INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Bradykinin (BK) is a peptide mediator that is formed during inflammation and tissue injury and there is evidence to suggest it plays a key role in the airway inflammation of asthma. For example, kinin activity has been measured in bronchoalveolar lavage fluid (BALF) from asthmatic subjects at rest and after allergen challenge (1, 2). BK itself is a potent bronchoconstrictor in asthmatic subjects (3, 4) and mediates the nasal blockage observed in patients with allergic rhinitis after allergen challenge (5). In animals, BK has been shown to have important effects in the lung including bronchoconstriction, plasma exudation, vascular dilatation, and sensitization of airway afferents (6). Two distinct receptors have been identified pharmacologically and through molecular cloning, termed B₁ and B₂ (9). Most of the biological activities of BK are mediated through the activation of constitutively expressed B₂ receptors (10). By contrast, B₁ receptors are not present in tissue under "normal" conditions, but are induced during inflammatory insults and after exposure to noxious stimuli (11). For example, functional and radioligand binding studies have demonstrated the appearance of B₁ receptors in tissues such as vascular and gastrointestinal smooth muscle, as well as in isolated cells including macrophages, platelets, vascular smooth muscle, and endothelial cells after exposure to lipopolysaccharide (LPS) or interleukin (IL)-1 (12). Stimulation of B₁ receptors elicits a variety of effects including vascular smooth muscle contraction and relaxation, prostanoids and interleukin-1 (IL-1) release, and increase in intracellular calcium level (14). Although stimulation of B₁ receptors with B₁ receptor agonists has no effect on airway tone in patients with asthma (4, 18), this caused a relaxant effect in isolated murine tracheal rings in vitro (19, 20).

Despite these observations, there still remain uncertainties regarding the functional role of BK B₁ receptors in the airways, particularly in terms of their role in bronchial hyperresponsiveness (BHR). In allergen-induced BHR, both B₁ and B₂ receptors have been implicated in the guinea pig (21), but in that study, modulation of these receptors after allergen challenge was not examined. We therefore determined whether BK B₁ and B₂ receptors are regulated after allergen exposure in sensitized rats, and their roles in allergen-induced BHR and bronchial inflammation were investigated. Thus, we examined the effect of a BK B₂ receptor antagonist, D-Arg[Hyp³, Thi⁵, D-Tic⁷, Oic⁸]BK (Hoe140), and a B₁ receptor antagonist, des-Arg¹⁰[Hoe140], on these parameters.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals, Sensitization Procedures, and Allergen Exposure

Pathogen-free inbred male Brown-Norway rats (Harlan Olac Ltd., Bicester, UK) (200 to 250 g, 9 to 13 wk old) kept in identical conditions were injected with 1 ml of 1 mg ovalbumin (OA) (Grade V, salt-free; Sigma, Dorset, UK) in 100 mg Al(OH)₃ (BDH, Dorset, UK) suspension in 0.9% (wt/vol) saline intraperitoneally on three consecutive days. Challenge with saline or OA aerosol exposure to rats was performed 21 d later. The animals were then placed in a 6.5-L Plexiglas chamber connected to a DeVilbiss PulmonSonic nebulizer (model No. 2511; DeVilbiss Health Care, U.K. Ltd., Middlesex, UK) that generated an aerosol mist of OA (1% wt/vol in 0.9% NaCl) pumped into the exposure chamber by the airflow supplied by a small animal ventilator (Harvard Apparatus Ltd., Kent, UK; 60 strokes/min; pumping volume, 10 ml). The exposure time was 15 min.

Expression of B₁ and B₂ Receptor Messenger RNA (mRNA)

We first examined the time-course of possible changes in the expression of B₁ and B₂ receptor mRNA in the following groups of rats: (1) naive: nonsensitized, saline-challenged, and killed 2 h later (n = 3). (2) SS: sensitized, saline-challenged, and killed at 2, 6, 12, and 24 h after challenge (n = 2 to 3 at each time point). (3) SO: sensitized, OA-challenged, and killed at 2, 6, 12, and 24 h for B₁, and 2, 8, 24, 48, and 96 h after challenge for B₂ receptor mRNA expression (n = 2 to 3 at each time point).

Rats were killed with a lethal dose of pentobarbital sodium (Expiral; Sanofi Animal Health Ltd., Herts, UK; 200 mg/kg, intraperitoneally) and lung tissues were collected for Northern blot analysis.

mRNA Analysis by Northern Blot Analysis

The lungs were cut into small cubes and snap-frozen in liquid nitrogen, and stored at 80° C for later assays for mRNA expression. Total RNA from lung tissue was extracted according to the method of Chomczynski and Sacchi (22), and mRNA was isolated using PolyATract mRNA isolation system kit (Promega, Southampton, UK). mRNA was size-fractionated on a 1% agarose/formaldehyde gel containing 20 mM morpholinosulfonic acid, 5 mM sodium acetate, and 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 7.0) and blotted onto Hybond-N membranes (Amersham, Middlesex, UK). Rat B₁ and B₂ receptor complementary DNA (cDNA) from recently cloned sequences of these receptors (23) and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 309-bp cDNA were labeled in random priming using [-³²P]deoxycytidine triphosphate (dCTP) (3,000 Ci/mmol; Amersham). The GAPDH cDNA was a 309-bp fragment amplified from reverse transcriptase (RT)-generated cDNA corresponding to rat GAPDH. Prehybridization and hybridization were carried out at 42° C with labeled probes (approximately 1.5 × 10⁶ counts per minute [cpm]/ml) in buffer containing 5× Denhardt's solution, 5× standard saline citrate (SSC), 50 mM TRIS-HCl (pH 7.5), 0.1% sodium dodecyl sulfate (SDS), 250 µl/ml sonicated denatured salmon sperm DNA, and 50% formaldehyde. After hybridization, each blot was washed to a stringency of 0.1× SSC/0.1% SDS for 30 min at 60° C and exposed for 1 to 14 d to Kodak X-OMAT S film. Autoradiographic bands were quantified by laser densitometry (Quantity One software; PDI, New York, NY). BK B₁ or B₂ receptor mRNA expression was expressed as a ratio of GAPDH mRNA expression.

Effect of B₁ Receptor Antagonist on BHR

We studied five groups of animals to investigate the effects of B₁ antagonist desArg¹⁰[Hoe140] (Peninsula, Merseyside, UK) on BHR: (1) Sensitized and saline-exposed animals (group SS, n = 8): Sensitized rats were injected with 0.9% saline 1 ml/kg intraperitoneally (control), and were exposed 30 min later to 0.9% NaCl aerosol. (2) Sensitized, OA-exposed animals (SO, n = 8): Sensitized animals were injected with 0.9% saline intraperitoneally (control), and 30 min later were exposed to 1% OA aerosol for 15 min. (3) Sensitized, saline-exposed, and BK B₁ antagonist-treated animals (SSB1, n = 10): The procedures were similar as for the SS group, apart from injection of desArg¹⁰[Hoe140] 200 nmol/kg intraperitoneally 30 min before exposure to 0.9% NaCl aerosol. (4) Sensitized, OA-exposed, and BK B₁ antagonist-treated animals (SOB1a, n = 7): The procedures were similar as for the SSB group, except that the aerosol exposure was to 1% OA. (5) Sensitized, OA-exposed, and BK B₁ antagonist-treated animals (SOB1b, n = 6): The procedures were the same as for the SOB1a group, but the dose of BK B₁ antagonist was 1 µmol/kg.

In all groups, BHR to acetylcholine (ACh) and bronchoconstriction to BK were examined 18 to 24 h after exposure to either 1% OA or 0.9% NaCl aerosol.

Effect of B₂ Receptor Antagonist on BHR

We studied the effects of BK B₂ receptor antagonist in four groups of rats: (1) Sensitized and saline-exposed animals (Group SS, n = 10): As previously defined in B₁ study. (2) Sensitized, OA-exposed animals (SO, n = 8): As previously defined. (3) Sensitized, saline-exposed, and BK B₁ antagonist-treated animals (SSB2, n = 6): The procedures were the same as in Group SSB1, but the injection 30 min before OA aerosol exposure was B₂-specific antagonist Hoe140, 200 nmol/kg. (4) Sensitized, OA-exposed, and BK B₂ antagonist-treated animals (SOB2, n = 8): All the procedures were the same as in Group SOB1a, except the injection before aerosol exposure of Hoe140, 200 nmol/kg.

Measurement of Bronchial Responsiveness to ACh and of Bronchoconstriction to BK

Rats were anesthetized with an intraperitoneal injection of 2 mg/kg midazolam (Roche Products Ltd., Welwyn Garden City, UK) and a subcutaneous injection of 0.4 mg/kg Hypnorm (Janssen Pharmaceuticals Ltd., Wantage, UK), which contains 0.315 mg/ml of fentanyl citrate and 10 mg/ml of fluanisone. A tracheal cannula (1.02 mm outer diameter) was inserted into the lumen of the cervical trachea through a tracheostomy. The animals were connected to a small-animal respirator (Harvard Apparatus, Edenbridge, Kent, UK) and ventilated with 10 ml/kg of air at a rate of 90 strokes/min. Transpulmonary pressure was measured with a pressure transducer (model FCO 40 ± 1,000 mm H₂O; Furness Controls, Bexhill, Sussex, UK) with one side attached to an air-filled catheter inserted into the right pleural cavity and the other side attached to a catheter connected to a side port of the intratracheal cannula. Airflow was measured with a pneumotachograph (model F1L; Mercury Electronics, Glasgow, Scotland) connected to a transducer (model FCO 40 ± 20 mm H₂O; Furness Controls). The signals from the transducers were digitized with a 12-bit analog-to-digital board (NB-MIO-16; National Instruments, Austin, TX) connected to a Macintosh II computer (Apple Computer, Cupertino, CA) and analyzed with software (Lab VIEW 2; National Instruments, Austin, TX) that was programmed to measure lung resistance (R_L) according to the method of von Neergard and Wirz (36). Aerosols were generated with an ultrasonic nebulizer (model 2511; PulmoSonic, DeVilbiss, Hazeltown, PA).

Animals were initially injected with propranolol (Inderal; Zeneca, Cheshire, UK; 1 mg/kg, intravenously) to block adrenergic effects and suxamethonium (Antigen Pharmaceuticals Ltd., Roscrea, Ireland) to stop spontaneous breathing. Baseline R_L was determined after inhalation of 0.9% saline aerosol (45 breaths of 10 ml/kg stroke volume at 90 strokes/min). Then, aerosols generated from increasing half-log₁₀ concentrations of ACh (acetylcholine chloride; Sigma, Dorset, UK) were administered by inhalation with the initial concentration of 10⁴ mol/L, increasing by half-log concentrations until concentration of 10¹ mol/L or the concentration resulting in more than 400% increase in R_L above baseline. Each concentration was administered for 45 breaths. The concentration of ACh needed to increase R_L 300% above baseline (PC₃₀₀) was calculated by interpolation on the log concentration-lung resistance curve.

After measurements of airway responsiveness to ACh, the animal was kept on the ventilator for 1 to 2 h to allow recovery from ACh effect before measurement of bronchial responsiveness to BK. When transpulmonary pressure was restored to baseline values, the animal was injected with BK B₁ receptor agonist desArg⁹-BK (30 nmol/kg; Peninsula, Merseyside, UK) to determine whether there was any bronchoconstrictor effects mediated through the BK B₁ receptor. Ten minutes later, 0.9% NaCl aerosol was inhaled again to determine the baseline R_L for measurement of bronchoconstriction to BK, then 1 mM BK (Sigma, Dorset, UK) aerosol was administered for 45 breaths. The maximal percentage increase in R_L above the baseline value was recorded as the bronchoconstrictor response to BK.

Bronchoalveolar Lavage (BAL) and Cell Counting

This is also described in detail elsewhere (24). Briefly, after an overdose of anesthetics, rats were lavaged with total 20 ml 0.9% sterile saline in 2-ml aliquots via the endotracheal tube. Total cell counts were determined using Kimura stain in a Neubauer chamber under an optical microscope (Olympus BH2; Olympus Optical Company Ltd., Tokyo, Japan). Differential cell counts from cytospin preparations stained by May-Grünwald stain were counted with at least 500 cells identified as macrophages, eosinophils, lymphocytes, and neutrophils according to standard morphology under ×400 magnification.

Materials

Sodium pentobarbital (Expiral) was purchased from Sanofi Animal Health Ltd., Herts, UK. Hoe140 was a gift from Hoechst AG, Frankfurt, Germany. DesArg¹⁰[Hoe140] and rat BK B₁ and B₂ receptor cDNA probes were kindly provided by Dr. P. McIntyre, Novartis Institute for Medical Research, London, UK.

Statistical Analysis

Data were presented as mean ± SEM. For multiple comparison of different groups, the Kruskall-Wallis test for analysis of variance (ANOVA) was used. If the Kruskall-Wallis test for ANOVA was significant, we then used the Mann-Whitney U test for comparison between two individual groups. The data analysis was performed using SPSS for Windows statistical software package (SPSS Inc., Chicago, IL). A p value of < 0.05 was considered to be significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Expression of BK B₁ and B₂ Receptor mRNA

B₁ receptor mRNA expression was increased in sensitized animals challenged with OA, with a peak of expression 2 to 6 h after challenge. B₁ mRNA was not changed in sensitized animals challenged with saline. Concentrations of B₂ mRNA were unaffected by sensitization or challenge with OA compared with naive animals (Figure 1).

View larger version (48K): [in this window] [in a new window]   Figure 1.   Northern blot analysis of mRNA expression for BK B1 ( panel A) and B2 ( panel B) receptors. The laser densitometric data were expressed as a ratio of GAPDH mRNA. On each upper panel, representative Northern blot bands are shown, together with the mean densitometric data (n = 2 to 3 in each group). Expression of B1 receptor mRNA in OA-sensitized and challenged animals peaked at 2 to 6 h and remained higher at 24 h after OA aerosol exposure when compared with baseline naive animals. There was no change in B2 receptor mRNA expression in saline- or allergen-exposed rats. h = hour; N = naive; SS = sensitized and saline-exposed; SO = sensitized and OA-exposed.

Effect of BK B₁ Antagonist DesArg¹⁰[Hoe140]

Bronchial responsiveness to ACh. Mean baseline lung resistances were 0.36 ± 0.02, 0.34 ± 0.03, 0.30 ± 0.02, 0.36 ± 0.02, and 0.37 ± 0.04 cm H₂O ml s¹, respectively, for the five groups studied. There was no significant difference between the five groups. The allergen-induced BHR to ACh in the sensitized animal challenged with OA was reflected in significantly greater R_L responses at ACh concentrations of 10^2.5, 10², and 10^1.5 M, compared with animals challenged with saline (Figure 2A), and also in greater mean logPC₃₀₀ (p < 0.005, Figure 2B). Treatment of sensitized and OA-exposed rats with desArg¹⁰[Hoe140] dose-dependently attenuated bronchial responsiveness, with logPC₃₀₀ at 1.76 ± 0.05 and 1.54 ± 0.1 M after the 0.2 and 1 µmol/kg doses, respectively, while having no effect on bronchial responsiveness to ACh in animals challenged with saline (Figures 2A and 2B).

View larger version (18K): [in this window] [in a new window]   Figure 2.   Bronchial responsiveness to ACh in B1 antagonist study. Baseline RL was determined after inhalation of 0.9% saline aerosol. Increasing half-log10 concentrations of ACh aerosol was administered with the initial concentration of 104 mol/L and the final concentration of 0.1 mol/L or the concentration resulting in more than 400% increase in RL above baseline values ( panel A). The concentration of ACh needed to increase RL 300% above baseline (PC300) was calculated by interpolation on the log concentration-lung resistance curve and expressed as logPC300 ( panel B). SS: sensitized and saline-exposed, n = 8 (open circles); SO: sensitized and 1% OA-challenged, n = 8 (closed circles); SSB: sensitized, B1 antagonist, desArg10[Hoe140] 200 nmol/kg treated and saline-exposed, n = 10 (open squares); SOB1a: sensitized, B1 antagonist, desArg10 [Hoe140] 200 nmol/kg treated and OA-exposed, n = 7 (closed squares); SOB1b: sensitized, B1 antagonist, desArg10[Hoe140] 1 µmol/ kg treated and OA-exposed, n = 6 (closed diamonds). *p < 0.02 compared with other groups; **p < 0.005 compared with SS and SSB groups; #p < 0.01 compared with SOB1a and SOB1b groups; NS = nonsignificant.

Bronchoconstriction to BK. OA-sensitized animals exposed to OA aerosol had a significantly greater response to BK than OA-sensitized, saline-exposed rats (51.9 ± 7.5 versus 203.4 ± 27.7%; p < 0.01). The B₁ antagonist desArg¹⁰[Hoe140] had no significant effect on the bronchoconstriction to BK (Figure 3). Administration of the B₁ agonist desArg⁹-BK before measurement of bronchoconstriction to BK had no significant effect on the baseline R_L measurements (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 3.   Bronchoconstriction to BK in B1 antagonist study. After measurement of bronchial reactivity to ACh, the animal was allowed to recover from bronchoconstriction in 1 to 2 h before exposure to 0.9% saline aerosol to determine again the baseline RL, followed by 1 mM BK aerosol 45 breaths. The maximal percent increase in RL after BK inhalation was recorded as the response to BK. Abbreviations as in Figure 2. *p < 0.01 compared with SS and SSB groups; NS = nonsignificant.

To exclude the possibility that the effect of the B₁ antagonist desArg¹⁰[Hoe140] may have worn off by the time the BK challenge was performed, we injected in a separate group of allergen-exposed sensitized rats (n = 6) two doses of the antagonist (200 nmol/kg intraperitoneally each) at 30 min before allergen exposure, and 18 to 24 h later after the termination of ACh challenge. BK challenge was then performed 30 min later. Controls were allergen-exposed, sensitized rats (n = 6) but treated with 0.9% NaCl administered intraperitoneally. Control rats demonstrated a 180.8 ± 18.4% increase in R_L after exposure to BK, and treatment with desArg¹⁰[Hoe140] did not cause a significant change in the bronchoconstrictor response to BK (147.5 ± 12.6). There was a significantly reduced bronchial responsiveness in the desArg¹⁰[Hoe140]- treated rats (log PC₃₀₀ in saline-treated: 1.96 ± 0.09, and in desArg¹⁰[Hoe140]-treated: 1.34 ± 0.04; p < 0.005).

BAL cell profile. Total cell number and numbers of macrophages were not significantly different in sensitized, OA- exposed rats, compared with sensitized, saline-exposed rats. However, the numbers of eosinophils, lymphocytes, and neutrophils increased significantly in the sensitized and OA-challenged SO group (p < 0.01). Treatment with desArg¹⁰[Hoe140] did not alter the numbers of eosinophils and neutrophils, although at the higher dose (1 µmol/kg), it produced a decrease in lymphocyte numbers after OA challenge (p < 0.05 compared with the SS group; Figure 4).

View larger version (21K): [in this window] [in a new window]   Figure 4.   BAL fluid cellular profiles in B1 antagonist study. BAL was performed with 20 ml 0.9% saline in 2-ml aliquots via endotracheal tube. Total cell counts were determined using Kimura stain in a Neubauer chamber under an optical microscope. Differential cell counts from cytospin preparations stained by May-Grünwald stain were counted with at least 500 cells identified as macrophages, eosinophils, lymphocytes, and neutrophils according to standard morphology under ×400 magnification. Mac = macrophages; Eos = eosinophils; Lym = lymphocytes; Neu = neutrophils. *p < 0.01 compared with SS and SSB groups; **p < 0.04 compared with SO group.

Effect of BK B₂ Receptor Antagonist Hoe140

There was no significant effect of Hoe140 on allergen-induced increase in logPC₃₀₀ in sensitized rats (Figure 5). However, Hoe140 significantly reduced bronchoconstriction to BK in sensitized rats and attenuated OA-induced BHR to BK (p < 0.03 for SSB2 versus SS group; p < 0.04 for SOB2, compared with SO group; Figure 6). Hoe140 had no effect on the inflammatory cell recruitment in BALF caused by OA sensitization and exposure (Figure 7).

View larger version (17K): [in this window] [in a new window]   Figure 5.   Bronchial responsiveness to ACh in B2 antagonist study. The procedures of sensitization, aerosol exposure, and measurement of bronchial reactivity were the same as in B1 antagonist study. Animals were classified into four groups: SS: sensitized and saline-exposed, n = 10 (open circles); SO: sensitized and 1% OA-challenged, n = 8 (closed circles); SSB2: sensitized, B2 antagonist, Hoe140-treated (200 nmol/kg, intraperitoneally) and saline-exposed, n = 6 (open triangles); SOB2: sensitized, B2 antagonist, Hoe140-treated (200 nmol/kg, intraperitoneally) and OA-exposed, n = 8 (closed triangles). Panel A shows the mean ACh concentration/lung resistance dose-response curve for each group; panel B shows the mean log PC300. Data shown as mean ± SEM. *p < 0.03 compared with SS group.

View larger version (16K): [in this window] [in a new window]   Figure 6.   Bronchial responsiveness to BK in B2 antagonist study. Abbreviations used similar to those of Figure 5. *p < 0.04 compared with SS and SOB2 groups; NS = nonsignificant.

View larger version (18K): [in this window] [in a new window]   Figure 7.   BALF cellular profiles in B2 antagonist study. Abbreviations used similar to those of Figure 5. *p < 0.05 compared with SS group.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We have shown for the first time an increase in B₁ receptor expression in the lungs of sensitized rats during airway inflammation induced by an allergen. OA challenge in sensitized animals induced an increase in B₁ mRNA concentrations with no change in B₂ mRNA levels. As previously described in this Brown-Norway rat model (24), exposure to OA induced BHR to ACh and bronchoconstriction to BK, and increased the numbers of eosinophils, neutrophils, and lymphocytes recovered in BALF. Pretreatment with a BK B₁ receptor antagonist prevented OA-induced BHR to ACh, and produced a small reduction in lymphocyte numbers. The B₂ receptor antagonist did not affect BHR to ACh but inhibited BK-induced bronchoconstriction. These results indicate that release of BK after allergen exposure may activate both BK B₁ and B₂ receptors, with B₁ receptors involved in induction of nonspecific BHR, whereas B₂ receptors but not B₁, as would be expected, are necessary for the transduction of BK-induced bronchoconstriction.

The majority of the bronchial effects of BK so far described are mediated through the activation of B₂ receptors. Using B₂ receptor antagonists, B₂ receptors have been shown to mediate BK-induced bronchoconstriction, airway microvascular leakage, mucus secretion, and excitation and sensitization of sensory fibers (6, 7, 25). These activities do not appear to involve B₁ receptors. In Brown-Norway rat lung membranes, we found exclusively binding sites for B₂ receptors in rat lung membrane with no evidence of B₁-binding sites using radioligand binding studies (26). The present study confirmed these findings, showing significant expression of B₂ mRNA in normal rat lung, but no detectable B₁ receptor mRNA. However, after allergen challenge, B₁ receptor mRNA expression is induced, whereas levels of B₂ receptor mRNA remain unchanged. The changes in receptor mRNA are mirrored by the effect of the B₁ and B₂ receptor antagonists that inhibited allergen-induced BHR to ACh and to BK, respectively. By contrast, IL-1- or ozone-treated rats do not demonstrate augmentation of mRNA expression of either B₁ or B₂ receptors (26, 27), and in the IL-1-exposed rats, an increase in bronchoconstriction to BK, which was mediated by B₂ receptors, was observed without BHR to ACh (26, 28).

The dose of B₂ antagonist Hoe140 was selected according to previous studies (25, 29, 30). We used a dose of Hoe140 that was higher than that used previously in order to achieve effective levels during the 24 h after allergen challenge in the present study. Inhibition of BK-induced bronchoconstriction by Hoe140 at 24 h indicates that the dose was effective. Hoe140 was also able to inhibit the bronchoconstriction induced by BK in rats exposed to allergen, although this inhibition was not as effective as that observed in the saline-challenged rats. There is relatively less information regarding the pharmacokinetic property of desArg¹⁰[Hoe140], which was used as a B₁ receptor antagonist. DesArg¹⁰[Hoe140] is a derivative of Hoe140 and shares the same metabolic stability of Hoe140 but has a relatively higher affinity for B₁ receptors (31). Although the selectivity of this antagonist for B₁ receptor is not large, our data indicate that most of the effects shown were mediated through the B₁ receptor. Thus, while there was a dose-dependent inhibition of OA-induced BHR by desArg¹⁰[Hoe140], there was no effect of Hoe140. In addition, we showed that desArg¹⁰[Hoe140] at the highest dose used, together with an additional dose administered 30 min before BK challenge, did not interfere with BK-induced bronchoconstriction that is mediated through B₂ receptor. This also indicated that there was no contribution of BK B₁ receptor in the bronchoconstrictor response to BK, even after allergen challenge. In addition, we found no bronchoconstrictor effect of the B₁ agonist des-Arg⁹-BK, even after allergen challenge. Our results are similar to those observed in asthmatic patients in whom B₁ agonists do not induce bronchoconstriction (4, 18). Thus, our data indicate that B₁ receptors are involved in allergen-induced BHR, whereas B₂ receptors mediate BK- induced bronchoconstriction.

The mechanisms by which induced B₁ receptors play a role in OA-induced BHR are unclear. Stimulation of B₁ receptors can elicit a variety of effects including vascular smooth muscle contraction and relaxation, prostanoid and IL-1 release from macrophages, and increase in intracellular calcium (14). A possible role of BK B₁ receptors on nerve endings has been postulated for chemotaxis of neutrophils (32). However, upregulated B₁ receptors do not appear to be involved in recruitment of inflammatory cells as the number of eosinophils and neutrophils in BAL fluid remained unchanged. However, the reduction in lymphocyte numbers may indicate some inhibition of T-cell regulation. In the Brown-Norway rat model, we have previously shown that there is an increase in expression of the mRNA for T-helper (Th2)-derived cytokines, IL-4, and IL-5 in the lung after allergen challenge (33), raising the possibility of B₁ receptor antagonists affecting Th2-cell activation. Although B₁ receptors do not appear to mediate bronchoconstriction, they may increase the responsiveness of airway smooth muscle through modulation of neural pathways. In the thermal hyperalgesia model in the rat, BK B₁ receptors were expressed, activation of which produced significant hyperalgesia, possibly on the nociceptive terminal itself (34). Other mechanisms may involve increased cytokine release from macrophages as has been previously shown (16). Information on the localization of B₁ receptors in the airways of allergen-exposed rats would be useful in determining the mechanisms involved.

Our results are somewhat at variance with those reported in other species. In the sheep, a B₂ receptor antagonist, NPC 567, inhibited Ascaris suum-induced BHR to carbachol and BK, neutrophil influx and release of leukotriene B4 and C4, and various products of the cyclooxygenase pathway in sheep (35). Another B₂ receptor antagonist had a small inhibitory effect on the BHR to histamine in guinea pigs after a single exposure to OA, but there was greater inhibitory effect of a B₁ receptor antagonist, des-Arg⁹[Leu⁸]-BK, against BHR to histamine and the neutrophilia in BALF (21). It is possible that these different results may result from species difference in the expression of B₁ receptors after allergen exposure.

In conclusion, BK B₁ receptors can be induced by OA exposure in OA-sensitized rats, and play a role in the induction of OA-induced BHR to ACh; in contrast, BK B₂ receptors are constitutively expressed and mediate the bronchoconstrictor effect of BK. Further studies are needed to elucidate the mechanisms of B₁ receptor upregulation by allergen exposure and those by which B₁ receptors lead to nonspecific BHR.