Reduction of tyrosine kinase activity and protein tyrosine dephosphorylation by anoxic stimulation in vitro.

Tyrosine-specific protein phosphorylation has been recently implicated in mediating pathological changes associated with cerebral ischemia. In the present study, acute hypoxia/ischemia (anoxia) was simulated in vitro by incubating rat hippocampal slices in glucose-free artificial cerebrospinal fluid saturated with 95% N2/5% CO2. A marked decrease in the level of tyrosine phosphorylation of many protein bands compared with the control was observed. Immunoprecipitation and western blot confirmed that the NR2A/2B subunits of the N-methyl-D-aspartate receptors are among the dephosphorylated proteins. Maximal dephosphorylation of bands corresponding to NR2A/2B was reached after 10 min, and no recovery was observed even after 1 h in normal, oxygenated artificial cerebrospinal fluid. The effect was partially blocked by dephostatin, a membrane-permeable inhibitor of protein tyrosine phosphatases, but was not affected by the presence of glutamate receptor antagonists, or by removing extracellular Ca2+ or chelating intracellular Ca2+. Enzyme assay showed that anoxic stimulation resulted in a selective reduction in protein tyrosine kinase activity without affecting protein tyrosine phosphatase activity. Thus the present work suggests that anoxic stimulation produces a selective inhibition of protein tyrosine kinase activity leading to tyrosine-dephosphorylation of several proteins including the N-methyl-D-aspartate receptors. The underlying mechanism may involve a novel signal transduction pathway, which may protect neurons from degeneration during ischemic stress.