Lack of role for nitric oxide (NO) in the selective destabilization of endothelial NO synthase mRNA by tumor necrosis factor-alpha.
The constitutive expression of endothelial nitric oxide (NO) synthase (cNOS) is essential for the physiological regulation of vascular tone and structure. The mechanism of downregulation of steady state cNOS mRNA in human umbilical vein endothelial cells exposed to tumor necrosis factor-alpha (TNF-alpha) was investigated by using Northern blot analysis of total cellular RNA. TNF-alpha produced a dose- and time-dependent decrease in cNOS mRNA expression that was near maximal at 10 U/mL and 6 hours of exposure, respectively. In contrast, steady state expression of endothelin-1 and plasminogen activator inhibitor-1 (PAI-1) mRNA was upregulated by TNF-alpha. The pharmacological generation of NO using sodium nitroprusside (10 mumol/L) and S-nitroso-acetylpenicillamine (100 to 400 mumol/L) had no effect on cNOS mRNA levels, and TNF-alpha-induced downregulation of cNOS was not prevented by coincubation with the inhibitors of NO synthesis N omega-nitro-L-arginine methyl ester (1 mmol/L) and NG-monomethyl L-arginine (10 mmol/L). Under control conditions, cNOS and PAI-1 mRNA were stable after treatment with actinomycin D for periods greater than 24 hours, whereas endothelin-1 message was rapidly degraded (half-life, < 1 hour). Pretreatment with TNF-alpha (30 U/mL) selectively reduced that half-life of cNOS mRNA to less than 12 hours without altering the stability of PAI-1 message. TNF-alpha-induced destabilization of cNOS mRNA could be partially prevented by coincubation with cycloheximide (1 mumol/L) but was not reproduced by addition of sodium nitroprusside.(ABSTRACT TRUNCATED AT 250 WORDS)
Mohamed
F
F
McGill University, Montreal, Quebec, Canada.
Monge
J C
JC
Gordon
A
A
Cernacek
P
P
Blais
D
D
Stewart
D J
DJ
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D001483:Base Sequence; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000032:analysis; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase; D012333:RNA, Messenger / Q000032:analysis; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014471:Umbilical Veins / Q000201:enzymology
D018076:DNA, Complementary; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases