Abstract 224: 20-hydroxyeicosatetraenoic Acid Mediated Amp Activated Protein Kinase Suppression Induces Endothelial Nitric Oxide Synthase Uncoupling
20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 product of arachidonic acid metabolism, is a potent vasoconstrictor thought to be involved in vascular dysfunction and the regulation of blood pressure. 20-HETE has been demonstrated to play an inhibitory role in eNOS activity and both animal and human studies have revealed strong associations between 20-HETE and endothelial dysfunction. However, the signalling mechanisms are largely unknown. Previous studies have shown that AMPK activity is involved in the Hsp90-eNOS association. Therefore we sought to investigate the effect of 20-HETE on eNOS and Hsp90 association, and whether this was mediated by AMPK. Treatment of human umbilical vein endothelial cells (HUVECs) with 10μM 20-HETE for 24h induces an increase in eNOS phosphorylation, which is not observed following acute treatment (30mins). This was accompanied by transient changes in Akt phosphorylation. Conversely, upstream AMPK activity increased rapidly following 20-HETE treatment (<1min) before decreasing by 30mins. Treatment with 20-HETE also resulted in an acute increase in the production of reactive oxygen species (ROS), which returned to control levels at >2hr post-20-HETE treatment. Immunoprecipitation of eNOS in cells treated with 20-HETE revealed a decrease in basal, VEGF and A23187 stimulated Hsp90 association with eNOS, indicating an uncoupling mechanism. Pre-treatment of the cells with AICAR (a chronic activator of AMPK) prevented the loss of Hsp90 association with eNOS following 20-HETE treatment. To further investigate the functional effect of Hsp90 dissociation, we utilised an endothelial-smooth muscle cell system to measure cGMP production. Chronic 20-HETE treatment appeared to reduce both basal and VEGF stimulated cGMP levels. Pre-treatment of the cells with AICAR protected against the 20-HETE induced decrease in cGMP levels, both basally and following VEGF stimulation. In conclusion, 20-HETE suppression of AMPK activity impairs eNOS-Hsp90 association, providing a mechanism for reduced NO bioactivity and endothelial dysfunction in diseases with elevated 20-HETE levels, such as hypertension.