Regulatory Peptides, 39 (1992) 137-145 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-0115/92/$05.00
137
REGPEP 01179
Neutral endopeptidase modulates endothelin- 1-induced airway smooth muscle contraction in guinea-pig trachea Giuseppe U. Di Maria, Michio Katayama, D. Benjamin Borson and Jay A. Nadel Cardiovascular Research Institute and Departments of Medicine and Physiology, University of California, San Francisco, CA (USA) (Received 12 November 1991; revised version received 17 February 1992; accepted 19 February 1992)
Key words: Enkephalinase; Peptide; Peptidase; Tachykinin metabolism
Summary This study was designed to evaluate the role of neutral endopeptidase (NEP) in modulating the airway smooth muscle contraction induced by endothelin-1 in isolated segments of guinea-pig trachea. Endothelin-1 (10-9_ 10-6 M) produced a concentration-dependent contraction that reached a maximum by 30 min. The NEP inhibitor leucine-thiorphan (10 -5 M) significantly increased the contractile response to endothelin-1. The addition of leucine-thiorphan to tracheal segments precontracted by 10-9 and 10- s M endothelin-1 increased isometric tension by 181 + 65~o (mean ± 1 S.E.M.; P < 0.05) and by 138 ± 49~o (P< 0.05), respectively. In contrast, the kininase II inhibitor captopril and the peptidase inhibitors leupeptin and bestatin had no effect. Preincubation of endothelin- 1 with 1 lag recombinant human NEP decreased the contractile activity of endothelin-1 by 72 ± 9~o, whereas no effect was observed using heat-inactivated NEP. We conclude that NEP modulates endothelin-induced contraction of airway smooth muscle in the guinea-pig trachea.
Correspondence: J.A. Nadel, Cardiovascular Research Institute and Departments of Medicine and Physiology, University of California, Box 0130, M 1325, San Francisco, CA 94143, USA.
138 Introduction
Endothelin-1 is a 21-residue peptide which has been isolated from the culture medium of vascular endothelial cells and found to be a potent vasoconstrictor [1]. Endothelin-1 is also produced by porcine and canine tracheal epithelial cells [2], and causes contraction of airway smooth muscle in guinea-pigs [3] and in humans [4]. High-affinity binding sites are found in airway smooth muscle [5,6]. An enzyme, neutral endopeptidase (NEP; also called enkephalinase, 3.4.24.11), exists on the surface of various cells in the airways including epithelium and airway smooth muscle. In the airways, NEP, by cleaving and thus inactivating a wide variety of bronchoactive peptides, limits the concentration of the peptide that reaches the receptor on the cell surface and thus modulates the effects of the peptide on airways [7,8]. Recently, it has been reported that endothelin-1 is also cleaved by NEP [9,10], and that the increased response to endothelin-1 caused by the removal of epithelium in guinea-pig trachea is not observed in the presence of the NEP inhibitor phosphoramidon [ 11]. Taken together these observations suggest that the contractile activity of endothelin-1 or airway smooth muscle can be modulated by NEP which is localized in various cell types in the airways. In the present study, we examined the effect of NEP inhibitor leucine-thiorphan and of recombinant NEP on endothelin-induced airway smooth muscle responses in guinea-pig trachea. The NEP inhibitor leucine-thiorphan, but not other peptidase inhibitors, potentiated the contractile responses to endothelin-1. The contractile activity of endothelin-1 was blunted by recombinant NEP. The results of this study suggest that NEP limits airway responses to endothelin-1.
Materials and Methods
Measurement of tracheal smooth muscle tension Male Hartley-outbred guinea-pigs (Charles River Breeding Laboratories, Wilmington, MA), weighing 500-700 g, were anesthetized with pentobarbital sodium (50 mg/ kg i.p.). The trachea was removed rapidly and immersed in oxygenated Krebs-Henseleit solution of the following composition (millimolar): NaCI, 118; KC1, 5.9; CaC1z, 2.5; MgSO4, 1.2; NaH2PO 4, 1.2; NaHCO3, 25.5; glucose, 5.6. After removal of loose connective tissue, transverse rings (5 mm long) were cut from the trachea and mounted in glass chambers filled with 14 ml of Krebs-Henseleit solution that was maintained at 37 °C and was aerated continuously by bubbling it with a mixture of 95 ~o 02-5 ~o CO2, which produced a pH of 7.4. Six tracheal rings were studied concurrently. The tracheal rings were connected to force-displacement transducers (Grass, FT03) for continuous recording of isometric tension on a Grass model 7D polygraph recorder. The rings were initially stretched to a tension of 10 g for 20 s and were then allowed to equilibrate for 1 h while resting tension was adjusted to 1 g. During equilibration, the Krebs-Henseleit solution was changed every 15 rain. We incubated the tracheal rings continuously with phentolamine (10 5 M), an ~-adrenergic receptor antagonist, and propranolol (10-6 M), a 13-adrenergic receptor antagonist. To determine the viability and reactivity of each tissue and to provide a basis for normalizing subsequent
139 changes in tension, tracheal rings were exposed to acetylcholine (10-3 M), and then rinsed to allow tension to return to resting values.
Effects of leucine-thiorphan on endothelin-induced contraction To determine the effect of leucine-thiorphan [ 12], a derivative of thiorphan and a potent NEP inhibitor [ 13], on endothelin-induced contraction, we first measured the response of tracheal rings from six guinea-pigs to single concentrations of endothelin- 1 ranging from 10 - 9 t o 1 0 - 6 M. After 30 min, when smooth muscle contraction induced by endothelin-1 was maximum, leucine-thiorphan (10-5 M) was added to the glass chamber and any further increase in tension was recorded when a stable plateau was achieved. We chose this strategy because preliminary studies showed that even with extensive washing, the contractions induced by endothelin-1 returned to baseline only very slowly. This is possibly due to the very hydrophobic nature of endothelin-1. Therefore, it was impossible to obtain repeated responses to this peptide. Because of this, we could not study the effects of leucine-thiorphan pre-incubation in tissues that already had a control response to endothelin-1. Therefore, to ensure within-tissue controls, we added leucine-thiorphan only after endothelin-1-induced contractions had become steady.
Comparison of effects of various peptidase and proteinase inhibitors on endothelin-induced contraction To determine whether only inhibition of NEP was responsible for potentiating contractile responses to endothelin-1, we evaluated the effect of other inhibitors in two groups of tracheal rings from four guinea-pigs. After contraction in response to 10 8 M endothelin-1 had reached a plateau, one group received sequentially (1) a combination of the following inhibitors (each inhibitor, 10-5 M): captopril, an inhibitor of kininase II [ 14]; leupeptin, a serine proteinase inhibitor [ 15]; and bestatin, an inhibitor of aminopeptidase [16]; (2) leucine-thiorphan (10-5 M), an inhibitor of NEP. In the second group of tissues precontracted by endothelin-1, we added sequentially the vehicles corresponding to the combination of inhibitors and to leucine-thiorphan. Either inhibitors or vehicles were added at 20 min intervals.
Effects of recombinant human NEP on the ability of endothelin-1 to induce tracheal smooth muscle contraction To determine whether the contractile response to endothelin-1 could be blunted by NEP, we compared the effects of endothelin-1 (3.10-8 M), endothelin-1 preincubated with recombinant human NEP, and endothelin-1 preincubated with NEP inactivated by heat in three groups of trachea rings from six guinea pigs. Endothelin- 1 was incubated overnight at 37°C, either with 1 ~tg of NEP or with the same amount of NEP which had been inactivated by boiling it for 1 h. Both NEP and heat-inactivated NEP were dissolved in 500 ~tl of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffer (pH 7.4).
140
Materials and reagents Endothelin-1 and leupeptin were obtained from Peninsula Laboratories (Belmont, CA), (+)-propranolol hydrochloride and acetylcholine chloride were obtained from Sigma (St. Louis, MO). Phentolamine was purchased from Ciba Geigy (Basel, Switzerland); and captopril and leucine-thiorphan were generous gifts from Squibb Pharmaceuticals (Princeton, N J). Endothelin-1 was dissolved in saline immediately prior to use. Leucine-thiorphan was dissolved in 100Yo ethanol, stored in the refrigerator, and diluted with normal saline before use. Recombinant human NEP was obtained from B. Bridenbaugh, Genentech, South San Francisco and was purified without detergents [ 17]. Statistical analysis Data were expressed as mean + S.E.M. Statistical analysis was performed using one-way analysis of variance and Newman-Keuls multiple range test. Unpaired Student's t-test was used to compare the effect of NEP on the contractile ability of endothelin-1. Significance was accepted at P < 0.05. Results
Effects of leucine-thiorphan on endothelin-induced contraction In each of six guinea-pigs, endothelin-1 alone caused a concentration-dependent increase in active tension of tracheal smooth muscle, with significant contractile effects Active tension (% of maximum) 80
60
40
20
9
8
7
6
Endothelin Concentration (- Log M) t.ig. 1. I:lliect of the peptidase inhibitor lcucine-thiorphan on the contractile rcsponscs to increasing concentrations of endothelin-1 in isolated segments of guinea-pig tracheal smooth muscle. Measurements were made 30 min after addition of endothelin-1, a time when contractile responses were maximum (open circles). Data are expressed as percentage of the control contraction induced by acetylcholine ( 1 0 - ) M) and are reported as mean + 1 S.E.M. (n = 6). When teucine-thiorphan was added (10 s M; closed circles), a further increase in active tension occurred which was significant at the lower molar concentrations of endothelin-1 ( P < 0.05).
141
occurring at 30 rain at concentrations of 10 - 7 M or greater ( P < 0.01) (Fig. 1). A further increase in tension of tracheal rings precontracted by endothelin-1 was obtained when leucine-thiorphan was added to the chamber. This increase in tension produced a leftward shift of the concentration-response curve to endothelin-1 (Fig. 1). Changes were significant at concentrations of 10- 9 M (P < 0.01), 1O-8 M (P < 0.01) and 10- 7 M ( P < 0.05) endothelin-1. In separate experiments, leucine-thiorphan alone (10-5 M) did not alter resting tension or the contraction induced by 10- 8 M acetylcholine (data not shown).
Effects of other peptidase and proteinase inhibitors on endothelin-induced contraction In a second group of studies, we examined the effect of various peptidase inhibitors on endothelin-induced contraction in trachea from four guinea-pigs. In control tissues, continued incubation with endothelin-1 alone (10-8 M) produced a maximum contraction at 30 rain. In separate tissues, incubation with a combination of captopril, leupeptin and bestatin (each inhibitor at 10 - 5 M) did not affect the contractile response to endothelin-1 compared to vehicle at the same time (Fig. 2). Subsequent addition of leucine-thiorphan (10-5 M) caused a marked increase in the contractile response to endothelin-1 compared either to the vehicle at the same time or to the control response to ET in the absence of inhibitors (P
137
REGPEP 01179
Neutral endopeptidase modulates endothelin- 1-induced airway smooth muscle contraction in guinea-pig trachea Giuseppe U. Di Maria, Michio Katayama, D. Benjamin Borson and Jay A. Nadel Cardiovascular Research Institute and Departments of Medicine and Physiology, University of California, San Francisco, CA (USA) (Received 12 November 1991; revised version received 17 February 1992; accepted 19 February 1992)
Key words: Enkephalinase; Peptide; Peptidase; Tachykinin metabolism
Summary This study was designed to evaluate the role of neutral endopeptidase (NEP) in modulating the airway smooth muscle contraction induced by endothelin-1 in isolated segments of guinea-pig trachea. Endothelin-1 (10-9_ 10-6 M) produced a concentration-dependent contraction that reached a maximum by 30 min. The NEP inhibitor leucine-thiorphan (10 -5 M) significantly increased the contractile response to endothelin-1. The addition of leucine-thiorphan to tracheal segments precontracted by 10-9 and 10- s M endothelin-1 increased isometric tension by 181 + 65~o (mean ± 1 S.E.M.; P < 0.05) and by 138 ± 49~o (P< 0.05), respectively. In contrast, the kininase II inhibitor captopril and the peptidase inhibitors leupeptin and bestatin had no effect. Preincubation of endothelin- 1 with 1 lag recombinant human NEP decreased the contractile activity of endothelin-1 by 72 ± 9~o, whereas no effect was observed using heat-inactivated NEP. We conclude that NEP modulates endothelin-induced contraction of airway smooth muscle in the guinea-pig trachea.
Correspondence: J.A. Nadel, Cardiovascular Research Institute and Departments of Medicine and Physiology, University of California, Box 0130, M 1325, San Francisco, CA 94143, USA.
138 Introduction
Endothelin-1 is a 21-residue peptide which has been isolated from the culture medium of vascular endothelial cells and found to be a potent vasoconstrictor [1]. Endothelin-1 is also produced by porcine and canine tracheal epithelial cells [2], and causes contraction of airway smooth muscle in guinea-pigs [3] and in humans [4]. High-affinity binding sites are found in airway smooth muscle [5,6]. An enzyme, neutral endopeptidase (NEP; also called enkephalinase, 3.4.24.11), exists on the surface of various cells in the airways including epithelium and airway smooth muscle. In the airways, NEP, by cleaving and thus inactivating a wide variety of bronchoactive peptides, limits the concentration of the peptide that reaches the receptor on the cell surface and thus modulates the effects of the peptide on airways [7,8]. Recently, it has been reported that endothelin-1 is also cleaved by NEP [9,10], and that the increased response to endothelin-1 caused by the removal of epithelium in guinea-pig trachea is not observed in the presence of the NEP inhibitor phosphoramidon [ 11]. Taken together these observations suggest that the contractile activity of endothelin-1 or airway smooth muscle can be modulated by NEP which is localized in various cell types in the airways. In the present study, we examined the effect of NEP inhibitor leucine-thiorphan and of recombinant NEP on endothelin-induced airway smooth muscle responses in guinea-pig trachea. The NEP inhibitor leucine-thiorphan, but not other peptidase inhibitors, potentiated the contractile responses to endothelin-1. The contractile activity of endothelin-1 was blunted by recombinant NEP. The results of this study suggest that NEP limits airway responses to endothelin-1.
Materials and Methods
Measurement of tracheal smooth muscle tension Male Hartley-outbred guinea-pigs (Charles River Breeding Laboratories, Wilmington, MA), weighing 500-700 g, were anesthetized with pentobarbital sodium (50 mg/ kg i.p.). The trachea was removed rapidly and immersed in oxygenated Krebs-Henseleit solution of the following composition (millimolar): NaCI, 118; KC1, 5.9; CaC1z, 2.5; MgSO4, 1.2; NaH2PO 4, 1.2; NaHCO3, 25.5; glucose, 5.6. After removal of loose connective tissue, transverse rings (5 mm long) were cut from the trachea and mounted in glass chambers filled with 14 ml of Krebs-Henseleit solution that was maintained at 37 °C and was aerated continuously by bubbling it with a mixture of 95 ~o 02-5 ~o CO2, which produced a pH of 7.4. Six tracheal rings were studied concurrently. The tracheal rings were connected to force-displacement transducers (Grass, FT03) for continuous recording of isometric tension on a Grass model 7D polygraph recorder. The rings were initially stretched to a tension of 10 g for 20 s and were then allowed to equilibrate for 1 h while resting tension was adjusted to 1 g. During equilibration, the Krebs-Henseleit solution was changed every 15 rain. We incubated the tracheal rings continuously with phentolamine (10 5 M), an ~-adrenergic receptor antagonist, and propranolol (10-6 M), a 13-adrenergic receptor antagonist. To determine the viability and reactivity of each tissue and to provide a basis for normalizing subsequent
139 changes in tension, tracheal rings were exposed to acetylcholine (10-3 M), and then rinsed to allow tension to return to resting values.
Effects of leucine-thiorphan on endothelin-induced contraction To determine the effect of leucine-thiorphan [ 12], a derivative of thiorphan and a potent NEP inhibitor [ 13], on endothelin-induced contraction, we first measured the response of tracheal rings from six guinea-pigs to single concentrations of endothelin- 1 ranging from 10 - 9 t o 1 0 - 6 M. After 30 min, when smooth muscle contraction induced by endothelin-1 was maximum, leucine-thiorphan (10-5 M) was added to the glass chamber and any further increase in tension was recorded when a stable plateau was achieved. We chose this strategy because preliminary studies showed that even with extensive washing, the contractions induced by endothelin-1 returned to baseline only very slowly. This is possibly due to the very hydrophobic nature of endothelin-1. Therefore, it was impossible to obtain repeated responses to this peptide. Because of this, we could not study the effects of leucine-thiorphan pre-incubation in tissues that already had a control response to endothelin-1. Therefore, to ensure within-tissue controls, we added leucine-thiorphan only after endothelin-1-induced contractions had become steady.
Comparison of effects of various peptidase and proteinase inhibitors on endothelin-induced contraction To determine whether only inhibition of NEP was responsible for potentiating contractile responses to endothelin-1, we evaluated the effect of other inhibitors in two groups of tracheal rings from four guinea-pigs. After contraction in response to 10 8 M endothelin-1 had reached a plateau, one group received sequentially (1) a combination of the following inhibitors (each inhibitor, 10-5 M): captopril, an inhibitor of kininase II [ 14]; leupeptin, a serine proteinase inhibitor [ 15]; and bestatin, an inhibitor of aminopeptidase [16]; (2) leucine-thiorphan (10-5 M), an inhibitor of NEP. In the second group of tissues precontracted by endothelin-1, we added sequentially the vehicles corresponding to the combination of inhibitors and to leucine-thiorphan. Either inhibitors or vehicles were added at 20 min intervals.
Effects of recombinant human NEP on the ability of endothelin-1 to induce tracheal smooth muscle contraction To determine whether the contractile response to endothelin-1 could be blunted by NEP, we compared the effects of endothelin-1 (3.10-8 M), endothelin-1 preincubated with recombinant human NEP, and endothelin-1 preincubated with NEP inactivated by heat in three groups of trachea rings from six guinea pigs. Endothelin- 1 was incubated overnight at 37°C, either with 1 ~tg of NEP or with the same amount of NEP which had been inactivated by boiling it for 1 h. Both NEP and heat-inactivated NEP were dissolved in 500 ~tl of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffer (pH 7.4).
140
Materials and reagents Endothelin-1 and leupeptin were obtained from Peninsula Laboratories (Belmont, CA), (+)-propranolol hydrochloride and acetylcholine chloride were obtained from Sigma (St. Louis, MO). Phentolamine was purchased from Ciba Geigy (Basel, Switzerland); and captopril and leucine-thiorphan were generous gifts from Squibb Pharmaceuticals (Princeton, N J). Endothelin-1 was dissolved in saline immediately prior to use. Leucine-thiorphan was dissolved in 100Yo ethanol, stored in the refrigerator, and diluted with normal saline before use. Recombinant human NEP was obtained from B. Bridenbaugh, Genentech, South San Francisco and was purified without detergents [ 17]. Statistical analysis Data were expressed as mean + S.E.M. Statistical analysis was performed using one-way analysis of variance and Newman-Keuls multiple range test. Unpaired Student's t-test was used to compare the effect of NEP on the contractile ability of endothelin-1. Significance was accepted at P < 0.05. Results
Effects of leucine-thiorphan on endothelin-induced contraction In each of six guinea-pigs, endothelin-1 alone caused a concentration-dependent increase in active tension of tracheal smooth muscle, with significant contractile effects Active tension (% of maximum) 80
60
40
20
9
8
7
6
Endothelin Concentration (- Log M) t.ig. 1. I:lliect of the peptidase inhibitor lcucine-thiorphan on the contractile rcsponscs to increasing concentrations of endothelin-1 in isolated segments of guinea-pig tracheal smooth muscle. Measurements were made 30 min after addition of endothelin-1, a time when contractile responses were maximum (open circles). Data are expressed as percentage of the control contraction induced by acetylcholine ( 1 0 - ) M) and are reported as mean + 1 S.E.M. (n = 6). When teucine-thiorphan was added (10 s M; closed circles), a further increase in active tension occurred which was significant at the lower molar concentrations of endothelin-1 ( P < 0.05).
141
occurring at 30 rain at concentrations of 10 - 7 M or greater ( P < 0.01) (Fig. 1). A further increase in tension of tracheal rings precontracted by endothelin-1 was obtained when leucine-thiorphan was added to the chamber. This increase in tension produced a leftward shift of the concentration-response curve to endothelin-1 (Fig. 1). Changes were significant at concentrations of 10- 9 M (P < 0.01), 1O-8 M (P < 0.01) and 10- 7 M ( P < 0.05) endothelin-1. In separate experiments, leucine-thiorphan alone (10-5 M) did not alter resting tension or the contraction induced by 10- 8 M acetylcholine (data not shown).
Effects of other peptidase and proteinase inhibitors on endothelin-induced contraction In a second group of studies, we examined the effect of various peptidase inhibitors on endothelin-induced contraction in trachea from four guinea-pigs. In control tissues, continued incubation with endothelin-1 alone (10-8 M) produced a maximum contraction at 30 rain. In separate tissues, incubation with a combination of captopril, leupeptin and bestatin (each inhibitor at 10 - 5 M) did not affect the contractile response to endothelin-1 compared to vehicle at the same time (Fig. 2). Subsequent addition of leucine-thiorphan (10-5 M) caused a marked increase in the contractile response to endothelin-1 compared either to the vehicle at the same time or to the control response to ET in the absence of inhibitors (P
