Objective— We have previously demonstrated that endothelial xanthine oxidase (XO) levels are dependent on the NADPH oxidase. We postulated that H₂O₂ may modulate the irreversible conversion of xanthine dehydrogenase (XDH) to XO and sought to examine mechanisms involved.

Methods and Results— H₂O₂ (100 μmol/L) decreased bovine aortic endothelial cell (BAEC) XDH protein expression, and metabolic labeling studies indicated that H₂O₂ stimulated conversion of XDH to XO. The decline in XDH was mimicked by the reactive oxygen species (ROS) generating compounds SIN-1 and Menadione, as well as by stimulating BAECs with angiotensin II (200 nmol/L). BAPTA-AM prevented the decline in XDH by H₂O₂, indicating that it was calcium-dependent. In keeping with calcium acting downstream of H₂O₂, the calcium ionophore A23187 (1 μmol/L) caused XDH-to-XO conversion, and this was not prevented by the antioxidants. In addition, XDH-to-XO conversion was blocked by 2-APB and NO donors and induced by thapsigargin and M-3M3FBS, implicating phospholipase C and endoplasmic reticulum calcium stores in this process.

Conclusions— Endothelial XO and XDH expression are strongly dependent on H₂O₂ and calcium. Stimulation of XDH conversion to XO may represent a feed-forward mechanism whereby H₂O₂ can stimulate further production of ROS.