Purpose
Overproduction of nitric oxide (NO) following brain ischemia and reperfusion (I/R) contributes to the pathophysiology of I/R injury. However, the scope of NO's actions during brain I/R are not completely understood. In cell culture, NO has been shown to induce endoplasmic reticulum (ER) stress and activate components of the unfolded protein response (UPR). The UPR is an ER stress response that inhibits protein synthesis to stop the further accumulation of misfolded proteins within the ER lumen, and increases transcription of ER-related genes to repair the ER. Translation arrest following the UPR is due to phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2 alpha) by the ER transmembrane eIF2 alpha kinase PERK. Brain I/R causes ER stress and activates PERK. Because NO has been shown to induce the UPR in cell culture, we here evaluated the role of NO on eIF2 alpha phosphorylation and PERK activation following cardiac arrest and resuscitation in several brain regions and several peripheral organs.
Methods
Transient global brain ischemia was induced by cardiac arrest, and reperfusion was by critical care cardiopulmonary resuscitation with all animals maintained normothermic throughout. Experimental groups (n=3 per group) were conducted in the presence and absence of 200 mg/kg of the nonspecific nitric oxide synthase (NOS) inhibitor L-NAME: nonischemic controls (NIC), 10 min ischemia plus 10 min (10R), 10R + L-NAME (10RN), 90 min (90R) reperfusion and 90R + L-NAME (90RN). PERK activation and eIF2 alpha phosphorylation were determined by Western blot in brain regions, heart, kidney, liver, lung, pancreas and skeletal muscle. Brain regions included: brainstem, midbrain, cerebellum, thalamus, hippocampus and cerebral cortex. The effect of the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) on PERK activation and eIF2 alpha phosphorylation in cultured neuroblastoma cells was also studied.
Results
Brain regions generally showed large increases in eIF2 alpha phosphorylation and PERK activation. We report here for the first time that I/R induce PERK activation and eIF2 alpha phosphorylation in kidney and liver. Heart showed evidence of eIF2 alpha phosphorylation. Pancreas, lung and skeletal muscle showed no change in eIF2 alpha phosphorylation with ischemia and reperfusion. Although SNAP activated PERK in cultured neuroblastoma cells, administration of L-NAME had no effect on levels of eIF2 alpha phosphorylation in all brain regions and responsive peripheral organs.
Discussion
Although NO activated PERK in cultured cells, activation of the UPR following I/R in brain and responsive peripheral organs appeared to occur independent of NO formation. Thus, it is likely that NO production is not proximal to PERK activation in the setting of ischemia and reperfusion injury. Further, our results showing that the UPR is active in kidney and liver, and to a small extent in heart, clarify peripheral contributions to the cellular pathophysiology of resuscitation from cardiac arrest.
Footnotes
Acknowledgements
Work supported by NIH grant NS044100 (D.J.D.).