CHARACTERIZATION OF INDUCIBLE NITRIC-OXIDE SYNTHASE EXPRESSION IN ENDOTOXEMIC RAT CARDIAC MYOCYTES IN-VIVO AND FOLLOWING CYTOKINE EXPOSURE IN-VITRO

Lipopolysaccharide (LPS) treatment results in widespread expression of the inducible isoform of nitric oxide (NO) synthase (iNOS). Although there is evidence for the expression of iNOS in heart tissue, regulati on of myocardial iNOS expression is not known. To determine the time c ourse and degree of iNOS induction in the adult heart, we examined iNO S mRNA expression and enzyme activity in (1) rat left ventricular tiss ue after LPS treatment in vivo, and (2) cultured, long-term rat cardia c myocytes maintained in serum and exposed to interleukin-1 beta, tumo r necrosis factor-alpha, interferon-gamma, and/or LPS. iNOS mRNA was d etected by Northern blot analysis and in situ hybridization. iNOS enzy me activity was measured in extracts of whole heart, and nitrate and n itrite (the stable endproducts of NO) accumulation was quantified in c ardiomyocyte culture media, iNOS mRNA was not detected in untreated he arts or cultured myocytes but was apparent within 3 h in both hearts o btained from LPS-treated animals and in cytokine-treated myocytes. In whole heart, iNOS mRNA expression peaked by 6 h after LPS and declined by 12 and 24 h, In situ hybridization demonstrated perinuclear locali zation of iNOS mRNA in both cardiac vascular smooth muscle and myocyte s with maximal expression at 6 h after LPS injection. In cardiac myocy tes, iNOS expression was maximal at. 12 to 24 h, persisted through 48 h, and was partially inhibited by dexamethasone, Interferon-gamma was the most potent single cytokine with regards to myocyte iNOS induction . Nitric oxide release in cytokine-stimulated cardiac myocytes was lar gely in the form of nitrate and was associated with increased glucose uptake and lactate release; the former finding indicates that NO inter acts with myocardial heme proteins and/or oxyradicals, while the latte r suggests inhibition of oxidative metabolism. Although non-myocardial cells may significantly contribute to iNOS expression in whole heart tissue, significant iNOS expression and NO production also take place within the myocyte, Induced NO production may regulate myocardial perf usion and impair myocardial function and metabolism. (C) 1995 Academic Press Limited