Peroxynitrite (ONOO-) is a cytotoxic product of the rapid reaction between nitric oxide and superoxide that may initiate inflammation. Isolated perfused tracheas from guinea pigs were incubated from the mucosal side for 15 min with peroxynitrite (1 to 100 muM). Thereafter, concentration-response curves to histamine and methacholine were constructed on the preparations. Peroxynitrite (10 muM) caused a significant hyperresponsiveness; the maximal contractions in response to histamine and methacholine were enhanced by 30% and 40%, respectively. In the peroxynitrite-treated group, clear epithelial damage as well as eosinophil destruction were detected. Moreover, 3, 5, and 10 days after intratracheal instillation of peroxynitrite (100 nmol), a significant rise in pulmonary resistance to histamine of anesthetized animals was observed. It is suggested that the generation of peroxynitrite from nitric oxide superoxide radicals during inflammatory processes induces epithelial damage, mediator release, and hence airway hyperresponsiveness. These findings may have clinical implications, because airway inflammation, epithelial damage, and hyperresponsiveness are characteristic features in patients suffering from asthma.

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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 guM) 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 gM) increased the cyclic GMP levels by 1100% without inducing a marked relaxation. 4 SNP (10 ,M) and S-nitroso-N-acetylpenicillamine (SNAP; a direct donor of nitric oxide; 10 tiM), relaxed the tissues by 75% and 25%, respectively, without any nitric oxide (NO) release by SNP (<1 pmol 100 PIu'), but a substantial NO release by SNAP (560 pmol 100 ,u1'1).5 It is concluded that the SNP-induced tracheal relaxations are probably not mediated by cyclic GMP and NO.

The effects of two mast cell stabilisers, sodium cromoglycate (SCG) and doxantrazole, on the formation of reactive oxygen species (ROS) were studied. Guineapig alveolar macrophages (AMs) generated lucigenin-dependent chemiluminescence (LDCL). This was increased when the cells were stimulated by phorbol myristate acetate (PMA) or zymosan (by 133% and 464%, respectively, in total LDCL over 60 min). SCG decreased PMA-induced LDCL at higher concentrations (10 mM, by 55%) than doxantrazole (1 mM, by 75%). SCG decreased radical production by AMs in response to zymosan in a concentration-dependent manner by ¡72%. Doxantrazole (0.1-1 mM) diminished total LDCL by 30-80%. In addition, glucose oxidase led to LDCL generation when incubated with glucose in a cell-free medium. This was inhibited by 47-83% in the presence of SCG or doxantrazole. SCG and doxantrazole inhibited the hydrogen peroxide-and peroxynitrite-induced LDCL by ¡92%. Moreover, these drugs slightly increased the survival rate of the AMs.It is concluded that doxantrazole-and sodium cromoglycate-inhibited lucigenindependent chemiluminescence production by guinea-pig alveolar macrophages is due to a direct scavenging effect on reactive oxygen species. Doxantrazole is y10-times more potent. Mast cell stabilisers may be effective in allergic asthma not only by preventing the allergen-induced mediator release, but also by preventing radical-induced lung damage. Eur Respir J 2002; 20: 867-872.

B Bo ov vi in ne e t tr ra ac ch he ea al l r re es sp po on ns si iv ve en ne es ss s i in n v vi it tr ro o: : r ro ol le e o of f t th he e e ep pi it th he el li iu um m a an nd d n ni it tr ri ic c o ox xi id de e G. Sadeghi-Hashjin* # , P.A.J. Henricks*, G. Folkerts*, A.K.C.P. Verheyen**, H.J. van der Linde*, F.P. Nijkamp* Bovine tracheal responsiveness in vitro: role of the epithelium and nitric oxide. G. Sadeghi-Hashjin, P.A.J. Henricks, G. Folkerts, A.K.C.P. Verheyen, H.J. van der Linde, F.P. Nijkamp. ERS Journals Ltd 1996. ABSTRACT: Airway epithelium releases inhibitory factors, such as nitric oxide (NO) and prostaglandin E 2 (PGE 2 ), 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 Gmonomethyl L-arginine (L-NMMA) on histamine responsiveness were investigated. The release of NO and PGE 2 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 (pD 2 ) 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 µM 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 PGE 2 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 E 2 released from bovine tracheal epithelium are probably too low to exert a significant effect on the histamine-induced contractions.