Effect of nitration on protein tyrosine phosphatase and protein phosphatase activity in neuronal cell membranes of newborn piglets.
Protein tyrosine phosphatase predominantly determines the status of protein tyrosine kinase-dependent phosphorylation of specific proteins and controls the survival and death of neurons. Previous studies have shown that protein tyrosine phosphatase activity is decreased during hypoxia in cortical membranes of the newborn piglet. We have also shown that nitric oxide (NO) free radicals are generated during hypoxia, and may result in modification of protein tyrosine phosphatase via peroxynitrite-mediated modification. The present study tests the hypothesis that the hypoxia-induced decrease in protein tyrosine phosphatase activity is NO-mediated. To test this hypothesis, in vitro experiments were conducted by measuring protein tyrosine phosphatase activity in the presence of an NO donor, sodium nitroprusside (SNP), or peroxynitrite. Since 3-nitrotyrosine is produced as a consequence of peroxynitrite reactions, we have also examined the effect of 3-nitrotyrosine on protein phophatase activity. Cerebral cortical P(2) membranes were prepared from seven normoxic newborn piglets and each sample was divided into three aliquots: a control group, a SNP group (exposed to 200 microM SNP), and a peroxynitrite group (exposed to 100 microM peroxynitrite). Protein tyrosine phosphatase activity was determined spectrophotometrically in the presence or absence of 2 microM bpV(phen), a highly selective inhibitor of protein tyrosine phosphatase. The protein tyrosine phosphatase activity was 198+/-25 nmol/mg protein/h in the normoxic group, 177+/-30 nmol/mg protein/h in the SNP group (p=NS versus normoxic) and 77+/-20 nmol/mg protein/h in the peroxynitrite group (p<0.001 versus normoxic). The results show that peroxynitrite but not SNP exposure results in decreased protein tyrosine phosphatase activity in vitro. Furthermore 3-nitrotyrosine (100 microm), a product of peroxynitrite, decreased the enzyme activity from 926+/-102 to 200+/-77 (p<0.001). We conclude that protein tyrosine phosphatase regulation is mediated by peroxynitrite. We propose that hypoxia-induced NO production leading to peroxynitrite formation is a potential mechanism of protein tyrosine phosphatase inactivation in vivo. The NO-induced decrease in protein tyrosine phosphatase and protein phosphatase activity, leading to Bcl-2 protein phosphorylation and loss of its antiapoptotic activity may be a NO-mediated mechanism of programmed cell death in the hypoxic brain.
Lopez
Carlos J
CJ
Department of Pediatrics, Drexel University College of Medicine, Philadelphia, PA, USA. md47@drexel.edu
Qayyum
Imran
I
Mishra
Om P
OP
Delivoria-Papadopoulos
Maria
M
D000818:Animals; D000831:Animals, Newborn; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D002540:Cerebral Cortex / Q000187:drug effects / Q000201:enzymology / Q000503:physiopathology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D002534:Hypoxia, Brain / Q000503:physiopathology*; D066298:In Vitro Techniques; D009474:Neurons / Q000187:drug effects / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D030421:Peroxynitrous Acid / Q000494:pharmacology; D017027:Protein Tyrosine Phosphatases / Q000187:drug effects / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000494:pharmacology
D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; D030421:Peroxynitrous Acid; D009599:Nitroprusside; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D017027:Protein Tyrosine Phosphatases