Inhibition of Neutral Endopeptidase Potentiates Bronchial Contraction Induced by Immune Response in Guinea Pigs In Vitro 1- 4

HIROTSUGU KOHROGI, TETSURO YAMAGUCHI, OSAMU KAWANO, IZUMI HONDA, MASAVUKI ANDO, and SHUKURO ARAKI

Introduction

Mammalian tachykinins (substance P and neurokinins A and B) exist in the central nervous system and in C fibers, also called unmyelinated afferent nerves or capsaicin-sensitive nerves (capsaicin being the pungent principle of red pepper). In the airways, tachykinins also exist in the bronchial C fibers of many species, including guinea pigs and humans (1, 2). There are many effects of tachykinins on the airway tissue: for example, they contract airway smooth muscle (3-7), increase bronchial gland secretion (8), increase vascular permeability (3), increase cholinergic neurotransmission (9), increase CI ion transport of the epithelium (10), and cause cough (11, 12). It is known that tachykinins are released from C fibers by capsaicin, histamine, bradykinin, and antidromic electrical stimulation (13). However, the roles of endogenous tachykinins and the releasing mechanism of tachykinins in airway inflammation are poorly understood. As small peptides, tachykinins are effectively cleaved by peptidases, especially neutral endopeptidase (NEP, also known as enkephalinase and membrane metalloendopeptidase; E.C. 3.4.24.11) (14-16). Recent in vivo and in vitrostudies show that inhibition of NEP potentiates the sensitivity and reactivity to tachykinins in the airways (6, 7, 11, 17-25). Furthermore, recombinant NEP inhibits cough induced by substance P and capsaicin in guinea pigs (26). These reports suggest that NEP in the airways normally cleaves tachykinins to inactive metabolites, thereby reducing their effects. Because the effect of chemical mediators released from mast cells by the immune response is an important part of the pathogenesis of bronchial asthma (27- 30) and because there is evidence that mast cells exist very close to unmyelinat636

SUMMARY To study the role of tachykinins and neutral endopeptidase (NEP), an enzyme that degrades tachyklnlns, In the immune response In the airways of guinea pigs sensitized to ovalbumin (OVA), we examined the bronchial contractile response to OVA by Inhibiting NEP in vitro. After Incubating bronchial tissues with the NEPInhibitors phosphoramidon and thlorphan, we added 10-5 % (10 ~g/ml) OVA. Phosphoramldon and thiorphan (10-5 M) significantly maintained the contraction that followed the peak contraction. In the next stages of the experiment, when the contraction Induced by 10-5 % OVAreached a plateau and began to relax, we added 10-5 M phosphoramidon. Phosphoramldon Inhibited the relaxation and significantly potentiated the contraction. In tissues treated with 10-5 M capsaicin to deplete tachyklnlns, phosphoramldon did not potentiate the OVA-induced contraction, but substance P (10-8 M) caused contraction. These results suggest that the immune response causes the release of tachyklnln-llke substances from capsaicin-sensitive nerves to Induce bronchial contraction In part. Toconfirm the mediators that cause the release of the tachyklnlnlike substances from the bronchus, we also examined whether phosphoramldon potentiates the effect of leukotrlene C. (LTC.), serotonin, histamine, and platelet-activating factor on bronchial contraction. When the contractions induced by these agonlsts reached a plateau and began to relax, we added phosphoramldon. Phosphoramldon Inhibited the relaxation and significantly potentiated the contractile response to 10-5 M LTC., and it significantly reduced the relaxing rate of the 10-8 M serotonin-induced contraction. However, It did not change the effect of histamine and plateletactivating factor. From these results, we conclude that (1) some chemical mediators released by the Immune response stimulate the release of tachyklnin-like substances, (2) NEP plays an important role In modulating the bronchial contraction Induced by substances In the Immune response, and (3) LTC. and serotonin are chemical mediators that release these substances. AM REV RESPIR DIS 1991; 144:636-641

ed nerves containing substance P in rat intestine (31), we focused on the interaction between chemical mediators released by the immune response and the vagal nerve, especially the bronchial C fibers. The goal of the present study is to determine whether chemical mediators released by the immune response stimulate the release of tachykinins from bronchial C fibers to induce bronchial smooth muscle contraction. To test this, we examined whether the NEP inhibitors phosphoramidon and thiorphan potentiate the bronchial contraction induced by the immune response. In addition, to test the chemical mediators responsible for releasing tachykinins, we also examined whether phospltoramidon potentiates the effect of such mediators as leukotriene C 4 (LTC4 ) , histamine, serotonin, and platelet-activating factor (PAF) on bronchial contraction.

Methods We used 52 male Hartley strain guinea pigs (350to 380 g). We injected 70 mg ovalbumin (OVA) dissolved in 1.8 ml normal saline in-

(Received in original form October 10, 1990 and in revised form March 25, 1991) I From the First Department of Internal Medicine, Kumamoto University Medical School, Kumamoto, Japan. 2 Supported by Scientific Grant No. 02670343 from the Ministry of Education, Science and Culture of Japan. 3 Presented in part at the 1990 World Conference on Lung Health, American Lung Association/American Thoracic Society, and International Union Against Tuberculosis and Lung Disease, Boston. • Correspondence and requests for reprints should be addressed to Hirotsugu Kohrogi, M.D., First Department of Internal Medicine, Kumamoto University Medical School, 1-1-1 Honjo, Kumamoto 860, Japan.

637

NEUTRAL ENDOPEPTIDASE AND TACHYKININS IN AIRWAYS IN IMMUNE RESPONSE

tramuscularly twiceat an interval of 1wk (32). g, enough tension to examine the effect of Guinea pigs were anesthetized with pentobarNEP inhibitors on OVA-inducedcontraction in sensitized strips, we added 10-5% OVA to bital (60 mg/kg) 3 wk after the second injection. Then, the trachea and lung were resect- both strips. Then weobserved the contractile response for 100 min. Phosphoramidon did ed. Weimmersed them in Krebs-Henseleit solution (NaCl, 118.3 mM; KCI, 4.7; MgS0 4 , not affect the resting tension, but thiorphan 1.2; KH 2 P 04 , 1.2; caci., 2.5; NaHC0 3 , 25; increased it in two of eight strips. Therefore, we continued the study of thiorphan in six and glucose, 11.1) and carefully isolated both pairs of strips that were stable. We expressed main bronchi from the lung. The main bronchi were cut open and placed as a strip of tis- the OVA-induced contraction as the percentsue in a lO-ml organ bath containing Krebs- age of the response to 10-3 M acetylcholine. Henseleit solution. The solution was mainEffect of Phosphoramidon on tained at 370 C and continuously aerated with Contraction Induced by the mixture of 95070 oxygen and 5% carbon Immune Response dioxide. One end of the bronchial strip was fixed Weprepared five pairs of bronchial strips, one by a silk thread to a glass hook in the bottom pair from each of five guinea pigs sensitized of the organ bath, and the other end was fixed to OVA.We added 10-5% OVA to both strips. by a silk thread to an isometric force-dis- After the plateau of OVA-induced contracplacement transducer (TB-611T; Nihonkoh- tion and the following relaxation, we added den Kogyo, Tokyo). The strip was placed be10-5 M phosphoramidon to one strip. We defined this point as 100% contraction. As the tween two rectangular platinum electrodes control, we added Krebs-Henseleit solution (6 x 40 mm) for electrical field stimulation. These electrodes were connected to an elec- to the other strip instead of phosphoramidon. tronic stimulator (SEN-3301; Nihonkohden Then, we observed the contractile response Kogyo). The bronchial strips were set up for during the following 100 min. tension measurement under a O.5-g load and Effect of Phosphoramidon on were equilibrated for more than 90 min in the Contraction Induced by the solution, which was replaced every 15 min. Immune Response in The responses were amplified (AP-621G; NiCapsaicin-treated Bronchial Tissue honkohden Kogyo) and recorded (RJG-4028; Nihonkohden Kogyo). We prepared six pairs of bronchial strips, one First, we stimulated the strips by electrical pair from each of six guinea pigs sensitized field stimulation for 20 s (pulse width, 1 ms; to OVA. As figure 3B shows, to deplete the intensity, 20 V; frequency 20 Hz) to examine endogenous tachykinins in the bronchial C noncholinergic, nonadrenergic contraction, fibers (21),we added capsaicin (10-5 M) to one which demonstrated the existence of tachyki- strip. After we observed the contraction and nins in the strips (3, 21). After washing the the following relaxation, wewashed the strips. strips with Krebs-Henseleit solution and al- As the control, we added the solvent of caplowing the contraction to return to the rest- saicin (0.1 % ethanol) to the other strip instead ing tension, we added 10-3 M acetylcholine of capsaicin (figure 3A). Then we added 10-5% OVA. After the plateau of the contracto the strip to examine the viability of bronchial smooth muscle. Then we washed the tion and the following relaxation, we added strips until they returned to the resting ten- phosphoramidon (10-5 M) to both strips. We sion (figure 3A). defined this point as 100%contraction. Then, To examine the effect of NEP inhibitors we observed the contractile responses during on bronchial contraction induced by the im- the following 100 min. Sequentially, after mune response or agonists, we performed par- washing the strips, wefinally added substance P (10-6 M) to examine whether tachyphylaxis allel studies on left and right bronchi from the same guinea pig. Werandomly chose one was induced by the tachykinins that were inibronchus to which we added one of the NEP tially released by capsaicin. To evaluate the inhibitors. contractile effect of substance P, we calculatBovine serum albumin did not contract sen- ed the ratio of substance P-induced contracsitized bronchial strips to OVA, and OVAdid tion to acetylcholine-induced contraction. not contract nonsensitized bronchial strips.

Effects of Pretreatment with Phosphoramidon and Thiorphan on Contraction Induced by the Immune Response We prepared six and eight pairs of bronchial strips, one from each of the guinea pigs sensitized to OVA, for phosphoramidon and thiorphan. We incubated one strip with 10-5 M phosphoramidon or thiorphan and the other strip with its solvent (Krebs-Henseleit solution or 0.1% ethanol) for 15min. Because preliminary studies showedthat 10-5% (10 ug/rnl) OVA caused contraction of more than 0.25

Effect of Phosphoramidon on Contraction Induced by LTC4

We prepared six pairs of bronchial strips, one pair from each of the six guinea pigs sensitized to OVA. We added LTC4 cumulatively from 10-10 to 10-7 M. After the plateau of the contraction at 10-7 M, we added phosphoramidon (10-5 M) to one strip. We defined this point as 100% contraction. As the control, weadded Krebs-Henseleit solution to the other strip instead of phosphoramidon. Then, we observed the contractile response during the following 100 min. To examine the effect of phosphoramidon

on contraction induced by LTC4 in normal guinea pig bronchus, we studied four pairs of bronchial strips, one pair from each of the four nonsensitized guinea pigs, by the same methods.

Effect of Phosphoramidon on Contraction Induced by Serotonin, Histamine, and PAF We prepared five to seven pairs of bronchial strips, one from each of five to seven guinea pigs sensitized to OVA for each series of experiments. We added serotonin or histamine from 10-7 M to higher concentrations until the contraction reached to 0.25 g or greater. After we confirmed the plateau of the contraction induced by serotonin or histamine, we added phosphoramidon (10-5 M). We defined this point as 100% contraction. PAF from 10-8 to 3 X 10-6 M caused slight relaxation (-0.05 to - 0.10 g) and was followed by a return to the resting tension. Therefore, we added phosphoramidon (10-5 M) when the tension returned to the baseline. As the control, we added Krebs-Henseleit solution instead of phosphoramidon. Then, weobserved the contractile response during the following 100 min.

Drugs The following pharmacologic agents were used: OVA, bovine serum albumin, acetylcholine, LTC4 , phosphoramidon, thiorphan, serotonin, histamine, PAF, and capsaicin (Sigma Chemical Co., St. Louis, MO). A stock solution of thiorphan (10-2 M), LTC4 (3.3 x 10-3 M), and PAF (10-2 M) was dissolved in ethanol, kept under nitrogen gas, and stored at -60 0 C. A stock solution (10-2 M) of capsaicin was dissolved in 100% ethanol and stored at 4 0 C. All agents were diluted in KrebsHenseleit solution just before the experiment. The concentrations given werefinal bath concentrations. Preliminary experimentation showed that the final bath concentration of ethanol in the bath did not alter the resting tension.

Analysis of Data Data were expressed as mean ± standard error of the mean (SEM). Statistical analysis of the data was made using the Student's unpaired t test to compare the active tensions (g) and the paired t test to compare the corrected values (%). A p value smaller than 0.05 was considered significant. Results

There was no difference between left and right main bronchus in response to electrical field stimulation: cholinergic contraction (0.49 ± 0.05 versus 0.47 ± 0.06 g; p > 0.05, Student's unpaired t test) and noncholinergic, nonadrenergic contraction (0.31 ± 0.08 versus 0.35 ± 0.03 g; p > 0.05), or in contractile response to 10-3 M acetylcholine (l.45 ± 0.07 versus 1.34 ± 0.10 g; p > 0.05).

KOHROGI, YAMAGUCHI, KAWANO, HONDA, ANDO, AND ARAKI

638

A

%

B

50 40

40

30 Contraction (% of 10. 3 MAch)

strips (102 ± 8 versus94 ± 9070; p >0.05), suggesting no tachyphylaxis to tachykinins in the capsaicin-treated strips.

%

50

Effect of Phosphoramidon on Contraction Induced by LTC4

30

Contraction (% of 10- 3 MAch)

20

10

20

10

·10

·10 20

40

60

80

,

, 20

100

40

60

80

100

Time (min)

Time (min)

Fig. 1. Effects of pretreatment with phosphoramidon and thiorphan on bronchial contraction induced by immune response. We incubated the bronchial strips sensitized to OVA with 10-5 M phosphoramidon (A) and 10-5 M thiorphan (B) for 15 min, and then we added 10-50/ 0 ovalbumin (OVA). The OVA-induced contractions are expressed as the percentage of response to 10-3 M acetylcholine (Ach). Each value is the mean ± SEM (n = 6 for both phosphoramidon and thiorphan). From 20 min after the additon of OVA, when the peak contraction was obtained, the OVA-induced contraction was significantly stronger in the strips pretreated with phosphoramidon (closed circles) or thiorphan (closed squares) than in the controls (open circles). Asterisks indicate p < 0.05; double asterisks indicate p < 0.01 (Student's paired t test).

%

200

150 % Contraction 100

50

o

" ----r--~--r---.-----r40 60 80 100 o 20 I

i

I

I

I

Time after Phosphoramidon (min)

Effects of Pretreatment with Phosphoramidon and Thiorphan on Contraction Induced by the Immune Response Phosphoramidon and thiorphan significantly maintained the contraction induced by the immune response (figure 1). From 20 min after the addition of OVA(10-5 070), when the peak contraction was obtained, the OVA-induced contraction was significantly stronger in the strips pretreated with phosphoramidon or thiorphan than in control strips.

Effect of Phosphoramidon on Contraction Induced by the Immune Response Phosphoramidon significantly potentiated the contraction induced by the immune response (figure 2). OVA (10-5 % ) caused contraction in the strips prepared from guinea pigs sensitized to OVA. When we added phosphoramidon or Krebs-Henseleit solution to the strips at the relaxing point (0.27 ± 0.03 versus0.31 ± 0.04 g; p > 0.05) after the maximal contraction (0.49 ± 0.25 versus 0.56 ± 0.25 g; p > O. 05), phosphoramidon

Fig. 2. Effect of phosphoramidon on bronchial contraction induced by immune response. The 10-5 % ovalbumin (OVA) caused contraction of the strips from guinea pigs sensitized to OVA. When the contraction began to relax, we added the NEP inhibitor phosphoramidon (10-5 M). We defined this point as 100% contraction and 0 min. Each value is the mean ± SEM (n = 5). Phosphoramidon inhibited the relaxation and significantly potentiated the contraction (closed circles) compared with control strips (open circles). Asterisk indicates p < 0.05; double asterisks indicate p < 0.01 (Student's paired t test).

inhibited the relaxation and significantly increased the tension compared with the control preparation, which continuously relaxed after the plateau (figures 2 and 3A).

Effect of Phosphoramidon on Contraction Induced by the Immune Response in Capsaicin-treated Bronchial Tissue Phosphoramidon did not potentiate the contraction induced by OVAin capsaicintreated strips. OVA (10-5 070) caused contraction in both the capsaicin-treated and nontreated strips (0.64 ± 0.29 versus 0.76 ± 0.21 g; p > 0.05). We added phosphoramidon at the relaxing point in the capsaicin-treated and nontreated strips (0.33 ± 0.08 versus 0.30 ± 0.05 g; p > 0.05). In the capsaicin-treated strips, phosphoramidon did not potentiate the contraction induced by OVA (figure 3B). However, in the nontreated strips, it significantly did (figures 3A and 4). There was no significant difference in the ratio of substance P-induced contraction to acetylcholine-induced contraction between capsaicin-treated and non-treated

Phosphoramidon significantly potentiated the Ll'Ca-induced contraction in sensitized strips (figure 5). We added phosphoramidon or Krebs-Henseleit solution to the strips at the stable plateau of the contraction induced by 10-7 M LTC4 (0.36 ± 0.05 versus 0.40 ± 0.05 g; p > 0.05). Phosphoramidon inhibited the relaxation and significantly increased the tension compared with the control preparation, which continuously relaxed after the plateau (figure 5). Phosphoramidon also significantly potentiated LTC4-induced contraction in nonsensitized strips (data not shown).

Effect of Phosphoramidon on Contraction Induced by Serotonin, Histamine, and PAF Phosphoramidon significantly reduced the relaxing rate in serotonin-induced contraction (figure 6). We added phosphoramidon or Krebs-Henseleit solution to the strips contracted by serotonin (10-6 M) at the relaxing point (0.35 ± 0.05 versus 0.28 ± 0.03 g; p > 0.05) after the maximal contraction (0.43 ± 0.06 versus 0.33 ± 0.05 g; p > 0.05). Phosphoramidon did not inhibit the relaxation, but the relaxing rate was significantly reduced (figure 6). In contrast, when we added phosphoramidon or Krebs-Henseleit solution to the strips contracted by histamine (10-6 or 10-5 M) at the relaxing point (0.49 ± 0.12 versus 0.48 ± 0.14 g; p > 0.05) after the maximal contraction (0.52 ± 0.13 versus 0.49 ± 0.13 g; p > 0.05), phosphoramidon did not change the contraction (figure 7). PAF caused a slight relaxation followed by a return to the resting tension. Phosphoramidon did not change the response to PAF (data not shown). Discussion

We found, in the guinea pig bronchus, that (1) the NEP inhibitors phosphoramidon and thiorpan potentiate the bronchial contraction induced by the immune response and by LTC4 in the maintenance phase, and (2) phosphoramidon reduces the relaxing rate of bronchial contraction induced by serotonin. NEP, a membrane-bound peptidase, is known to exist in the airway in many species of animals, including guinea pigs and humans (33, 34). Biochemical studies show that NEP cleaves tachykinins: sub-

639

NEUTRAL ENDOPEPTIDASE AND TACHYKININS IN AIRWAYS IN IMMUNE RESPONSE

,

Solvent

r

Phosphoramldon 5 10- M

t

A

Substance P 10-6 M

~0.59 10mln Capsaicin 10-5 M

B

__J~ A

EFS

(

A

~~

Phosphoramldon 10 -5M

t

Ach 10-3M

Fig. 3. Original tracing. First we stimulated the bronchial strips by EFS (electrical field stimulation for 20 s: 1 ms width; 20 V intensity; 20 Hz) to examine cholinergic contraction (rapid contraction: open triangle) and non-eholinergic, nonadrenergic contraction (sustained contraction: closed triangle) (3, 21). After washing the strips, 10-3 M acetylcholine (Ach) was added to examine the viability of smooth muscle and the strips were washed again. Capsaicin (10-5 M) was added to deplete the endogenous tachykinins (B), and we added the solvent of capsaicin for the control (A). Then we added 10-50/ 0 OVA.At the relaxing point, phosphoramidon (10-5 M) was added. Phosphoramidon did not potentiate the contraction in the capsaicin-treated strip (B), though it did in the nontreated strip (A). Finally, substance P (10-6 M) caused contraction in both strips, suggesting no tachyphylaxis to tachykinins in the capsaicintreated strip.

Fig. 4. Effect of phosphoramidon on bronchial contraction induced by the immune response in capsaicin-treated bronchial tissue. The 10-50/ 0 ovalbumin (OVA) caused contraction in both capsaicin-treated and nontreated strips. When the contraction began to relax, we added NEP inhibitor phosphoramidon (10-5 M). We defined this point as 100% contraction and 0 min. Each value is the mean ± SEM (n = 6). Phosphoramidon did not potentiate the contraction induced by OVA in the capsaicin-treated strips (closed circles), but it significantly did in the nontreated strips (open circles). Double asterisks indicate p < 0.01; triple asterisks indicate p < 0.001 (Student's paired t test).

0/0

140 120 100

80 % Contraction

60 40 20

o -20

r ,- - . -I - . , . I - - , -i - , - - i ---i ,-

o

20

40

60

80

100

Time after Phosphoramidon (min)

0/0

Fig. 5. Effect of phosphoramidon on bronchial contraction induced by LTC4 • After the plateau of the 10-7 M LTC4-induced contraction was confirmed, 10-5 M phosphoramidon was added (closed circles). This point is defined as 100% contraction and 0 min. Each value is the mean ± SEM (n = 6). Phosphoramidon significantly potentiated the LTC4 induced contraction compared with the control (open circles). Double asterisks indicate p < 0.Q1; triple asterisks indicate p < 0.001 (Student's paired t test).

140 120 100

% Contraction

stance P at Glrr'-Phe', Phe'-Phe", and Gly9-Leu 1o (14); neurokinin A at Ser 5Phe" and Gly'-Leu" (15); and neurokinin B at Asp4-Phe 5, Phe5-Phe6, Phe6-VaF, and Gly'-Leu" (16). Pharmacologic and physiologic studies show that NEP inhibitors potentiate exogenously administered and

80 60 40 20 0

I

i

I

,

I

I

0

20

40

60

80

100

Time after Phosphoramidon (min)

endogenously released tachykinin effect on airway tissues. For example, they potentiate the tachykinin-induced tracheobronchial smooth muscle contraction in vitro (6, 7, 18, 19,21), bronchoconstriction in vivo (20, 22-24), bronchial gland secretion (17), vascular permeabil-

ity (25), and cough response (11) in guinea pigs, ferrets, and humans. In addition, tachykinins perfused through the airways of guinea pigs are cleaved into smaller peptides, which are consistent with NEP action (35). Furthermore, recombinant NEP prevents cough induced by exogenously administered substance P and endogenously released tachykinins (26). This evidence strongly suggests that NEP plays an important role in regulating the many physiologic and pathophysiologic effects of tachykinins in the airways. In the present study, OVAdid not cause bronchial contraction in nonsensitized guinea pigs, and bovine serum albumin did not cause bronchial contraction in guinea pigs sensitized to OVA. In contrast, OVA caused bronchial contraction in guinea pigs sensitized to OVA, suggesting that the OVA-induced contraction is due to the immune response. Although thiorphan affected the resting tension in two of eight tissues, phosphoramidon had no direct effect on resting tension. Therefore, potentiation of the OVA-induced contraction in the maintenance phase by NEP inhibitors strongly suggests that the immune response causes the release of tachykinin-like substances from the airways and that the contractile effect of tachykinin-like substances on bronchial smooth muscle is potentiated by inhibiting the cleavage of the tachykinin-like substances due to endogenous NEP. Capsaicin stimulates bronchial C fibers and releases tachykinins (13, 36), causing bronchial contraction in guinea pigs (21) and humans (7). The effect of capsaicin is not reproducible in the second exposure because capsaicin depletes the tachykinins (20, 21). In our study, the immune response caused contraction in both capsaicin-treated and nontreated strips. The contraction was not potentiated by phosphoramidon in the capsaicin-treated strips, but it was potentiated in the nontreated strips. Because substance P caused similar contractions after the immune response in both groups of strips, tachyphylaxis to tachykinins in the capsaicin-treated strips was unlikely. Therefore, the failure of potentiation by phosphoramidon in the capsaicin-treated strips is probably due to the lack of tachykinins in the tissues. From these results, we suggest that the bronchial contraction induced by the immune response is partially mediated by endogenously released tachykinin-like substances. Pretreatment with NEP inhibitors did not affect peak contractions, which were induced within 10 min after the addition

640

KOHROGI, YAMAGUCHI, KAWANO, HONDA, ANDO, AND ARAKI

0/0

120 100 80 % Contraction

60 40 20 0 0

20

40

60

80

100

Time after Phosphoramidon (min)

Fig. 6. Effect of phosphoramidon on bronchial contraction induced by serotonin. When the 10-6 M serotonininduced contractionbegan to relax, NEP inhibitor phosphoramidon (10-5 M) was added. This point is defined as 100% contraction and 0 min. Each value is the mean ± SEM (n = 6). Phosphoramidon significantly reduced the relaxing rate of the serotonin-induced contraction (closed circles) compared with the control (open circles). Asterisks indicate p < 0.05; double asterisks indicate p < 0.01 (Student's paired t test).

0/0

120 100 80 % Contraction

60 40 20 0 -20 0

20

40

60

80

100

Fig. 7. Effect of phosphoramidon on bronchial contraction induced by histamine. When the histamine-induced (10-6 M, n = 2; 10-5 M, n = 4) contraction began to relax, NEP inhibitor phosphoramidon (10-5 M) was added. We defined this point as 100% contraction and omin. Each value is the mean ± SEM. Phosphoramidon (closed circles) did not change the contractile effect of histamine compared with control (open circles).

Time after Phosphoramidon (min)

of OVA. Furthermore, there was no significant difference in the OVA-induced peak contraction between capsaicintreated and nontreated tissues. These results suggest that chemical mediators released in the early phase do not stimulate the release of tachykinin-like substances and that those released in the maintenance phase do. It is known that histamine release precedes the release of LTC4 , LTD4 , and LTE 4 (LT: slow-reacting substance of anaphylaxis [SRS-A]) after antigen challenge in human lung tissues and in purified human lung mast cells (37, 38), suggesting that LT may be mediators responsible for the release of tachykinins. The immune response causes the release of many chemical mediators - LT, histamine, serotonin, PAF, and many others - from mast cells (27). In the present study, Ll'Ca-induced contraction was potentiated by phosphoramidon. This result suggests that LTC4 and/or its metabolites LTD 4 and LTE 4 are important chemical mediators that release tachykinin-like substances to induce bronchial smooth muscle contraction in the immune response. Phosphoramidon significantly reduced the relaxing rate of serotonin-induced contraction, suggesting that serotonin also stimulates the release of tachykininlike substances from bronchial C fibers. In fact, serotonin is known to stimulate the C fibers of the vagal nerves (39). The potentiation in the serotonin-induced

contraction was smaller than that in the Ll'Cz-induced contraction, suggesting that LTC4 is more stimulating than serotonin in releasing tachykinin-like substances from bronchial C fibers. Saria and coworkers measured the small amount of substance P- and neurokinin A-like immunoreactivity in perfused guinea pig lung by stimulating with capsaicin, bradykinin, histamine, dimethylphenyl piperazinium, and vagal nerve electrical stimulation (13). Phosphoramidon in our study did not potentiate the effect of histamine. We did not measure the amount of released tachykinins, and the histamine concentration used in our study was 10- to 100-fold lower than that in the study of Saria and coworkers. For these reasons, we speculate that histamine at lower concentrations does not stimulate bronchial C fibers to release enough tachykinin-like substances, which physiologically induce bronchial contraction in an organ bath. In inflammation of the airways, bradykinin and histamine may be important chemical mediators in the release of tachykinins. In addition to these inflammatory chemical mediators, we first suggest in this report that LTC4 and serotonin stimulate the release of tachykinin-like substances from the bronchus in guinea pigs.. Ray and coworkers along with Reynolds and McEvoy showed that hyperpnea-induced bronchoconstriction is potentiated by NEP inhibitor in normal guinea pigs and attenuated in tachy-

kinin-depleted animals, suggesting that hyperpnea stimulates the release of tachykinin-like substances (40, 41). Sestini and coworkers showed that the immune response by OVA increases CI ion transport of the epithelium in the rat trachea in vitro and that this response is reduced in tachykinin-depleted animals, suggesting that the immune response partially induces the release of tachykinin-like substances (42). They showed that PAF increases CI ion transport by releasing tachykinin-like substances. In our study, PAF did not release tachykinin-like substances to cause bronchial contraction. On the contrary, Didier and coworkers showed that vascular permeability induced by the immune response in rats that were passively immunized with monoclonal IgE-antidinitrophenol was not due to tachykinins in the trachea, because both tachykinin-depleted and normal rats similarly responded to antigen (43). The possible explanations for these gaps may be differences of species and/or sensitivity of tachykinin receptors responsible for CI ion transport of the epithelium, for contraction of the bronchial smooth muscle, and for vascular permeability. Another possible explanation is that NEP in the airways cleaves tachykinin-like substances too fast to detect their physiologic actions, on which the effect of many other chemical mediators are superimposed. In the present study, inhibition of NEP unmasked the contractile effect of tachykinin-like substances on the bronchial smooth muscle. Because mast cells are in intimate contact with unmyelinated nerves that contain substance P in rat intestines (31), it is possible that this relationship also exists in the airways. If so, chemical mediators released from mast cells may immediately and strongly stimulate the nerves to release tachykinins. It is known that airway inflammation or damage of the epithelium induced by viral infection or cigarette smoke reduces the activity of NEP, thereby increasing the sensitivity and reactivity to tachykinins (44, 45). Therefore, in inflamed airways, the endogenously released tachykinins resulting from immune response may act strongly for a long duration on smooth muscle, bronchial gland, epithelium, and cough receptors. In summary, the results of the present study suggest that (1) some chemical mediators released by the immune response stimulate the release of tachykinin-like substances, (2) NEP plays an important role in modulating the bronchial contraction induced by substances

NEUTRAL ENDOPEPTIDASE AND TACHYKININS IN AIRWAYS IN IMMUNE RESPONSE

in the immune response, and (3) LTC4 and serotonin are chemical mediators that release the substances. Acknowledgment We thank Miss Hiromi Moriyasu for her technical support. References 1. Hua X-Y, Theodorsson-Norheim E, Brodin E, Lundberg JM, Hokfelt T. Multiple tachykinins (neurokinin A, neuropeptide K and substance P) in capsaicin-sensitive sensory neurons in the guineapig. Regul Pep 1985; 13:1-19. 2. Lundberg JM, Hokfelt T, Martling C-R, Saria A, Cuello C. Substance P-immunoreactive sensory nerves in the lower respiratory tract of various mammals including man. Cell Tissue Res 1984; 235:251-61. 3. Lundberg 1M, Saria A, Brodin E, RosellS, Folkers K. A substance P antagonist inhibits vagally induced increase in vascular permeability and bronchial smooth muscle contraction in the guinea pig. Proc Nat! Acad Sci USA 1983; 80:1120-4. 4. Nilsson G, Dahlberg K, Brodin E, Sundler F, Strandberg K. Distribution and constrictor effect of substance P in guinea pig tracheobronchial tissues. In: von Euler US, Pernow B, eds. Substance P. New York: Raven Press, 1977; 75-81. 5. Uchida Y, Nomura A, Ohtsuka M, et al. Neurokinin A as a potent bronchoconstrictor. Am Rev Respir Dis 1987; 136:718-21. 6. Naline E, Devillier P, Drapeau G, et al. Characterization of neurokinin effects and receptor selectivity in human isolated bronchi. Am Rev Respir Dis 1989; 140:679-86. 7. Honda I, Kohrogi H, Yamaguchi T, Ando M, Araki S. Enkephalinase inhibitor potentiates substance P- and capsaicin-induced bronchial contractions in humans. Am Rev Respir Dis (In Press). 8. Baker AP, Hillegass LM, Holden DA, Smith WJ. Effect of kallidin, substance P, and other basic polypeptides on the production of respiratory macromolecules. Am Rev Respir Dis 1977; 115:811-7. 9. Grunstein MM, Tanaka Dr, Grunstein lS. Mechanism of substance P-induced bronchoconstricti on in maturing rabbit. 1 Appl Physiol1984; 57:1238-46. 10. Al-Bazzaz Fl, Kelsey lG, Kaage WD. Substance P stimulation of chloride secretion by canine tracheal mucosa. Am Rev Respir Dis 1985; 131:86-9. 11. Kohrogi H, Graf PD, Sekizawa K, Borson DB, Nadel lA. Neutral endopeptidase inhibitors potentiate substance P- and capsaicin-induced cough in awake guinea pigs. 1 Clin Invest 1988; 82:2063-8. 12. Katsumata U, Sekizawa K, Inoue H, Sasaki H, Takishima T. Inhibitory actions of procaterol, a beta-2 stimulant, on substance P-induced cough in normal subjects during upper respiratory tract infection. Tohoku 1 Exp Med 1989; 158:105-6. 13. Saria A, Martling C-R, Yan Z, TheodorssonNorheim E, Gamse R, Lundberg 1M. Release of multiple tachykinins from capsaicin-sensitive sensory nerves in the lung by bradykinin, histamine, dimethylphenyl piperazinium, and vagal nerve stimulation. Am Rev Respir Dis 1988; 137:1330-5. 14. Matsas R, Fulcher IS, Kenny Al, Turner Al. Substance P and [leu]enkephalin are hydrolyzed

by an enzyme in pig caudate synaptic membranes that is identical with the endopeptidase of kidney microvilli. Proc Natl Acad SciUSA 1983;80:3111-5. 15. Hooper NM, Kenny Al, Turner AJ. The metabolism of neuropeptides: neurokinin A (substance K) is a substrate for endopeptidase-24.11 but not for peptidyl dipeptidase A (angiotensin-converting enzyme). Biochem 1 1985; 231:357-61. 16. Hooper NM, Turner AJ. Neurokinin B is hydrolysed by synaptic membranes and by endopeptidase-24.11 ('enkephalinase') but not by angiotensin converting enzyme. FEBS Lett 1985; 190:133-6. 17. Borson DB, Corrales R, Varsano S, et al. Enkephalinase inhibitors potentiate substance P-induced secretion of 35S04-macromolecules from ferret trachea. Exp Lung Res 1987; 12:21-36. 18. Stimler-Gerard NP. Neutral endopeptidase-like enzyme controls the contractile activity of substance P in guinea pig lung. 1 Clin Invest 1987;79:1819-25. 19. Sekizawa K, Tamaoki 1, Graf PD, Basbaum CB, Borson DB, Nadel lA. Enkephalinase inhibitor potentiates mammalian tachykinin-induced contraction in ferret trachea. J Pharmacol Exp Ther 1987; 243:1211-7. 20. Thompson JE, Sheppard D. Phosphoramidon potentiates the increase in lung resistance mediated by tachykinins in guinea pigs. Am Rev Respir Dis 1988; 137:337-40. 21. Djokic TD, Nadel JA, Dusser DJ,Sekizawa K, Graf PD, Borson DB. Inhibitors of neutral endopeptidase potentiate electrically and capsaicininduced noncholinergic contraction in guinea pig bronchi. J Pharmacol Exp Ther 1989; 248:7-11. 22. Shore SA, Stimler-Gerard NP, Coats SR, Drazen JM. Substance P-induced bronchoconstriction in the guinea pig: enhancement by inhibitors of neutral metalloendopeptidase and angiotensinconverting enzyme. Am Rev Respir Dis 1988; 137:331-6. 23. Shore SA, Drazen 1M. Degradative enzymes modulate airway responses to intravenous neurokinins A and B. 1 Appl Physiol 1989; 67:2504-11. 24. Drazen 1M, Shore SA, Gerard NP. Substance P-induced effects in guinea pig lungs: effects of thiorphan and captopril. 1 Appl Physiol 1989; 66:1364-72. 25. Umeno E, Nadel JA, Huang H-T, McDonald DM. Inhibition of neutral endopeptidase potentiates neurogenic inflammation in the rat trachea. 1 Appl Physiol 1989; 66:2647-52. 26. Kohrogi H, Nadel JA, Malfrog B, Gorman C, Bridenbaugh R, Patton JS, Borson DB. Recombinant human enkephalinase (neutral endopeptidase) prevents cough induced by tachykinins in awake guinea pigs. 1 Clin Invest 1989; 84:781-6. 27. Holgate ST, Hardy C, Robinson C, Agius RM, Howarth PH. The mast cell as a primary effector cell in the pathogenesis of asthma. J Allergy Clin Immunol 1986; 77:274-82. 28. Adams GK III, Lichtenstein L. In vitro studies of antigen-induced bronchospasm: effect of antihistamine and SRS-A antagonist on response of sensitized guinea pig and human airways to antigen. J Immunol 1979; 122:555-62. 29. Dahlen S-E, Hansson G, Hedqvist P, Bjorck T, Granstrom E, Dahlen B. Allergen challenge of lung tissue from asthmatics elicits bronchial contraction that correlates with the release of leukotrienes C 4 , D 4 , and E 4 • Proc Nat! Acad Sci USA 1983; 80:1712-6. 30. Martin TR, Galli sr,Katona 1M, Drazen 1M.

641

Role of mast cells in anaphylaxis: evidence for the importance of mast cells in the cardiopulmonary alterations and death induced by anti-IgE in mice. 1 Clin Invest 1989; 83:1375-83. 31. Stead RH, Tomioka M, Quinonez G, Simon GT, Felten SY, Bienenstock 1. Intestinal mucosal mast cells in normal and nematode-infected rat intestines are in intimate contact with peptidergic nerves. Proc Natl Acad Sci USA 1987;84:2975-9. 32. Dunn CJ, Elliott GA, Oostveen lA, Richards 1M. Development of a prolonged eosinophil-rich inflammatory leukocyte infiltration in the guineapig asthmatic response to ovalbumin inhalation. Am Rev Respir Dis 1988; 137:541-7. 33. Ronco P, Pollard H, Galceran M, Delauche M, Schwartz JC, Verroust P. Distribution of enkephalinase (membrane metalloendopeptidase, E.C. 3.4.24.11) in rat organs: detection usinga monoclonal antibody. Lab Invest 1988; 58:210-7. 34. Johnson AR, Ashton J, Schulz WW, Erdos EG. Neutral metalloendopeptidase in human lung tissue and cultured cells. Am Rev Respir Dis 1985; 132:564-8. 35. Martins MA, Shore SA, Gerard NP, Gerard C, Drazen JM. Peptidase modulation of the pulmonary effects of tachykinins in tracheal superfused guinea pig lungs. J Clin Invest 1990;85:170-6. 36. Coleridge HM, Coleridge lCG. Impulse activity in afferent vagal C-fibers with endings in the intrapulmonary airways of dogs. Respir Physiol 1977; 29:125-42. 37. LewisRA, Wasserman SI, Goetzl El, Austen KF. Formation of slow-reacting substance of anaphylaxis in human lung tissue and cells before release. 1 Exp Med 1974; 140:1133-46. 38. Schleimer RP, MacGlashan DW Jr, Peters SP, Pinckard RN, Adkinson NF r-, Lichtenstein LM. Characterization of inflammatory mediator release from purified human lung mast cells. Am Rev Respir Dis 1986; 133:614-7. 39. Christian EP, Taylor GE, Weinreich D. Serotonin increases excitability of rabbit C-fiber neurons by two distinct mechanisms. 1 Appl Physiol 1989; 67:584-91. 40. Ray DW, Hernandez C, Leff AR, Drazen JM, Solway 1. Tachykinins mediate bronchoconstriction elicited by isocapnic hyperpnea in guinea pigs. J Appl Physiol 1989; 66:1108-12. 41. ReynoldsAM, McEvoyRD. Tachykininsmediate hypocapnea-induced bronchoconstriction in guinea pigs. 1 Appl Physiol 1989; 67:2454-60. 42. Sestini P, Bienenstock J, Crowe SE, et al. Ion transport in rat tracheal epithelium in vitro: role of capsaicin-sensitive nerves in allergic reactions. Am Rev Respir Dis 1990; 141:393-7. 43. Didier A, Kowalski ML, lay 1, Kaliner MA. Neurogenic inflammation, vascular permeability, and mast cells: capsaicin desensitization fails to influence IgE-anti-DNP induced vascular permeability in rat airways. Am Rev Respir Dis 1990; 141: 398-406. 44. Dusser Dl, lacoby DB, Djokic TD, Rubinstein I, Borson DB, Nadel lA. Virus induces airwayhyperresponsivenessto tachykinins: role of neutral endopeptidase. J Appl Physiol1989; 67:1504-11. 45. Dusser DJ, Djokic TD, Borson DB, Nadel lA. Cigarette smoke induces bronchoconstrictor hyperresponsiveness to substance P and inactivates airway neutral endopeptidase in the guinea pig: possible role of free radicals. 1 Clin Invest 1989; 84:900-6