Uncoupling effects of 2,4-dinitrophenol on electron transfer reactions and cell bioenergetics in rat brain in situ

We have examined the effects of uncoupling on electron transfer reactions and cell bioenergetics in rat brain in situ. The redox reactions of cyt aa3, cyt c and cyt b as well as the levels of phosphocreatine/inorganic phosphate ratio (PCr/Pi) were monitored in isolated perfused rat head, using optical and [31P]NMR techniques, respectively. In the first series of experiments anesthetized (pentobarbital) and mechanically ventilated rats underwent bilateral carotid arterial cannulation. The head (skull intact, muscle removed) was perfused with a perfluorochemical solution (FC-43), and the animal was then decapitated. By means of reflectance spectrophotometry, the redox reactions of cyt aa3, c and b were monitored before and after perfusing the head with uncoupler 2,4-dinitrophenol (DNP) as well as after complete anoxia. The second series of experiments was performed using [31P]NMR spectroscopy (24.33 MHz; 10.25 in. magnet). PCr/Pi ratios were first determined for living rats (in vivo) before and after exchange transfusion with FC-43 (Hct less than 0.5%), and then in isolated perfused head (in situ) before and after 2,4-DNP addition. Reduction of cyt aa3 (32 +/- 1.4%, mean +/- S.E.M.), cyt c (29 +/- 6%), and cyt b (19 +/- 2%) was induced by addition of 2,4-DNP. Compared to in vivo aerobic metabolism with a PCr/Pi ratio of 3.0-5.0, the value in the uncoupled state in situ was less than 0.1. We conclude that in contrast to in vitro, uncoupling of oxidative phosphorylation induces reduction of intramitochondrial cytochromes in situ.(ABSTRACT TRUNCATED AT 250 WORDS)