Coordinate induction of tetrahydrobiopterin synthesis and nitric oxide synthase activity in chicken macrophages: upregulation of GTP-cyclohydrolase I activity.
Biosynthesis of nitric oxide (NO) and tetrahydrobiopterin (BH4) was investigated during cytokine-mediated activation of chicken macrophages. Monocyte derived macrophages and HD11 cells, a chicken macrophage cell line, constitutively synthesize BH4. Treatment of these cells with chicken macrophage activation factor (ChMAF) causes up to 10-fold increases of intracellular BH4 and of nitrite concentrations in the cell culture supernatant. Elevated BH4 levels correlate with an increase in GTP-cyclohydrolase I (GTP-CH) activity. Kinetic studies show a joint upregulation of GTP-CH activity and NO synthase activity first detectable 4 hr after stimulation. A corresponding increase in the mRNA for GTP-CH was detected by Northern blot analysis with a chicken GTP-CH specific cDNA probe. These results demonstrate that cytokine-induced BH4 synthesis by chicken macrophages is at least partially regulated through increased GTP-CH gene expression. The functional relevance of BH4 formation for NO production is shown by experiments using 2,4-diamino-6-hydroxypyrimidine (DAHP) as a specific inhibitor of GTP-CH. Monocyte derived macrophages stimulated in the presence of DAHP show a significant decrease in NO synthesis. The effect of DAHP was reversed by adding sepiapterin, which allows synthesis of BH4 through a salvage pathway.
Kaspers
B
B
Institut für Physiologie, Physiologische Chemie und Tierernährung, Universität München. caspers@tiph.vetmed.uni-muenchen.de
Gütlich
M
M
Witter
K
K
Lösch
U
U
Goldberg
M
M
Ziegler
I
I
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000096:biosynthesis; D015152:Blotting, Northern; D002645:Chickens; D006136:GTP Cyclohydrolase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology*; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism
D012333:RNA, Messenger; D001708:Biopterins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D006136:GTP Cyclohydrolase; C003402:sapropterin