Possible involvement of tyrosine kinase activation in lipopolysaccharide-induced expression of Ca(2+)-insensitive but calmodulin-coupling nitric oxide synthase in rat glial cells.
To clarify the properties of an inducible type of nitric oxide synthase (i-NOS) in the brain, we examined whether lipopolysaccharide (LPS) induces NOS in glial cells cultured from neonatal rats. NOS activities (NO2- accumulation and L-[14C]citrulline formation) were detected by treatment with LPS at 10 micrograms/ml for 6-72 hr. L-[14C]citrulline formation by LPS-induced i-NOS was inhibited by NG-monomethyl-L-arginine (a NOS inhibitor) and diphenyleneiodonium (a flavo-protein inhibitor). The activity was not markedly changed in the presence or absence of Ca2+. The induction of i-NOS by LPS was abolished by cycloheximide, actinomycin D, or dexamethasone. In addition, the induction was inhibited by herbimycin A (a tyrosine kinase inhibitor), but was not by staurosporine, W-7, or FK-506. After LPS stimulation, 130 kDa proteins were reacted with anti-rat liver i-NOS antibody 5-72 hr. i-NOS induced from glial cells coupled tightly with endogenous calmodulin (CaM) even in the absence of Ca2+. These results suggest that LPS induces expression of 130-kDa i-NOS through an activation of tyrosine kinase, after which i-NOS couples with CaM, and that NO is formed for 6-72 hr in glial cells.
Kitamura
Y
Y
Department of Pharmacology, Hokkaido University, Sapporo, Japan.
Imaizumi
R
R
Kitayama
Y
Y
Esumi
H
H
Nomura
Y
Y
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D002478:Cells, Cultured; D002956:Citrulline / Q000378:metabolism; D004731:Endotoxins / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D007150:Immunohistochemistry; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D009457:Neuroglia / Q000187:drug effects / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019335:Nitroarginine / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D002147:Calmodulin; D004731:Endotoxins; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D019335:Nitroarginine; D002956:Citrulline; D009569:Nitric Oxide; C044101:endotoxin, Escherichia coli; D019001:Nitric Oxide Synthase; D011505:Protein-Tyrosine Kinases; D002118:Calcium