NO Donation Research Articles

Nitric oxide and mitochondria injuries in kidney tissue upon a simulation of cardiopulmonary bypass and circulatory arrest: an experimental study

Introduction: Surgical interventions under cardiopulmonary bypass and circulatory arrest are complicated by impaired microcirculation in tissues; during hypoxic period the mitochondria dysfunction becomes the leading pathogenetic factor of ischemia-reperfusion injuries. In this regard, the search for methods of organ protection seems to be an extremely urgent task. The using of nitric oxide can be a promising technique given its pluripotent properties.Objective: The study was aimed to examining of mitochondria injuries in kidney biopsies stipulated by cardiopulmonary bypass and circulatory arrest when simulating cardiac surgery, as well as to assessing the potentials of organ protection with nitric oxide in the experiment.Methods: The study was carried out on Altai breed sheep and included simulating of cardiopulmonary bypass and circulatory arrest. The first group (n = 6) was provided with intraoperative nitric oxide donation. The second group (n = 6) served as a control (without nitric oxide donation). Mitochondria injuries were assessed in kidney biopsies by measuring transmembrane potential and Ca2+-binding capacity of organelles, as well as by determining ATP and lactate concentrations.Results: Nitric oxide therapy was associated with less mitochondria dysfunction in kidney biopsies compared with the control group. There was a steady trend to the amelioration of the energy maintenance in the renal parenchyma in the nitric oxide donation group.Conclusion: A steady trend towards optimizing the energy supply of tissues under cardiopulmonary bypass and circulatory arrest was revealed in an experiment against the background of NO donation at a concentration of 80 ppm. Received 11 February 2024. Revised 28 March 2024. Accepted 29 March 2024. Funding: The The study was carried out within the framework of the state assignment “Protection of organs by nitric oxide in cardiovascular surgery: technological support (synthesis and delivery devices), mechanisms for implementing protective effects and impact on clinical outcomes” (topic No. 122123000017-3). Conflict of interest: The authors declare no conflict of interest. Contribution of the authors Conception and study design: A.M. Boyko, N.O. Kamenshchikov, Yu.K. Podoksenov, L.N. Maslov, B.N. Kozlov Data collection and analysis: A.M. Boyko, Yu.S. Svirko, V.A. Lugovskiy, A.V. Mukhomedzyanov, B.A. BazarbekovaStatistical analysis: I.V. KravchenkoDrafting the article: A.M. Boyko Critical revision of the article: N.O. Kamenshchikov, A.G. Miroshnichenko, Yu.K. Podoksenov, M.L. Diakova, K.A. Petlin, D.S. Panfilov, B.N. KozlovFinal approval of the version to be published: A.M. Boyko, N.O. Kamenshchikov, A.G. Miroshnichenko, Yu.K. Podoksenov, Yu.S. Svirko, V.A. Lugovskiy, M.L. Diakova, I.V. Kravchenko, A.V. Mukhomedzyanov, L.N. Maslov, B.A. Bazarbekova, K.A. Petlin, D.S. Panfilov, B.N. Kozlov

Kinetic regularities of NO donation by binuclear dinitrosyl iron complexes with thiolate ligands based on thiophenol derivatives in the presence of red blood cells

The kinetics of nitrogen oxide release by binuclear dinitrosyl iron complexes (B-DNICs) with thiolate ligands based on thiophenol and its several oxy, amino, and nitro derivatives was studied using a suspension of red blood cells simulating the internal medium of a blood vessel. The NO donating ability of the complexes was estimated by the kinetic parameters of first-order equation, which described the formation of intra-erythrocytic methemoglobin. Three typical kinetic profiles of NO donation were distinguished: pseudosaturation donation and donation with prolonged and explosive profiles. In the case of first-type NO donation typical of complexes with thiophenol and its nitro derivatives, the curves display a fast coming to saturation long before the complete release of all NO groups contained in the structure of the starting complex into a solution. Such a type of donation is likely due to the formation of long-lived nitrosyl intermediates in the system. The prolonged type of NO donation shown by the complex with hydroxyphenyl ligand is characterized by virtually constant rate of NO release into a solution without pronounced transition to saturation during experiment (10–12 min). In the case of explosive-type donation characteristic of the complex with aminophenyl ligands, a considerable portion of NO was fast released into a solution within 1–3 min. All complexes under study caused hemolysis of a 0.2% suspension of red blood cells. The complex with aminophenyl ligands exhibited the highest hemolytic activity.

Effect of albumin on the transformation of dinitrosyl iron complexes with thiourea ligands

Interaction and transformation of the mononuclear cationic dinitrosyl iron complex [Fe(SC(NH2)2)2(NO)2]+ (complex 1) upon binding with bovine serum albumin (BSA) have been explored using kinetic measurements, UV-Vis and fluorescence spectroscopy, and computational molecular modeling. BSA was found to bind up to five molecules of complex 1 per one protein molecule; as a result, the rate of NO release by complex 1 into solution decreases by a factor of 10. The binding constant of complex 1 with BSA measured by the quenching of intrinsic fluorescence of BSA is 5 × 105 М-1. Molecular docking calculations at pH = 7 have determined five-six low-energy binding sites for complex 1 at subunits I and II of BSA. The most stable protein-ligand complexes are located at the protein pockets near Cys34. The spectroscopic measurements and docking calculations have shown that the decomposition product of complex 1, the Fe(NO)2+ fragment, can form an adduct Fe(Cys34)(His39)(NO)2 (complex 2) with the coordination bonds of Fe with atoms S of Cys34 and ND of His39. The structure of complex 2 was supported by the density functional calculations of the absorption spectrum. Decomposition of complex 2 leads to nitrosylation of BSA at atom S of Cys34. Complexes 1 (bound with BSA), 2 and the nitrosylated BSA can serve as NO depot in plasma.

Influence of hemoglobin and albumin on the NO donation effect of tetranitrosyl iron complex with thiosulfate

The effects of deoxyhemoglobin (Hb) and albumin on the NO-donor activity of the anionic tetranitrosyl iron complex with thiosulfate ligands (1) were studied for the first time. It was shown that Hb significantly stabilizes complex 1; in its presence, NO generation from the complex proceeds at a noticeably slower rate. A similar effect is observed when complex 1 is bound to albumin, in which case complex 1 decomposes 27 times slower than in the absence of albumin in the solution. The observed effects provide a prolonged action of complex 1 as NO-donor, which may enhance its potential pharmacological efficacy.

Comprehensive study of nitrofuroxanoquinolines. New perspective donors of NO molecules

The goal of present work is the study of NO releasing mechanisms in nitrofuroxanoquinoline (NFQ) derivatives. Mechanisms of their structural non-rigidity and pathways of NO donation - spontaneous or under the action of sulfanyl radicals or photoirradiation - were considered in details, both experimentally and quantum chemically. Furoxan-containing systems of the discussed type are not capable of spontaneous or photoinduced decomposition under mild conditions, and sulfanyl (radical) induced processes are the most preferable. It was shown that appropriate modification of NFQ through [3 + 2] cycloaddition and subsequent aromatization is a powerful tool to design new prospective donors of NO molecule. Two newly obtained NFQ derivatives were proven to have unusually high NO activity in full accordance with the theoretical model. We hope that these examples will encourage community to seek for new NO active molecules among cycloadducts and modified furoxanes.

Quantum-chemical modeling of possible reactions of Roussin’s red esters with aryl ligands in DMSO solution

Possible reactions of Roussin’s red esters [Fe2(μ-SC6H4R)2(NO)4], where R = H, o-NH2, m-NO2, m-OH, or m-NH2, in DMSO solution were investigated by quantum chemical modeling. It was shown that in these systems, numerous reactions can occur, including NO donation, ligand substitution, and decomposition into mononuclear iron nitrosyl complexes. The resulting compounds are also NO donors.

Adaptive responses of rat descending vasa recta to ischemia

tested whether rat descending vasa recta (DVR) undergo regulatory adaptations after the kidney is exposed to ischemia. Left kidneys (LK) were subjected to 30-min renal artery cross clamp. After 48 h, the postischemic LK and contralateral right kidney (RK) were harvested for study. When compared with DVR isolated from either sham-operated LK or the contralateral RK, postischemic LK DVR markedly increased their NO generation. The selective inducible NOS (iNOS) inhibitor 1400W blocked the NO response. Immunoblots from outer medullary homogenates showed a parallel 2.6-fold increase in iNOS expression ( P = 0.01). Microperfused postischemic LK DVR exposed to angiotensin II (ANG II, 10 nM), constricted less than those from the contralateral RK, and constricted more when exposed to 1400W (10 µM). Resting membrane potentials of pericytes from postischemic LK DVR pericytes were hyperpolarized relative to contralateral RK pericytes (62.0 ± 1.6 vs. 51.8 ± 2.2 mV, respectively, P < 0.05) or those from sham-operated LK (54.9 ± 2.1 mV, P < 0.05). Blockade of NO generation with 1400W did not repolarize postischemic pericytes (62.5 ± 1.4 vs. 61.1 ± 3.4 mV); however, control pericytes were hyperpolarized by exposure to NO donation from S-nitroso- N-acetyl- dl-penicillamine (51.5 ± 2.9 to 62.1 ± 1.4 mV, P < 0.05). We conclude that postischemic adaptations intrinsic to the DVR wall occur after ischemia. A rise in 1400W sensitive NO generation and iNOS expression occurs that is associated with diminished contractile responses to ANG II. Pericyte hyperpolarization occurs that is not explained by the rise in ambient NO generation within the DVR wall.

Thiol-induced nitric oxide donation mechanisms in substituted dinitrobenzofuroxans

The goal of present work is the quantum chemical study of NO donation mechanism in dinitrobenzofuroxan aryl derivative. Mechanisms of its structural non-rigidity (1,3-N-oxidic and Boulton-Katritzky rearrangements) and minimum energy pathways of NO donation under the action of sulfanyl radical SH· were considered in details. DFT calculations were performed using B3LYP and UB3LYP functionals in the 6–311++G(d,p) basis set. Obtained results showed that a high experimentally proven NO-donor activity of dinitrobenzofuroxan aryl derivative is connected with its existence in the form of mixture of 1-N-oxide and 3-N-oxide, where the 3-N-oxide is more reactive towards SH·. The thiol-induced low-barrier mechanism of NO-donation is a result of para-aminophenyl substituent availability in position 7 of dinitrobenzofuroxan.

Nitric oxide donation by the binuclear tetranitrosyl iron complexes in the presence of erythrocytes

The kinetic regularities of nitric oxide donation in the presence of erythrocytes by representatives of a new class of synthetic nitric oxide donors, binuclear tetranitrosyl iron complexes (B-TNIC) [Fe2(SR)2(NO)4] with thiol-containing ligands, where R is pyrimidin-2-yl, 1-methylimidazol-2-yl, benzothiazol-2-yl, and penicillamine residue, were established. The NO-donating ability of B-TNIC was estimated from the apparent rate constants of the first order for the formation of intraerythrocyte methemoglobin with the variation of the initial concentration of the complex. During the standard experimental time (15—17 min), three of the four complexes released in the solution no more than a quarter of available NO groups. Their NO-donating ability turned out to be variable increasing with an increase in the initial concentration of the complex. At the same time, the complex bearing penicillamine residues as ligands donated almost all NO groups within approximately 1 min. Features of NO donation by the B-TNIC in the presence of erythrocytes are related to the formation in the system of an additional equilibrium pool of the membrane-associated complex, which is characterized by a reduced rate of hydrolytic dissociation with NO releasing to the solution. The NO-donating ability of the B-TNIC in the presence of erythrocytes is determined by the ratio of volumes of the free and membrane-associated pools of the complex.

Ruthenium dinitrosyl complexes – computational characterization of structure and reactivity

Elucidation of the electronic structure of a dinitrosyl dithiolate ruthenium complex in several formal oxidation states ranging from Ru(I) to Ru(III) has been undertaken. DFT and ab initio molecular dynamics simulations have shown clear evidence of asymmetry within the dinitrosyl moieties in all models though most noticeably in the excited states. The reaction pathway of a hyponitrite adduct formation was also examined and found to be more feasible in the excited states. These results, along with the recently reported study on the dinitrosyl dithiolate iron analog of these complexes, provide insight toward the mechanism of NO donation by dinitrosyl metal complexes.

Linked Thioredoxin-Glutathione Systems in Platyhelminth Parasites: ALTERNATIVE PATHWAYS FOR GLUTATHIONE REDUCTION AND DEGLUTATHIONYLATION

In most organisms, thioredoxin (Trx) and/or glutathione (GSH) systems are essential for redox homeostasis and deoxyribonucleotide synthesis. Platyhelminth parasites have a unique and simplified thiol-based redox system, in which the selenoprotein thioredoxin-glutathione reductase (TGR), a fusion of a glutaredoxin (Grx) domain to canonical thioredoxin reductase domains, is the sole enzyme supplying electrons to oxidized glutathione (GSSG) and Trx. This enzyme has recently been validated as a key drug target for flatworm infections. In this study, we show that TGR possesses GSH-independent deglutathionylase activity on a glutathionylated peptide. Furthermore, we demonstrate that deglutathionylation and GSSG reduction are mediated by the Grx domain by a monothiolic mechanism and that the glutathionylated TGR intermediate is resolved by selenocysteine. Deglutathionylation and GSSG reduction via Grx domain, but not Trx reduction, are inhibited at high [GSSG]/[GSH] ratios. We found that Trxs (cytosolic and mitochondrial) provide alternative pathways for deglutathionylation and GSSG reduction. These pathways are operative at high [GSSG]/[GSH] and function in a complementary manner to the Grx domain-dependent one. Despite the existence of alternative pathways, the thioredoxin reductase domains of TGR are an obligate electron route for both the Grx domain- and the Trx-dependent pathways. Overall, our results provide an explanation for the unique array of thiol-dependent redox pathways present in parasitic platyhelminths. Finally, we found that TGR is inhibited by 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7), giving further evidence for NO donation as a mechanism of action for oxadiazole N-oxide TGR inhibitors. Thus, NO donors aimed at TGR could disrupt the entire redox homeostasis of parasitic flatworms.

Synthesis, spectroscopic, thermal and biological activity studies on triazine metal complexes

The coordination behaviour of the triazine ligand with NNO donation sites, derived from 3-benzyl-7-hydrazinyl-4H-[1,3,4]thiadiazolo[2,3c][1,2,4]triazin-4-one (HL), towards some metal ions namely Mn(II), Fe(III), Co(II), Ni(II), Cu(II) and Zn(II) are reported. The metal complexes are characterized based on elemental analyses, IR, 1H NMR, solid reflectance, magnetic moment, molar conductance and thermal analyses (TG, DTG and DTA). The ionization constants of the organic ligand under investigation as well as the stability constants of its metal chelates are calculated spectrophotometrically at 25 °C. The chelates are found to have octahedral geometrical structures. The ligand (HL) and its binary chelates are subjected to thermal analyses (TG, DTG and DTA) and the different activation thermodynamic parameters are calculated from their corresponding DTG curves to throw more light on the nature of changes accompanying the thermal decomposition process of these compounds. The synthesized ligand and its metal complexes were found to have biological activity against the desert locust Schistocerca gregaria (Forsk.) (Orthoptera – Acrididae) and its adult longevities.

Structure Mechanism Insights and the Role of Nitric Oxide Donation Guide the Development of Oxadiazole-2-Oxides as Therapeutic Agents against Schistosomiasis

Schistosomiasis is a chronic parasitic disease affecting hundreds of millions of individuals worldwide. Current treatment depends on a single agent, praziquantel, raising concerns of emergence of resistant parasites. Here, we continue our explorations of an oxadiazole-2-oxide class of compounds we recently identified as inhibitors of thioredoxin glutathione reductase (TGR), a selenocysteine-containing flavoenzyme required by the parasite to maintain proper cellular redox balance. Through systematic evaluation of the core molecular structure of this chemotype, we define the essential pharmacophore, establish a link between the nitric oxide donation and TGR inhibition, determine the selectivity for this chemotype versus related reductase enzymes, and present evidence that these agents can be modified to possess appropriate drug metabolism and pharmacokinetic properties. The mechanistic link between exogenous NO donation and parasite injury is expanded and better defined. The results of these studies verify the utility of oxadiazole-2-oxides as novel inhibitors of TGR and as efficacious antischistosomal agents.

Effects of nitric oxide donor and nitric oxide synthase inhibitor on the resistance, exchange and capacitance functions of the canine intestinal vasculature

In the present study, we determined the vascular functions using a canine model of isolated intestinal segment perfused with constant flow. The effects of an NO donor, S-nitroso- N-acetylpenicillamine (SNAP) and an NO synthase inhibitor, N ω-nitro- l-arginine methyl ester ( l-NAME) on the vascular factors (resistance, exchange and capacitance) were evaluated. In condition of venous pressure at 0 mmHg, we determined and calculated arterial pressure (Pa) and capillary pressure (Pc). Vascular factors including total, pre- and post-capillary resistance ( R T, Ra and Rv), vascular compliance (VC) and capillary filtration coefficient ( K fc) were obtained. SNAP at doses 10 − 6 to 10 − 4 mol/l produced vasodilatory effects. It dose-dependently reduced the Pa, Pc, R T and Ra, as well as the Ra/Rv ratio. The Rv was slightly decreased. This agent increased the vascular capacity, VC and K fc. NO inhibition with l-NAME (10 − 6 to 10 − 4 mol/l) produced the opposite effects. The vasoconstrictory effects of l-NAME increased Pa, Pc, R T and Ra as well as the Ra/Rv ratio. It slightly raised the Rv. l-NAME reduced the vascular capacity, VC and K fc. The effects of l-NAME were also dose-dependent. This study has provided a detailed data of the vasodilatory and vasoconstrictory effects NO donation and inhibition on vascular factors in the intestinal vasculature.

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.

Nitric Oxide Donation Lowers Blood Pressure in Adrenocorticotrophic Hormone‐Induced Hypertensive Rats

Adrenocorticotrophic hormone (ACTH) elevates systolic blood pressure (SBP) and lowers plasma reactive nitrogen intermediates in rats. We assessed the ability of NO donation from isosorbide dinitrate (ISDN) to prevent or reverse the hypertension caused by ACTH. In the prevention study, male Sprague Dawley rats were treated with ACTH (0.2 mg/kg/day) or saline control for 8 days, with either concurrent ISDN (100 mg/kg/day) via the drinking water or water alone. Animals receiving ISDN via the drinking water were provided with nitrate‐free water for 8 hours every day. In the reversal study ISDN (100 mg/kg) or vehicle was given as a single oral dose on day 8. SBP was measured daily by the indirect tail‐cuff method in conscious, restrained rats. ACTH caused a significant increase in SBP compared with saline (P < 0.0015). In the prevention study, chronic administration of ISDN (100 mg/kg/day) did not affect the SBP in either group. In the reversal study, ISDN significantly lowered SBP in ACTH‐treated rats at 1 and 2.5 hours (132 ± 3 mmHg (1 h) and 131 ± 2 mmHg (2.5 h) versus 143 ± 3 mmHg (0 h), P < 0.002), but not to control levels. It had no effect in control (saline treated) rats. In conclusion, the lowering of SBP by NO donation is consistent with the notion that ACTH‐induced hypertension involves an impaired bioavailability or action of NO in vivo.

Vasoactive Responses of Veins Isolated from the Human Placental Chorionic Plate

The control of the blood flow within the fetoplacental circulation is poorly understood despite the essential role of the placenta in pregnancy. Our aim was to assess the vasoactive responses of veins from the placental chorionic plate. Biopsies were obtained from term placentae from uncomplicated pregnancies. Small veins from the chorionic plate were dissected free from surrounding tissue and studied using parallel wire myography. Human placental chorionic plate veins developed maintained constrictions to the thromboxane-mimetic U46619. Endothelium-dependent agonists did not promote venous relaxation. However, NO donation with the endothelial-independent agent, sodium nitroprusside, elicited significant relaxation. Venous constriction to U46619 and relaxation to sodium nitroprusside were modified by adjustment of media oxygen tension and normalization parameters. Human placental chorionic plate veins respond to vasoactive agents and may play a role in the control of the blood flow in the fetoplacental circulation.

Mechanism of S-nitrosation of recombinant human brain calbindin D28K.

Mass spectrometry and UV-vis absorption results support a mechanism for NO donation by S-nitrosoglutathione (GSNO) to recombinant human brain calbindin D(28K) (rHCaBP) that requires the presence of trace copper, added as either Cu,Zn-superoxide dismutase (CuZnSOD) or CuSO(4). The extent of copper-catalyzed rHCaBP S-nitrosation depends on the ratio of protein to GSNO and on the reaction time, and NO-transfer is prevented when copper chelators are present. CuZnSOD is an efficient catalyst of rHCaBP S-nitrosation, and the mechanism of CuZnSOD-catalyzed S-nitrosation involves reduction of the active-site Cu(II) by a number of the five free thiols in rHCaBP, giving rise to thiyl radicals. The Cu(I)ZnSOD formed catalyzes the reductive cleavage of GSNO present in solution to give GSH and release NO. rHCaBP thiyl radicals react with NO to yield the S-nitrosoprotein. Cu(II)ZnSOD is also reduced by GSH in a concentration-dependent manner up to 5 mM but not at higher GSH concentrations. However, unlike the rHCaBP thiyl radicals, GS(*) radicals dimerize to GSSG faster than their reaction with NO. The data presented here provide a biologically relevant mechanism for protein S-nitrosation by small S-nitrosothiols. S-nitrosation is rapidly gaining recognition as a major form of protein posttranslational modification, and the efficient S-nitrosation of CaBP by CuZnSOD/GSNO is speculated to be of neurochemical importance given that CaBP and CuZnSOD are abundant in neurons.

Inflammatory cytokines stimulate adrenomedullin expression through nitric oxide-dependent and -independent pathways.

A body of evidence indicates that the production of adrenomedullin (ADM) in vivo is activated in states of inflammation. Our aim was to characterize the intracellular signaling pathways along which inflammation leads to a stimulation of ADM expression. For this purpose, we characterized the effects of inflammatory cytokines, tumor necrosis factor-alpha (100 microg/L), interleukin-1beta (20 microg/L), and interferon-gamma (0.5 U/L) on ADM gene expression in rat aortic vascular smooth muscle cells (AVSMCs). We found that inflammatory cytokines induced a time-dependent 12-fold upregulation of ADM mRNA in AVSMCs that was paralleled by a substantial increase in inducible NO synthase mRNA expression. The stimulatory effect of cytokines on ADM gene expression was attenuated by NO deprivation induced by Nomega-nitro-L-arginine methyl ester (1 mmol/L) and was in part mimicked by the NO donor S-nitroso-N-acetylpenicillamine (100 micromol/L). The cGMP analog 8-bromo-cGMP (100 micromol/L) had no effect on ADM gene expression, and inhibition of cGMP production by 1H-oxodiazolo-quinoxalin-1 (ODQ, 200 micromol/L) was not able to abrogate the increase of ADM mRNA induced by NO donation using S-nitroso-N-acetylpenicillamine (100 micromol/L). The significant induction of ADM gene expression by inflammatory cytokines and NO donation was also observed in mesangial cells, endothelial cells, and hepatocytes. These findings suggest that NO is a direct activator of ADM gene expression in a variety of cell types and that inflammatory cytokines stimulate ADM expression via both NO-dependent and -independent mechanisms. The stimulatory effect of NO appears to not be related to the classic guanylate cyclase-cGMP pathway.

In vivo nitric oxide transfer of a physiological NO carrier, dinitrosyl dithiolato iron complex, to target complex

Dinitrosyl dithiolato iron complex (DNIC) has been identified as an endogenous NO carrier, yet in vivo mechanisms of NO donation remain undefined. Transnitrosylation, in which a coordinated NO group is transferred to another metal complex, has been observed in transition-metal-nitrosyl chemistry. In this study, we used three kinds of iron dithiocarbamate complexes (Fe-DTCs) as NO acceptors to elucidate in vivo transnitrosylation of diglutathionyl dinitrosyl iron complex [DNIC-(GS) 2]. Fe-DTCs were administered to mice after the injection of DNIC-(GS) 2 and electron paramagnetic resonance (EPR) spectra were measured both in the resected organs and in the upper abdomen of living mice. The spectral feature gradually changed from an initial DNIC-(GS) 2 signal to mononitrosyl iron dithiocarbamate one, suggesting that NO-Fe-DTC was formed through in vivo reaction of DNIC-(GS) 2 with Fe-DTC. The spectral results in in vitro and in vivo systems indicate that NO-Fe-DTCs can be formed not only by the transfer of coordinated NO-group(s) in DNIC-(GS) 2 but also by the abstraction of Fe-NO group in DNIC-(GS) 2 by free DTC ligands. Transnitrosylation proceeded more rapidly in blood than in liver and kidney; and more efficiently in kidney than in liver. Further, the ability to accept NO from DNIC was dependent on water-solubility of Fe-DTCs. Thus, in vivo transnitrosylation from DNIC to exogenous iron complex could be observed and this reaction was influenced by biological constituents and properties of iron complex. These results demonstrate that the transnitrosylation from DNIC to intrinsic NO acceptors like metalloproteins has a probable significance in in vivo NO transfer process.