NADPH oxidase is the source of ROS in STZ rat aorta : use of the novel highly selective NOX inhibitor VAS2870

Abstract

Abnormal vascular responsiveness in diabetes has been attributed to a number of changes in contractile and dilatory pathways, affected in part by the overproduction of reactive oxygen species (ROS). It has been reported that NADPH oxidase (NOX) is increased in endothelial cells of streptozotocin (STZ) treated rats, a model of diabetes. However pharmacological tools used in NOX studies are of limited use; the NOX inhibitor apocynin is non-selective and has off-target effects1 and may inhibit nitric oxide synthase. Here we use the novel, highly selective NOX inhibitor VAS28702 to confirm the source of ROS in aorta of STZ rats. Male Wistar rats (250 to 350g) were injected with 65mg/kg STZ; development of diabetes was confirmed after 5 days by testing blood glucose levels. Rats were killed by CO2 asphyxiation, and the thoracic aorta removed and cleaned for mounting in a 20ml organ bath. Aorta were tensioned to 1g, equilibrated and incubated with 10-5M VAS2870, 10-5M apocynin, 150U/ml superoxide dismutase (SOD) or vehicle control (0.1% DMSO) in Kreb’s buffer for 30 minutes, followed by an increasing concentration of phenylephrine (PE).