Low nanomolar concentrations of nitric oxide (NO) inhibit the
mitochondrial respiratory chain enzyme cytochrome oxidase (complex
IV) reversibly and in competition with molecular oxygen. We
have been investigating the cellular consequences of this interaction
using endogenously-generated and exogenously - applied NO.
We have found using endothelial cells that endogenous NO, either
basally produced or generated in response to stimulation with
bradykinin, reduces the rate of oxygen consumption by these
cells. This finding suggests that endogenous NO modulates oxygen
consumption under basal and stimulated conditions. We are studying
the bioenergetic consequences of this control mechanism.
We have also found that prolonged exposure to exogenous NO results
in persistent inhibition of mitochondrial respiration, which
is mainly localised at complex I. This persistent inhibition
seems to be the result of oxidative stress generated from mitochondrial
free-radical generation and involves S-nitrosylation. Indeed,
inhibition of the respiratory chain causes its reduction and
the subsequent generation of superoxide anions. It is likely
that these anions are initially converted by superoxide dismutase
to hydrogen peroxide, which is known to be a transcription factor
of several defence genes. However, if this inhibition is prolonged
it could result in the generation of peroxynitrite at the site
of superoxide anion production. Thus, persistent inhibition
of cytochrome oxidase could elicit a two-stage response - an
early one, in which the main consequence is the release of small
amounts of hydrogen peroxide, and a later one that involves
higher concentrations of hydrogen peroxide and the formation
of peroxynitrite.
We have also been studying the involvement of NO in apoptosis
and have shown that inhibition of mitochondrial respiration
by NO results in a relative mitochondrial hyperpolarisation,
an occurrence that requires the production of glycolytic ATP.
Our studies indicate that this hyperpolarisation is a protective
mechanism since neurons, which do not utilise the glycolytic
pathway and do not respond to NO by mitochondrial hyperpolarisation,
are more susceptible to NO-induced apoptosis than are glycolytically-active
astrocytes. Persistent inhibition of respiration by NO over
a prolonged time will eventually result in the collapse of membrane
potential, ATP depletion and, ultimately, cell death.
We are currently investigating the similarities and differences
between hypoxia-induced and NO-induced inhibition of mitochondrial
respiration, and are elucidating the specific genes involved
in the cellular defence against changes in oxygen availability
at the mitochondrial level.
