Nitric oxide synthases: regulation and function

Abstract

Nitric oxide (NO), the smallest signalling molecule known, is produced by three isoforms of NO synthase (NOS; EC 1.14.13.39). They all utilize l-arginine and molecular oxygen as substrates and require the cofactors reduced nicotinamide-adenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and (6R-)5,6,7,8-tetrahydrobiopterin (BH(4)). All NOS bind calmodulin and contain haem. Neuronal NOS (nNOS, NOS I) is constitutively expressed in central and peripheral neurons and some other cell types. Its functions include synaptic plasticity in the central nervous system (CNS), central regulation of blood pressure, smooth muscle relaxation, and vasodilatation via peripheral nitrergic nerves. Nitrergic nerves are of particular importance in the relaxation of corpus cavernosum and penile erection. Phosphodiesterase 5 inhibitors (sildenafil, vardenafil, and tadalafil) require at least a residual nNOS activity for their action. Inducible NOS (NOS II) can be expressed in many cell types in response to lipopolysaccharide, cytokines, or other agents. Inducible NOS generates large amounts of NO that have cytostatic effects on parasitic target cells. Inducible NOS contributes to the pathophysiology of inflammatory diseases and septic shock. Endothelial NOS (eNOS, NOS III) is mostly expressed in endothelial cells. It keeps blood vessels dilated, controls blood pressure, and has numerous other vasoprotective and anti-atherosclerotic effects. Many cardiovascular risk factors lead to oxidative stress, eNOS uncoupling, and endothelial dysfunction in the vasculature. Pharmacologically, vascular oxidative stress can be reduced and eNOS functionality restored with renin- and angiotensin-converting enzyme-inhibitors, with angiotensin receptor blockers, and with statins.

Figure 1

Important functions of the different NOS isoforms. (Top panel) Neuronal NOS is expressed in specific neurons of the central nervous system (CNS). It has been implicated in synaptic plasticity (i.e. phenomena such a long-term potentiation and long-term inhibition). These phenomena are involved in learning and memory formation. Neuronal NOS-derived NO also participates in central control of blood pressure. In the peripheral nervous system (PNS), neuronal NOS-derived NO acts as an atypical neurotransmitter, which mediates relaxing components of gut peristalsis, vasodilation, and penile erection. At least a minimal stimulation of soluble guanylyl cyclase in corpus cavernosum by nNOS-derived NO, and the subsequent formation of small amounts of cyclic GMP, is a prerequisite for the pro-erectile effects of the phosphodiesterase 5 inhibitors sildenafil (Viagra^®), vardenafil (Levitra^®), and tadalafil (Cialis^®). (Middle panel) Inducible NOS expression can be induced by cytokines and other agents in almost any cell type. This had initially been shown for macrophages (MΦ). The induction of inducible NOS in MΦ is essential for the control of intracellular bacteria such as Mycobacterium tuberculosis^, or the parasite Leishmania.^, However, inducible NOS is also up-regulated in various types of inflammatory disease, and the NO generated by the enzyme mediates various symptoms of inflammation.^, Finally, inducible NOS-derived NO is the predominant mediator of vasodilation and fall in blood pressure seen in septic shock. In fact, mice with a disrupted inducible NOS gene are protected from many symptoms of septic shock. (Bottom panel) Endothelial NOS-derived NO is a physiological vasodilator, but can also convey vasoprotection in several ways. NO released towards the vascular lumen is a potent inhibitor of platelet aggregation and adhesion to the vascular wall. Besides protection from thrombosis, this also prevents the release of platelet-derived growth factors that stimulate smooth muscle proliferation and its production of matrix molecules. Endothelial NO also controls the expression of genes involved in atherogenesis. NO decreases the expression of chemoattractant protein MCP-1 and of a number of surface adhesion molecules, thereby preventing leucocyte adhesion to vascular endothelium and leucocyte migration into the vascular wall. This offers protection against early phases of atherogenesis. Also the decreased endothelial permeability, the reduced influx of lipoproteins into the vascular wall and the inhibition of low-density lipoprotein oxidation may contribute to the anti-atherogenic properties of endothelial NOS-derived NO. Finally, NO has been shown to inhibit DNA synthesis, mitogenesis, and proliferation of vascular smooth muscle cells as well as smooth muscle cell migration, thereby protecting against a later phase of atherogenesis. Based on the combination of those effects, NO produced in endothelial cells can be considered an anti-atherosclerotic principle (for review, see Li and Förstermann).

Figure 2

Structure and catalytic mechanisms of functional NOS. (A) NOS monomers are capable of transferring electrons from reduced nicotinamide-adenine-dinucleotide phosphate (NADPH), to flavin-adenine-dinucleotide (FAD) and flavin-mononucleotide (FMN) and have a limited capacity to reduce molecular oxygen to superoxide (O₂^−•).^,, Monomers and isolated reductase domains can bind calmodulin (CaM), which enhances electron transfer within the reductase domain. NOS monomers are unable to bind the cofactor (6R-)5,6,7,8-tetrahydrobiopterin (BH₄) or the substrate l-arginine and cannot catalyze NO production.^, (B) In the presence of haem, NOS can form a functional dimer.^, Haem is essential for the interdomain electron transfer from the flavins to the haem of the opposite monomer.^, Due to differences in the calmodulin-binding domain, elevated Ca²⁺ is required for calmodulin binding (and thus catalytic activity) in nNOS and eNOS, whereas calmodulin binds to inducible NOS with high affinity even in the absence of Ca²⁺. When sufficient substrate l-arginine (l-Arg) and cofactor BH₄ are present, intact NOS dimers couple their haem and O₂ reduction to the synthesis of NO (fully functional NOS). l-Citrulline (l-Cit) is formed as the byproduct. For clarity, the flow of electrons is only shown from the reductase domain of one monomer to the oxygenase domain of the other monomer. NOS enzymes perform two separate oxidation steps, one to form N^ω-hydroxy-l-arginine and a second to convert this intermediate to NO. All NOS isoforms contain a zinc ion (Zn) coordinated in a tetrahedral conformation with pairs of CXXXXC motifs at the dimer interface. This site is of major importance for the binding of BH₄ and l-arginine. Electron transfer from the reductase domain (*) enables NOS ferric (Fe³⁺) haem to bind O₂ and form a ferrous (Fe²⁺)-dioxy species. This species may receive a second electron (**) preferentially from BH₄ or from the reductase domain. The nature of the resulting oxidized BH₄ has been identified as the trihydrobiopterin radical (BH₃^•) or the trihydropterin radical cation protonated at N5 (BH₃^•H⁺). The BH₃^• radical (or radical cation) can be recycled to BH₄ by the NOS itself (using an electron supplied by the flavins). Alternatively, there is evidence that reducing agents such as ascorbic acid (AscH, which is present in cells in millimolar concentrations) can reduce the BH₃^• radical back to BH₄ (Asc^·, ascorbate radical).

Figure 3

Regulation of endothelial NOS activity by intracellular Ca²⁺ and phosphorylation. An increase in intracellular Ca²⁺ leads to an enhanced binding of calmodulin (CaM) to the enzyme, which in turn displaces an auto-inhibitory loop and facilitates the flow of electrons from NADPH in the reductase domain to the haem in the oxygenase domain. Established functionally important phosphorylation sites in human endothelial NOS are Ser1177 and Thr495. In resting endothelial cells, Ser1177 is usually not phosphorylated. Phosphorylation is induced when the cells are exposed to oestrogens, vascular endothelial growth factor (VEGF), insulin, bradykinin or fluid shear stress. The kinases responsible for phosphorylation (green hexagons) depend on the primary stimulus. Oestrogen and vascular endothelial growth factor elicit phosphorylation of Ser1177 by activating serine/threonine kinase Akt. So far, Akt1 is the only kinase proven to regulate endothelial NOS function in vivo (framed green hexagon). Insulin probably activates both Akt and the AMP-activated protein kinase (AMPK), the bradykinin-induced phosphorylation of Ser1177 is mediated by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), and shear stress leads to phosphorylation of endothelial NOS mainly via protein kinase A (PKA). Phosphorylation of the Ser1177 residue increases the flux of electrons through the reductase domain and thus enzyme activity. The Thr495 residue of human endothelial NOS tends to be constitutively phosphorylated in endothelial cells. Thr495 is a negative regulatory site, and its phosphorylation is associated with a decreased electron flux and enzyme activity. The constitutively active kinase that phosphorylates endothelial NOS Thr495 is most probably protein kinase C (PKC, yellow hexagon). Phosphorylation of Thr495 reduces endothelial NOS activity (yellow block arrow). The phosphatase that dephosphorylates Thr495 appears to be protein phosphatase1 (PP1, black flag with black block arrow).

Figure 4

Potential mechanisms by which cardiovascular risk factors lead to oxidative stress and endothelial NOS uncoupling. (A) In many types of vascular disease, NADPH oxidases are up-regulated in the vascular wall and generate superoxide (O₂^−•). In experimental diabetes mellitus and angiotensin II-induced hypertension, this has been shown to be mediated by protein kinase C (PKC).^, Expression of endothelial NOS is also increased in vascular disease. H₂O₂, the dismutation product of O₂^−• can increase endothelial NOS expression via transcriptional and post-transcriptional mechanisms (SOD, superoxide dismutase). In addition, also protein kinase C activation can enhance endothelial NOS expression, and protein kinase C inhibitors reduce endothelial NOS expression levels in vascular disease. The products of NADPH oxidases and endothelial NOS, O₂^−• and NO·, rapidly recombine to form peroxynitrite (ONOO⁻). This can oxidize the essential cofactor of endothelial NOS (6R-)5,6,7,8-tetrahydrobiopterin (BH₄) to trihydrobiopterin radical (BH₃^•).^, BH₃^• can disproportionate to the quinonoid 6,7-[8H]-H₂-biopterin (BH₂). As a consequence, oxygen reduction and O₂ reduction by endothelial NOS are uncoupled from NO^· formation, and a functional NOS is converted into a dysfunctional O₂^−•-generating enzyme that contributes to vascular oxidative stress. The enhanced endothelial NOS expression (see above) aggravates the situation. (B) Oxidation of BH₄ to biologically inactive products such as the BH₃^• radical or BH₂ also reduces the affinity of the substrate l-arginine (l-Arg) to NO, and NOS catalyzes the uncoupled reduction in O₂, leading to the production of O₂^−• (and possibly also H₂O₂).