Tumor necrosis factor (TNF)-alpha has a biphasic effect on heart contractility and stimulates phospholipase A2 (PLA2) in cardiomyocytes. Because arachidonic acid (AA) exerts a dual effect on intracellular Ca2+ concentration ([Ca2+]i) transients, we investigated the possible role of AA as a mediator of TNF-alpha on [Ca2+]i transients and contraction with electrically stimulated adult rat cardiac myocytes. At a low concentration (10 ng/ml) TNF-alpha produced a 40% increase in the amplitude of both [Ca2+]i transients and contraction within 40 min. At a high concentration (50 ng/ml) TNF-alpha evoked a biphasic effect comprising an initial positive effect peaking at 5 min, followed by a sustained negative effect leading to 50-40% decreases in [Ca2+]i transients and contraction after 30 min. Both the positive and negative effects of TNF-alpha were reproduced by AA and blocked by arachidonyltrifluoromethyl ketone (AACOCF3), an inhibitor of cytosolic PLA2. Lipoxygenase and cyclooxygenase inhibitors reproduced the high-dose effects of TNF-alpha and AA. The negative effects of TNF-alpha and AA were also reproduced by sphingosine and were abrogated by the ceramidase inhibitor n-oleoylethanolamine. These results point out the key role of the cytosolic PLA2/AA pathway in mediating the contractile effects of TNF-alpha.
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