A study of the action of bradykinin and bradykinin analogues

Transcription

1 NS 214, pp Journal of Physiology (1994), A study of the action of bradykinin and bradykinin analogues in the human nasal airway C. E. Austin and J. C. Foreman* Department of Pharmacology, University College London, Gower Street, London WC1E 6BT 1. The aim of this study was to investigate the action of bradykinin on resistance to airflow and on vascular permeability in the human nasal airway, and to explore the receptor mediating these effects. 2. Aerosol administration of bradykinin (1-1,ug) caused a dose-related increase in nasal airway resistance (NAR) and an increase in albumin content of nasal lavage. 3. The bradykinin antagonists, [1-adamantane acetyl-d-arg', Hyp3, Thi58, D-Phe7]- bradykinin, 1 jug, and [D-Argo, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin, 1 jug, given 2 min before bradykinin, inhibited the increase in NAR and the increase of albumin content of nasal lavage caused by bradykinin. 4. The bradykinin antagonist, [D-Argo, Hyp3, D-Phe7]-bradykinin (1,ug) did not affect the increase in NAR produced by bradykinin, or the albumin content of nasal lavage. Increasing the dose of the antagonist to 1 jug did not change the increase in NAR induced by bradykinin. 5. The selective B1 kinin receptor agonist, [Des-Arg']-kallidin (1 jug) did not affect NAR or the albumin content of nasal lavage. 6. The receptor mediating increased NAR and the release of albumin induced by bradykinin in the human nasal airway appears not to be a B1 kinin receptor. The data are not entirely consistent with the effects of bradykinin in the human nasal airway being mediated by a B2 kinin receptor. It has previously been shown that in patients with allergic rhinitis, antigen challenge releases kinins into nasal lavage (Proud, Togias, Naclerio, Crush, Norman & Lichtenstein, 1983). This observation raises the question as to whether or not kinins are mediators of the symptoms of allergic rhinitis. Both bradykinin and kallidin (Lys-bradykinin) are found in nasal lavage following antigen challenge (Proud et al. 1983). Moreover, kinins appear in the nasal secretions of patients with naturally occuring allergic rhinitis (Svensson, Andersson, Persson, Venge, Alkner & Pipkorn, 199). Application of bradykinin to the nasal airway has been shown to cause an increase in nasal airway resistance and an increase in vascular permeability in the nasal airway (Proud, Reynolds, Lacapra, Kagey-Sobotka, Lichtenstein & Naclerio, 1988). The kinin receptor which is mediating these effects, may be B1 or B2, as classified by the relative potencies of bradykinin analogues (Regoli & Barabe, 198; Regoli, Rahleb, Dion & Drapeau, 199). The possible existence of a B3 receptor has also been suggested (Farmer, Burch, Meeker & Wilkins, 1989). In the human nasal airway, a B1 receptor agonist was found to be inactive in comparison with bradykinin and kallidin, and it was suggested that a B2 receptor mediates the effects of bradykinin in the human nasal airway (Rajakulasingam, Polosa, Holgate & Howarth, 1991). Our aim was to use antagonists to attempt to identify the kinin receptor involved in mediating the increase in nasal airway resistance and increase in vascular permeability of the human nasal airway. METHODS Subjects All subjects gave informed consent and the study was approved by the local Ethics Committee at University College London. For all studies, normal, healthy volunteers aged 21-4 years were used. Subjects suffering from a cold or *To whom correspondence should be addressed.

2 352 J. Physiol C. E. Austin and J C. Foreman reporting nasal symptoms were excluded. Subjects were taking no medication at the time of, or in the 2 weeks prior to the experiments. Experiments were performed in a laboratory with controlled temperature and humidity. Nasal resistance measurements Nasal airway resistance (NAR) was measured by active posterior rhinomanometry using a Mercury Electronics (Kilwinning, UK) NR6 Rhinomanometer. The instrument was programmed to calculate nasal resistance at a reference pressure of 75 Pa. The subject held a mask, with airtight seal, over the nose and mouth and breathed through the nose. Airflow was monitored by a pneumotachograph, and pressure within the oropharynx was monitored by an oral pressure cannula placed over the tongue and held through sealed lips. The instrument was programmed to give the mean NAR over four nasal cycles, and for each subject, three consecutive series of four breaths were used to calculate an overall mean of twelve breaths. Readings were only accepted if the coefficient of variation of the overall mean NAR for a subject was less than 2 %. Nasal lavage Nasal lavages were performed according to the method of Naclerio and co-workers (Naclerio et al. 1983). Subjects were in a sitting position with the head extended 3 deg from the horizontal. Five millilitres of warmed, sterile saline solution was instilled into one nostril while the subject abstained from breathing or swallowing. After 1 s the subject leaned forward and expelled the lavage fluid into a collection vessel, which was stored on ice until the completion of the experiment. Approximately 8 % of the lavage was recovered. In each experiment, three prewashes were collected over a 1 min period in order to remove pre-existing nasal secretions since we wished only to study the release of albumin induced by bradykinin. The third prewash was retained and served as a baseline. All samples were stored at -2 C until analysis. Albumin measurement Albumin was measured by single radial immunodiffusion. A protein standard serum was used to establish a calibration curve, and albumin from human sera was used as a control to confirm the function of the immunodiffusion plates. Nasal challenge Nasal challenge was undertaken using a nasal pump spray (Perfect-Valois, UK Ltd, Milton Keynes, UK), delivering 1,ul per activation, with a 98 % degree of accuracy. The device was placed in one nostril and activated once, then repeated for the second nostril. All solutions were at room temperature, and doses of substances were controlled by varying the concentration of the solutions. Fresh solutions of all challenge agents were made each day from stock solutions stored at -2 C. Bradykinin was dissolved in saline (NaCl, 154 mm) to achieve a range of concentrations from 41 to 1 mg ml-'. [D-Argo, Hyp3, D-Phe7]-bradykinin, [D-Argo, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin and [Des-Argt]-kallidin were dissolved in saline to give 1 mg ml' solutions. [1-Adamantane acetyl-d-argo, Hyp3, Thi5"8, D-Phe7]-bradykinin was dissolved in glacial acetic acid, neutralized with sodium hydroxide and made up to a 1 mg ml-' solution with distilled water. All solutions used were sterile. The vehicle controls used in the experiments were all sterile saline except in the experiments with [1-adamantane acetyl-d-argo, Hyp3, Thi5'8, D-Phe7]-bradykinin when the vehicle control was prepared from distilled water containing acetic acid neutralized with sodium hydroxide. Study design For each experiment, subjects were first challenged with vehicle, then by active posterior rhinomanometry, three NAR measurements, each of four nasal cycles, were taken every 5 min over a 15 min period to provide a baseline. On the first of two visits, subjects were given incremental doses of bradykinin (1-1 /ug) in each nostril, at 3 min intervals. After each challenge, NAR was measured at 2, 5, 1, 15 and 2 min. In a second group of subjects, the effect of bradykinin and [Des-Arg' ]-kallidin was studied on the release of albumin into the nasal cavity. Subjects were given incremental doses of either bradykinin or [Des-Argt]-kallidin (1-1 jug) in each nostril at 3 min intervals. Nasal lavage was performed 1 min after administration of each dose of kinin. On a second occasion, subjects were given [Des-Arg' ]- kallidin (1 /sg) in each nostril and NAR was measured every 5 min for 2 min. To study the action of the antagonists, another group of subjects was given the antagonist (1 /sg) in each nostril, 2 min prior to bradykinin, 1,ug, and NAR was measured as before. At a second visit, subjects underwent identical nasal treatments with the bradykinin antagonists and bradykinin, and a nasal lavage was performed 1 min after the challenge with bradykinin. Materials Bradykinin, [D-Argo, Hyp3, D-Phe7]-bradykinin, [1-adamantane acetyl-d-argo, Hyp3, Thi58, D-Phe7]-bradykinin and [Des-Arg']- kallidin were obtained from Peninsula Laboratories, St Helens, Merseyside, UK. [D-Argo, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin was obtained from Bachem (Saffron Walden, Essex, UK). Protein standard serum and the immunodiffusion plates were supplied by Behring Diagnostics (Milton Keynes, UK). Data analysis Changes in NAR in response to each substance were expressed as the percentage change from the vehicle control values. The response to each substance was quantified for each dose as the maximal change in NAR during the post-challenge measurements. The maximal response was expressed as an increase above baseline (vehicle) by subtracting the mean of the NAR values recorded after vehicle challenge from the mean NAR value after challenge with kinin, and converting the difference to a percentage of the baseline value. All data are expressed as means + standard error of mean. Repeated measures analysis of variance (ANOVA) was used to evaluate statistically the responses to bradykinin alone. Student's t test was used for the evaluation of the difference between values obtained after bradykinin challenge from those obtained after bradykinin challenge in the presence of the bradykinin antagonists. A probability value P< 5 was considered significant.

3 J. Physiol Bradykinin on the human nasal airway F Figure 1. Dose-response curve for bradykinin () and increased nasal airway resistance (NAR) The effect of 1,g [Des-Arg']-kallidin (V) is compared with that of bradykinin. The data are the means from 8 experiments for bradykinin and 5 experiments for [Des- Arg']-kallidin. Vertical bars represent S.E.M. Control (saline) values for NAR were Pa s cm-3 for bradykinin and Pa s cm-3 for [Des-Arg'4]- kallidin. z C Co a) cu C a) ' CUC ) L 2 F 15 F 1 F 5 F Kinin (,ug per nostril) RESULTS Figure 1 shows that bradykinin (1-1 jag) administered to each nostril, produced a dose-related increase in NAR (repeated measures ANOVA, P = -16, n = 8). Figure 2 demonstrates that over the same dose range, bradykinin also produced a dose-related increase in albumin content of nasal lavage (repeated measures ANOVA, P= -2, n = 1). The selective B1 kinin receptor agonist, [Des-Arg']- kallidin (1,ug) was ineffective compared with 1 ug bradykinin in causing an increase in NAR (Fig. 1) and did not affect the albumin content of nasal lavage (Fig. 2). The B2 kinin receptor antagonist [D-Arg', Hyp3, D-Phe7]-bradykinin (1 j#g) given 2 min prior to challenge with 1 jug bradykinin did not produce a significant reduction of the bradykinin-induced increase in NAR (Fig. 3) or the albumin content of the nasal lavage (Fig. 4). Increasing the dose of this antagonist to 1 jug also failed to produce any reduction of the bradykinin-induced increase in NAR. The increase in NAR induced by 1 jg bradykinin alone was % while the increase in NAR induced by the same dose of bradykinin after 1 jag of the antagonist was 58& % (n = 5, P= 277). The bradykinin antagonist [1-adamantane acetyl- D-Arg', Hyp3, Thi5'8, D-Phe7]-bradykinin, 1 jug, given 2 min prior to challenge with 1 jag bradykinin produced a significant (Student's paired t test, P= 5, n = 5) inhibition of NAR in response to bradykinin (Fig. 3), and a corresponding inhibition (P= 5, n = 5) of the albumin content of the lavage (Fig. 4). The antagonist itself produced no significant change in NAR (data not shown). Figures 3 and 4 show that the B2 kinin receptor antagonist [D-Arg', Hyp3, Thi5, D-Tic7, Oic']-bradykinin (1 jug) given 2 min prior to challenge with 1 jag 6 r 5 F Figure 2. Dose-response curve for bradykinin and [Des- Arg']-kallidin, and albumin content of nasal lavage fluid Bradykinin, ; [Des-Arg']-kallidin, V. The data are the means from 5-1 experiments and vertical bars show S.E.M. where larger than the symbol. 4 F E C.E :3 3 F 2 F 1 F Kinin (ug per nostril)

4 C. E. Austin and J C. Foreman 354 J. PhysioL z a: a ) cm ) L 12 F 1 F 8-6 F 4 F 2 I I BK BK + BK + BKA1 BKA2 BK + BKA3 Figure 3. The effect of bradykinin antagonists on the bradykinin-induced increase in NAR The antagonists [D- Argo, Hyp3, D-Phe7]-bradykinin (BKA1), [1- adamantane acetyl D- Argo, Hyp3, Thi5,, D-Phe7]-bradykinin (BKA2) and [D-Arg, Hyp3, Thi5, D-Tic7, Oic']-bradykinin (BKA3) were used. A dose of 1 pg of each antagonist was given 2 min prior to challenge with bradykinin, 1 pcg. The responses are expressed as mean percentage increase in NAR and the data are the means from 5 experiments for each antagonist and 15 experiments for bradykinin alone. Vertical bars represent S.E.M.; Statistically significant differences are shown by: *P= 5; tp= 19). bradykinin produced a significant inhibition of the bradykinin-induced increase in NAR (P=-9, n=5) and of the albumin content of the lavage (Student's paired t test, P = 37, n = 5). There was no significant difference (P = 16, n = 5) between the effectiveness of the active antagonists (BKA2 and BKA3). DISCUSSION We have shown that intranasally administered bradykinin induces a dose-related increase in NAR and an increase in albumin content of nasal secretion. A maximum effect does not appear to have been achieved even with a dose of 1 ug to each nostril. Significant effects were obtained with 1,g of bradykinin per nostril. In this respect, our data are similar to those of others (Proud et al. 1988; Rajakulasingam et al. 1991), where doses of between 2 and 2 pg produced dose-related effects. Kinin receptors have been classified into B1 and B2 types according to the relative potencies of a series of agonists and antagonists (Regoli & Barabe, 198; Regoli et al. 199). More recently, evidence has emerged indicating the possibility of a third type of kinin receptor, namely a B3 receptor (Farmer et al. 1989; Farmer, Burch, Kyle, Martin, Meeker & Togo, 1991; Field, Hall & Morton, 1992). This is based on the observation that, in some airways, neither B1 nor B2 receptor antagonists have any effect on the contractile effects of bradykinin; neither is [Des-Arg9]- bradykinin able to cause a response. Ligand binding studies corroborate these functional findings and support the view that these are kinin receptors which are neither B1 nor B2 in terms of previous definitions (Farmer et al. 1989). As yet, however, there are no specific agonists or antagonists available for these receptors. It has also been suggested that there are two subtypes of B2 receptor, B2A and B2B, based on evidence of differing affinities of B2 antagonists in two different B2 receptor-containing preparations (Rhaleb, Gobeil & Regoli, 1992). In that study [D-Arg, Hyp3, Thi5, D-TiC7, Oic']-bradykinin was equipotent on both preparations. In our study, the selective B1 receptor agonist, [Des-Arg']-kallidin did not cause an increase in '5 E.-.E D Figure 4. The effect of bradykinin antagonists on the bradykinin-induced increase in albumin content of nasal lavage fluid The antagonists [D-Arge, Hyp3, D-Phe7]-bradykinin (BKA1), [1-adamantane acetyl-d-argo, Hyp3, Thi5', D-Phe7]-bradykinin (BKA2) and [D-Arg, Hyp3, Thi5, D-Tic7, Oic5]-bradykinin (BKA3)were used. A dose of 1,ug of each antagonist was given 2 min prior to 1 pg bradykinin. The data are the means from 5 experiments and vertical bars represent S.E.M. Statistically significant differences are indicated by: *P= 5; tp= 37). Saline BK BKA1 BKA2 BKA3

5 J. Physiol Bradykinin on the human nasal airway 355 NAR and vascular permeability as measured by albumin release, and we conclude that the kinin receptor mediating these nasal effects is not of the B, subtype. The same conclusion was also reached in another study using [Des- Arg9]-bradykinin (Rajakulasingam et al. 1991). [D-Arg', Hyp3, D-Phe7]-bradykinin has been shown to be a competitive antagonist of B2 receptors in in vitro studies (Burch, Farmer & Steranka, 199) but we and others (Higgins, Barrow & Tyrell, 199; Pongracic, Naclerio, Reynolds & Proud, 1991) have found that it fails to block the bradykinin-induced effects in the human nasal airway. Although degradation of the antagonist cannot be ruled out, it seems unlikely that this explains its lack of effect in the human nasal airway. D-Phe7-substituted analogues of bradykinin are resistant to the action of kininase II (Togo, Burch, DeHaas, Connor & Steranka, 1989), though they are susceptible to carboxypeptidase N (Proud, Baumgarten, Naclerio & Ward, 1987). Moreover, if metabolism is a significant limitation to the effects of kinins in the nasal airway, then bradykinin itself would not be expected to be so active. Recently, acylation of the N terminus of [D-Argo, Hyp3, Thi5,8, D-Phe7]-bradykinin has been shown to yield a highly potent bradykinin antagonist. [1-Adamantane acetyl-d-argo, Hyp3, Thi58, D-Phe7]-bradykinin has been shown. to antagonize the vasodepressor action of bradykinin in the rat and presumably acts at B2 receptors (Lammek, Wang, Gavras & Gavras, 199), though an action on a different kinin receptor subtype has not been excluded. The antagonist was shown not to release catecholamines (Lammek et al. 199). We have shown that this antagonist blocks the bradykinin-induced increase of NAR and vascular permeability in the human nasal airway. Another novel, highly potent bradykinin antagonist, [D-Argo, Hyp3, Thi5, D-Tic7, Oic']-bradykinin (Hock et al. 1991; Wirth et al. 1991), was found to inhibit the bradykinin-induced increase in NAR and albumin release into the nasal airway. This antagonist has a large hydrophobic region contributed by D-Tic7,Oic8 and this is thought to lead to prolonged occupation of, and selectivity for, B2 receptors (Rhaleb et al. 1992). It is also highly stable against enzymatic degradation, since it is not a substrate for kininase II or carboxypeptidases and is only slowly degraded in human plasma (Hock et al. 1991). Our data show that one antagonist, [D-Arg, Hyp3, D-Phe7]- bradykinin, which has previously been shown to act at B2 receptors fails to inhibit the nasal actions of bradykinin that we have measured. In contrast two other novel bradykinin antagonists do inhibit the nasal action of bradykinin. [D-Argo, Hyp3, D-Phe7]-bradykinin is an antagonist of low potency and although we found it to be inactive even at a dose of 1 jug, we cannot exclude the possibility that it is active at the bradykinin receptor mediating increases in NAR. For the reasons discussed above, it seems unlikely that metabolism of the antagonist explains these effects but, again, this cannot be excluded. As pointed out above, there is published evidence which suggests the possibility that there is a B3 receptor or that the B2 class is subdivided. If it is accepted that [D-Arg', Hyp3, D-Phe7]-bradykinin is inactive in the human nasal airway, the activity of the two other antagonists that we have demonstrated would be consistent with heterogeneity of B2 receptors or the existence of another receptor. The kinin receptor mediating increased NAR and increased vascular permeability in the human nasal airway is not B1, and with the present pharmacological tools available, it can only be concluded that it appears to be B2: whether it is 'B3' or a subtype of the B2 receptor requires more potent and specific antagonists and agonists. REFERENCES BURCH, R. M., FARMER, S. G. & STERANKA, L. (199). Bradykinin receptor antagonists. Medical Research Reviews 1, FARMER, S. G., BURCH, R. M., KYLE, D. J., MARTIN, J. A., MEEKER, S. N. & TOGO, J. (1991). D-Arg[Hyp3, Thi5, D-Tic7, Tic8]- bradykinin, a potent antagonist of smooth muscle BK2 and BK3 receptors. British Journal of Pharmacology 12, FARMER, S. G., BURCH, R. M., MEEKER, S. A. & WILKINS, D. E. (1989). Evidence for a pulmonary B3 receptor. Molecular Pharmacology 36, 1-8. FIELD, J. L., HALL, J. M. & MORTON, I. K. M. (1992) Bradykinin receptors in the guinea-pig taenia caeci are similar to proposed B3 receptors in the guinea-pig trachea and are blocked by HOE 14. British Journal of Pharmacology 15, HIGGINS, P. G., BARROW, G. I. & TYRELL, D. A. J. (199). A study of the efficacy of the bradykinin antagonist NPC 567 in rhinovirus infection in human volunteers. Antiviral Research 14, HOCK, F. J., WIRTH, K., ALBUS, U., LINZ, W., GERHARDS, H. J., WIEMER, G., HENKE, S., BREIPOHL, G., KONIG, W., KNOLLE, J. & SCHOLKENS, B. A. (1991). Hoe 14 a new potent and long acting bradykinin-antagonist: in vitro studies. British Journal of Pharmacology 12, LAMMEK, B., WANG, Y., GAVRAS, A. & GAVRAS, H. (199). A new highly potent antagonist of bradykinin. Peptides 11, NACLERIO, R. M., MEIER, H. L., KAGEY-SOBOTKA, A., ADKINSON, N. F., MEYERS, D. A., NORMAN, P. S. & LICHTENSTEIN, L. M. (1983). Mediator release after nasal airway challenge with allergen. American Review of Respiratory Disease 128, PONGRACIC, J. A., NACLERIO, R. M., REYNOLDS, C. J. & PROUD, D. (1991). A competitive kinin receptor antagonist, [D-Arg, Hyp3, D-Phe7]-bradykinin, does not affect the response to nasal provocation with bradykinin. British Journal of Clinical Pha-rmacology 31, PROUD, D., BAUMGARTEN, C. R., NACLERIO, R. M. & WARD, P. E. (1987). Kinin metabolism in human nasal secretions during experimentally induced allergic rhinitis. Journal of Immunology 138, PROUD, D., REYNOLDS, C. J., LACAPRA, S., KAGEY-SOBOTKA, A., LICHTENSTEIN, L. M. & NACLERIO, R. M. (1988). Nasal provocation with bradykinin induces symptoms of rhinitis and a sore throat. American Review of Respiratory Disease 137,

6 356 C. E. Austin and J C. Foreman J. Physiol PROUD, D., ToGIAS, A., NACLERIO, R. M., CRUSH, S. A., NORMAN, P. S. & LICHTENSTEIN, L. M. (1983). Kinins are generated in vivo following nasal airway challenge of allergic individuals with allergen. Journal of Clinical Investigation 72, RAJAKULASINGAM, K., POLOSA, R., HOLGATE, S. T. & HOWARTH, P. H. (1991). Comparative nasal effects of bradykinin, kallidin and [Des-Arg9]-bradykinin in atopic rhinitic and normal volunteers. Journal of Physiology 437, REGOLI, D. & BARABE, J. (198). Pharmacology of bradykinin and related kinins. Pharmacological Reviews 32, REGOLI, D., RHALEB, N., DION, S. & DRAPEAU, G. (199). New selective bradykinin receptor antagonists and bradykinin B2 receptor characterisation. Trends in Pharmacological Sciences 11, RHALEB, N., GOBEIL, F. & REGOLI, D. (1992). Non-selectivity of new bradykinin antagonists for B1 receptors. Life Sciences 51, SVENSSON, C., ANDERSSON, M., PERSSON, C. G. A., VENGE, P., ALKNER, U. & PIPKORN, U. (199). Albumin, bradykinin and eosinophil cationic protein on the nasal mucosal surface in patients with hayfever during natural allergen exposure. Journal of Allergy and Clinical Immunology 85, ToGo, J., BURCH, R. M., DEHAAS, C. J., CONNOR, J. R. & STERANKA, L. R. (1989). D-Phe7-substituted peptide bradykinin antagonists are not substrates for kininase II. Peptides 1, WIRTH, K., HOCK, F. J., ALBUS, U., LINZ, W., ALPERMANN, H. G., ANAGNOSTOPOULOS, H., HENKE, S., BREIPOHL, G., K6NIG, W., KNOLLE, J. & SCHOLKENS, B. A. (1991). Hoe 14 a new potent and long acting bradykinin-antagonist: in vivo studies. British Journal of Pharmacology 12, Acknowledgements C.E.A is in receipt of a Science and Engineering Research Council Case Award with Pfizer Central Research, Sandwich, Kent as the industrial sponsors. We are grateful to the volunteers who participated in these experiments. Received 8 February 1993; accepted 16 November 1993.