Acidosis enhances translocation of protein kinase C but not Ca2+/calmodulin-dependent protein kinase II to cell membranes during complete cerebral ischemia

Systemic hyperglycemia and hypercapnia severely aggravate ischemic brain damage when instituted prior to cerebral ischemia. An aberrant cell signaling following ischemia has been proposed to be involved in ischemic cell death, affecting protein kinase C (PKC) and the calcium calmodulin kinase II (CaMKII). Using a cardiac arrest model of global brain ischemia of 10 min duration, we investigated the effect of hyperglycemia (20 mM) and hypercapnia (pCO2 300 mmHg) on the subcellular redistribution of PKC (α, β, γ) and CaMKII to synaptic membranes and to the microsomes, as well as the effect on PKC activity. We confirmed the marked translocation of PKC and CaMKII to cell membranes induced by ischemia, concomitantly with a decrease in the PKC activity in both the membrane fraction and cytosol. Hyperglycemia and hypercapnia markedly enhanced the translocation of PKC-γ to cell membranes while other PKC isoforms were less affected. There was no effect of acidosis on PKC activity, or on translocation of CaMKII to cell membranes. Our data strongly suggest that the enhanced translocation of PKC to cell membranes induced by hyperglycemia and hypercapnia may contribute to the detrimental effect of tissue acidosis on the outcome following ischemia. Copyright (C) 1999 Elsevier Science B.V

Visualization of free radical reactions in an aqueous sample irradiated by 290 MeV carbon beam

The detection of free radical reactions in a gelatin sample irradiated by heavy-ion beam was tested using electron paramagnetic resonance (EPR) spectroscopic and MRI methods. Geometry and the amount of free radical eneration in a sample are described. A reaction mixture containing glutathione and a nitroxyl radical, TEMPOL, was caked with gelatin, and then irradiated with a 290 MeV carbon beam. The amount of free radical generation in a solic sample was almost flat from the surface to the beam end, except for a small peak, the peak radioactivation profile, and then steeply decrased approaching the beam end. Total free radical reactions obtained with carbon-beam irradication were expected to be less than one-third of X-ray irradiation, when the same dose for a deeper target was considered. Both EPR and MRI are useful tools to visualize free radical generation in samples irradiated by a heavy-ion beam. The EPR-based method is more sensitive and quantitative than the MRI-based method; however, the MRI method can achieve high spatial resolution. This study gives the rationale for a redox regulation trial using antioxidant drugs to reduce the side effects on normal tissues in carbon-beam therapy

Delayed treatment with alpha-phenyl-N-tert-butyl nitrone (PBN) attenuates secondary mitochondrial dysfunction after transient focal cerebral ischemia in the rat

The present experiments were undertaken to explore the mechanisms of secondary brain damage in focal ischemia of long duration (2 h), followed by recirculation. Recirculation has previously been found to cause partial recovery and secondary deterioration of cellular bioenergetic state, the subsequent damage being ameliorated by a free radical spin trap, alpha-phenyl-N-tert-butyl nitrone (PBN), even when the drug was given 1 (or 3) h after the start of recirculation. Our objective was to assess whether the secondary deterioration of the cellular bioenergetic state is due to mitochondrial dysfunction and to study whether PBN acts by preventing secondary damage to mitochondria. Focal and perifocal ("penumbral") tissues were sampled after 2 h of ischemia and after 1, 2, and 4 h of recirculation; at the latter two times, vehicle- and PBN-injected animals were studied, PBN being given after 1 h of recirculation. Homogenates were prepared, and stimulated (+ADP), nonstimulated (-ADP), and uncoupled respiratory rates were measured polarographically. The results were similar in focus and penumbra, albeit more pronounced in the focus. Ischemia was associated with a decrease in ADP-stimulated and uncoupled respiration rates, with a marked fall in the respiratory control ratio, defined as ADP-stimulated divided by nonstimulated respiration. Recirculation (1 h) brought about partial recovery, but continued reflow (2 and 4 h) was associated with a secondary deterioration of respiratory functions. This deterioration was prevented by PBN, given 1 h after the start of recirculation. The results raise the question whether the secondary deterioration of the cellular bioenergetic state in focal ischemia-reperfusion is due to secondary mitochondrial dysfunction and whether the amelioration of the subsequent damage by PBN is partly or wholly due to the effect of the spin trap on the mitochondria