Figure 2.

Ras activity is necessary and sufficient for OGD preconditioning. (A) Activation of Ras is indicated by the ratio between GDP and GTP detected by thin layer chromatography. Ras is activated by 5 min of OGD in a NMDA receptor- and NO-dependent manner. Five minutes of OGD (–) induces robust activation of Ras. The activation of Ras by 5 min of OGD is blocked by the NMDA antagonist MK801 (10 μM) and the NOS inhibitor l-NAME (500 μM). Excess l-Arg (5 mM) reverses the l-NAME inhibition of Ras activation. The GC inhibitor 1H-[1,2,4]oxidiazolo[4,3-α]quinoxalin-1-one (ODQ) (10 μM) has no significant effect on Ras activation. Experiments were replicated three times with similar results. (B) Activation of Ras mediates OGD tolerance. OGD tolerance is blocked by the Ras inhibitor FPT Inh III (250 μM) and by expression of a dominant negative mutant Ras (DNRas) via adenovirus or herpes simplex virus vectors. DNRas does not have an effect on the neurotoxicity induced by 60 min of OGD. Viruses containing the lacZ gene were used to control for nonspecific effects of the recombinant viral systems. A constitutively active form of Ras (CARas) expressed by an adenoviral vector induces neuroprotection against 60 min of OGD. U, cultures infected with recombinant virus, but not exposed to OGD. Each data point is the mean ± SEM (n = 8) of at least two separate experiments. Each data point reflects a minimum of 16,000–30,000 neurons counted. Significance was determined by a balanced two-way ANOVA with a Student's t test: *, P = 0.001 comparing 60′ OGD to 5′/60′ OGD, lacZ-5′/60′ OGD, or CARas-60′ OGD; ^†, P = 0.001 when comparing 5′/60′ OGD to FPT Inh III-5′/60′ OGD, or DNRas-5′/60′ OGD.