Influence of ancillary ligand L in the nitric oxide photorelease by the [Ru(L)(tpy)NO] complex and its vasodilator activity based on visible light irradiation

The photochemical and pharmacological studies of the novel [Ru(L)(tpy)NO]³⁺ L = bpy (2,2′-bipyridine), NH · NHq (quinonediimine) and NH₂.NH₂cat (o-phenylenediamine) were investigated in aqueous medium. The synthesized nitrosyl ruthenium complexes showed nitric oxide (NO) release under light irradiation at 355 nm for [Ru(L)(tpy)NO]³⁺ complex with quantum yield of 0.14 ± 0.02, 0.47 ± 0.03 and 0.46 ± 0.02 mol Einstein⁻¹ for L = bpy, NH · NHq and NH₂ · NH₂cat, respectively, and 0.0065 ± 0.001 mol Einstein⁻¹ for light irradiation at 532 nm for [Ru(NH · NHq)(tpy)NO]³⁺ complex. The photochemical pathway at 355 nm light irradiation was described as a multi-step mechanism, although at 532 nm it was better attributed to a photo-induced electron transfer. The vasorelaxation induced by NO release produced by light irradiation in visible region from physiological solution of [Ru(NH · NHq)(tpy)NO]³⁺ complex was evaluated and compared with sodium nitroprusside (SNP). The results showed very similar vasodilator power between both species.     

The macrocyclic effect and vasodilation response based on the photoinduced nitric oxide release from trans-[RuCl(tetraazamacrocycle)NO](2+)

Irradiation of trans-[RuCl(cyclam)(NO)](2+), cyclam is 1,4,8,11-tetraazacyclotetradecane, at pHs 1-7.4, with near UV light results in the release of NO and formation of trans-[Ru(III)Cl(OH)(cyclam)](+) with pH dependent quantum yields (from approximately 0.01 to 0.16 mol Einstein(-1)) lower than that for trans-[RuCl([15]aneN(4))(NO)](2+), [15]aneN(4) is 1,4,8,12-tetaazacyclopentadecane, (0.61 mol Einstein(-1)). After irradiation with 355 nm light, the trans-[RuCl([15]aneN(4))(NO)](2+) induces relaxation of the aortic ring, whereas the trans-[RuCl(cyclam)(NO)](2+) complex does not. The relaxation observed with trans-[RuCl([15]aneN(4))(NO)](2+) is consistent with a larger quantum yield of release of NO from this complex.     

Photocytotoxic activity of a nitrosyl phthalocyanine ruthenium complex--a system capable of producing nitric oxide and singlet oxygen

The synthesis, structural aspects, pharmacological assays, and in vitro photoinduced cytotoxic properties of [Ru(NO)(ONO)(pc)] (pc = phthalocyanine) are described. Its biological effect on the B16F10 cell line was studied in the presence and absence of visible light irradiation. At comparable irradiation levels, [Ru(NO)(ONO)(pc)] was more effective than [Ru(pc)] at inhibiting cell growth, suggesting that occurrence of nitric oxide release following singlet oxygen production upon light irradiation may be an important mechanism by which the nitrosyl ruthenium complex exhibits enhanced biological activity in cells. Following visible light activation, the [Ru(NO)(ONO)(pc)] complex displayed increased potency in B16F10 cells upon modifications to the photoinduced dose; indeed, enhanced potency was detected when the nitrosyl ruthenium complex was encapsulated in a drug delivery system. The liposome containing the [Ru(NO)(ONO)(pc)] complex was over 25% more active than the corresponding ruthenium complex in phosphate buffer solution. The activity of the complex was directly proportional to the ruthenium amount present inside the cell, as determined by inductively coupled plasma mass spectroscopy. Flow cytometry analysis revealed that the photocytotoxic activity was mainly due to apoptosis. Furthermore, the vasorelaxation induced by [Ru(NO)(ONO)(pc)], proposed as NO carrier, was studied in rat isolated aorta. The observed vasodilation was concentration-dependent. Taken together, the present findings demonstrate that the [Ru(NO)(ONO)(pc)] complex induces vascular relaxation and could be a potent anti-tumor agent. Nitric oxide release following singlet oxygen production upon visible light irradiation on a nitrosyl ruthenium complex produces two radicals and may elicit phototoxic responses that may find useful applications in photodynamic therapy.     

Release of nitric oxide together with carbon-centered radicals from N-nitrosamines by ultraviolet light irradiation

Solutions of N-nitrosamines, N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosomorpholine and N-nitrosopyrrolidine in phosphate buffer (pH 7.4) were irradiated by ultraviolet (UV) light at room temperature. The N-nitrosamines were extensively degraded due to irradiation for 120 min in a time-dependent fashion as monitored by UV-absorption or high performance liquid chromatographic analysis. Carbon-centered radicals were generated from four N-nitrosamines during the short time irradiation of 10–60 s as monitored by electron spin resonance (ESR) technique using 5,5-dimethyl-1-pyrroline N-oxide and N-tert-butyl-α-phenylnitrone as spin traps. Nitric oxide (NO) was generated during the short time irradiation as monitored by ESR technique using cysteine-Fe(II) complex, N-methyl-d-glucamine dithiocarbamate and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. Significant amounts of nitrite (4–16%) from four N-nitrosamines and also a significant amount of nitrate (4%) was produced from N-nitrosodimethylamine during the irradiation time of 120 min. Released NO from the N-nitrosamines must be converted into nitrite through intermediary reactive nitrogen oxide species including nitrogen dioxide and dinitrogen trioxide in contact with dissolved oxygen.     

The bimolecular sensitization of nitric oxide release from weak interacting ruthenium units

This work reports on the bimolecular sensitization of nitric oxide release from cis-[Ru(bpy)₂(iso)NO](PF₆)₃ (1) (iso = isoquinoline and bpy = 2,2′-bipyridine) by irradiating the MLCT transition of the chloro analog cis-[Ru(bpy)₂(iso)Cl]PF₆ (2)_. The compounds displayed peaks in the ESI-MS spectra at m/z 749.1 and m/z 578.1 ascribed, respectively, to ([1(NO⁰)−2PF₆·CH₃OH]²⁺) and ([2−PF₆]⁺). In the cyclic voltammograms, the nitrosyl complex presented two redox waves related to the NO ligand at 0.48 and −0.37 V (versus Ag/AgCl, NO^+/0/−1 processes), while the sensitizer showed two reversible waves at 0.79 and −1.46 V (versus Ag/AgCl, Ru^2+/3+ and bpy ^0/−1, respectively). The most important feature of this system is that the nitrosyl compound does not have significant absorption in the visible region, while the sensitizer has an intense band centered at 496 nm. The irradiation of an equimolar mixture of the two compounds in an ethanol:water solution (v:v) with light of λ > 500 nm leads to NO release, as probed by amperometric measurements. The variational method was applied, showing that the two compounds self-assembly in solution with a 1:1 stoichiometry. Fluorescence spectra acquired at 77 K provided the E_0-0 for the system and, from the thermodynamic cycle it was estimated that the photoinduced electron transfer between the species has a ΔG value of −1.59 eV.     

Nitric oxide, nitrosyl iron complexes, ferritin and frataxin: A well equipped team to preserve plant iron homeostasis

Iron is a key element in plant nutrition. Iron deficiency as well as iron overload results in serious metabolic disorders that affect photosynthesis, respiration and general plant fitness with direct consequences on crop production.More than 25% of the cultivable land possesses low iron availability due to high pH (calcareous soils). Plant biologists are challenged by this concern and aimed to find new avenues to ameliorate plant responses and keep iron homeostasis under control even at wide range of iron availability in various soils. For this purpose, detailed knowledge of iron uptake, transport, storage and interactions with cellular compounds will help to construct a more complete picture of its role as essential nutrient. In this review, we summarize and describe the recent findings involving four central players involved in keeping cellular iron homeostasis in plants: nitric oxide, ferritin, frataxin and nitrosyl iron complexes. We attempt to highlight the interactions among these actors in different scenarios occurring under iron deficiency or iron overload, and discuss their counteracting and/or coordinating actions leading to the control of iron homeostasis.     

Triterpenoid biosynthesis and the transcriptional response elicited by nitric oxide in submerged fermenting Ganoderma lucidum

Ganoderic triterpenoid (GT) is a promising anti-tumour constituent in Ganoderma lucidum. The aim of this study was to investigate induction by and a possible signalling mechanism of nitric oxide (NO) for GT synthesis. Compared to the control, the biomass decreased by 43.5% at 120 h and the GT yield increased by 40.94% at 72 h in the presence of a 5 mM NO donor sodium nitroprusside supplement. The gene expression profiles of G. lucidum in response to NO were investigated by RNA-sequencing. Functional annotation and an enrichment analysis of the differentially expressed genes indicated that NO inhibited mycelial growth probably via the suppression of the glycolysis genes involved in carbohydrate metabolism. NO may function directly as a regulator of gene expression in the mevalonate pathway to induce GT biosynthesis, and the hyper-production of GT in response to NO could also be accomplished by a signalling function involving Ca²⁺ and a reactive oxygen species (ROS) pathway. The results of this study are useful for large-scale GT production and can facilitate further studies on the endogenous signalling pathways involved in the GT biosynthetic pathway.     

Estimation of nitric oxide level in vivo by microdialysis with water-soluble iron-N-methyl-d-dithiocarbamate complexes as NO traps: A novel approach to nitric oxide spin trapping in animal tissues

It was found that microdialysis, i.e., passage of aqueous solutions of iron-N-methyl-d-glucamine dithiocarbamate complexes through dialysis fibers implanted into heart, kidney and liver tissues of narcotized rats, was accompanied by effective binding of the complexes to nitric oxide from interstitial fluid. The walls of dialysis fibers used in this study were permeable for compounds with molecular weight not exceeding 5 kDa. The dialyzate samples collected every 20 min and containing diamagnetic nitrosyl Fe³⁺-MGD adducts were reduced to the paramagnetic state with sodium dithionite; their concentration was measured by the EPR method. The basic level of the adducts, which represented mononitrosyl iron complexes with MGD (MNIC–MGD), in the dialyzate samples of all tested organs were similar (1 μМ). Treatment of animals with the water-soluble nitroglycerine analog Isoket or a low-molecular dinitrosyl iron thiosulfate complex as a NO donor increased the concentration of MNIC–MGD with going out into a plateau. The novel approach allows determination of nitric oxide levels in tissue interstitial fluid from concentration of MNIC–MGD formed during microdialysis.     

The protein inhibitor of neuronal nitric oxide synthase (PIN): characterization of its action on pure nitric oxide synthases

Neuronal NO synthase (nNOS) was discovered recently to interact specifically with the protein PIN (protein inhibitor of nNOS) [Jaffrey, S.R. and Snyder, S.H. (1996) Science 274, 774–777]. We have studied the effects on pure NOS enzymes of the same GST-tagged PIN used in the original paper. Unexpectedly, all NOS isoenzymes were inhibited. The IC₅₀ for nNOS was 18±6 μM GST-PIN with 63 nM nNOS after 30 min at 37°C. Uncoupled NADPH oxidation was inhibited similarly, whereas cytochrome c reductase activity, the K_M for l-arginine, and dimerization were unaffected. We reconsider the physiological role of PIN in the light of these results.     

Reaction of nitric oxide with iron bleomycin bound to DNA: properties of the nitrosyl adduct and its reaction with O2

During the ESR spectroscopic titration of nitrosyl-iron bleomycin, ON---Fe(II)Blm, with DNA, its metal domain undergoes a change in environment as the DNA base pair to drug ratio increases to 50 to 1. The ¹⁵N---O stretching frequency of ON---Fe(II)Blm occurs at 1589 cm⁻¹, similar to that for nitrosyl hemoglobin and myoglobin. Upon addition of DNA (3 base pairs per drug molecule), this vibration is substantially broadened. Injection of O₂ into a solution of ON---Fe(II)BlmDNA converts the ESR signal of the nitrosyl species to low spin Fe(III) BlmDNA. NO is largely oxidized to NO₂⁻. The combination of these products suggests that the initial reaction of ON---Fe(II)Blm with O₂ generates Fe(III)Blm and peroxynitrite, O₂NO⁻. If peroxynitrite is formed in the reaction, it does not cause detectable DNA damage. The structural integrity of a supercoiled DNA plasmid, pBR322, is not compromised and no base propenals are produced during this reaction.     

Detection of nitrous oxide in the neuronal nitric oxide synthase reaction by gas chromatography-mass spectrometry

Using headspace gas chromatography-mass spectrometry, we detected significant amounts of nitrous oxide in the reaction products of the monooxygenase reaction catalyzed by neuronal nitric oxide synthase. Nitrous oxide is a dimerization product of nitroxyl anion; its presence in the reaction products indicates that the nitroxyl anion is a product of the neuronal nitric oxide synthase-catalyzed reaction.     

Substitution of chloride by nitrosyl ligand in a scorpionate ruthenium(III) compound: A theoretical study

A theoretical study of the ruthenium(III) complex [RuCl₂(pz₂CHSO₃)(en)] and of its nitrosyl-substituted product [Ru(NO)Cl(pz₂CHSO₃)(en)]⁺ is presented, based on density functional calculations. Several isomers of each compound differing in the position of the anionic tail of a bis(3,4-dimethyl-1-yl)methanesulfonate scorpionate ligand, pz₂CHSO₃⁻, relative to the monodentate ligands have been optimized. A two-step mechanism is proposed for the ligand substitution reaction that is consistent with the computational results and the weak coordination of the sulfonate group.     

Characterization of the mechanisms of action and nitric oxide species involved in the relaxation induced by the ruthenium complex

Nitric oxide (NO) plays an important role in the control of vascular tone. NO donors have therapeutic use and the most used NO donors, nitroglycerin and sodium nitroprusside have problems in their use. Thus, new NO donors have been synthesized to minimize these undesirable effects. Nytrosil ruthenium complexes have been studied as a new class of NO donors. trans-[RuCl([15]aneN(4))NO](2+), induces vasorelaxation only in presence of reducing agent. In this study, we characterized the mechanisms of vasorelaxation of trans-[RuCl([15]aneN(4))NO](2+) in denuded rat aorta and identified which NO forms are involved in this relaxation. We also evaluated the effect of this NO donor in decreasing the cytosolic Ca(2+) concentration ([Ca(2+)]c) of the vascular smooth muscle cells. Vasorelaxation to trans-[RuCl([15]aneN(4))NO](2+) (E(max): 101.8 +/- 2.3%, pEC(50): 5.03 +/- 0.15) was almost abolished in the presence of the NO* scavenger hydroxocobalamin (E(max): 4.0 +/- 0.4%; P < 0.001) and it was partially inhibited by the NO(-) scavenger L-cysteine (E(max): 79.9 +/- 6.9%, pEC(50): 4.41 +/- 0.06; P < 0.05). The guanylyl cyclase inhibitor ODQ reduced the E(max) (57.7 +/- 4.0%, P < 0.001) and pEC(50) (4.21 +/- 0.42, P < 0.01) and the combination of ODQ and TEA abolished the response to trans-[RuCl([15]aneN(4))NO](2+). The blockade of voltage-dependent (K(v)), ATP-sensitive (K(ATP)), and Ca(2+)-activated (K(Ca) K(+) channels reduced the vasorelaxation induced by trans-[RuCl([15]aneN(4))NO](2+). This compound significantly reduced [Ca(2+)]c (from 100% to 85.9 +/- 3.5%, n = 4). In conclusion, our data demonstrate that this NO donor induces vascular relaxation involving NO* and NO(-) species, that is associated to a decrease in [Ca(2+)]c. The mechanisms of vasorelaxation involve guanylyl cyclase activation, cGMP production and K(+) channels activation.     

The role of nitric oxide in orofacial pain

Nitric oxide (NO) is a free radical gas that has been shown to be produced by nitric oxide synthase (NOS) in different cell types and recognized to act as a neurotransmitter or neuromodulator in the nervous system. NOS isoforms are expressed and/or can be induced in the related structures of trigeminal nerve system, in which the regulation of NOS biosynthesis at different levels of gene expression may allow for a fine control of NO production. Several lines of evidence suggest that NO may play a role through multiple mechanisms in orofacial pain processing. This report will review the latest evidence for the role of NO involved in orofacial pain and the potential cellular mechanisms are also discussed.     

Pregnancy-associated increase in rat systemic arteries endothelial nitric oxide production diminishes vasoconstrictor but does not enhance vasodilator responses

Late pregnancy in rats is characterized by a decrease in arterial pressure and in isolated arterial vessels response to vasoconstrictors. In uterine arteries the pregnancy-associated attenuation of the response to vasoconstrictors has been attributed to an increase in basal and agonist-induced endothelial NO production. However, the role of NO in pregnancy-associated changes of systemic arteries reactivity to vasoactive agents remains to be fully elucidated. We examined whether pregnancy influences the reactivity of systemic arteries to vasodilator or vasoconstrictor agents through NO-dependent mechanisms. Thoracic aortic rings and mesenteric arterial bed of late pregnant rats showed refractoriness to phenylephrine-induced vasoconstriction that was abolished by NO synthase inhibition. The potency of L-NNA to enhance tension of aortic rings preconstricted with phenylephrine (10–20% of their maximal response) was significantly lower in preparations from pregnant animals. In phenylephrine-contracted aortas and mesenteric bed, the effects of the endothelium-dependent vasodilators acetylcholine, A23187 and bradykinin, were not influenced by pregnancy. Similarly, pregnancy did not affect the vasodilator responses of adenosine, isoproterenol, capsaicin, nitroprusside, forskolin, and Hoe234 in the mesenteric bed. NO synthase activity measured by determining the conversion of L−[³H]-arginine to L−[³H]-citrulline in aorta and mesenteric arteries homogenates was not altered by pregnancy. These findings show that endothelial-dependent and -independent vasodilators action as well as NO synthase activity in systemic arteries is uninfluenced by pregnancy, whereas pregnancy-associated hyporeactivity of systemic arteries to vasoconstrictors is related to an enhanced endothelial NO production either spontaneous or elicited directly or indirectly by vasoconstrictor agents. This interpretation implies that the enhanced NO production observed in systemic arteries during late pregnancy involves cellular pathways other than the ones involved in the response to endothelium-dependent vasodilators such as acetylcholine.     

Direct, real-time measurement of shear stress-induced nitric oxide produced from endothelial cells in vitro

Nitric oxide (NO) produced by the endothelium is involved in the regulation of vascular tone. Decreased NO production or availability has been linked to endothelial dysfunction in hypercholesterolemia and hypertension. Shear stress-induced NO release is a well-established phenomenon, yet the cellular mechanisms of this response are not completely understood. Experimental limitations have hindered direct, real-time measurements of NO under flow conditions. We have overcome these challenges with a new design for a parallel-plate flow chamber. The chamber consists of two compartments, separated by a Transwell® membrane, which isolates a NO recording electrode located in the upper compartment from flow effects. Endothelial cells are grown on the bottom of the membrane, which is inserted into the chamber flush with the upper plate. We demonstrate for the first time direct real-time NO measurements from endothelial cells with controlled variations in shear stress. Step changes in shear stress from 0.1 dyn/cm² to 6, 10, or 20 dyn/cm² elicited a transient decrease in NO followed by an increase to a new steady state. An analysis of NO transport suggests that the initial decrease is due to the increased removal rate by convection as flow increases. Furthermore, the rate at which the NO concentration approaches the new steady state is related to the time-dependent cellular response rather than transport limitations of the measurement configuration. Our design offers a method for studying the kinetics of the signaling mechanisms linking NO production with shear stress as well as pathological conditions involving changes in NO production or availability.     

Inhibition of nitric oxide synthase enhances contractile response of ventricular myocytes from streptozotocin-diabetic rats

The contractile hyporesponsiveness of the streptozotocin diabetic rat heart in vitro to β-adrenergic agonists is eliminated when the heart is perfused with N^G-nitro-l-arginine methyl ester (l-NAME), a non-selective inhibitor of nitric oxide synthase (NOS). The following study evaluated the hypothesis that an increased production of NO/cGMP within the diabetic myocyte inhibits the β-adrenergic-stimulated increase in calcium current and contractile response. Male Sprague-Dawley rats were given an intravenous injection of streptozotocin (60 mg/kg). After 8 weeks, L-type calcium currents were recorded in ventricular myocytes using the whole cell voltage-clamp method. Shortening of isolated myocytes was determined using a video edge detection system. cAMP and cGMP were measured using radioimmunoassay. Nitric oxide production was determined using the Griess assay kit. Basal cGMP levels and nitric oxide production were elevated in diabetic myocytes. Shortening of the diabetic myocytes in response to isoproterenol (1 μM) was markedly diminished. However, there was no detectable difference in the isoproterenol-stimulated L-type calcium current or cAMP levels between control and diabetic myocytes. Acute superfusion of the diabetic myocyte with l-NAME (1 mM) decreased basal cGMP and markedly enhanced the shortening response to isoproterenol but did not alter isoproterenol-stimulated calcium current. These data suggest that increased production of NO/cGMP within the diabetic myocyte suppressed β-adrenergic stimulated shortening of the myocyte. However, NO/cGMP apparently does not suppress shortening of the myocyte by inhibition of the β-stimulated calcium current.     

The roles of nitric oxide in murine cardiovascular development

Nitric oxide (NO) participates in a diverse array of biological functions in mammalian organ systems. Depending on the biochemical environment, the production of NO may result in cytoprotection or cytotoxicity. The paradoxical actions of NO arise from the complexities generated by the redox milieu, NO concentration/bioavailability, and tissue/cell context, which ultimately result in the wide range of regulatory roles observed. Additionally, in physiological versus pathological states, NO often displays diametrically opposing affects in several organ systems. Here, we will discuss the roles of NO during reproduction, organ system development, in particular, the cardiovascular system, and its potential implications in diabetes-induced fetal defects.     

Partial sequence and characterization of nitric oxide synthase gene from Hyphantria cunea

Nitric oxide synthase (NOS) gene has been partially sequenced from Hyphantria cunea and compared with those already determined from insects. Hyphantria cunea NOS possesses putative recognition sites for co‐factors heme, BH₄, CaM, FMN, FAD, and NADPH common to NOS. The deduced amino acid sequence of H. cunea NOS cDNA showed 70.3% identity to Manduca sexta NOS and 57.6–69.5% identity to NOS sequences from other insects. Nitric oxide synthase is expressed in all tissues of H. cunea, except in hemocytes. The NOS expression in midgut, fat body, epidermis, and Malpighian tubule strongly increased against Gram‐positive and Gram‐negative bacterial infection. These results suggest that NOS may play an important role in insect defense system against bacterial infection.