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 NO2-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 (mBH4), 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 II-1-induced iNOS mRNA expression and GTP cyclohydrolase activity is required in addition in addition to BH4 for optimal NO production by CM.
Oddis
C V
CV
Department of Pathology, University of Pittsburgh, Pennsylvania 15213, USA.
Finkel
M S
MS
D000818:Animals; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004790:Enzyme Induction; D006136:GTP Cyclohydrolase / Q000502:physiology*; D007375:Interleukin-1 / Q000494:pharmacology*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D016328:NF-kappa B / Q000502:physiology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000235:genetics; D011622:Pterins / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats
D007375:Interleukin-1; D016328:NF-kappa B; D011622:Pterins; D012333:RNA, Messenger; D009569:Nitric Oxide; C017523:6-methyltetrahydropterin; D019001:Nitric Oxide Synthase; D006136:GTP Cyclohydrolase