Requirement for nitric oxide activation of p21 ^ras /extracellular regulated kinase in neuronal ischemic preconditioning

Abstract

The mechanisms underlying neuronal ischemic preconditioning, a phenomenon in which brief episodes of ischemia protect against the lethal effects of subsequent periods of prolonged ischemia, are poorly understood. Ischemia can be modeled in vitro by oxygenglucose deprivation (OGD). We report here that OGD preconditioning induces p21 ras (Ras) activation in an N-methyl-D-aspartate receptor-and NO-dependent, but cGMP-independent, manner. We demonstrate that Ras activity is necessary and sufficient for OGD tolerance in neurons. Pharmacological inhibition of Ras, as well as a dominant negative mutant Ras, block OGD preconditioning whereas a constitutively active form of Ras promotes neuroprotection against lethal OGD insults. In contrast, the activity of phosphatidyl inositol 3-kinase is not required for OGD preconditioning because inhibition of phosphatidyl inositol 3-kinase with a chemical inhibitor or with a dominant negative mutant does not have any effect on the development of OGD tolerance. Furthermore, using recombinant adenoviruses and pharmacological inhibitors, we show that downstream of Ras the extracellular regulated kinase cascade is required for OGD preconditioning. Our observations indicate that activation of the Ras͞extracellular regulated kinase cascade by NO is a critical mechanism for the development of OGD tolerance in cortical neurons, which may also play an important role in ischemic preconditioning in vivo.

εPKC Is Required for the Induction of Tolerance by Ischemic and NMDA-Mediated Preconditioning in the Organotypic Hippocampal Slice

“…This hypothesis was supported by studies in vivo in which inhibition of NMDA receptors with MK-801 during IPC resulted in blockade of ischemic tolerance by IPC in gerbils (Kato et al, 1992b) and in vitro (Pringle et al, 1997b). Gonzalez-Zulueta et al (2000) showed that, during OGD preconditioning in neuronal cell cultures, the signaling cascade was initiated by activation of the NMDA receptors, calcium influx, and production of nitric oxide in an RAS-extracellular signal-regulated protein kinase (ERK)-dependent manner, leading to the development of neuronal tolerance to ischemia.…”

“…Furthermore, the extracellular and intracellular calcium chelation was also efficacious in blocking the induction of tolerance by NMDA preconditioning. We conjecture that a critical level of calcium is necessary to stimulate calcium-dependent enzymes, including PKC (Huang et al, 1999;Katsura et al, 1999) and nitric oxide synthase (Gonzalez-Zulueta et al, 2000;Nandagopal et al, 2001), that will lead to a signal transduction pathway that could ultimately play a role in neuroprotection.…”

The neuroprotective mechanism of brain ischemic preconditioning

“…Ras activates a number of pathways, but the most important seems to be MAPK, which transmits signals downstream to other protein kinases and gene regulatory proteins. Gonzalez-Zulueta and collaborators reported the essential role of NO activation in neuronal ischemic preconditioning [73] , and also found that preconditioning induced Ras activation in an NMDA receptor-and NO-dependent, but cGMP-independent manner. They also demonstrated that Ras activity was necessary and sufficient for ischemic tolerance induction in neurons.…”

Nitric Oxide Produced During Ischemia Is Toxic but Crucial to Preconditioning-Induced Ischemic Tolerance of Neurons in Culture

“…We first investigated whether sublethal oxygen-glucose deprivation (OGD) induced neuronal ischemic tolerance in murine cortical cultures containing both neurons and glia (days in vitro [13][14][15]. To do this, we have tried to determine both the sublethal and lethal exposure periods of OGD for cultured neurons (Fig.…”

“…A previous study (14) has revealed that the induction of neuronal ischemic tolerance is dependent on new protein synthesis, and the development of tolerance is blocked by cycloheximide. The authors postulated that the NO/p21 Ras /Raf/Erk pathway is critically involved in the PC-induced ischemic tolerance of neurons.…”