Response of toad brain respiratory chain enzymes to ouabain, elevated potassium, and electrical stimulus

Spectrophotometric and fluorometric techniques were used to monitor the proportion or reduced to oxidized cytochrome (cyt) and levels of reduced pyridine nucleotide in preparations of whole toad brain in vitro. In resting, well-oxygenated brain, levels of reduction for cyt a 3 ranged between 5% and 23%; for cyt a, 17–23%; for cyt c, 18–32%, and for cyt b, 25–42%. These levels of reduction cannot be due to functional hypoxia since hemoglobin in resting brains is 100% oxygenated. In brains treated with 10 −4M ouabain, a stimulant of brain respiration, the cytochromes first become more oxidized, then more reduced; ultimately thereis a tendency to return to the initial levels of reduction. In brains bathed with solutions containing 30 mM potassium, also a stimulant of brain respiration, the response is an immediate pulse of reduction in all cytochromes, followed by a tendency to return to the initial levels. Short trains of pulses of electrical field stimulation result in a biphasic change in the level of reduction of cyt a 3, an initial slight reduction being followed by a transient of increased oxidation. This response can be abolished by low-sodium bathing solution but not by ouabain. Cytochromes a, b and c show a simple oxidative response to electrical stimulation; the kinetics of this oxidative response are similar to those of the oxidative transient of the cyt a 3 response. Pyridine nucleotides, as measured by their fluorescence, respond to electrical stimulation with a transient oxidation which exhibits slower kinetics than the response of the cytochromes. The high resting levels of reduction of cyt a and cyt a 3, the reductiveresponse to ouabain and potassium, and the oxidative response of all cytochromes to electrical stimulation suggest a tighter coupling between oxygen utilization and neuronal function than would be expected if mitochondrial redox states simply reflected changes in phosphate acceptor potential resulting from activity of Na +-K + ATPase.