LACK OF ROLE FOR NITRIC-OXIDE (NO) IN THE SELECTIVE DESTABILIZATION OF ENDOTHELIAL NO SYNTHASE MESSENGER-RNA 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 mR NA in human umbilical vein endothelial cells exposed to tumor necrosis factor-alpha (TNF-alpha) was investigated by using Northern blot anal ysis of total cellular RNA. TNF-alpha produced a dose- and time-depend ent decrease in cNOS mRNA expression that was near maximal at 10 U/mL and 6 hours of exposure, respectively. In contrast, steady state expre ssion of endothelin-1 and plasmin ogen activator inhibitor-1 (PAI-1) m RNA was upregulated by TNF-alpha. The pharmacological generation of NO using sodium nitroprusside (10 mu mol/L) and S-nitroso-acetylpenicill amine (100 to 400 mu mol/L) had no effect on cNOS mRNA levels, and TNF -alpha-induced downregulation of cNOS was not prevented by coincubatio n with the inhibitors of NO synthesis N-omega-nitro-Larginine methyl e ater (1 mmol/L) and N-G-monomethyl L-arginine (10 mmol/L). Under contr ol conditions, cNOS and PAI-1 mRNA were stable after treatment with ac tinomycin D for periods greater than 24 hours, whereas endothelin-1 me ssage was rapidly degraded (half-life, <1 hour). Pretreatment with TNF -alpha (30 U/mL) selectively reduced the half-life of cNOS mRNA to les s than 12 hours without altering the stability of PAI-1 message. TNF-a lpha-induced destabilization of cNOS mRNA could be partially prevented by coincubation with cycloheximide (1 mu mol/L) but was not reproduce d by addition of sodium nitroprusside. These findings indicate that TN F-alpha downregulation of cNOS expression in human endothelial cells r esults predominantly from the selective destabilization of the mRNA by a mechanism involving the synthesis of new protein. However, NO produ ction by a TNF-alpha-inducible isoform of NOS did not appear to contri bute either to the decrease in steady state cNOS mRNA levels or the sh ortening of its half-life.