ATP and cytochrome c-dependent inhibition of caspase-9 activity in the cerebral cortex of newborn piglets.

The present study investigates the mechanism of activation of caspase-9 during hypoxia and tests the hypothesis that ATP and cytochrome c regulate the activity of caspase-9 in the cerebral cortex of newborn piglets. Cerebral tissue hypoxia was documented by decreased levels of high energy phosphates, ATP and phosphocreatine (PCr). Cytosolic fractions were prepared from cerebral cortices and passed through a G50 column, to remove endogenous ATP and cytochrome c. Caspase-9 activity was determined spectrofluorometrically using a specific fluorogenic substrate for caspase-9 at increasing concentrations of ATP (0-1.0 mM) or cytochrome c (0-3.0 microM). Caspase-9 activity (nmol/mg protein/h) was 1.26 +/- 0.15 in the normoxic and 2.13 +/- 0.14 in the hypoxic group (P < 0.05). The enzyme activity was inhibited by ATP or cytochrome c in both normoxic and hypoxic groups. The IC50 for ATP and cytochrome c increased 5-fold and 1.5-fold, respectively, following hypoxia, suggesting a hypoxia-induced modification of the ATP and cytochrome binding sites. The data demonstrate that ATP (1 mM) and cytochrome c (3.0 microM) inhibit caspase-9 activity by approximately 70%. On the basis of these observations, we propose a new and novel concept that the caspase-9 activity remains inhibited under the normoxic conditions and during hypoxia the decrease in ATP and decreases in the affinity for ATP and cytochrome c release the inhibitory block to activate the enzyme. Results of ATP- and cytochrome c-dependent inhibition of purified caspase-9 human recombinant show that the inhibitory effect by ATP and cytochrome c does not require Apaf-1. To our knowledge, this is a completely new concept and a new mechanism of regulation of caspase-9 activity that may lead to hypoxia-induced programmed cell death.