Abstract
Ritonavir (RTV), a prototypical protease inhibitor currently used as a key component of anti-HIV therapy, is known for its endothelial and hepatic toxicity. The effects of RTV and magnesium supplementation on cultured bovine endothelial cell (EC) and rat hepatic endothelial nitric oxide synthase (eNOS) status were investigated. RTV dose-dependently (5-30 µM) decreased EC viability after 48 h; high Mg (2mM) significantly attenuated the lost viability. ECs incubated with 15 µM RTV for 6 to 24h resulted in two- to fourfold elevation of oxidized glutathione and a 25% loss of total glutathione. At 24h, EC superoxide production due to RTV was detected by dihydroethidium staining and increased 41% when quantified by flow cytometry; altered glutathione status and superoxide levels were both substantially reversed by 2mM Mg. RTV reduced eNOS mRNA (-25% at 24 h) and led to decreased eNOS dimer/monomer ratios; nitric oxide-derived products decreased 40%; both changes were attenuated by Mg supplementation. In male Lewis-Brown Norway rats, RTV administration (75 mg/kg/day, 5 weeks) resulted in an 85% increase in plasma 8-isoprostane and a 23% decrease in hepatic eNOS mRNA; concomitantly, eNOS protein decreased 75%, whereas plasma nitrite level was reduced 48%. Dietary Mg supplementation (sixfold higher than control) prevented the eNOS mRNA decrease along with lowering 8-isoprostane and restored the eNOS protein and plasma nitrite levels comparable to controls. In conclusion, Mg attenuates RTV-mediated EC oxidative eNOS dysfunction and downregulation of hepatic eNOS expression; we suggest that Mg can serve as a beneficial adjunct therapeutic against RTV-mediated eNOS toxicity.