Stimulation of μ- and δ-Opiate Receptors and Tolerance of Isolated Heart to Oxidative Stress: the Role of NO-Synthase

Preliminary intravenous injection of -opiate receptor (OR) agonists DSLET (0.1 mg/kg) or DTLET (0.1 mg/kg) increased tolerance of isolated perfused myocardium to damage by oxidative stress simulated in vivo with FeSO₄ + ascorbic acid. This manifested itself by a decreased level of creatine phosphokinase (CPK) in the perfusate flowing out of the heart during the oxidative exposure. The preliminary systemic injection of -agonists DAMGO (0.1 mg/kg) or DALDA (0.1 mg/kg) failed to affect the release of CPK from the myocardium. The cardioprotective effect of the -agonist DSLET was completely abolished by preliminary intravenous injection of the -OR antagonist ICI 174,864 (2.5 mg/kg). The intravenous injection of the NO-synthase inhibitor L-NAME (50 mg/kg) also completely abolished the cardioprotective effect of -OR stimulation. The preliminary injection of DSLET but not of DAMGO prevented an increase in the level of diene conjugates and a decrease in the activity of superoxide dismutase (SOD) in the isolated myocardium tissue. Thus, the in vivo stimulation of -OR increased the tolerance of the heart to oxidative stress through activation of NO-synthase and SOD.     

Oxidative stress in pathogenesis of arterial hypertension in ISIAH rats]

The NO-synthase activity and the rate of NO production in the rat aortic wall and cerebellum were 2-1.5-fold higher in the ISIAH rats than in normotensive WAG rat strain. In contrast, the superoxide dismutase (SOD) activity was significantly reduced in the ISIAH rats. The blood level of reduced thiols was also much lower in the ISIAH rats. The findings suggest that oxidative stress may play a significant role in pathogenesis of stress-sensitive hypertension in the ISIAH rat strain.     

alpha-硫辛酸对2 型糖尿病早期肾病的保护作用研究

目的：探讨注射用alpha-硫辛酸对2 型糖尿病早期肾病（DN）的保护作用。方法：将56 例2 型糖尿病早期肾病患者随机分为治 疗组28 例,采用注射alpha-硫辛酸加常规治疗;对照组28 例,仅给予常规治疗。治疗前后测定尿微量白蛋白排泄率（UAER）、超氧化 物歧化酶（SOD）、丙二醛（MDA）、一氧化氮（NO）水平。结果：与治疗前比较,治疗组UAER及MDA均显著降低（P＜0.05）,SCr、 SOD及NO 显著升高（P＜0.05）。对照组SCr、UAER、SOD、MDA 及NO与治疗前比较差异无统计学意义（P＞0.05）。结论：注射用 alpha-硫辛酸能减少2型糖尿病肾病患者尿微量白蛋白的排泄,对早期肾病具有保护作用。     

Nitric oxide and oxidative stress in brain and heart of normal rats treated with doxorubicin: role of aminoguanidine

Doxorubicin (DOX) is a potent antitumor antibiotic drug known to cause severe cardiac toxicity. Moreover, its adverse effects were found to be extended to the cerebral tissue. Several mechanisms for this toxicity have been ascribed. Currently, one of the most accepted mechanisms is through free radicals; however, the exact role of nitric oxide (NO) is still unclear. Accordingly, a NO-synthase inhibitor with some antioxidant property, aminoguanidine (AG), was selected to examine its potential protective effect against DOX-induced toxicity. Male Wistar albino rats (150-200 g) were allocated into a normal control group, DOX-induced toxicity group, and DOX + AG-treated group. DOX was injected i.p. at a dose of 10 mg/kg divided into four equal injections over a period of 2 weeks. AG was injected i.p. at a dose of 100 mg/kg 1 h before each DOX injection. The animals were sacrificed 24 h after the last DOX injection and the following parameters were measured: serum lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) activities, cardiac and cerebral contents of malondialdehyde (MDA), conjugated diene (CD), glutathione (GSH), NO, and cytosolic calcium, as well as superoxide dismutase (SOD) and glutathione peroxidase (GSHP(X)) activities. Cardiotoxicity was manifested by a marked increase in serum LDH and CPK in addition to the sharp increase in MDA reaching eightfolds the basal level. This was accompanied by significant increase in CD, NO, cytosolic calcium, SOD, and GSHP(X) content/activity by 69, 85, 76, 125, and 41% respectively as compared to normal control. On the other hand, GSH was significantly depressed. In brain, only significant increase in MDA and GSHP(X) and decrease in GSH were obtained but to a lesser extent than the cardiac tissue. AG treatment failed to prevent the excessive release of cardiac enzymes; however, it alleviated the adverse effects of DOX in heart. AG administration resulted in marked decrease in the elevated levels of MDA, NO, SOD, and GSHP(X), however, MDA level was still pathological. The altered parameters in brain were restored by AG. It is concluded that, AG could not provide complete protection against DOX-induced toxicity. Therefore, it is recommended that, maintenance of the endogenous antioxidant, GSH, and regulation of calcium homeostasis must be considered, rather than NO formation, to guard against DOX-induced toxicity.     

Modulation of mast cell activity by a peptide agonist of the thrombin receptor: role of nitric oxide.

The effect of a thrombin receptor agonist peptide (TRAP-6) on the release of nitric oxide (NO) and platelet activating factor (PAF) from resting and calcium-ionophore (A23187)-activated rat peritoneal mast cells (RPMC) was studied using a platelet aggregation bioassay. RPMC spontaneously released NO, which inhibited TRAP-6-, ADP-, and PAF-stimulated platelet aggregation. This effect of NO was abolished by the addition of an NO binding agent, oxyhemoglobin (oxyHb), to the platelet suspension. The RPMC-induced suppression of platelet aggregation was completely inhibited by the NO-synthase inhibitor L-NAME. TRAP-6 and its high affinity analog haTRAP stimulated the rapid release of NO from RPMC. The effect of TRAP-6 was inhibited by pretreatment of the RPMC with L-NAME or with the inhibitor of the constitutive NO-synthase isoform (cNOS) calmidazolium. TRAP-6 inhibited PAF release from A23187-activated RPMC via an NO-dependent mechanism. Platelet aggregation induced by PAF release from activated RPMC was also confirmed in experiments using the PAF receptor antagonist ginkgolide B. Thus, TRAP-6 is a rapidly acting modulator of mast cell reactivity; it stimulates NO release and inhibits PAF secretion.     

Modulation of nitric oxide (NO) biosynthesis in lactobacilli

We characterized effects of nitric oxide synthase (NOS) substrate L-arginine and classical inhibitors of mammalian NOS on nitric oxide (NO) biosynthesis in probiotic bacteria Lactobacillus plantarum 8P-A3. NO-synthase origin of nitric oxide detected by fluorescent NO indicator 1,2-diaminoanthraquinone (DAA) was confirmed by induction of NO production by exogenous L-arginine. None of the used inhibitors of three isoforms of mammalian NOSs (L-NAME, L-NIL, nNOS inhibitor I) showed significant inhibitory effect of lactobacillar NO-synthase activity.     

Multiple low-dose and single high-dose treatments with streptozotocin do not generate nitric oxide

Streptozotocin (STZ) is a widely used diabetogenic agent that damages pancreatic islet beta cells by activating immune mechanisms, when given in multiple low doses, and by alkylating DNA, when given at a single high dose. Actually, STZ contains a nitroso moiety. Incubation of rat islets with this compound has been found to generate nitrite; moreover, photoinduced NO production from STZ has been demonstrated. These reports have suggested that direct NO generation may be a mechanism for STZ toxicity in diabetogenesis. Several other studies have denied such a mechanism of action. This study has shown that (1) the multiple low-dose (MLDS) treatment does not stimulate NO production at the islet level; in fact, nitrite + nitrate levels and aconitase activity (also in the presence of an NO-synthase inhibitor, namely NAME) remain unmodified; RT-PCR analysis demonstrates that this treatment does not stimulate iNOS activity; (2) the high-dose (HDS) treatment does not stimulate NO production; in fact nitrite + nitrate levels remain unmodified and iNOS mRNA levels are not altered, although aconitase activity is significantly decreased. Moreover, we have confirmed that the MLDS treatment is able to decrease SOD activity by day 11 and that STZ, given in a single high dose, transiently increases superoxide dismutase (SOD) values (24 h from the administration), then dramatically lowers SOD levels. On the basis of our results, we conclude that STZ, "in vivo" is unable to generate NO, both as a MLDS or HDS treatment, thus excluding that NO exerts a role in streptozotocin-dependent diabetes mellitus.     

Mechanism of the slow inotropic response of the mouse atrium mediated by the beta2-adrenoreceptor

The abundant beta2-adrenoceptors (AR) expression was revealed in the mouse atrial cardiomyocytes, albeit its function is poorly understood. Recently we revealed the slow developing (for 20-40 min) positive inotropic effect in the mouse atrium which was induced by the specific agonist of the beta2-adrenoceptors (5 mkM fenoterol) and the task of this study involved investigation of the found effect. It was shown that stimulation of beta2-AR is enough for rapid triggering of up-regulation of two signalling pathways that have the opposite influence on the contraction force. On one hand, activation of the adenylate cyclase--protein kinase A cascade occurs leading to increasing of intracellular Ca2+ concentration and amplitude of contraction; on the other hand, activation of the NO-synthase and enhance of NO production occurs which prevents the potentiating of the contraction force. During the first 15-20 minutes, superposition of these activation effects was revealed, which prevented the contraction strength increasing. Then the positive inotropic effects occurred due to the decreasing of NO production. It was shown that L-type Ca-channels and ryanodine receptors were the key targets incorporated in the beta2 adrenoreceptors signalling puzzle.     

Hyperglycemia reduces nitric oxide synthase and glycogen synthase activity in endothelial cells.

Hyperglycemia is considered a primary cause of diabetic vascular complications. A hallmark of vascular disease is endothelial cell dysfunction characterized by diminished nitric-oxide (NO)-dependent phenomena such as vasodilation, angiogenesis, and vascular maintenance. This study was designed to investigate the effects of a high level of D-glucose on endothelial NO response, oxidative stress, and glucose metabolism. Bovine aortic endothelial cells (BAECs) were pretreated with a high concentration of glucose (HG) (22 mmol/L) for at least 2 weeks and compared with control cells exposed to 5 mmol/L glucose (NG). The effect of chronic hyperglycemia on endothelial NO-synthase (eNOS) activity and expression, glycogen synthase (GS) activity, extracellular-signal-regulated kinase (ERK 1,2), p38, Akt expression, and Cu/Zn superoxide-dismutse (SOD-1) activity and expression were determined. Western blot analysis showed that eNOS protein expression decreased in HG cells and was accompanied by diminished eNOS activity. The activity of GS was also significantly lower in the HG cells than in NG cells, 25.0+/-17.4 and 89+/-22.5 nmol UDP-glucose.mg protein(-1)x min(-1), respectively. Western blot analysis revealed a 40-60% decrease in ERK 1,2 and p38 protein levels, small modification of phosphorylated Akt expression, and a 30% increase in SOD-1 protein expression in HG cells. Although SOD expression was increased, no change was observed in SOD activity. These results support the findings that vascular dysfunction due to exposure to pathologically high D-glucose concentrations may be caused by impairment of the NO pathway and increased oxidative stress accompanied by altered glucose metabolism.     

Selective inhibition of inducible NO-synthase by nonselective inhibitor

The study has shown that Nw-nitro-L-arginine, a nonselective nitric oxide (NO) inhibitor, in low non-vasoactive doses (10 mg/kg) exerted a protective effect in heat shock as demonstrated by a decrease in the mortality rate and prevention of acute hypotension in rats. The L-NNA in the same dose inhibited the basal NO production but left unaffected a carbachol-activated NO production. The findings suggest a possibility in principle of preferential inhibition of inducible NO-synthase in pathological conditions related to the NO overproduction using non-vasoactive doses of L-NNA the nonselective NO-synthase inhibitor.     

Arginase activity in the frog urinary bladder epithelial cells and its involvement in regulation of nitric oxide production

The activity of arginase converting arginine into ornithine and urea is of particular interest among many factors regulating NO production in the cells. It is known that by competing with NO-synthase for common substrate, arginase can affect the NO synthesis. In the present work, the properties of arginase from the frog Rana temporaria L. urinary bladder epithelial cells possessing the NO-synthase activity were characterized, and possible contribution of arginase to regulation of NO production by epithelial cells was studied. It has been shown that the enzyme had the temperature optimum in the range of 55-60 degrees C, K(m) for arginine 23 mM, and V(max) about 10 nmol urea/mg protein/min, and its activity was effictively inhibited by (S)-(2-boronoethyl)-L-cysteine (BEC), an inhibitor of arginase, at concentrations from 10(-6) to 10(-4) M. The comparison of arginase activity in various frog tissues revealed the following pattern: liver > kidney > brain > urinary bladder (epithelium) > heart > testis. The arginase activity in the isolated urinary bladder epithelial cells was 3 times higher than that in the intact urinary bladder. To evaluate the role of arginase in the regulation of NO production, epithelial cells were cultivated in the media L-15 or 199 containing different amounts of arginine; the concentration of NO2-, the stable NO metabolite, was determined in the culture fluid after 18-20 h of cells incubation. The vast majority of the produced nitrites are associated with the NOS activity, as L-NAME, the NOS-inhibitor, decreased their accumulation by 77.1% in the L-15 medium and by 80% in 199 medium. BEC (10(-4) M) increased the nitrite production by 18.0 % +/- 2.7 in the L-15 medium and by 24.2 +/- 3.5 in the 199 medium (p < 0.05). The obtained data indicate a relatively high arginase activity in the frog urinary bladder epithelium and its involvement in regulation of NO production by epithelial cells.     

Atrial natriuretic peptide influence on nitric oxide system in kidney and heart

Atrial natriuretic peptide (ANP) and nitric oxide (NO) induce diuresis, natriuresis and diminish vascular tone. Our previous studies showed NO system is involved in ANP hypotensive effect. The aim was to investigate ANP effects on renal and cardiac NO-synthase (NOS) activity. Rats were divided into two groups: group I, infused with saline (1 h, 0.05 ml/min); group II, received ANP bolus (5 microg/kg)+ANP infusion (1 h, 0.2 microg/kg x min). NADPH-diaphorase activity (NADPH-d) was determined in kidney and heart. NOS catalytic activity was determined in renal medulla and cortex and cardiac atria and ventricle by measuring the conversion of l-[U(14)C]-arginine to l-[U(14)C]-citrulline. In group I, NOS activity was determined in basal conditions and plus 1 microM ANP and in group II, NOS activity was determined in basal conditions. NADPH-d was higher in group II than in group I in glomeruli, proximal tubule, cortical and medullar collecting duct, right atria and left ventricle. NOS activity was increased by in vitro ANP addition and, in vivo, ANP infusion in all the studied tissues. ANP treatment increases renal and cardiac NO synthesis. This effect would be independent on the hemodynamic changes induced by ANP. The activation of NO pathway would be one of the mechanisms involved in diuretic, natriuretic and hypotensive effects of ANP.     

Nitric oxide formation in the oropharyngeal tract: possible influence of cigarette smoking.

Cigarette smoking reduces the level of nitric oxide (NO) in exhaled air by an unknown mechanism. The view that part of the effect of cigarette smoking on NO production should occur in the oropharyngeal tract is supported by several studies. We have therefore compared smokers and non-smokers regarding non-enzymatic formation of NO from nitrite in the oral cavity since this is a primary candidate target for cigarette smoke. We have also looked at NO synthase-dependent NO formation in the mucosa of the oropharyngeal tract as an alternative target for the inhibitory effect induced by cigarette smoke. Smokers exhaled 67% lower levels of NO than controls (p<0.01, n=15 each group). We could not detect any significant difference in salivary nitrite, nitrate or ascorbate between smokers and non-smokers. Mouthwash with the antibacterial agent chlorhexidine reduced salivary nitrite (-65%) and exhaled NO levels (-10%) similarly in the two groups. Immunohistochemical techniques revealed dense expression of inducible (but not endothelial or neuronal) NO synthase in the squamous epithelium of non-inflamed tonsillar and gingival tissue biopsies. In the same biopsies, significant Ca2+ -independent citrulline-forming activity was detected. We found no difference between smoking and non-smoking subjects regarding NO-synthase expression and in vitro activity. In another group of non-smoking subjects (n=10), spraying the oropharyngeal tract with the NO-synthase inhibitor NG-monomethyl-L-arginine (250 mg) significantly reduced exhaled NO levels for at least 30 min (-18%, p<0.01). Our data suggest that cigarette smoking does not affect non-enzymatic NO formation from nitrite in saliva. However, NO is also formed by inducible NO synthase in the squamous epithelium of the normal oropharyngeal tract. We suggest that cigarette smoking may down-regulate enzymatic NO formation in the oropharyngeal compartment as well as in the bronchial compartment.     

Gene transfer of CuZn superoxide dismutase enhances the synthesis of vascular endothelial growth factor

Nitric oxide (NO) and reactive oxygen species (ROS) are emerging as important regulators of angiogenesis. NO enhances VEGF synthesis in several cell types and is required for execution of VEGF angiogenic effect in endothelial cells. Similarly, hydrogen peroxide induces VEGF synthesis and recent studies indicate the involvement of ROS in signaling downstream of VEGF stimulation. VEGF synthesis can not only be enhanced by gene transfer of VEGF but also by overexpression of NO synthase genes. Here, we examined the possibility of augmentation of VEGF production by gene transfer of copper/zinc superoxide dismutase (CuZnSOD, SOD1). Overexpression of human SOD1 in mouse NIH 3T3 fibroblasts increased SOD activity, enhanced intracellular generation of H2O2 and significantly stimulated VEGF production as determined by increase in VEGF promoter activity, VEGF mRNA expression and VEGF protein synthesis. The stimulatory effect on VEGF synthesis induced by SOD1 gene transfer was reverted by overexpression of human catalase. The effect of H2O2 produced by engineered cells is mediated by activation of hypoxia-inducible factor response element (HRE) as well as Sp1 recognition site of VEGF promoter. This data suggest the feasibility of stimulation of angiogenesis by overexpression of SOD1.     

Effect of N-stearoylethanolamine on the level of stable NO metabolites in different pathological conditions which are accompanied by oxidative stress

The effects of N-stearoylethanolamine (NSE) at the level of stable NO metabolites--NO2- NO3- under different pathological conditions which were accompanied by oxidative stress and NO disbalance were studied. It was found that NSE promoted the increasing of NO metabolites level on the animal models with deficit of NO compared to control and evoked decreasing content of NO2- and NO3- under pathological conditions with NO overproduction. It is suppoused, that NSE effects could be determined, by its ability to modulate the the activity of NO-synthase izoformes.     

α-硫辛酸对2型糖尿病早期肾病的保护作用研究

目的：探讨注射用α-硫辛酸对2型糖尿病早期肾病（DN）的保护作用。方法：将56例2型糖尿病早期肾病患者随机分为治疗组28例,采用注射α-硫辛酸加常规治疗;对照组28例,仅给予常规治疗。治疗前后测定尿微量白蛋白排泄率（UAER）、超氧化物歧化酶（SOD）、／丙二醛（MDA）、一氧化氮（NO）水平。结果：与治疗前比较,治疗组UAER及MDA均显著降低（P〈0．05）,SCr、SOD及NO显著升高（P〈0．05）。对照组SCr、UAER、SOD、MDA及NO与治疗前比较差异无统计学意义（P〉0．05）。结论：注射用α-硫辛酸能减少2型糖尿病肾病患者尿微量白蛋白的排泄,对早期肾病具有保护作用。     

Nitric oxide and cardiac function

Nitric oxide (NO) participates in the control of contractility and heart rate, limits cardiac remodeling after an infarction and contributes to the protective effect of ischemic pre- and postconditioning. Low concentrations of NO, with production of small amounts of cGMP, inhibit phosphodiesterase III, thus preventing the hydrolysis of cAMP. The subsequent activation of a protein-kinase A causes the opening of sarcolemmal voltage-operated and sarcoplasmic ryanodin receptor Ca(2+) channels, thus increasing myocardial contractility. High concentrations of NO induce the production of larger amounts of cGMP which are responsible for a cardiodepression in response to an activation of protein kinase G (PKG) with blockade of sarcolemmal Ca(2+) channels. NO is also involved in reduced contractile response to adrenergic stimulation in heart failure. A reduction of heart rate is an evident effect of NO-synthase (NOS) inhibition. It is noteworthy that the direct effect of NOS inhibition can be altered if baroreceptors are stimulated by increases in blood pressure. Finally, NO can limit the deleterious effects of cardiac remodeling after myocardial infarction possibly via the cGMP pathway. The protective effect of NO is mainly mediated by the guanylyl cyclase-cGMP pathway resulting in activation of PKG with opening of mitochondrial ATP-sensitive potassium channels and inhibition of the mitochondrial permeability transition pores. NO acting on heart is produced by vascular and endocardial endothelial NOS, as well as neuronal and inducible synthases. In particular, while in the basal control of contractility, endothelial synthase has a predominant role, the inducible isoform is mainly responsible for the cardiodepression in septic shock.     

Influence of D2-receptor blockade on the nitrergic system activity of the nucleus accumbens

In Sprague-Dawley rats, by means of in vivo microdialysis combined with HPLC analysis it was shown that the infusion into the n. accumbens of the D2-receptor antagonist raclopride (20, 100 microM) did not affect extracellular level ofcitrulline (an NO co-product) in this brain area. The intraaccumbal infusion of NMDA, an NMDA receptor agonist (100 microM) caused a rise of the extracellular citrulline level in this brain area. This rise was prevented by intraaccumbal infusion of 0.5 mM 7-nitroindazole, a neuronal NO-synthase inhibitor, and it was significantly reduced by the infusion of raclopride (20, 100 microM) into this brain area. The data obtained suggest that the D2-receptors of the n. accumbens are implicated in the regulation of neuronal NO-synthase activity induced by local NMDA receptor stimulation.     

Chronic high blood flow potentiates shear stress-induced release of NO in arteries of aged rats

Aging impairs shear-stress-dependent dilation of arteries via increased superoxide production, decreased SOD activity, and decreased activation of endothelial nitric oxide (NO) synthase (eNOS). In the present study, we investigated whether chronic increases in shear stress, elicited by increases in blood flow, would improve vascular endothelial function of aged rats. To this end, second-order mesenteric arteries of young (6 mo) and aged (24 mo) male Fischer-344 rats were selectively ligated for 3 wk to elevate blood flow in a first-order artery [high blood flow (HF)]. An in vitro study was then conducted on first-order arteries with HF and normal blood flow (NF) to assess shear stress (1, 10, and 20 dyn/cm(2))-induced release of NO into the perfusate. In HF arteries of both age groups, shear stress-induced NO production increased significantly. In 24-mo-old rats, the reduced shear stress-induced NO production in NF arteries was normalized by HF to a level similar to that in NF arteries of 6-mo-old rats. The increased NO production in HF arteries of 24-mo-old rats was associated with increased shear stress-induced dilation, expression of eNOS protein, and shear stress-induced eNOS phosphorylation. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, reduced shear stress-induced eNOS phosphorylation and vasodilation. Superoxide production decreased significantly in HF compared with NF arteries in 24-mo-old rats. The decreased superoxide production was associated with significant increases in CuZn-SOD and extracellular SOD protein expressions and total SOD activity. These results suggest that stimulation with chronic HF restores shear-stress-induced activation of eNOS and antioxidant ability in aged arteries.