Nitric Oxide: Synthesis and Action

Abstract

Nitric oxide (NO) is a diatomic radical implicated in a variety of pathways including vascular homeostasis, neurotransmission and immune defence. NO is produced endogenously by three nitric oxide synthases (NOS) and can also be formed from nitrates and nitrites. Several mechanisms regulate NOS function, including protein–protein interactions, posttranslational modifications, cell localization, calcium levels, substrate availability, and the cell redox status. Picomolar NO concentrations activate guanylyl cyclase to increase cGMP levels and lead to vasodilation and memory formation. Excess NO concentrations cause posttranslational modifications of cellular components and formation of peroxynitrite leading to inflammation and cell death. Due to its involvement in multiple pathways and high reactivity with a myriad of downstream targets, tight regulation of NO production is crucial to maintain cellular function. Major advancements in understanding NO chemistry, production and downstream signalling have provided new avenues to target this pathway for the treatment of cardiovascular, neurodegenerative and autoimmune diseases.

Key Concepts

  • Nitric oxide is produced endogenously by three nitric oxide synthase (NOS) isozymes.
  • Low NO levels mediate cell signalling, while high NO levels mediate the immune response and defence against pathogens.
  • A tight regulation of NO production is necessary to maintain cellular function.
  • The guanylyl cyclase enzyme (GC1) is the primary receptor of NO in smooth muscle cells and mediates vasorelaxation. Better understanding of NO/GC1 signalling will lead to novel drugs targeting this pathway for improved cardiovascular health.
  • Nitrite, a product of nitrate reduction via oral bacteria, is a biologically relevant source of NO under hypoxic and acidic conditions.
  • Dysfunction in NO synthesis due to oxidative damage leads to NOS uncoupling and increased output of ROS, a well‐known trait of cardiovascular disease (CVD).

Keywords: nitric oxide; nitric oxide synthase; guanylyl cyclase; nitrate; nitrite; cardiovascular diseases

Figure 1. Overall mechanism and architecture of NOS enzymes. (a) General mechanism for NO production. (b) The overall architecture of NOS enzymes is very similar and contains an N‐terminal NOS oxygenase module (NOSox), an intervening Ca2+/calmodulin binding region (CaM) and a C‐terminal NOS reductase module (NOSred). (c) Specific features of each NOS isoform include a PDZ domain for NOSI, myristoylation and palmitoylation sites in NOSII. All three isoforms contain a C‐terminal tail (CT). NOSI and NOSIII also contain an autoinhibitory helix (AH) and a CD2A inhibitory element, as well as various phosphorylation sites. (d) X‐ray structure of the human NOSII oxygenase module. (e) X‐ray structure of Ca2+/CaM bound to a NOSIII‐derived peptide. (f) X‐ray structure of the rat NOSI reductase module.
Figure 2. Overall NOS pathways and regulation. (a) NOSI becomes activated with increased Ca2+ concentrations, phosphorylation and protein–protein interactions. NOSI‐derived NO improves blood flow and increases neuronal cell growth (1). Hyperactivated NOSI can lead to excitotoxicity and apoptosis (2). NOSI activity is shutdown through phosphorylation and proteasomal degradation (3). (b) NOSII expression is induced by various cytokines. Immune cells such as macrophages and neutrophils use NOSII to generate cytotoxic levels of NO to kill invading pathogens (1). Excessive NOSII activity can damage healthy cells leading to inflammation and cell death (2). NOSII is neutralised through proteasomal degradation and a negative feedback loop with NF‐κB (3). (c) NOSIII is activated similarly to NOSI. A variety of kinases, protein–protein interactions and increased Ca2+ levels enhance NOSIII activity. NOSIII‐derived NO inhibits platelet aggregation and dilates neighbouring blood vessels (1). Reactive oxygen species (ROS) and low BH4 levels can uncouple NOSIII activity and lead to superoxide formation, which in turn decreases available NO and exacerbates oxidative stress (2). Phosphorylation and proteasomal degradation turn this pathway off (3).
Figure 3. The NO–GC1–cGMP pathway and therapeutic targets. NO is generated in the endothelium and diffuses to smooth muscle cells. GC1 (blue) is the primary target for NO binding, which triggers an increase in cGMP generation. Second messenger cGMP activates PKG, which phosphorylates downstream targets for improved blood flow. Reactive oxygen species (ROS) and phosphodiesterase‐5 (PDE5) can inhibit this pathway (red arrows). Several pharmacological targets for this pathway are shown with green arrows.
Figure 4. The nitrate/nitrite/NO pathway. Nitrate is obtained primarily in the diet from leafy greens and cured meats. Oral bacteria oxidise nitrate to nitrite, which acts as an important source of NO under hypoxic and acidic conditions.
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Childers, Kenneth C, and Garcin, Elsa D(Sep 2017) Nitric Oxide: Synthesis and Action. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0000634.pub2]