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17325

Mechanism of Hyperoxia Induced Inactivation of Protein Tyrosine Phosphatase Activity In the Newborn Brain

Friday, October 19, 2012
Room R02-R05 (Morial Convention Center)
Ronald K. Sallas, MD, Qazi Ashraf, Anli Zhu and Maria Delivoria-Papadopoulos, Department of Pediatrics, Drexel University College of Medicine, Philadelphia, PA

Purpose

The present study tests the hypothesis that the hyperoxia-induced decrease in activity of protein tyrosine phosphatase (PTP) in cell membranes of the cerebral cortex of newborn piglets is mediated by nitric oxide (NO) derived from neuronal nitric oxide synthase (nNOS).

Methods

Fourteen piglets were assigned to normoxic (Nx, n=3) exposed to FiO2 of 0.21 for 2 hrs, hyperoxic (Hyx FiO2 = 1.0 for 120 min, pO2 > 400mmHg, n=6), and hyperoxic pre-treated with a selective inhibitor of nNOS, 7-nitro-indazole-sodium (Hyx+7-NINA, 1 mg/kg i.v., 30 mins prior to hyperoxia, n=5). ATP and phosphocreatine (Pcr) were measured biochemically to document cerebral energy status. PTP activity was determined spectrophotometrically using p-nitrophenyl phospahte (pNPP) as substrate and a highly selective inhibitor of PTP, bp V (phen){bis-peroxol (1, 10phenanthroline) oxovanadate}. The phosphate was assayed by a highly sensitive malachite green assay. The activity was expressed as nmole/mg protein/hr.

Results

The activity of PTP (nmoles/mg protein/hr) was 1.72 + 0.06 in Nx, 1.11 + 0.27 (p<0.05 vs Nx), and 1.46 + 0.21 in Hyx+7-NINA (p=NS vs Nx). The data show that administration of a highly selective inhibitor of nNOS, prior to hyperoxia, prevented the hyperoxia-induced decreased PTP activity.

Conclusion

We conclude that hyperoxia-induced decreased PTP activity is mediated by NO derived from nNOS. Since all members of the PTP family possess a cysteine residue at their active site, NO generated from nNOS during hyperoxia will decrease PTP activity by oxidation of sulfhydryl (-SH) group of the cysteine residue in the active site domain. The decrease in PTP activity will lead to increased tyrosine phosphorylation of anti-apoptotic proteins and results in loss of anti-apoptotic potential of the hyperoxic neuronal cell.