Chapter 31
Modulation of Vascular Function by AMPK: Assessment
of NO Bioavailability and Surrogates of Oxidative Stress
Swenja Kro ̈ller-Scho ̈n, Andreas Daiber, and Eberhard Schulz
Abstract
The endothelium plays a pivotal role in the development of vascular disease. Decreased bioavailability of
nitric oxide, a condition known as “endothelial dysfunction,” is considered an early step in this process
before atherosclerotic changes of the vessel wall occur. Endothelium-derived nitric oxide (lNO) may be
rapidly scavenged by superoxide anions; therefore, the equilibrium betweenlNO production on one hand
and its inactivation by oxidative stress on the other hand is of particular interest. Metabolic enzyme systems
such as AMP-activated protein kinase (AMPK) may affect the cellular production oflNO or reactive oxygen
species (ROS), while AMPK activity itself can also be modulated by ROS. Therefore, the analysis oflNO as
well as ROS levels is essential to understand how metabolism regulating enzymes like AMPK may modulate
vascular disease.
Key wordsReactive oxygen species, Nitric oxide, Endothelial function, Electron paramagnetic reso-
nance spectroscopy, Amplex red, Dihydroethidium staining, Peroxynitrite, Tyrosine nitration1 Introduction
In a healthy vessel, nitric oxide (lNO) produced by the endothelial
nitric oxide synthase (eNOS) causes vasodilation, while it prevents
the infiltration of inflammatory cells and thrombus formation. In
order to assess endothelial function, the easiest way is to analyze
vasodilation in response to endothelial-dependent stimulators such
as acetylcholine (e.g., by isometric tension studies of isolated vas-
cular rings). However, these functional studies require a high level
of handling experience especially in small vessels (e.g., mouse aorta)
in order to prevent injury during the preparation process. Another
option is the specific analysis oflNO bioavailability by electron
paramagnetic resonance (EPR) spectroscopy using Fe(II)
(DETC) 2 as spin trap [1]. EPR spectroscopy is used to study
compounds with unpaired electrons such as free radicals or para-
magnetic metal species. Since most radicals likelNO have a short
half-life, their reaction with another compound (as Fe(II)(DETC) 2Dietbert Neumann and Benoit Viollet (eds.),AMPK:MethodsandProtocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_31,©Springer Science+Business Media, LLC 2018
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