New insights into angiotensin, reactive oxygen and endothelial function

Angiotensin (Ang) II is an octapeptide in the blood that causes vasoconstriction, the release of aldosterone from the adrenal cortex, drives thirst behaviour in the brain, and initiates cell signalling by means of various intracellular mechanisms, notably in the endothelium. The endothelium modulates vascular tone by producing vasodilator and vasoconstrictor substances. Of these, the best characterized and potentially most important are nitric oxide (NO) and superoxide (O2 ). These small molecules are free radicals, meaning that they have one or more unpaired electrons in their outer orbitals. NO and O2 − exhibit opposing effects on vascular tone and chemically react with each other in a fashion that negates their individual effects and leads to the production of potentially toxic substances, such as peroxynitrite (ONOO) [1]. Peroxynitrite is an unstable valence isomer of nitrate and is also a free radical, resulting from the action of superoxide, a reactive oxygen species (ROS), on NO. These dynamic interactions have important implications, altering not only tissue perfusion but also contributing to the process of atherosclerosis. A superficial outline of peroxynitrite-related effects is shown in Figure 1. In endothelial cells and in vascular smooth muscle cells, a membrane-associated nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidase is the most important O2 − source. This oxidase is activated by stimulation with Ang II, suggesting that, under all conditions of an activated circulating and/or local renin–angiotensin system, endothelial dysfunction related to increased vascular O2 − production would be expected. The fact that Ang II stimulates the NADPH oxidase has been known for a decade. Many studies have shown that Ang II activates the enzyme and in turn how NADPH oxidases regulate numerous key Ang II-mediated effects. The topic has recently been reviewed by one of its originators [2]. All nucleated cells produce ROS, including superoxide anion, hydrogen peroxide (H2O2) and peroxynitrite. The NADPH oxidase enzyme complex was first worked out in neutrophils, where it plays an antimicrobial role. The enzyme complex has a series of subunits that vary to some degree between cell types. Precisely how Ang II activates the enzyme complex remains incompletely understood; however, Ang II, an AT1 receptor, mediates the process. In vascular smooth muscle cells (VSMC), the pivotal Nox1 subunit is activated, thereby beginning the ROS production–signalling cascade. ROS derived from NADPH oxidases serves a signalling function by inducing specific biochemical changes in numerous molecular targets. For example, the resultantly produced H2O2 can oxidize the thio group of protein cysteine residues that can inhibit protein tyrosine phosphatase activity. Figure 2 shows an abbreviated schema using VSMC as a model. However, Ang II has many more targets than VSMC. The AT1 receptor appears essentially ubiquitous. As a result, new functions and new mechanisms of action for Ang II crop up all the time, with surprising findings and results. Recently, Loot et al. explored a novel mechanism to show how Ang II results in impaired endothelial function [3]. The fact that Ang II does so is not new; however, the mechanism involved, namely the shutting down of the endothelial nitric oxide synthase (eNOS), is novel and introduces further twists into our knowledge of Ang II signalling that surely have ramifications above and beyond endothelial dysfunction. The signalling mechanisms involve the proline-rich tyrosine kinase 2 (PYK2). The group had observed earlier that fluid shear stress elicits the tyrosine phosphorylation of eNOS; however, the consequences of this modification on enzyme activity and the mechanisms were unclear. Fisslthaler and colleagues found that fluid shear stress induces the association of eNOS with PYK2 [4]. Furthermore, they were able to immunoprecipitate eNOS and PKY2 from PYK2-overexpressing HEK293 cells and observed that eNOS was tyrosine phosphorylated on Tyr657. They then performed a variety of functional studies in carotid arteries and other systems and showed that PYK2-induced phosphorylation at Tyr657 effectively shuts down the enzyme. Cleverly, they then produced a non-phosphorylatable mutant eNOS that was impervious to PYK2 phosphorylation at Tyr657. Their data indicated that PYK2 mediates the tyrosine phosphorylation of eNOS on Tyr657 in response to fluid shear stress and that this modifica-