Effects of lung volume on lung and chest wall mechanics in rats.

To investigate the effect of lung volume on chest wall and lung mechanics in the rats, we measured the impedance (Z) under closed- and open-chest conditions at various positive end-expiratory pressures (0-0.9 kPa) by using a computer-controlled small-animal ventilator (T. F. Schuessler and J. H. T. Bates. IEEE Trans. Biomed. Eng. 42: 860-866, 1995) that we have developed for determining accurately the respiratory Z in small animals. The Z of total respiratory system and lungs was measured with small-volume oscillations between 0.25 and 9.125 Hz. The measured Z was fitted to a model that featured a constant-phase tissue compartment (with dissipation and elastance characterized by constants G and H, respectively) and a constant airway resistance (Z. Hantos, B. Daroczy, B. Suki, S. Nagy, and J. J. Fredberg. J. Appl. Physiol. 72: 168-178, 1992). We matched the lung volume between the closed- and open-chest conditions by using the quasi-static pressure-volume relationship of the lungs to calculate Z as a function of lung volume. Resistance decreased with lung volume and was not significantly different between total respiratory system and lungs. However, G and H of the respiratory system were significantly higher than those of the lungs. We conclude that chest wall in rats has a significant influence on tissue mechanics of the total respiratory system.

Purification and characterization of pea seedling amine oxidase for crystallization studies.

Pea (Pisum sativum L.) seedling amine oxidase (EC 1.4.3.6) is the first amine oxidase to be crystallized that diffracts to atomic resolution (2.5 A). Extensive modifications of a published purification procedure were necessary to obtain protein that would give diffraction-quality crystals. Here we report the improved purification and also use this high-purity protein to reexamine some fundamental characteristics of pea seedling amine oxidase. The extinction coefficient at 280 nm (epsilon 1%(280)) and the molecular mass of the protein are investigated by a variety of techniques, yielding epsilon 1%(280) = 20 cm-1 and a mass 150 +/- 6 kD. In addition, the stoichiometry of the metal and organic cofactors, Cu(II) and 6-hydroxy dopa (Topa) quinone, respectively, is examined. The ratio of Cu(II):Topa:protein monomer is found to be 1:1:1.

Inhibition of NADH oxidation by pyridine derivatives.

The neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, an impurity in an illicit drug, is expressed after its oxidation to 1-methyl-4-phenylpyridinium by monoamine oxidase. The pyridinium is concentrated by carrier-mediated transport into the mitochondria where it inhibits NADH dehydrogenase and, hence, ATP synthesis. Some structurally related compounds have been tested for their effect on the oxidation of NAD+-linked substrates in intact mitochondria, and for the inhibition of the accumulation of the pyridinium into mitochondria and of NADH dehydrogenase activity in a membrane preparation. Some pyridine derivatives are more inhibitory to NADH dehydrogenase than is 1-methyl-4-phenylpyridinium but these are not concentrated into mitochondria by the uptake system. 4-Phenylpyridine, one of the most effective inhibitors, both occurs naturally and is an environmental pollutant.

Oxidation of analogs of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by monoamine oxidases A and B and the inhibition of monoamine oxidases by the oxidation products.

Twenty analogs of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were tested for their capacity to be oxidized by pure monoamine oxidase-A (MAO-A) prepared from human placenta and pure monoamine oxidase-B (MAO-B) prepared from beef liver. Several of the MPTP analogs were very good substrates for MAO-A, for MAO-B, or for both and had low Km values and high turnover numbers. These values were similar to or even better than those of kynuramine and benzylamine, good substrates for MAO-A and MAO-B, respectively. MPTP had relatively low Km values for oxidation by both MAO-A and MAO-B. In contrast, the turnover number for MPTP oxidation by MAO-B was considerably higher than the value for MAO-A. The corresponding pyridinium species of MPTP and several of the MPTP analogs inhibited MAO-A competitively with Ki values at micromolar concentrations; in contrast the pyridinium species inhibited MAO-B competitively at considerably higher concentrations (i.e., 100 microM or greater Ki values). The data provide information concerning the structural requirements for the oxidation of tetrahydropyridines by MAO-A and MAO-B and the inhibition of these enzymes by pyridiniums.

Structural dependence of the inhibition of mitochondrial respiration and of NADH oxidase by 1-methyl-4-phenylpyridinium (MPP+) analogs and their energized accumulation by mitochondria.

Nineteen structural analogs of 1-methyl-4-phenylpyridinium (MPP+) were studied for their capacity to inhibit the mitochondrial oxidation of NAD+-linked substrates and the aerobic oxidation of NADH in inner membrane preparations from cardiac mitochondria. In the majority of cases, a good correlation was found between the two inhibition effects monitored. A few compounds were effective inhibitors of NADH oxidase but had only marginal effects on mitochondrial respiration. From studies of their accumulation by mitochondria, it appears likely that the latter compounds are not effectively concentrated by intact mitochondria by the electrical gradient and, in part for this reason, cannot reach sufficiently high concentrations at the appropriate binding site of NADH dehydrogenase. In addition, evidence is presented that the penetration of pyridinium analogs to the inhibition site in the NADH dehydrogenase complex may also be rate limiting. The data support the thesis that, for a substituted tetrahydropyridine to be acutely neurotoxic, its pyridinium oxidation product must be actively accumulated in the mitochondria and must inhibit NADH-ubiquinone oxidoreductase in its membrane environment.

Importance of monoamine oxidase A in the bioactivation of neurotoxic analogs of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a potent dopaminergic neurotoxin that causes biochemical, pharmacological, and pathological deficits in experimental animals similar to those seen in human parkinsonian patients. All of the deficits can be prevented by treating mice with selective inhibitors of monoamine oxidase B (MAO-B), including deprenyl, prior to MPTP administration. We now report that the dopaminergic neurotoxicity of two potent MPTP analogs, namely the 2'-methyl and 2'-ethyl derivatives (2'-MeMPTP and 2'-EtMPTP), cannot be prevented by deprenyl pretreatment. However, the neurotoxicity of these two analogs can be prevented by pretreatment with a combination of deprenyl and the selective MAO-A inhibitor clorgyline at doses that are sufficient to almost completely inhibit both MAO-B and MAO-A activities. Moreover, the neurotoxicity of 2'-EtMPTP (but not of 2'-MeMPTP and MPTP) can be significantly attenuated by clorgyline alone. There was a parallel between the capacity of the MAO inhibitors to decrease the brain content of the pyridinium species after administration of the tetrahydropyridines and the capacity of the MAO inhibitors to protect against the neurotoxic action of the tetrahydropyridines. The data support the conclusion that both 2'-MeMPTP and 2'-EtMPTP are bioactivated to pyridinium species to a significant extent by MAO-A. Further, it appears that the formation of the pyridinium species plays an important role in the neurotoxic process.