Direct Effects on Airway Smooth Muscle Contractile Response Caused by Endothelin-1 in Guinea Pig Trachealis1 •2
STEVEN R. WHITE, DARREN P. HATHAWAY, JASON G. UMANS, and ALAN R. LEFF
Endothelin-I (ET-I), produced by cultured endothelial cells,is a potent constrictor of vascular smooth muscle (I). Endothelin also causes contraction in lower airways (2, 3). We recently demonstrated that intravenously administered ET-I is a potent constrictor of both central and lower airways in an in vivo preparation in guinea pigs (4). Intravenously administered ET-I elicited a biphasic response in tracheal smooth muscle (TSM) in vivoan initial relaxation response followed by sustained airway contraction that was independent of airway edemagenesis. The initial relaxation response was altered neither by removal of the epithelium nor by inhibition of cyclooxygenase; in contrast, subsequent TSM contraction was blocked substantially by either intervention (4). Although these data suggested an augmenting effect of the epithelium on contraction caused by ET-I, the effects of intravenous administration of ET-I in those studies was uncertain, and it is possible that other circulating mediators may have been activated by this "indirect" method of delivery. The objective of this study was to elucidate the direct effects of ET-I on TSM in situ using a preparation that preservesnormal anatomic relationships between epithelium and smooth muscle. We found that (1) topical application of ET-I elicits the same prolonged contraction of tracheal smooth muscle as that elicited by intravenously administered ET-I, (2) topically applied ET-I, unlike intravenously administered endothelin, does not cause an initial relaxation in tracheal smooth muscle tension, and (3) the epithelium does not augment the contraction elicited by topically applied ET-l. We found that direct, topical application of ET-I bypasses the modulating influences caused by intravenous administration and that intravascular delivery of ET-I is essential for the relaxation phase of the TSM response to ET-l. Eighteen male Hartley guinea pigs weighing rv 900 g were anesthetized intraperitoneally with pentobarbital (50 mg/kg) and intubated through a low cervical tracheostomy. Animals were ventilated mechanically with a volume ventilator (Model 681; Harvard Apparatus Co., South Natick, MA) using pure oxygen so that Pao, was> 300 mm Hg. Tidal volume was set at 6 ml/kg and respiratory rate was set to 60 breaths/min to maintain Paco, between 35 and 45 mm Hg and arterial blood pH between 7.35 and 7.45; we have previously determined these settings to be optimal in this preparation (5). Additional anesthesia was administered when required.
SUMMARY Endothelln·l (ET-l), a peptide derived from vascular endothelial cells, causes tracheal smooth muscle (TSM)relaxation followed by sustained contraction when administered Intravenously In guinea pigs by a mechanism that depends upon an Intact airway epithelium. To elucidate the potential role of the epithelium In modUlating the response to ET·l and the potential effects of local release of ET-l, we studied the effects of topical application of ET·l to a segment of TSM In situ. An epithelium smooth muscle preparation that does not disrupt normal anatomic relationships was used; smooth muscle contraction was measured Isometrically In vivo. Application of 10-10 mol/cm' ET-l to the epithelial surface In six animals caused 2.27 ± 0.45 g/cm active tension (AT) of the TSM segment after 30 min (p < 0.05 versus baseline); an Initial relaXBtlon response was not observed. Endothelln·l was dose·dependent and was 1,000 times more potent than acetylcholine in causing AT In TSM. Pretreatment with ET-l did not alter the subsequent response to acetylcholine. Contraction elicited by topical application of ET-l persisted> 3 h.ln five animals in which the eplthe· lIum was removed, 10-10 mol/cm' ET-l caused 4.45 ± 0.92 g/cm AT after 30 min (p < 0.05 versus Intact epithelium). These data suggest that topical application of ET-l elicits responses that are different from those elicited In the same preparation after Intravenously administered ET-l: (1) TSM contraction that Is nof preceded by a transient relaXBtlon phase, and (2) contraction that Is not reduced after removal of the epithelium. AM REV RESPIR DIS 1992; 145:491-493
A catheter was placed in the external jugular vein for intravenous administration of agonists. Bilateral cervical vagotomy was performed at the level of the third tracheal cartilage to eliminate potential confounding influences from parasympathetic and nonadrenergic, noncholinergic efferent transmission. A l.o-cm segment of midcervical trachea wasprepared for measurement of smooth muscle tension (5). A ventral midline incision was made in the trachea proximal to the endotracheal tube; transverse incisions then were made through the cartilage, taking care not to disturb either the local circulation or innervation to the segment within the surrounding dorsal tracheal sheath. The tracheal segment was attached by parallel sutures passed through the cartilage near the junction of the posterior membrane to a parallel bar on one side and a Model FT.03 force-displacement transducer (Grass Instruments, Quincy, MA) on the other. Contractile force was measured isometrically in situ after first setting the resting tension of the tracheal segment to 10g/cm; this tension corresponds to Lmax, the resting length at which maximal, reproducible contraction is obtained to intravenously administered acetylcholine in this preparation (5). Active force of contraction (total force minus initial resting force) was normalized by dividing by the longitudinal length of the segment and expressed as active tension (AT) in grams force per centimeter longitudinal segment length (g/cm) (4, 5). Resting tension was stable throughout each experiment, and AT could be determined to < 0.04 g/cm. All physiologic measurements were recorded continuously on a Gould 3800 chart recorder (Gould Instruments, Cleveland, OH). Doses of agonists were expressed as moles of agonist/body weight of the animal (mol/kg) when administered intravenously. Doses of agonists were expressed as moles of agonist per
surface area of the tracheal segment (mol/em') when administered topically (5). At the completion of each experiment, animals were killed with an overdose of pentobarbital followed by a saturated KCI solution administered intravenously. Histologic confirmation of epithelial integrity was obtained in all animals in which the epithelium was removed and in random samples of animals with an intact epithelium. Tracheal segments were excised upon completion of each experiment and fixed in 10070 formalin. Segments then were embedded in paraffin; sections 7 um thick were cut with a microtome and stained with hematoxylin-eosin (5). In four animals prepared as above, dose-response curves were generated in each according to the following protocol. (1) An intravenous dose-response curve was generated with 3 x 10-10 to 3 X 10-' mol/kg acetylcholine (ACh). (2) A topical doseresponse curve was generated with 6 x 10-.0 to 6 X 1(,' mol/em- ACh 20 min after completing the ET-l dose-response curve. (3) A topical dose-response
(Received in original form April 1. 1991 and in revised form August 23. 1991) I From the Section of Pulmonary and Critical Care Medicine and the Section of Nephrology, Department of Medicine and the Committee on Clinical Pharmacology, Division of the Biological Sciences, The University of Chicago, Chicago, Illinois. , Correspondence and requests for reprints should be addressed to Steven R. White, M.D., Section of Pulmonary and Critical Care Medicine, The University of Chicago, 5841 S. Maryland Ave., Box 98, Chicago, IL 60637.
491
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1,000 times more potent than an equivalent dose of topical acetylcholine before (opencircles) or after (closedcircles) endothelin-1 (p < 0.05 for 3 x 10-10 mol/em' endothelin-1 versus 6 x 10-10 mol/em' acetylcholine). Endothelin-1 did not alter the response of the tracheal segment to acetylcholine.
confirmed the complete removal of the epithelium without damage to the underlying smooth muscle in each animal in which this was done. In animals with an intact epithelium, histologic examination of randomly selected tracheal segments demonstrated that the epithelium and underlying smooth muscle remained completely intact in every segment that was examined. Endothelin-I was an extremely potent contractile agonist when applied directly to the epithelial surface of TSM. Compared with ACh, ET-1 was> 1,ODO-fold more potent in causing comparable AT (figure 2). In four animals, ET-1 (3 x 10-10 mol/ern 2) caused AT of 3.27 ± 1.06glcm versus 0.30 ± 0.08 g/cm for 6 x 10-10 mol/ern" ACh (p < 0.05). Topical ACh dose-response curves elicited after contraction with ET-1 were unchanged from pretreatment control values despite the additional AT elicited by ET-1 (figure 2). Differences in response were not significant at any point on the dose-response curve. Likewise, intravenous ACh dose-response curves generated from baseline tension were similar before and after ET-I, despite the additional AT elicited by ET-1 (figure 1). Intravenous administration of ACh (3 x 10-7 mol/kg) caused 0.47 ± 0.10 g/cm AT before and 0.48 ± 0.09 g/cm AT after topically applied ET-I. Endothelin-l applied directly to the tracheal epithelial surface caused the prolonged contraction of TSM. In three animals, contraction elicited by ET-1 (3 x 10-10 mol/ern') was 2.40 ± 0.51 g/cm after 40 min (p < 0.05 versus baseline tension) and still was 1.26 ± 0.55 g/cm after 3 h (p < 0.05 versus baseline tension). Epithelium removal increased the contractile response to ET-I (3 x 1O-1l mol/em") (figure 3). In six epithelium-intact animals, the maximal AT elicited by ET-1 was 2.23 ± 0.37 g/cm. In five other animals in which the epithelium was removed, the same dose of ET-1 caused 4.45 ± 0.92 g/cm maximal AT (p < 0.05). Epithelium removal did not cause damage
Time (min)
Fig. 3. Effect of removal of the epithelium on tracheal contraction elicited by topical endothelin-1. Tracheal smooth muscle contraction was greater in five animals in which the epithelium of the tracheal segment was removed (closed circles) versus six other animals with an intact epithelium (opencircles)(p < 0.05for maximal active tension in both groups).
to the underlying segment of trachealis. Removal of the epithelium followed by topical application of ET-1 did not significantly alter the subsequent response of the tracheal segment to intravenously administered ACh (figure 4). In animals with an intact epithelium, intravenously administered ACh (3 x 10-7 mollkg) caused 0.67 ± 0.11 g/cm AT before and 0.69 ± 0.16 g/cm AT after topically applied ET-l. In animals in which the epithelium was removed, the response to intravenously administered ACh was slightly greater. However, neither the dose required to elicit comparable responses nor the greatest differences were significant at any point. The response to intravenously administered ACh (3 x 10-7 mol/kg) was not impaired; AT was 0.66 ± 0.10g/cm before and 0.90 ± 0.18 g/cm after ET-l (p = 0.31) (figure 4).
,.. ,.. ,.. ,.. This study was undertaken to assess the direct effects of ET-1in a viable in situ preparation when applied directly to the target organ. These data demonstrate that (1) topically applied ET-1 elicits the same prolonged contraction of tracheal smooth muscle as that elicited by intravenously administered ET-1, (2) topical application of ET-I, unlike intravenous administration, does not cause an initial relaxation in tracheal smooth muscle tension, and (3) the epithelium does not modulate actively the contraction elicited by topical ET-l. These data demonstrate that, independent of the diffusion barrier of the overlying epithelium, endothelin acts directly on tracheal smooth muscle in the living state. The in situ preparation used in this study obviates some situations encountered in excised smooth muscle preparations. Alterations resulting from mechanical manipulation of and trauma to excised smooth muscle are reduced. Tissue hypoxia is avoided, as arterial circulation to the isolated airway is preserved. Finally, the in situ preparation permits direct comparison of two different modes of delivery of agonist to the smooth muscle-application of mediator to the intact mucosal surface of the epithelium versus systemic (in-
493
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Fig. 4. Preservation of tracheal smooth muscle responsiveness after epithelium removal and administration of endothelin-1 (ET-1). Left panel. Effect of ET-1 in control animals with an intact epithelium. In six guinea pigs in which the epithelium was intact, 3 x 10-11 mol/cm' topical ET-1 did not alter the subsequent response to intravenously administered acetylcholine (closed circles) compared with baseline (open circles). Right panel. Effect of ET-1 after epithelium removal. In five guinea pigs, removal of the epithelium followed by 3 x 10'" mollcm'topical ET-1 did not alter the subsequent response to intravenously administered acetylcholine (closed circles) compared with baseline (open circles). The increase in responsiveness after epithelium removal was not significant at the point of greatest difference.
direct) administration-which cannot be performed in vitro. Direct application of ET-l avoided potential systemic effects that may result from intravenous administration of ET-l (4), and preservesregional anatomic relationships that could not be accomplished with standard in vitro techniques. This method thus may (1) stimulate the effects of local release of ET-l in guinea pig airway in situ, (2) permit assessment of both epithelial influences and phasic responses, and (3) assess the response of airway smooth muscle after either topical application or intravenous administration of the same mediator. Endothelin-l administered directly was > 1,000times more potent than ACh in contracting TSM (figure 2). Contraction elicited by topically applied ET-l in TSM was prolonged; maximal AT > 500/0 persisted 3 h after topical application. In contrast to the response obtained previouslywith intravenously administered ET-l. (4), no initial relaxation phase was observed after topical application. This suggests that intravascular delivery is essential for development of the initial relaxationresponse. In contrast to the substantial augmentation of muscarinic responsiveness elicited by epithelial activation with other inflammatory peptides such as eosinophil major basic protein (5), there was no effect on muscarinic responsiveness of TSM contrac-
tion after exposure to topically applied ET-l (figure 2). We have demonstrated previously that intravenous administration of ET-l after removal of the epithelium in the same preparation elicits less contraction than in animals in which the epithelium remains intact (4). In those studies, it was suggested that this augmentation resulted from induction of synthesis of bronchoactive eicosanoids by the epithelium/lamina propria. We performed the experiments involving epithelial removal to test the hypothesis that the epithelium would modulate the contraction of topically applied ET-l in the same manner as it modulated the contraction of intravenously administered ET-l. This clearly was not the case, as the change in reactivity wasopposite to that elicited with intravenously administered ET-l. These data demonstrate that ET-l elicits contraction of airway smooth muscle in situ both after direct application and after intravenous (indirect) injection (4). Previous in vitro studies have demonstrated that removal of the epithelium augments the tracheal smooth muscle response to contractile stimuli (6,7). Although this has been postulated to be the result of removal of an inhibitory factor secreted by the epithelium (6, 8), such augmentation of contraction could also result from removal of an epithelial diffusion barrier (7).
Despite the additional active tone elicited by ET-l, responsiveness of the tracheal segment was not impaired by removal of the epithelium (figures 2 and 4). These in vivo experiments with ET-l differ substantially from epithelial smooth muscle responses demonstrated for other bronchoactive proteins (5), and they suggest that although the epithelium may modulate the indirect effects of ET-l, there are no substantial epithelial influences upon the direct response to ET-l. We conclude that ET-l is one of the most potent airway contractile agents yet identified. We find that epithelial modulation of the effects of ET-l upon TSM depends upon the route of administration. Our data demonstrate increased responsiveness of TSM to ET-l administered topically after epithelium removal in contrast to the response obtained by intravenous administration of ET-l (4). This effect is attributed to impaired diffusion of ET-l in the epithelium-intact state. However, the absence of both the relaxation and epithelialaugmenting responsesobservedwith intravenous administration suggests that intravenouslyadministered ET-l may initiate incidental effects through regional or systemic release of mediators that are not activated by the direct effects of ET-l on airway smooth muscle. References \. Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced byvascularendothelial cells.Nature 1988; 332:411-5. 2. Macquin-Mavier I, Levame M, Istin N, Harf A. Mechanisms of endothelin-mediated bronchoconstriction in the guinea pig. J Pharmacol Exp Ther 1989; 250:740-5. 3. Maggi CA, Patacchini R, Giuliani S, Meli A. Potent contractile effect of endothelin in isolated guinea-pig airways. Eur J Pharmacol 1989; 160: 179-82. 4. White SR, Hathaway DP, Umans JG, Tallet J, Abrahams C, Leff AR. Epithelial modulation of airwaysmooth muscle response to endothelin-l. Am Rev Respir Dis 1991; 144:373-8. 5. White SR, Ohno S, Munoz NM, et at. Epithelium-dependent contraction of airway smooth muscle caused by eosinophil MBP. Am J Physiol 1990; 259:L294-L303. 6. Flavahan NA, Aarhuus LL, Rimele TJ, Vanhoutte PM. Respiratory epithelium inhibits bronchial smooth muscle tone. J Appl Physiol 1985; 58:834-8. 7. Holroyde Me. The influence of epithelium on the responsiveness of guinea-pig isolated trachea. Br J Pharmacol 1986; 87:501-7. 8. Barnes PJ, Cuss FM, Palmer JB. The effect of airway epithelium on smooth muscle contractility in bovine trachea. Br J Pharmacol1985; 86:685-91.
STEVEN R. WHITE, DARREN P. HATHAWAY, JASON G. UMANS, and ALAN R. LEFF
Endothelin-I (ET-I), produced by cultured endothelial cells,is a potent constrictor of vascular smooth muscle (I). Endothelin also causes contraction in lower airways (2, 3). We recently demonstrated that intravenously administered ET-I is a potent constrictor of both central and lower airways in an in vivo preparation in guinea pigs (4). Intravenously administered ET-I elicited a biphasic response in tracheal smooth muscle (TSM) in vivoan initial relaxation response followed by sustained airway contraction that was independent of airway edemagenesis. The initial relaxation response was altered neither by removal of the epithelium nor by inhibition of cyclooxygenase; in contrast, subsequent TSM contraction was blocked substantially by either intervention (4). Although these data suggested an augmenting effect of the epithelium on contraction caused by ET-I, the effects of intravenous administration of ET-I in those studies was uncertain, and it is possible that other circulating mediators may have been activated by this "indirect" method of delivery. The objective of this study was to elucidate the direct effects of ET-I on TSM in situ using a preparation that preservesnormal anatomic relationships between epithelium and smooth muscle. We found that (1) topical application of ET-I elicits the same prolonged contraction of tracheal smooth muscle as that elicited by intravenously administered ET-I, (2) topically applied ET-I, unlike intravenously administered endothelin, does not cause an initial relaxation in tracheal smooth muscle tension, and (3) the epithelium does not augment the contraction elicited by topically applied ET-l. We found that direct, topical application of ET-I bypasses the modulating influences caused by intravenous administration and that intravascular delivery of ET-I is essential for the relaxation phase of the TSM response to ET-l. Eighteen male Hartley guinea pigs weighing rv 900 g were anesthetized intraperitoneally with pentobarbital (50 mg/kg) and intubated through a low cervical tracheostomy. Animals were ventilated mechanically with a volume ventilator (Model 681; Harvard Apparatus Co., South Natick, MA) using pure oxygen so that Pao, was> 300 mm Hg. Tidal volume was set at 6 ml/kg and respiratory rate was set to 60 breaths/min to maintain Paco, between 35 and 45 mm Hg and arterial blood pH between 7.35 and 7.45; we have previously determined these settings to be optimal in this preparation (5). Additional anesthesia was administered when required.
SUMMARY Endothelln·l (ET-l), a peptide derived from vascular endothelial cells, causes tracheal smooth muscle (TSM)relaxation followed by sustained contraction when administered Intravenously In guinea pigs by a mechanism that depends upon an Intact airway epithelium. To elucidate the potential role of the epithelium In modUlating the response to ET·l and the potential effects of local release of ET-l, we studied the effects of topical application of ET·l to a segment of TSM In situ. An epithelium smooth muscle preparation that does not disrupt normal anatomic relationships was used; smooth muscle contraction was measured Isometrically In vivo. Application of 10-10 mol/cm' ET-l to the epithelial surface In six animals caused 2.27 ± 0.45 g/cm active tension (AT) of the TSM segment after 30 min (p < 0.05 versus baseline); an Initial relaXBtlon response was not observed. Endothelln·l was dose·dependent and was 1,000 times more potent than acetylcholine in causing AT In TSM. Pretreatment with ET-l did not alter the subsequent response to acetylcholine. Contraction elicited by topical application of ET-l persisted> 3 h.ln five animals in which the eplthe· lIum was removed, 10-10 mol/cm' ET-l caused 4.45 ± 0.92 g/cm AT after 30 min (p < 0.05 versus Intact epithelium). These data suggest that topical application of ET-l elicits responses that are different from those elicited In the same preparation after Intravenously administered ET-l: (1) TSM contraction that Is nof preceded by a transient relaXBtlon phase, and (2) contraction that Is not reduced after removal of the epithelium. AM REV RESPIR DIS 1992; 145:491-493
A catheter was placed in the external jugular vein for intravenous administration of agonists. Bilateral cervical vagotomy was performed at the level of the third tracheal cartilage to eliminate potential confounding influences from parasympathetic and nonadrenergic, noncholinergic efferent transmission. A l.o-cm segment of midcervical trachea wasprepared for measurement of smooth muscle tension (5). A ventral midline incision was made in the trachea proximal to the endotracheal tube; transverse incisions then were made through the cartilage, taking care not to disturb either the local circulation or innervation to the segment within the surrounding dorsal tracheal sheath. The tracheal segment was attached by parallel sutures passed through the cartilage near the junction of the posterior membrane to a parallel bar on one side and a Model FT.03 force-displacement transducer (Grass Instruments, Quincy, MA) on the other. Contractile force was measured isometrically in situ after first setting the resting tension of the tracheal segment to 10g/cm; this tension corresponds to Lmax, the resting length at which maximal, reproducible contraction is obtained to intravenously administered acetylcholine in this preparation (5). Active force of contraction (total force minus initial resting force) was normalized by dividing by the longitudinal length of the segment and expressed as active tension (AT) in grams force per centimeter longitudinal segment length (g/cm) (4, 5). Resting tension was stable throughout each experiment, and AT could be determined to < 0.04 g/cm. All physiologic measurements were recorded continuously on a Gould 3800 chart recorder (Gould Instruments, Cleveland, OH). Doses of agonists were expressed as moles of agonist/body weight of the animal (mol/kg) when administered intravenously. Doses of agonists were expressed as moles of agonist per
surface area of the tracheal segment (mol/em') when administered topically (5). At the completion of each experiment, animals were killed with an overdose of pentobarbital followed by a saturated KCI solution administered intravenously. Histologic confirmation of epithelial integrity was obtained in all animals in which the epithelium was removed and in random samples of animals with an intact epithelium. Tracheal segments were excised upon completion of each experiment and fixed in 10070 formalin. Segments then were embedded in paraffin; sections 7 um thick were cut with a microtome and stained with hematoxylin-eosin (5). In four animals prepared as above, dose-response curves were generated in each according to the following protocol. (1) An intravenous dose-response curve was generated with 3 x 10-10 to 3 X 10-' mol/kg acetylcholine (ACh). (2) A topical doseresponse curve was generated with 6 x 10-.0 to 6 X 1(,' mol/em- ACh 20 min after completing the ET-l dose-response curve. (3) A topical dose-response
(Received in original form April 1. 1991 and in revised form August 23. 1991) I From the Section of Pulmonary and Critical Care Medicine and the Section of Nephrology, Department of Medicine and the Committee on Clinical Pharmacology, Division of the Biological Sciences, The University of Chicago, Chicago, Illinois. , Correspondence and requests for reprints should be addressed to Steven R. White, M.D., Section of Pulmonary and Critical Care Medicine, The University of Chicago, 5841 S. Maryland Ave., Box 98, Chicago, IL 60637.
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1,000 times more potent than an equivalent dose of topical acetylcholine before (opencircles) or after (closedcircles) endothelin-1 (p < 0.05 for 3 x 10-10 mol/em' endothelin-1 versus 6 x 10-10 mol/em' acetylcholine). Endothelin-1 did not alter the response of the tracheal segment to acetylcholine.
confirmed the complete removal of the epithelium without damage to the underlying smooth muscle in each animal in which this was done. In animals with an intact epithelium, histologic examination of randomly selected tracheal segments demonstrated that the epithelium and underlying smooth muscle remained completely intact in every segment that was examined. Endothelin-I was an extremely potent contractile agonist when applied directly to the epithelial surface of TSM. Compared with ACh, ET-1 was> 1,ODO-fold more potent in causing comparable AT (figure 2). In four animals, ET-1 (3 x 10-10 mol/ern 2) caused AT of 3.27 ± 1.06glcm versus 0.30 ± 0.08 g/cm for 6 x 10-10 mol/ern" ACh (p < 0.05). Topical ACh dose-response curves elicited after contraction with ET-1 were unchanged from pretreatment control values despite the additional AT elicited by ET-1 (figure 2). Differences in response were not significant at any point on the dose-response curve. Likewise, intravenous ACh dose-response curves generated from baseline tension were similar before and after ET-I, despite the additional AT elicited by ET-1 (figure 1). Intravenous administration of ACh (3 x 10-7 mol/kg) caused 0.47 ± 0.10 g/cm AT before and 0.48 ± 0.09 g/cm AT after topically applied ET-I. Endothelin-l applied directly to the tracheal epithelial surface caused the prolonged contraction of TSM. In three animals, contraction elicited by ET-1 (3 x 10-10 mol/ern') was 2.40 ± 0.51 g/cm after 40 min (p < 0.05 versus baseline tension) and still was 1.26 ± 0.55 g/cm after 3 h (p < 0.05 versus baseline tension). Epithelium removal increased the contractile response to ET-I (3 x 1O-1l mol/em") (figure 3). In six epithelium-intact animals, the maximal AT elicited by ET-1 was 2.23 ± 0.37 g/cm. In five other animals in which the epithelium was removed, the same dose of ET-1 caused 4.45 ± 0.92 g/cm maximal AT (p < 0.05). Epithelium removal did not cause damage
Time (min)
Fig. 3. Effect of removal of the epithelium on tracheal contraction elicited by topical endothelin-1. Tracheal smooth muscle contraction was greater in five animals in which the epithelium of the tracheal segment was removed (closed circles) versus six other animals with an intact epithelium (opencircles)(p < 0.05for maximal active tension in both groups).
to the underlying segment of trachealis. Removal of the epithelium followed by topical application of ET-1 did not significantly alter the subsequent response of the tracheal segment to intravenously administered ACh (figure 4). In animals with an intact epithelium, intravenously administered ACh (3 x 10-7 mollkg) caused 0.67 ± 0.11 g/cm AT before and 0.69 ± 0.16 g/cm AT after topically applied ET-l. In animals in which the epithelium was removed, the response to intravenously administered ACh was slightly greater. However, neither the dose required to elicit comparable responses nor the greatest differences were significant at any point. The response to intravenously administered ACh (3 x 10-7 mol/kg) was not impaired; AT was 0.66 ± 0.10g/cm before and 0.90 ± 0.18 g/cm after ET-l (p = 0.31) (figure 4).
,.. ,.. ,.. ,.. This study was undertaken to assess the direct effects of ET-1in a viable in situ preparation when applied directly to the target organ. These data demonstrate that (1) topically applied ET-1 elicits the same prolonged contraction of tracheal smooth muscle as that elicited by intravenously administered ET-1, (2) topical application of ET-I, unlike intravenous administration, does not cause an initial relaxation in tracheal smooth muscle tension, and (3) the epithelium does not modulate actively the contraction elicited by topical ET-l. These data demonstrate that, independent of the diffusion barrier of the overlying epithelium, endothelin acts directly on tracheal smooth muscle in the living state. The in situ preparation used in this study obviates some situations encountered in excised smooth muscle preparations. Alterations resulting from mechanical manipulation of and trauma to excised smooth muscle are reduced. Tissue hypoxia is avoided, as arterial circulation to the isolated airway is preserved. Finally, the in situ preparation permits direct comparison of two different modes of delivery of agonist to the smooth muscle-application of mediator to the intact mucosal surface of the epithelium versus systemic (in-
493
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Fig. 4. Preservation of tracheal smooth muscle responsiveness after epithelium removal and administration of endothelin-1 (ET-1). Left panel. Effect of ET-1 in control animals with an intact epithelium. In six guinea pigs in which the epithelium was intact, 3 x 10-11 mol/cm' topical ET-1 did not alter the subsequent response to intravenously administered acetylcholine (closed circles) compared with baseline (open circles). Right panel. Effect of ET-1 after epithelium removal. In five guinea pigs, removal of the epithelium followed by 3 x 10'" mollcm'topical ET-1 did not alter the subsequent response to intravenously administered acetylcholine (closed circles) compared with baseline (open circles). The increase in responsiveness after epithelium removal was not significant at the point of greatest difference.
direct) administration-which cannot be performed in vitro. Direct application of ET-l avoided potential systemic effects that may result from intravenous administration of ET-l (4), and preservesregional anatomic relationships that could not be accomplished with standard in vitro techniques. This method thus may (1) stimulate the effects of local release of ET-l in guinea pig airway in situ, (2) permit assessment of both epithelial influences and phasic responses, and (3) assess the response of airway smooth muscle after either topical application or intravenous administration of the same mediator. Endothelin-l administered directly was > 1,000times more potent than ACh in contracting TSM (figure 2). Contraction elicited by topically applied ET-l in TSM was prolonged; maximal AT > 500/0 persisted 3 h after topical application. In contrast to the response obtained previouslywith intravenously administered ET-l. (4), no initial relaxation phase was observed after topical application. This suggests that intravascular delivery is essential for development of the initial relaxationresponse. In contrast to the substantial augmentation of muscarinic responsiveness elicited by epithelial activation with other inflammatory peptides such as eosinophil major basic protein (5), there was no effect on muscarinic responsiveness of TSM contrac-
tion after exposure to topically applied ET-l (figure 2). We have demonstrated previously that intravenous administration of ET-l after removal of the epithelium in the same preparation elicits less contraction than in animals in which the epithelium remains intact (4). In those studies, it was suggested that this augmentation resulted from induction of synthesis of bronchoactive eicosanoids by the epithelium/lamina propria. We performed the experiments involving epithelial removal to test the hypothesis that the epithelium would modulate the contraction of topically applied ET-l in the same manner as it modulated the contraction of intravenously administered ET-l. This clearly was not the case, as the change in reactivity wasopposite to that elicited with intravenously administered ET-l. These data demonstrate that ET-l elicits contraction of airway smooth muscle in situ both after direct application and after intravenous (indirect) injection (4). Previous in vitro studies have demonstrated that removal of the epithelium augments the tracheal smooth muscle response to contractile stimuli (6,7). Although this has been postulated to be the result of removal of an inhibitory factor secreted by the epithelium (6, 8), such augmentation of contraction could also result from removal of an epithelial diffusion barrier (7).
Despite the additional active tone elicited by ET-l, responsiveness of the tracheal segment was not impaired by removal of the epithelium (figures 2 and 4). These in vivo experiments with ET-l differ substantially from epithelial smooth muscle responses demonstrated for other bronchoactive proteins (5), and they suggest that although the epithelium may modulate the indirect effects of ET-l, there are no substantial epithelial influences upon the direct response to ET-l. We conclude that ET-l is one of the most potent airway contractile agents yet identified. We find that epithelial modulation of the effects of ET-l upon TSM depends upon the route of administration. Our data demonstrate increased responsiveness of TSM to ET-l administered topically after epithelium removal in contrast to the response obtained by intravenous administration of ET-l (4). This effect is attributed to impaired diffusion of ET-l in the epithelium-intact state. However, the absence of both the relaxation and epithelialaugmenting responsesobservedwith intravenous administration suggests that intravenouslyadministered ET-l may initiate incidental effects through regional or systemic release of mediators that are not activated by the direct effects of ET-l on airway smooth muscle. References \. Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced byvascularendothelial cells.Nature 1988; 332:411-5. 2. Macquin-Mavier I, Levame M, Istin N, Harf A. Mechanisms of endothelin-mediated bronchoconstriction in the guinea pig. J Pharmacol Exp Ther 1989; 250:740-5. 3. Maggi CA, Patacchini R, Giuliani S, Meli A. Potent contractile effect of endothelin in isolated guinea-pig airways. Eur J Pharmacol 1989; 160: 179-82. 4. White SR, Hathaway DP, Umans JG, Tallet J, Abrahams C, Leff AR. Epithelial modulation of airwaysmooth muscle response to endothelin-l. Am Rev Respir Dis 1991; 144:373-8. 5. White SR, Ohno S, Munoz NM, et at. Epithelium-dependent contraction of airway smooth muscle caused by eosinophil MBP. Am J Physiol 1990; 259:L294-L303. 6. Flavahan NA, Aarhuus LL, Rimele TJ, Vanhoutte PM. Respiratory epithelium inhibits bronchial smooth muscle tone. J Appl Physiol 1985; 58:834-8. 7. Holroyde Me. The influence of epithelium on the responsiveness of guinea-pig isolated trachea. Br J Pharmacol 1986; 87:501-7. 8. Barnes PJ, Cuss FM, Palmer JB. The effect of airway epithelium on smooth muscle contractility in bovine trachea. Br J Pharmacol1985; 86:685-91.
