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

Objective: The role of arachidonate lipoxygenase activity in reoxygena tion induced cell injury in adult canine cardiac myocytes was investig ated. Methods: The production of hydroxyeicosatetraenoic acids (HETEs) , which are lipoxygenase metabolites, was measured with high pressure liquid chromatography in canine cardiac myocytes cultured under hypoxi c conditions and then reoxygenated. Free radical generation was evalua ted by electron paramagnetic resonance spectroscopy with a spin trappe r, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and luminol enhanced chemil uminescence emission. Cell injury was estimated in terms of morphologi cal changes and release of intracellular enzymes. Morphological damage to myocytes was quantified in terms of the percentage of hypercontrac ted ''round'' cells. The effects of nordihydroguaiaretic acid, AA-861, mepacrine, indomethacin, aspirin, alpha tocopherol, and 2-0-octadecyl ascorbic acid (CV-3611) on lipoxygenase metabolism, free radical gener ation and cell injury were also assessed. Results: Cardiac myocytes pr oduced 5-HETE and 12-HETE at less than 0. 1 ng.mg -1 protein under nor moxic conditions. Production of HETE was greatly increased at five hou rs 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 an d chemiluminescence emission were considerably increased after one to three hours of reoxygenation, although they increased only slightly af ter 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-86 1 (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. b ut not HETE production. Conclusion: The arachidonate lipoxygenase meta bolic pathway may have an important role in reoxygenation induced myoc ardial cell injury in adult cardiac myocytes, possibly because of the generation of free radicals.