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Objective and Design: There is increasing evidence for the involvement of reactive nitrogen species like peroxynitrite (ONOO-) in airway pathology, for example during allergic airway inflammation. Therefore, the effect of peroxynitrite exposure on airway responsiveness and inflammation was studied., Materials: Male BALB/c mice were treated intra-tracheally with authentic peroxynitrite and the peroxynitrite donor 3-morpholinosydnonimine (SIN-1). Control animals received decomposed solutions of peroxynitrite and SIN- 1., Methods: Airway inflammation was monitored by bronchoalveolar lavage, three and seven days after administration. Airway responsiveness to methacholine and acetylcholine was measured on day 1, 2, 3 and 7 post administration using whole body plethysmography., Results: Intra-tracheal administration of peroxynitrite 200 microM in 50 microl phosphate buffered saline (PBS) induced a significant increase in macrophages (>35%, p < 0.05) in the airway lumen three days after administration. In contrast, neither intra-tracheal administration of authentic peroxynitrite (up to 5 mM) nor the peroxynitrite donor SIN-1 (1 mM, both intra-tracheal and nebulized) changed airway responsiveness to methacholine. Moreover, peroxynitrite (5 mM) did not alter responsiveness to acetylcholine., Conclusion: Administration of peroxynitrite directly into the airways of BALB/c mice, induces airway inflammation, but not airway hyperresponsiveness. It is suggested that antioxidants in the epithelial lining fluid and/or the epithelium itself form an efficient barrier, which prevents peroxynitrite from reaching putative targets in the airway interstitium.

1. Nerve growth factor induces an airway hyperresponsiveness in vivo in guinea-pigs, as we have shown previously. Since antagonizing the neurokinin-1 (NK(1)) receptor can prevent this NGF-induced airway hyperresponsiveness and since sensory nerves release tachykinins, we investigated the role of sensory nerves in the NGF-induced airway hyperresponsiveness. 2. We used isolated tracheal rings from guinea-pigs to measure tracheal contractility. In these rings sensory nerve endings are present, but these endings lack any contact with their cell bodies. 3. In this in vitro system, NGF dose-dependently induced a tracheal hyperresponsiveness to histamine. The NK(1) receptor antagonist SR140333 could block the induction of tracheal hyperresponsiveness. 4. To further investigate the involvement of sensory nerve endings we used the cannabinoid receptor 1 (CB(1)) agonist R-methanandamide to inhibit excitatory events at the nerve terminal. The CB(1) receptor agonist was capable of blocking the tracheal hyperresponsiveness to NGF in the isolated system, as well as the airway hyperresponsiveness to NGF in vivo. 5. This indicates that NGF can induce an increase in airway responsiveness in the absence of sensory nerve cell bodies. NGF may act by increasing substance P release from sensory nerve endings, without upregulation of substance P in the neurons. Substance P in its turn is responsible for the induction of the NGF-induced airway hyperresponsiveness.

Acetylcholine administered to the inside of epithelium-denuded tracheal tubes did cause a potent contraction (2486+/-120 mg). In contrast, a response was hardly observed in tissues with an intact epithelial layer (674+/-81 mg), which was due to both the synthesis of nitric oxide and the activity of acetylcholinesterase, since the contractions to acetylcholine were significantly enhanced after preincubation with N(omega)-nitro-L-arginine methyl ester (L-NAME) or physostigmine (1374+/-65 and 1120+/-65 mg, respectively). In addition, the suppressive effect was caused by the barrier function of the epithelial layer, since preincubation of epithelium-denuded tissues with physostigmine significantly increased the pD2 value for acetylcholine (7.48+/-0.04) compared to intact tissues preincubated with physostigmine (6.32+/-0.10) and epithelium-denuded preparations without physostigmine (6.37+/-0.06). Increasing concentrations of physostigmine administered to the inside of tissues with epithelium did induce a potent spontaneous contraction (1440+/-350 mg) that was prevented by atropine. In contrast to what was expected, the contractile response was diminished in tracheal tubes without epithelium (665+/-221 mg). It is concluded that contractions of epithelium-denuded tissues are more pronounced to exogenous than to endogenous acetylcholine, and that the production and breakdown of this neurotransmitter is very rapid in intact guinea pig airways. Moreover, the release of nitric oxide and the barrier function of the epithelium did suppress the responsiveness to acetylcholine.

In this study, a tracheal perfusion apparatus was used to investigate the nature of the relaxing factor released by hyperosmolarity on the epithelial side of guinea pig trachea. NaCl induced concentration-dependent relaxation. This relaxation was not affected when the trachea was preincubated with a vasoactive intestinal peptide (VIP) receptor antagonist or with the nitric oxide synthesis inhibitor N(G)-monomethyl-L-arginine (L-NMMA). When the prostaglandin synthesis was prevented by preincubation with the phospholipase A(2)-inhibitor quinacrine, or the cyclooxygenase inhibitor indomethacin, the maximal relaxation induced by NaCl was suppressed by 50% (P<0.05). Moreover, the prostaglandin E(2) concentration was four times higher (P<0.05) in the organ bath during the relaxations, whereas the nitric oxide concentration remained unchanged. In conclusion, increased osmolarity on the airway surface leads to the release of prostaglandins, which are involved in part in the hyperosmolarity-induced relaxation of airway smooth muscle. This might be relevant for asthmatic patients since prostaglandin may modulate the bronchoconstrictive response to hyperosmolar stimuli and exercise.

1. This study investigates the role of tachykinins in a repeated challenge with dinitrobenzene sulphonic acid (DNS) on the tracheal vascular permeability in dinitrofluorobenzene (DNFB)-sensitized mice. 2. DNFB-contact sensitization was followed by an intranasal (i.n.) challenge with DNS. A second challenge with DNS was administered 24 h after the first challenge. To assess changes in tracheal vascular permeability, Evans blue dye accumulation in tracheal tissue was measured. 3. A repeated challenge with DNS in DNFB-sensitized mice led to a 2.8 fold increase in tracheal vascular permeability when compared to DNFB-sensitized and vehicle-challenged mice or a 2.5 fold increase when compared to DNFB-sensitized single DNS-challenged mice (P<0.001, ANOVA). 4. RP67580 (10-9 mol mouse-1 i.v.) reduced the increased tracheal vascular permeability induced by a second exposure to DNS in DNFB-sensitized mice completely when injected 15 min before the second challenge (P<0.001, ANOVA). 5. The increased tracheal vascular permeability response induced by the second exposure to DNS could be mimicked with i.n. application of capsaicin (10-10 mol mouse-1) or substance P (SP) (10-12 mol mouse-1) to DNFB-sensitized and single DNS-challenged mice. 6. These results suggest that both tachykinin NK1 receptors and sensory nerves are involved in the development of vascular hyperpermeability changes found in the trachea of DNFB-sensitized mice after a repeated DNS-challenge.

Eosinophil-derived cationic proteins play an essential role in the pathogenesis of bronchial asthma. We tested whether cationic proteins interfere with the cationic amino-acid transport in alveolar macrophages (AMPhi) and tracheal epithelial cells, and whether L-arginine-dependent pathways were affected. The effect of cationic polypeptides on cellular uptake of [(3)H]-L-arginine, nitrite accumulation, and the turnover of [(3)H]-L-arginine by nitric oxide (NO) synthase and arginase (formation of [(3)H]-L-citrulline and [(3)H]-L-ornithine, respectively) were studied. Poly-L-arginine reduced [(3)H]-L-arginine uptake in rat AMPhi and tracheal epithelial cells in a concentration-dependent manner (at 300 microgram/ml by 70%). Poly-L-lysine, protamine, and major basic protein (each up to 300 microgram/ml) tested in rat AMPhi inhibited [(3)H]-L-arginine uptake by 35 to 50%. During 6 h incubation in amino acid-free Krebs solution, rat AMPhi, precultured in the absence or presence of LPS (1 microgram/ml), accumulated 1.4 and 3.5 nmol/10(6) cells nitrite, respectively. Addition of 100 microM L-arginine increased nitrite accumulation by 70 and 400% in control and lipopolysaccharide-treated AMPhi, respectively. Nitrite accumulation in the presence of L-arginine was reduced by poly-L-arginine and poly-L-lysine (100 and 300 microgram/ml) by 60 to 85% and 20 to 30%, respectively. Poly-L-arginine, but not poly-L-lysine, inhibited nitrite accumulation already in the absence of extracellular L-arginine. Poly-L-arginine (300 microgram/ml) inhibited [(3)H]-L-citrulline formation by AMPhi stronger than that of [(3)H]-L-ornithine. We conclude that cationic proteins can inhibit cellular transport of L-arginine and this can limit NO synthesis. Poly-L-arginine inhibits L-arginine uptake more effectively than other cationic proteins and exerts additional direct inhibitory effects on NO synthesis.

Toluene diisocyanate (TDI) is a low-molecular-weight compound which is known to cause occupational asthma in 5 to 10% of exposed workers. Previously, we developed a murine model to investigate TDI-induced occupational asthma. Short-term exposure to TDI (skin sensitization twice daily on Day 0 and Day 1 and intranasal challenge on Day 8) led to a nonspecific tracheal hyperractivity 24 h after the challenge in TDI-sensitized mice when compared with nonsensitized mice whereas no TDI-specific IgE antibodies were found in the serum. Because 20% of subjects with TDI-induced occupational asthma exhibit an increase in serum IgE antibodies, we exposed mice for a longer period of time to investigate whether this procedure could induce TDI-specific antibody production in exposed mice. Long-term exposure (skin sensitization on 6 consecutive weeks followed by intranasal challenge on Week 7) resulted in the production of total IgE and IgG and TDI-specific IgE and IgG antibodies. Airway reactivity to various agonists was also measured in vitro and in vivo in long-term exposed mice. TDI-sensitized mice exhibited in vitro tracheal hyperreactivity to carbachol 3 h after the challenge when compared with the nonsensitized mice. Moreover, in vivo airway hyperresponsiveness to serotonin (5-hydroxytryptamine [5HT]) was found 3 h after the challenge in TDI-sensitized mice. Interestingly, in vivo airway hyperresponsiveness was not observed at any time point in the mice exposed to TDI according to the short-term protocol. In conclusion, by altering the exposure time and/or cumulative dosage of TDI different biological reactions can be elicited in exposed mice. This important finding might be a reflection of the diversity of symptoms found in patients suffering from TDI-induced asthma. Both the short-exposure and the long-exposure model will be useful to further investigate the mechanisms of action of TDI.

1. Motor responses to des-Arg9-bradykinin and bradykinin were studied in the isolated mouse trachea (precontracted with carbachol, 10 microM) and the urinary bladder of either Swiss, C57B1/6J or bradykinin B2 receptor knockout (Bk2r(-/-)) mice after 1-6 h in vitro. The expression of mRNA for the mouse B1 receptor in tracheal and urinary bladder tissues was also studied by using Northern blot analysis. 2. In isolated tracheae, des-Arg9-bradykinin produced a relaxant response that increased over time: no response was observed after 1 h of incubation, whereas after 6 h the maximum response (1 microM) was 68-84% of the relaxation produced by isoproterenol (1 microM) in the three mouse strains. The relaxant response to bradykinin (1 microM) observed at 1 h (38-51% of isoproterenol) was increased (62-65% of isoproterenol) after 6 h in Swiss and C57B1/6J mice, but was absent in Bk2r(-/-) mice. In the presence of cycloheximide, des-Arg9-bradykinin did not cause any response at 6 h. 3. Similar findings were obtained in the urinary bladder: at 1 h des-Arg9-bradykinin (1 microM) did not cause any motor effect, whereas at 6 h it caused a contraction that was 28-59% of that produced by carbachol (1 microM) in the three mouse strains. Cycloheximide blocked the response to des-Arg9-bradykinin. Bradykinin (1 microM) contracted urinary bladders at 1 h (34-35% of carbachol), as well as at 6 h (66-77% of carbachol) in Swiss and C57B1/6J strains, but was without effect in Bk2r(-/-) mice. 4. Northern blot hybridization with a specific cDNA probe against mouse B1 receptor mRNA using total RNA extracted from tracheae and urinary bladders freshly removed from Swiss and Bk2r(-/-) mice revealed minimal expression. However, marked hybridization was detected 150 min after in vitro exposure in both tissues. 5. Evidence is provided that in vitro exposure of mouse trachea and urinary bladder causes a time-dependent induction of B1 receptors that cause relaxation and contraction, respectively.

1. The endothelin (ET) receptor subtype that mediates niric oxide (NO)-dependent airway relaxation in tracheal tube preparations precontracted with carbachol and pretreated with indomethacin was investigated. The release of NO induced by ET from guinea-pig trachea using a recently developed porphyrinic microsensor was also measured. 2. ET-1 (1 pM-100 nM) contracted tracheal tube preparations pretreated with the NO-synthase inhibitor, L-NMMA, and relaxed, in an epithelium-dependent manner, preparations pretreated with the inactive enantiomer D-NMMA. The effect of L-NMMA was reversed by L-Arg, but not by D-Arg. 3. The selective ET(B) receptor agonists, IRL 1620 or sarafotoxin S6c, both (1 pM-100 nM) contracted tracheal tube preparations in a similar manner either after treatment with D-NMMA or with L-NMMA. In the presence of the ET(A) receptor antagonist, FR139317 (10 microM), ET-1 administration resulted in a contraction that was similar after either L-NMMA or D-NMMA. In the presence of the ET(B) receptor antagonist, BQ788 (1 microM), ET-1 relaxed and contracted tracheas pretreated with D-NMMA and L-NMMA, respectively. 4. Exposure of tracheal segments to ET-1 (1-1000 nM) caused a concentration-dependent increase in NO release that was reduced by L-NMMA. IRL1620 (1 microM) did not cause any significant NO release. FR139317 (10 microM), but not, BQ788 (1 microM), inhibited the NO release induced by ET-1. 5. These results demonstrate that in the isolated guinea-pig trachea activation of ET(B) receptors results in a contractile response, whereas activation of ET(A) receptors cause both a contraction, and an epithelium-dependent relaxation that is mediated by NO release.

The lucigenin-dependent chemiluminescence generation by guinea-pig isolated tracheal two rings preparations was studied. Tracheal preparations stimulated with phorbol myristate acetate (PMA) or opsonized zymosan generated chemiluminescence. The total amount of chemiluminescence generated in 120 min was 754+/-63 mV x min for PMA and 4832+/-396 mV x min for zymosan. Generation of chemiluminescence was decreased by more than 50% when the tissues were co-incubated with superoxide dismutase (100 U/ml). Also, addition of direct donors of nitric oxide diminished chemiluminescence generation by zymosan-activated tracheal rings significantly by about 50%. However, the presence of the precursor or of inhibitors of nitric oxide synthase did not influence zymosan-induced chemiluminescence. Removal of the epithelial layer from tracheal rings caused an approximately 90% decrease in chemiluminescence response. However, isolated epithelial cell suspensions did not generate chemiluminescence. Histologic examination showed that the number of eosinophils in the tracheal tissue was reduced from 56+/-7 to 18+/-8 per mm basal membrane when the epithelial layer was removed. These results indicated that (1) superoxide anion formation can take place in the guinea-pig trachea, (2) eosinophils in the epithelial and submucosal layers of guinea-pig trachea are likely candidates for superoxide generation although other cell types can also be involved, and (3) besides relaxing airway smooth muscle, nitric oxide donors may also affect superoxide in the airways.

In the present study, the effect of bradykinin on basal and precontracted mouse-isolated trachea was investigated. In basal conditions mouse-isolated tracheal rings do not respond to bradykinin. However, when the tracheal rings were precontracted with carbachol (10(-7) M) a relaxation with bradykinin (3 x 10(-9)-3 x 10(-7)) was found. The maximal response amounted 69.7+/-4.1% (n=15) with a pD2 value of 7.2+/-0.21. The selective bradykinin B2 receptor antagonist HOE 140 (10(-10)-10(-8) M) antagonized the bradykinin-induced relaxation, while the bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin (10(-6) M) had no influence. The selective bradykinin B1 receptor agonist des-Arg9-bradykinin (10(-6) M) caused a small relaxation (8.4+/-2.5%, n=6), which could be antagonized completely by the selective bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin (10(-6) M) while addition of the selective bradykinin B2 receptor antagonist HOE 140 (10(-8) M) was without effect. In the presence of indomethacin (10(-6) M) the relaxation of bradykinin was completely abolished. Pretreatment of the tracheal rings with capsaicin, or the presence of the selective NK1 receptor antagonist RP 67851 (10(-6) M) or the presence of the nitric oxide synthase inhibitor L-NAME (3 x 10(-4) M) had no effect on the bradykinin-induced relaxation. In conclusion, these results demonstrate that the mouse-isolated tracheal is a preparation in which bradykinin exerts a relaxant response via stimulation of bradykinin B2 receptors. This response is probably mediated by prostaglandins.

Airway epithelium releases inhibitory factors, such as nitric oxide (NO) and prostaglandin E2 (PGE2), which may counteract bronchoconstriction. We investigated whether epithelium-derived inhibitory substances exert a crucial influence on bovine tracheal responsiveness in vitro. Isotonic and isometric contractions in response to histamine of intact and epithelium-denuded tracheal smooth muscle strips were compared. In addition, the effects of L-arginine (L-arg), N(G)-nitro-L-arginine methyl esther (L-NAME), and N(G)-monomethyl L-arginine (L-NMMA) on histamine responsiveness were investigated. The release of NO and PGE2 from tracheal epithelium was measured. Removal of the epithelium from tracheal smooth muscle strips did not change the negative log of the concentration of histamine producing half the maximal effect (pD2) or the maximal effect (Emax). Incubation of the tissues for 25 min with L-arg or L-NAME did not influence basal tone or the contractions induced by histamine. However, incubation with L-NMMA increased the basal tone and caused a slight hyporesponsiveness to histamine. S-nitroso-N-acetyl-penicillamine (SNAP, a direct NO donor) reversed the contraction induced by histamine in a concentration-dependent manner. Stimulation of the epithelial layer by 0.1 microM histamine increased the release of NO 3-4 fold compared to basal levels; this effect was completely inhibited in the presence of L-NMMA. In addition, 1 mM histamine caused a significant increase in the release of PGE2 from the epithelial tissue. In conclusion, no functional inhibitory influence of the epithelium can be identified in bovine airways. The S-nitroso-N-acetyl-penicillamine-induced relaxation demonstrates the presence of a nitric oxide sensitive pathway in bovine airways. However, the amounts of nitric oxide and prostaglandin E2 released from bovine tracheal epithelium are probably too low to exert a significant effect on the histamine-induced contractions.

Toluene diisocyantate (TDI) is a low-molecular-weight compound that is known to cause occupational asthma in 5% to 10% of exposed workers. These patients exhibit marked airway hyperresponsiveness and granulocyte accumulation in the airways, and 10% to 20% were also determined to have TDI-specific IgE in their serum. In this study, we developed a murine model for TDI-induced asthma. After several sensitization and challenge regimens were tested, it was decided that optimal sensitization was observed after mice (BALB/c) were skin sensitized with TDI (1%) two times on two consecutive days and challenged intranasally 8 d later with TDI (1%). Sensitized mice exhibited tracheal hyperreactivity to carbachol 24 h after challenge (69% increase in maximal contractile response). In contrast, no differences between the control and TDI-treated groups was observed 2 and 48 h after challenge with 1% TDI. There appeared to be no elevation in TDI-specific IgE antibodies in the serum at all time points measured. In addition, no influx of leukocytes could be detected histologically in the trachea and lung tissue or airway lumen 2, 24, and 48 h after the challenge. Surprisingly, the tracheal hyperreactivity was associated with a marked increase in myeloperoxidase but not eosinophil peroxidase activity in the lung tissue and in the cells of the bronchoalveolar lavage fluid at 24 h after the challenge. To investigate the role of lymphocytes in the induction of tracheal hyperreactivity, mice were passively sensitized by intravenous injection of lymphoid cells from TDI-sensitized donor mice. Similar to active sensitization, adoptive transfer of lymphocytes from sensitized donors resulted in tracheal hyperreactivity 24 h after challenge of the recipients. In conclusion, these data show that TDI is capable of inducing lymphocyte-dependent but IgE-independent tracheal hyperreactivity in the mouse that is not associated with cellular infiltration in the airways. This model can be used to further investigate the possible mechanisms involved in the development of occupational asthma induced by TDl.

1. Sodium nitroprusside (SNP) completely relaxed the guinea-pig isolated, perfused trachea in a concentration-dependent manner. Although SNP was less potent by about 2 orders of magnitude, its maximal effect was 25% higher compared to isoprenaline. 2. SNP (3.2 microM) increased cyclic GMP levels by 300% and relaxed guinea-pig isolated, perfused trachea by 54%. The SNP-induced relaxations of the preparations were not affected by the guanylate cyclase inhibitor, methylene blue. Moreover, zaprinast, a cyclic GMP-specific phosphodiesterase inhibitor which was supposed to enhance SNP-induced relaxations, decreased the maximal relaxation by 22% (P < 0.001). 3. In contrast, 8Br-cyclic GMP (10 microM) increased the cyclic GMP levels by 1100% without inducing a marked relaxation. 4. SNP (10 microM) and S-nitroso-N-acetylpenicillamine (SNAP; a direct donor of nitric oxide; 10 microM), relaxed the tissues by 75% and 25%, respectively, without any nitric oxide (NO) release by SNP (< 1 pmol 100 microliters-1), but a substantial NO release by SNAP (560 pmol 100 microliters-1). 5. It is concluded that the SNP-induced tracheal relaxations are probably not mediated by cyclic GMP and NO.

To examine the role of cyclic 3', 5'-guanosine monophosphate (cGMP) in airway responsiveness the effects of substances known to interfere with nitric oxide (NO) or cGMP were investigated on guinea pig airways. Using a perfused organ bath system, it was possible to apply the chemicals from either the serosal or the mucosal side independently. In addition, levels of intracellular cGMP were determined in tissues after various treatments. Sodium nitroprusside (a donor of NO), zaprinast (a specific inhibitor of cGMP phosphodiesterase) and 8-bromo-cGMP (8-Br-cGMP) caused a concentration-dependent relaxation of guinea pig trachea. These results indicate that cGMP is an important second messenger mediating tracheal relaxations. The above mentioned drugs caused a more profound relaxation when applied to the serosal side compared to the mucosal side, suggesting a barrier function of the epithelial layer. Incubation on the mucosal side of the tissues with 100 microM pyrogallol (a generator of superoxide that may inactivate NO) increased the contractile response to histamine at concentrations 0.3-3.2 microM (P < 0.05). Treatment of the preparations with 1 mM cystamine (an inactivator of guanylate cyclase) caused a 5-fold increase in the sensitivity to histamine (P < 0.05), indicating the involvement of the NO/cGMP pathway in the development of airway hyperresponsiveness. Incubation of the tissues with 100 microM histamine elevated the intracellular cGMP levels 10-fold; this effect was completely prevented by incubation of the tissues with methylene blue (a potent inactivator of guanylate cyclase). Mucosal incubation of the tracheal tubes with 10 microM methylene blue induced an 8-fold increase in sensitivity to histamine (P < 0.01) and the Emax was slightly increased. 25 min after instillation of 0.4 mumol methylene blue into the airways of anaesthetized guinea pigs, the lung resistance in response to histamine was elevated up to 395 +/- 82% (P < 0.001). The present study revealed that inactivation of NO or guanylate cyclase enhances the histamine-induced contractions of guinea pig tracheas. Therefore, it is suggested that the NO/cGMP pathway may be implicated in the pathogenesis of airway hyperresponsiveness and that drugs which enhance cGMP levels in airway smooth muscle may be of significance in the treatment of airway obstruction and enhanced reactivity.

Bradykinin, applied locally to the airways, is a weak bronchoconstrictor agent in guinea pigs in vivo and it may cause constriction or dilatation of guinea pig airways smooth muscle in vitro. We examined the motor effect of bradykinin perfused through the lumen of isolated guinea pig tracheal tubes with or without nitric oxide (NO) synthase inhibitors. In the presence of NG-nitro-D-arginine methyl ester (D-NAME) or NG-monomethyl-D-arginine (D-NMMA) intraluminal bradykinin caused a moderate concentration-dependent relaxation. In contrast, in the presence of NG-nitro-L-arginine methyl ester (L-NAME) or NG-monomethyl-L-arginine (L-NMMA) tracheas developed a sustained increase in tone, and bradykinin caused a marked, concentration-dependent contraction, both effects being reversible by pretreatment with L-arginine, but not with D-arginine. The ability of bradykinin to relax (in the presence of D-NAME) or contract (in the presence of L-NAME) guinea pig tracheal tubes was not affected by indomethacin. Bradykinin contracted epithelium-denuded tracheas in the presence of either L-NAME or D-NAME. Both contraction and relaxation by bradykinin were blocked by the kinin B2 receptor antagonist, HOE 140. Baseline production of guanosine 3',5'-cyclic monophosphate (cyclic GMP) in strips of guinea pig trachealis in vitro was markedly reduced by L-NAME, but not by D-NAME. Bradykinin increased baseline cyclic GMP concentration. These results indicate that bradykinin releases NO or a NO-related molecule, which, possibly by increasing cyclic GMP concentrations, mediates relaxation and opposes contraction induced by bradykinin itself, and further, that bradykinin releases NO from the tracheal epithelium.

In this study, we investigated the importance of the beta 2-integrins for the development of tracheal hyperreactivity in a murine model for nonallergic asthma. The response was induced by skin sensitization with dinitrofluorobenzene (DNFB) followed by an intranasal challenge with the same hapten. Twenty-four hours after the challenge, tracheal hyperreactivity, a decrease in T cells in the blood, and increased neutrophil numbers in bronchoalveolar lavage fluid (BALF) and blood were observed. Monoclonal antibodies (mAbs) directed against the alpha-chains of LFA-1 (FD441.8) and Mac-1 (M1/70) were injected intravenously 2 h before and 2 h after the challenge. Treatment with anti-LFA-1 mAb totally inhibited the development of tracheal hyperreactivity measured 24 h after the challenge, whereas anti-Mac-1 mAb had only a partial effect on this response. The decrease in T cells in the blood, which was also evident 24 h after the challenge, was totally inhibited by treatment with anti-LFA-1, whereas anti-Mac-1 had little effect. The increase in the number of neutrophils in BALF at this time point was completely inhibited by both anti-LFA-1 and anti-Mac-1. In summary, evidence presented in this report highlights the possible importance of the adhesion molecule LFA-1 in the development of tracheal hyperreactivity. Our results suggest that LFA-1 present on T cells may play an integral role in this response.

Preincubation of guinea pig tracheas with the nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME, 120 microM) resulted in a significant upward shift of the histamine concentration-response curve with a concomitant inhibition of prostaglandin E2 production. Preincubation of the preparations with a 5-lipoxygenase inhibitor (AA-861, 2-(12-hydroxy-5,10-dodecadiynyl)-3,5,6-trimethyl-p-benzoquinone) or a leukotriene C4,D4,E4 receptor antagonist (FPL 55712, sodium 7-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)-2-hydroxy propoxy]-4-oxo-8- propyl-4H-1-benzopyran-2-carboxylate) totally blocked the L-NAME-induced tracheal hyperresponsiveness. A shift from cyclo-oxygenase to lipoxygenase products, in particular leukotrienes, is likely to be responsible for the L-NAME-induced tracheal hyperresponsiveness.

Guinea pigs and mice infected with the parasitic worms Toxocara canis developed airway inflammation and tracheal hyporesponsiveness. Preceding inflammatory cell infiltration a brief hyperreactive response occurred in guinea pigs to histaminergic receptor stimulation at 3 days post infection (p.i.) and in mice to acetylcholine receptor stimulation at 1 day p.i. Few but large eosinophilic inflammatory foci developed in guinea pigs at 14 days p.i. Mice demonstrated progressive multifocal inflammation from 7 days p.i. In addition to eosinophils mouse airways were infiltrated by lymphocytes, forming perivascular and (partial) peribronchial infiltrates in an oedematous submucosa. The inflammation persisted in mice for > or = 3 months and likewise persisted tracheal hyporeactivity. Incubation of tracheae of non-infected mice with pulmonary inflammatory cells caused a significant decrease in cholinergic receptor responsiveness. This downward shift was prevented by 60% when a cyclooxygenase inhibitor was added to the incubation medium but not when inhibitors of lipoxygenase and superoxide formation were added, suggesting the involvement of prostaglandin E2. This suggestion was supported by the finding of significantly increased prostaglandin E2 concentrations in the bronchoalveolar lavage fluid at 14 and 28 days p.i. It was concluded that tracheal hyporeactivity coincided with the presence of large numbers of eosinophils in the airways of both, guinea pigs and mice and that prostaglandin E2 involvement was conceivable.

In this study a new method is described for standardization of both length and weight of bovine tracheal smooth muscle strips to be used for measuring airway responsiveness in vitro. Tracheal smooth muscle was cut into strips 2.5-mm thick and 10-mm long with an average wet weight of 25.9 +/- 0.6 mg (n = 5). By use of preloads varying from 0.5 to 10 g, it was found that 5 g caused the highest contraction and relaxation of the tissues in response to pharmacological agents. Moreover, at a preload of 5 g the variations among the maximal responses induced by histamine, methacholine, KCl, and salbutamol were very small (standard errors being 6.8%, 5.0%, 2.5%, and 1.6% of the corresponding average maximal response) as well as were the variations in the pD2 values.

The pulmonary delayed-type hypersensitivity (DTH) reaction induced by picryl chloride is characterized by enhanced albumin concentration in the airway tissue in the early phase (2 h after challenge with picryl sulphonic acid). During the later phase (48 h after the challenge) enhanced tracheal reactivity to carbachol in vitro and cellular (mononuclear and polymorphonuclear leukocytes) accumulation into the airway tissue in vivo are prominent features. In this present study, the effects of a novel drug, LCB 2183, were examined in the pulmonary DTH reaction. LCB 2183 (25 mg/kg twice daily intragastric gavage) failed to inhibit the enhanced albumin accumulation in the early phase of this response. In contrast, LCB 2183 abolished the tracheal hyperreactivity and the leukocyte accumulation in the airways of picryl chloride-sensitized mice 48 h after the challenge. These results demonstrate that LCB 2183 could be effective in the treatment of airway hyperreactivity and pulmonary inflammation.

1. An experimental set up is used whereby the serosal (out)side or mucosal (in)side of the guinea-pig isolated tracheal tube can be stimulated selectively with drugs and reactivity measured. 2. Potassium induces a concentration-dependent (5-70 mM) monophasic contraction of tracheal tubes when added on the outside. In contrast, on the inside, potassium induces a concentration-dependent relaxation at low concentrations (5-40 mM) which was reversed into a contraction up to approximately basal tone at higher concentrations (50-70 mM). 3. Epithelium denudation reversed the potassium-induced relaxation into a contraction. Interestingly, in the 'half' epithelium-denuded trachea the contractions were significantly (P < 0.01) reduced by 46% compared to complete epithelium-denuded tissues. 4. Incubation with the nitric oxide (NO) synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 120 microM) for 30 min on the inside of the tracheal tube completely prevented the relaxation. However, L-NAME did not reverse the potassium-induced relaxation into a contraction. This indicates that potassium does not penetrate through the epithelial layer. 5. It is concluded that depolarization of smooth muscle cells leads to a monophasic contraction and that depolarization of the epithelium leads to a relaxation of tracheal smooth muscle. The epithelial layer has an important barrier function and can release relaxing factors like NO.

1. We investigated whether virus-induced airway hyperresponsiveness in guinea-pigs could be modulated by pretreatment with capsaicin and whether viral respiratory infections could potentiate ovalbumin-aerosol-induced tracheal hyperresponsiveness. 2. Animals were inoculated intratracheally with bovine parainfluenza-3 virus or control medium 7 days after treatment with capsaicin (50 mg kg-1, s.c.). Four days after inoculation, tracheal contractions were measured to increasing concentrations of substance P, histamine and the cholinoceptor agonist, arecoline. 3. In tracheae from virus-infected guinea-pigs, contractions in response to substance P, histamine and arecoline were significantly enhanced (P < 0.01) by 144%, 46% and 77%, respectively. Capsaicin pretreatment inhibited the hyperresponsiveness to substance P partly (62%) and to histamine and arecoline completely. 4. In another series of experiments animals were first sensitized with ovalbumin (20 mg kg-1, i.p.). After 14 days animals were exposed to either saline or ovalbumin aerosols for 8 days. After 4 aerosol exposures (4 days) animals were inoculated with either parainfluenza-3 virus or control medium. One day after the last ovalbumin aerosol, tracheal contraction in response to increasing concentrations of substance P, histamine and arecoline was measured. 5. Tracheae from ovalbumin-aerosol-exposed control inoculated animals showed a similar degree of airway hyperresponsiveness to saline-aerosol-exposed virus-treated guinea-pigs. Virus inoculation of ovalbumin-treated animals significantly potentiated the tracheal contractions to substance P compared to either of the treatments alone. The contractions in response to histamine and arecoline were only slightly enhanced. 6. In conclusion, sensory nerves and/or tachykinins are involved in virus-induced airway hyperresponsivenessin guinea-pigs and viral respiratory infections can potentiate the increase in tracheal responsiveness to bronchoconstrictor agonists after ovalbumin exposure.

The immunoinflammatory response to parasitic nematode infections and allergic diseases have some similarities, the most profound being the increases in eosinophils and serum total IgE concentration. Whether parasitic infections stimulate or inhibit allergic asthma is a matter of debate. We investigated the effect of Toxocara canis (T. canis) infection on airway function in BALB/c mice at various days post-infection. Within 24 h after infection, the trachea responded hyperreactive to carbachol stimulation. Eosinophils, and to a lesser degree lymphocytes, infiltrated the airways causing interstitial and alveolar inflammation (7 d post-infection). Concurrently with cell infiltration, the trachea became hyporesponsive to carbachol whereas the pulmonary resistance was increased and the dynamic compliance decreased. The hyporeactive response could be simulated in vitro by incubating normal tracheae with eosinophil-enriched bronchoalveolar lavage cells obtained from infected mice. The response depended on the number of cells added to the medium, a lower number causing a hyper- and a higher number a hyporeactive response. Anti-interleukin-5 (anti-IL-5) producing hybridoma cells given simultaneously with T. canis infection inhibited eosinophil infiltration in the airways but not that of lymphocytes. Anti-IL-5 treatment prevented tracheal hyporeactivity but not perivascular and peribronchial edema, increased pulmonary resistance, or decreased dynamic compliance. Treatment with isotype control antibody did not affect eosinophil number nor the observed changes in airway functions. It was concluded that T. canis-induced airway inflammation coincided with increased pulmonary resistance, decreased dynamic compliance, and perivascular/peribronchial edema. These phenomena were independent on the presence of eosinophils, whereas tracheal hyporeactivity was clearly associated with airway eosinophilia.

The aim of this study was to investigate the possible role of capsaicin-sensitive nerves in a pulmonary delayed-type hypersensitivity (DTH) reaction. Mice (Balb/c) were skin-sensitized with dinitrofluorobenzene (DNFB; 0.5%, 100 microliters) on two consecutive days and challenged intranasally 5 days later with dinitrobenzene sulphonic acid (0.6%, 50 microliters). Sensitized mice exhibited tracheal hyperreactivity to carbachol 24 and 48 h after challenge; however, no hyperreactivity was observed 2 h after challenge. At 24 h, but not at 48 h, hyperreactivity was associated with antigen-specific lymphocyte accumulation in bronchoalveolar lavage fluid (BALF). Capsaicin pretreatment, resulting in the depletion of sensory neuropeptides, virtually abolished hyperreactivity to carbachol 24 and 48 h after challenge in sensitized mice. Although at 24 h after challenge the lymphocyte population was elevated in the BALF collected from capsaicin-pretreated mice, this accumulation was not antigen-specific. Additionally, there was an increase in polymorphonuclear leukocyte accumulation (neutrophils and eosinophils) in the BALF collected from capsaicin-pretreated mice at this time point, but this increase was more profound in the sensitized group. In summary, tracheal hyperreactivity and cellular accumulation are prominent features of the lymphocyte-associated DTH reaction induced by DNFB in the mouse lung. Evidence presented in this report highlights the possible importance of sensory neuropeptides in pulmonary inflammation and airways hyperreactivity.

The role of substance P-containing sensory nerves in the development of tracheal hyperreactivity to histamine and arecoline was investigated in an allergic model of asthma. Male Hartley-strain guinea pigs were sensitized to ovalbumin (20 mg/kg i.p.) and 14 days later exposed to either saline or ovalbumin (2%) aerosols for 8 consecutive days. One day after the last aerosol exposure the animals were killed and the tracheas were removed. Isotonic muscle shortening in response to increasing doses of histamine and arecoline was measured. Capsaicin (50 mg/kg s.c.) or vehicle pretreatment was performed 7 days prior to sensitization. Tracheas from vehicle-pretreated sensitized and ovalbumin-aerosol exposed animals had increased reactivity to both histamine and arecoline compared to saline-aerosol exposed animals. Capsaicin pretreatment did not alter tracheal reactivity in the saline-aerosol exposed group. Capsaicin pretreatment prevented the increase in tracheal reactivity caused by both agonists in sensitized and ovalbumin-aerosol exposed guinea pigs. These results suggest that capsaicin-sensitive sensory nerves are important for the development of increased tracheal reactivity in an allergic model of asthma.

In the present study an animal model is described in which a sustained non-specific airway hyperresponsiveness is induced. Guinea pigs were inoculated intratracheally with parainfluenza type 3 (PI-3) virus or control solution. Two, 4, 8, and 16 days after inoculation the tracheae, bronchi, and lung strips were isolated and mounted in organ baths. Two days after inoculation no difference between the control solution and PI-3 virus group was observed, with respect to the histamine concentration/response curve obtained from tracheae, bronchi and lung strips of the respective groups. However, histamine concentration/response curves were significantly (P less than 0.01) shifted upwards in all parts of the airways 4, 8, and 16 days after PI-3 inoculation as compared with the control solution. The excessive contraction of the trachea was not specific for histamine, since an increase in the maximal response was obtained also for the cholinergic receptor agonist, arecoline on day 4 (32%, P less than 0.05), day 8 (24%, P less than 0.05), and day 16 (28%). Morphological examination of the central airways obtained from control solution-inoculated animals revealed no signs of inflammation. However, 2, 4, and 8 days, but not 16 days, after the viral infection, epithelial damage with loss of cilia and mucus-depleted goblet cells were observed. Thus, morphological changes were not directly associated with changes in airway responsiveness. Histological examination of the peripheral airways revealed an influx of inflammatory cells, as shown by typical lesions of patchy alveolitis and bronchiolitis. Bronchiolar epithelium was variously hyperplastic and dysplastic with degenerative changes, and the lumens of the bronchioli were occluded with mucus and inflammatory cells. In conclusion, the virus-induced airway hyperresponsiveness in guinea pigs shows similarities with the human situation, in which a sustained non-specific airway hyperresponsiveness is observed after a respiratory viral infection. In addition, the hyperresponsiveness seems to be accompanied by an influx of inflammatory cells in the airways but not with other morphological changes.

In asthma, a beta-adrenoceptor dysfunction may be the consequence of an active disease state rather than a fundamental abnormality. In the present study the possible involvement of T lymphocytes in beta-adrenergic impairment was investigated by studying the effects of lymphocyte-derived mediators of beta-adrenoceptor function of human peripheral blood mononuclear cells (PBMCs) and guinea pig trachea. Supernatants of phytohemagglutinin- or concanavalin A-activated PBMCs from either persons with asthma or healthy persons inhibited isoprenaline stimulated cyclic adenosine 3',5'-monophosphate (cAMP) production of PBMCs after 20 hours of preincubation. These supernatants also inhibited beta-adrenoceptor function of PBMCs from patients with asthma to the same extent. The isoprenaline stimulated cAMP production of PBMCs was not altered after a 2-hour preincubation period with human interleukin-1 (IL-1), IL-2, IL-3, IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon (IFN-gamma). In contrast, after 20 hours of preincubation, stimulated cAMP production of PBMCs was significantly diminished, with 63% by IL-1 (40 U/ml, p less than 0.01), with 36% by IL-2 (100 U/ml, p less than 0.05), with 37% by IFN-gamma (1000 U/ml, p less than 0.05), and with 21% by GM-CSF (100 U/ml, p less than 0.05). Preincubation of guinea pig tracheal segments with IL-1, IL-2, IL-4, or GM-CSF during 1 or 3 days did not affect the EC50 values or the maximal relaxation of isoprenaline dose response curves.

For the investigation of whether inflammatory cells were responsible for virus-induced airway hyperresponsiveness, tracheal spirals from healthy guinea pigs were incubated in organ baths with different numbers of bronchoalveolar cells obtained from guinea pigs 4 days after their inoculation with parainfluenza-3 (P-3) virus or control solution. Airway responsiveness was measured by performance of histamine concentration/response (C/R) curves on the tissues. Preparations incubated with 5 x 10(5) cells/ml obtained from guinea pigs treated with P-3 virus demonstrated a significant upward shift of the histamine C/R curve. The maximal contraction was increased by 26% as compared with the tissues incubated with the same number of cells from animals inoculated with control solution. When the number of cells was increased further to 5 x 10(6) cells/ml, no additional upward shift of the C/R curve was seen; the increase in maximal contraction was 24%. Tracheal spirals incubated with 5 x 10(4) cells/ml did not affect the histamine C/R curves. Addition of P-3 virus to the organ bath during the incubation period with the cells did not affect the histamine C/R curve either, irrespective of the inoculation solution or the number of bronchoalveolar cells used. The relative number of alveolar macrophages in bronchoalveolar lavage fluid decreased significantly from 86.3% +/- 2.6% in the control group to 71.8% +/- 3.3% in the P-3 virus group as a consequence of a significant increase in the percentage of monocytes, lymphocytes, and eosinophils. These results suggest that bronchoalveolar cells are causally involved in the virus-induced airway hyperresponsiveness.

An isometric method to measure the smooth muscle tone of murine tracheas in vitro was developed. Nine trachea rings from just beneath the larynx were prepared free of excess tissue with the help of a binocular microscope. These trachea parts were slipped onto supports in an organ bath containing Krebs' solution. Isometric tension was measured with a force displacement transducer connected to the upper-trachea support, and is expressed as changes in grams force. The cholinergic agonist carbachol contracted isolated tracheas. Serotonin also induced contractions, but was less potent than carbachol. Histamine induced tracheal contractions only at very high concentrations. Sympathomimetic beta adrenergic agonists relaxed carbachol-precontracted tracheas with the following order of potency: isoprenaline (beta 1 and beta 2 adrenoceptor agonist) greater than salbutamol (beta 2 adrenoceptor agonist) greater than prenalterol (beta 1 adrenoceptor agonist). Adrenaline and noradrenaline relaxed carbachol-precontracted tracheas, with adrenaline being the more potent relaxant. For the study of airway reactivity, this mouse trachea model has several advantages over immunopharmacologic models: The immune system of the mouse has been characterized extensively, and many reagents are available to study the immune system and, thus, possible interactions of this system with pharmacological mechanisms. Other animal models used in pharmacology are generally less well defined immunologically.

Pulmonary epithelial cells may be responsible for regulating airway smooth muscle function, in part by release of fatty acid-derived mediators. Incubation of isolated guinea pig tracheal epithelial cells with radiolabeled arachidonic acid (AA) leads to the production of 5- and 15-hydroxyeicosatetraenoic acid (5- and 15-HETE) and smaller amounts of leukotriene (LT) B4 and C4 and 12-hydroxyheptadecatrienoic acid (HHT). Epithelial cells also are able to release linoleic acid (LA) metabolites. Incubation with radiolabeled linoleic acid leads to the formation of 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE). The biological significance of these mediators produced by epithelial cells is discussed.

The influence of the linoleic acid metabolite 13-hydroxy-octadecadienoic acid (13-HODE) on guinea pig tracheal reactivity to both contractile and relaxant agonists was investigated in vitro. 13-HODE induced an increased contraction of tracheal rings to histamine, whereas methacholine responsiveness was not significantly affected. Relaxant responses to isoprenaline and prostaglandin E2 were not influenced either.

Cultured epithelial cells obtained from guinea pig tracheal preparations metabolized arachidonic acid into 5- and 15-hydroxy-eicosatetraenoic acid and into the prostaglandins E2 and F2 alpha. Linoleic acid was converted by the epithelial cells into 9-hydroxy-octadecadienoic acid (9-HODE) and smaller amounts of 13-HODE. It was further investigated whether linoleic acid metabolites are of importance for the regulation of airway smooth muscle function. 13-HODE caused an increase of maximal contraction of tracheal rings to histamine, while 9-HODE had no effect.

Guinea pigs were inoculated intra-tracheally with bovine parainfluenza type 3 virus (PI-3) to investigate whether airway hyperreactivity was associated with an increase in the number of broncho-alveolar cells. Two days after saline or PI-3 inoculation no difference in histamine reactivity of the isolated tracheal spirals was observed. The number of broncho-alveolar cells was also not changed. However, 4 days after PI-3 inoculation the histamine-induced contraction of the tracheal spirals was increased by 45% and the number of inflammatory cells in the airways by 150% as compared with the control group. In conclusion, PI-3 infection of the guinea pig respiratory airways induces tracheal hyperreactivity which is associated with an increase in broncho-alveolar cells.

Migrating larvae of the canine nematode Toxocara canis induce inflammatory responses in the lungs of infected mice. Histological examination of the lungs showed that mice, developed persistent multifocal inflammation in which eosinophils (eos), macrophages (M phi) and many lymphocytes were involved. Cuffing of blood vessels and bronchioles with mixed cell populations was also observed. Investigation into the response of isolated tracheae to cholinergic and beta-adrenergic receptor agonists demonstrated a significant decreased reactivity despite the pronounced involvement of eos and M phi. This hyporeactive state persisted for greater than or equal to 4 weeks p.i..

The effect of recombinant human interleukin-2 (rh IL-2, Genzyme, yeast derived) on the beta-adrenoceptor function of human peripheral blood mononuclear cells (PBMC), guinea pig splenic lymphocytes and isolated guinea pig tracheal spirals was investigated. Rh IL-2 (Genzyme, yeast derived) induces a dose dependent inhibition of the isoprenaline-stimulated cAMP production in PBMC and splenic lymphocytes after a two hour preincubation period. The inhibition is significant at 0.01 U/ml IL-2 and reaches a maximum at 1 U/ml amounting 81 +/- 8% and 76 +/- 6% for human PBMC and guinea pig splenic lymphocytes respectively. The sensitivity of isolated guinea pig tracheal spirals to isoprenaline is also significantly decreased after a two hour preincubation period with 1 U/ml rh IL-2 (Genzyme, yeast derived). In contrast, rh IL-2 (Cetus, bacteria derived) does not affect the beta-adrenoceptor function of human PBMC, guinea pig splenic lymphocytes and isolated tracheal spirals, after a two-hour preincubation period. Furthermore, human cell-line derived IL-2 (Jurkat, Genzyme) also does not influence human PBMC beta-adrenoceptor function. It can therefore be concluded that IL-2 does not affect lymphocyte and airway beta-adrenoceptor function after a two hour preincubation period. The inhibition of beta-adrenoceptor function by yeast derived rh IL-2 (Genzyme) is therefore probably not related to IL-2.

The beta-adrenergic receptor responsiveness of isolated guinea pig tracheal spirals can be negatively affected by intraperitoneal administration of the Gram-negative bacterium Haemophilus influenzae, four days prior to the experiment. The reduction in tracheal relaxation is accompanied by a decrease in beta-adrenergic receptor binding sites in splenic lymphocyte membranes and by a decrease in the fluidity of these membranes. The H. influenzae-induced dysfunction of both the respiratory airway and lymphocyte beta-adrenergic systems can be mimicked by modulating the amount of linoleic acid in the diet. This linoleic acid induced dysfunction of the beta-adrenergic system is also accompanied by a decrease in the plasma membrane fluidity of the splenic lymphocyte membranes of the guinea pigs. The role for plasma membrane fluidity in asthma is discussed in relation to current concepts for atopy.

In the present study, the effect of vaccination of guinea pigs with Bordetella pertussis was investigated, 4 days after treatment, on the cholinergic and beta-adrenergic receptor function in isolated tracheal spirals and the number of beta-adrenoceptor binding sites in guinea pig lung. It was found that B. pertussis caused an impairment in the beta-adrenoceptor function and a decrease in its number. Similar results were obtained with endotoxin. Leucocytosis promoting factor, however, was ineffective. These results indicate that endotoxin is the constituent responsible for the beta-adrenoceptor blocking effects of the bacterium. Also the combined whole cell diphtheria, B. pertussis and tetanus toxoid vaccine induced a beta-adrenoceptor blockade; the acellular vaccine was less effective. The results obtained with the B. pertussis vaccines are discussed in relation to the possible side-effects that sometimes occur after immunization of infants.

Haemophilus influenzae is a bacterium that can be isolated from the deeper airways of asthmatic patients. We investigated the effect of vaccination with H. influenzae on alpha and beta adrenoceptor function in guinea-pig tracheal spirals and lung parenchymal strips. The tracheal spirals from H. influenzae-vaccinated animals showed significantly less relaxation to isoproterenol as compared to controls, independent of whether the trachea was maximally contracted with carbachol or only exhibited an intrinsic tone. Furthermore, an increased contractile response to carbachol was observed in these spirals. To isoproterenol in the presence of a beta-2 adrenergic antagonist (H35/25), or to salbutamol alone, the tracheal preparations from H. influenzae-vaccinated animals also showed a decreased relaxation. These results suggest involvement of both beta-1 and beta-2 subtype adrenoceptors. On the other hand, lung parenchymal strips from vaccinated guinea-pigs relaxed significntly more to these drugs. This effect was not influenced by H35/25 but could be inhibited by phenoxybenzamine. Histamine-induced contraction did not differ between the groups. These results indicated that H. influenzae causes a partial blockade of the beta adrenoceptors in tracheal spirals and, therefore, may have important implications in asthmatic bronchitis. In contrast, parenchymal lung strips of the H. influenzae-pretreated group showed an increased relaxation.

Discrete lesions in the anterior hypothalamus (AHA) of the guinea pig brain reduce the anaphylactic contraction of the trachea in vitro after active in vivo sensitization by 40%. This difference in anaphylactic contraction does not correlate with a difference in homocytotropic antibodies but coincides with a decreased smooth muscle response to the anaphylactic mediators histamine and leukotriene C4. No difference in the beta-adrenoceptor function of the tracheal preparations can be found. The results suggest that AHA lesions afford protection against anaphylaxis in actively sensitized guinea pigs at least in part through a reduced smooth muscle response to anaphylactic mediators.

1. Pretreatment of guinea-pigs with endotoxin (1 mg kg-1 b.w., i.p., 4 days before the experiments) results in respiratory airway hyperreactivity in vitro. Dose-response curves with either arecoline or histamine on isolated tracheae from these animals display increased maximal contractions, and decreased EC50 values. 2. Tracheae denuded of epithelium respond with a similar hyperreactivity to histamine as observed in preparations from endotoxin pretreated animals. Removal of the epithelial layer of tracheae from endotoxin pretreated guinea-pigs did not additionally affect the histamine dose-response curve. 3. The cyclo-oxygenase inhibitor indomethacin (10 microM) induces histamine hyperreactivity which is equal in intact and epithelium-denuded tracheae from saline or endotoxin pretreated guinea-pigs. Similar results are obtained with the combined lipoxygenase/cyclo-oxygenase inhibitor nordihydroguaiaretic acid (10 microM). 4. Histamine (0.1 mM) induces an increase in prostaglandin E2 (PGE2) formation by the tracheal spiral in vitro, which is reduced by 34% by endotoxin pretreatment, and by about 60% following epithelium removal irrespective of endotoxin pretreatment. 5. Arachidonic acid (AA, 22 microM) stimulation of the guinea-pig trachea in vitro induces a relaxation, and an increase in PGE2 production. In preparations lacking the epithelium, AA induces a contraction which coincides with a 60% reduced increase in PGE2 formation. These effects are not altered by endotoxin pretreatment. 6. It is concluded that the endotoxin-induced respiratory airway hyperreactivity may be caused by a disturbed ability of epithelial cells to synthesize PGE2. The decreased formation of this prostaglandin is rather the consequence of a diminished liberation of AA from the phospholipid stores than a dysfunction of the cyclo-oxygenase enzyme.

Respiratory histaminergic and cholinergic receptor function was investigated in isolated tracheal spirals of guinea pigs receiving different diets. Comparison was made between saline treated (controls) and Haemophilus influenzae treated animals in non sensitized conditions, the latter being a model for bronchial hyperreactivity, and in sensitized conditions, being a model for allergen induced bronchial hypersensitivity. The different semi-synthetic diets (35 energy% fat), varying in linoleic acid content (5.85, 11.25 and 22.05 en% fat) and one diet with low linoleic acid (3.55 en%) in which linolenic acid was added additionally (5.30 en%), exerted profound effects on tracheal reactivity to histamine. In sensitized animals the maximal induced histamine contraction was significantly diminished in the dietary group receiving 5.85 en% linoleic acid as compared with the other dietary groups (35% decrease in the H. influenzae-treated, 20-30% decrease in saline treated animals). Results in non-sensitized animals were similar, though less pronounced. No effect on food intake or growth of the animals could be demonstrated during the six week experimental periods. The carbachol induced contraction of the tracheal spirals of sensitized animals receiving low linoleic acid was also significantly decreased as compared to the other dietary groups (30% for saline treated and 20-30% for H. influenzae-treated animals). No difference in carbachol responsiveness could be detected between the different dietary groups under non-sensitized conditions. The results are discussed in view of the current concepts for bronchial hyperreactivity, especially in relation to eicosanoid involvement.

The Gram-negative bacterium Haemophilus influenzae has been shown to cause a deterioration of the guinea pig pulmonary beta-adrenergic receptor system. In the present study we investigated further the mechanisms behind this effect. To this extent we evaluated the involvement of pulmonary macrophages (PM). Treatment of guinea pigs with killed H. influenzae bacteria resulted in the accumulation of a factor in the serum which could specifically stimulate PM. Thus stimulated, PM from nontreated animals caused a decrease of tracheal beta-adrenergic receptor function in vitro. This effect was evident by a decrease of the maximal response of the dose-response curves to isoprenaline, whereas the EC50 values did not change. Catalase and thiourea abolished the PM-induced effects, whereas superoxide dismutase did not, indicating that oxygen-centered radicals, in particular the highly reactive hydroxyl radical, may be responsible for the observed effects. In addition, dexamethasone also inhibited the decrease of tracheal beta-adrenergic receptor function. When activated PM, taken from animals that had been pretreated with killed H. influenzae bacteria 4 days beforehand, were stimulated with serum from a H. influenzae-treated animal, a potentiation of tracheal beta-adrenergic receptor function was observed.