Role Of Endogenous Nitric Oxide Research Articles

Role of endogenous nitric oxide in classic preconditioning in rat hearts

Ischemic preconditioning (IPC) protects the heart against subsequent sustained ischemia reperfusion (RP). Despite many triggers and signaling pathways, which seem to be involved in IPC, the IPC-mechanisms remain a controversial issue. One of them is endogenous production of nitric oxide (NO). To assess the role of NO in IPC and its relation with glycogen and glycolysis, the effects of inhibiting NO synthase with L-NAME (50 μM) were examined in IPC rat hearts perfused with medium containing 10 mM glucose. Left ventricular developed pressure-rate product (RPP) and end diastolic pressure (EDP), lactate and glycogen contents, and cell viability were measured. Global ischemia (25 min) was followed by 30 min RP. IPC consisted in one cycle of 3 min ischemia–5 min RP. IPC reduced EDP and improved RP recovery of RPP. L-NAME had no effects on the non-IPC group but abolished these effects of IPC. IPC reduced ischemic decrease of glycogen and the acceleration of glycolysis, and improved cell viability. L-NAME did not affect these effects of IPC. The results suggest that NO is ineffective on the noxious effects of ischemia-RP in non-IPC hearts and on the effects of IPC on cell viability, glycogenolysis and glycolysis whereas it is only involved in functional protection.

Role of endogenous nitric oxide (NO) and NO synthases in healing of indomethacin-induced intestinal ulcers in rats

We examined the roles of nitric oxide (NO) and NO synthase (NOS) isozymes in the healing of indomethacin-induced small intestinal ulcers in rats. Animals were given indomethacin (10 mg/kg, s.c.) and killed 1, 4 and 7 days after the administration. Indomethacin (2 mg/kg), N G-nitro- l-arginine methyl ester ( l-NAME: a nonselective NOS inhibitor: 10 mg/kg) and aminoguanine (a relatively selective iNOS inhibitor: 20 mg/kg) were given s.c. once daily for 6 days, the first 3 days or the last 3 days during a 7-day experimental period. Both indomethacin and l-NAME significantly impaired healing of these lesions, irrespective of whether they were given for 6 days, first 3 days or last 3 days. The healing was also impaired by aminoguanine given for the first 3 days but not for the last 3 days. Expression of iNOS mRNA in the intestine was up-regulated after ulceration, persisting for 2 days thereafter, and the Ca 2+-independent iNOS activity also markedly increased with a peak response during 1–2 days after ulceration. Vascular content in the ulcerated mucosa as measured by carmine incorporation was decreased when the healing was impaired by indomethacin and l-NAME given for either the first or last 3 days as well as aminoguanidine given for the first 3 days. These results suggest that endogenous NO plays a role in healing of intestinal lesions, in addition to prostaglandins, yet the NOS isozyme mainly responsible for NO production differs depending on the stage of healing: iNOS in the early stage and cNOS in the late stage.

Nitric Oxide in Asthma Therapy

The discovery of the delicate role of endogenous nitric oxide in the homeostasis of various cellular functions and the dynamic behaviour of the airways, has led to a new, rapidly progressing area of physiological science, that has direct bearing for our understanding of multiple airway diseases. The potentially protective effects of nitric oxide include: neuromodulation by mediating inhibitory non-cholinergic non-adrenergic nerve activity; smooth muscle relaxation, attenuating airway hyperresponsiveness to bronchoconstrictor stimuli and immune-suppression. NO itself or SNO can be administered directly to the airways, and the development of gene transfer therapy seems to become a realistic approach in the treatment of airway diseases. However, NO has also harmful effects, especially when it interacts with other molecules. At present, there are novel opportunities to modulate nitric oxide-synthesis aimed to restore the balance between the protective and deleterious effects of nitric oxide. This is potentially beneficial in both airway and alveolar diseases. Such interventions might be targeted in various ways, e.g. by using selective reactive nitrogen- and oxygen- scavengers, selective NO donors and selective nitric oxide synthase inhibitors. The possible therapeutical opportunities are reviewed in this paper. Nitric oxide has already made it from the bench to the bedside, and it is likely that new developments in this area will drastically change respiratory medicine during the coming 5-10 years.

Nitric oxide synthase inhibition in Thoroughbred horses augments O2 extraction at rest and submaximal exercise, but not during short‐term maximal exercise

Work is required to establish the role of endogenous nitric oxide (NO) in metabolism of resting and exercising horses. To examine the effects of NO synthase inhibition on O2 extraction and anaerobic metabolism at rest, and during submaximal and maximal exertion. Placebo and NO synthase inhibition (with Nomega-nitro-L-arginine methyl ester [L-NAME] administered at 20 mg/kg bwt i.v.) studies were performed in random order, 7 days apart on 7 healthy, exercise-trained Thoroughbred horses at rest and during incremental exercise leading to 120 sec of maximal exertion at 14 m/sec on a 3.5% uphill grade. At rest, NO synthase inhibition significantly augmented the arterial to mixed-venous blood O2 content gradient and O2 extraction as mixed-venous blood O2 tension and saturation decreased significantly. While NO synthase inhibition did not affect arterial blood-gas tensions in exercising horses, the exercise-induced increment in haemoglobin concentration and arterial O2 content was attenuated. In the L-NAME study, during submaximal exercise, mixed-venous blood O2 tension and haemoglobin-O2 saturation decreased to a greater extent causing O2 extraction to increase significantly. During maximal exertion, arterial hypoxaemia, desaturation of haemoglobin and hypercapnia of a similar magnitude developed in both treatments. Also, the changes in mixed-venous blood O2 tension and haemoglobin-O2 saturation, arterial to mixed-venous blood O2 content gradient, O2 extraction and markers of anaerobic metabolism (lactate and ammonia production, and metabolic acidosis) were not different from those in the placebo study. Endogenous NO production augments O2 extraction at rest and during submaximal exertion, but not the during short-term maximal exercise. Also, NO synthase inhibition does not affect anaerobic metabolism at rest or during exertion. It is unlikely that endogenous NO release modifies aerobic or anaerobic metabolism in horses performing short-term maximal exertion.

Role of endogenous nitric oxide in mucosal defense of inflamed rat stomach following iodoacetamide treatment

Nitric oxide (NO) plays a role in regulating the mucosal integrity of the stomach. However, its part in the mucosal defense of the inflamed stomach remains unclear. In the present study, we examined the effects of various NO synthase (NOS) inhibitors on gastric ulcerogenic and acid secretory responses following daily exposure of the stomach to iodoacetamide and investigated the role of each NOS isozyme in gastric protection from subchronic mucosal irritation. Gastric mucosal irritation was induced in rats by addition of 0.1% iodoacetamide to drinking water, and the gastric mucosa was examined on the 6th day. L-NAME (a nonselective NOS inhibitor: 20 mg/kg) or aminoguanidine (a selective iNOS inhibitor: 20 mg/kg) was given s.c. twice 24 h and 3 h before the termination of iodoacetamide treatment. Giving iodoacetamide in drinking water for 5 days produced minimal damage in the stomach with an increase in myeloperoxidase (MPO) activity and lipid peroxidation. Iodoacetamide treatment up-regulated the expression of iNOS mRNA and NO production in the stomach, without affecting nNOS expression. Both L-NAME and aminoguanidine markedly aggravated gastric lesions induced by iodoacetamide treatment, with a further enhancement in MPO activity and lipid peroxidation. Basal acid secretion as determined in pylorous-ligated stomachs was decreased following iodoacetamide treatment, but the response was significantly restored by both L-NAME and aminoguanidine. These results suggest that endogenous NO derived from both cNOS and iNOS is involved in mucosal defense of the inflamed stomach, partly by decreasing acid secretion, and contributes to maintaining mucosal integrity under such conditions.

Development of Genetically Engineered Mice Lacking All Three Nitric Oxide Synthases

Nitric oxide (NO) is produced in almost all tissues and organs, exerting multiple biological actions under both physiological and pathological conditions. NO is synthesized by three different isoforms of NO synthase (NOS): neuronal, inducible, and endothelial NOSs. Due to the substantial compensatory interactions among the NOS isoforms, the ultimate roles of endogenous NO in our body still remain to be fully elucidated. To address this point, we have successfully developed mice in which all three NOS genes are completely disrupted. NOS expression and activities were totally absent in the triply n/i/eNOS−/− mice before and after treatment with lipopolysaccharide. While the triply n/i/eNOS−/− mice were viable, their survival and fertility rates were markedly reduced as compared with wild-type mice. The phenotypes of those mice that we first noticed were polyuria, polydipsia, and renal unresponsiveness to vasopressin, characteristics consistent with nephrogenic diabetes insipidus. We subsequently observed that in those mice, arteriosclerosis is spontaneously developed with a clustering of cardiovascular risk factors. These results provide the first evidence that the systemic deletion of all three NOSs causes a variety of cardiovascular diseases in mice, demonstrating a critical role of the endogenous NOSs system in maintaining cardiovascular homeostasis.

Influence of nitric oxide on the activity of cuneate neurons in the rat

Some neurons of main and external cuneate nuclei are immunoreactive for nitric oxide (NO) synthase, suggesting a role for endogenous NO in the early stages of somatosensory processing. We tested this hypothesis by investigating the possibility that NO modulates cuneate discharge. We observed that both spontaneous and N-methyl-D-aspartate-evoked activities of cuneate neurons were decreased by NO precursor L-arginine. The inhibition of NO synthase, by application of N-nitro-L-arginine methyl ester, instead, abolished the depressant effect induced by L-arginine. Our data suggest a NO modulation of cuneate neurons and provide support for a physiologic role not only in increasing the signal-to-noise ratio in the excited cells but also in a form of surround inhibition.

Neuronal nitric oxide synthase controls enzyme activity pattern of mitochondria and lipid metabolism

Mitochondria are affected by endogenous nitric oxide (NO). Besides effects of NO on mitochondrial enzymes and the stimulation of mitochondrial H2O2 production, a NO-dependent increase in mitochondrial biogenesis in several tissues has been reported. It is still obscure whether NO generated by one specific or different NO synthase (NOS) isoenzymes determine such effects. Therefore, we analyzed the amount of mitochondria, respiratory chain enzyme complexes, and citrate synthase in the brain, muscle, heart, kidney, and liver by comparing wild-type (WT) mice and mice lacking the neuronal nitric oxide synthase isoform (nNOS-KO). Our results show that the activities of NADH:cytochrome c oxidoreductase and succinate cytochrome c oxidoreductase differ between WT and nNOS-KO mice. However, similar quantities of mitochondria were found in the homogenates of tissues in WT and nNOS-KO animals. Most impressive, higher activities and protein of citrate synthase were found in the brain, muscle, heart, kidney, and liver of nNOS-KO mice. Additionally, higher contents of fatty acid synthase and lipids were determined in the livers of nNOS-KO mice but not in the heart and brain. Furthermore, liver mitochondria from nNOS-KO mice consumed pyruvate at a higher rate and released more citric acid. Our data document a previously unrecognized role of endogenous NO in the regulation of lipid metabolism.

Inhibition of Nitric-Oxide Synthase Enhances Antigen-Induced Contractions and Increases Release of Cysteinyl-Leukotrienes in Guinea Pig Lung Parenchyma: Nitric Oxide as a Protective Factor

Nitric oxide (NO) in exhaled air is a biomarker of airway inflammation. However, the role of NO in the peripheral lung is not known. The aim of this study was to determine the role of endogenous NO in antigen-induced contractions of ovalbumin (OVA)-sensitized guinea pig lung parenchyma (GPLP). The contraction in this in vitro model of the peripheral lung closely resembles the corresponding response in human airways. Cumulatively increasing concentrations (10-10,000 microg/l) of OVA induced concentration-dependent contractions of the GPLP that were enhanced by the NO synthase (NOS) inhibitors N(omega)-nitro-L-arginine (L-NOARG; 100 microM), N(omega)-monomethyl-L-arginine (100 microM), N(omega)-nitro-L-arginine methyl ester (100 microM), and N-(3-(aminomethyl)benzyl)acetamidine (1400W; 1 microM). The enhancement induced by L-NOARG was reversed by coadministration with the 5-lipoxygenase inhibitor (R)-2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid (BAY x1005; 3 microM), whereas coadministration of L-NOARG with the cyclooxygenase inhibitor indomethacin (10 microM) did not change the effect of L-NOARG alone. L-NOARG (100 microM) did not affect the cumulative concentration-response relations for either leukotriene (LT) D4 (0.1-100 nM) or histamine (1-30 microM). The NO donor NONOate (0.001-100 microM) was ineffective in GPLP but potently relaxed precontracted guinea pig pulmonary artery. Furthermore, L-NOARG enhanced the release of LTE4 and decreased the release of prostaglandin E2 induced by OVA. In conclusion, endogenous NO exerts an inhibitory effect on antigen-induced contractions in the peripheral lung. The action of NO apparently involves inhibition of the release of mediators rather than direct relaxation of airway smooth muscle. The findings support the belief that endogenous NO has a protective anti-inflammatory effect in the airways.

Nephrogenic diabetes insipidus in mice lacking all nitric oxide synthase isoforms

Nitric oxide (NO) is produced in almost all tissues and organs, exerting a variety of biological actions under physiological and pathological conditions. NO is synthesized by three different isoforms of NO synthase (NOS), including neuronal, inducible, and endothelial NOSs. Because there are substantial compensatory interactions among the NOS isoforms, the ultimate roles of endogenous NO in our body still remain to be fully elucidated. Here, we have successfully developed mice in which all three NOS genes are completely deleted by crossbreeding singly NOS-/- mice. NOS expression and activities were totally absent in the triply NOS-/- mice before and after treatment with lipopolysaccharide. Although the triply NOS-/- mice were viable and appeared normal, their survival and fertility rates were markedly reduced as compared with the wild-type mice. Furthermore, these mice exhibited marked hypotonic polyuria, polydipsia, and renal unresponsiveness to an antidiuretic hormone, vasopressin, all of which are characteristics consistent with nephrogenic diabetes insipidus. In the kidney of the triply NOS-/- mice, vasopressin-induced cAMP production and membranous aquaporin-2 water channel expression were reduced associated with tubuloglomerular lesion formation. These results provide evidence that the NOS system plays a critical role in maintaining homeostasis, especially in the kidney.

Endogenous NO Blockade Enhances Tissue Factor Expression via Increased Ca 2+ Influx Through MCP-1 in Endothelial Cells by Monocyte Adhesion

Ca2+ plays an important role in tissue factor (TF) gene expression. We investigated the role of endogenous nitric oxide (NO) in the induction of TF expression in endothelial cells (ECs) by monocyte adhesion and the mechanisms of NO action. Inhibition of endogenous NO by Nomega-nitro-L-arginine methyl ester (L-NAME) enhanced TF promoter activity and protein expression induced in human coronary ECs by monocyte adhesion, as well as EC surface TF activity. L-NAME also induced monocyte chemoattractant protein-1 (MCP-1) expression, which was blocked by an NO donor, NOC18. Exogenous MCP-1 enhanced TF expression induced by monocyte adhesion, whereas adenovirus-mediated expression of the mutant MCP-1, 7ND, abolished the L-NAME enhancement of TF expression induced by monocyte adhesion. Monocyte attachment to L-NAME-treated ECs increased Ca2+ influx, which was prevented by NOC18, anti-MCP-1 antibody or 7ND. These results indicate that the binding of increased MCP-1 induced by endogenous NO blockade to CCR2 mediated the enhancement of Ca2+ influx only when monocytes adhered to ECs, which upregulated TF expression in ECs triggered by monocyte adhesion. MCP-1/CCR2 may play a role in Ca2+ influx-dependent TF regulation in the monocyte-EC interaction in the impairment of NO synthesis.

Partial nicotinic receptor blockade unmasks a modulatory role of nitric oxide on urethral striated neuromuscular transmission

The objective of this study was to investigate the possible modulatory role of endogenous nitric oxide (NO) production on the urethral striated muscle (USM) function in the sheep urethra. Significant NO synthase (NOS) activity was measured in both the particulate and cytosolic fractions of USM homogenates. NOS activity was calcium-dependent and showed greater inhibition by NOS inhibitors selective of the neural NOS isoform (nNOS). nNOS immunoreactivity was present in intramural nerves as well as in the sarcolemma of some striated fibers, being denser at the neuromuscular junction (NMJ). Double immunolabeling showed co-localization of nNOS with both alpha-bungarotoxin and choline acetyltransferase, at the USM endplates. For the first time, functional data support a role of NO on the USM contractility "in vitro," which became evident following partial nicotinic receptor inactivation with low concentrations of D-tubocurarine. Only under D-tubocurarine (0.25 microM) treatment, different NOS inhibitors, specially N(G)-propyl-L-arginine, as well as the guanylate cyclase inhibitor ODQ, all showed a significant enhancing effect on contractions induced by electrical field stimulation of intrinsic somatic nerves. These data suggest that local production of NO at the urethral NMJ may modulate release and/or action of acetylcholine on motor endplates by cyclic GMP-mediated effects. This modulatory action could be especially relevant when neuromuscular transmission at the USM is impaired.

Role of endogenous nitric oxide in endotoxin-induced alteration of hypoxic pulmonary vasoconstriction in mice

Pulmonary vasoconstriction in response to alveolar hypoxia (HPV) is frequently impaired in patients with sepsis or acute respiratory distress syndrome or in animal models of endotoxemia. Pulmonary vasodilation due to overproduction of nitric oxide (NO) by NO synthase 2 (NOS2) may be responsible for this impaired HPV after administration of endotoxin (LPS). We investigated the effects of acute nonspecific (N(G)-nitro-L-arginine methyl ester, L-NAME) and NOS2-specific [L-N6-(1-iminoethyl)lysine, L-NIL] NOS inhibition and congenital deficiency of NOS2 on impaired HPV during endotoxemia. The pulmonary vasoconstrictor response and pulmonary vascular pressure-flow (P-Q) relationship during normoxia and hypoxia were studied in isolated, perfused, and ventilated lungs from LPS-pretreated and untreated wild-type and NOS2-deficient mice with and without L-NAME or L-NIL added to the perfusate. Compared with lungs from untreated mice, lungs from LPS-challenged wild-type mice constricted less in response to hypoxia (69 +/- 17 vs. 3 +/- 7%, respectively, P < 0.001). Perfusion with L-NAME or L-NIL restored this blunted HPV response only in part. In contrast, LPS administration did not impair the vasoconstrictor response to hypoxia in NOS2-deficient mice. Analysis of the pulmonary vascular P-Q relationship suggested that the HPV response may consist of different components that are specifically NOS isoform modulated in untreated and LPS-treated mice. These results demonstrate in a murine model of endotoxemia that NOS2-derived NO production is critical for LPS-mediated development of impaired HPV. Furthermore, impaired HPV during endotoxemia may be at least in part mediated by mechanisms other than simply pulmonary vasodilation by NOS2-derived NO overproduction.

The effects of nitric oxide synthesis on the Na+,K+-ATPase activity in guinea pig kidney exposed to lipopolysaccharides

Endotoxins (lipopolysaccharides; LPS) are known to cause multiple organ failure, including renal dysfunction. LPS triggers the synthesis and release of cytokines and the vasodilator nitric oxide (NO*). A major contributor to the increase in NO* production is LPS-stimulated expression of inducible nitric oxide synthase (iNOS). This occurs in vasculature and most organs including the kidney. During endotoxemia, NO* and superoxide react spontaneously to form the potent and versatile oxidant peroxynitrite (ONOO-) and the formation of 3-nitrotyrosine (nTyr)-protein adducts is a reliable biomarker of ONOO- generation. Therefore, the present study was aimed at investigating the role of endogenous nitric oxide in regulating Na+,K(+)-ATPase activity in the kidney, and at investigating the possible contribution of reactive nitrogen species (RNS) by measuring of iNOS activity. In addition, the present study was aimed at investigating the relationship between nTyr formation with iNOS and Na+,K(+)-ATPase activities. Previously in our study, nTyr was not detectable in kidney of normal control animals but was detected markedly in LPS exposed animals. In this study, kidney Na+,K(+)-ATPase activity were maximally inhibited 6 h after LPS injection (P:0.000) and LPS treatment significantly increased iNOS activity of kidney (P:0.000). The regression analysis revealed a very close correlation between Na+,K(+)-ATPase activity and nTyr levels of LPS treated animals (r = -0.868, P = 0.001). Na+,K(+)-ATPase activity were also negatively correlated with iNOS activity (r = -0.877, P = 0.001) in inflamed kidney. These data suggest that NO* and ONOO- contribute to the development of oxidant injury. Furthermore, the source of NO* may be iNOS. iNOS are expressed by the kidney, and their activity may increase following LPS administration. In addition, NO* and ONOO- formation inhibited Na+,K(+)-ATPase activity. This results also have strongly suggested that bacterial LPS disturbs activity of membrane Na+,K(+)-ATPase that may be an important component leading to the pathological consequences such as renal dysfunction in which the production of RNS are increased as in the case of LPS challenge.

Involvement of nitric oxide generation in noise-induced temporary threshold shift in guinea pigs

The present study explored the role of endogenous nitric oxide (NO) in the temporary threshold shift caused by acoustic trauma. Guinea pigs were exposed to broadband white noise at a level of 105 ± 2 dB sound pressure level (SPL) for 10 min, causing a temporary threshold shift (TTS). The guinea pigs were divided into six groups (N-1 to N-6) according to survival days after noise exposure (0, 1, 2, 3, 7, 28 days). Auditory brainstem responses (ABR) were recorded before noise exposure, immediately after noise exposure and before sacrifice. Immediately after animals were sacrificed, the stria vascularis and the spiral ligament of the lateral wall of each individual cochlea were harvest as a unit and prepared for assay of NO. There was a significant correlation ( P < 0.001) between the NO concentration and final ABR threshold in the noise exposure groups. But the return of ABR threshold to pre-noise-exposed level is early than that of NO concentration. An average 16.2 dB threshold shift was found immediately after noise exposure. The threshold returned to the pre-noise-exposed level on the second post-exposure day. Comparing to unexposed control animals, the NO concentration increased nearly threefold immediately following noise exposure and decreased to twofold when the hearing threshold had returned to the pre-noise-exposed level. On the seventh post-exposure day the NO concentration was not different from that in unexposed control animals. Those findings indicate that endogenous NO is generated in the noise-induced temporal threshold shift and its concentration is correlated with the hearing loss.

Compensatory mechanisms influence hemostasis in setting of eNOS deficiency

The balance between thrombosis and hemorrhage is carefully regulated. Nitric oxide (NO) is an important mediator of these processes, as it prevents platelet adhesion to the endothelium and inhibits platelet recruitment. Although endothelial NO synthase (eNOS)-deficient mice have decreased vascular reactivity and mild hypertension, enhanced thrombosis in vivo has not been demonstrated. To determine the role of endogenous NO in hemostasis, a model of carotid arterial injury and thrombosis was performed using eNOS-deficient and wild-type mice. Paradoxically, the eNOS-deficient animals had a prolongation of time to occlusion compared with the wild-type mice (P < 0.001). Consistent with this finding, plasma markers suggesting enhanced fibrinolysis [tissue plasminogen activator (t-PA) activity and antigen and D-dimer levels] were significantly elevated in eNOS-deficient animals. Vascular tissue expression of t-PA and platelet activity levels were not altered. In endothelial cells, t-PA is stored in Weibel-Palade bodies, and exocytosis of these storage granules is inhibited by NO. Thus in the absence of NO, release of Weibel-Palade body contents (and t-PA) could be enhanced; this observation is also supported by increased von Willebrand factor levels observed in eNOS-deficient animals. In summary, although eNOS deficiency attenuates vascular reactivity and increases platelet recruitment, it is also associated with enhanced fibrinolysis due to lack of NO-dependent inhibition of Weibel-Palade body release. These processes highlight the complexity of NO-dependent regulation of vascular homeostasis. Such compensatory mechanisms may partially explain the lack of spontaneous thrombosis, minimally elevated baseline blood pressure, and normal life span that are seen in animals deficient in a pivotal regulator of vascular patency.

The effects of nitric oxide on striatal serotoninergic transmission involve multiple targets: an in vivo microdialysis study in the awake rat

The role of endogenous nitric oxide (NO) in N-methyl- d-aspartate (NMDA)-induced modulation of serotonin (5-HT) release in the striatum of freely moving rats has been studied using microdialysis technique. NMDA-induced increase in 5-HT release was significantly inhibited by selective nitric oxide synthase (nNOS) inhibitor S-methylthiocitrulline (S-Me-TC), ONOO − scavenger l-cysteine ( l-cys), and guanylate cyclase (GC) inhibitor 1 H[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ). These data suggest that modulation of 5-HT levels is linked to the formation of NO produced by NMDA receptor activation and that endogenously produced NO increases 5-HT concentrations both by stimulating formation of 3′–5′-cyclic monophosphate (cGMP) and conversion of ONOO −.

Nitric oxide protects against pancreatic subcellular damage in acute pancreatitis.

Oxidative stress involvement in damage to the pancreas in acute pancreatitis (AP) is well documented. However, little is known about oxidative damage occurring in the different subcellular fractions of pancreatic cells. The aim of this study was to ascertain the main targets of oxidative damage inside cells after AP and the role of endogenous nitric oxide (NO) in it. A model of cerulein-induced AP in rats was used and N-nitro-l-arginine methyl ester (l-NAME) was administered as an NO production inhibitor. After pancreatitis induction, indicative parameters of lipid peroxidation and protein oxidation together with some enzymatic and nonenzymatic endogenous free radical scavengers were assessed in serum and pancreatic subcellular fractions. In pancreatitic rats, malondialdehyde and protein carbonyl group concentrations were significantly increased (P < 0.05) in serum and some fractions. The increases were higher in l-NAME-treated rats (P < 0.05). Superoxide dismutase and catalase activities were also increased (P < 0.05) but were decreased (P < 0.05) with l-NAME. The alpha-tocopherol concentration diminished (P < 0.05) in serum and all the studied subcellular fractions and the decrease was stronger in l-NAME-treated rats. Our data suggest that microsomes followed by lysosomal + mitochondrial are the fractions most susceptible to oxidative damage in AP. Endogenous NO plays a protective role against oxidative damage to subcellular fractions.

Protection against concanavalin A-induced hepatocyte apoptosis by molsidomine is time-dependent.

Viral hepatitis and autoimmune liver diseases cause hepatocyte apoptosis. Concanavalin A (Con A)-induced hepatitis resembles human viral hepatitis and autoimmune hepatitis. The role of nitric oxide (NO) in liver injury was controversial in different liver injury models. We hypothesize both endogenous and exogenous NO protect liver against Con A-induced liver injury. Molsidomine is metabolized into SIN-1 by the liver, and SIN-1 subsequently generates NO. So, molsidomine was used as a NO donor in this study. To study a protective role of endogenous NO in Con A-induced liver injury, mice were pretreated with a specific inducible nitric oxide synthase (iNOS) inhibitor, L-N(6)-(1-iminoethyl)-lysine (L-NIL), before Con A challenge. To study a time-dependent protection against Con A-induced liver injury, animals were either given molsidomine, a NO donor, before or after Con A administration. Serum alanine aminotranferase (ALT) was analyzed. Liver samples were subjected to DNA fragmentation assay, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling stain, Western blot analysis, and caspase activity assays. Animals pretreated with L-NIL had significantly increased serum ALT levels compared with those challenged with Con A alone; but pretreatment with molsidomine dramatically decreased ALT levels in L-NIL-pretreated animals or in animals that received Con A alone. Administration of molsidomine 30 minutes before or 1, 2, and 3 hours after Con A injection significantly reduced serum ALT levels and attenuated hepatocyte apoptosis from caspase inactivation. The ALT reduction was associated with inhibition of both caspase-3 and caspase-8 activation and reduction of hepatocyte apoptosis. Endogenous NO plays an important protective role against Con A-induced liver injury by reducing hepatocyte apoptosis. Administration of a NO donor early after Con A injection protects the liver from injury. This is the first study demonstrating a time-dependent inhibition of liver injury induced by Con A administration.

Nitric oxide inhibits superoxide-stimulated urea permeability in the rat inner medullary collecting duct.

The inner medullary collecting duct (IMCD) contains relatively high nitric oxide (NO) synthetic capacity, but the effect of NO on IMCD transport remains unclear. We determined the effect of NO on basal and vasopressin (AVP)-stimulated urea (Purea) and water (Pf) permeabilities in isolated, perfused rat IMCD. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) increased cGMP production in IMCD, but neither SNAP (10(-4) M) nor 8-BrcGMP (10(-4) M), the cell-permeable analog of cGMP, affected basal or AVP-stimulated Purea. The free radical superoxide is produced by oxidases in the kidney and can interact with NO. To determine the effect of superoxide generation on transport, IMCDs were incubated with diethyldithiocarbamate (DETC; 10(-3) M), the inhibitor of superoxide dismutase (SOD). DETC significantly increased basal and AVP-stimulated Purea (control: 28.7 +/- 4.5 vs. DETC: 40.9 +/- 6.2 x 10(-5) cm/s; P < 0.001; n = 9). Preincubation of IMCD with SNAP or the SOD mimetic tempol completely inhibited DETC-stimulated Purea. DETC caused a significant increase in superoxide generation by IMCD, and this was blocked by SNAP. Incubation of IMCD with the NO synthase (NOS) substrate l-arginine blocked the stimulatory effect of DETC on Purea, and this was reversed by the neuronal NOS inhibitor 7-nitroindazole. In contrast, neither basal nor AVP-stimulated Pf was affected by NO donors or DETC. In summary, exogenous or endogenously produced NO does not affect basal urea transport in the IMCD but inhibits superoxide-stimulated Purea. In the inner medulla, superoxide generation by local oxidases may stimulate urea transport, and the role of endogenous NO may be to dampen this effect by decreasing superoxide levels.