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Updated September 1, 2023.
Nitric oxide (NO) is an essential molecule involved in several physiological and pathological processes within the mammalian body. The small, gaseous molecule NO diffuses rapidly and acts as a messenger in diverse functions, including vasodilation, neurotransmission, anti-tumor, and anti-pathogenic activities.
NO exhibits both cytotoxic and cytoprotective properties1. Sufficient NO levels are necessary for protecting internal organs, such as the liver, from ischemic injury. In contrast, sustained levels of NO production result in direct tissue toxicity and the vascular collapse associated with septic shock. Chronic expression of NO is associated with carcinomas and inflammatory conditions, including juvenile diabetes, multiple sclerosis, arthritis, and ulcerative colitis2.
NO is endogenously synthesized by a family of enzymes termed nitric oxide synthase (NOS), which oxidizes guanidine nitrogen of L-arginine, releasing citrulline and nitric oxide in the form of a free radical3. Once produced, NO diffuses rapidly across cell membranes. Its principal receptor is the specialized protein soluble guanylyl cyclase (sGC), which is, in fact, the only known receptor for the NO molecule.
Three isoforms of NOS have been identified: inducible (iNOS or NOS-2), endothelial (eNOS or NOS-3), and neuronal (nNOS or NOS-1) — each with separate functions. The inducible isoform (NOS-2) is calcium-independent and produces large amounts of NO gas, which can be cytotoxic. The neuronal enzyme (NOS-1) and the endothelial isoform (NOS-3) are calcium-dependent and produce low levels of NO gas as a signaling molecule4.
In inducible NOS (iNOS, NOS-2) signaling, NO is synthesized by NOS-2 as a result of the cell's response to cytokines and other agents of inflammation. The produced NO can follow two pathways. First, it can combine with dioxygen (O2-) from xanthine oxidase (XO) to form high levels of peroxynitrite (ONOO-). Peroxynitrite inhibits the phospholamban (PLB) protein of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)—a multiprotein complex responsible for transporting calcium from the cytosol into the sarcoplasmic reticulum.
PLB is known as a reversible inhibitor regulating the activity of the sarcoplasmic reticulum calcium pump. Chronic inhibition of PLB diminishes myofilament contractility, and PLB is known as a crucial regulator of cardiac muscle5.
Peroxynitrite can also cause DNA damage, including DNA strand breakage and base modification. It is involved in cell apoptosis and necrosis, and its generation is a pathological mechanism in stroke, diabetes, neurodegeneration, and cancer6.
In the second pathway, the NO molecule activates sGC. The sGC protein then releases cyclic guanosine monophosphate (cGMP), which acts on protein kinase G (PKG) to phosphorylate the actin-myosin sarcomere, decreasing calcium sensitivity and myofilament contractility.
In endothelial NOS (eNOS, NOS-3) signaling, the NO produced by NOS-3 activates the NO receptor sGC, which releases cGMP. The cGMP molecule phosphorylates PKG, which in turn donates a phosphoryl group to actin, decreasing its calcium sensitivity. A phosphoryl group can also be donated to a transmembrane calcium ion channel between the cell and caveolae, increasing the likelihood of channel opening.
A third target of the phosphoryl group from PKG is the phosphodiesterase-5 (PDE5) enzyme, well known for its role in blood vessel dilation. When PDE5 is inhibited, blood vessels are relaxed, and blood flow is increased. When activated, PDE5 inhibits the beta-adrenergic (beta-AR) receptors spanning the cell wall7.
The NOS-3 molecule also acts on potassium ion channels crossing the membrane at the caveolae, which is populated by superoxide dismutase (SOD) and the caveolin-3 protein, as well as potassium channels, calcium channels, and NOS-3 molecules.
Neuronal NOS (nNOS, NOS-1) signaling begins with NOS-1 as part of the SERCA protein complex. The NO produced from NOS-1 can follow two pathways. First, NO can combine with O2- to form ONOO-, which can covalently link to a cysteine on a protein target in a process termed S-nitrosylation8. In this case, the protein target is the calcium ion channels crossing the cell membrane to caveolae. Another target of the produced ONOO- is the SERCA protein itself, where it stimulates the release of calcium ions, which in turn act on calcineurin, a calcium-dependent protein phosphatase, which can close down channels that would allow more calcium to enter the cell.
Second, NO can activate a pathway through the sGC receptors. The sGC receptors detect subnanomolar concentrations of NO and rapidly transduce them into micromolar concentrations of cGMP, which activates PKG. Phosphorylation of the actin-myosin sarcomere by PKG decreases its calcium sensitivity and myofilament contractility.
Our poster highlights the nitric oxide signaling pathways mediated by each of the NOS isoforms – inducible, endothelial, and neuronal – demonstrating the effect of NO on intracellular calcium and potassium concentrations and the role of the NO receptor, sGC.