guanosine-triphosphate and Hypoxia-Ischemia--Brain

Hyperbaric oxygen treatment has been suggested as able to reduce hypoxia induced neuronal damage. The aim of the study was to compare the impact of different reoxygenation strategies on early metabolical (purine nucleotide content determined by HPLC) and morphological changes (index of cell injury after celestine blue/acid fuchsin staining) of hypoxically damaged rat neocortical brain slices. For this purpose slices (300 microm and 900 microm) were subjected to either 5 or 30 min of hypoxia by gassing the incubation medium with nitrogen. During the following reoxygenation period treatment groups were administered either 100% oxygen (O) or room air (A) at normobaric (1 atm absolute, NB-O; NB-A) or hyperbaric (2.5 atm absolute, HB-O; HB-A) conditions. After 5 min of hypoxia, both HB-O and NB-O led to a complete nucleotide status restoration (ATP/ADP; GTP/GDP) in 300 microm slices. However, reoxygenation after 30 min of hypoxia was less effective, irrespective of the oxygen pressure. Furthermore, administering hyperbaric room air resulted in no significant posthypoxic nucleotide recovery. In 900 microm slices, both control incubation as well as 30 min of hypoxia resulted in significantly lower trinucleotide and higher dinucleotide levels compared to 300 microm slices. While there was no significant difference between HB-O and NB-O on the nucleotide status, morphological evaluation revealed a better recovery of the index of cell injury (profoundly injured/intact cell-ratio) in the HB-O group. Conclusively, the posthypoxic recovery of metabolical characteristics was dependent on the duration of hypoxia and slice thickness, but not on the reoxygenation pressure. A clear restorative effect on purine nucleotides was found only in early-administered HB-O as well as NB-O in contrast to room air treated slices. However, these pressure independent metabolic changes were morphologically accompanied by a significantly improved index of cell injury, indicating a possible neuroprotective role of HB-O in early posthypoxic reoxygenation.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Brain; Brain Chemistry; Cell Count; Cell Survival; Chromatography, High Pressure Liquid; Coloring Agents; Energy Metabolism; Guanosine Diphosphate; Guanosine Triphosphate; Hyperbaric Oxygenation; Hypoxia-Ischemia, Brain; In Vitro Techniques; Male; Neocortex; Oxygen Inhalation Therapy; Purine Nucleotides; Rats; Rats, Wistar

An in vitro model of ischemia was obtained by subjecting PC12 cells differentiated with nerve growth factor to a combination of glucose deprivation plus anoxia. Immediately after the ischemic period, the protein synthesis rate was significantly inhibited (80%) and western blots of cell extracts revealed a significant accumulation of phosphorylated eukaryotic initiation factor 2, alpha subunit, eIF2(alphaP) (42%). Upon recovery, eIF2(alphaP) levels returned to control values after 30 min, whereas protein synthesis was still partially inhibited (33%) and reached almost control values within 2 h. The activities of the mammalian eIF2alpha kinases, double-stranded RNA-activated protein kinase, mammalian GCN2 homologue, and endoplasmic reticulum-resident kinase, were determined. None of the eIF2alpha kinases studied showed increased activity in ischemic cells as compared with controls. Exposure of cells to cell-permeable inhibitors of protein phosphatases 1 and 2A, calyculin A or tautomycin, induced dose- and time-dependent accumulation of eIF2(alphaP), mimicking an ischemic effect. Protein phosphatase activity, as measured with [(32)P]phosphorylase a as a substrate, diminished during ischemia and returned to control levels upon 30-min recovery. In addition, the rate of eIF2(alphaP) dephosphorylation was significantly lower in ischemic cells, paralleling both the greatest translational inhibition and the highest eIF2(alphaP) levels. The endogenous phosphatase activity from control and ischemic extracts showed different sensitivity to inhibitor 2 and fostriecin in in vitro assays, inhibitor-2 effect in ischemic cells being lower than in control cells. Together these results indicate that an eIF2alpha phosphatase, probably protein phosphatase 1, is implicated in the ischemia-induced eIF2(alphaP) accumulation in PC12 cells.

Topics: Adenosine Triphosphate; Animals; Cell Differentiation; Cell Survival; Enzyme Inhibitors; Eukaryotic Initiation Factor-2; Glutamic Acid; Guanosine Triphosphate; Hypoxia-Ischemia, Brain; Neurons; PC12 Cells; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 1; Proteins; Rats