The role of nitric oxide in cardiac depression induced by interleukin-1 beta and tumour necrosis factor-alpha.

1. Myocardial dysfunction during septic shock is associated with enhanced production of cytokines such as interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha). These cytokines depress cardiac mechanical function by a mechanism which is not well defined. 2. Bacterial endotoxin or cytokines cause the expression of Ca(2+)-independent nitric oxide (NO) synthase in cardiac myocytes, vascular endothelial cells and endocardial endothelial cells, causing enhanced production of NO. As NO has negative inotropic actions on cardiac muscle, we tested the sum effects of IL-1 beta plus TNF-alpha in the intact heart to determine whether enhanced expression of NO synthase activity in the cells that comprise the heart is involved in cardiac depression associated with cytokine stimulation. 3. Rat isolated working hearts perfused with IL-1 beta plus TNF-alpha showed a markedly greater depression in contractile function, measured as cardiac work, after 2 h of perfusion compared with time-matched control hearts. The depressant action of IL-1 beta plus TNF-alpha was first apparent after 1 h of perfusion; no early (15 min) cardiac depressant actions were seen. 4. The competitive inhibitor of Ca(2+)-dependent and Ca(2+)-independent NO synthases, NG-nitro-L-arginine methyl ester (L-NAME, 3 microM) when given concurrently with IL-1 beta plus TNF-alpha prevented the loss in contractile function such that these hearts after 2 h of perfusion had similar function to time-matched controls. L-NAME did not acutely reverse the loss of contractile function in hearts exposed for 2 h to IL-1 beta plus TNF-alpha. The protective action of L-NAME in the presence of cytokines was concentration-dependent and was not seen at a higher concentration (10 micro M) due to the significant reduction in coronary flow observed at this concentration.5. In contrast, when L-NAME (3 micro M) was given in the absence of IL-l beta plus TNF-alpha it depressed contractile function over the 2 h perfusion period by significantly reducing coronary flow.6. Inhibition of protein synthesis with cycloheximide (Cx) abolished the loss in function that occurred over 2h in both control and IL-1 beta plus TNF-a-treated hearts.7. Inducible, Ca2+-independent NO synthase activity was not observed in freshly isolated hearts but was observed in control hearts perfused for 2 h in vitro and was doubled in hearts perfused with IL-1 beta plus TNF-a. Cx prevented the expression of Ca2+-independent NO synthase in both control and cytokine-treated hearts.8. In summary, these results suggest that the depression of myocardial function by IL-l beta plus TNF-alpha is mediated, at least in part, by induction of Ca2+-independent NO synthase activity in the heart.

Analysis of myocardial plasmalogen and diacyl phospholipids and their arachidonic acid content using high-performance liquid chromatography.

A high-performance liquid chromatographic method for the assay of diacyl and plasmalogen (alk-1-enyl) phospholipid content and the determination of their fatty acid content from tissue homogenates is described. Myocardial phospholipids are rich in plasmalogens and have a high content of unsaturated fatty acids, including arachidonic acid, esterified in the sn-2 position. Using a three-stage HPLC assay we have analyzed the phospholipid subclass content and the amount of arachidonic acid esterified to these fractions extracted from isolated perfused rat hearts. After HPLC separation of total myocardial phospholipids, the phosphatidylcholine and phosphatidylethanolamine peak fractions are treated with phospholipase C to remove polar head groups and ultraviolet-absorbing benzoate derivatives are made. Separation and quantification of diacyl and plasmalogen content of the total phospholipids with nanomolar sensitivity is then achieved using isocratic elution with a silicic acid HPLC column. The separated plasmalogen and diacyl glycerobenzoates are then subjected to alkaline hydrolysis to remove fatty acids from the sn-2 position. The 2-(2,3-napthalimino)ethyltrifluoromethanesulfonate esters of the free fatty acids are then prepared and analyzed with subnanomolar sensitivity using reverse-phase chromatography with gradient elution. As plasmalogen-specific phospholipase A2 is activated during myocardial ischemia and comprises the majority of total phospholipase A2 activity in the heart, this methodology allows for a sensitive and complete determination of the changes in the mass of these phospholipids and their arachidonic acid content.

Lysoplasmenylethanolamine accumulation in ischemic/reperfused isolated fatty acid-perfused hearts.

Lysophospholipid accumulation has been implicated in the pathogenesis of irreversible injury during myocardial ischemia and reperfusion. Plasmalogens (phospholipids with a vinyl-ether bond in the sn-1 position) account for more than 50% of total myocardial sarcolemmal and sarcoplasmic reticulum phospholipids. Accumulation of plasmalogen choline and ethanolamine lysophospholipids (lysoplasmenylcholine and lysoplasmenylethanolamine) or the effects of exogenous fatty acids on lysoplasmalogen accumulation during ischemia and reperfusion have not been examined. Isolated working rat hearts perfused with buffer containing either 11 mM glucose or 11 mM glucose plus 1.2 mM palmitate were subjected to aerobic, ischemic, or ischemia/reperfusion protocols. Levels of lysoplasmenylcholine and lysoplasmenylethanolamine were quantified using a two-stage high-performance liquid chromatographic technique. In hearts perfused with glucose alone, no significant differences in levels of lysoplasmenylcholine or lysoplasmenylethanolamine were seen during ischemia or reperfusion. In fatty acid-perfused hearts, however, significant accumulation of lysoplasmenylethanolamine occurred during reperfusion but not during ischemia (723 +/- 112, 734 +/- 83, and 1,394 +/- 193 nmol/g dry wt for aerobic, ischemic, and ischemic/reperfused hearts, respectively; p less than 0.05 for ischemic/reperfused hearts versus aerobic or ischemic hearts). Lysoplasmenylcholine levels after ischemia and reperfusion did not differ significantly from aerobic values, regardless of whether fatty acids were present or absent from the perfusate. Aerobic and ischemic/reperfused rabbit hearts, in the presence of fatty acid, showed a similar profile in their lysoplasmalogen content. We conclude that differential lysoplasmenylethanolamine accumulation occurs during myocardial reperfusion when exogenous fatty acid concentrations are high. This may reflect the selective action of fatty acid intermediates on the metabolism of lysoplasmenylethanolamines.(ABSTRACT TRUNCATED AT 250 WORDS)