Nitric oxide in cerebral ischemic neurodegeneration and excitotoxicity

The observation that the free radical nitric oxide (NO) acts as a cell signaling molecule in key physiological processes such as regulation of blood pressure and immunological host-defense responses is probably one of the most important and exciting findings made in biology in the last decade. Likewise, in the brain NO has been implicated in a number of fundamental processes, including memory formation, sexual behavior and the control of cerebral blood flow. This has radically altered the accepted dogma of brain physiology and has placed NO at the center stage of neuroscience research. Evidence suggests that some of the actions of NO in the brain may be intimately linked to those of the classic excitatory neurotransmitter glutamate. The historical view that aberrations in glutamate signal transduction may underlie central neurodegeneration following, for example, cerebral ischemia, has implicated NO, by default, as a potential mediator of neuronal death. Indeed, with the advent of potent and specific compounds that interact with NO synthesizing (NOS) enzymes and with the NO signaling cascade, there is now ample evidence to suggest that NO can mediate neurodegeneration, although its involvement is paradoxical. Its cerebrovascular effects may act to limit ischemic damage by preserving tissue perfusion and preventing platelet aggregation, while NO produced in the parenchyma, either directly following the ischemic insult or at a later stage as part of a neuroinflammatory response, may be deleterious to the outcome of ischemia. Nonetheless, significant efforts are made into the potential therapeutic use of chemical NO donors and specific NOS inhibitors in the treatment of cerebral ischemia and other central neurodegenerative disorders. Here, the latest concepts and developments in our understanding of the role of NO in cerebral ischemic neurodegeneration are discussed.     

Nitric oxide signaling in pain and nociceptor sensitization in the rat

We investigated the role of nitric oxide (NO) in inflammatory hyperalgesia. Coinjection of prostaglandin E2 (PGE2) with the nitric oxide synthase (NOS) inhibitor NG-methyl-L-arginine (L-NMA) inhibited PGE2-induced hyperalgesia. L-NMA was also able to reverse that hyperalgesia. This suggests that NO contributes to the maintenance of, as well as to the induction of, PGE2-induced hyperalgesia. Consistent with the hypothesis that the NO that contributes to PGE2-induced sensitization of primary afferents is generated in the dorsal root ganglion (DRG) neurons themselves, L-NMA also inhibited the PGE2-induced increase in tetrodotoxin-resistant sodium current in patch-clamp electrophysiological studies of small diameter DRG neurons in vitro. Although NO, the product of NOS, often activates guanylyl cyclase, we found that PGE2-induced hyperalgesia was not inhibited by coinjection of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylyl cyclase inhibitor. We then tested whether the effect of NO depended on interaction with the adenylyl cyclase-protein kinase A (PKA) pathway, which is known to mediate PGE2-induced hyperalgesia. L-NMA inhibited hyperalgesia produced by 8-bromo-cAMP (a stable membrane permeable analog of cAMP) or by forskolin (an adenylyl cyclase activator). However, L-NMA did not inhibit hyperalgesia produced by injection of the catalytic subunit of PKA. Therefore, the contribution of NO to PGE2-induced hyperalgesia may occur in the cAMP second messenger pathway at a point before the action of PKA. We next performed experiments to test whether administration of exogenous NO precursor or donor could mimic the hyperalgesic effect of endogenous NO. Intradermal injection of either the NOS substrate L-arginine or the NO donor 3-(4-morphinolinyl)-sydnonimine hydrochloride (SIN-1) produced hyperalgesia. However, this hyperalgesia differed from PGE2-induced hyperalgesia, because it was independent of the cAMP second messenger system and blocked by the guanylyl cyclase inhibitor ODQ. Therefore, although exogenous NO induces hyperalgesia, it acts by a mechanism different from that by which endogenous NO facilitates PGE2-induced hyperalgesia. Consistent with the hypothesis that these mechanisms are distinct, we found that inhibition of PGE2-induced hyperalgesia caused by L-NMA could be reversed by a low dose of the NO donor SIN-1. The following facts suggest that this dose of SIN-1 mimics a permissive effect of basal levels of NO with regard to PGE2-induced hyperalgesia: (1) this dose of SIN-1 does not produce hyperalgesia when administered alone, and (2) the effect was not blocked by ODQ. In conclusion, we have shown that low levels of NO facilitate cAMP-dependent PGE2-induced hyperalgesia, whereas higher levels of NO produce a cGMP-dependent hyperalgesia.     

Nitric oxide synthase plays a signaling role in TCR-triggered apoptotic death

A functional role for stimulated nitric oxide (NO) production was tested in the TCR-triggered death of mature T lymphocytes. In purified peripheral human T cell blasts or the 2B4 murine T cell hybridoma, apoptotic cell death induced by immobilized anti-CD3 was blocked by inhibitors of NO synthase (NOS) in a stereospecific and concentration-dependent manner. This effect appeared to be selective since apoptotic death induced by anti-Fas Ab or the steroid dexamethasone was not affected by NOS inhibitors. TCR-stimulated expression of functional Fas ligand was attenuated in a stereospecific manner by NOS inhibitors, but these compounds did not inhibit TCR-stimulated IL-2 secretion or CD69 surface expression. Nitrosylated tyrosines, a stable marker for NO generation, were immunochemically detected in T cells using flow cytometry. TCR signals induced NO production, as measured by an increase in nitrotyrosine-specific staining. NOS enzymatic activity was detected in lysates of 2B4 cells, and Western blot analysis suggests that the activity is due to expression of the neuronal isoform of NOS. Thus, T cells have the capacity to generate NO upon Ag signaling, which may affect signal transduction, Fas ligand surface expression, and apoptotic cell death of mature T lymphocytes.     

Nitric oxide inhibition in an ovine model of heart failure

1. The role of nitric oxide (NO) in congestive heart failure was investigated by studying the acute haemodynamic, hormonal and renal effects of N(G)-monomethyl-L-arginine (L-NMMA(, a nitric oxide inhibitor, given as incremental bolus doses in six sheep before (normal) and after induction of heart failure (HF) by rapid left ventricular pacing (LVoff+). 2. 6-NMMA caused significant initial dose-dependent rises in left ventricular systolic pressure, mean arterial pressure (MAP), peripheral resistance (PR) and left atrial pressure and declines in cardiac output in both normal and HF states (maximum response in 2-6 min). These responses were all but abolished when L-arginine was given concurrently with L-NMMA. The dose-response curve for the L-NMMA-induced rise in MAP was shifted to the right following LVP (P < 0.05), which is consistent with previous observations of blunted NO synthase activity in HF. A subsequent decline in MAP and PR to below prebolus levels was observed 30-60 min after L-NMMA administration in the paced state. No significant hormonal or renal effects were observed. 3. In conclusion, the present study confirms the important haemodynamic role played by endogenous NO in the normal state and demonstrates a blunted pressor response to NO inhibition in this model of heart failure.     

Nitric oxide

Nitric oxide (NO) is a gas with diverse biological activities produced from arginine by NO synthases. It is capable of interacting with a number of molecules, most notably superoxide, forming peroxynitrite, which, in turn, can mediate bactericidal or cytotoxic reactions. Nitric oxide also mediates smooth muscle relaxation, neurotransmission, and modulation of inflammation in a number of organ systems and pathophysiologic conditions. Modulation of NO by administration of inhaled NO for respiratory distress syndromes and infusion of NO synthase inhibitors in bacterial sepsis are ongoing. Levels of exhaled NO are being evaluated for their utility in assessing inflammation in respiratory disorders such as asthma.     

Nitric oxide production and energy state in the heart after endotoxin administration

To evaluate nitric oxide (NO) production and the energy state of the heart after endotoxin administration, Wistar rats were injected i.p. with 10 mg/kg Escherichia coli lipopolysaccharide (endotoxin). Morphologic changes, plasma nitrite concentration, expression of inducible NO synthase (iNOS), and cardiac energy state, as reflected by several metabolites, were observed chronologically 0 (control), 4, 6 and 8 h after endotoxin administration. Electrocardiography (ECG) demonstrated arrhythmia after endotoxin administration. Biochemically, NO production increased in blood and iNOS increased in the heart. The amount of myocardial beta-ATP measured by 31P magnetic resonance spectroscopy (31P-MRS) increased transiently and then decreased. This transient increase might be a hyperdynamic response to endotoxin administration. At 4 and 6 h after endotoxin administration, pH measured by 31P-MRS was slightly decreased, but this decline was not statistically significant. On the other hand, the amount of lactate in heart samples increased in the 1H magnetic resonance spectra (1H-MRS). Ultrastructurally, in cardiovascular tissue, intracytoplasmic organelles were observed to be injured in blood vessels and cardiomyocytes associated with mast cell infiltration. These results suggest significant metabolic and morphologic abnormalities in the heart after endotoxin administration.     

Nitric oxide in invertebrates

Nitric oxide (NO) is considered an important signaling molecule implied in different physiological processes, including nervous transmission, vascular regulation, immune defense, and in the pathogenesis of several diseases. The presence of NO is well demonstrated in all vertebrates. The recent data on the presence and roles of NO in the main invertebrate groups are reviewed here, showing the widespread diffusion of this signaling molecule throughout the animal kingdom, from higher invertebrates down to coelenterates and even to prokaryotic cells. In invertebrates, the main functional roles described for mammals have been demonstrated, whereas experimental evidence suggests the presence of new NOS isoforms different from those known for higher organisms. Noteworthy is the early appearance of NO throughout evolution and striking is the role played by the nitrergic pathway in the sensorial functions, from coelenterates up to mammals, mainly in olfactory-like systems. All literature data here reported suggest that future research on the biological roles of early signaling molecules in lower living forms could be important for the understanding of the nervous-system evolution.     

Nitric oxide in respiratory diseases

Recent reports have described a pathogenic role of nitric oxide in several respiratory disease. It is specially useful in the adult respiratory distress syndrome, where it acts as a selective vasodilator and improves gas exchange, decreasing pulmonary shunting. Although it has a proven bronchodilator effect, its therapeutic role in diseases such as asthma and chronic limitation of airway flow is not well defined. This article review the metabolism, mechanisms of action, potential uses and adverse effects of nitric oxide in respiratory disease.     

Nitric oxide in vascular remodeling

Vascular remodeling is a series of structural changes in blood vessels. Therefore, it may be conceivable that any humoral factors and physical forces acting on the vascular wall are involved in the remodeling processes. Cells in the vascular wall respond to the humoral and physical factors and may induce extracellular matrix, cell adhesion molecules and other humoral factors. They even grow so that cellular and noncellular components deviate from the normal population. We discuss the relationship among nitric oxide (NO), pressure and growth of smooth muscles. Decreased NO may be a consequence as well as a cause of high pressure. Similarly, high pressure is a cause as well as a consequence of decreased NO. Remodeling could be a consequence of both high pressure and decreased NO. Thus, vascular remodeling is a complex dynamic state, where any causes and results are influenced by each other. Interaction of NO and pressure is one such complexity.     

Nitric oxide synthase in skeletal muscle fibers: a signaling component of the dystrophin-glycoprotein complex

The present review deals with the anatomical distribution, physiological importance, and pathological implications of the neuronal-type nitric oxide synthase (nNOS) in skeletal muscle. Throughout the body, nNOS is located beneath the sarcolemma of skeletal muscle fibers. In rodents, nNOS is enriched in type IIb muscle fibers, but is more homogenously distributed among type II and type I fibers in humans and subhuman primates. It is accumulated on the postsynaptic membrane at the neuromuscular junction. An increased concentration of nNOS is noted at the sarcolemma of muscle spindle fibers, in particular nuclear bag fibers, which belong to type I fibers. The association of nNOS with the sarcolemma is mediated by the dystrophin-glycoprotein complex. Specifically, nNOS is linked to alpha 1-syntrophin through PDZ domain interactions. Possibly, it also directly binds to dystrophin. The activity and expression of nNOS are regulated by both myogenic and neurogenic factors. Besides acetylcholine, glutamate has also been shown to stimulate nNOS, probably acting through N-methyl-D-aspartate receptors, which are colocalized with nNOS at the junctional sarcolemma. Functional studies have implicated nitric oxide as a modulator of skeletal muscle contractility, mitochondrial respiration, carbohydrate metabolism, and neuromuscular transmission. A clinically relevant aspect of nNOS is its absence from the skeletal muscle sarcolemma of patients with Duchenne muscular dystrophy (DMD). A concept is presented which suggests that, as a consequence of the disruption of the dystrophin-glyoprotein complex in DMD, nNOS fails to become attached to the sarcolemma and is subject to downregulation in the cytosol.     

Nitric oxide as a regulator of prostacyclin synthesis in cultured rat heart endothelial cells

The effects of nitric oxide (NO) and its second messenger cyclic guanosine monophosphate (cGMT) on prostacyclin (PGI2) synthesis were studied in cultured rat heart endothelial cells using three different non-enzymatic nitric oxide releasing substances as well as inhibitors of nitric oxide synthase and of soluble guanylate cyclase. Production of prostacyclin, measured as 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), was stimulated up to 1.7 fold in endothelial cells treated with the NO donors SIN-1 (3-morpholino sydnonimine), GEA 3162 (3-aryl-substituted oxatriazole imine) and GEA 3175 (3-aryl-substituted oxatriazole sulfonyl), chloride). In each case the synthesis of cGMP increase as much as 40-100 fold. An inhibitor of NO synthase, NG-nitro-L-arginine methyl ester (L-NAME), decreased the basal production of 6-keto-PGF1 alpha in non-stimulated endothelial cells, an effect that could be reversed by the NO donors SIN-1, GEA 3162 and GEA 3175. cGMP formation in the L-NAME treated endothelial cells was unaltered. The guanylate cyclase inhibitors, methylene blue (100 mumol/l) and LY83583 (100 mumol/l), caused a 1.5-10 fold increase in 6-keto-PGF1 alpha production while NO-donor-stimulated endothelial cGMP production was decreased by 10 to 90%. However, when SIN-1 was used as a stimulant, LY83583 had no significant effect on the production of cGMP. These findings support the hypothesis that NO stimulates prostacyclin production directly by activating cyclooxygenase. The results also suggest that NO could have an indirect effect on prostacyclin production via cGMP.     

Nitric oxide and pregnancy

We have hypothesized that an alteration in the production of endothelium-dependent factors by sex hormones is a potential unifying mechanism for both the decreased arterial contractility and the redistribution of cardiac output characteristic of normal pregnancy. Thus, the effect of pregnancy/ estradiol on any one vascular bed will reflect the number and distribution of estrogen receptors. In this article, we review what is known about the effects of pregnancy and estrogen on nitric oxide synthase. Pregnancy increases Ca(2+)-dependent NOS activity early in gestation. The timing of the increase parallels the increase in plasma estradiol concentration. The increase in maternal brain NOS during pregnancy is blocked by tamoxifen. cGMP content increases along a similar time course in most but not all tissues. The changes in cGMP more closely approximate the changes in blood flow during pregnancy. This suggests that multiple elements of the NO:cGMP pathway are altered by pregnancy. It also shows that cGMP content cannot always be used as a surrogate for NOS activity. Estradiol, but not progesterone or testosterone, increases CA(2+)-dependent NOS activity. NO accounts for some, but not all of the pregnancy-associated changes in maternal arterial contractile response. It is not involved in uterine quiescence. Nitric oxide synthase is developmentally regulated in the fetus and is likely important in regulating the distribution of fetal blood flow.     

Nitric oxide in learning and memory

The role of nitric oxide in the central nervous system is described. The main part of this article concerns the problem of learning and memory.     

Nitric oxide in the stress axis

Calcitonin gene-related peptide in sensory primary afferent neurons has an excitatory effect on postsynaptic neurons and potentiates the effect of substance P in the rat spinal dorsal horn. It has been established that calcitonin gene-related peptide expression in dorsal root ganglion neurons is depressed, and the effect of calcitonin gene-related peptide on dorsal horn neurons is attenuated, following peripheral nerve injury. We report here that a subpopulation of injured dorsal root ganglion neurons show increased expression of calcitonin gene-related peptide. Using in situ hybridization and the retrograde tracer, FluoroGold, we detected an increased number of medium- to large-sized rat dorsal root ganglion neurons projecting to the gracile nucleus that expressed alpha-calcitonin gene-related peptide messenger RNA following spinal nerve transection. Immunohistochemistry revealed a significant increase in calcitonin gene-related peptide immunoreactivity in the gracile nucleus and in laminae III-IV of the spinal dorsal horn. These results indicate that a subpopulation of dorsal root ganglion neurons express alpha-calcitonin gene-related peptide messenger RNA in response to peripheral nerve injury, and transport this peptide to the gracile nucleus and to laminae III-IV of the spinal dorsal horn. The increase of the excitatory neuropeptide, calcitonin gene-related peptide, in sites of primary afferent termination may affect the excitability of postsynaptic neurons, and have a role in neuronal plasticity following peripheral nerve injury.