Absence of Tachykinin Involvement in Leukotriene D 4 and Antigen-Induced Contraction of Guinea Pig Isolated Bronchus 1

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1 /97/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 283, No. 3 Copyright 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 283: , 1997 Absence of Tachykinin Involvement in Leukotriene D 4 and Antigen-Induced Contraction of Guinea Pig Isolated Bronchus 1 SUSAN M. CIBULSKY and DAVID C. THOMPSON Department of Pharmaceutical Sciences, University of Colorado School of Pharmacy, Denver, Colorado Accepted for publication August 15, 1997 ABSTRACT In guinea pig airways, contractions induced by leukotriene D 4 or antigen are thought to be mediated primarily by an action of the agonist or of released mast cell-derived mediators directly on the airway smooth muscle cell. An indirect contractile action mediated by endogenous tachykinins also has been described for both of these stimuli. The present study evaluated the contribution of endogenous tachykinins to ovalbumin- and leukotriene D 4 -induced contractions in the guinea pig bronchus by modulating the concentrations of tachykinins within the tissues and by using neurokinin receptor antagonists. Acute depletion of tachykinins with capsaicin had no effect on responses elicited by either stimulus. Similarly, tetrodotoxin treatment failed to influence leukotriene D 4 -induced contractions. Inhibitors of neutral endopeptidase (thiorphan) and angiotensin-converting Guinea pig central airways are well supplied with tachykinin-containing nerves (Lundberg et al., 1984; Manzini et al., 1989). A variety of pharmacological agents have been shown to stimulate the release of tachykinins from guinea pig airways. These include muscarinic and nicotinic cholinoceptor agonists (Kizawa and Takayanagi, 1985; Saria et al., 1988; Martins et al., 1991), bradykinin (Saria et al., 1988), histamine (Saria et al., 1988; Martins et al., 1991), plateletactivating factor (Martins et al., 1991) and leukotriene D 4 (Bloomquist and Kream, 1990; Martins et al., 1991). Given that the tachykinins, substance P and neurokinin A, are potent contractile agonists on airway smooth muscle (Saria et al., 1988), it is conceivable that contractions of airway smooth muscle by the aforementioned pharmacological agonists could be supplemented or mediated indirectly by tachykinins released from intrinsic airway nerves. Several studies support this contention. First-generation tachykinin antagonists abolish nicotine-induced contraction of the guinea pig bronchus (Kizawa and Takayanagi, 1985) and attenuate contractions of the guinea pig trachea caused by leukotriene D 4 (Bloomquist and Kream, 1990). Depletion of endogenous Received for publication April 30, This work was supported by National Heart, Lung and Blood Institute Grant HL enzyme (lisinopril) enhanced neurally mediated tachykininergic responses and potentiated leukotriene D 4. The latter effect persisted in the presence of tetrodotoxin or the neurokinin antagonists CP99994 and SR48968 and in tissues treated acutely with capsaicin. The potentiation was absent, however, from bronchi incubated with L-cysteine. Ovalbumin-induced contractions were unaltered by inhibition of neutral endopeptidase and angiotensin-converting enzyme. These observations suggest that tachykinins are not involved in mediation of leukotriene D 4 - or antigen-induced contractions of the guinea pig bronchus. The ability of protease inhibitors to potentiate leukotriene D 4 but not antigen-induced responses is therefore ascribed to inhibition of bioinactivation of leukotriene D 4 to leukotriene E 4. tachykinins by acute capsaicin treatment prevents the contractile activity of nicotine in the guinea pig bronchus (Kizawa and Takayanagi, 1985). Furthermore, inhibition of tachykinin metabolism enhances increases in guinea pig airway opening pressure induced by histamine and methacholine (Martins et al., 1991) and augments antigen- and leukotriene C 4 -induced contractions of the guinea pig bronchus (Kohrogi et al., 1991). Employing pharmacological tools used by previous investigators and newer, more selective neurokinin antagonists, the present study re-evaluates the contribution of an indirect tachykinin-dependent mechanism to the mediation of contractions induced by leukotriene D 4,anagonist for which there is the greatest functional evidence of tachykinin involvement. Given the contribution of leukotrienes to antigen-induced responses, we also assessed the role of tachykinins in ovalbumin-induced contractions in airway tissue obtained from sensitized guinea pigs. Materials and Methods Animal sensitization. Male guinea pigs ( g) were sensitized to ovalbumin using the method of Andersson (1980). In summary, ovalbumin was adsorbed to aluminum hydroxide (10 mg/ml) and administered as a single i.p. dose (40 g/kg). Fourteen to 21 days later, animals were used in experiments in which antigen-induced effects were examined. ABBREVIATIONS: KHS, Krebs-Henseleit solution; NEP, neutral endopeptidase; NK, neurokinin. 1102

2 1997 Tachykinin Modulation of Airway Contraction 1103 In experiments on the effects of leukotrienes, male Hartley guinea pigs that weighed 250 to 450 g, were anesthetized using sodium pentobarbital (100 mg/kg i.p.). Upon attainment of surgical anesthesia, the trachea and lungs were removed en bloc and placed in KHS of the following composition (mm): NaCl, 118.2; KCl, 4.74; CaCl 2, 2.54; KH 2 PO 4, 1.19; MgSO 4, 1.19; NaHCO 3, 26.2; D( )-glucose, Indomethacin (2.8 M) was included in the KHS to abolish the influence of cyclooxygenase products on tissue responses. Right and left main stem bronchial ring preparations, obtained immediately distal to the tracheal bifurcation, were isolated from each animal. Tissue segments were cleaned of extraneous connective tissue and mounted on stainless steel tissue hooks in 15-ml glass-jacketed organ baths containing KHS maintained at 37 C and gassed with 5% CO 2 in O 2. An initial load of 6 g was placed on bronchial segments. Tissues were allowed to equilibrate for 60 min, during which time the bathing solution was exchanged at 10-min intervals. Upon attainment of a stable base-line tone, a maximal contractile concentration of ACh (1 mm) was administered to all tissues. After the ACh response had stabilized, the tissues underwent a 60-min washout period during which the bathing solution was exchanged at 10-min intervals. Upon reattainment of a steady base-line tone, tissues were subjected to various treatments as noted below. (1) Depletion of endogenous tachykinins. Endogenous tachykinins were depleted using acute capsaicin treatment (Kizawa and Takayanagi, 1985; Thompson et al., 1987). One of the pair of airway preparations was exposed to capsaicin (10 M) for 60 min, the other to capsaicin vehicle (15 l of ethanol) for 60 min. Tissues then underwent a 60-min washout period during which the bathing solution was exchanged at 10-min intervals. (2) Protease inhibition. One of the pair of airway preparations was exposed to a neutral endopeptidase inhibitor, thiorphan (10 M), to an angiotensin-converting enzyme inhibitor, lisinopril (10 M), or to their combination, and the other of the pair was exposed to an equivalent volume of inhibitor vehicle. An incubation period of 30 min was then allowed before leukotriene D 4 or ovalbumin was applied to the tissues. (3) Inhibition of action potential conduction. One of the pair of airway preparations was exposed to tetrodotoxin (10 M), the other to an equivalent volume of inhibitor vehicle. An incubation period of 30 min was then allowed before leukotriene D 4 or ovalbumin was applied to the tissues. (4) Inhibition of neurokinin receptors. One of the pair of airway preparations was exposed to NK 1 antagonist, CP99994 (1 M) (Mc- Kee et al., 1993), and to NK 2 antagonist, SR48968 (10 M) (Advenier et al., 1992), and the other of the pair was exposed to an equivalent volume of inhibitor vehicle. An incubation period of 30 min was then allowed before leukotriene D 4 or ovalbumin was applied to the tissues. (5) Inhibition of conversion of leukotriene D 4 to leukotriene E 4. Tissues were treated with L-cysteine (3 mm) 30 min before commencement of the leukotriene D 4 concentration-response curve to prevent metabolism of leukotriene D 4 to leukotriene E 4 by tissue aminopeptidases (Snyder et al., 1984; Snyder and Krell, 1984). After the appropriate treatment regimen, a concentration-response curve to ovalbumin (antigen) or leukotriene D 4 was elicited by the cumulative addition of agonist to the bathing solution. Experimental protocols were designed on a paired basis such that one of each pair of tissues from each animal (e.g., left or right hilar bronchus) acted as a control (vehicle or no treatment) while the other of the pair was subjected to treatment. Experiments were designed such that equivalent numbers of each airway segment were assigned to control and test groups. No differences in responsiveness of left and right hilar bronchus were evident. Neurally mediated tachykininergic responses were induced in bronchial preparations treated with atropine (1 M), guanethidine (10 M), propranolol (1 M) (to abolish cholinergic and adrenergic neural influences) and thiorphan (10 M) and lisinopril (10 M) (to inhibit tachykinin degradation). Pseudo-cumulative frequency-response curves for electrical field stimulation ( Hz, pulse duration 0.3 ms, pulse amplitude 10 V, 5 s stimulation duration/60 s) were applied to preparations using rectangular wave pulses generated by a Grass S-88 stimulator in series with a signal conditioner (Stimu-Splitter, Med. Lab. Instruments, Fort Collins, CO). All responses were measured isometrically using force-displacement transducers (Grass FT.03c) and were displayed on a Maclab8 (ADI Instruments, Sydney, Australia)/Macintosh (Apple Computer Corp., Cupertino, CA) computer system. Materials. All reagents were of analytical grade. Drugs were obtained from the following sources: ACh perchlorate, capsaicin, L-cysteine, leukotriene D 4, leukotriene E 4, ovalbumin (grade V), tetrodotoxin, thiorphan (Sigma Chemical Co., St. Louis, MO); CP99994, lisinopril, SR48968 (Merck Frosst, Kirkland, Québec, Canada). Indomethacin was initially dissolved in 0.1 M Na 2 CO 3 and diluted 10,000-fold in KHS. Capsaicin, CP99994 and SR48968 were dissolved in ethanol and diluted in KHS. All other drugs were dissolved in distilled water and diluted in KHS. Statistical analysis. Data are presented as means and associated S.E.M. The negative logarithmic values of the concentrations of contractile agonist that produced a 40% maximal ACh response are denoted as log EC 40 ACh. This concentration was selected (rather than log EC 50 or pd 2 ) because the maximal effect of leukotriene D 4 was usually not obtained in experiments. Nevertheless, in the presence of L-cysteine (where a maximum did appear to be achieved), the maximal effect of leukotriene D 4 was 80% ACh maximum. Accordingly, the leukotriene D 4 log EC 40 ACh approximates the leukotriene D 4 log EC 50. Log EC 40 ACh values were determined by regression analysis and interpolation of the linear portions of individual concentration-response curves. Statistical comparisons of concentration-response curves were conducted using two-way analysis of variance (ANOVA). Post-hoc Student s unpaired t tests were used to compare responses at specific agonist concentrations. P.05 was considered significant. Results Initial studies were conducted to verify the efficacy of treatments. A combination of the protease inhibitors thiorphan and lisinopril enhanced neurally mediated tachykininergic responses in the guinea pig bronchus (fig. 1). These inhibitor-augmented responses were absent from tissues treated with the neurokinin receptor antagonists CP99994 and SR48968 (fig. 1). This same neurokinin antagonist combination induced a 57-fold rightward shift of the substance P concentration-response curve (control, ; treated, , mean log EC 50 S.E.M.). Acute capsaicin treatment also abolished neurally mediated tachykininergic responses (data not shown). Leukotriene D 4 induced concentration-dependent contraction of the guinea pig bronchus, threshold responses becoming manifest at about 1 nm. In bronchi from sensitized animals, ovalbumin evoked concentration-dependent contractions with a threshold of 0.1 ng/ml. Acute capsaicin exposure had no influence on either the leukotriene D 4 (P.18, ANOVA) or the ovalbumin (P.17, ANOVA) concentration-response relationships in the bronchus (fig. 2). Similarly, neither contractile responses induced by leukotriene D 4 (P.25, ANOVA) nor those induced by ovalbumin (P.50, ANOVA) were affected by CP99994 and SR48968 (fig. 3) or by tetrodotoxin (P.66, ANOVA for leukotriene D 4 ) (data not shown). By contrast, treatment of tissues with the combination of thiorphan and lisinopril resulted in marked potentiation of leukotriene D 4 at all concentrations except the maximal concentration tested, 0.1 M (fig. 4). Neither inhibitor alone, however, altered the leukotriene D 4 concentration-

3 1104 Cibulsky and Thompson Vol. 283 Fig. 1. Modulation of neurally mediated tachykininergic responses by protease inhibition and neurokinin receptor antagonism. Neurally mediated tachykininergic responses were induced by electrical field stimulation (EFS) in bronchi treated with inhibitors of adrenergic and cholinergic nerve function. Frequency-response curves for EFS were obtained in control preparations (open circles), in preparations incubated with thiorphan (10 M) and lisinopril (10 M) (closed circles) and in tissues incubated with thiorphan (10 M), lisinopril (10 M) and the neurokinin antagonists CP99994 (1 M) and SR48968 (10 M) (closed squares). Responses were expressed as a percentage of the response to ACh (1 mm). Data represent the mean with one S.E.M. from four experiments. response curve (P.71 for lisinopril; P.91 for thiorphan, ANOVA) (fig. 5). The combination of thiorphan and lisinopril failed to influence ovalbumin (P.88, ANOVA)- or ACh (P.73, ANOVA)-induced contractions (fig. 6). The mechanism of the potentiation of leukotriene D 4 by the combination of thiorphan and lisinopril was examined further in additional experiments. The potentiation persisted in tissues treated with tetrodotoxin (fig. 7) or with the neurokinin antagonists CP99994 and SR48968 (fig. 7) and in those tissues that had undergone acute capsaicin treatment (fig. 8). Conducting experiments in the presence of L-cysteine resulted in abolition of the potentiation (P.85, ANOVA) (fig. 9). It should be noted, however, that L-cysteine treatment alone served to enhance the potency of leukotriene D 4 independently of the presence of thiorphan and lisinopril (control log EC 40 ACh vs. L-cysteine control log EC 40 ACh ). Contractions induced by leukotriene E 4 were unaltered by thiorphan and lisinopril (P.53, ANOVA) (fig. 10). Discussion Leukotriene D 4 is a potent constrictor of airway smooth muscle. In the present study, leukotriene D 4 induced concentration-dependent contraction in the guinea pig bronchus in the range 1 to 100 nm. The contractile actions of leukotriene D 4 on the airway smooth muscle were thought to be mediated directly via a specific leukotriene receptor (Mong et al., 1985). However, a more recent study has implicated an additional indirect mechanism that involves the release of endogenous tachykinins, the contractile activity of which reinforces or supplements leukotriene D 4 receptor-mediated contraction of Fig. 2. Lack of effect of acute capsaicin treatment on contractions elicited by leukotriene D 4 and antigen. Leukotriene D 4 was administered in a cumulative manner to bronchial preparations (upper diagram). Antigen-induced responses were obtained by cumulative application of ovalbumin to bronchi from sensitized animals (lower diagram). Responses were obtained in control preparations (open symbols) and in preparations exposed previously to capsaicin (10 M) (closed symbols) and were expressed as a percentage of the response to ACh (1 mm). Data represent the mean with one S.E.M. from 5 to 6 experiments. the guinea pig trachea (Bloomquist and Kream, 1990). The tachykinins, substance P and neurokinin A, are localized in unmyelinated sensory nerves, afferent C-fibers, innervating the airways (Lundberg et al., 1984). A variety of stimuli are known to activate C-fibers in the airways in vivo (Coleridge and Coleridge, 1984), and many of these, including bradykinin, capsaicin and histamine, elicit tachykinin release from guinea pig airways in vitro (Saria et al., 1988; Martins et al., 1991). Given that tachykinins, such as substance P and neurokinin A, exert contractile effects on the guinea pig airway smooth muscle (Saria et al., 1988; Buckner et al., 1991), it is reasonable to expect that tachykinins released by contractile agonists contribute to the overall contractile response. The possible contribution of endogenous tachykinins to the manifestation of contractions elicited by leukotriene D 4 was investigated because there is evidence linking this agonist to tachykinins or tachykinin-containing nerves (Stewart et al., 1984; Bloomquist and Kream, 1990; Martins et al., 1991). In the present study, we evaluated the role of tachykinins by pharmacologically modulating the concentrations of endogenous tachykinins within guinea pig bronchus or by antago-

4 1997 Tachykinin Modulation of Airway Contraction 1105 Fig. 3. Neurokinin receptor blockade does not alter leukotriene D 4 or antigen-induced responses. Leukotriene D 4 was administered in a cumulative manner to bronchial preparations (upper diagram). Antigeninduced responses were obtained by cumulative application of ovalbumin to bronchi from sensitized animals (lower diagram). Responses were obtained in control preparations (open symbols) and in preparations treated with NK 1 antagonist (CP99994, 1 M) and NK 2 antagonist (SR48968, 10 M) (closed symbols) and were expressed as a percentage of the response to ACh (1 mm). Data represent the mean with one S.E.M. from 4 to 6 experiments. Fig. 4. Potentiation of leukotriene D 4 by protease inhibition. Leukotriene D 4 was administered in a cumulative manner to control preparations (open circles) and to preparations incubated with the protease inhibitors thiorphan (10 M) and lisinopril (10 M) (closed circles). Responses were expressed as a percentage of the response to ACh (1 mm). Data represent the mean with one S.E.M. from six experiments. *P.05, unpaired Student s t test, compared with the response at the same leukotriene D 4 concentration in control preparations. nizing neurokinin receptors that mediate tachykinin effects. The guinea pig bronchus was chosen because it possesses a denser tachykininergic nervous innervation than the guinea pig trachea (Manzini et al., 1989). The role of tachykinins in antigen-induced responses was also assessed, because contractions in the guinea pig airways are mediated primarily by leukotrienes and histamine (Undem et al., 1989), two mediators that have been shown to elicit the release of tachykinins (Saria et al., 1988; Bloomquist and Kream, 1990; Martins et al., 1991). Capsaicin administered acutely to guinea pig airway tissue results in the abolition of tachykinin-mediated nonadrenergic noncholinergic excitatory responses induced by electrical field stimulation (Kizawa and Takayanagi, 1985; Thompson et al., 1987). This treatment had no effect on the concentration-response relationships for antigen (ovalbumin) or leukotriene D 4, which suggests that endogenous tachykinins of C-fiber origin (or other agents subject to capsaicin-induced depletion) do not contribute to the contractile responses elicited by these agents. To evaluate more directly the contribution of tachykinins to ovalbumin- and leukotriene D 4 -induced contractions, we examined the inhibitory effects of newer, more selective antagonists of NK 1 (i.e., CP99994) and NK 2 (i.e., SR48968) receptors. In these studies, concentrations of CP99994 and SR48968 that abolished neurally mediated tachykininergic responses failed to influence contractions elicited by either ovalbumin or leukotriene D 4. These results contrast with those obtained by Bloomquist and Kream (1990), wherein the substance P antagonist [D-Pro 4, D- Trp 7,9 ]-substance P 4-11 was shown to inhibit leukotriene D 4 -induced contractile responses in the guinea pig trachea. These variant results may reflect a tissue-dependent difference in the role of tachykinins in leukotriene D 4 -induced contraction; Bloomquist and Kream utilized the trachea, whereas the present study was conducted in the bronchus. However, the possibility that [D-Pro 4, D-Trp 7,9 ]-substance P 4-11 acted as a leukotriene receptor antagonist was not excluded by Bloomquist and Kream (1990). The lack of inhibitory activity of CP99994 and SR48968 on leukotriene D 4 responses excluded such a possibility in the present study. The airway contractile actions of the tachykinins are modulated by tissue enzymes, including NEP and ACE, such that the potency of the endogenously released or exogenously applied tachykinins are enhanced in preparations in which these enzymes are inhibited (Djokic et al., 1988; Thompson et al., 1989; Warner et al., 1990). Alterations in tissue expression of NEP or ACE also appear to regulate the biological activity of the tachykinins. For example, viral or cigarette smoke exposure enhances the airway smooth muscle contractile activity of endogenous and exogenous tachykinins by down-regulating NEP expression (Dusser et al., 1989a,b). To enhance the potential for tachykinin involvement in contractions, experiments were repeated in the presence of thiorphan and lisinopril, inhibitors of NEP and ACE, respectively. Initial studies verified that neurally mediated tachykininergic responses in the guinea pig bronchus were enhanced by such treatment, as were contractile responses induced by substance P and neurokinin A (Thompson et al., 1989). In the presence of both of these inhibitors, leukotriene D 4 was po-

5 1106 Cibulsky and Thompson Vol. 283 Fig. 5. Individual protease inhibitors fail to potentiate leukotriene D 4. Leukotriene D 4 was administered in a cumulative manner to control preparations (open circles) and to preparations incubated with thiorphan (10 M) (closed circles, upper diagram) or lisinopril (10 M) (closed circles, lower diagram). Responses were expressed as a percentage of the response to ACh (1 mm). Data represent the mean with one S.E.M. from six experiments. tentiated, which suggests that tachykinins released by leukotriene D 4 contribute to the contractile response but normally do not do so as a result of rapid degradation by tissue proteases. It is notable that leukotriene D 4 -induced responses were not affected by the individual application of thiorphan or lisinopril. A similar requirement for the combination of both inhibitors for enhancement of neurally mediated tachykininergic responses in the guinea pig bronchus has been noted previously (Thompson et al., 1989). The potentiation of leukotriene D 4 by thiorphan and lisinopril provided circumstantial evidence in favor of a role for tachykinins. However, it is possible that other substances, such as calcitonin gene-related peptide, were protected from proteolysis by these inhibitors and contributed to the response (Martling et al., 1988; Tschirhart et al., 1990; Katayama et al., 1991). Accordingly, we further examined the role of neurally released tachykinins using other pharmacological tools shown in initial experiments to abolish neurally mediated tachykininergic responses. The potentiation of leukotriene D 4 was not prevented by either acute capsaicin desensitization or the selective neurokinin receptor antagonists, a result that argues strongly against tachykinins contributing to the enhancement of leukotriene D 4 -induced responses by Fig. 6. Lack of effect of protease inhibitors on antigen- or ACh-induced contractions. Antigen-induced responses were obtained by cumulative application of ovalbumin to bronchi from sensitized animals (upper diagram). ACh was administered in a cumulative manner to bronchial preparations (lower diagram). Responses were obtained in control preparations (open symbols) and in preparations treated with the protease inhibitors thiorphan (10 M) and lisinopril (10 M) (closed symbols) and were expressed as a percentage of the response to ACh (1 mm). Data represent the mean with one S.E.M. from 5 to 6 experiments. thiorphan and lisinopril. Clearly, had tachykinins been implicated in the potentiation, these treatments would have prevented it, particularly when the concentrations used were sufficient to prevent neurally mediated tachykininergic responses and/or elicit a 57-fold rightward shift of the substance P concentration-response curve. It should be noted that prevention by acute capsaicin desensitization of NEP inhibitor-induced enhancement of antigen-induced responses has been used as evidence of tachykinin involvement in the guinea pig bronchus (Kohrogi et al., 1991). The enhancing action of thiorphan and lisinopril appeared specific for leukotriene D 4 because contractions induced by ACh or antigen were unaffected by these inhibitors. In guinea pig airways, leukotriene D 4 can be metabolized to leukotriene E 4 by an aminopeptidase (Snyder et al., 1984; Aharony et al., 1985). This represents a bioinactivation step insofar as the contractile potency of leukotriene D 4 in guinea pig airways is enhanced when conversion to leukotriene E 4 is prevented by L-cysteine, an inhibitor of the aminopeptidase (Snyder et al., 1984; Snyder and Krell, 1984). Inhibition of leukotriene D 4 metabolism by thiorphan and lisinopril could

6 1997 Tachykinin Modulation of Airway Contraction 1107 Fig. 7. Inactivity of inhibition of action potential conduction or neurokinin antagonism on protease inhibitor-induced potentiation of leukotriene D 4. Leukotriene D 4 was administered in a cumulative manner to tissues treated with tetrodotoxin (10 M) (upper figure) and to tissues treated with NK 1 antagonist (CP99994, 1 M) and NK 2 antagonist (SR48968, 10 M) (lower figure). Preparations were incubated with vehicle (control, open circles) or with the protease inhibitors thiorphan (10 M) and lisinopril (10 M) (closed circles). Responses were expressed as a percentage of the response to ACh (1 mm). Data represent the mean with one S.E.M. from 4 to 6 experiments. *P.05, unpaired Student s t test, compared with the response at the same leukotriene D 4 concentration in control preparations. therefore underlie the observed potentiation. The present study reproduced the results of previous studies in that L- cysteine potentiated leukotriene D 4 in the bronchus. Under these conditions, however, thiorphan and lisinopril failed to affect leukotriene D 4 -induced contractions. L-Cysteine did not prevent the combination of thiorphan and lisinopril from enhancing substance P-induced contractions of the bronchus (data not shown), and this excludes the possibility that L- cysteine was interfering with thiorphan or lisinopril in a physicochemical manner. Finally, leukotriene E 4 -induced contractions were not altered by thiorphan and lisinopril, which makes it unlikely that the observed potentiation of leukotriene D 4 was due to protease inhibitor-induced potentiation of its metabolite, leukotriene E 4. Taken together, these results provide evidence that potentiation of leukotriene D 4 in the guinea pig bronchus involves inhibition of the conversion of leukotriene D 4 to leukotriene E 4. Leukotrienes mediate, at least in part, contractions elicited Fig. 8. Failure of acute capsaicin treatment to prevent protease inhibitor-induced potentiation of leukotriene D 4. Leukotriene D 4 was administered in a cumulative manner to bronchial preparations exposed previously to capsaicin (10 M). Responses were obtained in control preparations (open circles) and in preparations incubated with the protease inhibitors thiorphan (10 M) and lisinopril (10 M) (closed symbols) and were expressed as a percentage of the response to ACh (1 mm). Data represent the mean with one S.E.M. from eight experiments. Fig. 9. L-Cysteine treatment prevents protease inhibitor-induced potentiation of leukotriene D 4. Leukotriene D 4 was administered in a cumulative manner to bronchial preparations incubated with L-cysteine (3 mm) to prevent metabolism to leukotriene E 4. Responses were obtained in control preparations (open circles) and in preparations incubated with the protease inhibitors thiorphan (10 M) and lisinopril (10 M) (closed symbols) and were expressed as a percentage of the response to ACh (1 mm). Data represent the mean with one S.E.M. from five experiments. by antigen in sensitized guinea pig airways (Undem et al., 1989; Ro et al., 1991). Given the potentiating action of lisinopril and thiorphan on leukotriene D 4, a similar effect might have been anticipated on antigen-induced responses. Indeed, NEP inhibitors have been shown previously to enhance contractions induced by a single ovalbumin concentration (10 g/ml) in sensitized guinea pig bronchi (Kohrogi et al., 1991). In their studies, the NEP inhibitors, thiorphan and phosphoramidon, did not affect the peak of the antigen-induced contraction but rather augmented the waning phase of the antigen response, i.e., after attainment of the peak contraction. In the present study, thiorphan and lisinopril had no

7 1108 Cibulsky and Thompson Vol. 283 Fig. 10. Protease inhibition has no effect on leukotriene E 4 -induced contractions. Leukotriene E 4 was administered in a cumulative manner to control preparations (open circles) and to preparations incubated with the protease inhibitors thiorphan (10 M) and lisinopril (10 M) (closed circles). Responses were expressed as a percentage of the response to ACh (1 mm). Data represent the mean with one S.E.M. from six experiments. effect on contractions induced by the cumulative addition of antigen. These differing results may be related to the means of antigen application, i.e., single (Kohrogi and colleagues, 1991) vs. cumulative (present study) addition. However, cumulative addition involves the administration of an incrementally increasing antigen concentration to an established antigen-induced contraction. If tachykinins did contribute to maintenance of the antigen-induced contraction in the presence of NEP inhibitor, one would expect responses in the cumulative curve to be increased, because subsequent antigen additions would be made to an augmented response. An alternative explanation for the disparate findings involves the importance of leukotrienes in mediation of antigen-induced contractions in bronchi obtained from animals subjected to different sensitization regimens. In the present study, tissues were obtained from animals sensitized to produce IgE and IgG 1 antibodies (Andersson, 1980). By contrast, Kohrogi and colleagues (1991) sensitized animals using higher antigen doses (milligram range), a procedure that is likely to lead to the preferential development of IgG antibodies (Andersson, 1980). Antigen-induced leukotriene release is lower from tracheae obtained from IgE-sensitized guinea pigs than from those obtained from IgG-sensitized guinea pigs, even though the contractile responses do not differ substantially (Ro et al., 1991). The failure of L-cysteine to enhance the antigen-induced contraction in either IgE- or IgG 1 -sensitized tissues (Ro et al., 1991) brings into question the importance of leukotriene D 4 (at least) in mediation of these responses. The absence of an effect of thiorphan and lisinopril on antigen-induced contractions would be consistent with the results of Ro and colleagues (1991) if inhibition of metabolism underlies the enhancement of leukotriene D 4 by these protease inhibitors. Much interest has been shown in the possibility that an axon reflex exists in the airways such that stimulation of a nerve ending in the tachykinin-containing C-fiber network results in the spread of action potentials throughout the local C-fiber network and causes the release of tachykinins from the C-fiber nerve endings, which could then induce bronchoconstriction (in addition to other biological actions) (Barnes, 1986). Tetrodotoxin is a nerve toxin that abolishes action potentials by blockade of sodium channels (Catterall, 1980). In the present study, leukotriene D 4 -induced contractions were unaffected by tetrodotoxin treatment, as was the potentiation of leukotriene D 4 by the protease inhibitors. In the guinea pig trachea, tetrodotoxin inhibited maximal contractile responses induced by leukotriene D 4 (Bloomquist and Kream, 1990). It could be argued that the failure to identify such an effect in bronchi in the present study may be related to the use of insufficient concentrations of leukotriene D 4 to elicit a maximal response. However, 0.1 M leukotriene D 4 applied to L-cysteine-treated bronchi appeared to elicit a near-maximal contractile effect that was not affected by the protease inhibitors. If a tachykinin-dependent, tetrodoxinsensitive component contributed to the contraction, protease inhibition would have been expected to augment the response. In summary, the present results using more selective neurokinin antagonists argue that endogenous tachykinins do not contribute to contractions induced by antigen or leukotriene D 4 in the guinea pig bronchus, a tissue that is well endowed with tachykinin-containing nerves. Acknowledgments The authors wish to thank Dr. I. W. Rodger of Merck Frosst for the generous gift of CP99994 and SR48968, Dr. R. J. Altiere for constructive comments and Glenda Tate for technical assistance. References ADVENIER, C., ROUISSI, N., NGUYEN, Q. T., EMONDS-ALT, X., BRELIERE, J. C., NELIAT, G., NALINE, E.AND REGOLI, D.: Neurokinin A (NK 2 ) receptor revisited with SR 48968, a potent non-peptide antagonist. Biochem. Biophys. Res. Commun. 184: , AHARONY, D., DOBSON, P.T. AND KRELL, R.D.:In vitro metabolism of [ 3 H]- peptide leukotrienes in human and ferret lung: A comparison with the guinea pig. Bioch. Biophys. Res. Comm. 131: , ANDERSSON, P.: Antigen-induced bronchial anaphylaxis in actively sensitized guinea-pigs. Pattern of response in relation to immunization regimen. Allergy 35: 65 71, BARNES, P. J.: Asthma as an axon reflex. Lancet 1: , BLOOMQUIST, E. I. AND KREAM, R. M.: Release of substance P from guinea pig trachea by leukotriene D 4. Exptl. Lung Res. 16: , BUCKNER, C. K., GHANEKAR, S. V., KAYS, J. S., KRELL, R. D., FISHLEDER, R.I., WILL, J. A. AND VANN, J. M.: Pharmacological studies of tachykinin receptors mediating contraction of isolated airway smooth muscle. Ann. N.Y. Acad. Sci. 629: , CATTERALL, W. A.: Neurotoxins that act on voltage-sensitive sodium channels in excitable membranes. Ann. Rev. Pharmacol. Toxicol. 20: 15 43, COLERIDGE, J.C.G.AND COLERIDGE, H. M.: Afferent vagal C-fibre innervation of the lung and airways and its functional significance. Rev. Physiol. Biochem. Pharmacol. 99: 1 10, DJOKIC, T. D., NADEL, J. A., DUSSER, D. J., SEKIZAWA, K., GRAF,P.D.AND BORSON, B. A.: Inhibitors of neutral endopeptidase potentiate electrically and capsaicin-induced noncholinergic contraction in guinea pig bronchi. J. Pharmacol. Exp. Ther. 248: 7 11, DUSSER, D. J., DJOKIC, T. D., BORSON, D. B. AND NADEL, J. A.: Cigarette smoke induces bronchoconstrictor hyperresponsiveness to substance P and inactivates airway neutral endopeptidase in the guinea pig. Possible role of free radicals. J. Clin. Invest. 84: , 1989b. DUSSER, D. J., JACOBY, D. B., DJOKIC, T. D., RUBINSTEIN, I., BORSON, D. B. AND NADEL, J. A.: Virus induces airway hyperresponsiveness to tachykinins: Role of neutral endopeptidase. J. Appl. Physiol. 67: , 1989a. KATAYAMA, M., NADEL, J. A., BUNNETT, N. W., DI MARIA, G. U., HAXHUI, M.AND BORSON, D. B.: Catabolism of calcitonin gene-related peptide and substance P by neutral endopeptidase. Peptides 12: , KIZAWA, Y. AND TAKAYANAGI, I.: Possible involvement of substance P immunoreactive nerves in the mediation of nicotine-induced contractile responses in isolated guinea pig bronchus. Eur. J. Pharmacol. 113: , KOHROGI, H., YAMAGUCHI, T., KAWANO, O., HONDA, I. AND ANDO, M.: Inhibition of neutral endopeptidase potentiates bronchial contraction induced by immune response in guinea pigs in vitro. Am. Rev. Resp. Dis. 144: , LUNDBERG, J. M., HÖKFELT, T., MARTLING, C.-R., SARIA, A. AND CUELLO, C.: Substance P-immunoreactive sensory nerves in the lower respiratory tract of various mammals including man. Cell. Tissue Res. 235: , MANZINI, S., CONTI, S., MAGGI, C. A., ABELLI, L., SOMMA, V., DEL BIANCO, E. AND

8 1997 Tachykinin Modulation of Airway Contraction 1109 GEPPETTI, P.: Regional differences in the motor and inflammatory responses to capsaicin in guinea pig airways. Amer. Rev. Resp. Dis. 140: , MARTINS, M. A., SHORE, S. A. AND DRAZEN, J. M.: Release of tachykinins by histamine, methacholine, PAF, LTD 4, and substance P from guinea pig lungs. Am. J. Physiol. 261: L449 L455, MARTLING, C.-R., SARIA, A., FISCHER, J. A., HÖKFELT, T. AND LUNDBERG, J. M.: Calcitonin gene-related peptide and the lung: Neuronal coexistence with substance P, release by capsaicin and vasodilatory effect. Regul. Peptide 20: , MCKEE, K. T., MILLAR, L., RODGER, I. W. AND METTERS, K. M.: Identification of both NK 1 and NK 2 receptors in guinea-pig airways. Br. J. Pharmacol. 110: , MONG, S., WU, H.-L., SCOTT, M. O., LEWIS, M. A., CLARK, M. A., WEICHMAN,B.M., KINZIG, C. M., GLEASON, J. G. AND CROOKE, S. T.: Molecular heterogeneity of leukotriene receptors: Correlation of smooth muscle contraction and radioligand binding in guinea pig lung. J. Pharmacol. Exp. Ther. 234: , RO, J. Y., BUCKNER, C. K., BRENDEL, J. K., FISHLEDER, R. I. AND GRAZIANO, F. M.: Influence of indomethacin and L-cysteine on histamine and peptidoleukotriene release from superfused tracheas taken from guinea pigs passively sensitized with IgG1 and IgE antibodies. J. All. Clin. Immunol. 87: , SARIA, A., MARTLING, C.-R., YAN, Z., THEODORSSON-NORHEIM, E., GAMSE, R. AND LUNDBERG, J. M.: Release of multiple tachykinins from capsaicin-sensitive sensory nerves in the lung by bradykinin, histamine, dimethylpiperazinium, and vagal nerve stimulation. Amer. Rev. Resp. Dis. 137: , SNYDER, D. W., AHARONY, D., DOBSON, P., TSAI, B. S. AND KRELL, R. D.: Pharmacological and biochemical evidence for metabolism of peptide leukotrienes by guinea-pig airway smooth muscle in vitro. J. Pharmacol. Exp. Ther. 231: , SNYDER, D. W. AND KRELL, R. D.: Pharmacological evidence for a distinct leukotriene C 4 receptor in guinea-pig trachea. J. Pharmacol. Exp. Ther. 231: , STEWART, A. G., THOMPSON, D. C. AND FENNESSY, M. R.: Involvement of capsaicinsensitive afferent neurones in a vagal-dependent interaction between leukotriene D 4 and histamine on bronchomotor tone. Agents Actions 15: , THOMPSON, D. C., ALTIERE, R. J. AND DIAMOND, L.: Modulation of neurallymediated responses in airways by endogenous substance P. Amer. Rev. Resp. Dis. 135: A179, THOMPSON, D. C., DIAMOND, L. AND ALTIERE, R. J.: Differential susceptibility of tachykinins to enzyme inhibition in guinea pig airways. Amer. Rev. Resp. Dis. 139: A235, TSCHIRHART, E., BERTRAND, C., THEODORSSON, E. AND LANDRY, Y.: Evidence for the involvement of calcitonin gene-related peptide in the epithelium-dependent contraction of guinea-pig trachea in response to capsaicin. Naunyn- Schmiedeberg s Arch. Pharmacol. 342(2): , UNDEM, B. J., ADAMS III, G. K. AND BUCKNER, C. K.: Influence of electrical field stimulation on antigen-induced contraction and mediator release in the guinea pig isolated superfused trachea and bronchus. J. Pharmacol. Exp. Ther. 249: 23 30, WARNER, E. A., KRELL, R. D. AND BUCKNER, C. K.: Pharmacological studies on the differential influence of inhibitors of neutral endopeptidase on nonadrenergic, noncholinergic contractile responses of the guinea pig isolated hilar bronchus to transmural electrical field stimulation and exogenously applied tachykinins. J. Pharmacol. Exp. Ther. 254: , Send reprint requests to: David C. Thompson, Ph.D., Department of Pharmaceutical Sciences, University of Colorado School of Pharmacy, 4200 East 9th Avenue, Denver, Colorado,