]i transients and contraction with electrically stimulated adult rat cardiac myocytes. At a low concentration (10 ng/ml) TNF-␣ produced a 40% increase in the amplitude of both [Ca 2ϩ ]i transients and contraction within 40 min. At a high concentration (50 ng/ml) TNF-␣ 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 [Ca 2ϩ ]i transients and contraction after 30 min. Both the positive and negative effects of TNF-␣ were reproduced by AA and blocked by arachidonyltrifluoromethyl ketone (AACOCF3), an inhibitor of cytosolic PLA2. Lipoxygenase and cyclooxygenase inhibitors reproduced the highdose effects of TNF-␣ and AA. The negative effects of TNF-␣ 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-␣.heart; cytosolic Ca 2ϩ -dependent phospholipase A2; ceramidase; sphingosine

Background-The negative effect of tumor necrosis factor-␣ (TNF-␣) on heart contraction, which is mediated by sphingosine, is a major component in heart failure. Because the cellular level of glutathione may limit sphingosine production via the inhibition of the Mg-dependent neutral sphingomyelinase (N-SMase), we hypothesized that cardiac glutathione status might determine the negative contractile response to TNF-␣. Methods and Results-We examined the effects of TNF-␣ in isolated cardiomyocytes obtained from control rats or rats that were given the glutathione precursor N-acetylcysteine (NAC, 100 mg IP per animal). In cardiomyocytes obtained from control rats, 25 ng/mL TNF-␣ increased reactive oxygen species generation and N-SMase activity (500% and 34% over basal, respectively) and decreased the amplitude of [Ca 2ϩ ] i in response to electrical stimulation (22% below basal). NAC treatment increased cardiac glutathione content by 42%. In cardiomyocytes obtained from NAC-treated rats, 25 ng/mL TNF-␣ had no effect on reactive oxygen species production or N-SMase activity but increased the amplitude of [Ca 2ϩ ] i transients and contraction in response to electrical stimulation by 40% to 50% over basal after 20 minutes. This was associated with a hastened relaxation (20% reduction in t 1/2 compared with basal) and an increased phosphorylation of both Ser 16 -and Thr 17 -phospholamban residues (260% and 115% of maximal isoproterenol effect, respectively). Conclusions-It is concluded that cardiac glutathione status, by controlling N-SMase activation, determines the severity of the adverse effects of TNF-␣ on heart contraction. Glutathione supplementation may therefore provide therapeutic benefits for vulnerable hearts.

These findings support the conclusion that not only the change in total lipid content (DAG and TAG), but also FA composition is affected by rosiglitazone in an animal model of high-fat-diet-induced insulin resistance.