Cytokines and lipopolysaccharides induce inducible nitric oxide synthase but not enzyme activity in adult rat cardiomyocytes

There is evidence that nitric oxide (NO) formation in adult cardiomyocytes stimulated with lipopolysaccharide (LPS) is not commensurate with iNOS levels. Tetrahydrobiopterin (BH4) is a key factor in the stabilization and NO production by iNOS homodimer. Thus we hypothesized that BH4 is a limiting factor for NO production in adult cardiomyocytes in response to LPS and cytokines (TNF-α, IL-1, IFN-γ alone, or mixed). It was verified that LPS and cytokines induced iNOS expression which did not translate into increased nitrite or [14C]citrulline production. This response coincided with defective BH4 synthesis and low GTP cyclohydrolase activity. Furthermore, supplementation with BH4 and ascorbate failed to increase iNOS activity. This effect was related to preferential accumulation of BH2 rather than BH4 in these cells. Uncoupled iNOS activity in stimulated cells was examined using mitochondrial aconitase activity as an endogenous marker of superoxide anion radical (O2−) formation, and found not to be significantly inhibited. 2-Hydroxyethidium also was not significantly increased. We conclude that adult cardiomyocytes are an unlikely source of NO and O2− in inflammatory conditions. This finding adds a new and unexpected layer of complexity to our understanding of the responses of the adult heart to inflammation.