FREE-RADICAL GENERATION COUPLED WITH ARACHIDONATE LIPOXYGENASE REACTION RELATES TO REOXYGENATION INDUCED MYOCARDIAL-CELL INJURY

Objective: The role of arachidonate lipoxygenase activity in reoxygenation induced cell injury in adult canine cardiac myocytes was investigated. Methods: The production of hydroxyeicosatetraenoic acids (HETEs), which are lipoxygenase metabolites, was measured with high pressure liquid chromatography in canine cardiac myocytes cultured under hypoxic conditions and then reoxygenated. Free radical generation was evaluated by electron paramagnetic resonance spectroscopy with a spin trapper, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and luminol enhanced chemiluminescence emission. Cell injury was estimated in terms of morphological changes and release of intracellular enzymes. Morphological damage to myocytes was quantified in terms of the percentage of hypercontracted ''round'' cells. The effects of nordihydroguaiaretic acid, AA-861, mepacrine, indomethacin, aspirin, alpha tocopherol, and 2-0-octadecylascorbic acid (CV-3611) on lipoxygenase metabolism, free radical generation and cell injury were also assessed. Results: Cardiac myocytes produced 5-HETE and 12-HETE at less than 0. 1 ng.mg -1 protein under normoxic conditions. Production of HETE was greatly increased at five hours of reoxygenation after 45 minutes of hypoxia [5-HETE=12.0(SEM 0.5), 12-HETE=23.6(1.1) ng.mg-1 protein]. Both DMPO-OH adduct generation and chemiluminescence emission were considerably increased after one to three hours of reoxygenation, although they increased only slightly after 45 minutes of hypoxia. After five hours of reoxygenation, long rod cells gradually became deformed; 92.0% of the cells were converted to hypercontracted ''round'' cells. Cell injury and HETE production were significantly suppressed by nordihydroguaiaretic acid (10 muM), AA-861 (2 muM), and mepacrine (10 muM). Indomethacin (10 muM) and aspirin (50 muM) enhanced cell injury and HETE production. alpha Tocopherol and CV-3611 greatly suppressed cell injury and free radical generation. but not HETE production. Conclusion: The arachidonate lipoxygenase metabolic pathway may have an important role in reoxygenation induced myocardial cell injury in adult cardiac myocytes, possibly because of the generation of free radicals.