NADPH Role in Nitric Oxide Synthesis

Nitric oxide (NO) is recognized as a mediator in a broad array of biologic systems. NO is the endothelium-derived relaxing factor that causes vasodilation by relaxing vascular smooth muscle. It also acts as a neurotransmitter, prevents platelet aggregation, and plays an essential role in macrophage function. It has a very short half-life in tissues (3–10 seconds) because it reacts with O2 and and is converted into nitrates and nitrites including peroxynitrite (O=NOO−), a reactive nitrogen species (RNS). [Note: NO is a free radical gas that is often confused with nitrous oxide (N2O), the “laughing gas” that is used as an anesthetic and is chemically stable.]
1. Nitric oxide synthase Arginine, O2, and NADPH are substrates for cytosolic NO synthase ([NOS], Fig. 1). Flavin mononucleotide (FMN), FAD, heme, and tetrahydrobiopterin  are coenzymes, and NO and citrulline are products of the reaction. Three NOS isozymes, each the product of a different gene, have been identified. Two are constitutive (synthesized at a constant rate), calcium (Ca²⁺)–calmodulin (CaM)-dependent enzymes . They are found primarily in endothelium (eNOS) and neural tissue (nNOS) and constantly produce very low levels of NO for vasodilation and neurotransmission. An inducible, Ca²⁺-independent enzyme (iNOS) can be expressed in many cells, including macrophages and neutrophils, as an early defense against pathogens. The specific inducers for iNOS vary with cell type and include proinflammatory cytokines, such as tumor necrosis factor-α(TNF-α) and interferon-γ (IFN-γ), and bacterial endotoxins such as lipopolysaccharide (LPS). These compounds promote synthesis of iNOS, which can result in large amounts of NO being produced over hours or even days.

Figure 1: Synthesis and some actions of nitric oxide (NO). [Note: Flavin mononucleotide, flavin adenine dinucleotide, heme, and tetrahydrobiopterin are additional coenzymes required by NOS.] NADP(H) = nicotinamideadenine dinucleotide phosphate.
2. Nitric oxide and vascular endothelium NO is an important mediator in the control of vascular smooth muscle tone. NO is synthesized by eNOS in endothelial cells and diffuses to vascular smooth muscle, where it activates the cytosolic form of guanylyl cyclase (or, guanylate cyclase) to form cyclic guanosine monophosphate (cGMP). [Note: This reaction is analogous to the formation of cyclic adenosine monophosphate (cAMP) by adenylyl cyclase .] The resultant rise in cGMP causes activation of protein kinase G, which phosphorylates Ca²⁺ channels, causing decreased entry of Ca²⁺ into smooth muscle cells. This decreases the Ca²⁺–CaM activation of myosin light-chain kinase, thereby decreasing smooth muscle contraction and favoring relaxation.
Vasodilator nitrates, such as nitroglycerin, are metabolized to NO, which causes relaxation of vascular smooth muscle and, therefore, lowers blood pressure. Thus, NO can be envisioned as an endogenous nitrovasodilator. [Note: Under hypoxic conditions, nitrite (NO²⁻) can be reduced to NO, which binds to deoxyhemoglobin. The NO is released into the blood, causing vasodilation and increasing blood flow.]
3. Nitric oxide and macrophage bactericidal activity In macrophages, iNOS activity is normally low, but synthesis of the enzyme is significantly stimulated by bacterial LPS and by release of IFN-γ and TNF-α in response to infection. Activated macrophages form radicals that combine with NO to form intermediates that decompose, producing the highly bactericidal OH• radical.
4. Additional functions NO is a potent inhibitor of platelet adhesion and aggregation (by activating the cGMP pathway). It is also characterized as a neurotransmitter in the central and peripheral nervous systems.