Nitric Oxide Synthase Isozymes Research Articles

The potential mediation of nitric oxide in the activation of mitochondrion-dependent apoptosis and yak meat tenderness during postmortem aging

Nitric oxide (NO) may play a critical role in the regulation of various physiological and pathophysiological processes due to its effect on the apoptosis pathway. However, few reports have studied the effects of NO on apoptosis and yak meat tenderization. The present study was conducted to explore the molecular mechanisms of Ca2+-independent inducible NO synthase isozyme (iNOS)-derived NO in mitochondrial apoptosis and meat tenderization during postmortem aging. Results indicated that the aminoguanidi hydrochloride (AH) significantly reduced iNOS activities and NO production. Moreover, the inhibitory property of iNOS-derived NO dramatically decreased mitochondrial oxidative stress degree, which further reduced the production of mitochondrial Ca2+ and ATP. The inhibitory property of iNOS-derived NO also influenced the Bcl-2 family members, mitochondrial apoptotic cascade reaction, and meat tenderization by protecting against the degradation of myofibrils and the damage of mitochondrial microstructure. These results demonstrated that the iNOS-derived NO possessed a significant role in the mitochondrial apoptosis pathway and yak meat tenderization during aging.

Inhibition of Host Arginase Activity Against Staphylococcal Bloodstream Infection by Different Metabolites.

Staphylococcus aureus is a notorious bacterial pathogen that often causes soft tissue and bloodstream infections and invariably garners resistance mechanisms against new antibiotics. Modulation of the host immune response by metabolites is a powerful tool against bacterial infections, but has not yet been used against S. aureus infections. In this study, we identified four metabolite biomarkers: L-proline, L-isoleucine, L-leucine, and L-valine (PILV), through a metabolomics study using animal models of S. aureus bloodstream infection. The exogenous administration of each metabolite or of PILV showed anti-infective effects, and a higher protection was achieved with PILV in comparison to individual metabolites. During the staphylococcal infection, the expression of most host arginase and nitric oxide synthase (NOS) isozymes was simultaneously induced in mouse liver, kidney, and blood samples. However, the induction of arginase isozymes was dramatically stronger than that of NOS isozymes. This elevated arginase activity was inhibited by the metabolite biomarkers thus killing S. aureus, and PILV exhibited the strongest inhibition of arginase activity and bacterial inhibition. The suppression of arginase activity also contributed to the metabolite-mediated phagocytic killing of S. aureus in mouse and human blood. Our findings demonstrate the metabolite-mediated arginase inhibition as a therapeutic intervention for S. aureus infection.

Overexpression of inflammatory-related and nitric oxide synthase genes in olfactory bulbs from frontal lobe epilepsy patients

Neuroinflammation has been shown to constitute a crucial mechanism in the pathophysiology of epileptic brain and several genes of inflammatory mediators have been detected in surgically resected hippocampus tissue but not in non-related seizure brain regions. Interestingly, it has been reported an olfactory dysfunction in frontal lobe epilepsy (FLE). Our aim was to quantify the gene expression of inflammatory-related and nitric oxide synthase genes in olfactory bulbs (OB) tissue from FLE patients. RNA was isolated from OB resection of FLE patients and autopsy subjects without any neurological disease (n = 7, each). After cDNA synthesis, we performed qPCR for interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), nuclear factor κB p65 (RELA), Toll-like receptor 4 (TLR 4), its agonist high mobility group box 1 (HMGB 1) as well nitric oxide synthase isozymes (NOS 1, 2 and 3).We found a significant increase in gene expression of pro-inflammatory cytokines (IL-1β, IL-6 and TNFα), TLR4 receptor and in its agonist HMGB1 and the downstream transcription factor NFκB p65. Moreover, we observed an increase of both NOS1 and NOS3 and a slightly increase of NOS2; however, it was not significant. Our study describes the overexpression of inflammatory-related genes and NOS isozymes in OB from FLE patients. Even though, the number of patients was limited, our findings could point out that neuroinflammation and nitrosative stress-related genes in the OB could be produced in general manner in all brain regions and thus contribute in part, to the olfactory dysfunction observed in FLE patients.

Accessible Chiral Linker to Enhance Potency and Selectivity of Neuronal Nitric Oxide Synthase Inhibitors

The three important mammalian isozymes of nitric oxide synthase (NOS) are neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Inhibitors of nNOS show promise as treatments for neurodegenerative diseases. Eight easily-synthesized compounds containing either one (20a,b) or two (9a-d; 15a,b) 2-amino-4-methylpyridine groups with a chiral pyrrolidine linker were designed as selective nNOS inhibitors. Inhibitor 9c is the best of these compounds, having a potency of 9.7 nM and dual selectivity of 693 and 295 against eNOS and iNOS, respectively. Crystal structures of nNOS complexed with either 9a or 9c show a double-headed binding mode, where each 2-aminopyridine head group interacts with either a nNOS active site Glu residue or a heme propionate. In addition, the pyrrolidine nitrogen of 9c contributes additional hydrogen bonds to the heme propionate, resulting in a unique binding orientation. In contrast, the lack of hydrogen bonds from the pyrrolidine of 9a to the heme propionate allows the inhibitor to adopt two different binding orientations. Both 9a and 9c bind to eNOS in a single-headed mode, which is the structural basis for the isozyme selectivity.

Increased Ornithine-Derived Polyamines Cause Airway Hyperresponsiveness in a Mouse Model of Asthma

Up-regulation of arginase contributes to airways hyperresponsiveness (AHR) in asthma by reducing L-arginine bioavailability for the nitric oxide (NO) synthase isozymes. The product of arginase activity, L-ornithine, can be metabolized into polyamines by ornithine decarboxylase. We tested the hypothesis that increases in L-ornithine-derived polyamines contribute to AHR in mouse models of allergic airways inflammation. After measuring significantly increased polyamine levels in sputum samples from human subjects with asthma after allergen challenge, we used acute and subacute ovalbumin sensitization and challenge mouse models of allergic airways inflammation and naive mice to investigate the relationship of AHR to methacholine and polyamines in the lung. We found that spermine levels were elevated significantly in lungs from the acute model, which exhibits robust AHR, but not in the subacute murine model of asthma, which does not develop AHR. Intratracheal administration of spermine significantly augmented airways responsiveness to methacholine in both naive mice and mice with subacute airways inflammation, and reduced nitrite/nitrate levels in lung homogenates, suggesting that the AHR developed as a consequence of inhibition of constitutive NO production in the airways. Chronic inhibition of polyamine synthesis using an ornithine decarboxylase inhibitor significantly reduced polyamine levels, restored nitrite/nitrate levels to normal, and abrogated the AHR to methacholine in the acute model of allergic airways inflammation. We demonstrate that spermine increases airways responsiveness to methacholine, likely through inhibition of constitutive NO synthesis. Thus, inhibition of polyamine production may represent a new therapeutic target to treat airway obstruction in allergic asthma.

Modulation of gastric mucosal inflammatory responses to <i>Helicobacter pylori</i> by ghrelin: Role of cNOS-dependent IKK-<i>β</i> S-nitrosylation in the regulation of COX-2 activation

Disturbances in nitric oxide synthase (NOS) and cyclooxygenase (COX) isozyme systems, manifested by the excessive NO and prostaglandin (PGE2) generation, are well-recognized features of gastric mucosal inflammatory responses to H. pylori infection. In this study, we report that H. pylori LPS-induced enhancement in gastric mucosal inducible (i) iNOS expression and COX-2 activation was accompanied by the impairment in constitutive (c) cNOS phosphorylation, up-regulation in the inhibitory κB kinase-β (IKKβ) activation and the increase in the transcriptional factor, NF-κB, nuclear translocation. Further, we show that abrogation of cNOS control over NF-κB activation has lead to induction of iNOS expression and COX-2 activation through S-nitrosylation. Moreover, we demonstrate that the modulatory effect of peptide hormone, ghrelin, on the LPS-induced changes was reflected in the increase in Src/Akt-dependent cNOS activation through phosphorylation and the suppression of IKK-β activity through cNOS-mediated IKK-β protein S-nitrosylation. As a result, ghrelin exerted the inhibitory effect on NF-κB nuclear translocation, thus causing the repression of iNOS gene induction and the inhibition in COX-2 activation through iNOS-dependent S-nitrosylation. Our findings point to cNOS activation as a pivotal element in the signaling cascade by which ghrelin exerts modulatory control over proinflammatory events triggered in gastric mucosa by H. pylori infection.

Protective Role of Nitric Oxide Against Cardiac Arrhythmia - An Update

Nitric oxide (NO), a ubiquitous cell signalling molecule, plays an important role in the regulation of cardiovas- cular function. NO is produced by all cell types in the heart and is widely implicated in vasodilation, inflammation, ischemic preconditioning and cell death. Mounting evidence now suggests that NO also provides protection against car- diac arrhythmia. Pharmacological studies using NO precursors and NO donors have observed an anti-arrhythmic effect, while inhibitors of NO biosynthesis increase arrhythmia, or attenuate anti-arrhythmic actions in a variety of animal mod- els. Additionally, genetic studies from animals deficient in nitric oxide synthase (NOS) enzymes have found increased susceptibility to pharmacological or ischemia-induced arrhythmia while transgenic mice overexpressing certain NOS isozymes may be protected. In the human population polymorphisms in NOS enzymes, or differences in the levels of en- dogenous inhibitors of NO synthesis may also be important determinants of arrhythmic susceptibility. The precise mecha- nisms of NO-mediated protection from arrhythmia are still under investigation but may include reductions in calcium overload, regulation of gap junction/connexin expression, reductions in oxidative stress, and regulation of sympathetic ac- tivity. This review examines the pharmacological, genetic, and clinical evidence of a role for NO in reducing cardiac ar- rhythmia and discusses possible mechanisms of NO-mediated protection against arrhythmia.

Mapping the Binding and Calmodulin-Dependent Activation of Nitric Oxide Synthase Isozymes

Calmodulin (CaM) is a ubiquitous cytosolic Ca 2+ -binding protein that can bind and activate numerous intracellular target proteins including the nitric oxide synthase (NOS) enzymes. There are three different isoforms of NOS found in mammals - neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) - with all three isozymes capable of producing the free radical, nitric oxide (•NO). At elevated cellular Ca 2+ concentrations, CaM is able to bind and activate nNOS and eNOS. In contrast, the iNOS isozyme is transcriptionally regulated and binds to CaM in the absence of Ca 2+ . To further investigate the differences in the association of CaM to the Ca 2+ -dependent and Ca 2+ - independent NOS isoforms, a variety of CaM mutants were employed. These included CaM-troponin C chimeras, CaM EF hand pair proteins, CaM mutants incapable of binding to Ca 2+ , methionine oxidized CaM mutants, phosphomimetic CaM mutants and central linker CaM mutants. By characterizing these CaM proteins using a variety of biochemical techniques, we have mapped the binding and the CaM-dependent activation of the NOS enzymes to study their effect on the different electron transfer steps within the NOS enzymes. In addition, fluorescence and Forster resonance energy transfer (FRET) were used to monitor the orientation and conformation of CaM when bound to the NOS peptides and enzymes. Combining these cumulative results, a working model describing the CaM-dependent regulation of iNOS is proposed.

Binding kinetics of calmodulin with target peptides of three nitric oxide synthase isozymes

Efficient electron transfer from reductase domain to oxygenase domain in nitric oxide synthase (NOS) is dependent on the binding of calmodulin (CaM). Rate constants for the binding of CaM to NOS target peptides was only determined previously by surface plasmon resonance (SPR) ( Biochemistry 35, 8742–8747, 1996) suggesting that the binding of CaM to NOSs is slow and does not support the fast electron transfer in NOSs measured in previous and this studies. To resolve this contradiction, the binding rates of holo Alexa 350 labeled T34C/T110W CaM (Alexa-CaM) to target peptides from three NOS isozymes were determined using fluorescence stopped-flow. All three target peptides exhibited fast k on constants at 4.5 °C: 6.6 × 10 8 M − 1 s − 1 for nNOS 726–749, 2.9 × 10 8 M − 1 s − 1 for eNOS 492–511 and 6.1 × 10 8 M − 1 s − 1 for iNOS 507–531, 3–4 orders of magnitude faster than those determined previously by SPR. Dissociation rates of NOS target peptides from Alexa-CaM/peptide complexes were measured by Ca 2+ chelation with ETDA: 3.7 s − 1 for nNOS 726–749, 4.5 s − 1 for eNOS 492–511, and 0.063 s − 1 for iNOS 507–531. Our data suggest that the binding of CaM to NOS is fast and kinetically competent for efficient electron transfer and is unlikely rate-limiting in NOS catalysis. Only iNOS 507–531 was able to bind apo Alexa-CaM, but in a very different conformation from its binding to holo Alexa-CaM.

Subcellular distribution of nitric oxide synthase isoforms in the rat duodenum

To study the cell-type specific subcellular distribution of the three isoforms of nitric oxide synthase (NOS) in the rat duodenum. Postembedding immunoelectronmicroscopy was performed, in which primary antibodies for neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), were visualized with protein A-gold-conjugated secondary antibodies. Stained ultrathin sections were examined and photographed with a Philips CM10 electron microscope equipped with a MEGAVIEW II camera. The specificity of the immunoreaction in all cases was assessed by omitting the primary antibodies in the labeling protocol and incubating the sections only in the protein A-gold conjugated secondary antibodies. Postembedding immunoelectronmicroscopy revealed the presence of nNOS, eNOS, and iNOS immunoreactivity in the myenteric neurons, the enteric smooth muscle cells, and the endothelium of capillaries running in the vicinity of the myenteric plexus of the rat duodenum. The cell type-specific distributions of the immunogold particles labeling the three different NOS isozymes were revealed. In the control experiments, in which the primary antiserum was omitted, virtually no postembedding gold particles were observed. This postembedding immunoelectronmicroscopic study provided the first evidence of cell-type-specific differences in the subcellular distributions of NOS isoforms.

Ghrelin Suppression of <i>Helicobacter pylori</i>-Induced Gastric Mucosal Expression of iNOS is Mediated through the Inhibition of IKK-<i>β</i> Activation by cNOS-Dependent S-Nitrosylation

Excessive nitric oxide generation, caused by the disturbances in nitric oxide synthase (NOS) isozyme system, plays a key role in defining the extent of gastric mucosal inflammatory response to H. pylori infection. Here, we report that H. pylori LPS-induced enhancement in gastric mucosal inducible (i) iNOS expression and the impairment in constitutive (c) cNOS activity was associated with up-regulation in the inhibitory kB kinase-β (IKK?β) activation through phosphorylation, rise in IκB-α degradation, and the increase in the transcriptional factor, NF-κB, nuclear translocation. Further, we show that the countering effect of peptide hormone, ghrelin, on the LPS-induced disturbances in NOS isozyme system was reflected in the increase in Src/Akt-dependent cNOS activation through phosphorylation and the suppression of IKK-β activity through cNOSmediated IKK-β protein S-nitrosylation. As a consequence, ghrelin exerted the inhibitory effect on the LPS-induced rise in IκB-α degradation and NF-κB nuclear translocation, thus leading to iNOS gene suppression and the repression of iNOS induction. These results point to a central role of cNOS activation in controlling the signaling pathways of the LPS-triggered iNOS gene induction. Moreover, our findings suggest a molecular mechanism by which ghrelin suppresses the gastric mucosal proinflammatory consequences of H. pylori infection.

Ghrelin Protects against the Detrimental Consequences ofPorphyromonas gingivalis-Induced Akt Inactivation through S-Nitrosylation on Salivary Mucin Synthesis

Disturbances in nitric oxide synthase isozyme system and the impairment in salivary mucin synthesis are well-recognized features associated with oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis. In this study, using rat sublingual gland acinar cells, we report that P. gingivalis LPS-induced impairment in mucin synthesis and associated suppression in Akt kinase activity were accompanied by a decrease in constitutive nitric oxide synthase (cNOS) activity and an induction in inducible nitric oxide synthase (iNOS) expression. The LPS effect on Akt inactivation was manifested in the kinase S-nitrosylation and a decrease in its phosphorylation at Ser473. Further, we demonstrate that a peptide hormone, ghrelin, countered the LPS-induced impairment in mucin synthesis. This effect of ghrelin was reflected in the suppression of iNOS and the increase in Akt activation, associated with the loss in S-nitrosylation and the increase in phosphorylation, as well as cNOS activation through phosphorylation. Our findings suggest that induction in iNOS expression by P. gingivalis-LPS leads to Akt kinase inactivation through S-nitrosylation that detrimentally impacts cNOS activation through phosphorylation as well as mucin synthesis. We also show that the countering effect of ghrelin on P. gingivalis-induced impairment in mucin synthesis is associated with Akt activation through phosphorylation.

Examining the role of arginase in air pollution-induced exacerbation of asthma

Background The arginase isozymes (arginase 1 and 2), convert Larginine into L-ornithine and urea, and thus compete with the nitric oxide synthase isozymes for substrate. We have previously shown that arginase 1 expression is upregulated in human asthma and plays a functional role in airways hyperresponsiveness (AHR) in an animal model of allergic airways inflammation. Ambient particles and ozone are major constituents of urban air pollution and contribute to asthma exacerbations. However, the mechanisms underlying the exacerbation of allergic airways disease by air pollution remain to be elucidated. There is evidence that arginase expression is augmented in cigarette smoking asthmatics. We tested the hypothesis that arginase is involved in the exacerbation of respiratory symptoms in response to air pollution in animal models of allergic airways inflammation.

Cyclooxygenase and nitric oxide synthase in the presympathetic neurons in the paraventricular hypothalamic nucleus are involved in restraint stress-induced sympathetic activation in rats

Stress is one of the important factors to activate the sympathetic nervous system. We recently reported that central administration of corticotropin-releasing factor (CRF), known as a stress-related neuropeptide, increases the expression of both cyclooxygenase (COX) and nitric oxide synthase (NOS) in presympathetic neurons in the paraventricular hypothalamic nucleus (PVN). In the present study, therefore, we investigated whether brain COX and NOS can also mediate restraint stress (RS)-induced sympathetic activation by assessing the plasma catecholamine levels and neuronal activation of presympathetic neurons in the PVN. In addition, we examined effects of RS on the expression of both COX and NOS isozymes in the presympathetic PVN neurons. Intraperitoneal administration of an inhibitor for COX-1, COX-2 or inducible NOS (iNOS), but not for neuronal NOS (nNOS), reduced RS-induced elevation of plasma catecholamine levels and Fos expression in the presympathetic PVN neurons. Moreover, RS increased the expression of COX-1, COX-2 and iNOS in the presympathetic PVN neurons, whereas nNOS expression did not change. These results suggest that COX-1, COX-2 and iNOS in the presympathetic PVN neurons mediate acute RS-induced sympathetic activation.

Regulation of Interdomain Interactions by Calmodulin in Inducible Nitric-oxide Synthase

Nitric-oxide synthases (NOSs) catalyze the conversion of l-arginine to nitric oxide and citrulline. There are three NOS isozymes, each with a different physiological role: neuronal NOS, endothelial NOS, and inducible NOS (iNOS). NOSs consist of an N-terminal oxygenase domain and a C-terminal reductase domain, linked by a calmodulin (CaM)-binding region. CaM is required for NO production, but unlike other NOS isozymes, iNOS binds CaM independently of the exogenous Ca(2+) concentration. We have co-expressed CaM and the FMN domain of human iNOS, which includes the CaM-binding region. The Ca(2+)-bound protein complex (CaCaMxFMN) forms an air-stable semiquinone when reduced with NADPH and reduces cytochrome c when reconstituted with the iNOS FAD/NADPH domain. We have solved the crystal structure of the CaCaMxFMN complex in four different conformations, each with a different relative orientation, between the FMN domain and the bound CaM. The CaM-binding region together with bound CaM forms a hinge, pivots on the conserved Arg(536), and regulates electron transfer from FAD to FMN and from FMN to heme by adjusting the relative orientation and distance among the three cofactors. In addition, the relative orientations of the N- and C-terminal lobes of CaM are also different among the four conformations, suggesting that the flexibility between the two halves of CaM also contributes to the fine tuning of the orientation/distance between the redox centers. The data demonstrate a possible mode for precise control of electron transfer by altering the distance and orientation of redox centers in a protein displaying domain movement.

Biological Responses to Arsenic Compounds

Arsenic is a metalloid that generates various biological effects on cells and tissues. Depending on the specific tissue exposed and the time and degree of exposure, diverse responses can be observed. In humans, prolonged and/or high dose exposure to arsenic can have a variety of outcomes, including the development of malignancies, severe gastrointestinal toxicities, diabetes, cardiac arrhythmias, and death. On the other hand, one arsenic derivative, arsenic trioxide (As(2)O(3)), has important antitumor properties. This agent is a potent inducer of antileukemic responses, and it is now approved by the Food and Drug Administration for the treatment of acute promyelocytic leukemia in humans. The promise and therapeutic potential of arsenic and its various derivatives have been exploited for hundreds of years. Remarkably, research focused on the potential use of arsenic compounds in the treatment of human diseases remains highly promising, and it is an area of active investigation. An emerging approach of interest and therapeutic potential involves efforts to target and block cellular pathways activated in a negative feedback manner during treatment of cells with As(2)O(3). Such an approach may ultimately provide the means to selectively enhance the suppressive effects of this agent on malignant cells and render normally resistant tumors sensitive to its antineoplastic properties.

W1559 Pathogenic Importance of Endothelial Isozyme of Nitric Oxide Synthase (eNOS) in Aggravation of Gastric Lesions Induced By Indomethacin and Cold-Restraint Stress in Adjuvant-Induced Arthritic Rats

Background/Aim: Eicosanoids are involved in the regulatorymechanism of the gastricmucosal integrity under various conditions. We previously reported that COX-2/PGI2 plays a protective role in the gastric mucosal defense under ischemia/reperfusion (I/R)induced conditions, while indomethacin worsens these lesions, partly mediated by the up-regulation of leukotrienes and their receptors. However, it remains unknown whether thromboxane A2 (TXA2) plays any role in the pathogenesis of I/R-induced gastric injury. In the present study, we examined the role of TXA2 in the ulcerogenic response in the mouse stomach under I/Rinduced conditions. Methods: Under urethane anesthesia, the celiac artery was clamped (ischemia) for 30 min, followed by reperfusion by removal of the clamp. After reperfusion for 60 min, the area (mm2) of macroscopically visible lesions in the stomach was measured. SC-560 (COX-1 inhibitor: 5 mg/kg), rofecoxib (COX-2 inhibitor: 5 mg/kg), ozagrel [TX synthase (TXS) inhibitor: 200 mg/kg] or seratrodast [TXA2 receptor (TP) antagonist: 1-30 mg/kg] was administered PO 60 min before the onset of ischemia. Iloprost (PGI2 analogue: 1 μg/kg) was given IV 5 min before reperfusion. TXB2 and 6-keto-PGF1a contents in the gastric mucosa were measured by enzyme immunoassay. The expression of TP and TXS mRNAs were examined by RT-PCR. Results: I/R produced hemorrhagic lesions in the gastric mucosa, the lesion score being 9.3±2.1 mm2. The severity of I/R-induced gastric lesions was significantly decreased by the pretreatment with ozagrel, seratrodast or iloprost. By contrast, the gastric ulcerogenic response under I/R conditions was markedly worsened by pretreatment with rofecoxib, and this response was abrogated by co-administration of iloprost. The expressions of TP and TXS mRNAs were observed in the normal mouse stomach, and these expressions remained unchanged under I/R-induced conditions. I/R significantly increased both TXB2 and 6-keto-PGF1a contents in the gastric mucosa. The increase of TXB2 content under I/R conditions was significantly attenuated by SC-560 but not rofecoxib, while the increased PGI2 biosynthetic response was totally attenuated by rofecoxib but not SC-560. I/R by itself had no effect on the TXB2/ 6-keto-PGF1a ratio in the gastric mucosa, yet the ratio was markedly increased by rofecoxib and decreased by ozagrel, respectively. Conclusion: These results suggest that 1) COX-1/TXA2 plays a crucial role in the development of I/R-induced gastric lesions, in addition to COX-2/PGI2, and 2) the aggravation by rofecoxib of I/R-induced gastric lesions may be accounted partly for by the imbalance of TXA2/PGI2 ratio in the stomach.

Differential roles of nitric oxide synthase isozymes in cardiotoxicity and mortality following chronic doxorubicin treatment in mice

The roles of individual nitric oxide synthases (NOS) in anthracycline-related cardiotoxicity are not completely understood. We investigated the effects of a chronic treatment with doxorubicin (DOX) on knockouts of the individual NOS isozymes and on transgenic mice with myocardial overexpression of eNOS. Fractional shortening (FS) was reduced in untreated homozygous nNOS and iNOS knockouts as well as in eNOS transgenics. DOX-induced FS decrease in wild-type mice was attenuated only in eNOS knockouts, which were found to overexpress nNOS. No worsening of contractility was observed in DOX-treated eNOS transgenics and iNOS knockouts. Although the surviving DOX-treated nNOS knockouts exhibited no further impairment in contractility, most (70%) animals died within 7 weeks after treatment onset. In comparison to untreated wild-type hearts, the nitric oxide (NO) level was lower in hearts from DOX-treated wild-type mice and in all three untreated knockouts. DOX treatment had no effect on NO in the knockouts. These data indicate differential roles of the individual NOS in DOX-induced cardiotoxicity. Protection against DOX effects conferred by eNOS deletion may be mediated by a compensatory overexpression of nNOS. NOS inhibition-based prevention of anthracycline-induced cardiotoxicity should be eNOS-selective, simultaneously avoiding inhibiting nNOS.

Nitric oxide synthase isozymes in spinally projecting PVN neurons are involved in CRF-induced sympathetic activation

In the brain, corticotropin-releasing factor (CRF) has been shown to activate the sympatho-adrenomedullary outflow, but the central mechanisms of action are still not fully understood. Previously, we reported that inducible nitric oxide synthase (iNOS) is involved in central CRF-induced elevation of plasma catecholamines in rats. Nitric oxide is mainly synthesized by neuronal NOS (nNOS) and iNOS in many areas in the brain. Of these areas, the paraventricular hypothalamic nucleus (PVN) contains neurons projecting to the intermediolateral cell column (IML) of the spinal cord, thereby directly affecting the sympathetic activity. Therefore, in the present study, we investigated the effect of intracerebroventricularly (i.c.v.) administered CRF on plasma catecholamine levels and expression of NOS isozymes (iNOS and nNOS) and Fos (a marker for neuronal activation) in the spinally projecting PVN neurons, using rats microinjected with a monosynaptic retrograde tracer into the IML. CRF (1.5 nmol/animal, i.c.v.) effectively elevated plasma catecholamine levels. The spinally projecting neurons labeled with a tracer were detected in the dorsal cap, ventral part and posterior part of the PVN. CRF significantly increased the number of spinally projecting neurons triple-labeled with Fos and iNOS in all of these PVN subnuclei. On the other hand, CRF significantly increased the number of spinally projecting neurons triple-labeled with Fos and nNOS only in the ventral part of the PVN. These results suggest that in spinally projecting PVN neurons, iNOS mainly contributes to the centrally administered CRF-induced activation of the sympatho-adrenomedullary outflow in rats.

Functionally important role for arginase 1 in the airway hyperresponsiveness of asthma

l-Arginine metabolism by the arginase and nitric oxide (NO) synthase (NOS) families of enzymes is important in NO production, and imbalances between these pathways contribute to airway hyperresponsiveness (AHR) in asthma. To investigate the role of arginase isozymes (ARG1 and ARG2) in AHR, we determined the protein expression of ARG1, ARG2, the NOS isozymes, and other proteins involved in l-arginine metabolism in lung tissues from asthma patients and in acute (3-wk) and chronic (12-wk) murine models of ovalbumin-induced airway inflammation. Expression of ARG1 was increased in human asthma, whereas ARG2, NOS isoforms, and the other l-arginine-related proteins (i.e., cationic amino acid transporters 1 and 2, agmatinase, and ornithine decarboxylase) were unchanged. In the acute murine model of allergic airway inflammation, augmentation of ARG1 expression was similarly the most dramatic change in protein expression. However, ARG2, NOS1, NOS2, and agmatinase were also increased, whereas NOS3 expression was decreased. Arginase inhibition in vivo with nebulized S-(2-boronoethyl)-l-cysteine attenuated the methacholine responsiveness of the central airways in mice from the acute model. Further investigations in the chronic murine model revealed an expression profile that more closely paralleled the human asthma samples: only ARG1 expression was significantly increased. Interestingly, in the chronic mouse model, which generates a remodeling phenotype, arginase inhibition attenuated methacholine responsiveness of the central and peripheral airways. The similarity in arginase expression between human asthma and the chronic model and the attenuation of AHR after in vivo treatment with an arginase inhibitor suggest the potential for therapeutic modification of arginase activity in asthma.