The effect of aspirin and two nitric oxide donors on the infarcted heart in situ

Nitric oxide (NO) donors are heterogeneous substances which release NO, a biologically active compound. NO released by nitric oxide donors has important effects on the circulation by causing vasodilation, diminishing myocardial contractile force, inhibiting platelet aggregation, and counteracting the effects of thromboxane A2. In the infarcted heart, activation of the inducible form of nitric oxide synthase (iNOS) and the formation of prostacyclin and thromboxane A2 by cyclooxygenase (COX) were increased. Myocardial infarction also resulted in increased myocardial NO production. Aspirin (acetylsalicylic acid. ASA) at low concentration (35 mg/kg/day) fails to change iNOS production, in contrast to higher dose (150 mg/kg/day) which, as previously shown, inhibits iNOS activity. ASA at all doses also suppresses myocardial prostanoid formation because of inhibition of COX. Recently, two NO donors have been synthesized: NCX 4016 and Diethylenetriamine/NO (DETA/NO). NCX 4016 combines an NO-releasing moiety with a carboxylic residue via an esteric bond. We describe here that NCX 4016 (65 mg/kg/day) increased prostacyclin and thromboxane A2 production in the infarcted heart muscle, overcoming the inhibitory effects of ASA. As a result of nitric oxide release, oxidation products of NO (NO2- and NO3-; NOx) in arterial blood rose following administration of NCX 4016. On oral administration, NCX 4016 did not change systemic arterial pressure. The effects of a single NO donor, DETA/NO (1.0 mg/kg/day) on the infarcted heart were also investigated On intravenous administration, the compound increased NO concentration in arterial blood slightly but to a lesser degree than NCX 4016. Like NCX 4016, it raised myocardial production of prostacyclin and thromboxane A2 in the infarcted heart. However, it caused a severe fall in blood pressure. These findings demonstrate that newly-synthesized NO donors release nitric oxide in situ and increase myocardial production of prostanoids. NCX 4016 has therapeutic potential because it can be orally administered, lacks hypotensive effects, increases blood levels of nitric oxide and myocardial prostacyclin production.     

Exogenous,basal, and flow-induced nitric oxide production and endothelial cell proliferation

The role of nitric oxide (NO) from endogenous and exogenous sources in regulating large vessel and microvascular endothelial cell proliferation was investigated. Exogenous NO liberated from five different chemical donors inhibited bovine aortic, bovine retinal microvascular, and human umbilical vein endothelial cell proliferation in a dose-dependent manner as determined by ³H-thymidine incorporation. The potency of the donors varied as a function of the donors' half-lives. Donors with half-lives greater than 30 min were more effective than donors with significantly shorter half-lives. Coincubation of endothelial cells with 0.4 mM deoxyadenosine and 0.4 mM deoxyguanosine reduced the percentage of inhibition due to an NO donor. These data are consistent with a ribonucleotide reductase-dependent mechanism of inhibition. Inhibition of basal NO production with four different inhibitors of nitric oxide synthase (NOS) did not modify proliferation. Laminar flow with a wall shear stress of 22 dyn/cm²inhibited the proliferation of subconfluent bovine aortic endothelial cells. The addition of a NOS inhibitor did not abrogate the flow-induced inhibition of proliferation, suggesting that flow-stimulated release of NO from endothelial cells did not account for flow-induced inhibition of proliferation. Taken together, these data suggest that relatively large concentrations of exogenous NO inhibit endothelial cell proliferation, while endogenous levels of NO are inadequate to inhibit proliferation. J. Cell. Physiol. 171:252–258, 1997. © 1997 Wiley-Liss, Inc.     

Nitric oxide donors, sodium nitroprusside and S-nitroso-N-acetylpenicillamine, stimulate myoblast proliferation in vitro

Summary Nitric oxide (NO) is an inter- and intracellular messenger involved in a variety of physiologic and pathophysiologic conditions. The effect of two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) and their effect on myoblast proliferation was examined. Both donors stimulated an increase in myoblast cell number over a range (1–10 μM) of donor concentrations. However, 50 μM SNAP inhibited myoblast proliferation. Cell numbers from cultures treated with degraded 10 μM SNAP were equivalent to the control. Therefore, it appears NO can stimulate as well as inhibit myoblast proliferation.     

iNOS/NO signaling regulates apoptosis induced by glycochenodeoxycholate in hepatocytes

Inducible nitric oxide synthase (iNOS) and nitric oxide (NO) can ameliorate apoptosis induced by toxic glycochenodeoxycholate (GCDC) in hepatocytes. However, the underlying molecular mechanisms are not yet understood in detail. This study is to clarify the function of iNOS/NO and its mechanisms during the apoptotic process. The apoptosis was brought about by GCDC in rat primary hepatocytes. iNOS/NO signaling was then investigated. iNOS inhibitor 1400 W enhanced the GCDC-induced apoptosis as reflected by caspase-3 activity and TUNEL assay. Exogenous NO regulated the apoptosis subsequent to NO donor S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP). The GCDC-induced apoptosis was decreased with 0.1 mM SNAP or 0.15 mM SNP, while it was increased with 0.8 mM SNAP or 1.2 mM SNP. The endogenous iNOS inhibited apoptosis, but the exogenous NO played a dual role during the GCDC-induced apoptosis. There was a potential iNOS/Akt/survivin axis that inhibited the hepatocyte apoptosis in low doses of NO donors. In contrast, high doses of NO donors activated CHOP through p38MAP-kinase (p38MAPK), upregulated TRAIL receptor DR5, and suppressed survivin. Consequently the high doses of NO donors promoted the apoptosis in hepatocytes. Our data suggest that the iNOS/NO signaling can modulate Akt/survivin and p38MAPK/CHOP pathways to mediate the GCDC-induced the apoptosis in hepatocytes. These signaling pathways may serve as targets for therapeutic intervention in cholestatic liver disease.     

Bmx, a member of the Tec family of nonreceptor tyrosine kinases, is a novel participant in pharmacological cardioprotection

Previous studies have indicated that PKC-epsilon is a central regulator of protective signal transduction in the heart. However, the signaling modules through which PKC-epsilon exerts its protective effects have only begun to be understood. We have identified a novel participant in the PKC-epsilon signaling system in cardioprotection, the nonreceptor tyrosine kinase Bmx. Functional proteomic analyses of PKC-epsilon signaling complexes identified Bmx as a member of these complexes. Subsequent studies in rabbits have indicated that Bmx is activated by nitric oxide (NO) in the heart, concomitant with the late phase of NO donor-induced protection, and provide the first analysis of Bmx expression/distribution in the setting of cardioprotection. In addition, increased expression of Bmx induced by NO donors was blocked by the same mechanism that blocked cardioprotection: inhibition of PKC with chelerythrine. These findings indicate that a novel type of PKC-tyrosine kinase module (involving Bmx) is formed in the heart and may be involved in pharmacological cardioprotection by NO donors.     

Physicochemistry of dinitrosyl iron complexes with thiolate ligands underlying their beneficial effect in endometriosis

Exogenous dinitrosyl iron complexes (DNIC) with thiolate ligands as NO and NO⁺ donors are capable of exerting both regulatory and cytotoxic effects on diverse biological processes similarly to those characteristic of endogenous nitric oxide. Regulatory activity of DNIC (vasodilatory, hypotensive, suppressing thrombosis, increasing erythrocyte elasticity, accelerating skin wound healing, inducing penile erection, etc.) is determined by their capacity of NO and NO⁺ transfer to biological targets of the latter (heme- and thiol-containing proteins, respectively) due to higher affinity of the proteins for NO and NO⁺ than that of DNIC. Cytotoxic activity of DNIC is provided by rapid DNIC decomposition under action of iron-chelating compounds, resulting in appearance of NO and NO⁺ in cells and tissues in high amounts. The latter mechanism is suggested to cause the blocking effect of DNIC as cytotoxic effectors on the development of benign endometrial tumors in rats with experimental endometriosis. It is also proposed that a similar mechanism can operate to cause at least a delay of malignant tumor proliferation under action of DNIC.     

Caspase-mediated apoptosis in neuronal excitotoxicity triggered by nitric oxide.

BACKGROUND: Excitotoxicity and excess generation of nitric oxide (NO) are believed to be fundamental mechanisms in many acute and chronic neurodegenerative disorders. Disturbance of Ca2+ homeostasis and protein nitration/nitrosylation are key features in such conditions. Recently, a family of proteases collectively known as caspases has been implicated as common executor of a variety of death signals. In addition, overactivation of poly-(ADP-ribose) polymerase (PARP) has been observed in neuronal excitotoxicity. We therefore designed this study to investigate whether triggering of caspase activity and/or activation of PARP played a role in cerebellar granule cell (CGC) apoptosis elicited by peroxynitrite (ONOO-) or NO donors. MATERIALS AND METHODS: CGC from wild-type or PARP -/- mice were exposed to various nitric oxide donors. Caspase activation and its implications for membrane alterations, Ca2+ homeostasis, intracellular proteolysis, chromatin degradation, and cell death were investigated. RESULTS: CGC exposed to NO donors undergo apoptosis, which is mediated by excess synaptic release of excitotoxic mediators. This excitotoxic mechanism differs from direct NO toxicity in some other neuronal populations and does not involve PARP activation. Inhibition of caspases with different peptide substrates prevented cell death and the related features, including intracellular proteolysis, chromatin breakdown, and translocation of phosphatidylserine to the outer surface of the cell membrane. Increased Ca2+ influx following N-methyl-D-aspartate (NMDA) receptor (NMDA-R) activation was not inhibited by caspase inhibitors. CONCLUSIONS: In CGC, NO donors elicit apoptosis by a mechanism involving excitotoxic mediators, Ca2+ overload, and subsequent activation of caspases.     

The genetic activity of NO-containing agents is regulated by complex formation with intracellular iron

This work is a part of a directional search for new crystal donors of nitric oxide (NO), which are promising for complex chemotherapy. The relationships between the physico-chemical properties of NO donors, their genotoxic and mutagenic activities, and the dependence on intracellular iron were studied. New crystal NO donors (di- and trinitrosyl iron complexes with synthetic ligands) were examined for the first time and compared with known NO donors containing natural ligands. All but one compound induced expression of the Escherichia coli sfiA gene belonging to the SOS regulon and exerted a mutagenic effect on Salmonella typhimurium TA1535. These effects were fully or significantly inhibited by the iron(II)-chelating agent o-phenanthrolin, depending on the mono- or binuclear structure of the ligands. The rate of donating free NO in solution did not positively correlate with the genotoxic activity of the crystal NO donors. The genetic activity of all NO donors proved to depend on intracellular iron.     

Nano carriers for nitric oxide delivery and its potential applications in plant physiological process: A mini review

Nitric oxide (NO) plays a key role in plant growth and defense. Since NO is a small molecule, devoid of charge and relatively lipophilic, it easily crosses cell membranes, acting as an important signaling messenger. Recently, several papers described the beneficial effects due to application of small molecular weight NO donors in plants. Exogenous NO donors break seed dormancy, stimulate plant germination and greening, control iron homeostasis in plants, and improve plant tolerance to salinity, metal toxicity, temperature and drought stress. However, these NO donors are thermally and photochemically unstable. A promising strategy that has been successfully used in biomedical applications is the combination of NO donors with nanomaterials. The encapsulation of NO donors in nanoparticles/nanotubes is able to control the release of therapeutic amounts of NO, thus improving its beneficial effects. Although nanomaterials have been used successfully to carry agrochemicals in plants, the delivery of NO is still to be studied. In this context, the present review highlights the advantages of applications of NO donors in plants, the uses of nanotechnology in agriculture, and the necessity to develop new strategies based on the combination of NO and nanomaterials in agriculture. Therefore, this review hopes to open up new perspectives in the area of nanobiotechnology, NO and agriculture.     

Relaxant effects of sodium nitroprusside and NONOates in goat middle cerebral artery: delayed impairment by global ischemia-reperfusion.

Global cerebral ischemia and subsequent reperfusion induce early impairment of the vasodilator responses to hypercapnia and vasoactive substances. Nitric oxide (NO) is involved in the regulation of cerebral blood flow (CBF) in both health and disease. The present study was designed to assess possible changes in the cerebrovascular reactivity to NO donors induced by cerebral ischemia-reperfusion in goats. Female goats (n = 9) were subjected to 20 min global cerebral ischemia under halothane/N2O anesthesia. Sixteen additional goats were sham-operated as a control group. One week later the effects of ischemia-reperfusion on relaxations to NO donors sodium nitroprusside (SNP), diethylamine/NO (DEA/NO), diethylenetriamine/NO (DETA/NO), and spermine/NO (SPER/NO) were studied in rings of middle cerebral artery (MCA) isolated in an organ bath for isometric tension recording. SNP, DEA/NO, DETA/NO, and SPER/NO induced concentration-dependent relaxations of MCA precontracted with KCl (DEA/NO > SPER/NO > SNP > DETA/NO) or with endothelin-1 (DEA/NO > SNP > SPER/NO > DETA/NO). Relaxations were always higher in endothelin-1-precontracted arteries. One week after cerebral ischemia concentration-response curves to SNP and DEA/NO were displaced to the right, indicating a reduction in relaxant potency of NO donors. The classical nitrovasodilator SNP and NONOates induce relaxation of isolated goat MCA which is partially inhibited by arterial depolarization. Global cerebral ischemia followed by reperfusion induces delayed impairment of the relaxant effects of NO on cerebrovascular smooth muscle, which results in reduced vasodilatory potency of NO donors in large cerebral arteries.     

The role of nitric oxide in regulation of the cardiovascular system in reptiles

The roles that nitric oxide (NO) plays in the cardiovascular system of reptiles are reviewed, with particular emphasis on its effects on central vascular blood flows in the systemic and pulmonary circulations. New data is presented that describes the effects on hemodynamic variables in varanid lizards of exogenously administered NO via the nitric oxide donor sodium nitroprusside (SNP) and inhibition of nitric oxide synthase (NOS) by l-nitroarginine methyl ester (l-NAME). Furthermore, preliminary data on the effects of SNP on hemodynamic variables in the tegu lizard are presented. The findings are compared with previously published data from our laboratory on three other species of reptiles: pythons (), rattlesnakes () and turtles (). These five species of reptiles possess different combinations of division of the heart and structural complexity of the lungs. Comparison of their responses to NO donors and NOS inhibitors may reveal whether the potential contribution of NO to vascular tone correlates with pulmonary complexity and/or with blood pressure. All existing studies on reptiles have clearly established a potential role for NO in regulating vascular tone in the systemic circulation and NO may be important for maintaining basal systemic vascular tone in varanid lizards, pythons and turtles, through a continuous release of NO. In contrast, the pulmonary circulation is less responsive to NO donors or NOS inhibitors, and it was only in pythons and varanid lizards that the lungs responded to SNP. Both species have a functionally separated heart, so it is possible that NO may exert a larger role in species with low pulmonary blood pressures, irrespective of lung complexity.     

Astrocytes protect neurons from nitric oxide toxicity by a glutathione-dependent mechanism

Nitric oxide (NO) contributes to neuronal death in cerebral ischemia and other conditions. Astrocytes are anatomically well positioned to shield neurons from NO because astrocyte processes surround most neurons. In this study, the capacity of astrocytes to limit NO neurotoxicity was examined using a cortical co-culture system. Astrocyte-coated dialysis membranes were placed directly on top of neuronal cultures to provide a removable astrocyte layer between the neurons and the culture medium. The utility of this system was tested by comparing neuronal death produced by glutamate, which is rapidly cleared by astrocytes, and N-methyl-D-aspartate (NMDA), which is not. The presence of an astrocyte layer increased the LD(50) for glutamate by approximately four-fold, but had no effect on NMDA toxicity. Astrocyte effects on neuronal death produced by the NO donors S-nitroso-N-acetyl penicillamine and spermine NONOate were examined by placing these compounds into the medium of co-cultures containing either a control astrocyte layer or an astrocyte layer depleted of glutathione by prior exposure to buthionine sulfoximine. Neurons in culture with the glutathione-depleted astrocytes exhibited a two-fold increase in cell death over a range of NO donor concentrations. These findings suggest that astrocytes protect neurons from NO toxicity by a glutathione-dependent mechanism.     

Influence of nitric oxide donors and peroxynitrite on the contractile effect and concentration of norepinephrine

Nitric oxide (NO) and peroxynitrite (ONOO) are said to destroy norepinephrine (NE). We studied the role of NE decomposition by NO donors and ONOO as they affect the contractile activity of NE in rat denuded thoracic aorta. First, we determined the relaxing effect of NO donors (SNAP, PROLI/NO, Sodium nitrite, SIN-1) and ONOO after precontraction by NE (1 microM). SNAP and SIN-1 (EC(50) 50-110 nM) were more active than PROLI/NO, Sodium nitrite or ONOO (EC(50) 19-30 microM). The relaxing effect of NO donors and ONOO were decreased by ODQ (10 microM), a guanylate cyclase inhibitor. Second, we compared the contractile activity of NE before and after preincubation with NO donors or ONOO in presence of ODQ. NE (1 microM) was incubated with NO donors or ONOO at the concentrations of 0.1 mM in both Krebs solution or phosphate buffer (pH 7.4; 0.1 M) for 10 minutes at 37 degrees C. NE evoked the aorta contraction in the same concentrations before and after preincubation with NO donors. In contrast, ONOO decreased effect of NE, EC(50) was measured at 4.3+/-0.3 nM and 13.4+/-1.6 nM, before and after preincubation of NE with ONOO respectively. Third, we measured the NE concentration using the HPLC method. We revealed that the concentration of NE after preincubation with NO donors was unaltered. However HPLC measurement revealed that NE concentration after preincubation with ONOO was reduced 2-3-fold. Therefore, under these experimental conditions ONOO, but not NO donors, was capable of destroying NE.     

Intracellular iron ions regulate the genetic activity of NO-donating agents

This work is a part of a directional search for new crystal donors of nitric oxide (NO), which are promising for complex chemotherapy. The relationships between the physico-chemical properties of NO donors, their genotoxic and mutagenic activities, and the dependence on intracellular iron were studied. New crystal NO donors (di-and trinitrosyl iron complexes with synthetic ligands) were examined for the first time and compared with known NO donors containing natural ligands. All but one compound induced expression of the Escherichia coli sfiA gene belonging to the SOS regulon and exerted a mutagenic effect on Salmonella typhimurium TA1535. These effects were fully or significantly inhibited by the iron(II)-chelating agent o-phenanthrolin, depending on the mono-or binuclear structure of the ligands. The rate of donating free NO in solution did not positively correlate with the genotoxic activity of the crystal NO donors. The genetic activity of all NO donors proved to depend on intracellular iron.     

Reversible S-nitrosation and inhibition of HIV-1 protease.

Nitric oxide (*NO) is a short-lived free radical with many functions including vasoregulation, synaptic plasticity, and immune modulation and has recently been associated with AIDS pathology. Various pathophysiological conditions, such as viral infection, trigger inducible nitric oxide synthase (iNOS) to synthesize NO in the cell. NO-derived species can react with thiols of proteins and form nitrosothiol adducts. HIV-1 protease (HIV-PR) contains two cysteine residues, Cys67 and Cys95, which are believed to serve a regulatory function. We have found that HIV-PR is inactivated by nitric oxide produced in vitro by NO donors and by iNOS. Sodium nitroprusside inhibited HIV-PR by 70%, and S-nitroso-N-acetylpenicillamine completely inhibited the enzyme. Furthermore, iNOS generated sufficient NO to inhibit HIV-PR activity by almost 90%. This inactivation was reversed by the addition of reducing agents. Treatment of HIV-PR with NO donors and ritonavir (a competitive peptide inhibitor) indicates that NO exerts its effect through a site independent of the active site of HIV-PR. Using electrospray ionization mass spectrometry, we found that NO forms S-nitrosothiols on Cys67 and Cys95 of HIV-PR which directly correlate with a loss of activity. These data indicate that NO may suppress HIV-1 replication by directly inhibiting HIV-PR.     

Nitric oxide acts as an antioxidant and delays programmed cell death in barley aleurone layers

Nitric oxide (NO) is a freely diffusible, gaseous free radical and an important signaling molecule in animals. In plants, NO influences aspects of growth and development, and can affect plant responses to stress. In some cases, the effects of NO are the result of its interaction with reactive oxygen species (ROS). These interactions can be cytotoxic or protective. Because gibberellin (GA)-induced programmed cell death (PCD) in barley (Hordeum vulgare cv Himalaya) aleurone layers is mediated by ROS, we examined the effects of NO donors on PCD and ROS-metabolizing enzymes in this system. NO donors delay PCD in layers treated with GA, but do not inhibit metabolism in general, or the GA-induced synthesis and secretion of alpha-amylase. alpha-Amylase secretion is stimulated slightly by NO donors. The effects of NO donors are specific for NO, because they can be blocked completely by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The antioxidant butylated hydroxy toluene also slowed PCD, and these data support our hypothesis that NO is a protective antioxidant in aleurone cells. The amounts of CAT and SOD, two enzymes that metabolize ROS, are greatly reduced in aleurone layers treated with GA. Treatment with GA in the presence of NO donors delays the loss of CAT and SOD. We speculate that NO may be an endogenous modulator of PCD in barley aleurone cells.     

Expression and roles of Cl- channel ClC-5 in cell cycles of myeloid cells

This study investigated the effect of exogenous nitric oxide (NO) on endothelial glucocorticoid receptor (GR) function. The NO donor diethylenetriamine NONOate (DETA, 50-500microM) caused concentration dependent nuclear localization of transfected chimeric green fluorescent protein GFP-GR and elevated expression of secreted alkaline phosphatase (SEAP) from a glucocorticoid response element (GRE) promoter construct in bovine aortic endothelial cells. Other weaker NO donors (S-nitroso-N-acetylpenicillamine and spermine NONOate) failed to induce GFP-GR nuclear localization, but all the NO donors activated GRE-SEAP expression, a response unaffected by the antioxidant N-acetyl-L-cysteine. Overall, exogenous NO from high concentration donors can directly activate GR, suggesting a potential feedback mechanism for NO to regulate endothelial inducible nitric oxide synthase (iNOS) expression.     

Nitric oxide modulates ionic transport in the isolated intestine of the eel, Anguilla anguilla

We investigated the role of NO (nitric oxide) in the isolated intestine of the sea water adapted eel, by testing the effect of various donors on I(sc) (short-circuit current), due to net Cl(-) absorption in the control conditions. We found that the endogenous NO-synthase substrate l-arginine as well as two different NO donors, SNP (sodium nitroprusside) and SIN-1 (3-morpholinosydnonimine), produced a slow and gradual decrease of I(sc). The effect of SNP was reduced by the pretreatment with ODQ (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one), a specific inhibitor of the soluble guanylyl cyclase, suggesting the involvement of cGMP (cyclic GMP) in some physiological actions of NO. The effect of the NO donors on I(sc) was similar to that observed when the tissues were perfused with solution in which the HCO(3)(-) buffer was substituted with Hepes buffer. In addition the NO donors produced a negligible effect on I(sc) when the tissues were perfused with Hepes buffer or in the presence of bilateral SITS(4-Acetoamido-4'-iso-thiocyanatostilbene-2,2'disulphonic acid), an inhibitor of the HCO(3)(-) transport mechanisms, operating on both cell membranes of the eel enterocyte and responsible for HCO(3)(-) uptake by the cell. Based on these observations we suggest that NO regulates I(sc) and hence the transepithelial ion transport indirectly by modulating the endocellular concentration of HCO(3)(-) and/or H(+). In addition it is likely that NO modulates the permeability of the paracellular pathway since SNP produced also an increase of the tissue conductance and a decrease of the magnitude of the dilution potential.     

Visible light-induced nitric oxide release from a novel nitrobenzene derivative cross-conjugated with a coumarin fluorophore

Nitric oxide (NO) is a well-known free-radical molecule which is endogenously biosynthesised and shows various functions in mammals. To investigate NO functions, photocontrollable NO donors, compounds which release NO in response to light, are expected to be potentially useful. However, most of the conventional NO donors require harmful ultra-violet light for NO release. In this study, two dimethylnitrobenzene derivatives conjugated with coumarins were designed, synthesized and evaluated as photocontrollable NO donors. The optical properties and efficiency of photo-induced NO release were dependent upon the nature of the conjugation system. One of these compounds, Bhc-DNB (1), showed spatiotemporally well-controlled NO release in cultured cells upon exposure to light in the less-cytotoxic visible wavelength range (400–430 nm).