Lack of a role for bradykinin in allergen-induced airway microvascular leakage and bronchoconstriction in the guinea pig

We have investigated the role of bradykinin in allergen-induced airway microvascular leakage and bronchoconstriction in sensitized guinea pigs. We used a selective bradykinin B2 receptor antagonist, HOE140, which has been shown to prevent the airway effects induced by bradykinin. Lung resistance (R_L) was measured for 6 min after challenge with allergen. Extravasation of Evans blue dye into airway tissues was used as an index of the airway microvascular leakage. Aerosolized ovalbumin (5 mg/ml, 30 breaths) induced a significant increase in R_L and leakage of dye in the trachea, main bronchi and intrapulmonary airways in the ovalbumin-sensitized guinea pigs. HOE140 given by inhalation (200 M, 60 breaths) had no effect on the airway microvascular leakage and bronchoconstriction induced by the allergen. I.v. HOE140 (200 nmol/kg) did not significantly inhibit these airway responses. We conclude that bradykinin-mediated mechanism may not play a significant role in airway microvascular leakage or bronchoconstriction induced by allergen.accepted by I. Ahnfelt-Rønne     

Effect ofβ(in2)-adrenoceptor agonists against platelet-activating factor-induced airway microvascular leakage and bronchoconstriction in the guinea pig

We have investigated the effect of inhaled formoterol (0.75 mg/ml, 60 breaths) and salbutamol (25 mg/ml, 60 breaths) against airway microvascular leakage and bronchoconstriction induced by inhaled platelet-activating factor (PAF) and histamine in anaesthetized guinea pigs. Lung resistance (R _L) was measured for 6 min after challenge, followed by measurement of extravasation of Evans blue dye into airway tissues, used as an index of airway microvascular leakage. PAF (0.1, 0.4 and 1 mM; 30 breaths) caused a significant increase inR _L and extravasation of dye, but the responses were smaller than those induced by histamine (5 mM, 30 breaths). Both formoterol and salbutamol caused a small but significant inhibition of extravasation in the distal intrapulmonary airways induced by PAF (0.1 and 0.4 mM for formoterol and 0.1 mM for salbutamol), and only formoterol reduced the increase inR _L induced by 1 mM PAF. These drugs also inhibited both airway effects of histamine to a higher degree. In conclusion, formoterol and salbutamol can partly inhibit airway microvascular leakage and bronchoconstriction induced by inhaled PAF and histamine. The inhibitory potency of(in2)-adrenoceptor agonists may be dependent on the inflammatory mediator inducing the airway effects.     

Involvement of tachykinin receptors in oedema formation and plasma extravasation induced by substance P,neurokinin A,and neurokinin B in mouse ear

The involvement of tachykinin receptors in skin inflammation induced by substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) was investigated in mouse ears. Intradermal injection of tachykinins (0.1–100 pmol/site) into the ear skin produced oedema formation. RP 67580 (ED₅₀:0.34 mg/kg, i.v.) and SR 140333 (ED₅₀:0.19 mg/kg, i.v.), the non-peptide NK₁ receptor antagonists, inhibited SP-induced oedema. SR 140333 was also effective in preventing NKA- and NKB-induced oedema. SR 48968 (1 mg/kg, i.v.), a non-peptide NK₂ antagonist, induced a significant inhibition of NKA-induced oedema but had no effect on the response to SP and NKB. SR 142801 (3 mg/kg, i.v.), a non-peptide NK₃ antagonist, prevented only NKB-induced oedema. In contrast, phosphoramidon (0.1 and 0.5 mg/kg, i.v.), an endopeptidase inhibitor, enhanced the oedema response to tachykinins. SR 140333, SR 48968, and SR 142801 blocked the enhancement by phosphoramidon of the response to SP, NKA, and NKB, respectively. Plasma extravasation in ear skin was induced by i.v. injection of tachykinins (0.7–17.6 nmol/kg). RP 67580 (ED₅₀:0.15 mg/kg, i.v. for SP) and SR 140333 (ED₅₀:14.3 g/kg, i.v. for SP) inhibited tachykinin-induced plasma extravasation in ear skin. However, SR 48968 and SR 140281 had no effect on the vascular response to tachykinins. Chlorpheniramine (4 mg/kg, i.v.), a histamine H₁ blocker, inhibited the response to local SP but not to i.v. SP. These results suggest that in addition to the NK₁ receptors, functional NK₂ and NK₃ receptors may participate in the oedema response to local NKA and NKB in the ear skin. However, it appears that NK₁ receptors on blood vessels are involved predominantly in plasma extravasation induced by i.v. tachykinins in the ear.accepted by G. Letts     

Protective activity of ketoprofen lysine salt against the pulmonary effects induced by bradykinin in guinea-pigs

We investigated the capacity of ketoprofen lysine salt (KLS) to counteract the pulmonary effects of some mediators of airway inflammation. The protective ffect of KLS and its R-isomer against bradykinin (BK) induced plasma extravasation in the airways and bronchoconstriction was evaluated in anaesthetized guinea-pigs, in parallel with the capacity of KLS to inhibit the production of thromboxane A₂ (TXA₂). Moreover, we studied the ability of KLS to modulate leukotriene C₄ (LTC₄) and acetylcholine (ACH) induced bronchoconstriction and the associated production of TXA₂. Nimesulide (NIM) was used as the reference compound.KLS dose-dependently inhibited the bronchoconstriction and the associated production of TXA₂ induced by BK, with closely related ID₅₀ values of 31.2 and 34.0g/kg i.v., respectively. The protection was evident 10 min after KLS administration and, at 100 g/kg i.v., lasted up to 2h, Moreover, KLS dose-dependently inhibited the increase in capillary permeability induced by BK, with a potency (ID₅₀ 23.4 g/kgi.v.) slightly higher than that shown against the bronchoconstriction. KLS also prevented the bronchoconstriction and TXA₂ production triggered by LTC₄, but not ACH induced bronchoconstriction. In all the models studied, KLS was about 10 times more potent than NIM.These data demonstrate the capacity of KLS to counteract the bronchoconstriction induced by BK and LTC₄ and to a large extent the airway inflammation induced by BK. Blockade of prostanoid production is likely to account for this protective effect, since the R-isomer of KLS was devoid of significant activity.accepted by M. J. Parnham     

Involvement of substance P as a mediator in capsaicin-induced mouse ear oedema

We examined the involvement of substance P (SP) in mouse ear oedema induced by topical application of capsaicin (250 µg/ear). Reapplication of capsaicin at 4h, 24h, and 48h after initial treatment did not induce a second oedema response. Oedema induced after the second application was significantly (p<0.01 orp<0.001) suppressed for up to 30 days but was observed when capsaicin was applied 40 days after initial treatment. Topical pretreatment of ears with capsaicin at 4h, 24h and 48h before i.v. injection of SP (5 µg/kg) did not cause a significant inhibition of plasma extravasation in ear skin. NK₁ receptor antagonists such as RP 67580 (ED₅₀:0.19 mg/kg, i.v.), spantide II (ED₅₀:0.33 mg/kg, i.v.), and GR 82334 (ED₅₀:0.26 mg/kg, i.v.), inhibited capsaicin-induced ear oedema, whereas SR 48968 (2.0 mg/kg, i.v.), a NK₂ receptor antagonist, had no effect. Furthermore, RP 67580 (0.5 kg/mg, i.v.) inhibited the oedema response induced by reapplication of capsaicin at 50 days after initial treatment. These results indicate that tachyphylaxis of capsaicin-induced oedema is reversible and suggest that this response may be due mainly to a reduction of SP in sensory neurones but not to any loss of responsiveness of NK₁ receptors. We also conclude that SP and NK₁ receptors are involved predominantly in the development of capsaicin-induced mouse ear oedema.     

Influence of B2 receptor antagonists on bradykinin-induced vasodilation and edema formation in isolated rabbit hindlimbs

In search of new possibilities to prevent acute inflammatory vascular reaction, we examined the effect of two selective B₂ receptor antagonists, CP 0127 ([Bissuccimidohexane (L-Cys⁶)^–1] and HOE 140 (D-Arg[Hyp³, Thi⁵, D-Tic⁷, Oic⁸]BK), on changes in perfusion pressure and on edema formation caused by bradykinin (BK) in the isolated perfused rabbit hindlimbs. CP 0127 and HOE 140 were added to the perfusion fluid 2 min prior to the first BK-administration (5 × 10^–9 mol/l). A second BK-stimulation was performed after 30 minutes. The antagonists were tested in groups of 6 experiments each at concentrations of 10^–6 mol/l, 5 × 10^–9 mol/l and 10^–10 mol/l. CP 0127 was also tested in a concentration of 10^–8 mol/l. The application of BK resulted in an acute decrease of the mean arterial pressure and in a continual edema formation, reflected by an increase of organ weight (controls,n=6). Pretreatment with CP 0127 as well as with HOE 140 attenuated dose-dependently the BK-induced vasodilation (p < 0.005) and edema formation. The current results indicate that CP 0127 and HOE 140 are able to reduce BK-induced effects on vascular tone and edema formation.     

Lipoxygenase metabolites mediate increased airways resonsiveness to histamine after acute platelet activating factor exposure in the guinea-pig

Platelet activating factor (Paf, 0.02 g/kg, i.v. bolus) caused an acute increase in airways responsiveness to histamine in anaesthetized guinea-pigs prepared for recording airways resistance (R _L ) and dynamic compliance (C _dyn ). Aspirin pretreatment (10 mg/kg, i.v.) attenuated the return of airways responsiveness to prechallenge levels. Pretreatment with the combined cyclooxygenase/lipoxygenase inhibitors BW 755C (20 mg/kg, i.v.) and ETYA (20 mg/kg, i.v.), or with the putative cysteinyl-containing leukotriene antagonist FPL 55712 (0.25 mg/kg/min, i.v.), or a Paf antagonist SRI 63441 (2.5 mg/kg, i.v.), prevented Paf-induced increased airways responsiveness. Inhibitors of leukotriene synthesis, BW 755C and ETYA, or action, FPL 55712, had variable effects of Paf-induced bronchoconstriction. These data suggest that lipoxygenase metabolites, possibly leukotrienes, may mediate an acute increase in airways responsiveness to histamine after Paf exposure.     

Role of tachykinins in enhancement of bradykinin-induced bronchoconstriction by captopril

In anesthetized, mechanically ventilated guinea pigs, infusion of captopril (1 mg/kg/h), an angiotensin converting enzyme inhibitor, significantly enhanced bronchoconstriction induced by intravenous injection of bradykinin (BK; 0.1–30 nmol/kg). Pretreatment of guinea pigs with capsaicin (100 g/kg) slightly suppressed the bronchoconstriction by BK alone and almost all of the enhancement of BK-induced bronchoconstriction by captopril was suppressed. Intravenous injection of substance P (SP; 0.1–100 nmol/kg), neurokinin A (NKA; 0.1–30 nmol/kg) and neurokinin B (NKB; 0.1–30 nmol/kg) also induced dose-dependent bronchoconstriction but captopril treatment enhanced only the bronchoconstriction induced by SP. BK degradation in bronchoalveolar lavage fluid (BALF) in vitro was significantly suppressed by captopril (p<0.05). Captopril infusion to guinea pigs significantly increased the levels of BK, SP, and NKA in BALF after BK injection (p<0.05). FK224, an NK1 and NK2 receptor antagonist and SR 48968, an NK2 receptor antagonist, significantly suppressed the broncoconstriction induced by BK alone (p<0.01 and p<0.05, respectively) as well as the enhancement by captopril (p<0.01). It can be concluded that the enhancement of BK-induced bronchoconstriction by captopril was attributable to inhibition of the degradation of BK itself and thereby enhanced release of NKA and partly of SP from sensory nerves by BK.accepted by M. J. Parnham     

Effect of the tachykinin receptor antagonists,SR 140333, FK 888, and SR 142801, on capsaicin-induced mouse ear oedema

We examined the effect of SR 140333, a nonpeptide NK₁ receptor antagonist, FK 888, a peptide NK₁ antagonist, and SR 142801, a non-peptide NK₃ antagonist, on ear oedema induced by topical application of capsaicin (250 g/ear) in mice. SR 140333 (ED₅₀: 39 g/kg, i.v.) dose-dependently inhibited the oedema response to capsaicin, whereas FK 888 (1.0 mg/kg, i.v.) and SR 142801 (3.0 mg/kg, i.v.) had no effect. Furthermore, SR 140333 significantly (p<0.001) suppressed ear oedema in response to intradermal injection of substance P (SP) (100 pmol/site) by i.v. administration (0.1 mg/kg), and co-injection (50 pmol/site). In contrast, FK 888 (1.0 mg/kg, i.v. and 500 pmol/site) was ineffective in the response to SP. The present results suggest that the difference in effects of the two NK₁ receptor antagonists on the oedema response to capsaicin is due to species differences in affinities for the NK₁ receptor in the mouse skin. Moreover, it seems unlikely that the NK₃ receptor is involved primarily in capsaicin-induced mouse ear oedema.accepted by G. W. Carter     

Modulation of allergic and immune complex-induced lung inflammation by bradykinin receptor antagonists

Objective: The effect of bradykinin (B₁ or B₂) receptor antagonists was studied in allergic and immune-complex-induced lung inflammation.Methods: Lungs of BALB/c mice were examined 24 h after induction of lung inflammation, either allergic (ovalbumin-sensitized submitted to two aerosol of antigen, one week apart) or immune-complex induced (intratracheal instillation of IgG antibodies followed by intravenous antigen). The bradykinin B₂ receptor antagonist, HOE-140 or bradykinin B₁ receptor antagonist, R-954 were given intraperitoneally (100 g/kg), 30 min before induction.Results: In allergic inflammation, pre-treatment with R-954 reduced eosinophil infiltration into the lungs, mucus secretion and the airway hyperreactivity to methacholine. Pre-treatment with HOE-140 increased eosinophil infiltration but did not affect the other parameters. In immune-complex inflammation, HOE-140 increased neutrophil infiltration but not their activation nor the hemorrhagic lesions. R-594 pre-treatment did not change the parameters examined.Conclusion: These results show important modulatory effects of bradykinin B₁ and B₂ receptor antagonists in both models of lung inflammation.Received 6 May 2003; returned for revision 22 July 2003; accepted by N. Boughton-Smith 8 October 2003Received 6 May 2003; returned for revision 22 July 2003; accepted by N. Boughton-Smith 8 October 2003     

Histamine receptor blocking effects of cimetidine in the airways

We investigated the modification of histamine-induced bronchoconstriction by the H₂-antagonist cimetidine in conscious sheep. One hundred breaths of 5% histamine aerosol increased mean (SD) pulmonary resistance (R _L) by 5.6 (1.4) cmH₂O/1/sec. This increase inR _L was completely blocked by intravenous clemastine (0.5 mg), a specific H₁-antagonist, indicating that the histamine-induced bronchoconstriction was mediated by H₁-receptors. Intravenous cimetidine caused a dose-dependent enhancement of the histamine response between 1 and 1000 mg with a mean peak ΔR _L of 15.3 (5) cmH₂O/1/sec (P<0.05) at the 1000 mg dose, while it blocked the histamine response at a dose of 2400 mg [ΔR _L=1.9 (2) cmH₂O/1/sec,p=NS]. This paradoxic effect was not related to an anticholinergic mechanism as intravenous cimetidine (2400 mg) failed to block carbachol-induced (25 breaths of 1% solution) bronchoconstriction. We conclude that in the ovine airway, cimetidine is a selective H₂-histamine receptor blocker at lower tissue concentrations, and a combined H₂- and H₁-histamine receptor blocker at high tissue concentrations.     

Role of eicosanoids in airflow obstruction and airway plasma exudation induced by trimellitic anhydride-conjugate in guinea-pigs 3 and 8 weeks after sensitization

Abstract. Trimellitic anhydride (TMA) is a low molecular weight chemical which can cause occupational asthma. We studied the role of eicosanoids in airway responses to TMA at different times after sensitization in actively sensitized guinea-pigs. Sensitization was performed by two intradermal injections of free TMA (0.1 ml of 0.3% TMA in corn oil). At 3 and 8 weeks after sensitization, the guinea-pigs were anaesthetized and challenged with intratracheal instillation of 0.5% TMA conjugated to guinea-pig serum albumin (TM A-GPSA; 50 μl). Lung resistance (RL) was measured to assess airflow obstruction, and the tissue content of Evans Blue dye was measured to assess airway plasma exudation. Intratracheal instillation of TMA-GPSA induced a slowly progressing increase in R_L, reaching a peak at approximately 3.5 min after the challenge (6.0 ± 2.0cm H₂O/ml/s in the 3-week group and 3.8 + 0.6 in the 8-week group). Pretreatment before challenge with pyrilamine (anti-histamine: 2 mg/kg. intravenously) slowed the onset of the increase in R_L following challenge with TMA-GPSA, and significantly attenuated the peak response. A combination of pyrilamine and IC1-192, 605 (thromboxane receptor antagonist; 0.5 mg/kg, intravenously) completely abolished the increase in R_Lin both week groups. A combination of pyrilamine and ICI-198, 615 (leukotrieneC₄/D₄/ E₄ receptor antagonist: 0.5 mg/kg, intravenously) did not further attenuate the increase in R_L compared with pretreatment with pyrilamine alone, but the induced Evans Blue dye extravasation was completely inhibited in the 3-week group, whereas a remaining extravasation was observed in the 8-week group. We conclude that the bronchoconstrictor response to TMA-GPSA in actively sensitized guinea-pigs is mediated by histamine and thromboxane A₂ both early and late after sensitization, whereas leukotrienes and histamine partially mediate TMA-induced airway plasma exudation.     

Modulation of neurally mediated airway microvascular leakage in guinea-pig airways byβ 2 agonists

The effect of two {ie29-2} agonists, salbutamol (100 g/kg i.v.) and broxaterol (100 g/kg i.v.), on airway microvascular leakage induced by vagal stimulation was studied in anaesthetised guinea pigs. Airway microvascular leakage was measured by Evans blue extravasation. Broxaterol, but not salbutamol, inhibited Evans blue dye extravasation at all airway levels, an effect prevented by pretreatment with propranolol (1 mg/kg). Neither of the {ie29-3} had any effect on substance P-induced Evans blue dye extravasation. Broxaterol inhibits the prejunctional release of tachykinins from airway sensory nerves by stimulation of -receptors. The mechanism by which -adrenoceptor agonists prevent airway microvascular leakage deserves further study.     

Bradykinin Stimulates MMP-2 Production in Guinea Pig Tracheal Smooth Muscle Cells

The implication of bradykinin (BK) receptors in the release of the matrix metalloproteinase-2 (MMP-2; gelatinase A) was studied in guinea pig tracheal smooth muscle cells (GP-TSMC). Bradykinin (10^–8–10^–4 M) induced a time- and concentration-dependent upregulation of MMP-2 production from cultured GP-TSMC. Pretreatment of the GP-TSMC with the bradykinin B₂ receptor (BKB-R) antagonist Hpp-HOE-140 (Hpp-D-Arg⁰-Hyp³-Thi⁵-D-Tic⁷-Oic⁸-BK; 10^–8–10^–4 M) significantly inhibited the BK-stimulated upregulation of MMP-2 in GP-TSMC in a concentration-related manner. Conversely, GP-TSMC pretreated with the selective bradykinin B₁ receptor (BKB₁-R) antagonist R-954 (Ac-Orn[Oic², -MePhe⁵, D-Nal⁷, Ile⁸]desArg⁹BK; 10^–8–10^–4 M) did not show any change in the response to BK. Moreover, the selective BKB₂-R agonist Lys⁰BK (kallidin; 10^–8–10^–4 M) stimulated whereas the selective BKB₁-R agonist desArg⁹BK (DBK; 10^–8–10^–4 M) had no effect on MMP-2 release from GP-TSMC. Further, the nonselective cyclooxygenase (COX) enzyme inhibitor indomethacin (IND; 10^–5 M), the glucocorticosteroid dexamethasone (DEX; 1 ng/mL) and the protein synthesis inhibitors, cycloheximide (CHX; 10^–6 M) and actinomycin D (ACT-D; 10^–8 M) also inhibited BK-induced MMP-2 release from GP-TSMC. These results provide the first evidence for the involvement of BK in the release of MMP-2 from airway smooth muscle cells through activation of the BKB₂-R. Such response is mostly mediated by the induction of COX and the subsequent production of endogenous prostaglandins (PGs). It could therefore be suggested that MMP-2 might play a role in the process of airway remodeling.     

IL-1β acutely increases pulmonary SP and permeability without associated changes in airway resistance and ventilation in anesthetized rats

Bronchopulmonary C-fibers (PCFs), when activated, promote substance P (SP) release, increase microvascular leak, and produce bronchoconstriction and apnea. IL-1β administered systemically or locally into the pulmonary parenchyma stimulates PCFs. Thus, we tested whether right atrial bolus injection or aerosol inhalation of IL-1β, to mainly stimulate PCFs, would acutely affect pulmonary SP level and vascular permeability, airway resistance (R_L), and ventilation in anesthetized rats. Our results showed that 30 min after IL-1β injection (2–6 μg kg⁻¹), SP levels and Evans blue extravasation in bronchoalveolar lavage fluid were markedly increased and these responses were eliminated or largely reduced in neonatal capsaicin-treated rats. In contrast, neither injection nor inhalation of IL-1β (5–15 μg ml⁻¹) significantly altered R_L and ventilation. Additionally, the capsaicin-evoked (4 μg kg⁻¹, i.v.) apneic response was unaffected by IL-1β treatment. Our data suggest that IL-1β, as administered in this study, can acutely increase pulmonary SP and vascular permeability, likely via stimulating PCFs, with little impact on R_L and ventilation.     

Involvement of prostaglandin E(2) derived from enteric glial cells in the action of bradykinin in cultured rat myenteric neurons

We characterized bradykinin (BK)-induced changes in the intracellular Ca(2+) concentration ([Ca(2+)]i) and membrane potential in cultured rat myenteric neurons using ratiometric Ca(2+) imaging with fura-2 and the whole-cell patch-clamp technique, respectively. BK evoked a dose-dependent increase of [Ca(2+)]i that was abolished by HOE 140, a B2 receptor antagonist but not by [Lys-des-Arg(9)]-BK, a B1 receptor antagonist. [Lys-des-Arg(9)]-HOE140, a B1 receptor agonist, failed to cause a [Ca(2+)]i response. Double staining with antibodies against the B2 receptor together with PGP9.5 or S100 indicated that B2 receptors were expressed in neurons and glial cells. The BK-evoked [Ca(2+)]i increase was suppressed by indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and potentiated by prostaglandin E(2) (PGE(2)). The release of PGE(2) from cultured myenteric plexus cells was increased by BK. BK induced a large increase in [Ca(2+)]i in neurons when myenteric plexus cells were cultured at the high density but not at the low density, and caused a small increase in [Ca(2+)]i in neurons when proliferation of enteric glial cells was suppressed. BK evoked a slow and sustained depolarization in myenteric neurons, which was sensitive to indomethacin. These results indicated that BK caused a [Ca(2+)]i increase and depolarization in rat myenteric neurons through the activation of B2 receptors, which was partly associated with PGE(2) released from glial cells in response to BK. It is suggested that a neuron-glial interaction plays an important role in the effect of BK in the rat myenteric plexus.     

Modulation of bradykinin responses in airway smooth muscle by epithelial enzymes

We have studied the effect of epithelium removal on responses of guinea pig trachea to bradykinin (BK). BK (1 nM–10 M) gave a concentration-dependent relaxation when epithelium was present (E+: EC₅₀=10±3 nM). Epithelium removal resulted in a biphasic response to BK with relaxation at low concentrations (E–: EC₅₀=3.0±1.0 nM) and a recontraction to baseline at higher concentrations (EC₅₀=2.0±1 M). Phosphoramidon (10 M), an inhibitor of neutral endopeptidase (NEP), which cleaves BK into inactive peptides, potentiated relaxation (EC₅₀=1.0±0.9 nM and 0.1±0.1 nM in E+ and E respectively) and contraction in trachea with intact epithelium (EC₅₀=0.08±0.03 M). Inhibition of cyclooxygenase by indomethacin (5 M), inhibited relaxation to BK in E+ tracheal segments, resulting in a slight contraction (EC₅₀=1.0 M), whereas a potent contractile response was observed in E–segments (EC₅₀ 1.6 M, maximal contraction >1 g). In the presence of both indomethacin and phosphoramidon BK caused contraction, even in the presence of epithelium (EC₅₀=0.2±0.11 M), and the response in the absence of epithelium was similar to the response observed in trachea with intact epithelium (EC₅₀=0.25±0.1 M). The contractile effect of BK on airway smooth muscle may be inhibited by a protective role of epithelium, due to release of relaxant prostanoids and by degradation by epithelial NEP. In asthma, bronchoconstrictor responses to BK may be partly explained by loss of airway epithelium.     

New highly potent bradykinin B2 receptor antagonists

pA₂ values of new B₂ receptor antagonists ranging from 7.51 to 8.86 were measured on the rabbit jugular vein, while lower values were observed in the other preparations (for instance, the hamster urinary bladder). The most potent antagonists were those containing a hydroxyproline (Hyp) in position 3, ad-Arg at the N-terminal and a Leu instead of a Phe in position 8, with or without other chemical changes.d-Arg[Hyp³,d-Phe⁷, Leu⁸]-BK was found to be competitive, selective for B₂ receptors and specific for kinins since it was without effect against substanceP and angiotensin II in the rabbit jugular vein. The essential feature for obtaining B₂ receptor antagonists appears to be the replacement or reorientation of Phe⁸ of bradykinin. The rabbit jugular vein provides a sensitive bioassay in which the potency and specificity of B₂ receptor antagonists can be adequately evaluated.     

Effect of des arginine9-bradykinin and other bradykinin fragments on the synthesis of prostacyclin and the binding of bradykinin by vascular cells in culture

Bradykinin (BK) fragments, des arg¹-BK, des arg¹,pro²-BK, des phe⁸,arg⁹-BK and des pro⁷,phe⁸,arg⁹-BK were synthesized and along with des arginine⁹-BK (daBK), tested for their ability to induce prostacyclin synthesis in homogeneous cultures of cells from the calf pulmonary artery. Of the fragments daBK was the only peptide, in addition to bradykinin (BK), to activate the synthesis of prostacyclin (PGI₂) and platelet activating factor (PAF) in endothelial cells and PGI₂ in fibroblasts and smooth muscle cells. Half-maximal activation of PGI₂ synthesis differed with the cell type. The other fragments tested did not directly affect PGI₂ synthesis. These fragments also did not inhibit daBK or BK activation of PG synthesis.BK bound to endothelial cells with a dissociation constant (K_d) of 2.1 nM and a B_max of 47.9 fmoles/10⁶ cells. The K_d for the binding of BK to smooth muscle cells and fibroblasts was somewhat higher, 4.9 nM and 7.9 nM, respectively. None of the fragments tested, including daBK, altered the binding of BK. Des arg⁹[leu⁸]-BK, reported to be a competitive antagonist of the bradykinin B₁ receptor, inhibited daBK induced PG of PAF synthesis in endothelial cells but had little effect of BK binding or BK induced PG synthesis. Finally, the BK antagonist [thi^5,8, d-phe⁷]-BK blocked both BK binding and the ability of either BK or daBK to induce PG synthesis, thus substantiating that the binding of these kinins is a step in the activation of PG synthesis.This work was supported by NIH grants AG05007, HL25776 and HL07035.     

Ability of inhaled procaterol, a β32 adrenoceptor agonist, to attenuate eicosanoid-induced airflow obstruction and airway microvascular leakage

Beta-2 adrenoceptor agonists are widely used as bronchodilators in the treatment of asthma mainly via inhalation. In the present study, we evaluated the ability of inhaled procaterol, a β2 adrenoceptor agonist, to reduce eicosanoid-induced airway micro-vascular leakage, and compared the ability with its inhibitory effect against bron-choconstriction. Tracheostomized guinea-pigs were given aerosolized procaterol (10 or 100 μg/ml) for 10min under spontaneous breathing. Immediately after the end of inhalation, the animals were mechanically ventilated. Fourteen minutes after the end of inhalation, Evans blue dye (20mg/kg) was given i.v. One minute later, 2nmol/kg leukotriene D4 (LTD4), 50 nmol/kg U-46619, a thromboxane A2 mimetic, or vehicle was administered i.v. LTD4- or U-46619-induced increase in lung resistance was measured for 6 min. After removing the lungs, the amount of extravasated Evans Blue due in the lower airways was examined as an index of microvascular leakage. Inhaled procaterol significantly attenuated increases in both lung resistance and Evans Blue dye extravasation caused by these eicosanoids. The degree of inhibition was almost complete for lung resistance (approximately 90%), but it was only partial (range 18.5–61.2%) for the dye extravasation. No significant changes in mean systemic blood pressure and in heart rate was observed after an inhalation of 10μg/ml procaterol. These results suggest that inhaled β2 adrenoceptor agonists may reduce airway microvascular leakage caused by inflammatory mediators such as eicosanoids without affecting systemic circulation. However, these agonists may attenuate airway microvascular leakage only partially even in doses which can inhibit bronchoconstric-tion almost completely.