NF-kappa B and GTP cyclohydrolase regulate cytokine-induced nitric oxide production by cardiac myocytes

We previously reported that interleukin-1 beta (IL-1) alone stimulated nitric oxide (NO) production by neonatal rat cardiac myocytes (CM) in culture. The present studies were undertaken to explore the signal transduction pathways involved in IL-1-induced NO production by CM. Translocation from the cytosol to the nucleus of nuclear factor-kappa B (NF-kappa B) and activation of guanosine 5'-triphosphate (GTP) cyclohydrolase [rate-limiting enzyme in tetrahydrobiopterin (BH4) synthesis] have been implicated in IL-1 signaling. Accordingly, the effects of the NF-kappa B inhibitor pyrolidine dithiocarbamate (PDTC) and the GTP cyclohydrolase inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP) on IL-1-induced NO production by CM were studied. PDTC and DAHP inhibited IL-1-induced NO production by CM (6.7 +/- 0.6 vs. 0.9 +/- 0.3 and 0.3 +/- 0.1 nmol . 1.25 x 10(5) cells(-1). 48 h(-1), respectively, P < 0.01, n = 12 for each). Immunohistochemical staining revealed that PDTC blocked IL-1-stimulated nuclear translocation of NF-kappa B. The membrane-permeable analogue of the NO synthase cofactor BH4, methyl-BH4 (mBH(4)), only partially reversed DAHP inhibition of NO2- formation (6.7 +/- 0.6 vs. 2.4 +/- 0.3 nmol . 1.25 X 10(5) cells(-1). 48 h(-1), P < 0.01, n = 12). Semiquantitative reverse transcription polymerase chain reaction revealed no inducible NO synthase (iNOS) mRNA production in cells treated with IL-1 + PDTC. CM treated with IL-1 + DAHP did express iNOS mRNA. We report for the first time that nuclear translocation of NF-kappa B is essential for Il-1-induced iNOS mRNA expression and GTP cyclohydrolase activity is required in addition to BH4 for optimal NO production by CM.