Thrombin stimulates calcium‐dependent production of mitochondrial ROS in pulmonary microvascular endothelial cells

Abstract

Vascular cells respond to various stimuli via enhanced production of reactive oxygen species (ROS). In endothelial cells, proteins that produce ROS include the flavoenzyme NADPH oxidase of cell membranes and the mitochondrial electron transport chain. Thrombin, an enzyme linked to atherosclerosis and restenosis injury, can trigger cellular oxidative stress, although the mechanism for its effects in endothelial cells is unclear. To determine the mechanism by which thrombin evokes endothelial cell signaling, murine pulmonary microvascular endothelial cells (MPMVECs) were simultaneously loaded with the calcium indicator dye Fluo-4/AM and the mitochondrial membrane potential indicator tetramethylrhodamine, ethyl ester, perchlorate (TMRE) and visualized by confocal microscopy. Thrombin stimulated a large calcium release from the intracellular stores that preceded mitochondrial membrane potential loss. Subsequently, mitochondrial superoxide production was enhanced as evidenced by an increase in fluorescence of the mitochondrial superoxide-sensitive fluorophore, MitoSOX Red. Thrombin treatment also increased nuclear HE fluorescence, an indicator of cellular superoxide production, and was unaffected in the prescence of the NADPH oxidase inhibitor Apocynin and the anion channel blocker DIDS. In contrast, Apocynin and DIDS ablated the HE fluorescence increase observed in response to Angiotensin II, a known stimulator of NADPH oxidase. Thus, thrombin stimulates ROS production in endothelial cells primarily via mitochondria, and may play an important role in the vascular response in areas adjacent to injury. [HL-60290 & HL-07748]