INTRODUCTION
Nitric oxide (NO) is a gaseous signaling molecule that readily diffuses across cell membranes and regulates a wide range of physiologic and pathophysiologic processes including cardiovascular, inflammation, immune, and neuronal functions. Nitric oxide should not be confused with nitrous oxide (N2O), an anesthetic gas.
*The author acknowledges the contribution of the previous authors of this chapter, George Thomas, PhD, & Peter Ramwell, PhD.
DISCOVERY OF ENDOGENOUSLY GENERATED NITRIC OXIDE
The first observations of the biologic role of endogenously generated NO were made in rodent macrophages and neutrophils: In vitro exposure of these cells to endotoxin lipopolysaccharide resulted in the accumulation of significant amounts of nitrite and nitrate in the cell culture medium. Furthermore, injection of endotoxin in animals elevated urinary nitrite and nitrate, the two oxidation products of NO.
The second observation was made by investigators in 1980 who found that the ability of acetylcholine to elicit relaxation of isolated strips of rabbit aorta was entirely dependent on the presence of the endothelium. If the endothelium was removed, the vessel still exhibited normal relaxation responses to nitroglycerin, but not to acetylcholine or carbachol. They discovered that following stimulation with acetylcholine or carbachol, the endothelium released a short-lived molecule that resulted in relaxation and dilation of surrounding vascular smooth muscle. The synthesis of this factor was not affected by cyclooxygenase inhibitors, indicating that it was distinct from endothelium-derived prostacyclin. They named this vasodilator endothelium-derived relaxing factor (EDRF), since it promoted relaxation of precontracted smooth muscle preparations. In 1987, by comparing the pharmacologic and biochemical properties of the suspect molecule, three independent groups reported that EDRF and NO are the same molecule. It was later reported that other vasodilator molecules may be a part of EDRF, but it is clear that NO provides the major part of its activity. Subsequent studies revealed that NO was generated by many cells and was, like the eicosanoids (see Chapter 18), found in almost all tissues. The finding that NO is endogenously generated and elicits specific biologic effects explains why pharmacologic reagents that release NO (nitrates, nitrites, nitroprusside; see Chapters 11 and 12) are potent vasodilators.
NITRIC OXIDE SYNTHESIS, SIGNALING MECHANISMS, & INACTIVATION
Synthesis
NO, written as NO· to indicate an unpaired electron in its chemical structure, or simply NO, is a highly reactive signaling molecule that is made in a wide variety of cells, most prominently neurons, skeletal muscle, endothelial cells, and certain immune system cells. In these cells, NO is synthesized by one or more of three closely related NO synthase (NOS, EC 1.14.13.49) isoenzymes, each of which is encoded by a separate gene and named for the initial cell type in which it was isolated (Table 19-1). These enzymes, neuronal NOS (nNOS or NOS-1), macrophage or inducible NOS (iNOS or NOS-2), and endothelial NOS (eNOS or NOS-3), despite their names, are each expressed in a wide variety of cell types, often with an overlapping distribution. These isoforms generate NO from the amino acid L-arginine in an O2- and NADPH-dependent reaction (Figure 19-1). This enzymatic reaction involves enzyme-bound cofactors, including heme, tetrahydrobiopterin, and flavin adenine dinucleotide. In the case of nNOS and eNOS, NO synthesis is evoked by agents and processes that increase cytosolic calcium concentrations. Binding of calcium-calmodulin complexes to eNOS and nNOS leads to enzyme activation. On the other hand, iNOS is not regulated by calcium, but is inducible. In macrophages and several other cell types, inflammatory mediators induce the transcriptional activation of the iNOS gene, resulting in accumulation of iNOS and generation of increased quantities of NO.
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Figure 19-1. Nitric oxide generation from L-arginine and nitric oxide donors and the formation of cGMP. L-NMMA inhibits nitric oxide synthase. Some of the nitric oxide donors such as furoxans and organic nitrates and nitrites require a thiol cofactor such as cysteine or glutathione to form nitric oxide. |
Signaling Mechanisms
NO mediates its effects by covalent modification of proteins. There are three major effector targets of NO (Figure 19-1):
1. Metalloproteins¾ NO interacts with metals, especially iron in heme. Soluble guanylyl cyclase (sGC), an enzyme that generates cyclic GMP from guanosine triphosphate (GTP), contains heme, which binds readily to NO. NO binding to heme results in activation of sGC and elevation in intracellular cGMP levels. cGMP activates protein kinase G (PKG), which phosphorylates specific proteins. NO exerts vasodilatory effects (Chapter 12), which are largely mediated by NO-dependent elevations in cGMP and PKG activity. Several other metalloproteins are targets of NO. The affinity of NO for iron is also responsible for its inhibitory effect on enzymes that contain iron-sulfur clusters such as the tricarboxylic acid cycle enzyme aconitase. NO inhibits mitochondrial respiration by inhibition of cytochrome oxidase. Inhibition of the heme-containing cytochrome P450 enzymes by NO is a major pathogenic mechanism in inflammatory liver disease.
2. Thiols¾ NO reacts with thiols (compounds containing the -SH group) to form nitrosothiols. In proteins, the thiol moiety is found in the amino acid cysteine. Upon exposure to NO, certain proteins are found to accumulate nitrosothiols, which can activate or inhibit the activity of these proteins. This posttranslational modification, termed S-nitrosylation, is reversed by chemical reduction by intracellular reducing agents. The formation of nitrosothiols is not mediated by direct reaction of NO with thiols, but rather requires either metals or oxygen to catalyze the formation of this adduct. Indeed, NO undergoes both oxidative and reductive reactions, resulting in the formation of a variety of oxides of nitrogen that can nitrosylate thiols, nitrate tyrosines (below), or which are stable oxidation products (Table 19-2). H-ras, a regulator of cell proliferation, is activated by S-nitrosylation, while the metabolic enzyme glyceraldehyde-3-phosphate dehydrogenase is inhibited when it is S-nitrosylated. Glutathione, a major intracellular sulfhydryl-containing compound, also interacts with NO under physiologic conditions to generate S-nitrosoglutathione, a more stable form of NO. Nitrosoglutathione may serve as an endogenous long-lived adduct or carrier of NO. Vascular glutathione is decreased in diabetes mellitus and atherosclerosis, and this may account for the increased incidence of cardiovascular complications in these conditions.
3. Tyrosine nitration¾ NO reacts very efficiently with superoxide to form peroxynitrite (ONOO-), a powerful oxidant that leads to DNA damage, irreversible nitration of tyrosine, and oxidation of cysteine to disulfides or to various oxides (SOX). In several diseases, cellular degeneration, due to apoptotic mechanisms or due to ischemia, leads to excess superoxide production, and a consequent increase in peroxynitrite levels. Numerous proteins have been found to contain nitrotyrosines, and this modification can be associated with either activation or inhibition of protein function. However, it is not yet clear whether tyrosine nitration has essential roles in either physiologic signaling or in the pathology of any disease. Protein tyrosine nitration is also used as a marker for the presence of oxidative and nitrosative stress. Peroxynitrite-mediated protein modification is regulated by the cellular content of glutathione, which can protect against tissue damage by scavenging peroxynitrite. Factors that regulate the biosynthesis and decomposition of glutathione may have important consequences on the toxicity of NO.
Inactivation
The lability of NO is related to its rapid reactions with metals and reactive oxygen species. Thus, NO reacts with heme and hemoproteins, including oxyhemoglobin, which catalyzes NO oxidation to nitrate. NO reactions with hemoglobin may also result in partial S-nitrosylation of hemoglobin, resulting in transport of NO throughout the vasculature. NO is also inactivated by superoxide, and scavengers of superoxide anion such as superoxide dismutase may protect NO, enhancing its potency and prolonging its duration of action.
PHARMACOLOGIC MANIPULATION OF NITRIC OXIDE
Inhibitors of Nitric Oxide Synthesis
The primary strategy to inhibit the generation of NO in cells is to use NOS inhibitors. The majority of these inhibitors are arginine analogs that bind to the NOS arginine-binding site. Since each of the NOS isoforms has high sequence similarity, most of these inhibitors do not exhibit selectivity for any of the NOS isoforms. In many disorders, such as inflammation and sepsis (see below), inhibition of the iNOS isoform is desired, whereas in neurodegenerative conditions, nNOS-specific inhibitors are needed. However, administration of nonselective NOS inhibitors leads to concurrent inhibition of eNOS, which impairs its homeostatic signaling and also results in vasoconstriction and potential ischemic damage. Thus, newer NOS isoform-selective inhibitors are being designed that exploit subtle differences in substrate-binding sites between the isoforms, as well as newer inhibitors that prevent NOS dimerization, the conformation required for enzymatic activity. The efficacy of NOS isoform-selective inhibitors in medical conditions is under investigation.
Nitric Oxide Donors
NO donors, which release NO or related NO species, are used to elicit smooth muscle relaxation. Different classes of NO donors have differing biologic properties, related to the nature of the NO species that is released and the mechanism that relates to their release.
1. Organic nitrates¾ Nitroglycerin, which dilates veins and coronary arteries, is metabolized to NO by mitochondrial aldehyde reductase, an enzyme enriched in venous smooth muscle, accounting for the potent venodilating activity of this molecule. Other organic nitrates, such as isosorbide dinitrate are metabolized to an NO-releasing species through a currently unidentified enzyme. Organic nitrates have less significant effects on aggregation of platelets, which appear to lack the enzymatic pathways necessary for rapid metabolic activation. Organic nitrates are limited by the loss of therapeutic effect during continuous administration. This nitrate tolerance may derive from NO-mediated inhibition of mitochondrial aldehyde reductase.
2. Organic nitrites¾ Organic nitrites, such as the volatile antianginal isoamylnitrite, also require metabolic activation to elicit vasorelaxation, although the responsible enzyme has not been identified. Nitrites are arterial vasodilators and do not exhibit the rapid tolerance seen with nitrates.
3. Sodium nitroprusside¾ Sodium nitroprusside, which is used for rapid pressure reduction in arterial hypertension, generates NO in response to light as well as chemical or enzymatic mechanisms in cell membranes. See Chapter 11 for additional details.
4. Hybrid NO donors¾ A new strategy involves the incorporation of NO-donating moieties onto currently available cardiovascular drugs. This approach is being tested with drugs such as aspirin and the angiotensin-converting enzyme inhibitor captopril. SNOCap, which incorporates a nitrosothiol moiety on captopril, is currently being examined for its efficacy in cardiovascular disorders.
5. NO gas inhalation¾ NO itself can be used therapeutically. Inhalation of NO results in reduced pulmonary artery pressure and improved perfusion of ventilated areas of the lung. Inhaled NO has been used for acute respiratory distress syndrome, acute hypoxemia, and cardiopulmonary resuscitation with evidence for short-term improvements in pulmonary function.
6. Alternate strategies¾ Another mechanism to enhance the activity of NO is to enhance the downstream NO signaling pathway. Sildenafil, an inhibitor of type 5 phosphodiesterase, results in prolongation of the duration of NO-induced cGMP elevations in a variety of tissues (see Chapter 12).
NITRIC OXIDE IN DISEASE
VASCULAR EFFECTS
NO has a significant effect on vascular smooth muscle tone and blood pressure. Numerous endothelium-dependent vasodilators, such as acetylcholine and bradykinin, act by increasing intracellular calcium levels, which induces NO synthesis. Mice with a knockout mutation in the eNOS gene display increased vascular tone and elevated mean arterial pressure, indicating that eNOS is a fundamental regulator of blood pressure. The effects of vasopressor drugs are increased by inhibition of NOS.
Apart from being a vasodilator, NO protects against thrombosis and atherogenesis through several mechanisms. A major mechanism involves the inhibition of proliferation and migration of vascular smooth muscle. In animal models, myointimal proliferation following angioplasty can be blocked by NO donors, by NOS gene transfer, and by NO inhalation.
The antithrombotic effects of NO are also mediated by NO-dependent inhibition of platelet aggregation. Both endothelial cells and platelets themselves contain eNOS, which acts to regulate thrombus formation. Thus, endothelial dysfunction and the associated decrease in NO generation may result in abnormal platelet function. As in vascular smooth muscle, cGMP mediates the effect of NO in platelets. NO may have an additional inhibitory effect on blood coagulation by enhancing fibrinolysis via an effect on plasminogen.
NO also reduces endothelial adhesion of monocytes and leukocytes, key features of the early development of atheromatous plaques. This effect is due to the inhibitory effect of NO on the expression of adhesion molecules on the endothelial surface. In addition, NO may act as an antioxidant, blocking the oxidation of low-density lipoproteins and thus preventing or reducing the formation of foam cells in the vascular wall. Plaque formation is also affected by NO-dependent reduction in endothelial cell permeability to lipoproteins. The importance of eNOS in cardiovascular disease is supported by experiments showing increased atherosclerosis in animals deficient in eNOS by pharmacologic inhibition. Atherosclerosis risk factors, such as smoking, hyperlipidemia, diabetes, and hypertension, are associated with decreased endothelial NO production, and thus enhance atherogenesis.
SEPTIC SHOCK
As mentioned previously, increased urinary excretion of nitrate, the oxidative product of NO, is a feature of gram-negative bacterial infection. Lipopolysaccharide components from the bacterial wall induce synthesis of iNOS, resulting in exaggerated hypotension, shock, and, in some cases, death. This hypotension is reversed by NOS inhibitors such as L-NMMA (Table 19-3) in humans as well as in animal models. A similar reversal of hypotension is produced by compounds that prevent the action of NO (such as methylene blue), as well as by scavengers of NO (such as hemoglobin). Furthermore, knockout mice lacking a functional iNOS gene are more resistant to endotoxin than wild-type mice. However, thus far there has been no correlation between the hemodynamic effects of relatively nonselective NOS inhibitors and survival rate in gram-negative sepsis. The absence of benefit may reflect the inability of the NOS inhibitors to differentiate between NOS isoforms or may reflect concurrent inhibition of beneficial aspects of iNOS signaling.
INFLAMMATION
The host response to infection or injury involves the recruitment of leukocytes and the release of inflammatory mediators, including NO. Numerous cytokines, such as tumor necrosis factor and interleukin-1, as well as bacterial-derived mediators, induce the transcription of iNOS in leukocytes, fibroblasts, and other cell types, accounting for enhanced levels of NO. NO is an important microbicide and may have important roles in tissue adapting to inflammatory states. However, overproduction of NO may exacerbate tissue injury in both acute and chronic inflammatory conditions. NO generated during inflammation is involved in the vasodilation associated with acute inflammation and can interact with superoxide to generate peroxynitrite and subsequently modify proteins, lipids, and nucleotides. In experimental models of acute inflammation, inhibitors of iNOS have a dose-dependent protective effect, suggesting that NO promotes edema and vascular permeability. NO has a detrimental effect in chronic models of arthritis; dietary L-arginine supplementation exacerbates arthritis, whereas protection is seen with iNOS inhibitors. Psoriasis lesions, airway epithelium in asthma, and inflammatory bowel lesions in humans all demonstrate elevated levels of NO and iNOS. Synovial fluid from patients with arthritis contains increased oxidation products of NO, particularly peroxynitrite. Recent studies have shown that NO stimulates the synthesis of inflammatory prostaglandins by activating cyclooxygenase isoenzyme II (COX-2). Thus, inhibition of the NO pathway may have a beneficial effect on inflammatory diseases, including joint diseases.
However, NO also appears to play an important protective role in the body via immune cell function. When challenged with foreign antigens, TH 1 cells (see Chapter 56) respond by synthesizing NO. Inhibition of NOS and knockout of the iNOS gene can markedly impair the protective response to injected parasites in animal models.
THE CENTRAL NERVOUS SYSTEM
NO has been proposed to have a major role in the central nervous system¾as a neurotransmitter, as a modulator of ligand-gated receptors, or both. NO synthesis is induced at postsynaptic sites in neurons upon activation of the NMDA subtype of glutamate receptor, which results in calcium influx and activation of nNOS. In several neuronal subtypes, eNOS is also present and activated by neurotransmitter pathways that lead to calcium influx. NO synthesized postsynaptically may function as a retrograde messenger and diffuse to the presynaptic terminal to enhance the efficiency of neurotransmitter release through a cGMP or S-nitrosylation-dependent mechanism. It has been suggested that a major role for NO is in the regulation of synaptic plasticity, the molecular process that underlies learning and behavior.
THE PERIPHERAL NERVOUS SYSTEM
Nonadrenergic, noncholinergic (NANC) neurons are widely distributed in peripheral tissues, especially the gastrointestinal and reproductive tracts (see Chapter 6). Considerable evidence implicates NO as a mediator of certain NANC actions, and some NANC neurons appear to release NO. Penile erection is thought to be caused by the release of NO from NANC neurons; it is well documented that NO promotes relaxation of the smooth muscle in the corpora cavernosa¾the initiating factor in penile erection¾and inhibitors of NOS have been shown to prevent erection caused by pelvic nerve stimulation in the rat. Thus, impotence is a possible clinical indication for the use of a NO donor, and trials have been carried out with nitroglycerin ointment and the nitroglycerin patch. An established approach is to inhibit the breakdown of cGMP by the phosphodiesterase (PDE isoform 5) present in the smooth muscle of the corpora cavernosa with drugs such as sildenafil (see Chapter 12).
RESPIRATORY DISORDERS
NO has been shown to improve cardiopulmonary function in adult patients with pulmonary artery hypertension and is approved for this indication (see Preparations Available). It is administered by inhalation. It has also been administered by inhalation to newborns with pulmonary hypertension and acute respiratory distress syndrome. The current treatment for severely defective gas exchange in the newborn is with extracorporeal membrane oxygenation (ECMO), which does not directly affect pulmonary vascular pressures. NO inhalation decreases pulmonary arterial pressure and improves blood oxygenation. Thus, when pulmonary resistance is elevated, it is possible to exploit the vasodilator properties of NO by administering it via inhalation of a few parts per million. Adults with respiratory distress syndrome also appear¾in open trials¾to benefit from NO inhalation. NO may have an additional role in relaxing airway smooth muscle and thus acting as a bronchodilator. For these reasons, NO inhalation therapy is being widely tested in both infants and adults with acute respiratory distress syndrome. The adverse effects of this use of NO are being assessed.
PREPARATIONS AVAILABLE
Nitric Oxide (INOmax)
Inhalation: 100, 800 ppm gas
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