Stable isotope measurement of in vivo nitric oxide production in health and disease: an updated systematic review and meta-analysis.

Whole-body basal nitric oxide production is impaired in postprandial endothelial dysfunction in healthy rats

BackgroundLow bioavailability of nitric oxide (NO) is implicated in the pathophysiology of sickle cell disease (SCD). We designed a nested pilot study to be conducted within a clinical trial testing the effects of a daily ready-to-use supplementary food (RUSF) fortified with arginine (Arg) and citrulline (Citr) vs. non-fortified RUSF in children with SCD. The pilot study evaluated 1) the feasibility of a non-invasive stable isotope method to measure whole-body NO production and 2) whether Arg+Citr supplementation was associated with increased whole-body NO production. SubjectsTwenty-nine children (70% male, 9–11years, weight 16.3–31.3 kg) with SCD. MethodsSixteen children received RUSF+Arg/Citr (Arg, 0.2 g/kg/day; Citr, 0.1 g/kg/day) in combination with daily chloroquine (50 mg) and thirteen received the base RUSF in combination with weekly chloroquine (150 mg). Plasma amino acids were assessed using ion-exchange elution (Biochrom-30, Biochrom, UK) and whole-body NO production was measured using a non-invasive stable isotopic method. ResultsThe RUSF+Arg/Citr intervention increased plasma arginine (P = .02) and ornithine (P = .003) and decreased the ratio of asymmetric dimethylarginine to arginine (P = .01), compared to the base RUSF. A significant increase in whole-body NO production was observed in the RUSF-Arg/Citr group compared to baseline (weight-adjusted systemic NO synthesis 3.38 ± 2.29 μmol/kg/hr vs 2.35 ± 1.13 μmol/kg/hr, P = .04). No significant changes were detected in the base RUSF group (weight-adjusted systemic NO synthesis 2.64 ± 1.14 μmol/kg/hr vs 2.53 ± 1.12 μmol/kg/hr, P = .80). ConclusionsThe non-invasive stable isotopic method was acceptable and the results provided supporting evidence that Arg/Citr supplementation may increase systemic NO synthesis in children with SCD.

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

Nitric oxide (NO) is involved in many physiological and pathological processes in the human body. At least two major pathways produce NO: (1) the L-arginine-NO-oxidative pathway in which NO synthase (NOS) enzymes convert L-arginine to NO; (2) the nitrate-nitrite-NO reductive pathway in which NO is produced from the serial reduction of nitrate and nitrite. The deficiency of NO is involved in the pathophysiology of cardiometabolic disorders. Intervention with foods containing nitrate and nitrite can potentially prevent or treat some chronic diseases, including cardiovascular diseases and diabetes. A better understanding of the NO cycle would help develop effective strategies for preventing or treating the disorders in which NO homeostasis is disturbed. This review summarizes quantitative aspects of NO production, emphasizing the nitrate-nitrite-NO pathway. Available data indicates that total NO production by NOS-dependent L-arginine-NO pathway is about 1000 μmol.day-1. Of about 1700 μmol.day-1 ingested nitrate, ~25 % is extracted by the salivary glands and of which ~20 % is converted nitrite. It means that about 5 % of ingested nitrate is converted to nitrite in the oral cavity; assuming that all produced nitrite is reduced to NO in the stomach, it can be calculated that contribution of the nitrate-nitrite-NO pathway to the whole-body NO production is about 85 μmol.day-1 (1700 ×0.05=85) or approximately 100 μmol.day-1. The lower contribution of the nitrate-nitrite-NO pathway does not mean that this pathway has lower importance in the whole-body NO homeostasis. Even in the adequate L-arginine supply, NOS-dependent NO production is insufficient to meet all NO functions, and the nitrate-nitrite-NO pathway must provide the rest. In conclusion, the contribution of the nitrate-nitrite-NO pathway in the whole human body NO production is <10 %, and the nitrate-nitrite-NO pathway is complementary to the NOS-dependent NO production.

Associations between Aging and Vitamin D Status with Whole-Body Nitric Oxide Production and Markers of Endothelial Function

Background: Rats with chronic renal failure have a low nitric oxide (NO) production and a diminished NO excretion. The supplementation of L-arginine has an inhibitory effect on the progression of renal insufficiency. Methods: The present study was designed to determine whether chronic renal failure patients have a low NO production. Plasma and urine nitrate (NO₃) and nitrite (NO₂), stable metabolites of NO, were measured in 83 consecutive patients with chronic renal failure. The 83 chronic renal failure patients were divided into three groups: group 1, mild renal failure (creatinine clearance >60 ml/min/1.73 m²); group 2, moderate renal failure (creatinine clearance >30 <60 ml/min/1.73 m²), and group 3, severe renal failure (creatinine clearance <30 ml/min/1.73 m²). Thirty-three healthy volunteers served as controls. Results: The daily urinary NO excretion was significantly lower in patients with moderate and severe renal failure as compared with those with mild renal failure and normal controls. The lowest values were found in the severe renal failure group. When the 24-hour urinary NO excretion or NO per milligram creatinine and the NO clearance were correlated with the renal function in all patients as a group, these parameters were directly correlated with the creatinine clearance and inversely correlated with the serum creatinine level. The plasma NO concentration was not different between the three chronic renal failure groups, but higher than in the controls. Plasma NO in renal failure patients was not correlated with the creatinine clearance or serum creatinine levels. Conclusions: Chronic renal failure is a state of NO deficiency. Treatment strategies to increase NO production (L-arginine supplementation or other NO compounds) may prove to be useful in maintaining the renal function and slow the progression of renal disease.

Endothelin-1 inhibits sodium reabsorption in the thick ascending limb (THAL) via stimulation of nitric oxide (NO) production. The mechanism whereby endothelin-1 stimulates THAL NO is unknown. We hypothesized that endothelin-1 stimulates THAL NO production by activating phosphatidylinositol 3-kinase (PI3K), stimulating Akt activity, and phosphorylating NOS3 at Ser1177. This enhances NO production and inhibits sodium transport. We measured 1) NO production by fluorescence microscopy using DAF2-DA, 2) Akt activity using a fluorescence resonance energy transfer-based Akt reporter, 3) phosphorylated NOS3 and Akt by Western blotting, and 4) NKCC2 activity by fluorescence microscopy. In isolated THAL, endothelin-1 (1 nmol/liter) increased NO production from 0.23 +/- 0.24 to 2.81 +/- 0.32 fluorescence units/min (p < 0.001; n = 5) but failed to stimulate NO production in THALs isolated from NOS3-/- mice. Wortmannin (150 nmol/liter), a PI3K inhibitor, reduced endothelin-1-stimulated NO by 83% (0.49 +/- 0.13 versus 3.31 +/- 0.49 fluorescence units/min for endothelin-1 alone; p < 0.006; n = 5). Endothelin-1 stimulated Akt activity by 0.16 +/- 0.02 arbitrary units as measured by fluorescence resonance energy transfer (p < 0.001; n = 5) and increased phosphorylation of Akt at Ser473 by 56 +/- 11% (p < 0.002; n = 7). Dominant-negative Akt blocked endothelin-1-induced NO by 60 +/- 8% (p < 0.001 versus control; n = 6), and an Akt inhibitor had a similar effect. Endothelin-1 increased phosphorylation of NOS3 at Ser1177 by 89 +/- 24% (p < 0.01; n = 7) but had no effect on Ser633. Endothelin-1 inhibited NKCC2 activity, an effect that was blocked by dominant-negative Akt and NOS inhibition. We conclude that endothelin-1 stimulates THAL NO production by activating PI3K, stimulating Akt activity, and phosphorylating NOS3 at Ser1177. This enhances NO production and inhibits sodium transport.

A reduction in nitric oxide (NO) synthesis has been suggested to play a role in pregnancy-induced hypertension. We have recently reported that normal pregnancy in the rat is associated with significant increases in whole-body NO production and renal protein expression of neuronal and inducible NO synthase. The purpose of this study was to determine whether whole-body and renal NO production is reduced in a rat model of pregnancy-induced hypertension produced by chronically reducing uterine perfusion pressure starting at day 14 of gestation. Chronic reductions in uterine perfusion pressure resulted in increases in arterial pressure of 20 to 25 mm Hg, decreases in renal plasma flow (<23%) and glomerular filtration rate (<40%), but no difference in urinary nitrite/nitrate excretion relative to control pregnant rats. In contrast, reductions in uterine perfusion pressure in virgin rats resulted in no significant effects on arterial pressure. Renal endothelial (<4%) and inducible (<11%) NO synthase protein expression did not decrease significantly in the chronically reduced uterine perfusion pressure rats relative to normal pregnant rats; however, significant reductions in neuronal NO synthase were observed (<30%). The results of this study indicate that the reduction in renal hemodynamics and the increase in arterial pressure observed in response to chronic decreases in uterine perfusion pressure in pregnant rats are associated with no change in whole-body NO production and a decrease in renal protein expression of neuronal NO synthase.

Laminar shear stress (LSS) is known to increase endothelial nitric oxide (NO) production, which is essential for vascular health, through expression and activation of nitric oxide synthase 3 (NOS3). Recent studies demonstrated that LSS also increases the expression of argininosuccinate synthetase 1 (ASS1) that regulates the provision of L-arginine, the substrate of NOS3. It was thus hypothesized that ASS1 might contribute to vascular health by enhancing NO production in response to LSS. This hypothesis was pursued in the present study by modulating NOS3 and ASS1 levels in cultured endothelial cells. Exogenous expression of either NOS3 or ASS1 in human umbilical vein endothelial cells increased NO production and decreased monocyte adhesion stimulated by tumor necrosis factor-α (TNF-α). The latter effect of overexpressed ASS1 was reduced when human umbilical vein endothelial cells were co-treated with small interfering RNAs (siRNAs) for ASS1 or NOS3. SiRNAs of NOS3 and ASS1 attenuated the increase of NO production in human aortic endothelial cells stimulated by LSS (12 dynes·cm(-2)) for 24 h. LSS inhibited monocyte adhesion to human aortic endothelial cells stimulated by TNF-α, but this effect of LSS was abrogated by siRNAs of NOS3 and ASS1 that recovered the expression of vascular cell adhesion molecule-1. The current study suggests that the expression of ASS1 harmonized with that of NOS3 may be important for the optimized endothelial NO production and the prevention of the inflammatory monocyte adhesion to endothelial cells.

Impaired nitric oxide production in children with MELAS syndrome and the effect of arginine and citrulline supplementation

Nitric oxide (NO) has a critical role in neuronal function; however, high levels lead to cellular injury. While guanidino-methylated arginines (MA) including asymmetric dimethylarginine (ADMA) and N(G)-methyl-l-arginine (NMA) are potent competitive inhibitors of nitric oxide synthase (NOS) and are released upon protein degradation, it is unknown whether their intracellular concentrations are sufficient to critically regulate neuronal NO production and secondary cellular function or injury. Therefore, we determine the intrinsic neuronal MA concentrations and their effects on neuronal NOS function and excitotoxic injury. Kinetic studies demonstrated that the K(m) for l-arginine is 2.38 microm with a V(max) of 0.229 micromol mg(-1) min(-1), while K(i) values of 0.67 microm and 0.50 microm were determined for ADMA and NMA, respectively. Normal neuronal concentrations of all NOS-inhibiting MA were determined to be approximately 15 microm, while l-arginine concentration is approximately 90 microm. These MA levels result in >50% inhibition of NO generation from neuronal NOS. Down-modulation or up-modulation of these neuronal MA levels, respectively, dramatically enhanced or suppressed NO-mediated excitotoxic injury. Thus, neuronal MA profoundly modulate NOS function and suppress NO mediated injury. Pharmacological modulation of the levels of these intrinsic NOS inhibitors offers a novel approach to modulate neuronal function and injury.

Background: Major Depressive disorder (MDD) is associated with chronic low-grade inflammation with elevated proinflammatory cytokines. Notably, elevated inflammation is a known contributor to cardiovascular dysfunction and disease. Interleukin (IL)-6 has been shown to be elevated in rodent models of depression. Additionally, preliminary data from our lab suggests that there is a greater proportion of IL-6 producing CD8+ T cells in adults with MDD. Although IL-6 has been reported to be increased in adults with MDD and known to have a direct effect on vascular endothelial cells, the exact mechanism by which IL-6 interacts with the endothelium is unknown. Therefore, we tested the hypothesis that IL-6 treatment will decrease nitric oxide (NO) production and increase mitochondrial ROS (mitoROS) in endothelial cells. Methods: Human aortic endothelial cells (HAECs) were used to study the effects of IL-6 on NO production. The HAECs were treated with vehicle or 10 ng/mL IL-6 for 16 hours, and then stimulated with Acetylcholine to induce NO production and the real time NO production was measured using a commercially available assay. We also measured mitoROS in HAECs treated with or without IL-6. Results are expressed as mean ± SEM (n=3-6 replicates) and group differences were assessed by one-way ANOVA followed by Tukey’s post-hoc test. Results: L-6 was found to significantly lower NO production in the cells (Control: 276.24 ± 7.01; IL-6: 215.19 ± 6.07 MFI, p=0.022). Acetylcholine stimulation resulted in greater NO production in the control cells (Control: 276.24 ± 7.01; Control + Ach: 354.28 ± 6.26 MFI, p=0.0005) whereas this increase was abolished in the IL-6 treated cells (IL-6: 215.19 ± 6.07; IL-6 + Ach: 225.21 ± 5.16 MFI, p=0.955). Additionally, we also found greater mitoROS in the IL-6 treated cells (Control: 683.02 ± 32.98; IL-6: 840.86 ± 38.63 MFI, p=0.002). Conclusion: These preliminary results suggest that IL-6 reduces NO production in endothelial cells, which may be in part due to elevated mitoROS. This work is supported by grants from NIH K01AG061271, R01AG060395 and AHA940023 (DWT). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BackgroundOxidative stress due to the overproduction of nitric oxide (NO) and other oxygen reactive species (ROS), play a main role in the initiation and progression of the OA disease and leads to the degeneration of mitochondria. Therefore, the goal of this work is to describe the difference in telomere length of peripheral blood leukocytes (PBLs) and Nitric Oxide (NO) production between mitochondrial DNA (mtDNA) haplogroup J and non-J carriers, as indirect approaches of oxidative stress.MethodsThe telomere length of PBL was analyzed in DNA samples from 166 healthy controls (114 J and 52 non-J) and 79 OA patients (41 J and 38 non-J) by means of a validated qPCR method. The NO production was assessed in 7 carriers of the haplogroup J and 27 non-J carriers, by means of the colorimetric reaction of the Griess reagent in supernatants of cultured chondrocytes. Inducible nitric oxide synthase (iNOS) mRNA from these samples was analyzed by qPCR. Appropiated statistical analyses were performedResultsCarriers of the haplogroup J showed a significantly longer telomere length of PBLs than non-J carriers, regardless of age, gender and diagnosis (p = 0.025). Cultured chondrocytes carrying the mtDNA haplogroup J also showed a lower NO production than non-J carriers (p = 0.043). No significant correlations between age and telomore length of PBLs were detected neither for carriers of the haplogroup J nor for non-J carriers. A strong positive correlation between NO production and iNOS expression was also observed (correlation coefficient = 0.791, p < 0.001).ConclusionThe protective effect of the mtDNA haplogroup J in the OA disease arise from a lower oxidative stress in carriers of this haplogroup, since this haplogroup is related to lower NO production and hence longer telomere length of PBLs too.

The amino acid arginine is the sole precursor for nitric oxide (NO) synthesis. We recently demonstrated that an acute reduction of circulating arginine does not compromise basal or LPS-inducible NO production in mice. In the present study, we investigated the importance of citrulline availability in ornithine transcarbamoylase-deficient spf(ash) (OTCD) mice on NO production, using stable isotope techniques and C57BL6/J (wild-type) mice controls. Plasma amino acids and tracer-to-tracee ratios were measured by LC-MS. NO production was measured as the in vivo conversion of l-[guanidino-(15)N(2)]arginine to l-[guanidine-(15)N]citrulline; de novo arginine production was measured as conversion of l-[ureido-(13)C-5,5-(2)H(2)]citrulline to l-[guanidino-(13)C-5,5-(2)H(2)]arginine. Protein metabolism was measured using l-[ring-(2)H(5)]phenylalanine and l-[ring-(2)H(2)]tyrosine. OTC deficiency caused a reduction of systemic citrulline concentration and production to 30-50% (P < 0.001), reduced de novo arginine production (P < 0.05), reduced whole-body NO production to 50% (P < 0.005), and increased net protein breakdown by a factor of 2-4 (P < 0.001). NO production was twofold higher in female than in male OTCD mice in agreement with the X-linked location of the OTC gene. In response to LPS treatment (10 mg/kg ip), circulating arginine increased in all groups (P < 0.001), and NO production was no longer affected by the OTC deficiency due to increased net protein breakdown as a source for arginine. Our study shows that reduced citrulline availability is related to reduced basal NO production via reduced de novo arginine production. Under basal conditions this is probably cNOS-mediated NO production. When sufficient arginine is available after LPS stimulated net protein breakdown, NO production is unaffected by OTC deficiency.

Different mouse strains possessing the Nramp1r allele, which were theoretically expected to have relatively high nitric oxide (NO) production after cytokine stimulation, were used to analyze the genetic factors associated with NO production. After gamma interferon and lipopolysaccharide stimulation, the strains NZB/N, DBA/2N, AKR/N, and A/J showed significantly low NO production; NJL, 129/J, MOG, SJL/J, CBA/N, and NOD/Shi had moderate amounts; and C3H/He and SPR had the highest levels as compared to the other mice. The F1 progeny of A/J x C3H/He and AKR/N x C3H/He showed significantly higher NO production, whereas the F1 progeny of DBA/2N x C3H/He produced a relatively low amount. Furthermore, the backcross progeny from their F1 showed variations in NO production, and therefore it was speculated that the regulation of NO production is polygenic. Genetic typing experiments related to the NO production in the backcross progeny demonstrated significant deviations to some genetic microsatellite markers. Sequencing of the iNOS promoter regions of the Nramp1r strains to examine the relationship with NO production revealed that MOG and SPR strains had substitutions within the NF-kappaB and the gamma-IRE transcription binding factor, respectively.