Nitroxyl and its anion in aqueous solutions: spin states,protic equilibria,and reactivities toward oxygen and nitric oxide

The thermodynamic properties of aqueous nitroxyl (HNO) and its anion (NO(-)) have been revised to show that the ground state of NO(-) is triplet and that HNO in its singlet ground state has much lower acidity, pKa((1)HNO/(3)NO(-)) approximately 11.4, than previously believed. These conclusions are in accord with the observed large differences between (1)HNO and (3)NO(-) in their reactivities toward O(2) and NO. Laser flash photolysis was used to generate (1)HNO and (3)NO(-) by photochemical cleavage of trioxodinitrate (Angeli's anion). The spin-allowed addition of (3)O(2) to (3)NO(-) produced peroxynitrite with nearly diffusion-controlled rate (k = 2.7 x 10(9) M(-1) x s(-1)). In contrast, the spin-forbidden addition of (3)O(2) to (1)HNO was not detected (k < 3 x 10(5) M(-1) x s(-1)). Both (1)HNO and (3)NO(-) reacted sequentially with two NO to generate N(3)O as a long-lived intermediate; the rate laws of N(3)O formation were linear in concentrations of NO and (1)HNO (k = 5.8 x 10(6) M(-1) x s(-1)) or NO and (3)NO(-) (k = 2.3 x 10(9) M(-1) x s(-1)). Catalysis by the hydroxide ion was observed for the reactions of (1)HNO with both O(2) and NO. This effect is explicable by a spin-forbidden deprotonation by OH(-) (k = 4.9 x 10(4) M(-1) x s(-1)) of the relatively unreactive (1)HNO into the extremely reactive (3)NO(-). Dimerization of (1)HNO to produce N(2)O occurred much more slowly (k = 8 x 10(6) M(-1) x s(-1)) than previously suggested. The implications of these results for evaluating the biological roles of nitroxyl are discussed.     

The decomposition of peroxynitrite to nitroxyl anion (NO-) and singlet oxygen in aqueous solution

The mechanism of decomposition of peroxynitrite (OONO(-)) in aqueous sodium phosphate buffer solution at neutral pH was investigated. The OONO(-) was synthesized by directly reacting nitric oxide with superoxide anion at pH 13. The hypothesis was explored that OONO(-), after protonation at pH 7.0 to HOONO, decomposes into (1)O(2) and HNO according to a spin-conserved unimolecular mechanism. Small aliquots of the concentrated alkaline OONO(-) solution were added to a buffer solution (final pH 7.0-7.2), and the formation of (1)O(2) and NO(-) in high yields was observed. The (1)O(2) generated was trapped as the transannular peroxide (DPAO(2)) of 9, 10-diphenylanthracene (DPA) dissolved in carbon tetrachloride. The nitroxyl anion (NO-) formed from HNO (pKa 4.5) was trapped as nitrosylhemoglobin (HbNO) in an aqueous methemoglobin (MetHb) solution. In the presence of 25 mM sodium bicarbonate, which is known to accelerate the rate of decomposition of OONO(-), the amount of singlet oxygen trapped was reduced by a factor of approximately 2 whereas the yield of trapping of NO(-) by methemoglobin remained unaffected. Because NO(3)(-) is known to be the ultimate decomposition product of OONO(-), these results suggest that the nitrate anion is not formed by a direct isomerization of OONO(-), but by an indirect route originating from NO(-).     

On the acidity and reactivity of HNO in aqueous solution and biological systems

The gas phase and aqueous thermochemistry and reactivity of nitroxyl (nitrosyl hydride, HNO) were elucidated with multiconfigurational self-consistent field and hybrid density functional theory calculations and continuum solvation methods. The pK(a) of HNO is predicted to be 7.2 +/- 1.0, considerably different from the value of 4.7 reported from pulse radiolysis experiments. The ground-state triplet nature of NO(-) affects the rates of acid-base chemistry of the HNO/NO(-) couple. HNO is highly reactive toward dimerization and addition of soft nucleophiles but is predicted to undergo negligible hydration (K(eq) = 6.9 x 10(-5)). HNO is predicted to exist as a discrete species in solution and is a viable participant in the chemical biology of nitric oxide and derivatives.     

Diatoms respire nitrate to survive dark and anoxic conditions

Diatoms survive in dark, anoxic sediment layers for months to decades. Our investigation reveals a correlation between the dark survival potential of marine diatoms and their ability to accumulate NO(3)(-) intracellularly. Axenic strains of benthic and pelagic diatoms that stored 11-274 mM NO(3)(-) in their cells survived for 6-28 wk. After sudden shifts to dark, anoxic conditions, the benthic diatom Amphora coffeaeformis consumed 84-87% of its intracellular NO(3)(-) pool within 1 d. A stable-isotope labeling experiment proved that (15)NO(3)(-) consumption was accompanied by the production and release of (15)NH(4)(+), indicating dissimilatory nitrate reduction to ammonium (DNRA). DNRA is an anaerobic respiration process that is known mainly from prokaryotic organisms, and here shown as dissimilatory nitrate reduction pathway used by a eukaryotic phototroph. Similar to large sulfur bacteria and benthic foraminifera, diatoms may respire intracellular NO(3)(-) in sediment layers without O(2) and NO(3)(-). The rapid depletion of the intracellular NO(3)(-) storage, however, implies that diatoms use DNRA to enter a resting stage for long-term survival. Assuming that pelagic diatoms are also capable of DNRA, senescing diatoms that sink through oxygen-deficient water layers may be a significant NH(4)(+) source for anammox, the prevalent nitrogen loss pathway of oceanic oxygen minimum zones.     

Sialin (SLC17A5) functions as a nitrate transporter in the plasma membrane

In vivo recycling of nitrate (NO(3)(-)) and nitrite (NO(2)(-)) is an important alternative pathway for the generation of nitric oxide (NO) and maintenance of systemic nitrate-nitrite-NO balance. More than 25% of the circulating NO(3)(-) is actively removed and secreted by salivary glands. Oral commensal bacteria convert salivary NO(3)(-) to NO(2)(-), which enters circulation and leads to NO generation. The transporters for NO(3)(-) in salivary glands have not yet been identified. Here we report that sialin (SLC17A5), mutations in which cause Salla disease and infantile sialic acid storage disorder (ISSD), functions as an electrogenic 2NO(3)(-)/H(+) cotransporter in the plasma membrane of salivary gland acinar cells. We have identified an extracellular pH-dependent anion current that is carried by NO(3)(-) or sialic acid (SA), but not by Br(-), and is accompanied by intracellular acidification. Both responses were reduced by knockdown of sialin expression and increased by the plasma membrane-targeted sialin mutant (L22A-L23A). Fibroblasts from patients with ISSD displayed reduced SA- and NO(3)(-)-induced currents compared with healthy controls. Furthermore, expression of disease-associated sialin mutants in fibroblasts and salivary gland cells suppressed the H(+)-dependent NO(3)(-) conductance. Importantly, adenovirus-dependent expression of the sialinH183R mutant in vivo in pig salivary glands decreased NO(3)(-) secretion in saliva after intake of a NO(3)(-)-rich diet. Taken together, these data demonstrate that sialin mediates nitrate influx into salivary gland and other cell types. We suggest that the 2NO(3)(-)/H(+) transport function of sialin in salivary glands can contribute significantly to clearance of serum nitrate, as well as nitrate recycling and physiological nitrite-NO homeostasis.     

Partitioning of atmospherically relevant ions between bulk water and the water/vapor interface

Recently, surface-sensitive spectroscopy data and molecular dynamics simulations have generated intense interest in the distribution of electrolyte ions between bulk water and the air/water interface. A partitioning model for cations and anions developed for biopolymer surface is extended here to interpret the effects of selected acids, bases, and salts on the surface tension of water. Data for electrolytes were analyzed by using a lower-bound value for the number of water molecules in the surface region [0.2 H(2)O A(-2) (approximately two layers of water)], obtained by assuming that both Na(+) and SO(4)(2-) (i.e., Na(2)SO(4)) are fully excluded from this region. Surface-bulk partition coefficients of atmospherically relevant anions and the proton are determined. Notably, we find that H(+) is most strongly surface-accumulated, I(-) is modestly accumulated, NO(3)(-) is evenly distributed, and OH(-) is weakly excluded.     

Plasma nitric oxide is associated with the occurrence of moderate to severe acute graft-versus-host disease in haemopoietic stem cell transplant recipients

BACKGROUND AND OBJECTIVES: Nitric oxide (NO) has been implicated as one of the mediators of acute graft-versus-host disease (GVHD) but reports on its measurement during haemopoietic stem cell transplantation (HSCT) in human are scarce. The present study was conducted to measure the plasma NO in HSCT recipients in order to delineate its relationships with acute GVHD. DESIGN AND METHODS: Thirty-nine randomly selected patients undergoing HSCT were recruited. Thirty-one patients received allogeneic transplants (ALLO) from HLA-identical siblings (n=20), haploidentical parent (n=1) and matched unrelated donors (n=10). Eight patients received autologous (AUTO) HSCT. Plasma levels of nitrite/nitrate (NO(2)(-)/NO(3)(-)), the end-product of NO, were measured by chemiluminescence and the results were correlated with the occurrence and severity of acute GVHD. RESULTS: Baseline NO(2)(-)/NO(3)(-) levels before HSCT were similar in the ALLO and AUTO patients (17.4 vs 21.1 microL, p>0.05). Significant increases in plasma NO(2)(-)/NO(3)(-) (> 2 times the baseline level) were found in all 13 patients with acute GVHD > or = grade 2, in 15 out of 18 patients with acute GVHD grade < or = 1 and 3 out of 8 patients receiving autologous HSCT. The increase in NO(2)(-)/NO(3)(-) among the three groups of patients was significantly different (135.5 vs 56.3 vs 36.6 micromol/L, p < 0.001). The average NO production, calculated as the area under the curve, was also significantly differently among the three groups of patients (44.5 vs 30.0 vs 23.8 micromol/L, p < 0.001). INTERPRETATION AND CONCLUSIONS: Plasma NO in HSCT recipients is quantitatively associated with the occurrence of acute GVHD and its role remains to be determined.     

Nitroxyl anion donor, Angeli's salt, does not develop tolerance in rat isolated aortae

The nitroxyl anion (HNO) is emerging as a novel regulator of cardiovascular function with therapeutic potential in the treatment of diseases such as heart failure. It remains unknown whether tolerance develops to HNO donors, a limitation of currently used nitrovasodilators. The susceptibility of the HNO donor, Angeli's salt (AS), to the development of vascular tolerance was compared with the NO donors, glyceryl trinitrate (GTN) and diethylamine/NONOate (DEA/NO) in rat isolated aortae. Vasorelaxation to AS was attenuated (P<0.01) by the HNO scavenger l-cysteine, whereas the sensitivity to GTN and DEA/NO was decreased (P<0.01) by the NO. scavenger carboxy-[2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidozoline-1-oxy-3-oxide]. The soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one impaired responses to GTN>or=AS>DEA/NO. Pretreatment with 10, 30, and 100 micromol/L of GTN for 60 minutes induced a 4- (P<0.05), 13- (P<0.01), and 48-fold (P<0.01) decrease in sensitivity to GTN, demonstrating tolerance development. In contrast, pretreatment with AS or DEA/NO (10, 30, and 100 micromol/L) did not alter their subsequent vasorelaxation. All of the nitrovasodilators (30 micromol/L) displayed a similar time course of vasorelaxation and cGMP accumulation over a 60-minute period. Unlike vasorelaxation, the magnitude of peak cGMP accumulation differed substantially: DEA/NO>AS>GTN. GTN did not induce cross-tolerance to either AS or DEA/NO. In contrast, pre-exposure to DEA/NO, but not AS, caused a concentration-dependent attenuation (P<0.01) of GTN-mediated relaxation, which was negated by the protein kinase G inhibitor guanosine 3',5'-cyclic monophosphorothioate, 8-(4-chlorophenylthio)-,Rp-isomer, triethylammonium salt. In conclusion, vascular tolerance does not develop to HNO, nor does cross-tolerance between HNO and GTN occur. Thus, HNO donors may have therapeutic advantages over traditional nitrovasodilators.     

Increased basal levels of plasma nitric oxide in Type 2 diabetic subjects. Relationship to microvascular complications

To assess the underlying mechanisms of decreased endothelial function and advanced vascular complications in patients with Type 2 diabetes, we determined basal levels of plasma nitric oxide (NO(x): NO(2)(-) and NO(3)(-)) using a newly developed high-performance liquid chromatography (HPLC)-Griess method in hospitalized 129 diabetic and 76 nondiabetic subjects, and examined their clinical characteristics. Serum lipid peroxide and advanced glycation end products (AGEs) as markers of oxidative stress were also measured, and intima-media thickness (IMT) of the carotid artery was evaluated as a marker of atherosclerosis. In diabetic subjects, microvascular complications were newly evaluated during their admission. There were no differences in age or sex between the diabetic and nondiabetic subjects. Although there was no difference in basal plasma NO(2)(-) levels between the two groups, the basal levels of plasma NO(3)(-) in diabetic subjects were significantly higher than those in nondiabetic subjects. Plasma NO(x) levels in neither diabetic nor nondiabetic subjects correlated with serum lipids, HbA1c, or IMT. In diabetic subjects, plasma NO(3)(-) levels were related not only to the presence of hypertension but also to advanced microvascular complications. Moreover, plasma NO(3)(-) levels were positively correlated with both serum lipid peroxide and AGEs. Multiple regression analysis revealed that serum AGEs level was strongly associated with plasma NO(3)(-) level. Thus, the findings are consistent with the hypothesis that decreased endothelium-dependent vasodilation in diabetic subjects is associated with the impaired action of NO secondary to its inactivation resulting from increased oxidative stress, rather than decreased NO production from vascular endothelium, and that abnormal NO metabolism is related to advanced diabetic microvascular complications.     

Positive inotropic and lusitropic effects of HNO/NO- in failing hearts: independence from beta-adrenergic signaling

Nitroxyl anion (HNONO(-)), the one-electron reduced form of nitric oxide (NO), induces positive cardiac inotropy and selective venodilation in the normal in vivo circulation. Here we tested whether HNO/NO(-) augments systolic and diastolic function of failing hearts, and whether contrary to NO/nitrates such modulation enhances rather than blunts beta-adrenergic stimulation and is accompanied by increased plasma calcitonin gene-related peptide (CGRP). HNO/NO(-) generated by Angelis' salt (AS) was infused (10 microg/kg per min, i.v.) to conscious dogs with cardiac failure induced by chronic tachycardia pacing. AS nearly doubled contractility, enhanced relaxation, and lowered cardiac preload and afterload (all P < 0.001) without altering plasma cGMP. This contrasted to modest systolic depression induced by an NO donor diethylamine(DEA)NO or nitroglycerin (NTG). Cardiotropic changes from AS were similar in failing hearts as in controls despite depressed beta-adrenergic and calcium signaling in the former. Inotropic effects of AS were additive to dobutamine, whereas DEA/NO blunted beta-stimulation and NTG was neutral. Administration of propranolol to nonfailing hearts fully blocked isoproterenol stimulation but had minimal effect on AS inotropy and enhanced lusitropy. Arterial plasma CGRP rose 3-fold with AS but was unaltered by DEA/NO or NTG, supporting a proposed role of this peptide to HNO/NO(-) cardiotropic action. Thus, HNO/NO(-) has positive inotropic and lusitropic action, which unlike NO/nitrates is independent and additive to beta-adrenergic stimulation and stimulates CGRP release. This suggests potential of HNO/NO(-) donors for the treatment of heart failure.     

Reduction of nitrite to nitric oxide during ischemia protects against myocardial ischemia-reperfusion damage

Nitric oxide (NO.) is thought to protect against the damaging effects of myocardial ischemia-reperfusion injury, whereas xanthine oxidoreductase (XOR) normally causes damage through the generation of reactive oxygen species. In the heart, inorganic nitrite (NO(2)(-)) has the potential to act as an endogenous store of NO., liberated specifically during ischemia. Using a detection method that we developed, we report that under ischemic conditions both rat and human homogenized myocardium and the isolated perfused rat heart (Langendorff preparation) generate NO. from NO(2)(-) in a reaction that depends on XOR activity. Functional studies of rat hearts in the Langendorff apparatus showed that nitrite (10 and 100 microM) reduced infarct size from 47.3 +/- 2.8% (mean percent of control +/- SEM) to 17.9 +/- 4.2% and 17.4 +/- 1.0%, respectively (P < 0.001), and was associated with comparable improvements in recovery of left ventricular function. This protective effect was completely blocked by the NO. scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO). In summary, the generation of NO. from NO(2)(-), by XOR, protects the myocardium from ischemia-reperfusion injury. Hence, if XOR is presented with NO(2)(-) as an alternative substrate, the resultant effects of its activity may be protective, by means of its production of NO. , rather than damaging.     

Effects of Cl(-) substitution on electrophysiological properties, Ca(2+) influx and prolactin secretion of rat lactotropes in vitro

In this study, we compared the effects of different chloride (Cl(-)) substitutes - methane sulfonate (CH(3)SO(-)(3)), bromide (Br(-)), nitrate (NO(-)(3)), thiocyanate (SCN(-)) and perchlorate (ClO(-)(4)) - on the secretory activity and calcium current activation of rat lactotropes in primary culture. We observed that CH(3)SO(-)(3) decreased basal prolactin (PRL) secretion. Br(-) had no effect, whereas the more lyotropic anions, such as NO(-3), SCN(-) and C1O(-4), increased basal PRL secretion. The latter three substitutes induced a significant shift in the voltage dependence of T-type calcium channel activation towards hyperpolarized values. However, this shift alone cannot explain the increase in secretion. Anion permeability studies also demonstrated that the organic anion CH(3)SO(-3) was less permeant than Cl(-), whereas monovalent inorganic anions were more permeant, with the following anion permeability sequence: SCN(-) > ClO(-4) > NO(-3) > Br(-). In conclusion, deprivation of Cl(-) ions has converse consequences on basal and induced secretion; permeating anions result in a transient increase in intracellular Ca(2+) ions. This process involves voltage-dependent Ca(2+) channels. We propose that an alteration in intracellular anion concentrations may influence the activation of internal effectors such as G proteins or channel proteins and, therefore, interfere with exocytosis. These effects are correlated with an external action of lyotropic anions, particularly NO(-3), ClO(-4) and SCN(-), on the gating properties of T-type calcium channels, probably through changes in cell surface charges. The results demonstrate the modulatory effect of anions on the secretory activity of rat lactotropes and underline the specific role played by chloride in stimulus-secretion coupling.     

支气管哮喘患者诱导痰炎性指标与气道反应性的关系

目的分析诱导痰中硝酸盐/亚硝酸盐(NO_3~-/NO_2~-)浓度和嗜酸粒细胞(EOS)计数与气道反应性的相关性,探讨上述指标对评估病情、调整支气管哮喘(简称哮喘)治疗方案的指导意义。方法 2003年2月至2004年6月华西医院哮喘门诊收集轻至中度非急性发作期哮喘患者35例,其中轻度9例,中度26例;经吸入糖皮质激素(ICS)和长效β_2受体激动剂(LABA)联合治疗1年,随访期间记录哮喘症状积分,测定气道反应性[以比气道传导率下降35%所需的激发剂浓度(PC_(35)sGaw)表示]、诱导痰中 EOS 计数和 NO_3~-/NO_2~-浓度。15名健康志愿者作为对照组,测定其诱导痰 EOS,NO_3~-/NO_2~-浓度。结果 35例患者中26例完成1年或以上的治疗和随访。26例患者 PC_(35)sGaw 治疗前为0.08g/L,治疗3个月为1.40g/L,随后7个月维持在2.64 g/L 水平。在治疗第3个月时,诱导痰NO_3~-/NO_2~-水平从治疗前的(734±72)×10~(-3)g/L 下降至(230±41)×10~(-3)g/L,差异有统计学意义(q=6.26,P<0.05),治疗7个月时 NO_3~-/NO_2~-水平降至(137±27)×10~(-3)g/L,与健康对照[(136±20)×10~(-3)g/L]比较差异无统计学意义(q=3.77,P>0.05)。治疗3个月后 EOS 计数为0.014±0.007,与健康对照(0.016±0.008)比较差异无统计学意义(q=2.94,P>0.05);随访期间任一时点PC_(35)sGaw 与 EOS 计数无相关(r_1=0.237,r_2=0.536,r_3=0.675,P 均>0.05)。5个月内 PC_(35)sGaw 与NO_3~-/NO_2~-水平呈负相关(r_1=-0.872,r_2=-0.653,r_3=-0.639,r_4=-0.656,P 均<0.05)。结论 PC_(35)sGaw 与诱导痰 NO_3~-/NO_2~-是反映哮喘气道炎症较为敏感的指标,可作为评价疗效及调整治疗方案的指标。     

Stimulation by nizatidine, a histamine H(2)-receptor antagonist, of duodenal HCO(3)(-)secretion in rats:relation to anti-cholinesterase activity

AIM:To examine whether nizatidine stimulates duodenal HCO(3)(-) secretion in rats by inhibiting AChE activity.METHODS:Under pentobarbital anesthesia, a proximal duodenal loop was perfused with saline, and the HCO(3)(-) secretion was measured at pH 7.0 using a pH-stat method and by adding 10mM HCl. Nizatidine, neostigmine, carbachol or famotidine was administered i.v. as a single injection.RESULTS:Intravenous administration of nizatidine (3-30mg/kg) dose-dependently increased duodenal HCO(3)(-) secretion, and the effect at 10mg/kg was equivalent to that obtained by carbachol at 0.01mg/kg. This nizatidine action was observed at the same dose range that inhibited acid secretion and enhanced gastric motility, mimicked by i.v. injection of neostigmine(0.03mg/kg), and significantly attenuated by bilateral vagotomy and prior s.c. administration of atropine but not by indomethacin, a cyclooxygenase inhibitor, or NG-nitro-L-arginine methyl ester, a NO synthase inhibitor. The HCO(3)(-) secretory response to acetylcholine (0.001mg/kg) was significantly potentiated by the concurrent administration of nizatidine (3mg/kg,i.v.). The IC(50) of nizatidine for AChE of rat erythrocytes was 1.4·10(-6) M, about 12 times higher than that of neostigmine. Neither famotidine (> 10(-3) M, 30mg/kg, i.v.) nor cisapride (> 10(-3) M,3mg/kg, i.v.) had any influence on AChE activity or duodenal HCO(3)(-) secretion. Duodenal damage induced by acid perfusion (100mM HCl for 4h) in the presence of indomethacin was significantly prevented by nizatidine and neostigmine, at the doses that increased the HCO(3)(-)secretion.CONCLUSION:Nizatidine stimulates duodenal HCO(3)(-) secretion, in both vagal dependent and atropine sensitive manners, and the action is associated with the anti-AChE activity of this agent.     

Evidence against a role for NADPH oxidase modulating hepatic vascular tone in cirrhosis

BACKGROUND & AIMS: Increased hepatic vascular resistance in cirrhosis is in part due to reduced nitric oxide (NO) bioavailability. This is related to insufficient NO synthesis from endothelial nitric oxide synthase and to enhanced NO scavenging by superoxide radicals (O(2)(-)). Nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase is an important source of O(2)(-) that increases vascular tone in different cardiovascular disorders. Thus, our aims were to study the molecular and biochemical state of NADPH-oxidase in cirrhotic livers and to investigate its possible role in modulating hepatic vascular tone in cirrhosis. METHODS: NADPH-oxidase expression and enzymatic activity were determined in control (n = 8) and CCl(4)-cirrhotic (n = 8) rat livers. Additional control (n = 6) and CCl(4)-cirrhotic (n = 10) rats were treated with apocynin (a selective NADPH-oxidase inhibitor) or its vehicle. Mean arterial pressure, portal pressure, and superior mesenteric arterial blood flow were measured in vivo. Moreover, hepatic endothelial function was evaluated in isolated and perfused rat livers by dose-response curves to acetylcholine. In addition, in 6 control and 6 cirrhotic human livers NADPH-oxidase activity and expression were evaluated. RESULTS: Rat cirrhotic livers had no increased NADPH-oxidase protein expression or activity in relation to control livers. NADPH-oxidase inhibition did not modify splanchnic or systemic hemodynamics in control or cirrhotic rats and did not improve the impaired endothelial-dependent vasodilatory response to acetylcholine of cirrhotic livers. Human cirrhotic livers also did not exhibit increased NADPH-oxidase expression or activity. CONCLUSIONS: Our study shows that NADPH-oxidase activity is decreased in the cirrhotic livers and therefore cannot explain increased hepatic O(2)(-), endothelial dysfunction, and increased vascular tone in cirrhotic livers.     

Impact of anthropogenic atmospheric nitrogen and sulfur deposition on ocean acidification and the inorganic carbon system

Fossil fuel combustion and agriculture result in atmospheric deposition of 0.8 Tmol/yr reactive sulfur and 2.7 Tmol/yr nitrogen to the coastal and open ocean near major source regions in North America, Europe, and South and East Asia. Atmospheric inputs of dissociation products of strong acids (HNO(3) and H2SO(4)) and bases (NH(3)) alter surface seawater alkalinity, pH, and inorganic carbon storage. We quantify the biogeochemical impacts by using atmosphere and ocean models. The direct acid/base flux to the ocean is predominately acidic (reducing total alkalinity) in the temperate Northern Hemisphere and alkaline in the tropics because of ammonia inputs. However, because most of the excess ammonia is nitrified to nitrate (NO(3)(-)) in the upper ocean, the effective net atmospheric input is acidic almost everywhere. The decrease in surface alkalinity drives a net air-sea efflux of CO(2), reducing surface dissolved inorganic carbon (DIC); the alkalinity and DIC changes mostly offset each other, and the decline in surface pH is small. Additional impacts arise from nitrogen fertilization, leading to elevated primary production and biological DIC drawdown that reverses in some places the sign of the surface pH and air-sea CO(2) flux perturbations. On a global scale, the alterations in surface water chemistry from anthropogenic nitrogen and sulfur deposition are a few percent of the acidification and DIC increases due to the oceanic uptake of anthropogenic CO(2). However, the impacts are more substantial in coastal waters, where the ecosystem responses to ocean acidification could have the most severe implications for mankind.     

Asymmetric dimethylarginine (ADMA) in the aqueous humor of diabetic patients

Asymmetric dimethylarginine (ADMA) is an endogenous NO synthase (NOS) inhibitor whose production is enhanced by oxidative stress. Recent studies have shown that ADMA may also directly stimulate the production of reactive oxygen species (ROS) by up-regulation of the renin-angiotensin system independently of NOS inhibition. In this study, to investigate the clinical association of ADMA with diabetic retinopathy, we evaluated the levels of ADMA and NO oxides (NO2- and NO3-) in serum and aqueous humor obtained during cataract surgery from non-diabetic subjects (n = 21) and diabetic patients (n = 17). We found that the ADMA existed in aqueous humor and its level was similar to that in serum. The ADMA levels in both serum and aqueous humor were higher in diabetic patients, especially those with severe retinopathy, than in the non-diabetic group (serum ADMA: 0.67 +/- 0.26 vs. 0.53 +/- 0.08 micromol/l, p<0.05; aqueous humor ADMA: 0.55 +/- 0.20 vs. 0.32 +/- 0.16 micromol/l, p<0.05). Also, the aqueous humor level of ADMA, but not the serum level, was correlated with HbA1c on analysis of all the patients (R = 0.33, p<0.05 by simple regression analysis). However, a correlation between the ADMA levels in serum and aqueous humor was not observed in either the non-diabetic group or the diabetic group. Furthermore, serum and aqueous humor levels of NOx did not differ between the two groups, and no correlation with ADMA levels was observed in either group. These results suggest that ROS production may be enhanced in the eyes of diabetics. Since ADMA may act to potentiate ROS production independently of its inhibition of NOS, further investigation is required to clarify the possible contribution of ADMA to the development or progression of retinopathy.     

Reactions of the NO redox forms NO+, *NO and HNO (protonated NO-) with the melatonin metabolite N1-acetyl-5-methoxykynuramine

The different NO redox forms, NO+, *NO and HNO (=protonated NO-), were compared for their capabilities of interacting with the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK), using NO+SbF6-, PAPA-NONOate and Angeli's salt as donors of the respective NO species. Particular attention was paid to stability and possible interconversions of the redox forms. *NO formation was followed by measuring the decolorization of 2-(trimethylammonio-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (TMA-PTIO), at different pH values, at which NO+ is, in aqueous solution, either highly unstable (pH 7.4) or relatively stable (pH 2.0). *NO donation by PAPA-NONOate, as indicated by TMA-PTIO decolorization, was similar at either pH and 3-acetamidomethyl-6-methoxycinnolinone (AMMC) was formed as the major product from AMK, at pH 7.4 more efficiently than at pH 2.0. At pH 2.0, TMA-PTIO decolorization by NO+SbF6- was much weaker than by PAPA-NONOate, but AMMC was produced at substantial rates, whereas neither TMA-PTIO decolorization nor AMMC formation was observed with the NO+ donor at pH 7.4. As NO+ is also stable in organic, especially aprotic solvents, NO+SbF6- was reacted with AMK in acetonitrile, ethanol, butanol, and ethyl acetate. In all these cases, AMMC was the only or major product. In ethyl acetate, N1-acetyl-5-methoxy-3-nitrokynuramine (AMNK) was also formed, presumably as a consequence of organic peroxides emerging in that solvent. Presence of tert-butylhydroperoxide in an ethanolic solution of NO+SbF6- and AMK also resulted in AMNK formation, in addition to AMMC and two red-fluoresecent, to date unknown products. However, hydrogen peroxide enhanced *NO-dependent AMMC production from AMK and also from N1-acetyl-N2-formyl-5-methoxykynuramine. HNO donation by Angeli's salt (Na2N2O3) also caused AMMC formation from AMK at pH 7.4, with a somewhat lower efficiency than PAPA-NONOate, but no AMNK nor any other product was detected. Therefore, all three NO congeners are, in principle, capable of nitrosating AMK and forming AMMC, but in biological material the reaction with NO+ is strongly limited by the extremely short life-time of this redox form.     

The reduction potential of nitric oxide (NO) and its importance to NO biochemistry

A potential of about -0.8 (+/-0.2) V (at 1 M versus normal hydrogen electrode) for the reduction of nitric oxide (NO) to its one-electron reduced species, nitroxyl anion (3NO-) has been determined by a combination of quantum mechanical calculations, cyclic voltammetry measurements, and chemical reduction experiments. This value is in accord with some, but not the most commonly accepted, previous electrochemical measurements involving NO. Reduction of NO to 1NO- is highly unfavorable, with a predicted reduction potential of about -1.7 (+/-0.2) V at 1 M versus normal hydrogen electrode. These results represent a substantial revision of the derived and widely cited values of +0.39 V and -0.35 V for the NO/3NO- and NO/1NO- couples, respectively, and provide support for previous measurements obtained by electrochemical and photoelectrochemical means. With such highly negative reduction potentials, NO is inert to reduction compared with physiological events that reduce molecular oxygen to superoxide. From these reduction potentials, the pKa of 3NO- has been reevaluated as 11.6 (+/-3.4). Thus, nitroxyl exists almost exclusively in its protonated form, HNO, under physiological conditions. The singlet state of nitroxyl anion, 1NO-, is physiologically inaccessible. The significance of these potentials to physiological and pathophysiological processes involving NO and O2 under reductive conditions is discussed.