Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation

Abstract

Nitric oxide (NO) is a small free radical with critical signaling roles in physiology and pathophysiology. The generation of sufficient NO levels to regulate the resistance of the blood vessels and hence the maintenance of adequate blood flow is critical to the healthy performance of the vasculature. A novel paradigm indicates that classical NO synthesis by dedicated NO synthases is supplemented by nitrite reduction pathways under hypoxia. At the same time, reactive oxygen species (ROS), which include superoxide and hydrogen peroxide, are produced in the vascular system for signaling purposes, as effectors of the immune response, or as byproducts of cellular metabolism. NO and ROS can be generated by distinct enzymes or by the same enzyme through alternate reduction and oxidation processes. The latter oxidoreductase systems include NO synthases, molybdopterin enzymes, and hemoglobins, which can form superoxide by reduction of molecular oxygen or NO by reduction of inorganic nitrite. Enzymatic uncoupling, changes in oxygen tension, and the concentration of coenzymes and reductants can modulate the NO/ROS production from these oxidoreductases and determine the redox balance in health and disease. The dysregulation of the mechanisms involved in the generation of NO and ROS is an important cause of cardiovascular disease and target for therapy. In this review we will present the biology of NO and ROS in the cardiovascular system, with special emphasis on their routes of formation and regulation, as well as the therapeutic challenges and opportunities for the management of NO and ROS in cardiovascular disease.

FIGURE 1.

Oxygen and oxidoreductase enzymes regulate nitric oxide (NO) homeostasis. The gradient in the concentration of oxygen shifts the function of globins from oxidizing, NO-scavenging proteins to nitrite-reducing, NO-generating proteins.

FIGURE 2.

Architecture of nitric oxide synthases (NOS). A: the arrangement of the domains in the NOS monomer. The oxygenase/heme domain (red) is connected to the reductase domain by a flexible linker, containing a calmodulin (CaM) binding sequence. The reductase domain includes a flavin mononucleotide (FMN)-binding domain (orange) that shuttles electrons from NADPH/FAD to the heme group and a FAD-containing domain (yellow) that uses NADPH as an electron source. B: the binding of CaM (blue) to NOS promotes electron transfer from the FMN domain of one monomer to the heme domain of the other monomer. C: three-dimensional structure model of NOS. The figure is assembled from the separated structures of the human endothelial NOS oxygenase domain (PDB:4D1O) (574), the CaM binding peptide bound to CaM (PDB:2N8J) (740), and the structure of the neuronal NOS reductase (PDB:1TLL) (338).

FIGURE 3.

Chemical structure of the nitric oxide synthase (NOS) substrate l-arginine, tetrahydrobiopterin, and selected NOS inhibitors.

FIGURE 4.

Architecture of soluble guanylyl cyclase (sGC). Top: arrangement of sGC domains in the sequence of α and β subunits. Bottom: model for the interaction of the N-termini domains of the α and β subunits of human sGC. Domains are colored according to the top panel. Catalytic GTP cyclase domains are not shown. The model for the human sGC protein is based on the model for Manduca sexta sGC derived from chemical cross-linking, small-angle X-ray scattering, and homology modeling (312, 313, 662).

FIGURE 5.

Soluble guanylyl cyclase (sGC) function in healthy and endothelial dysfunction states. Oxidative stress conditions cause oxidation of BH₄ to BH₂, superoxide production in the endothelial cells, and promote oxidation and heme loss in smooth muscle cell sGC.

FIGURE 6.

Chemical structure of selected soluble guanylyl cyclase (sGC) stimulators and activators.

FIGURE 7.

Structure of the NADPH oxidase (NOX) core protein membrane and cytosolic domains. The sequence comprises six transmembrane helices and a cytosolic flavoprotein/dehydrogenase domain. The transmembrane helices are indicated by colors: I, red; II, orange; III, yellow; IV, green; V, cyan; and VI, dark blue. The location of loops B and C is indicated. The dehydrogenase domain is shown in purple. The cofactors heme and FAD are shown as red and yellow sticks, respectively. Figure was drawn with PyMOL based on the structures for the transmembrane (PDB: 5O0T) and dehydrogenase (PDB: 5O0X) domains of NOX5 (613).

FIGURE 8.

Putative assembly architecture of NADPH oxidase (NOX)1, NOX2, NOX4, and NOX5 and their regulatory proteins.

FIGURE 9.

Cellular distribution of NADPH oxidase (NOX) species in the blood vessels. Predominant forms in each cell type are shown in bold.

FIGURE 10.

Chemical structures of selected NADPH oxidase (NOX) inhibitors.

FIGURE 11.

Superoxide generation by nitric oxide synthase (NOS) monomer and dimer in the presence or absence of calmodulin (CaM).

FIGURE 12.

Progression of nitric oxide (NO) synthesis, superoxide, and peroxynitrite during the development of endothelial nitric oxide synthase (eNOS) uncoupling.

FIGURE 13.

Mitochondrial electron transfer chain. Main sites of superoxide production are indicated.

FIGURE 14.

Structures of selected mitochondria-targeted compounds.

FIGURE 15.

Architecture of xanthine dehydrogenase (XDH)/zanthine oxidase. A and B indicate the arrangement of the domains in the XDH/xanthine oxidoreductase (XOR) monomer. The middle, FAD-containing domain (yellow), is connected to the N-terminal domain containing two iron/sulfur clusters (Fe₂S₂) (green) and the molybdopterin domain (pink) by two flexible linkers. Posttranslational changes modify the protein activity from a XDH (A), NADH-producing enzyme, to the xanthine oxidase (XO) (B), hydrogen peroxide/superoxide-generating enzyme. C: three-dimensional structure of the XDH monomer (PDB:1FO4) (268).

FIGURE 16.

Structures of xanthine oxidase substrates and inhibitors.

FIGURE 17.

Cross-talk between mitochondria, NADPH oxidase (NOX), and other reactive oxygen species (ROS) sources. Mitochondrial ROS are released to the cytosol via the mitochondrial permeability transition pore (mPTP). These can cause activation of PKC and phosphatidylinositol 3-kinase (PI3K) kinases and Rac1 GTPase with subsequent activation of NOX enzymes and further ROS production. Alternatively, NOX can release ROS that activate protein kinase C, isoform ε (PKCε) and in turn activate the mitochondrial ATP-dependent potassium channels (K_ATP), increasing mitochondrial ROS. Cytosolic ROS can increase ROS-generating activity of endothelial nitric oxide synthase (eNOS) and xanthine oxidase (XO).