First printed in R&D Systems' 2000 Catalog.
The role of nitric oxide (NO) as a biological signaling and effector molecule was virtually unknown until the late 1980s. The seemingly ubiquitous involvement of this molecule has resulted in an explosion of interest in the field resulting in more than twenty thousand publications in the last five years. This review is intended to provide a brief introduction to NO biology and will touch upon only a limited number of the most well-established areas in the field.
NO is a short-lived molecule (t1/2=seconds) capable of diffusing across membranes and reacting with a variety of targets. Reaction with O2 in aqueous solutions produces the relatively unreactive nitrate (NO3-) and nitrite (NO2-) ions as products.1-4 In the presence of superoxide (O2-), however, NO reacts extremely rapidly to produce the very reactive and toxic peroxynitrite (ONOO-) which subsequently decomposes into additional highly reactive intermediates. NO ultimately exerts its biological effects by reacting either directly or through other reactive nitrogen intermediates with a variety of targets such as heme groups, Fe-S or Zn-S clusters, sulfhydryl groups or various other chemical substrates.2-8 This diversity of potential targets is reflected in the large number of different systems that utilize NO as a mediator and provides ample opportunity for abnormal regulation and development of pathological effects.
|Figure 1. Nitric oxide synthases catalyze the production of NO and L-citrulline from L-arginine, O2, and NADPH-derived electrons.|
Nitric oxide is produced by a group of enzymes called nitric oxide synthases (NOS).9-13 These enzymes catalyze the production of NO and L-citrulline from L-arginine, O2, and NADPH-derived electrons. Mammalian systems contain three well-characterized isoforms of the enzyme: neuronal NOS (nNOS, also called NOS1), inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3). The names reflect characteristics of the activity or the original tissues in which the enzymes were first described, but it is now known that each of these isoforms is expressed in a variety of tissues and cell types.14-15
The three main isoforms share structural similarities and have nearly identical catalytic mechanisms.9-13 They all require a number of cofactors and prosthetic groups for activity including FAD, FMN, heme, calmodulin, and tetrahydrobiopterin. The homodimeric form is required for NO production, and the subunits have molecular masses of approximately 160 (nNOS), 135 (eNOS), and 130 kDa (iNOS). Three distinct domains are necessary for catalytic activity. Starting at the C-terminus there is 1) a reductase domain, 2) a calmodulin-binding domain, and 3) an oxygenase domain. The reductase domain contains the FAD and FMN moieties and shares extensive amino acid (aa) homology with cytochrome P-450 reductase. This domain transfers electrons from NADPH to the oxygenase domain. The oxygenase domain actually catalyzes the conversion of arginine into citrulline and NO and contains the binding sites for heme, tetrahydrobiopterin, and arginine. Calmodulin binding is required for activity of all of the NOS isoforms. This cofactor normally functions as a sensor of intracellular Ca2+ concentration exerting its effect only in response to elevated intracellular Ca2+ levels. Calmodulin functions differently in each of the NOS isoforms as described below.
The NOS isoforms display a number of differences related to their individual functions. Transcriptional regulation and post-translational regulation of catalytic activity is distinct for each isoform. Isoform-specific domains direct subcellular localization thus influencing the functional significance of the enzyme. The discussion below will very briefly address some of the unique properties and functions of each isoform.
nNOS is expressed in the central and peripheral nervous systems and in skeletal muscles.16-19 In the brain, NO acts as a neuromodulator to influence functions such as behavior and memory formation. In the peripheral nervous system, NO acts as a neurotransmitter participating in functions such as smooth muscle control, gastrointestinal motility, and neuroendocrine function. NO functions in skeletal muscles as a signal transducer to regulate both metabolism and muscle contractility.
Although nNOS is usually reported to be a constitutive enzyme, its expression is developmentally regulated and also influenced by certain physiologic and pathophysiologic stimuli such as shear stress and nerve injury.20,21 The functions attributed to nNOS require rapid and localized production of NO and timely termination of biosynthesis. The primary form of regulation is mediated through the obligatory binding of calmodulin, which occurs only in response to transient increases in intracellular Ca2+. Thus, nNOS generates NO in short bursts following appropriate activation signals. The physiological concentrations of NO generated by this isoform are in the picomolar range.22
A 300 aa segment at the amino terminus of classical nNOS is not shared with the other isoforms and functions in the specific regulation of nNOS.9,10,16,19 This domain contains a PDZ motif that binds to several other proteins and directs nNOS to sites of signal transduction. In neuronal tissues, Ca2+ influx triggered by the N-methyl-D-aspartate (NMDA) receptors is an important inducer of nNOS activity. Association of nNOS with the NMDA receptors occurs through the interaction of the PDZ motif with the postsynaptic density proteins PSD-93 and PSD-95, which in turn bind to the NMDA receptors. In skeletal muscle, nNOS is anchored to the sarcolemma via association of its PDZ motif with the syntrophin protein. In this location, nNOS is activated by membrane depolarization and thereby exerts its control on muscle contractility. The amino-terminal segment of nNOS also has a binding site for a widely-expressed and highly conserved protein called PIN (protein inhibitor of NO synthase) that inhibits catalytic activity by destabilizing homodimer formation. Differences within this amino-teminal domain also result from a number of distinct mRNA splice variants that are expressed selectively in certain tissues.19 The significance of these splice variants is poorly understood at this time.
Inappropriate regulation of nNOS has been implicated in a number of neurologic diseases. Neuronal injury resulting from stroke and certain neurotoxins are known to be caused by release of excess glutamate which causes neurotoxicity upon activation of NMDA receptors.16-18 The role of NO was clearly demonstrated when cultured neurons from nNOS knockout mice were shown to be resistant to glutamate toxicity. These knockout mice also have much less tissue damage in response to focal ischemia (reduced blood supply). Neurotoxicity appears to result from the action of superoxide and peroxynitrite as evidenced by results with superoxide dismutase knockout mice and transgenic mice that overexpress superoxide dismutase. The source of superoxide could be nNOS itself. Unlike the other isoforms, the reductase activity of nNOS can be uncoupled from NO production under certain conditions and catalyze the reduction of O2 to superoxide.9 Some evidence suggests that nNOS may also be involved in neurodegenerative diseases such as Huntington’s disease and Parkinson’s disease.16,17
The earliest medical applications of NO relate to the function of eNOS in the cardiovascular system. Nitroglycerin was first synthesized by Alfred Nobel in the 1860s, and this compound was eventually used medicinally to treat chest pain. The mechanism by which nitrovasodilators relax blood vessels was not well defined but is now known to involve the NO signaling pathway.
Cells that express eNOS include vascular endothelial cells, cardiomyocytes and others.13,14 In blood vessels, NO produced by the eNOS of endothelial cells functions as a vasodilator thereby regulating blood flow and pressure.17,24 Mutant eNOS knockout mice have blood pressure that is 30% higher than wild-type littermates.17 Within cardiomyocytes, eNOS affects Ca2+ currents and contractility.25 Expression of eNOS is usually reported to be constitutive though modest degrees of regulation occur in response to factors such as shear stress, exercise training, chronic hypoxia, and heart failure.21,26
The unique N-terminal sequence of eNOS is about 70 residues long and functions to localize the enzyme to membranes.13,27 Upon myristoylation at one site and palmitoylation at two other sites within this segment, the enzyme is exclusively membrane-bound.13 Palmitoylation is a reversible process that is influenced by some agonists and is essential for membrane localization.28 Within the membrane, eNOS is targeted to the caveolae, small invaginations characterized by the presence of proteins called caveolins. These regions serve as sites for the sequestration of signaling molecules such as receptors, G proteins and protein kinases. The oxygenase domain of eNOS contains a motif that binds to caveolin-1, and calmodulin is believed to competitively displace caveolin resulting in eNOS activation.21 Bound calmodulin is required for activity of eNOS, and this binding occurs in response to transient increases in intracellular Ca2+. 9,10 Thus, eNOS occurs at sites of signal transduction and produces short pulses of NO in response to agonists that elicit Ca2+ transients. Physiological concentrations of eNOS-derived NO are in the picomolar range.22
Within the cardiovascular system, eNOS generally has protective effects. Studies with nNOS and eNOS knockout mice clearly indicate that eNOS plays a protective role in cerebral ischemia by preserving cerebral blood flow.17 During inflammation and atherosclerosis, low concentrations of NO prevent apoptotic death of endothelial cells and preserve the integrity of the endothelial cell monolayer.29,30 Likewise, NO also acts as an inhibitor of platelet aggregation, adhesion molecule expression, and vascular smooth muscle cell proliferation.24,31,32 Therefore, eNOS-related pathologies usually result from impaired NO production or signaling. Altered NO production and/or bioavailablility have been linked to such diverse disorders as hypertension, hypercholesterolemia, diabetes, and heart failure.26
Isolation of iNOS was first reported in macrophages where its activity was found to be inducible in response to stimuli such as proinflammatory cytokines or endotoxin. Expression of iNOS has now been reported in a large number of cell types, and in most circumstances, the enzyme is inducible.14,15 The iNOS gene is under transcriptional control although activity is also influenced by a variety of other control mechanisms that affect mRNA stability, translation and degradation of the protein, and availability of substrate and cofactors.9,10,13,14 This enzyme is found in the cytoplasm.
All NOS isoforms require bound calmodulin for activity; however, iNOS is unusual in that it binds calmodulin tightly even at very low Ca2+ concentrations.9,10 iNOS activity is not responsive to changes in intracellular Ca2+ levels and thus this isoform is capable of a high output and long-lasting release of NO far exceeding that of the other isoforms. iNOS produces physiological concentrations of NO in the nanomolar range whereas the Ca2+-dependent isoforms produce picomolar concentrations of NO.22
One of the best described functions of iNOS is its role in the macrophage-mediated response to infectious agents.33,34 While there is evidence that NO plays a regulatory role in the immune system signaling cascade, macrophage-derived NO has been more clearly defined as an effector molecule that kills or inactivates target cells. The effect is probably mediated by a number of highly reactive nitrogen and oxygen intermediates. The phagocyte NADPH oxidase produces superoxide which rapidly combines with NO to yield peroxynitrite and several other toxic decay products.33 These reactive intermediates were shown to cause a variety of DNA alterations including deamination and strand breaks. Reactive nitrogen intermediates also cause protein modifications that involve nitrosylation of cysteine and tyrosine residues and inactivation of metal-containing enzymes. These modifications affect a number of metabolic enzymes and membrane transporters leading to cytostatic or cytotoxic effects against some invading pathogens. The cytotoxic effect of NO has also been demonstrated against cancer cells as well as the macrophages that produce NO.35 These mammalian cell targets are killed by an apoptotic mechanism, but the exact signaling pathway leading to cell death is not understood.
The overproduction of NO by iNOS is implicated in a number of pathologies. Septic shock, which is often fatal, is caused by the overactivation of macrophages in response to bacterial infections in the blood.15 The resulting overproduction of NO leads to a severe drop in blood pressure and subsequent dysfunction of multiple organs. NO generated by iNOS is present in a number of inflammatory conditions including rheumatoid arthritis, Crohn’s disease and asthma.24 Because of its role as both an immune mediator and an effector molecule, NO can have deleterious or beneficial roles in inflammatory conditions depending on the setting.7,34
NO seems to be everywhere exerting its biological functions. The unique chemical properties of NO permit it to act in very diverse ways, in many cell types, and without mutual interference. Enormous strides have been made in the last ten years in the field of NO biology, and yet a vast amount or work remains to reach a full understanding of NO and its multitude of effects.