Lack of Role for Nitric Oxide (NO) in the Selective Destabilization of Endothelial NO Synthase mRNA by Tumor Necrosis Factor–α

Abstract

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–α (TNF-α) was investigated by using Northern blot analysis of total cellular RNA. TNF-α 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-α. The pharmacological generation of NO using sodium nitroprusside (10 μmol/L) and S-nitroso-acetylpenicillamine (100 to 400 μmol/L) had no effect on cNOS mRNA levels, and TNF-α–induced downregulation of cNOS was not prevented by coincubation with the inhibitors of NO synthesis N^ω-nitro-l-arginine methyl ester (1 mmol/L) and N^G-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-α (30 U/mL) selectively reduced the half-life of cNOS mRNA to less than 12 hours without altering the stability of PAI-1 message. TNF-α–induced destabilization of cNOS mRNA could be partially prevented by coincubation with cycloheximide (1 μmol/L) but was not reproduced by addition of sodium nitroprusside. These findings indicate that TNF-α downregulation of cNOS expression in human endothelial cells results predominantly from the selective destabilization of the mRNA by a mechanism involving the synthesis of new protein. However, NO production by a TNF-α–inducible isoform of NOS did not appear to contribute either to the decrease in steady state cNOS mRNA levels or the shortening of its half-life.