Bradykinin-induced Airway Microvascular Leakage and Bronchoconstriction Are Mediated via a Bradykinin B2 Receptor 1 •2

MASAKAZU ICHINOSE and PETER J. BARNES

Introduction

Bradykinin (Bk), a nine-amino acid peptide, is formed from plasma precursors as part of the inflammatory response (1,2) and may be an important mediator of inflammatory airway diseases such as asthma (3, 4). Asthmatic patients have elevated kinin concentrations in plasma (5), and in nasal and bronchoalveolar lavage fluid after antigen challenge (6, 7). Both intravenous and inhaled Bk cause bronchoconstriction in asthmatic but not in normal subjects (8-10). Bradykinin causes not only bronchoconstrictor response but also airway microvascular leakage (11, 12), which may be relevant to asthma (13, 14). Two distinct Bk receptors have been described; B1 receptors are selectively activated by des Arg-Bk and to a lesser extent lys-Bk (kallidin), whereas B, receptors are more potently stimulated by Bk itself (1). A third Bk receptor has been suggested in airway smooth muscle, but no selective agents or antagonists have been described (15). The purpose of this study was to investigate the receptor type that was involved in Bk-induced airway plasma extravasation and bronchconstrictor response. Weexamined the inhibitory effect ofthe Bk B1 antagonist des ArgS-Leus-Bk (1) and the Bs antagonist n-Arg-[Hyp 3 Thisos-D-Phe 7]-Bk on intravenous Bk (NPC 349) (16)in anesthetized, mechanically ventilated guinea pigs. Methods Animal Preparation Male Dunkin-Hartley guinea pigs (Charles River, Kent, UK) weighing 350 to 400 g were anesthetized with urethane (2 g/kg intraperitoneally). Body temperature (rectal) was maintained at about 37° C by placing the animal on a heated blanket (Homeothermic blanket system; Harvard Apparatus Ltd., Edenbridge, UK). The carotid artery and jugular vein were cannulated to monitor systemic blood pressure and to inject drugs, 1104

SUMMARY We have Investigated the effects of bradykinin (Bk) antagonists on Bk-Induced airway microvascular leakage and bronchoconstrlctlon In mechanically ventilated guinea pigs by simultaneous measuring extravasation of Evans blue dye and airway opening pressure (Pao). Bradykinin (1 ~/kg Intravenously) significantly Increased leakage of dye In trachea, main bronchi, and Intrapulmonary airways, and Increased Pao, Indicating airway narrowing. The selective Bk B 2 antagonist o-Arg-[Hyp3-Thls"-D-Phe1-Bk (NPC 349; 400 1J.9/kg Intravenously) did not alter basal leakage, but when given 2 min before Bk, significantly Inhibited the Bk-Induced plasma extravasation by 99.8% In the trachea (p < 0.05), by 75.9% In main bronchi (p < 0.05), by 83.5% In proximal Intrapulmonary airways (p < 0.05), and by 91.5% In distal Intrapulmonary airways (p < 0.05). NPC 349 also reduced the Increase In Pao Induced by Bk by 73.5% (p < 0.05) without affecting the basal Pao. However, NPC 349 had no Inhibitory effect on Bk-Induced leak or bronchoconstrlctlon when given 30 min before Bk. By contrast, the B, antagonist des Arg8_l.eu'-Bk (500 1J.g/kg Intraveneously) had no effect on the Bk-Increased plasma extnMIsatIon and Pao. we conclude that Bk Increases a11'W11Y plasma IeaIcBge and bronchoconstrlctlon via activation of B2 receptors. AM REV RESPIR DIS 1990j 142:1104-1107

respectively. The trachea was cannulated immediately distal to the larynx, and the lungs were mechanically ventilated with a small animal constant volume respirator (Model 501718; Harvard Apparatus Ltd.) at a frequency of 60 strokes/min and at a tidal volume of 10 mllkg. We monitored airway opening pressure (Pao) by a differential pressure transducer (Farnell Electronic Components Ltd., Leeds, UK). All animals were pretreated with propranolol (1 mg/kg intravenously) 30 min before experimentation to block the bronchodilator effects of catecholamines, which may be released after Bk administration (17, 18).

lower part of trachea (Tr), main bronchi (MB), and central (cIPA, the proximal 3 mm portion) and peripheral intrapulmonary airways (pIPA, the remaining distal portion) (19, 20). The tissues were blotted dry, placed in preweighed tubes, and reweighed, and their dye content was extracted in formamide at 37° C for 16 h. Dye concentration wasquantified from light absorbance at 620 nm (philips spectrophotometer, Cambridge, UK) and its tissue content (nanograms dye/milligram wet weight tissue) wascalculated from a standard curve of dye concentrations in the range of 0.5 to 10 ug/ml,

Measurement of Airway Microvascular Leak

Initially, westudied the effect of each antagonist on basal Evans blue dye leakage and Pao in three groups of animals. At time zero, saline (vehicle for both antagonists; 1 m1Ikg intraveneously, Group 1~ n = 5), NPC 349 (400 ug/kg intravenously, Group 2, n = 5), or des Arg 9 _ Leus-Bk (500 ug/kg intravenously, Group 3, n = 5) was given, followed 1 min later by Evans blue dye (30 mg/kg intravenously). After 1 min,

Protocol

Vascular permeability was quantified by the extravasation of Evans blue dye, which correlates well with extravasation of radiolabeled albumin in the skin (19) and airways (20). The tissue content of Evans blue dye after experimental intervention was determined by perfusing the systemic circulation with saline to remove intravascular dye (20,21). After inductionof leakage (5 min after saline or Bk injection), the thorax was opened and a bluntended, 13-gauge needle passed through a left ventriculotomy into the aorta. The ventricles were cross-clamped and blood was expelled through an incision in the right atrium at 80 mm Hg pressure with about 100 ml saline (pH 5.5)until the perfusate wasclear. The lungs were then removed. The connective tissues, vasculature, and parenchyma were gently scraped, and the airways weredivided into four components:

(Received in original form March 1. 1990 and in revised form May 24. 1990) 1 From the Department of Thoracic Medicine, National Heart and Lung Institute, Brompton Hospital, London SW3 6LY, United Kingdom. 2 Correspondence and requests for reprints should be addressed to Professor Peter J. Barnes, Department of Thoracic Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK.

B. RECEPTOR AND A1FfWAY RESPONSE

1105 150

150

saline (vehicle for Bk; I ml/kg intravenously) was injected and the animal wasperfused with saline 5 min later in order to remove intervascular dye. The do ses of Bk antagonists were , chosen from a previous study (22). In another group of animals, we studied the inhibitory effect of each antagonist on Bkinduced Evans blue dye extravasation and Pao. At time zero, saline (I ml/kg intravenously, Group I, n = 5), NPC 349 (400 ug/kg intravenously, Group 2, n = 5), or des Argr-Leus-Bk (500 ug/kg, Group 3, n = 5) was given, followed I min later by Evans blue dye injection. After a further I min, Bk (I ug/kg intravenously) was injected, and the animal was perfused 5 min later. The duration of th e inhibitory effect ofNPC 349 was investigated by administering NPC 349 (400 ug/kg intravenously) 30 min before Evans blue dye injection in a further group of animals (n = 5).

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Statistical A nalyses Data were exp ressed as mean ± SE , and th e Mann-Whitney U test (two tai led) wa s used to test the null hypothesis. P robability value s of p < 0.05 were considered significant.

Results

Plasma Extra vasation Table 1 shows the baseline plasma extravasation. There was no significant difference between any group. Bk (l ug/kg in travenously) increased Evans blue extravasation in Tr from 27.2 ± 3.8 to 71.6 ± 8. 0, in MB from 36.2 ± 4.4 to 114 ± 12.4, in clPA from 33.6 ± 8.1 to 119 ± 5.7, and in plPA from 37.6 ± 6.8 to 142 ± 9.7 ng /mg tissue (mean ± SE, n = 5). NPC 349 (400 ug/kg, intra-

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Fig. 1. Histogram ill ustrating bradykinin (1 llQIkg intravenous ly}-induced plasma exudation in guinea pig airways (Tr = trachea; MB = main bronchi; clPA and plPA = central and peripheral intrapulmonary airways, respectively) . Response to Bk after saline (control; 1 ml/kg intravenously, 1 min before Evans blue dye injection) and after NPC 349 (400 IIgJkg intravenously, 1 and 30 min before Evans blue dye injection. Mean ± SE of five animals are shown . Significa nt differences from control values: Aster isk indicates p < 0.05 (MannWhitney U test, two tailed) .

venously) alone had no sign ificant effect on Evans blue extravasation (table I), bu t when NPC 349 was given 2 min before Bk injection, it significantly inhibited the Bk-induced Evans blue dye extravasation at all airway levels (figure 1). By contrast , NPC 349 given 30 min before Bk injection had no effect on the Bk-induced plasma extravasation (figure 1). The percentage inhibition of the difference between extravasation after saline control and basal extravasation was 99.8% in trachea, 75.9010 in main bronchi, 83.5% in proximal intra pulmonary airways, and 91.5% in distal intrapulmonary airways (p < 0.05 at all airway levels). No significant inhibitory effect on Bk-induced Evans blue dye extravasation was ob-

TABLE 1

Plasma Extra vasat ion '

Saline (1 mVkg intravenously) NPC 349 (400 IIgJkg intravenously)

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Fig. 2. Effect of des Argl-Leu' -Bk (500 I'gJkg intravenously, 1 min before Evans blue injection) pretreatment on bradykinin (1 IIQ/kgintravenouslYr induced plasma exudation in gui nea pig airways. Abbreviations are the same as in figure 1. Data are mean ± SE of five animals. There were no significant differences between the values of cont rol and des Arg'-Leu'·Bk pretreated animals (Mann-Whitney U test, two-tailed).

served after des Argr-Leu'-Bk (500 ug/kg intravenously 2 min before Bk) (figure 2).

EFFECT OF Bk ANTAGONISTS ON BASAL EVANS BLUE DYE EXTRAVASATION

des Arg (500 IIg1kg intravenously)

100

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Leu -Bk

Bradykinin (Bk), des Argt-Leut-Bk , and D-Arg[Hyp 3-Thi5 '".D-Phe']-Bk (NP C 349) were kindly donated by Dr. H . P. Rang, Sandoz Institute for Medical Research (London, UK). Urethane, formamide, and Evans blue dye were obtained from Sigma Chemical Co. (Poo le, Do rset, UK), propranolol hydrochloride from Imperial Chemical Industries p ic. (Macclesfield, UK) and saline (0.9«1,10 sodium chloride) from Traveno l Laboratories (Thetford, UK ).

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Tr

MB

clPA

plPA

27.2 ± 3.8 25.6 ± 7.1 25.6 ± 3.7

36.2 ± 4.4 37.0 ± 10.9 27.0 ± 5.5

33.6 ± 8.1 31.4 ± 9.6 31.6 ± 9.4

37.6 ± 6.8 35.6 ± 7.0 38.0 ± 6.2

Definition of abbreviations: Tr - trachea; MB = main bronch i; clPA and plPA • central and peripheral intrapUlmonary airways ;

des Arg • des Arg'-Leu' -bradykinin . Values are mean :I: SE of five animals. Each agent was administe red 1 min before Evans blue dye injection. No signif icant difference was observed between value of saline and of each antagonist. • Evans blue dye in nanograms/milligram tissue.

Airway-opening Pressure The effects of NPC 349 and de s Arg"Leus-Bk pret reatments on Bk-induced increase in Pao are shown in figure 3. (Bk (1 ug/kg intravenously) increased Pao from 9.7 ± 0.8 to 25.3 ± 3.8 ern H 2 0 (n = 5). NPC 349 given 1 min before Bk significantly (p < 0.05) inhibited the increase in Pao induced by Bk (9.4 ± 0.7 to 13.4 ± 0.5 em H 2 0 ), but NPC 349 given 30 min before Evans blue injection and des Argt-Leut-Bk had no effect on the Bk-induced increase in Pao.

Blood Pressure The mean baseline blood pressure was 33.6 ± 2.5 mm Hg (n = 5) and fell to 14.4 ± 2.1 mm Hg after Bk (1 ug/kg intravenously). After NPC 349 1 and 30 min before intravenous Bk and des Arg"Leut-Bk , blood pressure fell from 34.4 ± 3.0' to 16.0 ± 2.3, from 32.8 ± 4.2 to 16.3 ± 1.2, and from 35.2 ± 5.0 to 16.3 ± 1.8 mm Hg (n = 5), respectively. Discussion Our data show that intravenous Bk induces airway Evans blue dye extravasation and bronchoconstriction in anesthetized, mechanically ventilated guinea pigs. Both effects are blocked by the Bk Bs-antago-

ICHINOSE AND BARNES

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