Constitutive Nitric Oxide Synthase Isoforms Research Articles

How to say NO to the Right vs. Left heart E‐C coupling

There are constitutive NO synthase isoforms, neuronal NOS (nNOS) and endothelial NOS (eNOS) in myocardium, regulating the contractility and calcium dynamics. Physiological and anatomical differences between the right (RV) and left ventricle (LV) are evident. Although there is a lot of study of the regulatory roles of NOS in LV myocytes (LVCMs), only a few study in RV myocytes (RVCMs). Here we compared the roles of nNOS and eNOS in the RVCMs and LVCMs isolated by using Langendorff perfusion system from S-D rats. The sarcomere shortening (∆SL) and increased amount of calcium transient (∆[Ca²⁺]i) paced by 2 Hz field stimulation were simultaneously measured using the Ionoptix system. An nNOS-specific inhibitor, S-methyl-L-thiocitrulline (SMTC), and the non-specific NOS inhibitor, L-NG-Nitroarginine methyl ester (L-NAME) were used to dissect the roles of NOS isoforms. RVCMs showed smaller ∆[Ca²⁺]i than LVCMs. The treatment with SMTC and L-NAME significantly decreased the ∆[Ca²⁺]i of RVCMs, while only slightly decreased in LVCMs. Perplexingly, the ∆SLof RVCMs was increased by SMTC, whilenot altered in LVCMs. The mismatched changes in ∆SL and ∆[Ca²⁺]i between RVCMs and LVCMs suggested differential changes in the calcium sensitivity of contraction. In fact, the Ca2+ sensitivity was increased by SMTC in RVCMs while not in LVCMs. In addition, the diastolic sarcomere length (SLD) of RVCMs were decreased by SMTC and L-NAME while not in LVCMs. As for the decreased myofilament calcium sensitivity through sGC-cGMP-PKG pathway, altered phosphorylation of troponin I (TnI) was suggested. In fact, the SMTC treatment decreased the phosphorylated TnI in RVCMs, while not LVCMs. Finally, differential distribution of splice variants of nNOS were supposed; the phosphorylated nNOSβ was higher in the myofilament from RVCMs than LVCMs. Taken together, we firstly provide evidence that the inhibition of nNOSβ in RVCMs increases myofilament calcium sensitivity through decreased phosphorylation of TnI.

Roles of nNOS and eNOS in rat heart ; comparison between the left and right ventricle myocytes

Right ventricle (RV) has physiological as well as anatomical differences compared with left ventricle (LV). There are constitutive NO synthase isoforms, neuronal NOS (nNOS) and endothelial NOS (eNOS) in myocardium, regulating the calcium dynamics and contractility. Here we compared the roles of nNOS and eNOS in the RV and LV myocytes from S‐D rats. After isolating the myocytes using Langendorff perfusion system, their sarcomere shortening and [Ca2+]i were simultaneously measured using the Ionoptix system while paced by 2 Hz field stimulation. nNOS‐specific inhibitor, S‐methyl‐L‐thiocitrulline (SMTC) and the non‐specific NOS inhibitor, L‐NG‐Nitroarginine methyl ester (L‐NAME) were used to pharmacologically dissect the roles of NOS isoforms.According to the immunoblot assay, the protein amounts of nNOS and its phosphorylated form were not different between RV and LV. Although the amount of eNOS was lower in RV than LV, its phosphorylated form was similar. RV myocytes showed slower contraction (longer TPeak) and slower relaxation (longer Trelax) than LV myocytes. The treatment with SMTC or L‐NAME shortened TPeak and Trelax of RV myocytes, abolishing the kinetic differences of ΔSL. It was notable that the Trelax of LV myocytes became slightly longer by SMTC. Paradoxically, the length of sarcomere shortening (ΔSL) was increased while the amplitude of calcium transient (Δ[Ca2+]i) was decreased by SMTC in RV myocytes. However, SMTC did not affect the ΔSL and Δ[Ca2+]i of LV myocytes. Our study suggest that the slower contraction and relaxation of RV myocytes might be partly due to differential responses to the nNOS‐dependent intracellular signaling. The opposite direction of changes in the amplitudes of ΔSL and Δ[Ca2+]i by SMTC suggest that the nNOS in RV myocytes is responsible for negative inotropy despite the positive effects on the calcium transient, which is not evident in LV myocytes.

Isoform profile of NOS enzyme in structure of rats’ solitary-vagal complex in arterial hypertension of various origin

The aim of this study was to characterize the nitric oxide isoforms profile in rats’ nucleus tractus solitarii (NTS) and dorsal motor nucleus (DMN) of vagus nerve in arterial hypertension (AH) of various origin (essential (EAH) – rats of SHR line, and endocrine-salt AH). Materials and methods. The study was performed on 30 aged male rats. Among them, 20 Wistar rats were divided into two groups – control (10 rats) and 10 rats with simulated endocrine-salt AH (ESAH) and 10 SHR rats. An immunohistochemical method was used to study the of nitric oxide synthase isoforms expression features in DMN and NTS. The following parameters were determined: the content of immunoreactive material (IRM) for the studied peptides (U if ), the relative area of the IRM (%) and the IRM concentration in 1 μm 2 (U if /μm 2 ). Results. It was found that in rats with both models of AH, the expression indices of all three nitric oxide synthase (NOS) isoforms in the studied structures increased. In our opinion, this is due to the activation of the studied structures in AH conditions. This must be considered as an important element of high blood pressure compensating. It is achieved through the implementation by a complex of mechanisms like reducing of the sympathetic tone and increasing of the parasympathetic tone by activating a system of secondary messengers; improving neurotrophy due to the high activity of constitutive NOS isoforms; iNOS-mediated NO overproduction, as a factor in compensating for its bioavailability in conditions of local ischemia in AH. Conclusions. Regardless of the AH etiopathogenesis in both experimental groups, the expression of all three NOS isoforms increases in the DMN and NTS structures. In rats with EAH in the DMN and NTS structures, the expression indices of NOS isoforms have their own characteristics. So in the first structure, the largest changes in the indices of the immunoreactive material content and concentration are observed for eNOS, and the relative area for iNOS. At the same time, in the NTS structure, the largest changes in the content indices are observed for iNOS, and the concentration and relative area for eNOS. In rats with ESAH in the DMN structure, the highest changes in the indices of IRM content and concentration are observed for the endothelial isoform of NOS, and the relative area – for inducible. In the NTS structure, the IRM content changed the most for nNOS, and the concentration and relative area for iNOS.

Cardiovascular risk and the effect of nitric oxide synthase inhibition in female rats: The role of estrogen

It is known that autonomic modulation is responsive to ovarian hormone levels and that estrogen increases nitric oxide (NO) bioavailability. However, little is known about the interaction of nitric oxide synthase (NOS) isoforms with autonomic modulation, oxidative stress and cardiovascular risk in females. This study aimed to investigate cardiovascular, autonomic and oxidative parameters after selective NOS inhibition. A spectral analysis of systolic arterial pressure (SAP) and heart rate variability (HRV) was performed. NO levels, total antioxidant capacity (TRAP), lipid hydroperoxides (LOOH) and paraoxonase 1 (PON1) activity were measured in the plasma of rats treated with L-NG-nitroarginine methyl ester (L-NAME), S-methylisothiourea (SMT) or saline. Wistar rats, ovariectomized (OVX) with or without estradiol treatment (1mg/kg/day) or with a false ovariectomy (SHAM), were submitted to artery and vein catheterization. Cardiovascular parameters were evaluated before and after the administration of saline or NOS inhibitors. After 2h, plasma samples were collected for biochemical measurement. At baseline, cardiovascular and autonomic parameters were not different among the groups. L-NAME, the constitutive NOS isoform (cNOS) inhibitor, promoted an increase in mean arterial pressure (MAP) and a reduction in the low frequency band (LF) of SAP of SHAM rats, but this increase was smaller in OVX animals, which also showed a reduction in PON1 activity. The decreased activity of PON1 caused by L-NAME was prevented in the OVX+E group. SMT, an inducible NOS isoform (iNOS) inhibitor, promoted an increase in MAP and in the LF of SAP, in interbeat interval (IBI) parameters at LFnu and in LF/HF ratio of HRV in all groups, but the OVX+E had lower levels of NO when compared with the OVX group. Our data suggest that while cNOS contributes to maintaining the activity of PON1 in OVX rats, iNOS activity maintains the levels of NO in OVX+E rats.

Cross-Talk Between NO Synthase Isoforms in Neuro-Inflammation: Possible Implications in HIV-Associated Neurocognitive Disorders

Inducible nitric oxide synthase (iNOS) is expressed in several cell types, particularly in inflammatory cells, in response to diverse proinflammatory stimuli, including viral proteins as HIV Tat and gp120. This response is preceded by an early decline in basal nitric oxide (NO) levels, dependent on a signaling leading to inhibition of the constitutive isoform of NO synthase (cNOS). This process requires critical levels of arachidonic acid (AA), generated by Ca<sup>2+</sup>-dependent activation of cytosolic phospholipase A2, and is mediated by the downstream tyrosine kinase-dependent phosphorylation of cNOS. Lowering basal NO levels are necessary for the activation of nuclear factor-κB, and thus for the expression of a variety of genes regulated by this transcription factor, which include iNOS. Notably, NO and AA, two small lipid soluble molecules, can trigger the above responses also in distal cells. Thus, AA produced at the very early stages of the inflammatory response is a likely critical signal switching the regulation of the "NO tone" from physiological (i.e., mediated by cNOS) to pathological (i.e., mediated by iNOS). This later phase of the inflammatory response is often accompanied by the onset of deleterious effects in the tissue, in which a critical role is played by iNOS-derived NO (directly or indirectly, i.e., via formation of peroxynitrite) as well as by products of the AA cascade. In this review, the authors discuss the implications of the crosstalk between the NOS isoforms in HIV-associated neuro-pathogenesis highlighting the role of NO and AA as mediators of cytotoxicity.

Nitric oxide and heat shock protein 90 co-regulate temperature-induced bleaching in the soft coral Eunicea fusca

Coral bleaching represents a complex physiological process that is affected not only by environmental conditions but by the dynamic internal cellular biology of symbiotic dinoflagellates (Symbiodinium spp.) and their cnidarian hosts. Recently, nitric oxide (NO) has emerged as a key molecule involved with the expulsion of Symbiodinium from host cnidarian cells. However, the site of production remains under debate, and the corresponding signaling pathways within and between host and endosymbiont remain elusive. In this study, using freshly isolated Symbiodinium from the soft coral Eunicea fusca, I demonstrate that thermally induced stress causes an upregulation in Symbiodinium heat shock protein 90 (Hsp90). In turn, Hsp90 shows a concomitant ability to enhance the activity of a constitutively expressed isoform of NO synthase. The resulting production of NO constitutes a signaling molecule capable of inducing Symbiodinium expulsion. Using nitric oxide synthase (NOS) and Hsp90 polyclonal antibodies, thermal stress-induced Hsp90 was shown to co-immunoprecipitate with a constitutive isoform of NOS. The specific blocking of Hsp90 activity, with the Hsp90 inhibitor geldanamycin, was capable of inhibiting NO production implicating the involvement of a coordinated regulatory system. These results have strong evolutionary implications for Hsp90–NOS chaperone complexes among biological kingdoms and provide evidence for a new functional role in symbiotic associations.

Effects of chronic nitric oxide synthase inhibition on TNB-induced colitis in rats.

Nitric oxide (NO) synthesis is increased in ulcerative colitis, but the role of NO in colitis is poorly understood. The present study employed Nw-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, in rats to evaluate the effect of NO on 2,4,6-trinitrobenzenesulphonic acid (TNB)-induced colitis. L-NAME solutions were placed in subcutaneous, osmotic mini-pumps which continuously released L-NAME at 0.042, 0.208, 0.417, or 1.667 mg kg-1 h-1. L-NAME dose-dependently enhanced lesions in TNB-induced colitis. The two higher doses of L-NAME significantly increased colonic mucosal damage, although there was slight, nonsignificant reduced lesion formation with the lowest dose of L-NAME. 0.042 mg kg-1 h-1. A single dose of L-NAME at 100 mg kg-1 subcutaneously injected daily in TNB-treated rats also increased lesions, and these ulcerogenic actions of L-NAME were reversed by L-arginine but not by D-arginine (both at 500 mg kg-1, s.c.). Only the highest dose of L-NAME (mini-pump) significantly depressed myeloperoxidase (MPO) activity. Faecal occult bleeding showed a close relationship with severity of colitis. These findings suggest that there may exist a balance between NO protective and aggressive effects. In TNB-induced colitis, antagonism of endogenous NO generation was intensified, whereas slight inhibition of NO synthesis reduced lesions. Variations in responses, related to timing or dose changes in L-NAME, may reflect the differences in inducible vs constitutive NO synthase isoforms.

Expression of Constitutive Nitric Oxide Synthase Isoforms in Varicose Vein Wall; Preliminary Results

There are conflicting findings in literature about the structural changes of the primary varicose veins. NO (a potent vasodilatator) is synthesized by nitric oxide synthase (NOS). From 3 known NOS isoforms the two are constitutional: eNOS (endothelial NOS) and nNOS (neuronal NOS). 10 varicose and 10 control vein samples were processed by standard light microscopy and immuno-histochemica techniques using rabbit polyclonal antibodies against eNOS and nNOS. Antibodies expression was evaluated semiquantitatively and proved morphometrically by 2D-image analysis. total area of NOS isoforms expressions was determined by color analysis and color digital subtraction. The results showed discontinuous and significantly lower expression of both NOS isoforms the in the tunica media of varicose veins compared with the control group. For the statistical analysis the unpaired t-test was used. Our results suppose lower NO levels in varicose vein wall, deducing that varicose dilatation is due to other mechanism, and they contradict the results of previously published similar works.

Nitric oxide and human sperm motility : an immunohistochemical study

Defective sperm function is a common cause of human male infertility. Human spermatozoa are very susceptible to oxidative stress, probably because they contain high levels of polyunsaturated fatty acids and are exposed to ROS produced by contaminating neutrophils or by the spermatozoa themselves. In addition, due to the cytoplasm scarcity, they lack an adequate reserve of antioxidant enzymes. It has been previously shown that human spermatozoa produce nitric oxide (NO) as well as superoxide anion and consequently peroxynitrite, which can rapidly react with proteins, lipids, and nucleic acids. NO is a short-lived free radical produced by three isoforms of NO synthase (NOS): endothelial (eNOS), neuronal (nNOS), and inducible (iNOS), which are responsible for the conversion of L-arginine to L-citrulline and NO. NO plays an important role in sperm physiology, although at high concentrations it could lead to cytotoxicity accompanied by decreased sperm motility. In recent studies it has been evidenced the presence of a significant negative linear correlation between NO concentration and sperm motility, as well as between peroxynitrite production and the kinetic features of spermatozoa. It has been previously demonstrated that the spermatozoa of fertile men express both eNOS and nNOS, which appear to be involved in sperm motility, metabolism, capacitation and acrosome reaction. However, the specific role of NOS isoforms in gametes is not well understood, even if iNOS seems to negatively affect the sperm function through the production of large amounts of NO, and consequently the nitration of protein tyrosine by peroxynitrite. The aim of the present study was to assess the presence and localization of NOS isoforms in human spermatozoa isolated from controls and asthenozoospermic infertile patients, by means of immunohistochemical techniques, in order to characterize the pattern of NOS expression and to evaluate the correlation with sperm function. In addition we studied the immunoexpression of citrulline, to achieve the microscopic visualization of NOS catalytic activity, and nitrotyrosine, since the protein tyrosine nitration represents a shift from the physiological role of NO toward pathogenetic pathways. The main results of our study are that the immunohistochemical expression of constitutive NOS isoforms is lower in the asthenozoospermic group, while the immunoexpression of iNOS and nitrotyrosine is higher in comparison with normozoospermic fertile subjects, thus suggesting a possible pathogenetic role of NO in the reduction of sperm motility.

Nitric Oxide Synthase Enzymes in the Airways of Mice Exposed to Ovalbumin: NOS2 Expression Is NOS3 Dependent

Objectives and Design. The function of the airway nitric oxide synthase (NOS) isoforms and the lung cell types responsible for its production are not fully understood. We hypothesized that NO homeostasis in the airway is important to control inflammation, which requires upregulation, of NOS2 protein expression by an NOS3-dependent mechanism. Materials or Subjects. Mice from a C57BL/6 wild-type, NOS1−/−, NOS2−/−, and NOS3−/− genotypes were used. All mice strains were systemically sensitized and exposed to filtered air or ovalbumin (OVA) aerosol for two weeks to create a subchronic model of allergen-induced airway inflammation. Methods. We measured lung function, lung lavage inflammatory and airway epithelial goblet cell count, exhaled NO, nitrate and nitrite concentration, and airway NOS1, NOS2, and NOS3 protein content. Results. Deletion of NOS1 or NOS3 increases NOS2 protein present in the airway epithelium and smooth muscle of air-exposed animals. Exposure to allergen significantly reduced the expression of NOS2 protein in the airway epithelium and smooth muscle of the NOS3−/− strain only. This reduction in NOS2 expression was not due to the replacement of epithelial cells with goblet cells as remaining epithelial cells did not express NOS2. NOS1−/− animals had significantly reduced goblet cell metaplasia compared to C57Bl/6 wt, NOS2−/−, and NOS3−/− allergen-exposed mice. Conclusion. The airway epithelial and smooth muscle cells maintain a stable airway NO concentration under noninflammatory conditions. This “homeostatic” mechanism is unable to distinguish between NOS derived from the different constitutive NOS isoforms. NOS3 is essential for the expression of NOS2 under inflammatory conditions, while NOS1 expression contributes to allergen-induced goblet cell metaplasia.

Cerebral blood flow regulation by nitric oxide in neurological disorders

There has been a rapid increase in the amount of information on the physiological and pathophysiological roles of nitric oxide (NO) in the brain. This molecule, which is formed by the constitutive isoforms of NO synthase, endothelial (eNOS) and neuronal (nNOS), plays an obligatory role in the regulation of cerebral blood flow and cell viability and in the protection of nerve cells or fibres against pathogenic factors associated with Alzheimer's disease, Huntington's disease, seizures, and migraine. Cerebral blood flow is impaired by decreased formation of NO from endothelial cells, autonomic nitrergic nerves, or brain neurons and also by increased production of reactive oxygen species (ROS). The NO-ROS interaction is an important topic in discussing blood flow and cell viability in the brain. Excessive production of NO by inducible NOS (iNOS) and nNOS in the brain participates in neurotoxicity. Recent studies on brain circulation have provided useful information about the involvement of impaired NO availability or uncontrolled NO production in cerebral pathogenesis, including Alzheimer's disease, seizures, vascular headaches, and inflammatory disorders. Insight into the role of NO in the brain will contribute to our better understanding of cerebral hemodynamic dysfunction and will aid in developing novel therapeutic measures in diseases of the central nervous system.

Cerebral Blood Flow Regulation by Nitric Oxide: Recent Advances

Nitric oxide (NO) is undoubtedly quite an important intercellular messenger in cerebral and peripheral hemodynamics. This molecule, formed by constitutive isomers of NO synthase, endothelial nitric-oxide synthase, and neuronal nitric-oxide synthase, plays pivotal roles in the regulation of cerebral blood flow and cell viability and in the protection of nerve cells or fibers against pathogenic factors associated with cerebral ischemia, trauma, and hemorrhage. Cerebral blood flow is increased and cerebral vascular resistance is decreased by NO derived from endothelial cells, autonomic nitrergic nerves, or brain neurons under resting and stimulated conditions. Somatosensory stimulation also evokes cerebral vasodilatation mediated by neurogenic NO. Oxygen and carbon dioxide alter cerebral blood flow and vascular tone mainly via constitutively formed NO. Endothelial dysfunction impairs cerebral hemodynamics by reducing the bioavailability of NO and increasing the production of reactive oxygen species (ROS). The NO-ROS interaction is an important issue in discussing blood flow and cell viability in the brain. Recent studies on brain circulation provide quite useful information concerning the physiological roles of NO produced by constitutive isoforms of nitric-oxide synthase and how NO may promote cerebral pathogenesis under certain conditions, including cerebral ischemia/stroke, cerebral vasospasm after subarachnoid hemorrhage, and brain injury. This information would contribute to better understanding of cerebral hemodynamic regulation and its dysfunction and to development of novel therapeutic measures to treat diseases of the central nervous system.

Effects of Aminoguanidine, an Inhibitor of Inducible Nitric Oxide Synthase, on Nitric Oxide Production and Its Metabolites in Healthy Control Subjects, Healthy Smokers, and COPD Patients

Nitric oxide (NO) is produced by resident and inflammatory cells in the respiratory tract by the enzyme NO synthase (NOS), which exists in three isoforms: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS. NO production is increased in patients with COPD, and the production of NO under oxidative stress conditions generates reactive nitrogen species that may amplify the inflammatory response in COPD. To examine the role of increased NO in COPD, we administered a relatively selective iNOS inhibitor, aminoguanidine, by nebulization in a double-blind, placebo-controlled study in COPD patients, healthy smokers, and healthy nonsmoking subjects. We investigated whether aminoguanidine had any effect on exhaled NO produced in the central lung (flux of NO from the airways [Jno] and peripheral lungs (concentration of NO in peripheral lung [Calv], on NO metabolites (nitrite [NO(2)(-)]/nitrate [NO(3)(-)], peroxinitrite [ONOO(-)], nitrotyrosine), and on a marker of oxidative stress (8-isoprostane) in exhaled breath condensate (EBC) and in sputum. Aminoguanidine administration resulted in a significant reduction in Jno compared with administration of the saline solution control in healthy subjects, smokers, and COPD patients. Calv in smokers and in COPD patients was not completely inhibited 1 h after aminoguanidine inhalation, in marked contrast to previous results in asthma. Moreover, ONOO(-) and NO(2)(-)/NO(3)(-) levels were also increased in EBC and in sputum of smokers and COPD and were not completely inhibited following aminoguanidine inhalation. 8-Isoprostane levels were also increased in smokers and in COPD patients but were not reduced after aminoguanidine inhalation. These results suggest that the constitutive NOS isoform as well as iNOS might be involved in NO release and contribute to the high Calv and ONOO(-) production in patients with COPD. Clinicaltrials.gov Identifier: NCT00180635.

Immunohistochemical Localization of Nitric Oxide Synthase (NOS) in Mouse Mammary Gland during Reproductive Cycle

Nitric oxide (NO) has been proposed as a mediator of postnatal mammary gland development. We investigated the immunohistochemical localization of three isoforms of nitric oxide synthase (NOS) enzymes, neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS), in the mouse mammary gland during reproductive cycle. The NOS isoforms detected in normal mouse mammary glands were the constitutive forms of NOS (nNOS and eNOS). nNOS was localized to the alveoli, myoepithelia, lactiferous ducts and blood vessel endothelia, while eNOS was localized in the alveoli, lactiferous ducts and blood vessel endothelia. The strongest immunoreactivity for both constitutive NOS isoforms was observed in pregnant mice. The differential staining intensity of NOS enzymes in the mammary gland led us to conclude that nitric oxide in the mouse mammary gland is mainly synthesized by constitutive NOS isoforms, and suggest that NO has functional roles in post-pubertal growth and differentiation of the mammary gland.

INHIBITORY EFFECTS OF ENDOGENOUS l‐ARGININE ANALOGUES ON NITRIC OXIDE SYNTHESIS IN PLATELETS: ROLE IN PLATELET HYPERAGGREGABILITY IN HYPERTENSION

1. An increase in plasma concentrations of endogenous L-arginine analogues, which are inhibitors of nitric oxide (NO) synthesis, may be involved in platelet activation and the increased risk of thrombosis in essential hypertension. Nitric oxide is synthesised in platelets from the amino acid L-arginine by inducible and constitutive isoforms of NO synthase (NOS), which leads to increased levels of cGMP. 2. In the present study, we investigated basal intraplatelet cGMP levels, platelet aggregation and pro-inflammatory biomarkers in hypertensive patients. The effects of endogenous (N(G)-monomethyl-L-arginine (L-NMMA) and asymmetric dimethylarginine (ADMA); both at 1 mmol/L) and exogenous (aminoguanidine and N(G)-nitro-L-arginine; both at 1 mmol/L) L-arginine analogues and the neutral amino acid L-leucine (1 mmol/L) in inhibiting NOS activity in platelets were also investigated. 3. Twelve healthy controls and 18 hypertensive patients participated in the study. Platelet aggregation induced by collagen was increased in hypertensive patients (95 +/- 5%) compared with controls (72 +/- 5%). Basal NOS activity and intraplatelet cGMP levels were reduced in hypertensive platelets. Moreover, ADMA, L-NMMA and L-leucine were effective inhibitors of NO synthesis in both hypertensive and control platelets. Essential hypertension led to an inflammatory response, with increased plasma concentrations of fibrinogen, C-reactive protein and cytokines. 4. These findings provide evidence that, in essential arterial hypertension, the enhanced plasma levels of endogenous L-arginine analogues ADMA and L-NMMA, potent inhibitors of L-arginine transport and NO synthesis in platelets, may play a role in increased platelet aggregation via a cGMP-dependent mechanism.

Nitrergic Modulation of Gastrointestinal Function During Early Endotoxemia

After bacterial infection, the host reacts by signalling to the central nervous system where a cascade of physiologic, neuroendocrine and behavioural processes is orchestrated, collectively termed the acute phase response. Endotoxemia following Gram-negative bacterial infection induces a wide array of effects, including fever, loss of appetite and changes in gastrointestinal function that attempt to eliminate the challenge and restore homeostasis. Systemic administration of low doses of endotoxin (5-40 microg/kg) to rats is associated with changes in gastrointestinal motor function, inhibition of gastric acid secretion and increase in the gastric mucosal resistance to damage. These changes are rapid in onset (observed within one hour), not related to vascular dysfunction, and appear to be mediated by mechanisms that involve the peripheral and the central nervous system. Nitric oxide (NO) plays a central role in the physiology of the gastrointestinal tract and its response to illness. Accumulated evidence supports an increase of NO synthesis in the brainstem, as well as in the gastric myenteric plexus thirty minutes after endotoxin administration. Such a synthesis is due to constitutive nitric oxide synthase (NOS) and occurs before the induction of NOS takes place. In this review we provide experimental evidence supporting the hypothesis that activation of a physiologic mechanism, mediated by the autonomic and the central nervous systems as well as constitutive NOS isoforms, is involved in acute changes of gastrointestinal function during early endotoxemia.

Nitric Oxide Synthase (NOS) as Therapeutic Target for Asthma and Chronic Obstructive Pulmonary Disease

In the respiratory tract, NO is produced by residential and inflammatory cells. NO is generated via oxidation of L-arginine that is catalysed by the enzyme NO synthase (NOS). NOS exists in three distinct isoforms: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). NO derived from the constitutive isoforms of NOS (nNOS and eNOS) and other NO-adduct molecules (nitrosothiols) are able to modulate bronchomotor tone. NO derived from the inducible isoform of NO synthase, up-regulated by different cytokines via NF-kappaB-dependent pathway, seems to be a pro-inflammatory mediator with immunomodulatory effects. The production of NO under oxidative stress conditions secondarily generates strong oxidising agents (reactive nitrogen species) that may amplify the inflammatory response in asthma and COPD. Moreover, NO can be exhaled and levels are abnormal in stable atopic asthma and during exacerbations in both asthma and COPD. Exhaled NO might therefore be a non-invasive tool to monitor the underlying inflammatory process. It is suggested that NOS regulation provides a novel target in the prevention and treatment of chronic inflammatory diseases of the airways such as asthma and COPD.

Nitric oxide in the kidney: functions and regulation of synthesis

In the kidney nitric oxide (NO) has numerous important functions including the regulation of renal haemodynamics, maintenance of medullary perfusion, mediation of pressure-natriuresis, blunting of tubuloglomerular feedback, inhibition of tubular sodium reabsorption and modulation of renal sympathetic neural activity. The net effect of NO in the kidney is to promote natriuresis and diuresis. Significantly, deficient renal NO synthesis has been implicated in the pathogenesis of hypertension. All three isoforms of nitric oxide synthase (NOS), namely neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2) and endothelial NOS (eNOS or NOS3) are reported to contribute to NO synthesis in the kidney. The regulation of NO synthesis in the kidney by NOSs is complex and incompletely understood. Historically, many studies of NOS regulation in the kidney have emphasized the role of variations in gene transcription and translation. It is increasingly appreciated, however, that the constitutive NOS isoforms (nNOS and eNOS) are also subject to rapid regulation by post-translational mechanisms such as Ca(2+) flux, serine/threonine phosphorylation and protein-protein interactions. Recent studies have emphasized the role of post-translational regulation of nNOS and eNOS in the regulation of NO synthesis in the kidney. In particular, a role for phosphorylation of nNOS and eNOS at both activating and inhibitory sites is emerging in the regulation of NO synthesis in the kidney. This review summarizes the roles of NO in renal physiology and discusses recent advances in the regulation of eNOS and nNOS in the kidney by post-translational mechanisms such as serine/threonine phosphorylation.

Nitric oxide in obstructive uropathy: role of endothelial nitric oxide synthase.

Nitric oxide (NO) ameliorates fibrosis in experimental obstructive uropathy. Previously, we demonstrated that renal fibrosis was decreased after 2 weeks of unilateral ureteral obstruction in inducible nitric oxide synthase (iNOS) knock-out mice given L-arginine supplemented drinking water. We proposed that the 2 constitutive isoforms of nitric oxide synthase (NOS) mediated down-regulation of renal fibrosis in response to prolonged ureteral obstruction. To determine the specific role of endothelial NOS (eNOS) versus neuronal NOS in modulating renal fibrosis due to obstructive uropathy, we evaluated renal injury following unilateral ureteral obstruction in C57BL/6J mice subjected to biochemical inhibition of the constitutive isoforms of NOS and in eNOS knockout mice. Four groups of C57BL/6J mice were studied. Complete unilateral ureteral obstruction was created by ligating the right ureter at age 8 weeks. A single daily intraperitoneal injection of 30 mg./kg. S-methyl-L-thiocitrulline (SMLT), a selective neuronal and endothelial NOS inhibitor was started 24 hours before ureteral obstruction and administered to half of the study animals. SMLT treated mice and control animals were further subdivided to receive either regular tap water or 1% L-arginine (weight/volume) supplemented water after unilateral ureteral obstruction. Animals were sacrificed on postoperative day 3, 7 or 14. In addition, eNOS knockout mice with unilateral ureteral obstruction were given tap water or L-arginine supplemented water to drink and sacrificed after 14 days. Urine specimens from the bladder and the obstructed renal pelvis along with serum were collected. Nitrite level in each fluid was determined. Renal morphology and cortical thickness were assessed in the normal and obstructed kidneys. Interstitial fibrosis was evaluated using trichrome stain. SMLT was well tolerated by C57BL/6J mice. Serum nitrite levels and nitrite excretion in bladder urine were similar in all SMLT treated groups throughout the duration of unilateral ureteral obstruction. A reduction of pelvic urine nitrite levels by 89%, 68% and 48% versus bladder urine nitrite levels was observed after 3, 7 and 14 days of unilateral ureteral obstruction (p <0.05). Administration of SMLT resulted in a significant increase in bladder urine nitrite level at 7 days and in pelvic urine nitrite levels at 14 days. No significant histological differences in the obstructed kidney were seen after 3, 7 or 14 days of unilateral ureteral obstruction in SMLT treated versus control mice regardless of whether they received tap water or L-arginine supplemented drinking water. In eNOS knockout mice with unilateral ureteral obstruction for 14 days L-arginine supplementation had no effect on pelvic urine nitrite levels and did not alter renal histopathology or cortical thickness. NO production is decreased in the obstructed kidney in mice with unilateral ureteral obstruction. Biochemical inhibition of constitutive NOS did not modulate renal injury after 14 days of unilateral ureteral obstruction. In contrast to previous findings with iNOS knockout mice, dietary supplementation with L-arginine had no effect on the degree of fibrosis in the obstructed kidney in SMLT treated C57BL/6J or eNOS knockout mice. We conclude that NO derived from eNOS within the kidney has a pivotal role in protecting against renal fibrosis in response to unilateral ureteral obstruction.

Constitutive nitric oxide synthases are responsible for the nitric oxide production in the ischemic aged cerebral cortex

Aged brain shows reduced biological plasticity to meet emergency conditions such as ischemia, a process in which nitric oxide (NO) and apoptosis have been shown to play important roles. Using a model of transient global ischemia, we have analyzed the NO system and the p53, bax and bcl-2 response in the cerebral cortex of aged rats. Although immediately after ischemia the NO level is maintained, the reperfusion period increases NO concentrations together with the following: (i) greater bulk-protein nitration mainly due to a 50-kDa immunoreactive band; (ii) an increase in p53 protein; and (iii) an up-regulation of Bax together with a down-regulation of Bcl-2. These results match up with induced endothelial nitric oxide synthase expression immediately after ischemia and in neuronal nitric oxide synthase with the reperfusion. However, inducible nitric oxide synthase was not altered with ischemia/reperfusion. Altogether, these data suggest that NO production in cerebral cortex of aged ischemic animals is due to the constitutive NO synthase isoforms. This response is accompanied by the increased expression of pro-apoptotic proteins.