Abstract 257: Oxidative Stress Induces Endothelial Dysfunction: Role of Ubiquitin-Proteasome System
Endothelial Nitric oxide (NO) production is dependent on adequate cellular tetrahydrobiopterin (BH4), a key cofactor for endothelial NO syntheses (eNOS). Reduction of BH4 levels is reported in diseased vessels and plays a causal role in the development of eNOS uncoupling. However, the mechanisms that lead to BH4 reduction are not entirely understood. Here we report that angiotensin-II (Ang II) reduced tetrahydrobiopterin (BH4) and eNOS uncoupling by peroxynitrite ONOO−)-triggered proteasome activation. Compared to control, exposure of human umbilical vein endothelial cells (HUVEC) to angiotensin II (Ang II, 100nM) for 6h significantly decreased the levels of total biopterins (BH4 plus BH2) and BH4 (−22.0 4.6%, n= 3, p<0.01), indicating decreased synthesis of biopterins. In parallel, Ang II but not vehicle significantly reduced the levels of GTP-cyclohydrolase (GTPCH), a rate-limiting enzyme for de novo synthesis of biopterins, and dihydrofolate reductase (DHFR), a crucial enzyme for BH4 recycle from BH2. Ang II but not vehicle significantly increased the 26S proteasome activity. In addition, administration of proteasome inhibitors, MG132, abolished the Ang II-induced reduction of both GTPCH and DHFR. While Ang II significantly increased O2−, the scavenging of O2 − by Tempol (SOD mimetic), or inhibition of NOS with N-nitro-L-arginine methyl ester hydrochloride (L-NAME) (1mM) significantly attenuated Ang II-induced both 26S proteasome activation and the reduction of both GTPCH and DHFR, suggesting that Ang II via endogenous ONOO− causes 26S proteasome-dependent degradation of both GTPCH and DHFR. Moreover, inhibition of NAD(P)H oxidase with either apocynin or by overexpression of p67phox-dominant negative mutants ablated Ang II-induced proteasome activation and degradation of both GTPCH and DHFR. Finally, treatment of mice aorta ex vivo with MG132 (0.5 μM for 1h followed by Ang II for 12h) reversed the Ang II-induced reduction of GTPCH, DHFR and BH4, and increased acetylcholine-induced endothelium-dependent relaxation. We conclude that Ang II activates NAD(P)H oxidase to release O2. − and ONOO−, which activate 26S proteasome resulting in increased degradation of both GTPCH and DHFR, two key enzymes in controlling the levels of BH4.