Nitric Oxide: A Physiologic Messenger

1 February 1994 | Volume 120 Issue 3 | Pages 227-237

Purpose: To review the physiologic role of nitric oxide, an unusual messenger molecule that mediates blood vessel relaxation, neurotransmission, and pathogen suppression.

Data Sources: A MEDLINE search of articles published from 1987 to 1993 that addressed nitric oxide and the enzyme that synthesizes it, nitric oxide synthase.

Study Selection: Animal and human studies were selected from 3044 articles to analyze the clinical importance of nitric oxide. Descriptions of the structure and function of nitric oxide synthase were selected to show how nitric oxide acts as a biological messenger molecule.

Data Extraction: Biochemical and physiologic studies were analyzed if the same results were found by three or more independent observers.

Data Synthesis: Two major classes of nitric oxide synthase enzymes produce nitric oxide. The constitutive isoforms found in endothelial cells and neurons release small amounts of nitric oxide for brief periods to signal adjacent cells, whereas the inducible isoform found in macrophages releases large amounts of nitric oxide continuously to eliminate bacteria and parasites. By diffusing into adjacent cells and binding to enzymes that contain iron, nitric oxide plays many important physiologic roles. It regulates blood pressure, transmits signals between neurons, and suppresses pathogens. Excess amounts, however, can damage host cells, causing neurotoxicity during strokes and causing the hypotension associated with sepsis.

Conclusions: Nitric oxide is a simple molecule with many physiologic roles in the cardiovascular, neurologic, and immune systems. Although the general principles of nitric oxide synthesis are known, further research is necessary to determine what role it plays in causing disease.

A noxious, unstable gas is an unlikely candidate to act as a biological messenger. However, in the last 7 years, nitric oxide, a byproduct of automobile exhaust, electric power stations, and lightning, was discovered in the body, where it participates in various functions, including suppression of pathogens, vasodilation, and neurotransmission [1-8]. We describe what is known about nitric oxide, focusing on its clinical relevance.

Unusual Properties of Nitric Oxide Metabolism

Nitric oxide is an unusual messenger. It is a small molecule composed of one atom each of nitrogen and oxygen, not to be confused with nitrous oxide, N_2O, an inhalational anesthetic commonly referred to as laughing gas. Nitric oxide is an uncharged molecule with an unpaired electron. These characteristics of nitric oxide make it an ideal messenger molecule: Uncharged, nitric oxide can diffuse freely across membranes. With an unpaired electron, it is called a radical molecule, which is highly reactive (having a half-life of 2 to 30 seconds); after transmitting a signal spontaneously, it decays into nitrite.

Nitric oxide is made by nitric oxide synthase in an unusual reaction that converts arginine and oxygen into citrulline and nitric oxide. The mechanism of nitric oxide synthesis is not completely understood, but it involves the transfer of electrons between various cofactors, including flavin adenine dinucleotide, flavin mononucleotide, nicotinamide adenine dinucleotide phosphate (NADPH), tetrahydrobiopterin, and heme. Finally, one atom of oxygen from oxygen binds with the terminal guanidine nitrogen from arginine to form nitric oxide [9, 10].

Although several nitric oxide synthase isoforms have been isolated [11-17], all are homologous and divided into two categories with different regulation and activities (Figure 1). The constitutive isoforms in neuronal or endothelial cells are always present [11, 18]. These nitric oxide synthase isoforms are inactive until intracellular calcium levels increase, the calcium-binding protein calmodulin binds to calcium, and the calcium-calmodulin complex binds to and activates nitric oxide synthase [19-22]. The constitutive nitric oxide synthase isoforms then synthesize small amounts of nitric oxide until calcium levels decrease. This intermittent production of small amounts of nitric oxide transmits signals. In contrast, the inducible nitric oxide synthase isoform is normally absent from macrophages and hepatocytes, but when these cells are activated by specific cytokines, an inducible nitric oxide synthase enzyme is produced; once produced, it always synthesizes large amounts of nitric oxide. Induced nitric oxide synthase is transcriptionally regulated [16, 23]. The continuous production of large amounts of nitric oxide kills or inhibits pathogens.

View larger version (22K): [in this window] [in a new window]   Figure 1. Diagrammatic representation of the structure of cloned forms of nitric oxide synthase with sites for cofactor binding. CAL = binding site for calmodulin; FMN = flavin mononucleotide; FAD = flavin adenine dinucleotide; NADPH = reduced form of nicotinamide adenine dinucleotide phosphate; P = site for phosphorylation by cyclic adenosine monophosphate-dependent protein kinase. The sites for heme, arginine, and tetrahydrobiopterin binding are unknown.

 

Other mechanisms of regulating nitric oxide synthase enzymes recently were discovered. Neuronal nitric oxide synthase can be phosphorylated by protein kinases to decrease its activity [24]. The subcellular location of nitric oxide synthase in endothelial cells can also be changed by its phosphorylation [25]. Although the constitutive isoforms are regulated by calcium, the inducible isoforms also appear to bind calmodulin, although calcium has little effect on their activity [26]. The role of calmodulin in inducible nitric oxide synthase function is unknown.

Most molecules that transmit signals between cells, such as hormones, neurotransmitters, and growth factors, act through specific protein receptors that are often associated with the plasma membrane. In contrast, nitric oxide diffuses out of the cell that generates it and into target cells, where it interacts with specific molecular targets (Table 1). The best-characterized receptor of nitric oxide is iron, contained in certain proteins as a heme group or as an iron-sulfur complex. Nitric oxide exerts some of its effects by binding to iron-containing enzymes and either activating or inactivating the enzymes. When nitric oxide binds to the iron in the heme group of guanylate cyclase, the enzyme is activated. Guanylate cyclase then produces cyclic guanosine monophosphate (cGMP), and the increase in cGMP activates other cellular processes (Figure 2). By changing the activity of guanylate cyclase, nitric oxide dilates arteries, signals neurons, and kills cells.

View this table: [in this window] [in a new window]   Table 1. Molecular Targets of Nitric Oxide*

 

View larger version (17K): [in this window] [in a new window]   Figure 2. Nitric oxide arterial smooth muscle. A messenger molecule such as acetylcholine binds to the acetylcholine receptor on an endothelial cell, activating inward calcium currents. Calcium binds to calmodulin and activates endothelial cell nitric oxide synthase, which converts arginine plus oxygen into citrulline and nitric oxide. Nitric oxide diffuses out of the endothelial cell into an adjacent smooth muscle cell and activates guanylate cyclase by binding to the iron in its heme group. The increase in cyclic guanosine monophosphate (cGMP) causes smooth muscle relaxation, and thus vasodilation. GTP = guanosine triphosphate.

 

Another unusual way in which nitric oxide affects cells is by facilitating transfer of an ADP-ribose group to an accepting molecule (a process called ADP ribosylation). Normally an ADP-ribose group is attached to a protein target by an enzyme, for example, when cholera toxin ADP-ribosylates a guanosine triphosphate-binding protein. However, when nitric oxide diffuses into a cell, it can cause auto-ADP ribosylation, that is, ADP ribosylation of a target without enzyme catalysis. For example, nitric oxide inactivates the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase by attaching an ADP-ribose group to it, thereby blocking the production of adenosine triphosphate from glycolysis [27-32].

Nitric Oxide in the Cardiovascular System

Although the effect of exogenously administered nitrates on the vasculature has been studied for decades, the first clue that endothelial cells released a substance that could cause vasodilation was found only recently. In 1980, Furchgott and Zawadski [33] discovered that sections of the aorta would relax in response to agonists only if the inner linings of endothelial cells were intact. However, aortic rings with no endothelial cells could not relax. Endothelial cells thus released an agent that relaxed vascular smooth muscle; within several years, it was discovered that this "endothelial-derived relaxation factor" was nitric oxide [34].

The Role of Nitric Oxide in Mediating an Active State of Vasodilation

The vasculature is in a constant state of active dilation mediated by nitric oxide. Endothelial cells continuously release small amounts of nitric oxide, producing a basal level of vascular smooth muscle relaxation. When inhibitors of nitric oxide synthase, such as nitro-arginine methyl ester or n-mono-methyl-arginine, are infused into animals [35-44] or humans [45], nitric oxide production is inhibited, vascular smooth muscle contracts, and blood pressure increases.

Nitric oxide dilates blood vessels by directly relaxing vascular smooth muscle cells (see Figure 2). An agonist such as acetylcholine binds to its receptor on endothelial cells, causing a transient increase in intracellular calcium. Calcium binds to calmodulin, and the calcium-calmodulin complex activates endothelial nitric oxide synthase, which makes nitric oxide. Nitric oxide then diffuses out of the endothelial cell and into adjacent smooth muscle cells, where it binds to the heme group of guanylate cyclase. Guanylate cyclase is activated to produce cGMP, which, through a cascade of protein kinases, induces smooth muscle relaxation. Because the half-life of nitric oxide in biological fluids is between 2 and 30 seconds, the effect of nitric oxide spontaneously diminishes and the vessel constricts unless more nitric oxide is produced.

Nitric Oxide: An Autoregulator and Neural Regulator of Blood Flow

Nitric oxide automatically regulates blood flow in response to local changes in some regions of the vasculature. Ischemia and reperfusion cause vasodilation only in the affected tissue, and this response is mediated by nitric oxide. For example, a study of ischemia in patients' arms and legs showed an increase in nitric oxide production [46]. However, whereas some investigators found that ischemia and reperfusion increase nitric oxide production in animals [47, 48], others observed a decrease in nitric oxide [49-51]. Shear-stress, an increase in blood flow through a vessel, is another physical stimulus to which endothelial cells respond by increasing nitric oxide production [52, 53]. In animal studies, increases in blood flow but not pressure caused an increase in nitric oxide production and dilation in excised hearts and vessels [54-59]. Factors locally released by adjacent tissue, such as bradykinin or acetylcholine, can also induce nitric oxide release in some vessels but not in others. A basal level of nitric oxide regulates blood flow in the brain [60-62], heart [59, 63-67], lung [68], gastrointestinal tract [69], and kidney [38, 42, 43, 70]. Thus, nitric oxide is an endogenous autoregulator of blood flow.

The release of nitric oxide into the vasculature is also controlled by the autonomic nervous system. Parasympathetic nerves containing nitric oxide synthase terminate in the adventitia of certain large vessels, such as the cerebral and retinal arteries [71]. Nitric oxide is released from the nerves and diffuses into the muscular media from the outside of the vessel, causing vasorelaxation.

The Role of Nitric Oxide in Hypertension and Vasospasm

Because nitric oxide plays a central role in regulating blood pressure, defects in the regulation of nitric oxide synthase could lead to vasospasm or hypertension, although this has not been proved. Clearly the endothelium is abnormal in persons with hypertension of unknown cause ("essential hypertension"), because acetylcholine causes less vasodilation in hypertensive than in healthy persons [72-74]. In contrast, the vascular smooth muscle is normal in persons with hypertension because intravenous nitroprusside, which releases nitric oxide, causes equal amounts of vasodilation in both hypertensive and healthy persons [74, 75]. Abnormal endothelial nitric oxide synthase could cause this defective response to acetylcholine in persons with hypertension. In fact, the blood pressure of healthy persons increases when nitric oxide synthase inhibitors are infused, but the blood pressure of persons with hypertension changes much less [74, 75]. These studies imply that persons with hypertension produce less nitric oxide. However, direct measurement in hypertensive persons' arteries shows normal quantities of nitric oxide synthase (Personal communication. Michel T, Brigham and Women's Hospital, Boston, Massachusetts). Perhaps endothelial nitric oxide synthase in persons with hypertension has an abnormal structure or regulation.

Endothelial dysfunction is characteristic of other diseases, including diabetes and atherosclerosis [76-80]. Some atherosclerotic arteries paradoxically constrict when the vasodilator acetylcholine is infused. Defects in the cascade of events leading to production of nitric oxide might cause less relaxation. Nitric oxide not only regulates the systemic blood pressure but also the local blood flow to specific vascular beds [81], including the brain [60-62], kidney [38, 42, 43, 70], lung [68], heart [59, 63, 64, 66, 67], and gastrointestinal tract [69]. A local defect in nitric oxide production could cause vasospasm in particular organs. Perhaps nitric oxide is involved in hepatorenal syndrome, Prinzmetal angina, Raynaud disease, or preeclampsia.

However, nitric oxide is not the only vasodilator, and defects in other relaxation pathways could also cause endothelial dysfunction and contractile abnormalities. Agonists such as acetylcholine, bradykinin, and thrombin bind to endothelial cells, inducing the release of vasodilators including nitric oxide, endothelial-derived hyperpolarizing factor, and prostacyclin. These compounds relax smooth muscle cells in different ways. Nitric oxide activates guanylate cyclase. Endothelial-derived hyperpolarizing factor activates a potassium channel, and the potassium flux hyperpolarizes and thus relaxes the smooth muscle cell. Prostacyclin can activate both adenylate cyclase and guanylate cyclase, thereby causing an increase in cyclic adenosine monophosphate and cGMP and ultimately relaxation. Another endothelial cell agonist is lysophosphatidylcholine, a component of oxidized low-density lipoprotein that activates an endothelial pathway that leads to relaxation of smooth muscle. This pathway probably does not involve nitric oxide [82-84] but rather an endothelial cell-surface receptor coupled to a guanosine triphosphate-binding protein. Lysophosphatidylcholine disrupts the interaction of this receptor and its coupled guanosine triphosphate-binding protein [85].

Although no diseases have been described in which excessive amounts of nitric oxide are produced, rare cases of abnormally low blood pressure, such as idiopathic orthostatic hypotension, may be associated with overproduction of nitric oxide. As discussed below, nitric oxide mediates the hypotension of sepsis. It may be involved in local shunting of blood through the liver in cirrhosis [86, 87], although some studies have not found excessive amounts of nitric oxide in the cirrhotic liver [88, 89].

Effects of Nitric Oxide on Cardiac Contractility and Platelet Clotting

Nitric oxide influences areas of the cardiovascular system besides blood vessels. It exerts a negative chronotropic effect on the heart and a negative inotropic effect on cardiac muscle cells similar to its relaxation of smooth muscle cells. Our laboratory has shown that nitric oxide synthase can be induced during viral myocarditis (unpublished data). Reversible left ventricular dysfunction known to occur during myocarditis may be caused by an excess of nitric oxide that relaxes cardiac muscle and resolves as nitric oxide synthase produces less nitric oxide.

Nitric oxide reduces clotting by acting directly on platelets. The nitric oxide produced by endothelial cells can diffuse not only toward the vessel wall but also into the vessel lumen, where it enters platelets. Nitric oxide synthase has also been found inside platelets [90]. It reduces clotting by inhibiting platelet aggregation [91-95] and adhesion [96-101]. The molecular basis of this action is unknown, but possible mechanisms include nitric oxide activating platelet guanylate cyclase and adenylate cyclase [99, 102-106], inhibiting platelet phospholipase C [107], or attaching ADP-ribose to platelet glyceraldehyde 3-phosphate dehydrogenase [108]. Thus, endothelial generation of nitric oxide not only regulates blood pressure but also blood clotting.

Nitric Oxide: An Immune Effector Capable of Eliminating Many Pathogens

In contrast to the neuronal and endothelial nitric oxide synthase, the inducible macrophage nitric oxide synthase produces a large amount of nitric oxide. In large quantities, nitric oxide kills almost any nearby cell. It kills or inhibits the growth of many pathogens, including bacteria [109-113], fungi [114], and parasites [115-122] (Table 2). In particular, nitric oxide eliminates intracellular pathogens such as Mycobacterium tuberculosis, and recent studies from our laboratory (unpublished) and others [123, 124] suggest that nitric oxide blocks viral replication as well. Nitric oxide also kills tumor cells [125]. In addition, nitric oxide might damage normal host cells in autoimmune diseases in which nitric oxide synthase is inappropriately induced. Thus, it is a nonspecific effector capable of killing or inhibiting the growth of many targets, both pathogens and host cells.

View this table: [in this window] [in a new window]   Table 2. Pathogens and Cellular Targets of Nitric Oxide*

 

Mechanisms whereby Nitric Oxide Kills Its Targets

Because nitric oxide has so many molecular targets, it can damage cells in many ways, although precisely how it does so is unclear. Nitric oxide inhibits adenosine triphosphate production at three separate steps, blocking glycolysis by transferring ADP-ribose to glyceraldehyde-3-phosphate dehydrogenase, disrupting the Krebs cycle by binding to the heme group of cis-aconitase, and inhibiting oxidative phosphorylation by binding to the heme group of ubiquinone reductase. Nitric oxide inhibits DNA synthesis by inactivating ribonucleotide reductase [126, 127] and can damage DNA directly by deamination [128]. While continuously secreting large amounts of nitric oxide, activated macrophages avoid the toxic effects of nitric oxide by mechanisms that are also unknown.

Transcriptional Regulation of Macrophage Nitric Oxide Synthase

The production of nitric oxide must be controlled precisely, because excessive amounts could damage the host and too little could lead to immunodeficiency. In contrast to the neuronal and endothelial nitric oxide synthase enzymes that are always present but only active when intracellular calcium levels increase, nitric oxide synthase is normally absent in quiescent macrophages; however, once it is synthesized it is always active. Macrophage nitric oxide synthase is thus transcriptionally regulated [16, 23]. The sequence of signals leading to nitric oxide production begins with an infection that induces immune cells to release cytokines, which in turn signal macrophages to make nitric oxide synthase (Figure 3). For example, a viral infection induces CD4⁺ helper T lymphocytes or natural killer lymphocytes to produce interferon-, or lipopolysaccharide, from the cell wall of infecting bacteria cause cells to make tumor necrosis factor-. Specific combinations of cytokines, such as interferon- along with either tumor necrosis factor- or interleukin-1 ß, activate intracellular transcription factors inside macrophages, such as nuclear factor- B, to initiate production of nitric oxide synthase [129, 130]. Nuclear factor- B itself can be activated by oxygen radicals, suggesting a novel positive feedback loop in which nitric oxide might activate transcription factors, which in turn could increase nitric oxide synthesis by causing additional nitric oxide synthase expression [131]. Once synthesized, macrophage nitric oxide synthase produces nitric oxide continuously. In fact, macrophages activated in culture can make nitric oxide synthase and produce nitric oxide for days, but macrophages activated in vivo must eventually curtail nitric oxide production to avoid damaging the host. This is probably achieved by other inhibitory cytokines, such as transforming growth factor-ß [132-136] and mRNA-destabilizing sequences [12, 137].

View larger version (17K): [in this window] [in a new window]   Figure 3. Induction of nitric oxide synthase in macrophages. Lipopolysaccharide (LPS) is released from the wall of infecting bacteria, causing host cells to release tumor necrosis factor- (TNF-). Tumor necrosis factor- binds to macrophages and activates intracellular proteins such as nuclear factor- B (NF- B), which induce the transcription of nitric oxide synthase. Once synthesized, macrophage nitric oxide synthase continuously makes nitric oxide, which can kill bacteria. MAC-NOS = macrophage nitric oxide synthase.

 

Nitric Oxide in the Human Immune System

The role of nitric oxide in the immune system is better defined in animals than in humans. Although nitric oxide synthase can be induced easily in murine macrophages (and also in murine neutrophils, hepatocytes, fibroblasts, glial cells, smooth muscle cells, and endothelial cells [138-144]), it cannot be induced reproducibly in human macrophages. However, nitric oxide synthase can be induced in human hepatocytes in culture. The best evidence to show that humans make inducible nitric oxide synthase comes from clinical trials in which patients who were given intravenous interleukin-2 excreted nitric oxide byproducts in their urine [145, 146]. Perhaps inhibitory cytokines, such as transforming growth factor-ß, are present more often in humans than in animals, thus preventing the widespread induction of nitric oxide synthase. However, because nitric oxide synthase can be induced in so many different types of animal cells, every human cell might also induce nitric oxide synthase given the correct set of cytokines.

The Role of Nitric Oxide in Mediating Septic Hypotension

We would expect excessive amounts of nitric oxide to cause hypotension, and it appears to be responsible for the hypotension associated with sepsis. During bacterial infections, the lipopolysaccharide component released from bacterial walls stimulates production of the inducible isoform of nitric oxide synthase in many cells, including macrophages, hepatocytes, and endothelial cells. Release of massive amounts of nitric oxide into the blood stream overwhelms the arterial smooth muscle and causes excess dilation and hypotension. Treatment of sepsis in animals with inhibitors of nitric oxide synthase reverses hypotension within minutes [147-149]. A few patients with sepsis have been treated with nitric oxide synthase inhibitors. Their mean arterial pressure increased from about 50 to 90 mm Hg within minutes as their systemic vascular resistance doubled [150, 151], which is consistent with the hypothesis that excessive nitric oxide causes hypotension during sepsis. However, the patients died soon thereafter, despite treatment, because the course of sepsis clearly involves much more than nitric oxide-induced hypotension.

Immunodeficiency syndromes in which patients cannot produce nitric oxide have not been identified. This is in contrast to chronic granulomatous disease, in which patients are susceptible to bacterial infections because they cannot produce another molecule with an unpaired electron, superoxide [152, 153]. One case report identified high levels of an arginine analog in the serum of a patient with renal failure [154]. Perhaps an excess of this compound inhibits nitric oxide synthase, causing the hypertension and immunodeficiency often seen with chronic renal failure. It is tempting to speculate that the immune system of the malnourished patient is compromised in part because of an inability to produce nitric oxide. Arginine, a substrate of nitric oxide synthase, is an essential amino acid, and a diet lacking arginine might reduce nitric oxide synthesis. Nitric oxide suppresses M. tuberculosis and M. leprae, intracellular pathogens that especially affect malnourished patients. Determination of how diet affects intracellular arginine concentrations, coupled with observation of the effect of arginine-deficient diets on mice infected with M. tuberculosis, might lead to the treatment of malnourished patients with tuberculosis with a simple diet rich in arginine.

The role of nitric oxide in the immune system is unique, because nitric oxide is nonspecific and damages any cell or pathogen. In contrast, antibodies or cytotoxic T lymphocytes act by first recognizing specific pathogens or infected cells and then destroying them. Furthermore, recent work suggests that only one inducible nitric oxide synthase isoform exists [155] and that it can be induced in many, if not all, cells. Thus, nitric oxide functions as a widespread, nonspecific immune system by itself, and it may have developed as a primitive immune system before the complex and specific interactions of lymphocytes evolved.

Nitric Oxide as a Neurotransmitter

Abundant evidence indicates that nitric oxide is a neurotransmitter in the peripheral and central nervous systems. Immunohistochemical studies have localized nitric oxide synthase to discrete neuronal populations, such as those in the myenteric plexus throughout the gastrointestinal tract [156-158]. Electric stimulation of gut segments produces relaxation corresponding to the relaxant phase of peristalsis and associated with nitric oxide release. Nitric oxide synthase inhibitors block this relaxation, indicating that nitric oxide is the neurotransmitter of these neurons [159, 160]. Nitric oxide released from neurons in the gastrointestinal tract causes gastric relaxation in response to a food bolus, as well as relaxation of the ileocolonic junction and anal sphincter [161-163]. Pelvic plexus neurons contain nitric oxide synthase, and nitric oxide is a mediator of penile erection [164]. Nitric oxide synthase neurons also innervate the adventitia of blood vessels [158]. Thus, nitric oxide is a neurotransmitter in the peripheral nervous system.

In the brain, nitric oxide synthase occurs in discrete neuronal groups [158], but the specific pathways in which it is involved are unclear. Because nitric oxide synthase neurons are found in the cerebellum, nitric oxide might be involved in coordination and balance. Nitric oxide synthase neurons are also found in the accessory olfactory bulb, where they may cause olfactory signal processing.

Nitric Oxide and the Transmission of Signals between Neurons

Neurons release nitric oxide, which diffuses into adjacent neurons in a series of steps. The presynaptic neuron is triggered by glutamate binding to the n-methyl-d-aspartate subtype receptor. This receptor possesses a calcium channel that opens, and the resulting influx of calcium binds to calmodulin to activate neuronal nitric oxide synthase. Nitric oxide is produced and diffuses out of the presynaptic neuron into the postsynaptic neuron, where it binds to the heme group of guanylate cyclase, activating the enzyme to produce cGMP. Given physiologic conditions, small amounts of nitric oxide allow glutamate to increase cGMP levels in the brain. However, the massive release of glutamate during stroke triggers formation of large amounts of nitric oxide that are neurotoxic to adjacent neurons.

The neurons in the central nervous system that contain nitric oxide synthase stain with the dye nitroblue tetrazolium in the presence of NADPH and were designated NADPH-diaphorase neurons [156, 165-167]. These cells are selectively resistant to the neurologic damage of stroke and to neurodegenerative conditions such as Huntington and Alzheimer diseases [168]. These nitric oxide synthase neurons appear to mediate neurotoxicity in vascular stroke [169]. Strokes are associated with a massive release of the excitatory transmitter glutamate. Large doses of glutamate and its derivative n-methyl-d-aspartate cause neurologic damage comparable to that occurring in stroke [170]. Further, drugs that block the n-methyl-d-aspartate subtype of the glutamate receptor alleviate stroke damage even when administered after occluding cerebral arteries. Nitric oxide appears to mediate this neurotoxicity, because nitric oxide synthase inhibitors block neurotoxic effects of glutamate in tissue culture and reduce stroke damage in mice, rats, and cats after cerebral artery ligation [171, 172]. Because nitric oxide synthase inhibition also constricts blood vessels, the resulting cerebral ischemia may counteract the beneficial effects of nitric oxide synthase inhibition, perhaps accounting for the failure of very large doses of nitric oxide synthase inhibitors to decrease stroke damage in some animal studies [173].

Nitric Oxide as a Factor in Penile Erection

The mechanism of penile erection is a good example of nitric oxide functioning both as a neurotransmitter and as a vasodilator. Penile erection results when cavernosal and pelvic nerves induce vasodilation of arteries that supply the corpora cavernosa, but until recently the neurotransmitter causing erection was unknown. Acetylcholine and norepinephrine were ruled out as neurotransmitters of this nonadrenergic, noncholinergic system. The cavernous nerves contain neuropeptides, such as vasoactive intestinal peptide, but evidence of this peptide's role in penile erection is minimal.

Several lines of evidence show that nitric oxide transmits the neurons responsible for erection. First, isolated strips of smooth muscle from the corpora relax when electrically stimulated; this relaxation is blocked by nitric oxide synthase inhibitors [174-178]. Next, immunohistochemical studies show that nitric oxide synthase is localized to nerves in the pelvic plexus, the cavernous nerve, and processes extending into the corpus cavernosa, innervating the adventitial layers of penile arteries [164]. Finally, electric stimulation of the pelvic nerves of live rats causes a physiologic erection that can be blocked completely by intravenous nitric oxide synthase inhibitors [164].

This mechanism of erection involves nitric oxide both as a neurotransmitter and as a vasodilator. After a neural stimulus for erection is introduced, nerves from the pelvic plexus synthesize and release nitric oxide into the corpus cavernosum. The smooth muscle of the corpus cavernosum relaxes in response, and the corpora fill with blood and swell, producing an erection. Whether nitric oxide synthase is involved in erectile diseases is unknown. A lack of nitric oxide might cause impotence, and an excess could lead to priapism.

Nitric Oxide Therapy

Although nitrates have been prescribed to treat angina pectoris for more than a century, it was not until recently that their mechanism of action was understood. Nitrates form nitric oxide, which causes direct vasodilation of the coronary arteries. Some nitrates, such as nitroprusside, spontaneously decompose to release nitric oxide; others, such as nitroglycerin and isosorbide dinitrate, are metabolized by cells into nitric oxide.

Nitrates have been used as vasodilators to treat hypertension, angina, hypertensive crises, and other emergencies requiring immediate afterload reduction. Inhaled nitric oxide relaxes pulmonary arteries, reducing pulmonary vascular resistance, and it is metabolized swiftly so that systemic vascular resistance is not affected. It has been used to treat pulmonary hypertension of neonates by dilating pulmonary arteries [179]. It has also been used to treat the adult respiratory distress syndrome by reducing pulmonary artery pressure and intrapulmonary shunting [180]. Nitric oxide reduces the size of myocardial infarctions in animals [50, 181], although whether it does this by vasodilation, by reducing inflammatory cell infiltration, or by removing other radical molecules is unknown. It is even possible that nitric oxide delivered in a stable preparation directly to the corpus cavernosum could induce erection, thereby treating impotence. Perhaps inhaled nitric oxide could be used as a bronchodilator to treat asthma or chronic obstructive pulmonary disease by directly relaxing constricted bronchial smooth muscle.

Nitric oxide synthase inhibitors successfully treat the hypotension of sepsis, although they do not reduce mortality. Whether inhibitors can treat other conditions involving an excess of nitric oxide is unresolved. Nitric oxide synthase inhibitors reduce the infarction size of strokes in some animal studies but not in others [171-173]. However, the widespread use of nitric oxide synthase inhibitors is limited because nitric oxide is involved in many different physiologic functions. Blocking nitric oxide synthesis from neuronal nitric oxide synthase to reduce the severity of stroke might also block nitric oxide from endothelial nitric oxide synthase, leading to hypertension and clotting. The commonly used nitric oxide synthase inhibitors are compounds related to the nitric oxide synthase substrate arginine, such as n-mono-methyl-arginine or n-nitro-arginine methyl ester. These compounds nonselectively block all nitric oxide synthase isoforms. Widespread treatment of patients with nitric oxide synthase inhibitors awaits development of compounds that inhibit specific nitric oxide synthase isoforms.

Conclusion

Top Conclusion Author & Article Info References

In 1987, only 7 articles were written about endogenously produced nitric oxide, but since then more than 3000 have been published. Nitric oxide, once considered a noxious byproduct of combustion, has been shown to be an endogenous messenger that plays various physiologic roles. As this explosion in research continues, the molecular targets of nitric oxide will be more precisely defined, and perhaps novel methods by which it regulates enzymes, such as amino acid nitrosylation, will be discovered. Research into the regulation of nitric oxide synthase should lead to a better understanding of its role in the pathogenesis of various diseases. The development of nitric oxide synthase inhibitors to block specific isoforms of nitric oxide as well as stable compounds that release it is also likely. Finally, the study of nitric oxide may lead to an understanding of how other small molecules, such as carbon monoxide, serve as biological messengers.

Abbreviations

ADP: adenosine diphosphate

cGMP: cyclic guanosine monophosphate

NADPH: nicotinamide adenine dinucleotide phosphate

Author and Article Information

Top Conclusion Author & Article Info References

From The Johns Hopkins University School of Medicine, Baltimore, Maryland.
Requests for Reprints: Solomon H. Snyder, MD, Departments of Neuroscience, Pharmacology, and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205.
Grant Support: By the National Institutes of Health through grants PSA K1102451 (Dr. Lowenstein), MH18501, DA00266, and Research Scientist Award DA-00074; and a grant from the W.M. Keck Foundation (Dr. Snyder).

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