Effect of neuronal NOS inhibitor, 7‐nitro‐indazole, on cytosolic caspase 9 activity in vitro

Abstract

Previous studies have shown that nitric oxide synthase (NOS) inhibitors prevent or reduce the hypoxia‐induced neuronal injury. The present study tests the hypothesis that neuronal NOS inhibitor, 7‐nitro‐indazole (7‐NI) will inhibit the cysteine protease, caspase 9, the initiator of programmed cell death. The effect of 7 NI on caspase 9 activity was determined in the cytosolic fraction of the cerebral cortex of normoxic (Nx, n = 4) and hypoxic (Hx, n = 4) newborn piglets. The animals were exposed to either 21% (Nx) or 7% (Hx) oxygen for 60 min. Cerebral tissue hypoxia was documented by the ATP and phosphocreatine levels. Cytosols were prepared and caspase 9 activity determined using a specific fluorogenic substrate (‐Ac‐Leu‐Glu‐His‐Asp‐AFC) for caspase 9 and monitored at 400 nm excitation and 505 nm emission wavelength. Caspase 9 activity in Nx group was 629 ± 49 as compared to 960 ± 105 pmoles AFC/min/mg protein (p < 0.002, 50.2% increase) in the Hx group. In presence of 10, 50 and 100 μm 7‐NI, caspase 9 activity decreased to 459 ± 41, 252 ± 36 and 256 ± 29 pmoles/min/mg protein, respectively, in Nx group. In Hx group, the activity decreased to 578 ± 49, 402 ± 37, 393 ± 35 pmoles/min/mg protein, respectively. The data demonstrate that 7‐NI inhibits caspase 9 in both the Nx and Hx groups. It is concluded that 7‐NI is an inhibitor of caspase 9. It is proposed that 7 NI administration will be neuroprotective due to its properties as inhibitor of nNOS as well as of caspase 9 and prevent the hypoxia‐induced neuronal death.Acknowledgements: Supported by NIH‐HD‐38079.