We will continue to study the effects of age on vascular arachidonic acid metabolism. Oxidative damage to the endothelium is consistent with the free radical theory of aging and may account for the reduced capacity to form prostacyclin associated with aging. We propose that radicals produced from superoxide, H2O2, or lipid hydroperoxides have differential effects on cyclooxygenase, prostacyclin synthetase and lipoxygenase such that altered ratios of PGI2/PGE2 and 12-HPETE/PGs may be expressed: i.e. low dose peroxide may activate lipoxygenase and inhibit cyclooxygenase or prostacyclin synthetase. Higher doses may inhibit both lipoxygenase and cyclooxygenase while even higher doses lead to morphologic changes and cell lysis. In previous studies in rabbits we have demonstrated that arachidonic acid metabolism by vascular cyclooxygenase produces radicals which autoinactivate both this protein and prostacyclin synthetase. The rate of inactivation is more rapid in aortas from older animals suggesting a reduced capacity to detoxify the radical produced. Similarly rat aortae were found to be 1) almost completely resistant to autoinaction during perfusion with arachidonate and 2) approximately 2-2.5 times more resistant to inhibition caused by H2O2. In addition we observed a striking differential in dose of H2O2 which results in inhibition of PGI2 formation (ID50 = 35 uM) and that which causes endothelial cell lysis (0.5 - 1 mM). Further in contrast to exogenously applied 15-HPETE which preferentially inhibits prostacyclin synthetase, H2O2 specifically inhibits cyclooxygenase. Interestingly, the time course of H2O2-mediated inhibition in less than 1 min with no further damage to cyclooxygenase observed with longer exposure at low dose. Thus, we plan to study the effects of radical producers on arachidonate metabolism in endothelial and smooth muscle cells in tissue culture, and in perfused vascular beds. A number of peroxides will be tested to evaluate their possible differential effects on the activities of lipoxygenase, cyclooxygenase and prostacyclin synthetase. We will continue studying differences in sensitivity between young and old rabbits to both autoinactivation and exogenous radical inhibition. We will also continue to explore differences between rats and rabbits in terms of the disparate capacities to detoxify radicals. We will begin to study neutrophil-endothelial cell interactions as a model of damage occurring during aging and atherosclerosis.
