Nitric Oxide Signaling in Pain and Nociceptor Sensitization in the Rat

The significance of nitric oxide (NO) in man was first investigated in the late 1980s, and NO has subsequently received great attention from biologists. Initially, this highly reactive gaseous molecule was seen as a mere noxious air pollutant. Closer investigation of its function in physiological processes, however, revealed that it took part in many different biologic processes. This multifunctionality led to its declaration as the molecule of the year in 1992. We now know NO to be a smooth-muscle relaxant in blood vessels, an inhibitor of platelet aggregation, a neurotransmitter, and a mediator in local defense (2, 3). In the airways, NO is an important molecule with different functions such as stimulation of ciliary motility, mediation in inflammation, bacteriostatic and virostatic activity, and regulation of bronchial airway tone and even pulmonary vascular tone (4–7). Further studies on other systems will probably reveal more processes in which NO plays a key role. Studies in healthy adults indicate that NO in nasal air is mainly produced in the epithelial cells of the nasal cavity, particularly in the paranasal sinuses (8). Many factors, such as smoking, drugs, physio-logical factors, and nasal and paranasal disorder, influence the level of NO measured in nasal air (6, 9, 10). The measurement technique is also of great importance (10, 11). NO measurement has begun to be used in experimental clinical settings, in order to clarify the clinical value of NO in diagnostic problems and therapeutic strategies for disorders such as primary ciliary dyskinesia (PCD) and various forms of sinusitis and allergy. The use of NO as a noninvasive diagnostic and therapeutic tool is the ultimate goal. Many cells within the (upper and lower) respiratory tract can produce NO, including endothelial cells, epithelial cells, neutrophils, and (alveolar) macrophages (12). First, l-arginine is taken up by the cells via cationic transporters (CAT) (Fig. 1). CAT1 is constitutively expressed (housekeeping), while CAT2 is induced by cytokines. Second, l-arginine is N-hydroxylated into NG-hydroxy-l-arginine (NOHA). Subsequently, a three-electron oxidation takes place, resulting in NO and l-citrulline. While NO diffuses to the lumen, l-citrulline can be reconverted to l-arginine via arginosuccinate inside the cell (13). NO metabolic pathway (13) (reproduced with permission). This pathway of generation of NO is regulated by a family of enzymes called nitric oxide synthases (NOS). Three isoforms of NOS have now been identified in man and are differentially distributed in organs and tissues (14). Constitutively expressed nitric oxide synthase (cNOS) consists of two isoforms, nNOS (NOS type 1) and eNOS (NOS type 3), respectively expressed in neurons and vascular endothelium. The activity of nNOS and eNOS is regulated by intracellular calcium/calmodulin concentrations. These isoforms have been localized in human alveolar type II cells and in transformed and primary cultures of human bronchial epithelial cells (15). Inducible NOS (iNOS or NOS type 2) is probably present in every (epithelial) cell, and is activated by proinflammatory cytokines and/or bacterial products (2). The inducible form of NOS is calcium independent. LPS alone increases the production of NO in human epithelial cells, but IFN-γ acts synergistically to enhance this response (15). Immunohistochemical and mRNA in situ hybridization show that NO synthase is expressed apically in the paranasal sinus epithelium, in contrast to the epithelium of the nasal cavity, where only weak NO synthase activity was found (16). The NOS of the paranasal sinuses most closely resembles the inducible isoform but has different characteristics from iNOS expressed elsewhere. These isoforms seem to be constantly expressed and active, and to be resistant to steroids. These properties are associated with constitutive, rather than with inducible, isoforms of NOS (16). A new nonenzymatic pathway has been discovered in man that produces NO by reduction of inorganic nitrite under specific conditions (17). These nonenzymatic reactions take place in the stomach, on the surface of the skin, in the ischemic heart, and in infected nitrite-containing urine. NO generated by this mechanism is likely to play a role in similar biologic events, as when produced from l-arginine by NO synthases. The exact origin of NO measured in nasal air and the relative contribution from other sources are not fully known. Not only is there the production within the nasal cavity and the paranasal sinuses, but there is also a contribution from other sources such as the ambient air and, more important, the lower respiratory tract (6–8, 10, 18, 19). Most studies indicate that the main production of nasal NO is in the paranasal sinuses (16, 20, 21). The first indication is the observation that there is a transient decrease in nasal NO measured from one nostril when air is continuously removed from one maxillary sinus, while air injected into the same sinus results in a transient elevation of nasal NO. This suggests a continuous flow of NO from the maxillary sinus to the nasal cavity (20). Another indication is the reduction of NO release from the paranasal sinuses by instillation of NO synthase inhibitor (L-NAME) into the maxillary sinus. Administration of L-NAME in the nasal cavity results in only a slight reduction of nasal NO levels (20). In patients who have impaired ostial patency, significantly lower nasal NO levels are measured. Impairment of ostial patency and thus lower nasal NO levels are seen in disorders such as Kartagener's syndrome and cystic fibrosis. In these cases, there is probably a lower contribution of NO flowing from the paranasal sinuses into the nose, in addition to a possibly decreased production of NO (8, 22). Moreover, nasal NO levels are high in man and other primates with paranasal sinuses, while, in contrast, the baboon, a primate which lacks paranasal sinuses, has very low nasal NO levels (21). The strong constitutive expression of iNOS in the sinus epithelium and the lack of expression in the nasal epithelium are another indication (16). There are indications that nasal NO levels in children rise until the age of 10 years, when they reach the normal value as in adults. This may be a sign of increasing pneumatization of the developing paranasal sinuses in growing children (16, 23). The role of bacteria in the production of nasal NO has also been suggested; however, most studies showed nasal NO release to be independent of the presence of bacteria, since systemic antibiotics had no effect on the nasal NO values of healthy adults, and the sterile nasal cavities of neonates delivered by cesarean section had measurable nasal NO levels (7, 24, 25). As, in recent years, a wide variety of physiological processes in which NO is involved have been thoroughly investigated, it became clear that NO is important within the system where it is produced. Although initially considered a noxious air pollutant, many scientists now agree on the important roles of NO in different organ systems, such as those of a neurotransmitter in the nervous system, a smooth-muscle relaxant, and an inhibitor of platelet aggregation in the cardiovascular system (6, 16, 26). In the airways, NO seems to be of great importance in local host defense and is a major mediator in many physiological and pathophysiological events, although the exact role of this pluripotent gas is far from fully known. It participates in host defense and inflammation, and as an airborne messenger in bronchial tonus and pulmonary vascular resistance. The role of NO in inflammation is contradictory. Some studies indicate a harmful role of NO in inflammation, whereas others indicate a positive influence (18). There is evidence that NO production is enhanced at sites of inflammation, leading to local increased NO levels, as in asthma, cystitis, and inflammatory bowel disease (18, 27). The harmfulness of NO may be due to extensive production of NO by iNOS in some inflammatory circumstances such as pertussis and asthma, leading to autotoxicity in the affected area (18). However, basal NO production in the upper respiratory tract by a continuous expressed iNOS, leading to fairly high NO levels, has no destructive effect on local airway epithelium, and is even physiological (16). On the contrary, NO production in the upper respiratory tract seems to serve as an important protection against local attack, not as a mere inflammatory mediator, but as a regulator of various protective activities in host defense. A remarkable illustration of the positive role of NO in inflammation was given by McCafferty et al., who found worse inflammation in iNOS knockout mice than in wild-type mice in an animal model of colon inflammation (28). The enhanced production of NO during local aggression against the airway epithelium suggests a role of NO in host defense. NO concentration in normal paranasal sinuses and even in the nasal cavity exceeds greatly NO concentrations that are bacteriostatic (i.e., 100 ppb) (6, 16, 29). Children who have low NO production, as in primary ciliary dyskinesia (PCD) and cystic fibrosis, also have recurrent airway infections, a fact which may be an indication of the (host) protective effect of NO. NO may also have virostatic activities, as indicated in a mouse model (30). There are also indications that NO is active against fungi and parasites, and it may also protect against tumor cells (31). NO is also a regulator of ciliary beat frequency in the upper airway epithelium (4, 5, 32). The lack of NO in nasal air in diseases caused by profound ciliary dysfunction, such as PCD, strongly suggests a relation between NO and ciliary motility with clinical implications. For example, in infection, increased NO production can lead to enhanced ciliary activity, resulting in an effective clearance of aggressive organisms and potentially noxious metabolic products. This can have beneficial results in host defense. Other findings suggest that NO enhances blood flow in the human nasal mucosa (33). Although its possible protective effect is not clear yet, further studies on this subject may elucidate the meaning of this finding. NO produced in the upper respiratory tract follows the airstream to the lower airways and lungs with inhalation. This supports the hypothesis that NO derived from the upper airways has physiological effects in the lung and acts as an aerocrine messenger. There is some evidence that inhaled (exogenous) NO, at concentrations as low as 100 ppb, significantly decreases pulmonary vascular resistance and improves arterial oxygenation in subjects with severe pulmonary disease (33). Other studies suggest that NO helps to decrease the bronchial tonus, although this might be a central rather than a peripheral airway effect (7). NO in gas phase at low concentrations, as in the human airways, is fairly stable and therefore can be detected and quantified. The most widely used technique for measurement of NO in exhaled air is the chemiluminescence method. This highly sensitive technique is based on the emission of electromagnetic radiation from excited NO2*. NO reacts with an excess of ozone (O3), resulting in NO2 with an electron in an excited state (NO2*), which returns to its basic energy by emitting a photon. The quantity of light emitted is proportional to the NO concentration and can be displayed online on-screen. The lower limit of measurement is 1 ppb. Nasal NO measurement is based on the same method as exhaled NO, but sampling can be done directly or indirectly from the nose (6, 10). Other methods that have been used to measure NO in human exhaled air are mass spectrometry and gas chromatography–mass spectrometry (6). The measurement technique that is used in a particular experiment is very important for the eventual value of the nasal NO level (10, 34). Even in the same population the NO level is dependent on the measurement technique (11). The most important factors are ambient NO; the method of measuring (i.e., sampling while breathholding or tidal breathing, soft palate closure, etc.); and the characteristics of the chemiluminescence analyzer, the sampling flow, and the intranasal flow (10, 11). For comparison of different values, it is important to have a notion of these factors. In 1997, the European Respiratory Society Task Force tried to determine a standard method in order to obtain more comparable and reliable values (10). However, scientists continue to use different experimental settings, and one should be aware of this in order to interpret and compare NO values from different studies. The values of oral and nasal NO in the exhaled air of controls measured by the chemiluminescence method vary among laboratories: oral NO ranges from 4 to 160 ppb, while nasal NO varies from 200 to 2000 ppb (12, 22, 23, 35–38). Another remarkable feature is that NO levels are always higher in the upper respiratory tract than in the lower airways in normal subjects (6, 8, 10, 12, 22, 24, 36, 38). The variety of NO values in different studies is due to different factors such as measurement techniques, physiological variations, and pathologic changes (9–11, 16, 23, 34, 39–41). A summary of the influences on nasal NO is given in Table 1. Nasal NO levels rise from birth until the age of 10 years, when they reach the normal adult level. This finding supports the paranasal origin of nasal NO, as in children development of paranasal sinuses results in higher nasal NO levels until the age of 10 years, when they reach their final constitution (16, 23, 43). Interestingly, Schedin et al. found nasal NO already present at birth, including those neonates delivered by cesarean section (25). When nasal NO levels were correlated with body surface, the concentration in children around 10 years of age was approximately twice as high as the nasal NO concentration in adults. The following two possible explanations have been proposed: 1)the surface of paranasal sinuses in children develops faster than the body surface 2)children excrete a larger proportion of NO in the nasal mucosa (16). Another study found that nasal NO levels in adults between 20 and 90 years of age were similar (23). Artlich et al. related levels of nasal NO to the body surface in preterm children and found that the NO excretion is similar to that of adults (about 3 nl/kg/min−1). They concluded that the lower NO levels in preterm children are due to the smaller volume of ventilated sinuses and smaller epithelial surface at that age (43). Mammals without sinuses have no age-related increase in nasal NO (44). Recently, Qian et al. contradicted Lundberg et al.'s conclusions. They showed that intranasal flow had a great influence on the result of NO measurement (16, 34). As there are many differences in ventilation and measurement techniques between children and adults, intranasal flow will not always be comparable. More work needs to be done to make measurements in children and adults more comparable, in order to draw conclusions about age-dependent NO differences (34). There is no evidence that nasal NO levels are sex-related (10, 34, 39). Variation in nasal NO levels in relation to the menstrual cycle has not yet been studied. Several studies show that nasal NO decreases during physical exercise (6, 10, 45). Lundberg et al. (6) showed that nasal NO decreased by 47% after 1 min of physical exercise. A maximal reduction of 76% was found at the end of the exercise period; thereafter, NO levels slowly increased. They reached normal basal levels in about 15–20 min. There are several possible reasons for this decrease in nasal NO. Firstly, changes in nasal cavity volume could result in lower NO levels by dilution of nasal air (46). This possibility has been rejected by a recent finding that nasal NO is independent of nasal cavity volume (47). Secondly, NO could be destroyed by reactive agents produced in the nasal mucosa during physical exercise. Thirdly, changes in NO could be caused by a reduction of blood flow in the nasal mucosa with a concomitant decrease in substrate supply to the highly producing NOS type 2 in the paranasal sinuses (6, 46). Smoking control subjects have somewhat lower exhaled NO and nasal NO values than age- and sex-matched nonsmokers. The reason for this could be related to the toxic effect of inhaled smoke on the downregulation in NOS and/or the disruption of NO-producing cells (6, 10, 23). When evaluating the effect of drugs on nasal NO, one should be aware of interactions among drugs, patients, and diseases. It is not always easy to determine whether the changes in nasal NO are caused by the drug or by the disease itself. Topical and systemic glucocorticoids showed no effect on the nasal NO levels in healthy people (6, 8, 48, 49). Antibiotics in healthy persons do not alter nasal NO levels (6, 8). Topical nasal decongestants, such as oxymetazoline, result in a decrease of nasal NO levels (6, 10, 40, 47, 50). The reason for this may be a reduction, caused by vasoconstriction, in substrate supply to the high-output NOS type 2 in the sinuses. Histamine seems to have no influence on nasal NO levels (51). Nasal NO levels in people suffering from an upper respiratory tract infection (URTI) do not differ from nasal NO levels in healthy people. Specifically, Ferguson & Eccles (50) and Lindberg et al. (23) found no significant differences in nasal NO levels during and after an episode of URTI. Lindberg et al. (23) found similar nasal NO levels in patients with URTI and healthy controls (23). Baraldi et al. reached the same conclusion when comparing children with and without URTI (41). The effect of allergic rhinitis on nasal NO is not consistent. Some researchers report higher nasal NO levels in patients with allergic rhinitis (9, 40, 42). This may be due to an upregulation of iNOS by local infection, resulting in higher NO production (9). Kharitonov et al. found that nasal NO levels in patients suffering from allergic rhinitis and treated with topical nasal glucocorticoids are even lower than nasal NO levels in controls (9). This led to the hypothesis that iNOS in nasal epithelial cells gives rise to increased nasal NO levels in allergic rhinitis and contributes to the normal NO production in basal circumstances, since topical nasal glucocorticoids normally do not reach the sinus cavity and decrease nasal NO values in allergic rhinitis to levels lower than nasal NO levels in controls. According to this hypothesis, iNOS in the nasal cavity, as its activity is altered by glucocorticoids, must be different from iNOS found in the paranasal sinuses, which is not influenced by glucocorticoids (9, 52). Lundberg et al. (36) and Henriksen et al. (53) found no alterations in nasal NO levels in patients with allergic rhinitis. The cause of these discrepancies is not very clear. One could speculate that the upregulation of iNOS in the nose leads to higher nasal NO levels in rhinitis, as is the case in local infections in the lower airways, such as asthma (9, 52, 54). In contrast, swelling of the nasal mucosa in rhinitis can lead to occluded sinus ostia, which results in a reduced passage of NO from the paranasal sinuses to the nasal cavity, where it is measured (40). An interesting finding supporting this view was made by Arnal et al. (40), who found increased nasal NO levels in patients with allergic rhinitis. But patients without symptoms at the moment of the measurement had even higher nasal NO levels than patients with symptoms. One could postulate that nasal NO levels in patients with symptoms are lower because of a reduced contribution of the NO produced in the paranasal sinuses, as a result of obstructed sinus ostia. In patients without symptoms, ostial patency is mostly better leading to a higher of NO from the paranasal sinuses into the nasal cavity (40). in the nasal NO level measure may be the result of in the may even This must be taken into when a given nasal NO value is Nasal NO levels seem not to be influenced by asthma (18, 22, 36, One can that asthma the upper respiratory tract to a than the lower airways, where increased NO levels are of glucocorticoids can NO levels by reduction of iNOS NO levels are considered to be a of airway measurement of NO levels in the lower airways could indicate the of (6, 22). Nasal NO levels seem to be decreased in patients suffering from but not studies are consistent. Lindberg et al. patients with sinusitis and found that nasal NO production was reduced by more than in comparison with healthy subjects (23). In contrast, Arnal et al. found no significant differences in their study of patients with sinusitis Lindberg et al. found similar nasal NO levels in patients after sinusitis and healthy subjects (23). nasal NO levels were measured by Baraldi et al. in children with These decreased nasal NO levels increased after with systemic nasal NO levels were to the levels of healthy children (41). It has not yet been whether low nasal NO levels in sinusitis result from reduced passage of NO via the sinus ostia, or whether the NO production is reduced in those patients (41). A low production of NO as a cause of low nasal NO levels in sinusitis is by the study of Lindberg et al., who found nasal NO levels to be low sinus or by sinus as by (23). In contrast to Lindberg et al.'s Baraldi et al. found only a reduced NO level in children with a of the sinus in the air derived from the nostril (41). The effect of nasal has not been The of nasal NO and nasal has been in only one Arnal et al. increased nasal NO levels in patients with nasal and to whereas patients with nasal without had significantly lower nasal NO The nasal NO concentration in patients with allergic was significantly higher than in patients with For a similar of sinus nasal NO was higher in allergic than in This that is an important in relation to the level of nasal NO in In the nasal NO concentration was correlated with the of alterations of the paranasal sinuses. This that the of the paranasal sinuses by the decreases the nasal NO with a similar of of the nasal NO levels was that sites of production other than the sinuses also to the nasal NO. It has been that also may to the NO production, as they also iNOS in their epithelial cells (2). can that the of paranasal sinus and the allergic strongly influence the nasal NO level in nasal studies report very low nasal NO levels in patients suffering from cystic (12, 22, This may be the result of reduced NO production by destroyed epithelial cells or reduced NOS An increased NO into the sinus and a reduced NO passage from the sinuses to the nasal cavity may be another possible (12, 22, Kartagener's syndrome is a and They are part of In patients with PCD, nasal NO levels are (8, 12, 52). explanations are reduced NO production by a reduced from the nasal and paranasal and reduced passage of NO via the sinus (8, 12, 22, 52). In studies on PCD, significantly lower nasal NO levels in than in disease controls. nasal NO values, however, do not We found that the in have no significant influence on the nasal NO level et al., of NO can an interesting and diagnostic and therapeutic However, to be done in order to make it a in This noninvasive measurement can be even in It could be used as an easy for the of In the therapeutic may It is to that drugs will be used to or decrease NO production in such a that it can have a positive influence on However, there is to be in the various physiological and pathologic factors, such as that nasal NO, particularly the should on and on measurements more reliable and comparable. NO is a gaseous the significance of which in man to be investigated in the late the it has the attention of many who have revealed its significance in various physiological and pathologic processes. It has functions in the cardiovascular system, the nervous system, and the upper and lower In the airways, NO levels in the upper respiratory tract are higher ppb) than those in the lower respiratory tract The chemiluminescence which is based on a of NO with resulting in the emission of is the most widely used measurement technique for NO. NO has a major influence on airway by mediation in ciliary activity, inflammation, host bronchial and pulmonary vascular resistance. It is also considered to be an aerocrine messenger between the upper and lower such as physical smoking, and some drugs influence physiological nasal NO concentrations. conditions such as allergic rhinitis, nasal cystic fibrosis, and lead to altered nasal NO concentrations. of nasal NO can be at and can be used to for disease or to the effects of However, the clinical of the measurement of nasal NO in different physiological and pathologic conditions to be it can be used as a diagnostic on the function of NO in and is its in diagnostic and therapeutic of some

Bacterial pathogens typically upregulate the host's production of nitric oxide synthase (NOS) and nitric oxide (NO) as antimicrobial agents, a response that is often mediated by microbe-associated molecular patterns (MAMPs) of the pathogen. In contrast, previous studies of the beneficial Euprymna scolopes/Vibrio fischeri symbiosis demonstrated that symbiont colonization results in attenuation of host NOS/NO, which occurs in high levels in hatchling light organs. Here, we sought to determine whether V. fischeri MAMPs, specifically lipopolysaccharide (LPS) and the peptidoglycan derivative tracheal cytotoxin (TCT), attenuate NOS/NO, and whether this activity mediates the MAMPs-induced light organ morphogenesis. Using confocal microscopy, we characterized levels of NOS with immunocytochemistry and NO with a NO-specific fluorochrome. When added exogenously to seawater containing hatchling animals, V. fischeri LPS and TCT together, but not individually, induced normal NOS/NO attenuation. Further, V. fischeri mutants defective in TCT release did not. Experiments with NOS inhibitors and NO donors provided evidence that NO mediates apoptosis and morphogenesis associated with symbiont colonization. Attenuation of NOS/NO by LPS and TCT in the squid-vibrio symbiosis provides another example of how the host's response to MAMPs depends on the context. These data also provide a mechanism by which symbiont MAMPs regulate host development.

Nitric oxide (NO) plays important role in alleviating abiotic stresses in plants, including those caused by arsenic (As). Here, we examined the effects of endogenous and exogenous NO in Spirodela intermedia W. Koch (Lemnaceae) under As exposure. For this purpose, we evaluated the As content, reactive oxygen species (ROS) levels, membrane damage and enzymatic antioxidant system. The levels of endogenous NO and the activity of nitrate reductase (NR) were also addressed. The As treatment triggered the production of high endogenous levels of NO and a pronounced activation of the antioxidant enzymes; however, it was not sufficient to completely avoid the increment in ROS content and membrane damage. In contrast, exogenous NO decreased the As levels in plants exposed to As and NO donor, mitigating the ROS production and membrane damage, while maintaining a lower activity of the antioxidant enzymes compared with As-treated plants. Exogenous NO further downregulated the NR activity by a negative feedback, while As boosted the NR activity, consistent with the high endogenous levels of NO observed upon As treatment. Our results suggest that both endogenous and exogenous NO play critical roles in alleviating the As-induced oxidative stress in S. intermedia by reducing As uptake, and possibly by acting as an antioxidant molecule.

The effects of endogenous and exogenous nitric oxide (NO) on electrical activity were investigated in circular smooth muscle preparations isolated from the guinea-pig stomach antrum. The actions of endogenous NO were evaluated from the effects of inhibition of NO synthesis by N(omega)-nitro-L-arginine (nitroarginine), while those of exogenous NO were assessed from the effects of SIN-1, an NO donor. Antral circular smooth muscle generated slow potentials periodically at a frequency of about 1 cycle per min (cpm), and unitary potentials were also generated in a random fashion in the interval between slow potentials. Application of nitroarginine (10(-5) M) increased the frequency of slow potentials, with no significant alteration of the resting membrane potential and amplitude of slow potentials. Frequency analysis of unitary potentials revealed that nitroarginine also increased the spectral density at 0.01-1 Hz frequency. The refractory period for the generation of slow potentials evoked by depolarizing pulses was about 10 s, but was decreased to 6 s by nitroarginine. In the presence of nitroarginine, SIN-1 (10(-9)-10(-7) M) reduced the amplitude and frequency of slow potentials: low concentrations (<10(-8) M) reduced only the frequency of slow potentials, while higher concentrations (10(-8)-10(-7) M) reduced both the amplitude and frequency of slow potentials, in a concentration-dependent manner, before abolishing the slow potentials. The power spectrum of the unitary potentials indicated that SIN-1 (>10(-8) M) reduced the spectral density at 0.01-1 Hz frequency. The refractory period for the generation of slow potentials was increased again to about 10 s by SIN-1. Thus, the excitatory effects of nitroarginine could be antagonized by SIN-1, suggesting that the inhibitory effects of endogenous NO are comparable to those of exogenous NO produced by SIN-1. The results also suggested that the effects of NO on smooth muscle are insignificant and NO selectively inhibits the activity of intramuscular interstitial cells of Cajal (ICC-IM).

Although nitric oxide (NO) is thought to be beneficial in hepatic ischemia-reperfusion (I/R), the mechanisms for this effect are not well established. To investigate the effects of endogenous NO and exogenous NO supplementation on hepatic I/R injury and their pathogenic mechanisms, serum ALT and hyaluronic acid (endothelial cell damage), and hepatic malondialdehyde and H2O2 (oxidative stress), myeloperoxidase activity (leukocyte accumulation), and endothelin (vasoconstrictor peptide opposite to NO) were determined at different reperfusion periods in untreated rats and rats receiving L-NAME, L-NAME+L-arginine, and spermine NONOate (exogenous NO donor). After reperfusion every parameter increased in untreated animals. Endogenous NO synthesis inhibition by L-NAME increased hepatocyte and endothelial damage as compared to untreated rats, which was reverted and even improved by the addition of L-arginine. Spermine NONOate also improved this damage. However, different mechanisms account for the beneficial effect of endogenous and exogenous NO. Oxidative stress decreased by both L-NAME and L-NAME+L-arginine, but remained unmodified by spermine NONOate. Myeloperoxidase increased by L-NAME and this effect was reverted by the addition of L-arginine, whereas no change was observed with spermine NONOate. Endothelin levels were not modified by L-NAME and L-NAME+L-arginine, but decreased with spermine NONOate. These results suggest that, although both endogenous and exogenous NO exert a protective role in experimental hepatic I/R injury, the mechanisms of the beneficial effect of the two sources of NO are different.

Pulmonary vasodilation during cardiopulmonary transition at birth is mediated by endothelium derived nitric oxide (NO), which is generated by the endothelial isoform of NO synthase (NOS), or eNOS. Inhaled NO therapy is currently often used to treat neonatal pulmonary hypertension. Some patients receiving inhaled NO are difficult to wean from the gas after prolonged treatment, suggesting that long-term NO exposure modifies the function of the NO-cGMP signalling cascade. Although NO is known to acutely and reversibly inhibit eNOS activity, the effects of prolonged NO exposure on eNOS expression are not known. We therefore determined the effects of long-term NO exposure on eNOS expression in early passage ovine fetal pulmonary artery endothelial cells (PAEC). To first examine the effects of endogenous NO, PAEC were treated with 2 mM nitro-L-arginine methyl ester (L-NAME). L-NAME caused a decrease in eNOS protein expression that was evident within 8h and maximal by 16h (67% decrease); the effect persisted for at least 48h. The effects of exogenous NO were examined in PAEC treated with the NO donor spermine NONOate or the control parent compound spermine (10-8M to 10-6M). Exogenous NO caused increases in both eNOS protein and cell lysate NOS activity that were also evident within 8h, maximal at 16h (223% of control), and persistent for at least 48h. However, eNOS mRNA abundance was not altered by changes in the level of endogenous or exogenous NO, indicating that the effect of NO on eNOS expression is mediated at the level of translation. This effect is more rapid than previously demonstrated transcriptional regulation of PAEC eNOS by oxygen(≥ 24h). Thus, both endogenous and exogenous NO cause positive feedback, translational upregulation of eNOS gene expression in fetal PAEC. To our knowledge, this is the first demonstration of eNOS regulation mediated at the level of translation. Furthermore, these findings suggest that difficulties with prolonged requirements for inhaled NO in certain patients are not due to changes in the capacity for endogenous NO production.

In this issue of the Journal, Xie and co-workers (1) demonstrate that introducing nitric oxide (NO) synthase type II (NOSII, iNOS) into tumor cells produces cytotoxicity not only in the transfected cells but also in bystander tumor cells. Here, we discuss the potential implications of these experiments in the light of present knowledge on the complex role of NO in tumor biology. The growth of solid tumors is regulated by interactions between endothelial cells of the tumor vasculature, tumorinfiltrating immune cells (such as T lymphocytes and macrophages), and the tumor cells themselves. In these cellular interactions, the unusual biologic messenger molecule and cytotoxin, NO, may play important pathobiologic roles in addition to its many physiologic functions (2). Endogenous NO production from L-arginine has been directly or indirectly demonstrated in all of these cell types. In most cases, the inducible NO synthase gene (Nos2)—one of three known human Nos genes (3)— was switched on (presumably by NF-kB-dependent mechanisms). Its gene product, the high-output NOS-II isoform, unlike its constitutively expressed low-output counterparts (NOS-I and -III), is not regulated by the intracellular concentration of free calcium (3) and is chronically active. Such continuously high exposure of cells to endogenous NO as well as exogenous NO donors will inhibit proliferation and induce cell death (4). High NO levels inhibit mitochondrial respiration, the citric acid cycle, glycolysis, and DNA replication. Locally high levels of reactive oxygen species (ROS), stemming, for example, from host immune cells or from an insufficient oxygen supply to the tumor tissue, may exacerbate these toxic effects by generating even more reactive compounds, such as peroxynitrite (ONOO). The latter compound arises from the diffusion-limited interaction of NO and O2 − and is even more reactive than NO, but it is stable enough to diffuse to and thus harm tumor cells. Apart from these tumoricidal effects, NO has facilitated tumor growth and vascularization in a few experimental models, and, in certain human carcinomas, endogenous NO production was positively associated with tumor grade (5). Thus, NO has a complex, at least dual, action on tumor growth that may depend on the local concentrations of NO, additional factors such as the presence of ROS, and the type of tumor and its susceptibility to NO. In those models where NO had a permissive effect on tumor growth, NOS activity was up to two orders of magnitude lower than that associated with NO-dependent tumor toxicity and apoptosis (5). The dual action of NO on tumor cell proliferation is reminiscent of what has been repeatedly demonstrated for ROS (6,7). Maximal growth promotion by ROS is observed when cells maintain a low but sufficient oxidant signal for the induction of growth-competence genes (8,9), as may be the case with moderate NO concentrations. At high ROS concentrations, the equilibrium balance between the cellular antioxidant defense and oxidant levels is shifted, i.e., toward lipid peroxidation and DNA fragmentation. Besides this concentration dependence, the mechanisms of action of NO may also diverge considerably (Fig. 1). Signaling functions at comparatively low NO concentrations involve soluble guanylyl cyclases as the principal molecular target and subsequent increases in the intracellular level of the second messenger molecule, cyclic guanosine monophosphate (cGMP) (10). The tumor-relevant consequences of these increases in cGMP may be the stimulation of (neo)vascularization and angiogenesis. Moreover, endogenous NO may not only promote the growth of existing tumors, but it may also be tumorigenic itself. NO can cause mutations by mediating the deamination of S-methylcytosine to thymine and also by increasing the formation of DNA strand breaks (11). Increased NO formation has been implicated in several tumor types associated with chronic infections and inflammations, such as gastric, duodenal, esophageal, bladder, and liver cancers (12,13). What are the therapeutic implications of this dual role of NO? One apparent goal may be to enhance NO synthesis to a maximum level only in tumor tissue so that cell proliferation is impaired and cell death is induced. This may be achieved in principle by simply substituting NO in the form of so-called NO donors. Indeed, NO is an effective hypoxic radiosensitizer of tumor tissue (14), while a nonselective NOS inhibitor increases tumor survival (15). Alternatively, endogenous NO formation may be induced in the tumor cell by different cytokines (tumor necrosis factor-a and interleukin 2). Tumor necrosis factor-a has been shown to induce both NO and superoxide (O2 ) production, providing an effective means of local ONOO formation (16). However, cytokines induce NOS-II expression systemically in the blood vessel wall, resulting in massive NOand cGMPmediated vasodilatation and hypotension, which has been recognized as the primary limiting factor in high-dose cytokine therapy (17). A more direct approach is to transfer cytokine or NO synthase

Restoring Oxygen Carrying Capacity of Sickle Erythrocytes with Nitric Oxide Donors.

Nitric Oxide

Nitric oxide: physiology and pharmacology.

Bioinspired Design of Reversible Fluorescent Probes for Tracking Nitric Oxide Dynamics in Live Cells

Effects of endogenous and exogenous nitric oxide on endothelin-1 production in cultured vascular endothelial cells

It was inevitable that important relationships between two of the most intensely studied topics in biomedical research, apoptosis and nitric oxide (NO), would become apparent. Apoptosis is essential to normal development as well as physiological cell turnover. Although apoptosis in excess can manifest as tissue damage, a failure to undergo apoptosis constitutes pathological cellular overgrowth. It is now evident that NO and its reaction products can either promote or prevent apoptosis in a multitude of settings. The ubiquitous distribution of the NO synthases and the remarkable diffusibility and diverse chemical reactivity of NO in biological systems make this molecule unique among the regulators of apoptosis. Understanding the factors that govern the consequences of NO exposure on cell viability and identifying the conditions in which NO regulation of apoptosis contribute to pathology are topics of considerable interest and potential importance. In this article, we will review the recent observations on NO as a regulator of apoptosis. Apoptosis, or programmed cell death, is distinguished from lytic or necrotic cell death by specific biochemical and structural events (see recent review in Reference 11 ). Apoptogenic signals trigger cell-specific signaling pathways, including protease activation, followed by the appearance of morphological changes characteristic of cells undergoing apoptosis, including condensation of nuclei and cytoplasm, blebbing of the cytoplasmic membranes, and finally fragmentation into apoptotic bodies that are phagocytosed by neighboring cells. The elucidation of the signaling events in apoptosis is occurring at a rapid pace and includes the identification of the key roles of cysteine proteases (known as caspases), Bcl-2 family members, and mitochondria. Caspases, the mammalian counterpart of ced-3 in Caenorhabditis elegans , are a family of cysteine proteases now known to contain at least 14 homologs. Ectopic expression of any of the caspase family proteases can cause apoptosis; however, not all caspase family …

Glioma Stem Cell Proliferation and Tumor Growth Are Promoted by Nitric Oxide Synthase-2

Direct measurement of actual levels of nitric oxide (NO) in cell culture conditions using soluble NO donors