 0.82 ± 0.22 g/cm (95070 CI, -1.38 to - 0.25 g/cm) for animals having an intact epithelium (p = NS) (figures 2 and 3). Pretreatment with either indomethacin or BW 755C attenuated the tracheal contractile response to ET-l. Tracheal contraction caused by 10-8 mol/kg ET-1 was 1.65 ± 0.28 g/cm in control animals versus 0.44 ± 0.30 g/cm (95070 CI, - 0.41 to 1.29g/cm, p = NS versus baseline tension) in animals pretreated with indomethacin (p < 0.001) (figure 4). Active tension elicited by 10-8 mol/kg ET-1 after BW 755C (-0.19 ± 0.25 g/cm; 95070 CI, - 0.52 to 0.44 g/cm) did not differ from zero baseline resting tension but did
differ substantially from active tension at the same dose ofET-1 in untreated control animals (P < 0.002) (figure 4). No animal pretreated with BW 755C had a contractile response > 0.3 g/cm. In contrast to the substantial diminution of contraction caused by inhibition of cyclooxygenase, the tracheal relaxation response was not altered significantly by indomethacin (figures 3 and 4). In animals receiving indomethacin, maximal relaxation after ET-l was -1.02 ± 0.13 g/cm (95070 CI, -1.39 to -0.65 g/cm; p = NS versus untreated control animals). Maximal tracheal relaxation after ET-l was -1.41 ± 0.16 g/cm in animals treated with BW 755C (95% CI, -1.92 to -0.89 g/cm, p = NSversus untreated control) (figures 3 and 4). Endothelin-I also caused substantial dose-related constriction of lower airways. The RL increased from 0.148 ± 0.030 em H10/L/s (baseline control) to 0.992 ± 0.274 em H 10/L/s after 10-8 mol/kg ET-l (P < 0.005) (figure 5). Both indomethacin and BW 755C blocked completely the increase in RLelicited by ET-1 (figure 5). The RLafter 10-8 mol/kg
WHITE, HATHAWAY, UMANS, TALLET, ABRAHAMS, AND LEFF
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Fig. 4. Effect of cyclooxygenase inhibition on tracheal smooth muscle contraction elicited by endothelin-1. Left panel. Relaxation phase of the response. Pretreatment with either in~ome~h.acin (~/o~ed squares) or B~ 755C (opensquares) did not alter significantly tracheal smooth muscle relaxation eh~lted Wlt~ Intra~enously adm,"lster~d endothelin-t compared with that in control animals (open circles); AT == active tension. R/~~t panel. Contra~lle phase of the response. Tracheal smooth muscle contractile response to intravenously administered endothehn-1 was attenuated substantially by prior administration of either indomethacin (n == 5) (closedsquares, Indo) or BW 755C (n == 4) (open squares) compared with that in control animals (n == 6) (open circles).
ET-l in animals pretreated with indomethacin was0.129 ± 0.039 em H 20/L/s (p == NS versus baseline); RL after 10-8 mol/kg ET-l in animals pretreated with BW 755Cwas0.247 ± 0.028em H 20/L/s (p == NS versusbaseline). At 10-8 mol/kg ET-l, the RL response differed significantly from untreated control animals for both indomethacin (p < 0.02) and BW 755C (p < 0.05). The relaxation response to intravenously administered ET-l was not related to changes in reflex sympathetic activation caused by ET-l. In six animals treated with propranolol, the initial relaxation phase of the tracheal response was not altered significantly from that of control animals not treated with propranolol (figures 3 and 6). Maximal relaxation after ET-l was -0.61 ± 0.15g/cm (950/0 CI, -1.01 to - 0.22 g/cm) for animals pretreated with propranolol versus - 0.82
± 0.22 g/cm for untreated control animals (p == NS) (figures 3 and 6). Intravenous administration of ECH did not elicit either a significant contraction of the tracheal segment (figures 3 and 7) or a substantial increase in RLin any animal. However, intravenouslyadministered ECH elicited an initial relaxation response of the tracheal segment that was similar to that caused by ET-l. The maximal relaxation response after ET-l was -0.68 ± 0.15 g/cm (950/0 CI, -1.17 to - 0.19 g/cm; p == NS) (figures 3 and 7). Discussion
The objective of this study was to elucidate the potential role of ET-I in the contraction of airway smooth muscle insitu. A new finding in this study is that tracheal smooth muscle contraction elicit0.40
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Fig. 5. Effect of endothelin-1 on lung resistance: attenuation by cyclooxygenase inhibition. Intravenouslyadministered endothelin-1 increased substantially lung resistance (RL) from baseline resistance (n == 6) (open circles, ContrOl). Pretreatment with either indomethacin (n == 5) (closedsquares) or BW755C (n == 4) (open squares)blocked completely the lower airways response to endothelin·1.
(log mol/kg i.v.)
Fig. 6. Effect of 13-adrenergic blockade on tracheal smooth muscle relaxation elicited by endothelin-1. Pretreatment with propranolol (n == 6) (closed circles) did not alter significantly the initial relaxation phase of the tracheal smooth muscle response elicited with intravenously administered endothelin-1 compared with that in control animals (n == 6) (open circles); AT == active tension.
ed by intravenously administered ET-I wasaugmented substantially by the presence of an intact airway epithelium. Removal of the epithelium attenuated significantly the contractile response of the isolated tracheal segment to ET-l (figure 2). Epithelial modulation was specific to the contractile response as epithelial removal did not alter the initial relaxation response elicited by ET-l (figures 2 and 3). Previous in vitrostudies have demonstrated that removal of the epithelium augments the tracheal smooth muscle response to contractile stimuli (1-3,20). Although this has been postulated to be the result of removal of an inhibitory factor secreted by the epithelium (2,21), such augmentation of contraction could also result from removal of an epithelial diffusion barrier (22, 23). Prior studies of excised airway smooth muscle and epithelium have been generated in a milieu substantially different from the living state. The in situ preparation used in this study obviates some situations encountered in excisedsmooth muscle preparations. Alterations resulting from mechanical manipulation of' and trauma to excisedsmooth muscle are reduced. Tissue hypoxia is avoided, as arterial circulation to the isolated airway is preserved. We have demonstrated previously (7, 8) that cellular inflammatory mediators such as Major Basic Protein of eosinophils elicit an epitheliumdependent contraction in both canine and guinea pig tracheal smooth muscle. In those studies, as wellas in this investigation, epithelial removal alone had no effect upon the contractile response to ACh. Our data provide additional evidence that the epithelium could secrete a contractile factor in response to stimulation by a local mediator; such a contractile factor in turn could modulate airway smooth muscle contraction. Another new finding in this study was the presence of a biphasic response to ET-l in tracheal smooth muscle (figures 1 and 2). The initial relaxation phase elicited by ET-I in the isolated tracheal segment was not altered either by removal of the epithelium (figures 3 and 4) or by blockade of cyclooxygenase (figures 3 and 4). Unlike the contractile phase, the relaxation phase of the tracheal response could be elicited by a small fragment of the C-terminal portion of the ET-l polypeptide (figures 3 and 7). Further, the relaxation phase was not abolished by pretreatment with propranolol. Tothe extent that ET-l elicits hypotension (10, 17), our data demonstrate that reflex sympathetic secretion of catecholamines is not
EPITHELIAL-DEPENDENT TRACHEAL SMOOTH MUSCLE CONTRACTION BY ENDOTHELlN·1
-1.20 +--------.---r---------.--..,-----, -8.5 -10.0 -9.5 -9.0 -8.0
(log mol/kg l.v.)
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Fig. 7. Effect of endothelin-C-terminal hexapeptide on tracheal smooth muscle contraction. Left panel. Relaxation phase of the response. Intravenously administered ECH (closed circles) elicited initial relaxation of the isolated tracheal segment that was similar to that caused by equivalent doses of intravenously administered endothelin-1 (open circles); AT = active tension. Right panel. Contractile phase of the response. Intravenously administered endothelin-G-terminal hexapeptide (n = 4) (closed circles) did not elicit contraction of the isolated tracheal segment compared with equivalent doses of intravenously administered endothelin-1 (n = 6) (open circles).
the cause of the initial relaxation response of airway smooth muscle and, since the to intravenously administered ET-l. Our contractile responses in the tracheal segdata suggest that ET-l has at least two ment and lower airwayswerequalitatively discrete effects on airway smooth mus- similar, indicates that ET-l may cause incle: (1) an early, direct effect causing creased lung resistance by a mechanism relaxation, which may be elicited by the that does not depend either upon the deC-terminal residues of the endothelin velopment of airway edema or changes protein, and (2) a later contractile effect, in vascular smooth muscle tone. which requires both synthesis of a prosIn our preparation, resting tension was taglandin mediator (seebelow) and more relatively high compared with active tenthan the terminal six C-residues of the sion. However, because of posterior atendothelin peptide. tachments, substantially greater resting Tracheal contraction and lung resis- tensions are required in vivo to produce tance changes in the lower airways in re- a resting tension comparable to optimal sponse to ET-l were blocked by inhibi- resting length (Lmax) in vitro (8, 15). tion of cyclooxygenase (figures 4 and 5). High resting tensions may be required in Recent studies have demonstrated that tracheal smooth muscle or peripheral papretreatment with indomethacin attenu- renchymal lung strips (24) when the ates the increase in inspiratory pressure elastance of the tissue is fairly high relaand respiratory compliance elicited by en- tive to the muscle content. In living sysdothelin in guinea pigs in vivo (14, 24). tems, other factors may account for the Our data demonstrate that inhibition of relatively low active tension compared cyclooxygenase by two different agents with resting tension, including the blocks both the contractile response of posterior attachments of the tracheal tracheal smooth muscle and changes in posterior membrane and elastance of the lung resistance in the lower airways elicit- split cartilage and other noncontractile ed by ET-l. However, cyclooxygenase in- elements in the isolated tracheal segment. hibition sufficient to block completely We have previously demonstrated this the contractile response did not inhibit phenomenon in both guinea pigs and the early relaxation response of tracheal other species (8, 15, 25). In this in situ smooth muscle to ET-l. These studies did preparation, resting tension is stable to not identify the specific prostaglandin or 0.02 g/cm and the sensitivity of measurethromboxane responsible for this effect. ment of active tension does not vary over The tracheal relaxation response to ET-l extended periods of time. This permits was not altered by ~-adrenergic blockade detection of a small incremental signal (figures 3 and 6). of ~ 0.04g/cm, demonstrating both specIn these studies, airway contraction ificity and precision in this preparation. was assessed both isometrically in an isoThe precise role of ET-l in the patholated central airway and by determina- genesis of airway hyperreactivity remains tion of lung resistance in intact lower air- to be established. Our data and those ways. Unlike measurements of lung re- from prior investigations (13, 14,26) indisistance, isometric forcegeneration is not cate that ET-l is one of the most potent geometry-dependent (15). Isometric force airway contractile agents yet identified. generation caused by ET-l therefore Inflammation is a hallmark of asthma reflects directly augmented contractility (5), and many inflammatory mediators
increase bronchovascular permeability sufficiently to allow leakage of macromolecules from the vascular space into the adjacent tissues and even into the airway lumen (6, 27, 28). Because it is a relativelysmall molecule (molecular weight, 1"\.12,500 daltons), ET-l could be extravasated under these conditions. Weconclude that ET-l is a potent bronchoreactive substance that has both direct and epithelium-mediated bronchoconstrictive effects on airway smooth muscle. The contractile effects elicitedby ET-l are modulated through de novo synthesis of eicosanoids in the airway and the presence of an intact epithelium. Our data indicate that an intact molecule may be essential for the contractile response elicited by ET-l in that the C-terminal fragment is not sufficient to cause contraction. In contrast, transient relaxing effects of ET-l may be mediated by the first six amino acids of the peptide and are altered neither by ~adrenergic blockade nor by either cyclooxygenase or 5-lipoxygenase blockade. References 1. Butler GB, Adler KB, Evans IN, Morgan OW, Szarek JL. Modulation of rabbit airway smooth muscle responsiveness by respiratory epithelium. Am Rev Respir Dis 1987; 135:1099-1104. 2. Flavahan NA, Aarhuus LL, Rimele TJ, Vanhoutte PM. Respiratory epithelium inhibits bronchial smooth muscle tone. J Appl Physiol 1985; 58:834-8. 3. Laitenen LA, Heino M, Laitenen A, KavaT, Haahtela T. Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 1985; 131:599-606. 4. Empey OW, Laitenen LA, Jacobs L. Mechanisms of bronchial hyperreactivity in normal subjects after upper respiratory tract infection. Am Rev Respir Dis 1976; 133:131-7. 5. Austen KF, Orange RP. Bronchial asthma: the possible role of the chemical mediators of immediate hypersensitivity in the pathogenesis of subacute chronic disease. Am Rev Respir Dis 1975; 112: 423-36. 6. Fick RB, Metzger WJ, Richerson HB, et 0/. Increased bronchovascular permeability after allergen exposure in sensitive asthmatics. J Appl Physiol 1987; 63:1147-55. 7. Brofman JD, White SR, Blake JS, Munoz NM, Gleich GJ, Leff AR. Epithelial augmentation of trachealis contraction caused by MBP of eosinophils, J Appl Physiol 1989; 66:1867-73. 8. White SR, Ohno S, Munoz NM, et 0/. Epithelium-dependent contraction of airway smooth muscle caused by eosinophil MBP. Am J Physiol 1990; 259:L294-303. 9. Yanagisawa M, Kurihara H, Kimura S, et 0/. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411-5. 10. Minkes RK, MacMillan LA, BeHan JA, Kerstein MD, McNamara DB, Kadowitz PJ. Analysis of regional responses to endothelin in hindquarters vascular bed of cats. Am J Physiol 1989; 256: H598-602. 11. Inoue A, Yanagisawa M, Kimura S, et 0/. The human endothelin family: three structurally and
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