Effects of linoleic and oleic acid anilides on prostacyclin synthesis and fibrinolytic profile of human endothelial cells in culture: relevance to the toxic oil syndrome.

We evaluated the effects of fatty-acid anilides (FAA) on prostacyclin (PGI2) synthesis and on the fibrinolytic properties of human umbilical vein endothelial cells. Preincubation of endothelial cells with oleic- and linoleic-anilides (OAA and LAA, respectively) resulted in a time- and concentration-dependent inhibition of ionophore A23187- and thrombin-induced PGI2 synthesis. However, no significant effects of FAA on arachidonic acid-induced PGI2 synthesis were found, except with 1000 microM LAA which inhibited cyclooxygenase activity after 24 h. In general terms, OAA showed similar inhibitory effects on PGI2 production as did LAA, but with a shifted time course, since the production of PGI2 at 24 h for OAA was similar to that observed for LAA at 2 h. The release of labeled arachidonic acid from cell membranes was significantly reduced (75-85%), after 24 h, with both FAA. The effect of 100 microM LAA on thrombin-induced PGI2 production was rapid (within 15 min) and irreversible after 60 min. The recovery of PGI2 synthesis after LAA treatment was blocked by cycloheximide, suggesting a decrease of phospholipase(s) activity or cessation of enzyme synthesis. Moreover, this reduced PGI2 synthesis was not associated with [3H]adenine release. Our data indicate that FAA induce a significant impairment of stimulated PGI2 synthesis and arachidonic acid release in endothelial cells, acting primarily as inhibitors of phospholipase(s) rather than of cyclooxygenase. Finally, both LAA and OAA induce an anti-fibrinolytic activity in these cells where major changes are observed in the plasminogen activator inhibitor and the urine-type plasminogen activator.

Influence of fatty acid anilides present in toxic oils on the metabolism of exogenous arachidonic acid in cultured human endothelial cells.

The effect of fatty acid anilides (FAA) on the exogenous arachidonic acid (AA) metabolism and toxicity of isolated human endothelial cells was studied to clarify their possible role in the etiology of toxic oil syndrome. Confluent cells were incubated with and without linoleic acid anilide (LAA), oleic acid anilide (OAA) and two unrelated samples for 2-24 h prior to the addition of [l-14C]AA alone or with calcium ionophore A-23187. The eicosanoids produced were analyzed by RP-HPLC. A dual stimulatory and inhibitory effect on the conversion of exogenous AA as a function of preincubation time with anilides (100 and 1000 microM) was observed. Treated cells significantly increased (1-3-fold) the production of the main cyclooxygenase-derived prostanoids (6-keto-PGF1 alpha and PGF2 alpha) formed by these cells, with a maximum stimulatory effect after 2-3 h, only when AA was used alone. However, afterwards a time- and dose-dependent decrease in prostanoid formation was observed with LAA (P < 0.05 at 24 h), either in the absence or presence of ionophore A-23187 in the incubation mixture. This inhibitory effect on cyclooxygenase was not observed with OAA, which still stimulate after 24 h of treatment. The changes in prostanoid synthesis were not followed with a parallel release in the lactate dehydrogenase activity in the medium (except with unrelated samples). Moreover, anilide treatment increased the appearance of cytosolic lipid droplets or vacuoles after 2 and 5 h of contact with LAA and OAA, respectively. From these results, it was suggested that anilides impair prostanoid synthesis in endothelial cells; their stimulatory effect could be explained by an unspecific effect on cell membrane, not related to cell toxicity and the inhibitory effect by an inhibition of the cyclooxygenase activity. These observations further contribute to our understanding of the possible role of anilides in the etiology of the toxic oil syndrome.