In numerous experimental systems, the neurohormone melatonin has been shown to protect against oxidative stress, an effect which appears to be the result of a combination of different actions. In this study, we have investigated the possible contribution to radical scavenging by substituted kynuramines formed from melatonin via pyrrole ring cleavage. N1-Acetyl-5-methoxykynuramine (AMK), a metabolite deriving from melatonin by mechanisms involving free radicals, exhibits potent antioxidant properties exceeding those of its direct precursor N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and its analog N1-acetylkynuramine (AK). Scavenging of hydroxyl radicals was demonstrated by competition with ABTS in a Fenton reaction system at pH 5 and by competition with DMSO in a hemin-catalyzed H2O2 system at pH 8. Under catalysis by hemin, oxidation of AMK was accompanied by the emission of chemiluminescence. AMK was a potent reductant of ABTS cation radicals, but, in the absence of catalysts, a poor scavenger of superoxide anions. In accordance with the latter observation, AMK was fairly stable in a pH 8 H2O2 system devoid of hemin. Contrary to AFMK, AMK was easily oxidized in a reaction mixture generating carbonate radicals. In an oxidative protein destruction assay based on peroxyl radical formation, AMK proved to be highly protective. No prooxidant properties of AMK were detected in a sensitive biological test system based on light emission by the bioluminescent dinoflagellate Lingulodinium polyedrum. AMK may contribute to the antioxidant properties of the indolic precursor melatonin.

Precision-cut lung slices (PCLS) allow comparison of the airway responses of different species under identical experimental conditions. The aim of this study was to establish and characterise PCLS from guinea pigs (GPs) and to compare them with human PCLS.GP PCLS were prepared according to previously published procedures with the exception that the agarose solution and the initial incubation medium contained isoproterenol to avoid post mortem airway contraction.The median effective concentrations (EC50, expressed as nM) for agonist-induced bronchoconstriction in GP and human PCLS, respectively, were: leukotriene D 4 (1.8, 5.0); thromboxane (16, 1.3); serotonin (69, unresponsive); histamine (217, 2,170); and methacholine (231, 234). Allergen-induced bronchoconstriction of passively sensitised PCLS was attenuated by histamine or thromboxane-prostanoid receptor antagonists and was almost completely prevented by their combination with leukotriene receptor antagonists. Airways pre-contracted with methacholine were relaxed by the b-agonist salbutamol or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Simultaneous studies of airways and vessels are possible with, for example, EC50 values for endothelin-1 of 37 nM (pulmonary arteries), 10 nM (pulmonary veins) and 9.6 nM (airway).When compared with previous findings in rat and mouse, these data show that guinea pig lungs are a more appropriate model for human airway pharmacology than lungs from rats or mice.

The etiology of airway hyperresponsiveness associated with asthma requires an understanding of the regulatory mechanisms mediating human airway smooth muscle cell (SMC) contraction. The objective of this study was to determine how human airway SMC contraction (induced by histamine) and relaxation (induced by formoterol) are regulated by Ca 21 oscillations and Ca 21 sensitivity. The responses of human small airways and their associated SMCs were studied in human lung slices cut from agarose-inflated lungs. Airway contraction was measured with phase-contrast video microscopy. Ca 21 signaling and Ca 21 sensitivity of airway SMCs were measured with two-photon fluorescence microscopy and Ca 21 -permeabilized lung slices. The agonist histamine induced contraction of human small airways by stimulating both an increase in intracellular Ca 21 concentration in the SMCs in the form of oscillatory Ca 21 waves and an increase in Ca 21 sensitivity. The frequency of the Ca 21 oscillations increased with histamine concentration, and correlated with increased contraction. Formoterol induced airway relaxation at low concentrations by initially decreasing SMC Ca 21 sensitivity. At higher concentrations, formoterol additionally slowed or inhibited the Ca 21 oscillations of the SMCs to relax the airways. The action of formoterol was only slowly reversed. Human lung slices provide a powerful experimental assay for the investigation of small airway physiology and pharmacology. Histamine induces contraction by simultaneously increasing SMC Ca 21 signaling and Ca 21 sensitivity. Formoterol induces long-lasting relaxation by initially reducing the Ca 21 sensitivity and, subsequently, the frequency of the Ca 21 oscillations of the airway SMCs.Keywords: lung slice; smooth muscle cell; two-photon microscopy; hyperresponsiveness; histamine Because increased airway smooth muscle cell (SMC) contraction is a major characteristic of airway hyperresponsiveness (AHR) associated with asthma (1), a prerequisite to address AHR is an understanding of the mechanisms that regulate airway SMC contraction. It is well established that airway SMC contraction is regulated by the phosphorylation of the regulatory myosin light chain (rMLC) (2, 3) to induce its interaction with adjacent actin filaments via the classical mechanism of cross-bridge cycling to generate force (4). (5)(6)(7)(8). Although the frequency of these Ca 21 oscillations has been found to increase with agonist concentration, and that this correlates with increased contraction, the frequency of the Ca 21 oscillations generated by similar agonist concentrations differs considerably between species (9).To induce SMC relaxation, the increase in [Ca 21 ] i is generally reversed to inactivate MLCK, but the cessation of the cross-bridge cycle requires that the rMLC is dephosphorylated; this reaction is mediated by MLC phosphatase (MLCP). With the exception of mice (10), the activity of MLCP appears to be Ca 21 independent, but is regulated by the same agonists that stimulate increases in [Ca 21 ] ...

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A wide range of industrial chemicals can induce respiratory allergic reactions. Hence, there is an urgent need for methods identifying and characterizing the biological action of chemicals in the lung. Here, we present an easy, reliable alternative method to measure lung function changes ex vivo after exposure to chemical allergens and compare this to invasive in vivo measurements after sensitization with the industrial chemicals trimellitic anhydride (TMA) and 2,4-dinitrochlorobenzene (DNCB). Female BALB/c mice were sensitized epicutaneously with the respiratory allergen TMA and the contact sensitizer DNCB. The early allergic response to TMA and DNCB was registered in vivo and ex vivo on day 21 after inhalational challenge with dry standardized aerosols or after exposure of precision-cut lung slices (PCLS) to dissolved allergen. Airway hyperresponsiveness (AHR) to increasing doses of methacholine (MCh) was measured on the next day in vivo and ex vivo. Bronchoalveolar lavage (BAL) was performed for immunological characterization of local inflammation. TMA-sensitized mice showed AHR to MCh in vivo (ED(50): 0.06 microg MCh vs. 0.21 microg MCh in controls) and in PCLS (EC(50): 0.24 microM MCh vs. 0.4 microM MCh). TMA-treated animals showed increased numbers of eosinophils (12.8 x 10(4) vs. 0.7 x 10(4)) and elevated eotaxin-2 concentrations (994 pg/ml vs. 167 pg/ml) in BAL fluid 24 h after allergen challenge. In contrast, none of these parameters differed after sensitization with DNCB. The present study suggests that the effects of low molecular weight allergens, like TMA and DNCB, on ex vivo lung functions tested in PCLS reflect the in vivo situation.