Heat-activated sustaining nitric oxide release from zwitterionic diazeniumdiolate loaded in thermo-sensitive liposomes

Zwitterionic diazeniumdiolate, a nitric oxide precursor, was dissolved in basic buffer solution (pH = 9.0) and encapsulated in thermo-sensitive liposomes composed of phospholipids of different sensitive temperatures. The basic intra-liposomal environment dramatically delayed spontaneous NO release. When the liposomes were placed in physiological buffer solution and temperatures were increased to the sensitive temperatures of the phospholipids’ membranes, extra-liposomal protons started to influx to collapse the pH gradient and subsequently induce a significant NO release. Moreover, the presence of a stronger pH gradient when the liposomes were applied to a more acidic environment caused a higher proton influx driving force to trigger the influx of protons and, subsequently, NO release. In this work, we demonstrate that thermo-sensitive liposomes can be used to create a stable pH gradient in a nanoenvironment entrapping zwitterionic diazeniumdiolate for sustained and heat/acid-activated NO release.     

Release of nitric oxide from novel diazeniumdiolates monitored by laser magnetic resonance spectroscopy.

We describe a technique in which for the first time laser magnetic resonance spectroscopy (LMRS) is used online to monitor the release of nitric oxide from synthetic NO donors. LMRS is a spectroscopic method for selective, sensitive (to 1 ppb), and time-resolved NO gas detection in the far-infrared and midinfrared spectral regions. We used two partly novel sets of diazeniumdiolates, the first set derived from N,N-diethylamine (-->DEA-NO), piperidine ((-->PIPE-NO), 2-methylpiperidine (-->MEPIPE-NO), and 2-ethylpiperidine (-->EPIPE-NO) and the second set derived from 2-, 3-, and 4-piperidine carboxylic acids (-->PIPECO-NO, NIPECO-NO, ISONIPECO-NO). We monitored the acid-catalyzed NO liberation from these compounds as influenced by parameters such as pH, temperature, concentration, and molecular structure. PIPECO-NO turned out to be the fastest donor of the group. The 3- and 4-substituted isomer derivatives were only negligibly faster releasers than unsubstituted PIPE-NO, which on the other hand showed to be faster than the higher homologues MEPIPE-NO and EPIPE-NO. The results demonstrate that varying neighboring groups affect the functional diazeniumdiolate group differently. A vicinal carboxyl group increases and alkyl groups decrease the rate of NO release.     

L-精氨酸脂质体对慢性低氧高二氧化碳大鼠一氧化氮及其合酶基因表达的影响

目的:探讨L-精氨酸脂质体(L-Arg)对慢性低氧高二氧化碳大鼠一氧化氮代谢及内皮型一氧化氮合酶基因(ecNOSmRNA)表达的影响.方法:将40只健康雄性SD大鼠随机分为4组,正常对照组(NC组),低氧高二氧化碳4周组(HH组),低氧高二氧化碳加L-Arg4周组(HL组)和低氧高二氧化碳加L-Arg脂质体4周组(HP组).采用硝酸还原酶法测定血浆一氧化氮代谢产物(NOx-)含量,组织原位杂交、光镜和图像分析等方法观察肺细小动脉显微结构以及肺细小动脉ecNOSmRNA的表达变化.结果:①HH组mPAP和RV/(LV S)高于NC组,HP组均明显低于HL组与HH组;②HH组的血浆NO含量显著低于NC组(P＜0.01),HL组与HP组均明显高于HH组(P＜0.01);③HH组的肺细小动脉ecNOSmRNA的平均吸光度值低于NC组(P＜0.05);而HP组明显高于HH组和HL组(P＜0.01);④HP组的肺细小动脉管壁面积/管总面积比值(WA/TA)和中膜厚度(PAMT)均明显低于HH组(P＜0.01),且低于HL组(P＜0.05).结论:L-Arg脂质体较L-Arg有更明显的降低慢性低氧高二氧化碳大鼠肺动脉压和减轻肺血管重建的治疗作用,其机制可能与L-Arg脂质体促进L-Arg的跨膜转运有关.     

Effect of nitric oxide on the transport and release of oxygen in fetal blood

It is well known that nitric oxide (NO), the most important vasodilator agent, plays an important role in lowering vascular resistance in the human umbilical-placental circulation and that its deficiency is related to the pathogenesis of pre-eclamptic disorder. Besides it has recently been demonstrated that human hemoglobin (HbA) is able to transport nitric oxide, as S-nitrosohemoglobin (SNO-Hb), from the arterial to the venous blood. In the present study we examine the functional properties of the adult and fetal nitrosated hemoglobins to see if the double transport of oxygen and NO may influence the fetal oxygenation and the relation between maternal and fetal blood. Our results show that S-nitrosation significantly increases the oxygen affinity of the adult Hb (HbA) with respect to native protein (no-nitrosated) while the functional properties of HbF are less influenced. The oxygen affinity modification, found for SNO-HbA, was ascribed to the nitrosation of cysteine beta 93: really, the same residue is also present in the gamma chains of fetal hemoglobin, while the increase of affinity is less evidenced; hence, it is probable that the 39 aminoacidic substitutions between beta and gamma chains allay the effects of S-nitrosation. As regards the physiological modulators (protons, chloride ions, 2,3-diphosphoglyceric acid, and temperature), they influence the oxygen affinity of the two hemoglobins S-nitrosated, in equal mode with respect to the native forms determining the same variation on the oxygen affinity. Hence, our results evidence the fact that the NO release by SNO-HbA "in vivo" would be limited to regions of extremely low oxygen tension (such as hypoxic regions), while in fetus, SNO-HbF would unload nitric oxide and oxygen at pressure values close to normal.     

Effect of desflurane-induced preconditioning following ischemia-reperfusion on nitric oxide release in rabbits

Nitric oxide (NO) is the mediator of ischemic preconditioning against myocardial infarction. Desflurane produces anesthetic preconditioning to protect the myocardium against infarction. In the model of myocardial ischemia-reperfusion injury in rabbits, we evaluated desflurane-induced ischemic preconditioning and studied its mechanism of NO synthesis. Thirty-two male adult New Zealand white rabbits were anesthetized with intravenous (IV) 30 mg/kg pentobarbital followed by 5 mg/kg/hr infusion. All rabbits were subjected to 30 minutes (min) long lasting left anterior descending coronary artery (LAD) occlusion and three hours (hr) of subsequent reperfusion. Before LAD occlusion, the rabbits were randomly allocated into four groups for preconditioning treatment (eight for each group). The control group did not receive any preconditioning treatment. The desflurane group received inhaled desflurane 1.0 MAC (minimal end-tidal alveolar concentration) for 30 min that was followed by a 15 min washout period. The L-NAME-desflurane group received L-NAME (NG-nitro-L-arginine methyl ester; non-selective Nitric Oxide Synthetase (NOS) inhibitor) 1 mg/kg IV 15 min before 1.0 MAC inhaled desflurane for 30 min. The L-NAME group received L-NAME 1 mg/kg IV. Infarct volume, ventricular arrhythmia, plasma lactate dehydrogenase (LDH), creatine kinase (CK) activity and myocardial perfusion were recorded simultaneously. We have found that hemodynamic values of the coronary blood flow before, during, and after LAD occlusion were not significantly different among these four groups. For the myocardial ischemia-reperfusion injury animals, the infarction size (mean +/- SEM) in the desflurane group was significantly reduced to 18 +/- 3% in the area at risk as compared with 42 +/- 7% in the control group, 35 +/- 6 in the L-NAME group, and 34 +/- 4% in the L-NAME-desflurane group. The plasma LDH, CK levels, and duration of ventricular arrhythmia were also significantly decreased in the desflurane group during ischemia-reperfusion injury. Our results indicate that desflurane is an anesthetic preconditioning agent, which could protect the myocardium against the ischemia-reperfusion injury. This beneficial effect of desflurane on the ischemic preconditioning is probably through NO release since L-NAME abrogates the desflurane preconditioning effect.     

Effect of heterogeneous ventilation and nitric oxide production on exhaled nitric oxide profiles.

Elevated exhaled nitric oxide (NO) in the breath of asthmatic subjects is thought to be a noninvasive marker of lung inflammation. Asthma is also characterized by heterogeneous bronchoconstriction and inflammation, which impact the spatial distribution of ventilation in the lungs. Since exhaled NO arises from both airway and alveolar regions, and its level in exhaled breath depends strongly on flow, spatial heterogeneity in flow patterns and NO production may significantly affect the exhaled NO signal. To investigate the effect of these factors on exhaled NO profiles, we developed a multicompartment mathematical model of NO exchange using a trumpet-shaped central airway segment that bifurcates into two similarly shaped peripheral airway segments, each of which empties into an alveolar compartment. Heterogeneity in flow alone has only a minimal impact on the exhaled NO profile. In contrast, placing 70% of the total airway NO production in the central compartment or the distal poorly ventilated compartment can significantly increase (35%) or decrease (-10%) the plateau concentration, respectively. Reduced ventilation of the peripheral and acinar regions of the lungs with concomitant elevated NO production delays the rise of NO during exhalation, resulting in a positive phase III slope and reduced plateau concentration (-11%). These features compare favorably with experimentally observed profiles in exercise-induced asthma and cannot be simulated with single-path models. We conclude that variability in ventilation and NO production in asthmatic subjects impacts the shape of the exhaled NO profile and thus impacts the physiological interpretation.     

Androgen-dependent nitric oxide release in rat penis correlates with levels of constitutive nitric oxide synthase isoenzymes.

Androgens are known to influence penile erection and nitric oxide synthase (NOS) activity in cavernosal tissue homogenates. The present study was an assessment of the effects of castration and androgen replacement on the in vivo release of nitric oxide (NO), and of the simultaneously recorded intracavernosal pressure (ICP) changes elicited by electrostimulation of the cavernosal nerves (SCN) in the anesthetized rat. The extracellular levels of NO in the corpora were monitored electrochemically using porphyrin microsensors. The content of NOS isoenzymes in corporal homogenates was determined by immunoblotting. The responses of castrated rats with or without testosterone (T) implants were compared to those of intact animals. Castration virtually abolished both the NO and the ICP responses to SCN. There was a concomitant significant decrease in the content of both the neuronal (nNOS) and the endothelial (eNOS) isoenzymes in the cavernosal tissue. All these effects of castration were prevented by T replacement. The NO response to SCN was positively correlated with the levels of nNOS and eNOS, especially when the values of the two isoforms were added (r = 0.71, P < 0.001). These data suggest that the facilitatory action of androgens on penile erection involves the up-regulation of both constitutive NOS isoenzymes in the corpora cavernosa.     

Sequence-specific endonuclease Bam HI. Effect of hydrophobic reagents on sequence recognition and catalysis

The specificity of cleavage of Bam HI is altered in the presence of hydrophobic reagents, such as glycerol and M2SO. The enzyme with altered specificity, designated Bam HI.1, generated digestion patterns of various DNAs, which were distinct from those generated by Bam HI. Cleavage sites recognized in phiX174 RF DNA in the presence of these hydrophobic reagents are not related to the Bam HI palindrome. Bam HI.1 appears to be an endogenous form of Bam HI that can be expressed by altering the hydrophobicity of the reaction.     

Dynamics of nitric oxide release in the cardiovascular system

The endothelium plays a critical role in maintaining vascular tone by releasing nitric oxide (NO). Endothelium derived NO diffuses to smooth muscles, triggering their relaxation. The dynamic of NO production is a determining factor in signal transduction. The present studies were designed to elucidate dynamics of NO release from normal and dysfunctional endothelium. The nanosensors (diameter 100-300 nm) exhibiting a response time better than 100 micros and detection limit of 1.0 x 10(-9) mol L(-1) were used for in vitro monitoring of NO release from single endothelial cells from the iliac artery of normotensive (WKY) rats, hypertensive (SHR) rats, and normal and cholesterolemic rabbits. Also, the dynamics and distribution of NO in left ventricular wall of rabbit heart were measured. The rate of NO release was much higher (1200 +/- 50 nmol L(-1) s(-1)) for WKY than for SHR (460 +/- 10 nmol L(-1) s(-1)). Also, the peak NO concentration was about three times higher for WKY than SHR. Similar decrease in the dynamics of NO release was observed for cholesterolemic rabbits. The dynamics of NO release changed dramatically along the wall of rabbit aorta, being highest (0.86 +/- 0.12 micromol L(-1)) for the ascending aorta, and lowest for the iliac aorta (0.48 +/- 0.15 micromol L(-1)). The distribution of NO in the left ventricular wall of rabbit heart was not uniform and varied from 1.23 +/- 0.20 micromol L(-1) (center) to 0.90 +/- 0.15 micromol L(-1) (apex). Both, the maximal concentration and the dynamics of NO release can be useful diagnostic tools in estimating the level of endothelial dysfunction and cardiovascular system efficiency.     

The direct release of nitric oxide by gypenosides derived from the herb Gynostemma pentaphyllum.

Herbal medicines are increasingly being utilized to treat a wide variety of disease processes. Gypenosides are triterpenoid saponins contained in an extract from Gynostemma pentaphyllum and are reported to be effective in the treatment of cardiovascular diseases; however, the mechanism underlying the therapeutic effect is not known. We tested the hypothesis that gypenosides extracted from G. pentaphyllum elicit vasorelaxation through the direct release of endothelium-derived nitric oxide. Nitric oxide production in bovine aortic endothelial cells grown under standard tissue culture conditions was quantitated using a chemiluminescence method. Arterial vasomotion was assayed using isolated porcine coronary artery rings under standard isometric recording conditions. The extract of G. pentaphyllum at 0.1-100 microg/ml elicited concentration-dependent vasorelaxation of porcine coronary rings that was antagonized by the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester. Indomethacin had no significant effect on G. pentaphyllum-induced relaxation. The G. pentaphyllum extract elicited a concentration-dependent increase in nitric oxide production from cultured bovine aortic endothelial cells. At the concentrations utilized, there was no morphological evidence for cellular toxicity. These results demonstrate that extracts of G. pentaphyllum directly stimulate nitric oxide release, but not prostanoid production. Nitric oxide production in response to gypenosides may be one mechanism whereby this herbal medicine elicits its therapeutic effects.     

Rates of release of nitric oxide from HbSNO and internal electron transfer

The discovery that hemoglobin (Hb) in erythrocytes contains a fraction of beta-Cys-93 thiols as the nitrosylated derivative (HbSNO) led to the suggestion that this species is involved in transporting and releasing nitric oxide, which is the signal for local vasodilation. The release of NO from HbSNO requires an electron transfer to facilitate release and to regenerate the cysteine thiol via one-electron reduction in the absence of added thiols. An alternative mechanism, which has received much attention, transfers the nitrosyl group to an external thiol, which in turn would have to be reduced. The observed first order rate constant for the spontaneous oxidation of the ferrous heme of deoxy HbSNO is 1.0 x 10(-4)s(-1) in the absence of thiols. Under the same conditions, native Hb is stable. The oxidation of HbSNO occurs with the same rate constant that can be derived for the rate reported for the formation of HbNO from HbSNO. These similarities suggest that both processes involve the same reaction: internal electron transfer and direct release of nitric oxide.     

Effect of cytochalasins on surfactant release from alveolar type II cells

Cytochalasins enhanced surfactant secretion from primary cultures of [³H]choline-labeled type II epithelial cells from the rat. Cytochalasins A, B, C, D and dihydrocytochalasin B enhanced secretion of phosphatidyl-[³H]choline ([³H]PC) in a dose-dependent manner with EC₅₀ values of 1, 2, 0.5, 0.1 and 1 μM for cytochalasins A, B, C, D and dihydrocytochalasin B, respectively. Only cytochalasin A caused significant cytotoxicity as determined by release of the intracellular enzyme lactate dehydrogenase (EC 1.1.1.17). Dose responses of surfactant release induced by cytochalasins B, C and D were biphasic; maximal release was observed between 0.1–1.0 μM for cytochalasins C and D between 1 and 10 μM for cytochalasin B. Secretion decreased toward control levels at concentrations of cytochalasin above these maximal concentrations. Increased rates of [³H]PC release were noted between 1 and 3 h after exposure to cytochalasin D. Increased rates of surfactant release induced by cytochalasin D were additive to release induced by the β-adrenergic agonist, terbutaline, or forskolin, although cytochalasin D had no direct effect on cytosolic cyclic AMP levels. Changes in cell shape and microfilament organization were observed by phase-contrast microscopy and fluorescence microscopy using rhodamine-conjugated phalloidin after exposure of the isolated type II cells to cytochalasin D. Disruption of microfilaments associated with lamellar bodies of the purified type II cells occurred after treatment with cytochalasin D. Cytochalasin D augmented surfactant release from purified type II cells and disrupted the microfilament structure of those cells, supporting the hypothesis that alterations in microfilaments are associated with surfactant release.     

Effect of nitric oxide on exercise-induced proteinuria in rats.

Temporary proteinuria occurring after exercise is a common finding, and it is explained predominantly by alterations in renal hemodynamics. In this study, we investigated whether nitric oxide (NO), which is known to have an effect on renal hemodynamics and to increase during exercise, has a role in postexercise proteinuria. In the first step of this study, the effect of acute NO synthase blockage on exercise proteinuria was evaluated. The urinary protein levels in animals that performed acute exhaustive treadmill running exercise were considerably elevated compared with the control animals. Significantly elevated urinary protein levels were also detected in animals that received Nomega-nitro-L-arginine methyl ester before exhaustion, compared with both control and exhausted groups, and mixed-type proteinuria was detected in electrophoresis, as in all exhausted animals. In the second step of the study, a NO donor (isosorbide mononitrate) was given to rats 1 h before exhaustive exercise. Mixed-type proteinuria and the elevation in urinary protein levels that occur as a consequence of exhaustive exercise were prevented by NO donor treatment. Finally, in the third step of our study, a calcium channel blocker (diltiazem), another vasodilator, was applied to the rats 1 h before exhaustive exercise. Urinary protein levels were not different in exhausted rats with or without calcium channel blocker treatment. On the other hand, in both groups, urinary protein levels were higher than in the control group. The tail-cuff blood pressure alterations caused by vasodilator drug applications before exercise were not different for NO donor and calcium channel blocker groups. These results suggest that endogenous NO might prevent the postexercise proteinuria from becoming more severe by affecting hemodynamic changes that occur during exercise.     

Shear stress-induced nitric oxide release triggers the liver regeneration cascade.

The trigger of the liver regeneration cascade is currently unknown and has been the subject of debate. We hypothesize that, following 2/3 partial hepatectomy (PHX), an increase in the blood flow-to-liver mass ratio results in shear stress-induced nitric oxide (NO) release, which triggers the liver regeneration cascade. Portal venous pressure (PVP), reflecting shear stress in the liver, increased to the same extent following PHX and selective portal vein branch ligation (PVL), a hemodynamic model of PHX, suggesting similar amounts of shear stress in both models. Two indices of the initiation of the liver regeneration cascade were used: proliferative factor (PF) activity in blood 4 h after PHX or PVL and hepatic c-fos mRNA expression 15 min. after PHX or PVL. PF activity and c-fos mRNA expression were increased to similar extents after PHX and PVL, suggesting a similar stimulus in both models. PF activity and c-fos mRNA expression were inhibited by administration of the nitric oxide synthase antagonist, l-NAME, and the NO donor, SIN-1, reversed the inhibition in both models. These results provide support for the hypothesis that a hemodynamic change results in increased shear stress in the liver causing generation of NO, which then triggers the liver regeneration cascade.     

一氧化氮的释放对海马脑片CA1区痫样放电的影响

用自制的一氧化氮（ＮＯ）敏感电极－Ｎａｆｉｏｎ－壳聚糖合镍修饰铂电极（Ｎａｆｉｏｎ－ＣＴＳ（Ｎｉ）－Ｐｔ）连续测定了青霉素致痫海马脑片ＣＡ１区锥体层神经元ＮＯ的释放,并同时观察了ＮＯ合酶抑制剂７－ｎｉｔｒｏ－ｉｎｄａｚｏｌｅ（７－ＮＩ）及Ｎ＾ω－ｎｉｔｒｏ－Ｌ－ａｒｇｉｎｉｎｅ（Ｌ－ＮＮＡ）对诱发痫波及ＮＯ释放量的影响。研究观察到：（１）在青霉素致痫脑片模型上,诱发的痫波随青霉素浓度的增加而增多,     

Effect of airways constriction on exhaled nitric oxide.

While airway constriction has been shown to affect exhaled nitric oxide (NO), the mechanisms and location of constricted airways most likely to affect exhaled NO remain obscure. We studied the effects of histamine-induced airway constriction and ventilation heterogeneity on exhaled NO at 50 ml/s (Fe(NO,50)) and combined this with model simulations of Fe(NO,50) changes due to constriction of airways at various depths of the lung model. In 20 normal subjects, histamine induced a 26 +/- 15(SD)% Fe(NO,50) decrease, a 9 +/- 6% forced expiratory volume in 1 s (FEV(1)) decrease, a 19 +/- 9% mean forced midexpiratory flow between 25% and 75% forced vital capacity (FEF(25-75)) decrease, and a 94 +/- 119% increase in conductive ventilation heterogeneity. There was a significant correlation of Fe(NO,50) decrease with FEF(25-75) decrease (P = 0.006) but not with FEV(1) decrease or with increased ventilation heterogeneity. Simulations confirmed the negligible effect of ventilation heterogeneity on Fe(NO,50) and showed that the histamine-induced Fe(NO,50) decrease was due to constriction, with associated reduction in NO flux, of airways located proximal to generation 15. The model also indicated that the most marked effect of airways constriction on Fe(NO,50) is situated in generations 10-15 and that airway constriction beyond generation 15 markedly increases Fe(NO,50) due to interference with the NO backdiffusion effect. These mechanical factors should be considered when interpreting exhaled NO in lung disease.     

Synergistic action of rutin and orthovanadate on nitric oxide release from mouse macrophage cells

The cooperative action of sodium orthovanadate (a putative protein-phosphotyrosine phosphatase inhibitor) and rutin (an effective superoxide scavenger) on the nitric oxide (NO) production of J774A.1 mouse macrophage cells has been investigated. Orthovanadate alone caused a mild but significant increase in NO production of the cells at its highest concentration used (500 microM). Orthovanadate and rutin together caused a significant increase in the nitrite level of the supematants of the J774A.1 cells after a 24-hour incubation period, in a concentration dependent manner. The optimal doses for orthovanadate and rutin were 50 microM and 100 microM, respectively. This cooperative action of rutin and orthovanadate was totally inhibitable by catalase, reduced glutathion, N-acetylcystein, cycloheximide, pyrrolidine dithiocarbamate (a putative NF-kappaB inhibitor), genistein and tyrphostin-AG126 (two protein tyrosine-kinase inhibitors). Superoxide dismutase had no inhibitory effect. Orthovanadate and rutin (only together) could induce the oxidation of 2',7'-dichlorofluorescein-diacetate, a marker of hydrogen peroxide. This effect was inhibitable by reduced glutathion, a hydrogen peroxide specific scavenger. These findings suggest, that orthovanadate can induce the production of NO by J774A.1 macrophages not only by inhibition of protein tyrosine-phosphatases, but, using it with rutin, by increasing the level of hydrogen peroxide in the cells.