Evidence for the participation of nitric oxide in pemphigus

Pemphigus is an inflammatory autoimmune disorder of the skin. Nitric oxide (NO) is an inflammatory mediator linked to a variety of physiological and pathophysiological phenomena that include skin tumors, psoriasis, urticaria, and atopic dermatitis. Inflammatory cells present in pemphigus lesions are important sources of NO production. We investigated whether NO is involved in pemphigus. A prospective cohort study was conducted at the Dermatology Service of the Hospital Universitário Walter Cantídio of the Federal University of Ceará. All patients seen at the outpatient clinic between August 2000 and July 2001, with a clinically and histologically confirmed diagnosis of pemphigus were included. The median age was 42.5 years (range: 12-69 years) with a male to female ratio of 3:2. Total serum nitrite levels, used as a marker for NO production, were determined by the Griess reaction. Skin biopsies from pemphigus and breast surgery (control) patients were used for the detection of the inducible NO synthase (iNOS) by immunohistochemistry. Twenty-two (22) patients with pemphigus and eight (8) controls who did not differ in demographic characteristics were included. Total serum nitrite levels were significantly higher (>7 µmol/L) in pemphigus patients compared to controls (<6 µmol/L), regardless of the severity of the clinical activity of pemphigus (P < 0.0001). All pemphigus biopsies presented increased immunostaining for iNOS that was not detected in normal skin samples. These data are the first to demonstrate that pemphigus patients display increased serum NO levels that are associated with increased iNOS expression in the affected skin.

Antagonic effect of the inhibition of inducible nitric oxide on the mortality of mice acutely infected with Escherichia coli and Bacteroides fragilis

Sepsis, the leading cause of death in intensive care units, is associated with overproduction of nitric oxide (NO) due to inducible NO synthase (iNOS), responsible for some of the pathologic changes. Aminoguanidine (AG) is a selective iNOS inhibitor with reported inconsistent actions in sepsis. To investigate the influence of iNOS, we studied models of acute bacterial sepsis using acute challenges with aerobic (Escherichia coli) and anaerobic (Bacteroides fragilis) bacteria in the presence of AG. Six-week-old, 23 g, male and female BALB/c and C57Bl/6j mice, in equal proportions, were inoculated (ip) with bacteria in groups of 4 animals for each dose and each experiment in the absence or presence of AG (50 mg/kg, ip, starting 24 h before challenge and daily until day 6) and serum nitrate was measured by chemiluminescence. Both types of bacteria were lethal to mice, with an LD50 of 6 nephelometric units (U) for E. coli and 8 U for B. fragilis. Nitrate production peaked on the second day after E. coli inoculation with 8 and 6 U (P < 0.05), but was absent after non-lethal lower doses. After challenge with B. fragilis this early peak occurred at all tested doses after 24 h, including non-lethal ones (P < 0.05). AG-treated mice challenged with E. coli presented higher survival (P < 0.05) and increased LD50. AG-treated mice challenged with B. fragilis had lower LD50 and higher mortality. Control AG-treated animals presented no toxic effects. The opposite effect of iNOS blockade by AG in these models could be explained by restriction of oxygen for immune cells or an efficient action of NO in anaerobic localized infections. The antagonic role of NO production observed in our bacterial models could explain the reported discrepancy of NO action in sepsis.

New nitric oxide donors based on ruthenium complexes

Nitric oxide (NO) donors produce NO-related activity when applied to biological systems. Among its diverse functions, NO has been implicated in vascular smooth muscle relaxation. Despite the great importance of NO in biological systems, its pharmacological and physiological studies have been limited due to its high reactivity and short half-life. In this review we will focus on our recent investigations of nitrosyl ruthenium complexes as NO-delivery agents and their effects on vascular smooth muscle cell relaxation. The high affinity of ruthenium for NO is a marked feature of its chemistry. The main signaling pathway responsible for the vascular relaxation induced by NO involves the activation of soluble guanylyl-cyclase, with subsequent accumulation of cGMP and activation of cGMP-dependent protein kinase. This in turn can activate several proteins such as K+ channels as well as induce vasodilatation by a decrease in cytosolic Ca2+. Oxidative stress and associated oxidative damage are mediators of vascular damage in several cardiovascular diseases, including hypertension. The increased production of the superoxide anion (O2-) by the vascular wall has been observed in different animal models of hypertension. Vascular relaxation to the endogenous NO-related response or to NO released from NO deliverers is impaired in vessels from renal hypertensive (2K-1C) rats. A growing amount of evidence supports the possibility that increased NO inactivation by excess O2- may account for the decreased NO bioavailability and vascular dysfunction in hypertension.

Effects of pneumonectomy on nitric oxide synthase expression and perivascular edema in the remaining lung of rats

Pneumonectomy is associated with high mortality and high rates of complications. Postpneumonectomy pulmonary edema is one of the leading causes of mortality. Little is known about its etiologic factors and its association with the inflammatory process. The purpose of the present study was to evaluate the role of pneumonectomy as a cause of pulmonary edema and its association with gas exchange, inflammation, nitric oxide synthase (NOS) expression and vasoconstriction. Forty-two non-specific pathogen-free Wistar rats were included in the study. Eleven animals died during or after the procedure, 21 were submitted to left pneumonectomy and 10 to sham operation. These animals were sacrificed after 48 or 72 h. Perivascular pulmonary edema was more intense in pneumonectomized rats at 72 h (P = 0.0131). Neutrophil density was lower after pneumonectomy in both groups (P = 0.0168). There was higher immunohistochemical expression of eNOS in the pneumonectomy group (P = 0.0208), but no statistically significant difference in the expression of iNOS. The lumen-wall ratio and pO2/FiO2 ratio did not differ between the operated and sham groups after pneumonectomy. Left pneumonectomy caused perivascular pulmonary edema with no elevation of immunohistochemical expression of iNOS or neutrophil density, suggesting the absence of correlation with the inflammatory process or oxidative stress. The increased expression of eNOS may suggest an intrinsic production of NO without signs of vascular reactivity.

Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection

Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO). In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS) in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT). Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test) and allodynia (von Frey hair test). Control animals did not present any alteration (sham-animals). The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 µg in 50 µL), blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48% by day 30) in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed’s lamina X) and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469%). Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20%), reaching the greatest increase (60%) 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

Nitric oxide synthesis and biological functions of nitric oxide released from ruthenium compounds

During three decades, an enormous number of studies have demonstrated the critical role of nitric oxide (NO) as a second messenger engaged in the activation of many systems including vascular smooth muscle relaxation. The underlying cellular mechanisms involved in vasodilatation are essentially due to soluble guanylyl-cyclase (sGC) modulation in the cytoplasm of vascular smooth cells. sGC activation culminates in cyclic GMP (cGMP) production, which in turn leads to protein kinase G (PKG) activation. NO binds to the sGC heme moiety, thereby activating this enzyme. Activation of the NO-sGC-cGMP-PKG pathway entails Ca2+ signaling reduction and vasodilatation. Endothelium dysfunction leads to decreased production or bioavailability of endogenous NO that could contribute to vascular diseases. Nitrosyl ruthenium complexes have been studied as a new class of NO donors with potential therapeutic use in order to supply the NO deficiency. In this context, this article shall provide a brief review of the effects exerted by the NO that is enzymatically produced via endothelial NO-synthase (eNOS) activation and by the NO released from NO donor compounds in the vascular smooth muscle cells on both conduit and resistance arteries, as well as veins. In addition, the involvement of the nitrite molecule as an endogenous NO reservoir engaged in vasodilatation will be described.

Contrasting effects of nitric oxide and corticotropin- releasing factor within the dorsal periaqueductal gray on defensive behavior and nociception in mice

The anxiogenic and antinociceptive effects produced by glutamate N-methyl-D-aspartate receptor activation within the dorsal periaqueductal gray (dPAG) matter have been related to nitric oxide (NO) production, since injection of NO synthase (NOS) inhibitors reverses these effects. dPAG corticotropin-releasing factor receptor (CRFr) activation also induces anxiety-like behavior and antinociception, which, in turn, are selectively blocked by local infusion of the CRF type 1 receptor (CRFr1) antagonist, NBI 27914 [5-chloro-4-(N-(cyclopropyl)methyl-N-propylamino)-2-methyl-6-(2,4,6-trichlorophenyl)aminopyridine]. Here, we determined whether i) the blockade of the dPAG by CRFr1 attenuates the anxiogenic/antinociceptive effects induced by local infusion of the NO donor, NOC-9 [6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine], and ii) the anxiogenic/antinociceptive effects induced by intra-dPAG CRF are prevented by local infusion of Nω-propyl-L-arginine (NPLA), a neuronal NOS inhibitor, in mice. Male Swiss mice (12 weeks old, 25-35 g, N = 8-14/group) were stereotaxically implanted with a 7-mm cannula aimed at the dPAG. Intra-dPAG NOC-9 (75 nmol) produced defensive-like behavior (jumping and running) and antinociception (assessed by the formalin test). Both effects were reversed by prior local infusion of NBI 27914 (2 nmol). Conversely, intra-dPAG NPLA (0.4 nmol) did not modify the anxiogenic/antinociceptive effects of CRF (150 pmol). These results suggest that CRFr1 plays an important role in the defensive behavior and antinociception produced by NO within the dPAG. In contrast, the anxiogenic and antinociceptive effects produced by intra-dPAG CRF are not related to NO synthesis in this limbic midbrain structure.

Partial baroreceptor dysfunction and low plasma nitric oxide bioavailability as determinants of salt-sensitive hypertension: a reverse translational rat study

This study determined whether clinical salt-sensitive hypertension (cSSHT) results from the interaction between partial arterial baroreceptor impairment and a high-sodium (HNa) diet. In three series (S-I, S-II, S-III), mean arterial pressure (MAP) of conscious male Wistar ChR003 rats was measured once before (pdMAP) and twice after either sham (SHM) or bilateral aortic denervation (AD), following 7 days on a low-sodium (LNa) diet (LNaMAP) and then 21 days on a HNa diet (HNaMAP). The roles of plasma nitric oxide bioavailability (pNOB), renal medullary superoxide anion production (RMSAP), and mRNA expression of NAD(P)H oxidase and superoxide dismutase were also assessed. In SHM (n=11) and AD (n=15) groups of S-I, LNaMAP-pdMAP was 10.5±2.1 vs 23±2.1 mmHg (P<0.001), and the salt-sensitivity index (SSi; HNaMAP−LNaMAP) was 6.0±1.9 vs 12.7±1.9 mmHg (P=0.03), respectively. In the SHM group, all rats were normotensive, and 36% were salt sensitive (SSi≥10 mmHg), whereas in the AD group ∼50% showed cSSHT. A 45% reduction in pNOB (P≤0.004) was observed in both groups in dietary transit. RMSAP increased in the AD group on both diets but more so on the HNa diet (S-II, P<0.03) than on the LNa diet (S-III, P<0.04). MAP modeling in rats without a renal hypertensive genotype indicated that the AD*HNa diet interaction (P=0.008) increases the likelihood of developing cSSHT. Translationally, these findings help to explain why subjects with clinical salt-sensitive normotension may transition to cSSHT.

Effects of nitric oxide on magnocellular neurons of the supraoptic nucleus involve multiple mechanisms

Physiological evidence indicates that the supraoptic nucleus (SON) is an important region for integrating information related to homeostasis of body fluids. Located bilaterally to the optic chiasm, this nucleus is composed of magnocellular neurosecretory cells (MNCs) responsible for the synthesis and release of vasopressin and oxytocin to the neurohypophysis. At the cellular level, the control of vasopressin and oxytocin release is directly linked to the firing frequency of MNCs. In general, we can say that the excitability of these cells can be controlled via two distinct mechanisms: 1) the intrinsic membrane properties of the MNCs themselves and 2) synaptic input from circumventricular organs that contain osmosensitive neurons. It has also been demonstrated that MNCs are sensitive to osmotic stimuli in the physiological range. Therefore, the study of their intrinsic membrane properties became imperative to explain the osmosensitivity of MNCs. In addition to this, the discovery that several neurotransmitters and neuropeptides can modulate their electrical activity greatly increased our knowledge about the role played by the MNCs in fluid homeostasis. In particular, nitric oxide (NO) may be an important player in fluid balance homeostasis, because it has been demonstrated that the enzyme responsible for its production has an increased activity following a hypertonic stimulation of the system. At the cellular level, NO has been shown to change the electrical excitability of MNCs. Therefore, in this review, we focus on some important points concerning nitrergic modulation of the neuroendocrine system, particularly the effects of NO on the SON.

Central release of nitric oxide mediates antinociception induced by aerobic exercise

Nitric oxide (NO) is a soluble gas that participates in important functions of the central nervous system, such as cognitive function, maintenance of synaptic plasticity for the control of sleep, appetite, body temperature, neurosecretion, and antinociception. Furthermore, during exercise large amounts of NO are released that contribute to maintaining body homeostasis. Besides NO production, physical exercise has been shown to induce antinociception. Thus, the present study aimed to investigate the central involvement of NO in exercise-induced antinociception. In both mechanical and thermal nociceptive tests, central [intrathecal (it) and intracerebroventricular (icv)] pretreatment with inhibitors of the NO/cGMP/KATP pathway (L-NOArg, ODQ, and glybenclamide) prevented the antinociceptive effect induced by aerobic exercise (AE). Furthermore, pretreatment (it, icv) with specific NO synthase inhibitors (L-NIO, aminoguanidine, and L-NPA) also prevented this effect. Supporting the hypothesis of the central involvement of NO in exercise-induced antinociception, nitrite levels in the cerebrospinal fluid increased immediately after AE. Therefore, the present study suggests that, during exercise, the NO released centrally induced antinociception.

Nitric oxide levels are not changed in saliva of patients infected with hepatitis C virus

The aim of this investigation was to determine nitric oxide metabolite levels in saliva samples from hepatitis C virus-positive patients in an attempt to test the hypothesis if increased levels of nitric oxide metabolites correlates with the presence of HCV-RNA in saliva. Saliva of 39 HCV-positive patients and 13 HCV-negative patients, without clinical or laboratorial evidence of liver disease were tested for nitric oxide metabolites. HCV-RNA was detected in serum and saliva by a RT-PCR method and nitric oxide level was determined by evaluation of its stable degradation products, nitrate and nitrite. No differences were found between the concentration of nitrite in saliva from HCV patients and controls, in despite of the presence or not of HCV RNA in saliva. Patients with HCV and cirrhosis had higher concentrations of nitrite but not significantly different from the control group or the groups of anti-HCV patients without cirrhosis. Increased levels of nitrite were not detected in anti-HCV positive patients, an indirect indication that chronic sialoadenitis are infrequent in these patients or occurs with low intensity not sufficient to increase nitric oxide metabolite levels in saliva.

Antioxidant factors, nitric oxide levels, and cellular damage in leprosy patients

Introduction The immune response caused by Mycobacterium leprae is a risk factor for the development of oxidative stress (OS) in leprosy patients. This study aimed to assess OS in leprosy patients before the use of a multidrug therapy. Methods We evaluated the nitric oxide (NO) concentration; antioxidant capacity; levels of malondialdehyde, methemoglobin and reduced glutathione; and the activity of catalase and superoxide dismutase (SOD) in leprosy patients. Results We observed lower SOD activity in these leprosy patients; however, the NO levels and antioxidant capacity were increased. Conclusions The infectious process in response to M. leprae could primarily be responsible for the OS observed in these patients.

Inhaled nitric oxide in the management of persistent pulmonary hypertension of the newborn: a meta-analysis

OBJECTIVES: To evaluate the use of inhaled nitric oxide (NO) in the management of persistent pulmonary hypertension of the newborn. METHODS: Computerized bibliographic search on MEDLINE, CURRENT CONTENTS and LILACS covering the period from January 1990 to March 1998; review of references of all papers found on the subject. Only randomized clinical trials evaluating nitric oxide and conventional treatment were included. OUTCOMES STUDIED: death, requirement for extracorporeal membrane oxygenation (ECMO), systemic oxygenation, complications at the central nervous system and development of chronic pulmonary disease. The methodologic quality of the studies was evaluated by a quality score system, on a scale of 13 points. RESULTS: For infants without congenital diaphragmatic hernia, inhaled NO did not change mortality (typical odds ratio: 1.04; 95% CI: 0.6 to 1.8); the need for ECMO was reduced (relative risk: 0.73; 95% CI: 0.60 to 0.90), and the oxygenation was improved (PaO2 by a mean of 53.3 mm Hg; 95% CI: 44.8 to 61.4; oxygenation index by a mean of -12.2; 95% CI: -14.1 to -9.9). For infants with congenital diaphragmatic hernia, mortality, requirement for ECMO, and oxygenation were not changed. For all infants, central nervous system complications and incidence of chronic pulmonary disease did not change. CONCLUSIONS: Inhaled NO improves oxygenation and reduces requirement for ECMO only in newborns with persistent pulmonary hypertension who do not have diaphragmatic hernia. The risk of complications of the central nervous system and chronic pulmonary disease were not affected by inhaled NO.

Myocardial repair with long-term and low-dose administration of a nitric oxide synthesis inhibitor. Myofibroblasts, type III collagen and fibronectin

OBJECTIVE: To study the healing process of the myocardium in hypertensive rats undergoing inhibition of nitric oxide synthesis. METHODS: Two groups of animals were studied: one received L-NAME, 12mg/kg/day, and the other was a control group. The presence of type III collagen, fibronectin, and alpha-smooth muscle actin-positive cells was assessed by immunohistochemistry. RESULTS: Fibronectin was seen in both early and late lesions, while type III collagen was seen mainly in areas of incomplete healing, situated among myocytes and around the intramyocardial branches of the coronary arteries. Areas representing early and late lesions showed a population of spindle-shaped cells. Immunohistochemistry showed that these cells were positive for alpha-smooth muscle actin. CONCLUSION: In the myocardium of hypertensive rats, the alpha-smooth muscle actin-positive cells are related to the accumulation of type III collagen and fibronectin in the areas of myocardial damage.