Biphasic oxidation of mitochondrial NAD(P)H

Area of Science:

• Mitochondrial biochemistry
• Cellular redox homeostasis
• Oxidative stress response

Background:

• Mitochondrial pyridine nucleotides (NAD(P)H) are crucial for maintaining mitochondrial structural integrity.
• Their redox state is sensitive to oxidative stress, impacting cellular function.

Purpose of the Study:

• To investigate the biphasic oxidation of endogenous NAD(P)H in rat liver mitochondria induced by tert-butylhydroperoxide.
• To elucidate the mechanisms underlying the second phase of NAD(P)H oxidation.

Main Methods:

• Induction of oxidative stress using tert-butylhydroperoxide in isolated rat liver mitochondria.
• Monitoring of NAD(P)H oxidation, inner membrane potential, and ATP synthesis.
• Assessment of mitochondrial swelling and permeabilization.
• Inhibition studies using cyclosporin A, EGTA, butylhydroxytoluene, and trifluoperazine.

Main Results:

• A rapid, biphasic oxidation of mitochondrial NAD(P)H was observed.
• The first phase rapidly oxidized ~85% of NAD(P)H.
• The second phase, occurring after inner membrane potential collapse and swelling, involved ATP synthesis disruption and inner membrane permeabilization.
• The second phase was inhibited by cyclosporin A and EGTA, and delayed by butylhydroxytoluene and trifluoperazine.

Conclusions:

• The second phase of NAD(P)H oxidation is linked to mitochondrial permeabilization.
• This phase likely involves extramitochondrial NADH oxidation via the outer membrane electron transport system.
• Subsequent NADPH oxidation occurs via a transhydrogenase reaction in permeabilized mitochondria.