Effect of acetylcysteine on experimental corneal wounds in dogs.

The effects of 3, 10 and 20% concentrations of acetylcysteine on experimental corneal wound healing in dogs were evaluated. Experimental corneal wounds were induced surgically, up to the depth of the anterior third of the stroma, in both eyes of 18 dogs. One of the eyes was treated topically with 0.9% NaCl solution three times a day. The contralateral eye was treated topically with acetylcysteine (3, 10 and 20% concentrations) in each of 6 cases separately. Corneal wounds were measured by fluorescein staining every day. The mean time of healing in the 3% group was significantly different from control eyes (6.17 +/- 1.94 days). It was 7.19 +/- 0.75 days in the 20% group and 7 +/- 2 days in the 10% group. The last two groups were not significantly different from the controls (9.67 +/- 3.01 days and 8.17 +/- 3.60 days, respectively).

Sodium cromoglycate and doxantrazole are oxygen radical scavengers.

The effects of two mast cell stabilisers, sodium cromoglycate (SCG) and doxantrazole, on the formation of reactive oxygen species (ROS) were studied. Guinea-pig 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 < or = 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 < or = 92%. Moreover, these drugs slightly increased the survival rate of the AMs. It is concluded that doxantrazole- and sodium cromoglycate-inhibited lucigenin-dependent chemiluminescence production by guinea-pig alveolar macrophages is due to a direct scavenging effect on reactive oxygen species. Doxantrazole is approximately 10-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.

Histopathological studies on the effects of peroxynitrite on the lungs and trachea of rabbits.

Peroxynitrite (ONOO-) is the reaction product between nitric oxide and the superoxide anion. It is a biological oxidant and cytotoxic anion, produced in vivo, which might cause inflammation and damage to the lungs. This study was designed to investigate whether direct contact with peroxynitrite could cause pathological changes in the airways. Twenty New Zealand rabbits were divided into four experimental groups. They were anesthetised by intravascular injection of thiopental sodium, and peroxynitrite (0.5 mL of a 10 mM solution) was inoculated intratracheally. At 2 h, 24 h, 48 h, and 72 h postinoculation trachea and lungs were isolated for histopathology. The histopathological observations in the lungs were congestion, serous exudation, infiltration of leukocytes and degeneration, which were found to be time-dependent. Changes were similar at 48 and 72 h. Degenerative changes were not progressive. The changes in the trachea were congestion, oedema, leukocyte infiltration, and degeneration of tracheal epithelium. This study shows that peroxynitrite formation in the respiratory tract induces lung inflammation and degenerative changes.

Parasiticidal effects of peroxynitrite on ovine liver flukes in vitro.

Peroxynitrite (ONOO-) is a cytotoxic anion, produced by interaction between nitric oxide and superoxide in vivo in some inflammatory cells. This study investigated its effects on Fasciola hepatica and Dicrocoelium dendriticum isolated from ovine livers and kept in bile at room temperature. Peroxynitrite was synthesized using a quenched flow reactor and assayed spectrophotometrically. It was applied at different concentrations (10(-3.5) to 10(-2.3 M)) to the flukes kept in bile. The viability of the peroxynitrite-treated flukes was compared with a control group (n=5-7 per group). Control F. hepatica and D. dendriticum lived for 226+/-11and 208+/-14min, respectively. Life times were decreased by peroxynitrite at all concentrations used (P<0.001). At the highest concentration of peroxynitrite, F. hepatica and D. dendriticum lived only for 6.1+/-0.4 and 4.1+/-4.1+/-0.2min, respectively. Correlation between peroxynitrite concentration and parasite viability was significant in the case of F. hepatica (r= -0.842; P= 0.0035). A single application of peroxynitrite can decrease the life span of ovine liver flukes. A failure in the activation of hepatic macrophages in infected animals may lead to a decreased production of free radicals and, thus, peroxynitrite. Such a failure is likely to deprive the body of a defence tool against multicellular parasites.

Role of the epithelial layer in the generation of superoxide anion by the guinea-pig isolated trachea.

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.

Peroxynitrite: a two-faced metabolite of nitric oxide.

The discovery that nitric oxide (NO) reacts with superoxide (O2.-) forming peroxynitrite (ONOO-) (1) and the proof that this reaction occurs in vivo (2,3) holds enormous implications for the understanding of free radicals in biological systems. Not only in mammalian defense mechanisms against microorganisms, but also in pathophysiology during overexposure of tissues to radicals or other highly reactive species. Peroxynitrite is a highly reactive compound with harmful effects on cells and could therefore be an important microbicidal compound. Furthermore, the reaction of superoxide with NO interferes with NO signalling mechanisms. NO is not only released in response to inflammatory agents by inflammatory cells, but is also an important messenger molecule in paracrine mechanisms and neurotransmission. Whether peroxynitrite formation is a negative side effect of NO and superoxide release, or a functional characteristic is yet to be determined, and will be discussed in this review.

Bovine tracheal responsiveness in vitro: role of the epithelium and nitric oxide.

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.

Relaxation of guinea-pig trachea by sodium nitroprusside: cyclic GMP and nitric oxide not involved.

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.

Peroxynitrite induces airway hyperresponsiveness in guinea pigs in vitro and in vivo.

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.

Induction of guinea pig airway hyperresponsiveness by inactivation of guanylate cyclase.

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.