Differential regulation of cytokine production by nitric oxide

Nitric oxide (NO) has recently been identified as a potent and pleiotropic intracellular mediator produced by and acting on many cells of the body. Although considerable attention has been devoted to the regulation of NO by inflammatory cytokines, and also to the role of NO as an important effector molecule in immune function, there is very little information on the role of this mediator in modulating T-cell-dependent cytokine production. In this study we show that physiological levels of NO (either produced by activated macrophages or by the addition of exogenous NO donors) can selectively down-regulate interleukin-3 (IL-3) production by spleen cells from contact-sensitized mice, while leaving IL-2 activity unaffected. Thus NO may have an important role as an immunomodulatory as well as effector molecule in the immune system.     

Nitric oxide in renal health and disease

Nitric oxide (NO) is a labile radical gas that is widely acclaimed as one of the most important molecules in biology. Through covalent modifications of target proteins and redox reactions with oxygen and superoxide radical and transition metal prosthetic groups, NO plays a critical role in many vital biological processes, including the control of vascular tone, neurotransmission, ventilation, hormone secretion, inflammation, and immunity. Moreover, NO has been shown to influence a host of fundamental cellular functions, such as RNA synthesis, mitochondrial respiration, glycolysis, and iron metabolism. NO is formed from L-arginine by NO synthases (NOSs), a family of related enzymes encoded by separate unlinked genes. The different NOS isozymes exhibit disparate tissue and intrarenal distributions and are governed by unique regulatory mechanisms. In the kidney, NO participates in several vital processes, including the regulation of glomerular and medullary hemodynamics, the tubuloglomerular feedback response, renin release, and the extracellular fluid volume. While NO serves beneficial roles as a messenger and host defense molecule, excessive NO production can be cytotoxic, the result of NO's reaction with reactive oxygen and nitrogen species, leading to peroxynitrite anion formation, protein tyrosine nitration, and hydroxyl radical production. Indeed, NO may contribute to the evolution of several commonly encountered renal diseases, including immune-mediated glomerulonephritis, postischemic renal failure, radiocontrast nephropathy, obstructive nephropathy, and acute and chronic renal allograft rejection. Moreover, impaired NO production has been implicated in the pathogenesis of volume-dependent hypertension. This duality of NO's beneficial and detrimental effects has created extraordinary interest in this molecule and the need for a detailed understanding of NO biosynthesis.     

Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide

There has been confusion as to what role(s) nitric oxide (NO) has in different physiological and pathophysiological mechanisms. Some studies imply that NO has cytotoxic properties and is the genesis of numerous diseases and degenerative states, whereas other reports suggest that NO prevents injurious conditions from developing and promotes events which return tissue to homeostasis. The primary determinant(s) of how NO affects biological systems centers on its chemistry. The chemistry of NO in biological systems is extensive and complex. To simplify this discussion, we have formulated the "chemical biology of NO" to describe the pertinent chemical reactions under specific biological conditions. The chemical biology of NO is divided into two major categories, direct and indirect. Direct effects are defined as those reactions fast enough to occur between NO and specific biological molecules. Indirect effects do not involve NO, but rather are mediated by reactive nitrogen oxide species (RNOS) formed from the reaction of NO either with oxygen or superoxide. RNOS formed from NO can mediate either nitrosative or oxidative stress. This report discusses various aspects of the chemical biology of NO relating to biological molecules such as guanylate cyclase, cytochrome P450, nitric oxide synthase, catalase, and DNA and explores the potential roles of NO in different biological events. Also, the implications of different chemical reactions of NO with cellular processes such as mitochondrial respiration, metal homeostasis, and lipid metabolism are discussed. Finally, a discussion of the chemical biology of NO in different cytotoxic mechanisms is presented.     

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 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.     

Physiological and physiopathological aspects of nitric acid in mammalian tissues

In recent years, nitric oxide (NO), a single but highly reactive molecule has become known as the central point of many researchs. NO is synthesized by the enzyme nitric oxide synthase (NOS) in mammals from the amino-acid L-arginine. The products of L-arginine oxidation by NOS are L-citrulline and NO. Nitric oxide has a very short half life, is lipid soluble, reacts easily with several enzymatic systems, and is produced by a wide amount of cells. At least, three kinds of enzymes NOS have been described: two of them are calcium-dependent and continuously present in select cells (constitutive NOS, cNOS). One cNOS isoform is present in the cytosol of neuronal cells, while the other isoform is present in membrane-bound form, in endothelial cells. cNOS produces small quantities of NO, following stimulation by specific agonist. NO produced by cNOS frequently mediates cellular communications and cellular signaling. A third isoform is calcium-independent, is not present in unstimulated cells, and produces large quantities of NO following stimulation of the appropriate cell with cytokines or LPS (inducible NOS, iNOS). NO is a mediator of both physiological and pathological process. It acts directly on its targets, one of them, maybe the most important, is the soluble guanylate cyclase, and produces a variety of biological effects, ranged from cytoprotection to cytotoxicity. An analysis of the biochemistry and physiology of NO is the focus of this review, together with its biological action and potential therapeutical implications.     

Procyanidins extracted from Pinus maritima (Pycnogenol): scavengers of free radical species and modulators of nitrogen monoxide metabolism in activated murine RAW 264.7 macrophages

Nitrogen monoxide (NO) has diverse physiological roles and also contributes to the immune defense against viruses, bacteria, and other parasites. However, excess production of NO is associated with various diseases such arthritis, diabetes, stroke, septic shock, autoimmune, chronic inflammatory diseases, and atheriosclerosis. Cells respond to activating or depressing stimuli by enhancing or inhibiting the expression of the enzymatic machinery that produce NO. Thus, maintenance of a tight regulation of NO production is important for human health. Phytochemicals have been traditionally utilized in ways to treat a family of pathologies that have in common the disregulation of NO production. Here we report the scavenging activity of Pycnogenol (the polyphenols containing extract of the bark from Pinus maritima) against reactive oxygen and nitrogen species, and its effects on NO metabolism in the murine macrophages cell line RAW 264.7. Macrophages were activated by the bacterial wall components lipopolysaccharide (LPS) and interferon (IFN-gamma), which induces the expression of large amounts of the enzyme nitric oxide synthase (iNOS). Preincubation of cells with physiological concentrations of Pycnogenol significantly decreased NO generation. It was found that this effect was due to the combination of several different biological activities, i.e., its ROS and NO scavenging activity, inhibition of iNOS activity, and inhibition of iNOS-mRNA expression. These data begin to provide the basis for the conceptual understanding of the biological activity of Pycnogenol and possibly other polyphenolic compounds as therapeutic agents in various human disorders.     

Applications of electron paramagnetic resonance spectroscopy to study interactions of iron proteins in cells with nitric oxide

Nitric oxide and species derived from it have a wide range of biological functions. Some applications of electron paramagnetic resonance (EPR) spectroscopy are reviewed, for observing nitrosyl species in biological systems. Nitrite has long been used as a food preservative owing to its bacteriostatic effect on spoilage bacteria. Nitrosyl complexes such as sodium nitroprusside, which are added experimentally as NO-generators, themselves produce paramagnetic nitrosyl species, which may be seen by EPR. We have used this to observe the effects of nitroprusside on clostridial cells. After growth in the presence of sublethal concentrations of nitroprusside, the cells show they have been converted into other, presumably less toxic, nitrosyl complexes such as (RS)2Fe(NO)2. Nitric oxide is cytotoxic, partly due to its effects on mitochondria. This is exploited in the destruction of cancer cells by the immune system. The targets include iron-sulfur proteins. It appears that species derived from nitric oxide such as peroxynitrite may be responsible. Addition of peroxynitrite to mitochondria led to depletion of the EPR-detectable iron-sulfur clusters. Paramagnetic complexes are formed in vivo from hemoglobin, in conditions such as experimental endotoxic shock. This has been used to follow the course of production of NO by macrophages. We have examined the effects of suppression of NO synthase using biopterin antagonists. Another method is to use an injected NO-trapping agent, Fe-diethyldithiocarbamate (Fe-DETC) to detect accumulated NO by EPR. In this way we have observed the effects of depletion of serum arginine by arginase. In brains from victims of Parkinson's disease, a nitrosyl species, identified as nitrosyl hemoglobin, has been observed in substantia nigra. This is an indication for the involvement of nitric oxide or a derived species in the damage to this organ.     

Dietary copper in the physiology of the microcirculation

Dietary copper has long been known to be essential for cardiovascular homeostasis. However, the role of copper and cuproenzymes in the normal control of vascular physiology is not well understood. Most studies in the cardiovascular system have focused on copper deficiency-induced defects in the heart or large vessels. Recently, attention has also focused on the effects of copper deficiency in the microcirculation or the small blood vessels that control blood flow, nutrient and waste exchange, and peripheral vascular resistance. Studies in the microcirculation demonstrate that copper is important in mechanisms of macromolecular leakage, platelet-endothelial interactions and vascular smooth muscle reactivity. There is a significantly greater leakage of proteins from postcapillary venules in copper-deficient rats in response to mast cell-released histamine. This response appears to be the result of increased numbers of mast cells and thereby increased available histamine. Copper deficiency also causes an inhibition of in vivo thrombogenesis, which appears to be related to an inhibition of platelet adhesion. Subsequent studies have demonstrated that this is probably caused by a diminished concentration of the adhesion molecule von Willebrand factor. Nitric oxide (NO)-mediated arteriole vasodilation is also compromised in copper-deficient rats. This functional deficit to NO can be reversed by the addition of Cu, Zn-superoxide dismutase (SOD), suggesting that degradation of NO by superoxide anion occurs during copper deprivation. These observations demonstrate that dietary copper is necessary for several microvascular control mechanisms affecting inflammation, microhemostasis and regulation of peripheral blood flow.     

Stress, adaptation, and nitric oxide

The biological role of nitric oxide (NO) has been studied for more than ten years. Nevertheless, the number of investigations in this field continues to increase. It is now suggested that NO is a previously unrecognized, very important regulator of physiological functions and cell metabolism in the body. Through the application of the methods of molecular biology, more and more data are being accumulated on the regulatory role of NO in the mechanism of gene expression and protein biosynthesis. The data presented in this review show an important role of NO in stress and adaptive responses of organisms and thereby expand existing notions on the biological role of this unique molecule. This review substantiates the idea that the system of NO generation is a newly discovered stress-limiting system. The action of this NO-ergic system is based on the capability of NO to limit key links of the stress reaction and to enhance the potency of endogenous defense systems of the organism. The role of NO is considered at the major stages of adaptation: 1) at the urgent stage related with the stress reaction; 2) at the stage of the transition from urgent to long-term adaptation; and 3) at the stage of long-term adaptation characterized by the formation of stable protective effects. It is demonstrated that pharmacological "imitation" of the activated NO-ergic system by administration of NO donors to the organism provides in many instances an efficient protection against stress damage and enhances the adaptive capacity of the organism.     

Toxicity of nitrogen dioxide: an introduction

Many questions, needed to advance our understanding of the mechanism of injury from high-level NO2, remain unanswered to date. This is partly due to the limited interest in the toxicity of high-level exposures, and partly due to the public pressure and interest to study the effects of low- (environmental) levels. However, the effects of exposure to high-level NO2 are of great interest to the military since high levels of NO2 may be found in combat situations. It is also important to the civilian section in occupational settings where accidents may occur as in silo filler accidents. To fill this gap in knowledge, the Department of Respiratory Research, Division of Medicine at Walter Reed Army Institute of Research took the initiative and convened a panel of experts in a symposium to discuss in depth the effects of exposure to high-level nitrogen dioxide. The symposium goals were to address the issues beginning from the chemistry of NO2 molecule, to the dosimetry of its uptake (isolated lung), to the biological effects of exposure in vivo in small animals (rats), large animals (sheep), and finally in the most relevant species, humans.     

Nitric oxide and lipid peroxidation

Nitric oxide (NO) is a free radical produced enzymatically in biological systems from the guanidino group of L-arginine. Its large spectrum of biological effects is achieved through chemical interactions with different targets including oxygen (O2), superoxide (O2o-) and other oxygen reactive species (ROS), transition metals and thiols. Superoxide anions and other ROS have been reported to react with NO to produce peroxynitrite anions that can decompose to form nitrogen dioxide (NO2) and hydroxyl radial (OHo). Thus, NO has been reported to have a dual effect on lipid peroxidation (prooxidant via the peroxynitrite or antioxydant via the chelation of ROS). In the present study we have investigated in different models the in vitro and in vivo action of NO on lipid peroxidation. Copper-induced LDL oxidation were used as an in vitro model. Human LDL (100 micrograms ApoB/ml) were incubated in oxygene-saturated PBS buffer in presence or absence of Cu2+ (2.5 microM) with increasing concentrations of NO donnors (sodium nitroprussiate or nitroso-glutathione). LDL oxidation was monitored continuously for conjugated diene formation (234 nm) and 4-hydroxynonenal (HNE) accumulation. Exogenous NO prevents in a dose dependent manner the progress of copper-induced oxidation. Ischaemia-reperfusion injury (I/R), characterized by an overproduction of ROS, is used as an in vivo model. Anaesthetized rats were submitted to 1 hour renal ischaemia following by 2 hours of reperfusion. Sham-operated rats (SOP) were used as control. Lipid peroxidation was evaluated by measuring the HNE accumulated in rats kidneys in presence or absence of L-arginine or D-arginine infusion. L-arginine, but not D-arginine, enhances HNE accumulation in I/R but not in SOP (< 0.050 pmol/g tissue in SOP versus 0.6 nmol/g tissue in I/R), showing that, in this experimental conditions, NO produced from L-arginine, enhances the toxicity of ROS. This study shows that the pro- or antioxydant effects of NO are different in vivo and in vitro and could be driven by environmental conditions such as pH, relative concentrations of NO and ROS, ferryl species.     

NO, thiols and disulfides

The chemical nature of the messenger molecule, nitric oxide (NO), and especially its reactivity towards thiol groups and disulfides, could explain, at least partly, its intervention in so many different biological processes. NO can be regarded as the smallest molecule suitable for electron transport in biological systems. The S-nitrosation reaction and its reverse reaction represent the most convenient general way to store, to transport and finally to release NO. Nitric oxide is also particularly convenient for playing a role in interconversions of thiol groups and disulfides in chain radical or oxidation-reduction processes, and to be subsequently engaged in complex sequences of reactions accounting for different biological situations.     

Role of soluble guanylate cyclase in the molecular mechanism underlying the physiological effects of nitric oxide

In this review the molecular mechanisms underlying the antihypertensive and antiaggregatory actions of nitric oxide (NO) are discussed. It has been shown that these effects are directly connected with the activation of soluble guanylate cyclase and the accumulation of cyclic 3;,5;-guanosine monophosphate (cGMP). The mechanism of guanylate cyclase activation by NO is analyzed, especially the role and biological significance of the nitrosyl--heme complex formed as a result of interaction of guanylate cyclase heme with NO and the role of sulfhydryl groups of the enzyme in this process. Using new approaches for studying the antihypertensive and antiaggregatory actions of nitric oxide in combination with the newly obtained data on the regulatory role of guanylate cyclase in the platelet aggregation process, the most important results were obtained regarding the molecular bases providing for a directed search for and creation of new effective antihypertensive and antiaggregatory preparations. In studying the molecular mechanism for directed activation of soluble guanylate cyclase by new NO donors, a series of hitherto unknown enzyme activators generating NO and involved in the regulation of hemostasis and vascular tone were revealed.     

Dominance of cyclooxygenase-2 in the regulation of pancreatic islet prostaglandin synthesis

Dramatic, scientifically important discoveries in prostaglandin (PG) pharmacology and physiology have taken place over the past decade. Chief among these discoveries is the identification of two separate forms of cyclooxygenase (COX), a constitutive and an inducible form, both of which exist in most tissues. The pancreatic islet is an exception to this rule because it continually and dominantly expresses the inducible form, COX-2. It has also been learned that nonsteroidal anti-inflammatory drugs affect the two forms of COX with different potencies, a finding with far-reaching clinical implications. An equally important finding is that PGE2, which is known to negatively modulate glucose-induced insulin secretion, has at least four different subtypes of receptors with different mechanisms of action and metabolic consequences. These recent changes in our understanding of the molecular regulation of PG synthesis call for a reconsideration of previous hypotheses involving PGE2 as a regulator of beta-cell function in physiological and pathophysiological states.     

Angiotensin II signal transduction pathways

It has been 100 years since the discovery of renin by Tigerstedt and Bergman. Since that time, numerous discoveries have advanced our understanding of the renin-angiotensin system, including the observation that angiotensin II is the effector molecule of this system. A remarkable aspect of angiotensin II is the many different physiological responses this simple peptide induces in different cell types. Here, we focus on the signal transduction pathways that are activated as a consequence of angiotensin II binding to the AT1 receptor. Classical signaling pathways such as the activation of heterotrimeric G proteins by the AT1 receptor are discussed. In addition, recent work examining the role of tyrosine phosphorylation in angiotensin II-mediated signal transduction is also examined. Understanding how these distinct signaling pathways transduce signals from the cell surface will advance our understanding of how such a simple molecule elicits such a wide variety of specific cellular responses.     

Nitric oxide, the biological mediator of the decade: fact or fiction?

Nitric oxide (NO), an atmospheric gas and free radical, is also an important biological mediator in animals and humans. Its enzymatic synthesis by constitutive (c) and inducible (i) isoforms of NO synthase (NOS) and its reactions with other biological molecules such as reactive oxygen species are well characterized. NO modulates pulmonary and systemic vascular tone through its vasodilator property. It has antithrombotic functions and mediates some consequences of the innate and acute inflammatory responses; cytokines and bacterial toxins induce widespread expression of iNOS associated with microvascular and haemodynamic changes in sepsis. Within the lungs, a diminution of NO production is implicated in pathological states associated with pulmonary hypertension, such as acute respiratory distress syndrome: inhaled NO is a selective pulmonary vasodilator and can improve ventilation-perfusion mismatch. However, it may have deleterious effects through modulating hypoxic pulmonary vasoconstriction. Inhibitors of NOS may be of benefit in inotrope-refractory septic shock, but toxicity of newly developed selective iNOS inhibitors have prevented clinical trials of efficacy. An expanding literature on the origins and measurement of NO in exhaled breath implicates NO as a potentially useful marker of disease activity in respiratory tract inflammation in the future. This report reviews some aspects of research into the clinical importance of nitric oxide.     

Nitric oxide as a peripheral and central mediator in temperature regulation

In animals including humans nitric oxide (NO) serves as a biological messenger both peripherally at neuroeffector junctions and in the central nervous system where it modulates neuronal activity. Evidence for the involvement of NO in homeostatic control is accumulating also for temperature regulation in homeotherms. In the periphery an auxiliary role in the vasomotor control of convective heat transfer to heat dissipating surfaces and modulation of thermoregulatory heat generation, especially in brown adipose tissue as the site of nonshivering thermogenesis, are discussed as NO actions. At the central level a thermolytic role of NO in thermoregulation as well as in fever is assumed, however, experimental data opposing this view suggest that topical specificity may be important. At the level of single neurons, the observed interrelationships between thermosensitivity and responsiveness to NO are still not consistent enough to reconcile these data with the effects of NO-donors and inhibitors of NO-synthase on temperature regulation.     

Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a powerful experimental system to study biochemical, cell biological and molecular biological aspects of lipid synthesis. Most but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of this unicellular eukaryote have been cloned, and many gene products have been functionally characterized. Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes, turnover and degradation of complex lipids, regulation of lipid biosynthesis, and linkage of lipid metabolism to other cellular processes. Here we summarize current knowledge about lipid biosynthetic pathways in S. cerevisiae and describe the characteristic features of the gene products involved. We focus on recent discoveries in these fields and address questions on the regulation of lipid synthesis, subcellular localization of lipid biosynthetic steps, cross-talk between organelles during lipid synthesis and subcellular distribution of lipids. Finally, we discuss distinct functions of certain key lipids and their possible roles in cellular processes.     

Osteoporosis: current modes of prevention and treatment

Alzheimer's disease (AD), the most common cause of dementia, has become a major public health concern as our population ages. In recent years, AD has attracted the attention of a wide range of biological disciplines, and substantial progress has been made in understanding the mechanisms of neurodegeneration in AD. Four different genes have now been associated with AD and are providing insights into the pathogenesis of the disease. The roles of beta-amyloid, tau, hormonal changes, inflammation, and oxidative stress in the neurodegeneration of AD are also being delineated. Based on these discoveries, rational therapeutic strategies are developing rapidly. The authors review these and other recent advances in the neurobiology and pharmacotherapy of AD.