Mechanisms related to reduction of radical in mouse lung using an L-band ESR spectrometer

Reduction of radicals in mouse lung was characterized in whole animals usin g an L-band ESR technique and nitroxide radicals as probes. An aqueous solu tion of nitroxide radical was immediately instilled intratracheally to mous e after euthanasia. Nitroxide radicals without charged groups were reduced significantly in the lung, while radicals with charged groups were only sli ghtly reduced. Permeation rates across lung plasma membrane were not rate l imiting of the stage of reduction of the noncharged nitroxides. Michaelis p arameters, apparent Km and apparent Vmax, were obtained from the Lineweaver -Burk plots of the reduction. Among noncharged nitroxides with constant app arent Vmax, radicals with a larger n-octanol/water partition coefficient sh owed a lower apparent Km, thereby suggesting that the concentration of thes e nitroxides in the membrane contributes to apparent Km. The reduction rate of noncharged nitroxide, hydroxy-TEMPO, was influenced by noncharged SH re agents instilled together with the nitroxide; dithiothreitol stimulated the reduction, while the oxidized reagent inhibited it. The Lineweaver-Burk pl ots of the nitroxide reduction in the presence of various concentrations of dithiothreitol suggest the possibility that the reduction system for hydro xy-TEMPO is based on a kind of ping pong bi-reactant mechanism, and that th e reduction system utilizes SH as an electron donor. Endogenous glutathione contributed partially to the reduction. (C) 1999 Elsevier Science Inc.