92

10.5

10.7

Mechanisms of Ischemia/Reperfusion Injury

MYOCARDI~_ F~d-~-USION: FlqDIOOL T~c~PING COR- RELATES TO DYSFUNCTION ~ ENE]~GY DEPRESSION I.E. Blasig, A. Tosaki ~, A.Y. Steinschneider =, E.K. Ruuge =, B. Ebert, Acad. Sci., 1136 Berlin, GDR; ~Inst. Henry Beaufour, 92350 Le Plessis Robinson, France; =Acad. Med. Sci., 121552 Moscow, USSR.

The myocardial reperfusion injury is accom- panied by the generation of free radicals, but, their pathogenetic relevance is not clear. Isolated rat hearts underwent total and global ischemia. During the following reperfusion DMPO was added to the effluent in which the formation of oxy-radical adducts (DMPO-OH) was measured by ESR spectrometry. The adducts were observed within seconds after the onset of re- flow, maximum after 3 min. The experiments provides the f i r s t evidence that the concentra- tion of the radicals traced within this inter- val inversely correlates to the degree of the functional recovery (developed pressure, con- t r ac t i l i t y , heart rate, coronary and aortic flow), p<O.05, n=lO. In a second study an in- verse correlation was also found concerning the myocardial content of high-energy phosphates, p<O.O01, n=l l . (No relationship to inorganic phosphate, end-diastolic pressure, ar- rhythmias.) The effluent spin trapping is a new approach for the investigation of the patho- genetic role of free radicals during reperfu- sion. It is concluded that the radicals de- tected in the perfusate, during the initial phase of reperfusion, reflect the disturbances of the affected heart and may contribute to the contractile and energetic failure induced.

MITOCHONDRIAL GLUTATHIONE IN ISCHEMIA-REPERFUSION INJURY OF ISOLATED AND PERFUSED RAT HEART. A. Bindoli and M.P. Rigobello Centro Studio Fisiologia Mitocondriale (CNR) and Dipartimento di Chimica Biologica, Universit~ di Padova, Padova ( I ta ly) .

Glutathione is an essential component of mitochon- drial metabolism and i ts major function is to pro- tect against oxidative stress. The changes of mi- tochondrial glutathione of isolated and perfused rat heart in various experimental conditions are evaluated. Glutathione concentration of heart ho- mogenate was around 9 nanomol/mg protein, while that of the mitochondria was 1.2 nanomol/mg pro- tein corresponding to about 3% of total glutathio- ne content. When the hearts were perfused with 0.1 mM H202 or diamide the total heart glutathio- ne decreased respectively of 68% and 88%, while, on the contrary, the mitochondrial glutathione was almost completely preserved. After ischemia, followed by a 5 min reperfusion, both total and mitochondrial glutathione decreased of about 40% indicating that the mitochondrial pool, at varian- ce with what occurs with the oxidizing agents, is not specifically preserved; i t was also observed that mitochondrial glutathione leaks out in the cytosol during the ischemic period and is not fur- ther affected by the reperfusion. The reported da- ta indicate that mitochondrial glutathione pool can be relat ively elevated even during periods of cytoplasmic glutathione depletion due to oxidi- zing species; on the contrary this pool is not specifically preserved during ischemia and/or reperfusion.

H Y P O X I A - R E O X Y G E N A T I O N T O X I C I T Y UPON HUMAN ENDOTHELIAL CELLS IN CULTURE Carine MICHIELS, Thierry ARNOULD, Jos~ REMACLE Laboratoire de Biochimie Cellulaire, Facult~s Universitaires ND de la Paix, 61 rue de Bruxelles, 5000 NAMUR, Belgium

Ischemia-reperfusion is observed in various diseases such as myocardium infarct. Different theories have been proposed to explain the reperfusion injury amongst which the free radical generation plays a crucial role. A hypoxia (H)- reoxygenation (R) model upon human endothelial cells in culture was developed in order to mimic the in vlvo situation. Different parameters were compared under H or H/R and we found that oxygen readmission led to damage amplification after a short hypoxia period. In order to estimate the importance of various causes of toxicity, the effects of various protective molecules were compared. Different antioxidant molecules, iron-chelating agent, xanthine oxldase inhibitors and energy-supplying molecules were very efficient protectors. Synergy could also be observed between the antioxidants and the energy-supplying molecules or the xanthine oxidase inhibitors. The toxic effect of 02"(-) could be lowered by the presence of SOD in the culture medium while glutathione peroxidase was the most efficient enzyme when injected into the cells. The production of O2"(-) and of H202 by endothelial cells was directly evidenced during the R period. In addition, a xanthine oxidase activity could be observed by a direct assay in cells after H but not in control cells thus confirming the previous conclusions of xanthine oxidase as a potent source of free radicals in these conditions. These results stress once more the importance of free radical production during the reoxygenation step. They also show that hydrroperoxides are probably the most harmful species for the cells and that the energetic state of these cells is a critical factor affecting the ability of the cells to defend themselves against these toxic molecules. C.M. is Research Associate of FNRS (Fonds National de la Recherche Scientifique, Belgium).

OXIDATIVE STRESS IN THE BRAIN: A NEW MODEL James D. Adams, Lori K. Klaidman, Carl P. LeBel and Ifeoma N. Odunze School of Pharmacy, University of Southern California, Los Angeles, CA 90033, U.S.A.

t-Butylhydroperoxide given intracerebroventricularly produces a massive oxidative stress in the brain. Glutathione disulfide levels increase intracellularly by as much as 90 fold, which may allow a very sensitive means of identification of the occurence of oxidative stress. Glutathione levels decrease at the same time due to the action of glutathione peroxidase. The activity of this enzyme is low in the brain with the lowest activity occuring in the midbrain. The low rate of release of glutathione disulfide from the brain and the low activity of glutathione disulfide reductase in the brain may, in part, explain the high intracellular levels of glutathione disulfide during oxidative stress. Aging alters the ability of the brain to detoxify an oxidative stress, in that 8 month old mice retain glutathione disulfide in their brains much more than 2 month old mice. The midbrain may be especially susceptible to oxidative stress due to lower levels of glutathione peroxidase and glutathione disulfide reductase.

10.6

10.8