Control Of Nitric Oxide Production Research Articles

030 Light-controlled relaxation of rat penile corpus cavernosum by a novel NO donor, NO-Rosa

Phosphodiesterase-5 inhibitors are not always effective for patients with erectile dysfunction (ED) who also have diabetes mellitus or have had a radical prostatectomy, because of the reduction in nitric oxide (NO) production. The control of NO production may be useful as a therapy for ED. In this study, we focused on a light-controllable NO releaser. Previously, we developed a blue (470–500 nm) light-controllable NO releaser, NOBL-1. Subsequently, we improved NOBL-1, because blue light has low tissue permeability. We have now succeeded in the development of a yellow-green (530–590 nm) light-controllable NO releaser, NO-Rosa. We investigated whether we could control the relaxation of the corpus cavernosum smooth muscle by using an NO-Rosa derivative and light irradiation. Fluorescence imaging of the NO release from NO-Rosa in HEK-293 cells was performed using DAF-FM. Adult male Wistar-ST rats were used for the isometric tension study. The corpus cavernosum was treated by the addition of L-NAME (100 μM) to inhibit endogenous NO production and pre-contracted with noradrenaline (10 μM). The response of the corpus cavernosum smooth muscle to irradiation with yellow-green light, before and after the addition of an NO-Rosa derivative (10 μM), was measured and recorded.

Impaired functions of neural stem cells by abnormal nitric oxide-mediated signaling in an in vitro model of Niemann-Pick type C disease

Nitric oxide (NO) has been implicated in the promotion of neurodegeneration. However, little is known about the relationship between NO and the self-renewal or differentiation capacity of neural stem cells (NSCs) in neurodegenerative disease. In this study, we investigated the effect of NO on self-renewal of NSCs in an animal model for Niemann-Pick type C (NPC) disease. We found that NO production was significantly increased in NSCs from NPC1-deficient mice (NPC1-/-), which showed reduced NSC self-renewal. The number of nestin-positive cells and the size of neurospheres were both significantly decreased. The expression of NO synthase (NOS) was increased in neurospheres derived from the brain of NPC1-/- mice in comparison to wild-type neurospheres. NO-mediated activation of glycogen synthase kinase-3beta (GSK3beta) and caspase-3 was also observed in NSCs from NPC1-/- mice. The self-renewal ability of NSCs from NPC1-/- mice was restored by an NOS inhibitor, L-NAME, which resulted in the inhibition of GSK3beta and caspase-3. In addition, the differentiation ability of NSCs was partially restored and the number of Fluoro-Jade C-positive degenerating neurons was reduced. These data suggest that overproduction of NO in NPC disease impaired the self-renewal of NSCs. Control of NO production may be key for the treatment of NPC disease.

Nitric Oxide in the Pulmonary Vasculature

Homeostasis in the pulmonary vasculature is maintained by the actions of vasoactive compounds, including nitric oxide (NO). NO is critical for normal development of the pulmonary vasculature and continues to mediate normal vasoregulation in adulthood. Loss of NO bioavailability is one component of the endothelial dysfunction and vascular pathology found in pulmonary hypertension (PH). A broad research effort continues to expand our understanding of the control of NO production and NO signaling and has generated novel theories on the importance of pulmonary NO production in the control of the systemic vasculature. This understanding has led to exciting developments in our ability to treat PH, including inhaled NO and phosphodiesterase inhibitors, and to several promising directions for future therapies using nitric oxide-donor compounds, stimulators of soluble guanylate cyclase, progenitor cells expressing NO synthase (NOS), and NOS gene manipulation.

Subcellular targeting and trafficking of nitric oxide synthases

Unlike most other endogenous messengers that are deposited in vesicles, processed on demand and/or secreted in a regulated fashion, NO (nitric oxide) is a highly active molecule that readily diffuses through cell membranes and thus cannot be stored inside the producing cell. Rather, its signalling capacity must be controlled at the levels of biosynthesis and local availability. The importance of temporal and spatial control of NO production is highlighted by the finding that differential localization of NO synthases in cardiomyocytes translates into distinct effects of NO in the heart. Thus NO synthases belong to the most tightly controlled enzymes, being regulated at transcriptional and translational levels, through co- and post-translational modifications, by substrate availability and not least via specific sorting to subcellular compartments, where they are in close proximity to their target proteins. Considerable efforts have been made to elucidate the molecular mechanisms that underlie the intracellular targeting and trafficking of NO synthases, to ultimately understand the cellular pathways controlling the formation and function of this powerful signalling molecule. In the present review, we discuss the mechanisms and triggers for subcellular routing and dynamic redistribution of NO synthases and the ensuing consequences for NO production and action.

Fatty acid control of nitric oxide production by macrophages

Modulation of macrophage functions by fatty acids (FA) has been studied by several groups, but the effect of FA on nitric oxide production by macrophages has been poorly examined. In the present study the effect of palmitic, stearic, oleic, linoleic, arachidonic, docosahexaenoic and eicosapentaenoic acids on NF-κB activity and NO production in J774 cells (a murine macrophage cell line) was investigated. All FA tested stimulated NO production at low doses (1–10 μM) and inhibited it at high doses (50–200 μM). An increase of iNOS expression and activity in J774 cells treated with a low concentration of FA (5 μM) was observed. The activity of NF-κB was time-dependently enhanced by the FA treatment. The inhibitory effect of FA on NO production may be due to their cytotoxicity, as observed by loss of membrane integrity and/or increase of DNA fragmentation in cells treated for 48 h with high concentrations. The results indicate that, at low concentrations FA increase NO production by J774 cells, whereas at high concentrations they cause cell death.

Endothelial nitric oxide synthase polymorphism, nitric oxide production, salt sensitivity and cardiovascular risk factors in Hispanics

Mutations in the endothelial nitric oxide synthase (eNOS) gene may be associated with abnormal nitric oxide (NO) production and cardiovascular diseases. In this study, we investigated the prevalence of two eNOS polymorphisms, the Glu298Asp variant on exon 7, and the 4a/b variable number of tandem repeats (VNTR) on intron 4, and their association with blood pressure (BP), NO production, salt sensitivity and cardiovascular risk factors in healthy Venezuelans. The prevalence of both polymorphisms in Venezuelans was comparable to that described for Caucasians, but significantly different from that known for African-Americans and Japanese. The 4a/b genotype was associated with reduced levels of NO metabolites (25% decrease), larger BP lowering in response to salt restriction (9.0 vs 4.8 mmHg, P<0.05), greater prevalence of salt sensitivity (39% in 4a/b and 27% in 4b/b; P<0.05) and with higher LDL-cholesterol levels. The Glu298T polymorphism did not affect NO production, nor it was associated with salt sensitivity. Glu298Asp polymorphism was positively associated with higher weight, triglycerides and LDL-cholesterol. Neither polymorphism was associated with changes in fasting or postload serum glucose, BP, obesity and albuminuria. In conclusion, the prevalence of eNOS polymorphisms is strongly determined by ethnic factors. The 4a/b gene polymorphism could be a genetic susceptibility factor for the BP response to salt intake and for the genetic control of NO production. The reduced NO production in subjects with the 4a/b genotype may be responsible for the increased sensitivity of their BP to salt.

IL-10 up-regulates nitric oxide (NO) synthesis by lipopolysaccharide (LPS)-activated macrophages: improved control of Trypanosoma cruzi infection.

We examined the effects of IL-10 on tumour necrosis factor-alpha (TNF-alpha) and NO production by LPS-activated macrophages and on the ability of these cells to control Trypanosoma cruzi infection. We first observed that the addition of rIL-10 to macrophages of the J774 cell line decreased their synthesis of TNF-alpha but increased their release of NO in a dose-dependent manner. In parallel, treatment of J774 cells with rIL-10 resulted in a better control of T. cruzi infection involving up-regulation of NO synthesis, as it was not observed in presence of N-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NO synthase. The enhancing effect of rIL-10 on NO production was not observed on peritoneal macrophages from wild-type C57Bl/6 mice, but well on macrophages from IL-10 knock-out mice. The control of NO production by endogenous IL-10 was confirmed by the demonstration that neutralization of IL-10 secreted by LPS-activated macrophages from wild-type mice inhibited their production of NO and, in parallel, their ability to control T. cruzi infection. Taken together, these data demonstrate that both exogenous and endogenous IL-10 up-regulate the production of NO by LPS-activated macrophages and improve thereby their ability to clear T. cruzi infection.

Shear stress-mediated NO production in inner medullary collecting duct cells.

Recent evidence suggests that nitric oxide (NO) within the inner medullary collecting duct (IMCD) functions to regulate sodium and water reabsorption. Because fluid shear stress has been shown to increase NO production in endothelial and vascular smooth muscle cells, experiments were designed to determine whether a similar mechanism exists in IMCD cells. Cultured IMCD-3 cells derived from murine IMCD were subjected to 60 min of pulsatile shear stress. Nitrite production (2,3-diaminonaphthalene fluorometric assay) increased 12-, 16-, and 23-fold at 3.3, 10, and 30 dyn/cm(2), respectively, compared with static control cultures. Preincubation with the non-isoform-specific NO synthase inhibitor nitro-L-arginine methyl ester reduced nitrite production by 83% in response to 30 dyn/cm(2). Western blotting and immunofluorescence analysis of static IMCD-3 cell cultures revealed the expression of all three NO synthase isoforms (NOS-1 or neuronal NOS, NOS-2 or inducible NOS, and NOS-3 or endothelial NOS) in IMCD-3 cultures. These results indicate that NO production is modulated by shear stress in IMCD-3 cells and that fluid shear stress within the renal tubular system may play a role in the regulation of sodium and water excretion by control of NO production in the IMCD.

Amlodipine promotes kinin-mediated nitric oxide production in coronary microvessels of failing human hearts

Recently, we found that amlodipine can release nitric oxide (NO) from canine coronary microvessels, which raises the question of whether amlodipine can also promote coronary NO production in failing human hearts. The goal of this study was to define the effect of amlodipine on NO production in failing human hearts and to determine the role of kinins in the control of NO production induced by amlodipine. Six explanted human hearts with end-stage heart failure were obtained immediately at transplant surgery. Coronary microvessels were isolated as previously described, and nitrite, the stable metabolite of NO in aqueous solution, was measured using the Griess Reaction. Amlodipine (10 −10 to 10 −5 mol/L) significantly increased nitrite production in coronary microvessels in a dose-dependent manner. The increase in nitrite in response to the highest dose of amlodipine (79%) was similar in magnitude to either that of the angiotensin-converting enzyme inhibitor ramiprilat (74%) or the neutral endopeptidase inhibitors phosphoramidon (61%) and thiorphan (72%). Interestingly, the increase in nitrite production induced by amlodipine was entirely abolished by N ω-nitro- l-arginine methyl ester and also HOE-140 (a bradykinin-2 antagonist) and dichloroisocoumarin (a serine protease inhibitor that blocks kallikrein activity). These results indicate that amlodipine can promote coronary NO production in failing human hearts and that this effect is dependent on a kinin-mediated mechanism.

Role of endothelial kinins in control of coronary nitric oxide production.

The purpose of the present study was to determine whether interventions that promote kinin production or decrease kinin inactivation affect nitric oxide production in isolated canine coronary microvessels. Accordingly, bradykinin (10[-8] to 10[-5] mol/L), ramiprilat (10[-10] to 10[-8] mol/L), A23187 (10[-8] to 10[-6] mol/L), kallikrein (1 to 20 U/mL), and kininogen (0.5 to 10 microg/mL) were used to stimulate endothelium-dependent nitric oxide production. Receptor antagonists, serine protease inhibitors, and a kinin antibody were used to inactivate local kallikrein-kinin activity. Nitrite, the metabolite of nitric oxide in aqueous solution, was measured using the Griess reaction. All the agonists significantly increased nitrite release. For instance, the highest dose of bradykinin, ramiprilat, A23187, kallikrein, and kininogen markedly increased nitrite production, from 60+/-10 to 156+/-12, 153+/-11, 161+/-15, 176+/-15, and 168+/-16 pmol/mg (all P<.05), respectively. The increased nitrite production caused by these agents was not only blocked by N omega-nitro-L-arginine methyl ester (L-NAME) and HOE 140 (which blocks B2 kinin receptor) but by the kinin antibody also. For instance, nitrite production elicited by bradykinin, ramiprilat, A23187, and kininogen was reduced to 95+/-8, 87+/-8, 94+/-11, and 85+/-11 pmol/mg (all P<.05), respectively, by the kinin antibody. Carbachol-induced nitrite production (from 66+/-8 to 144+/-13) was blocked by L-NAME but not by HOE 140 or the kinin antibody. These results suggest that either increasing kininogen to promote endogenous kinin formation or inhibiting angiotensin-converting enzyme to decrease kinin breakdown, increases nitric oxide production in isolated coronary microvessels. These data indicate that a microvessel kallikrein-kinin system has an important role in the control of nitric oxide production in coronary microvessels.

Control of Nitric Oxide Production by Endogenous TNF-α in Mouse Retinal Pigmented Epithelial and Muller Glial Cells

Since the induction of nitric oxide synthase (NOS) by lipopolysaccharide (LPS) has been suggested to be partially dependent of the synthesis of tumor necrosis factor α (TNFα), we have investigated in vitro the production of NO in retinal cells from mice deficient in Lymphotoxin α (LTα)/TNFα. Treatment of retinal Müller glial (RMG) and retinal pigmented epithelial (RPE) cells from both wild-type and knockout mice with LPS and interferon γ (IFNγ) induced NO synthesis as determined by nitrite release into the media and was correlated to an increase in NOS-2 mRNA levels, evaluated by RT-PCR. However, the level of nitrite and the accumulation of mRNA was always less in cells from LTα/TNFα knockout mice than in wild-type mice. Simultaneous addition of TNFα restored the level of NO synthesis by RMG and RPE cells from LTα/TNFα knockout mice stimulated with LPS and IFNγ to wild type levels. Transforming growth factor β (TGFβ) blocked LPS/IFNγ-induced NO production in RMG and RPE cells from wild-type and LTα/TNFα knockout mice. Our results demonstrate that induction of NO synthesis in RMG and RPE cells by LPS and IFNγ is dependent in part on endogenous TNFα while inhibition of NO production by TGFβ does not require a modulation of TNFα synthesis.

Differences in Expression of Nitric Oxide Synthase Isoforms Between Neonatal and Juvenile Brain and Microvessels. 262

Autoregulation of cerebral blood flow (CBF) is an essential homeostatic mechanism. In contrast to the adult, neonatal CBF autoregulation operates within a narrow range of systemic blood pressures, and is pressure passive beyond this range. Nitric oxide (NO), one of the biochemical mediators involved in CBF autoregulation, is a potent vasorelaxant whose production is catalyzed by at least three distinct NO synthases (NOS). In order to understand the molecular basis of CBF in the newborn, we examined the expression and distribution of 3 NOS isoforms (bNOS, iNOS, ecNOS) in various sections of the brain and microvasculature of newborn (45 day old) and juvenile (6 week old) pigs (n=3). Western blots of equal amounts of total protein lysates probed with antibodies specific to each NOS were scanned by laser densitometer and revealed different patterns of expression among the tissues and differences between newborn and juvenile. The newborn brain cortex expressed 5fold higher levels of bNOS and 10fold lower levels of ecNOS than in the juvenile. iNOS levels were unchanged. Neonatal cerebral microvessels showed 3fold lower levels of bNOS and approximately equal levels of ecNOS in cerebral microvessels from juvenile pigs. We conclude that there is a net deficiency of these NOS isoforms in the immature brain and microvasculature of the newborn piglet and that the differences in expression leads to immature control of NO production contributing to altered neonatal CBF autoregulation.

Control of Nitric Oxide Production by Transforming Growth Factor-β1: Mechanistic Insights and Potential Relevance to Human Disease

Studies on the multifunctional nature of the transforming growth factor-β (TGF-β) family of cytokines and the enzyme nitric oxide synthase (NOS) have suggested that they mediate a wide variety of vital processes in evolutionarily divergent organisms. Numerous mechanistic studies have investigated the consequences of the regulation of NO by the TGF-β's for mammalian physiology. Studies with several cell typesin vitroindicate that TGF-β1 negatively controls the expression of the enzyme responsible for the prolonged production of large amounts NO, the inducible nitric oxide synthase (NOS2; iNOS), by reducing the expression and activity of NOS2 at multiple levels. Recent studies with TGF-β1 null mice or mice which overexpress TGF-β1 suggest that this cytokine may be a primary negative regulator of NOS2in vivo.The interaction between NOS2 and TGF-β1 may represent a central homeostatic mechanism in mammalian physiology with implications for a variety of human diseases.

Circulating levels of nitric oxide in fertile women in relation to the menstrual cycle

To investigate nitric oxide (NO) production in the two phases of normal menstrual cycle. Prospective clinical study. Normal human volunteers in an academic research environment. Fifteen normally cycling women. Follicle growth monitoring by ultrasound, serum, and plasma sampling in the midfollicular phase, at ovulation, and in the midsecretory phase. Plasma concentration of NO stable oxidation products and serum concentrations of E2 and P. Plasma concentration of NO metabolites resulted higher in the follicular phase with respect to the secretory phase and peaked at midcycle. The results strongly support the existence of an E2 control of NO production and release. The significant reduction in NO metabolites observed in the secretory phase suggests a possible opposing action of P on either NO production or release.

Control of nitric oxide production by endogenous TGF-β1 and systemic nitric oxide in retinal pigment epithelial cells and peritoneal macrophages

Both in vivo and in vitro experiments demonstrate that transforming growth factor-beta1 (TGF-beta1) suppresses expression of the inducible form of nitric oxide synthase (iNOS). In this study, we examined the effects of exogenous and endogenous TGF-beta1 on retinal pigment epithelial (RPE) cells and resident peritoneal macrophages ex vivo using cells from TGF-beta1 null (TGF-beta1-/-) mice or age-matched wild-type (TGF-beta1+/+) or heterozygous (TGF-beta1+/-) littermates. RPE cells from both TGF-beta1-/- mice and TGF-beta1+/+ littermates produced NO and were immunocytochemically positive for iNOS protein only following treatment with interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS); however, RPE cells from TGF-beta1-/- mice produced 40% more NO than cells from TGF-beta1+/+ mice. In contrast, resident peritoneal macrophages from both TGF-beta1+/+ and TGF-beta1-/- mice expressed iNOS protein without stimulation and in the absence of detectable production of NO. The expression of iNOS was increased by treatment with IFN-gamma, resulting in detectable levels of NO. Macrophages from TGF-beta1+/+ mice appeared to produce NO in a manner inversely proportional to the serum content of NO2- and NO3- of the mice from which the cells were obtained; no such correlation existed in TGF-beta1+/- or TGF-beta1-/- mice. Treatment of RPE cells or macrophages from both TGF-beta1+/+ and TGF-beta1-/- mice with exogenous TGF-beta1 decreased both iNOS protein and NO production. These findings demonstrate a novel role of endogenous TGF-beta1 in coupling systemic NO production to the production of NO by macrophages, and demonstrate that endogenous and exogenous TGF-beta1 can act differently to suppress NO production.

Modulation of neuroeffector transmission by endogenous nitric oxide: a role for acetylcholine receptor-activated nitric oxide formation, as indicated by measurements of nitric oxide/ nitrite release

Nitric oxide (NO) synthase inhibitors enhanced nerve-mediated contractile responses in guinea pig ileum longitudinal muscle, likely via a prejunctional effect on substance P-like neuroeffector transmission. Supporting a modulatory role for NO, application of NO through administration of acid sodium nitrite evoked marked inhibitory effects on responses to transmural nerve stimulation. Substance P-like responses to nerve stimulation were abolished by substance P receptor antagonists and were enhanced by atropine, indicating a cholinergic influence on substance P-like neuroeffector transmission. Since acetylcholine can evoke release of NO from endothelium, the possible role of acetylcholine in NO release in ileum was examined. The release of NO/nitrite, determined by chemiluminescence, was inhibited by N G-monomethyl-L-arginine (L-NMMA), by calcium removal, by tetrodotoxin or by atropine, indicating a nerve-mediated control of NO production. A basis for the NO release is likely to be spontaneous neuronal activity, where release of acetylcholine, with subsequent muscarinic receptor activation, contributes to stimulation of NO formation.