Photoactive ruthenium nitrosyls: Effects of light and potential application as NO donors

Previous studies in our laboratory have shown that mixed lineage kinase 3 (MLK3) can be activated following global ischemia. In addition, other laboratories have reported that the activation of MLK3 may be linked to the accumulation of free radicals. However, the mechanism of MLK3 activation remains incompletely understood. We report here that MLK3, overexpressed in HEK293 cells, is S-nitrosylated (forming SNO-MLK3) via a reaction with S-nitrosoglutathione, an exogenous nitric oxide (NO) donor, at one critical cysteine residue (Cys-688). We further show that the S-nitrosylation of MLK3 contributes to its dimerization and activation. We also investigated whether the activation of MLK3 is associated with S-nitrosylation following rat brain ischemia/reperfusion. Our results show that the administration of 7-nitroindazole, an inhibitor of neuronal NO synthase (nNOS), or nNOS antisense oligodeoxynucleotides diminished the S-nitrosylation of MLK3 and inhibited its activation induced by cerebral ischemia/reperfusion. In contrast, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (an inhibitor of inducible NO synthase) or nNOS missense oligodeoxynucleotides did not affect the S-nitrosylation of MLK3. In addition, treatment with sodium nitroprusside (an exogenous NO donor) and S-nitrosoglutathione or MK801, an antagonist of the N-methyl-D-aspartate receptor, also diminished the S-nitrosylation and activation of MLK3 induced by cerebral ischemia/reperfusion. The activation of MLK3 facilitated its downstream protein kinase kinase 4/7 (MKK4/7)-JNK signaling module and both nuclear and non-nuclear apoptosis pathways. These data suggest that the activation of MLK3 during the early stages of ischemia/reperfusion is modulated by S-nitrosylation and provides a potential new approach for stroke therapy whereby the post-translational modification machinery is targeted.

Nitric oxide (NO) can induce apoptosis (programmed cell death) at micromolar or higher doses. Although cell death via NO-induced apoptosis has been studied quite extensively, the targeted delivery of such doses of NO to infected or malignant tissues has not been achieved. The primary obstacle is indiscriminate NO release from typical systemic donors such as glycerin trinitrate: once administered, the drug travels throughout the body, and NO is released through a variety of enzymatic, redox, and pH-dependent pathways. Photosensitive NO donors have the ability to surmount this difficulty through the use of light as a localized stimulus for NO delivery. The potential of the method has prompted synthetic research efforts toward new NO donors for use as photopharmaceuticals in the treatment of infections and malignancies. Over the past few years, we have designed and synthesized several metal nitrosyls (NO complexes of metals) that rapidly release NO when exposed to low-power (milliwatt or greater) light of various wavelengths. Among them, the ruthenium nitrosyls exhibit exceptional stability in biological media. However, typical ruthenium nitrosyls release NO upon exposure to UV light, which is hardly suitable for phototherapy. By following a few novel synthetic strategies, we have overcome this problem and synthesized a variety of ruthenium nitrosyls that strongly absorb light in the 400-600-nm range and rapidly release NO under such illumination. In this Account, we describe our progress in designing photoactive ruthenium nitrosyls as visible-light-sensitive NO donors. Our research has shown that alteration of the ligands, in terms of (i) donor atoms, (ii) extent of conjugation, and (iii) substituents on the ligand frames, sensitizes the final ruthenium nitrosyls toward visible light in a predictable fashion. Density functional theory (DFT) and time-dependent DFT (TDDFT) calculations provide guidance in this "smart design" of ligands. We have also demonstrated that direct attachment of dye molecules as light-harvesting antennas also sensitize ruthenium nitrosyls to visible light, and TDDFT calculations provide insight into the mechanisms of sensitization by this technique. The fluorescence of the dye ligands makes these NO donors "trackable" within cellular matrices. Selected ruthenium nitrosyls have been used to deliver NO to cellular targets to induce apoptosis. Our open-design strategies allow the isolation of a variety of these ruthenium nitrosyls, depending on the choices of the ligand frames and dyes. These designed nitrosyls will thus be valuable in the future endeavor of synthesizing novel pharmaceuticals for phototherapy.

Regulation of NF-κB activation and nuclear translocation by exogenous nitric oxide (NO) donors in TNF-α activated vascular endothelial cells

Direct measurement of actual levels of nitric oxide (NO) in cell culture conditions using soluble NO donors

Nitric oxide (NO) is a free radical gas and a short-lived messenger which has many paracrine functions. Direct assessment of NO production is very difficult in vivo. However, the paranasal cavities generate a high amount of NO which diffuses in the nasal cavity where it can be easily measured. Several studies have suggested alterations of the NO production in heart failure. Thus, we assessed nasal NO concentration in normal subjects and in heart failure patients. The nasal NO concentration averaged 227 +/- 10 ppb in the control group (n = 20), and 210 +/- 10, 198 +/- 20 and 159 +/- 54 ppb in New York Heart Association (NYHA) class II (n = 30), III (n = 28) and IV (n = 7) patients, respectively (mean +/- standard error [SE], not significant using analysis of variance [ANOVA]). Nasal NO level was not influenced by age, sex or etiology of the heart failure or by treatment with frusemide, angiotensin-converting enzyme inhibitor or digoxin. However, treatment with NO-releasing drugs (nitrates or molsidomine) significantly decreased the nasal NO level in heart failure patients. A two-way ANOVA revealed that treatment with a NO-releasing drug influenced nasal NO concentration (P = 0.0005), whereas NYHA class did not (P = 0.23), with a trend towards an interaction between the two parameters (P = 0.09): the inhibitory effect of NO-releasing drug on nasal NO concentration was more pronounced in severe heart failure. In an additional group of 12 patients (NYHA class II or III), the nasal NO concentration was 174 +/- 19 ppb during NO-releasing drug treatment and increased to 231 +/- 27 ppb 3 days after withdrawal of the nitrates (P = 0.0007 using paired t-test). Conversely, the nasal NO concentration in another group of seven patients (NYHA class II or III) was 219 +/- 32 ppb without nitrate treatment and decreased to 188 +/- 28 ppb 7 days after nitrate addition (P = 0.02 using paired t-test). In contrast, the nasal NO concentration in another group of ten ischemic patients without heart failure was 203 +/- 25 ppb without nitrate treatment and was similar (207 +/- 28 ppb) 7 days after nitrate addition (not significant using paired t-test). In conclusion, nasal NO production is normal in heart failure, except in patients receiving NO-releasing drugs. Nasal NO concentration could be useful for investigating the mechanism(s) by which exogenous NO donors decrease endogenous NO production.

Introduction: In pancreatic ischemia/reperfusion (IR) injury (IRI) the role of nitric oxide (NO) is not completely understood. Using a rat model of normothermic in situ IRI, the effect of endogenous and exogenous NO donors on post-ischemic tissue oxygenation and tissue damage was investigated. Methods: IR was induced by 2-hour normothermic in situ ischemia of a pancreatic tail segment pedunculated on the splenic vessels with 2 h of reperfusion in an untreated, an L-arginine- and a sodium-nitroprusside-treated group (Wistar rats, n = 7/group). Animals without ischemia served as controls. Tissue oxygenation (pO_2ti) was monitored using a pO₂-sensitive Clark-type electrode. Histological investigation was performed following a semiquantitative score (edema, vacuolization, PMN infiltration, necrosis). Plasma lipase was another marker of organ damage. Results: The administration of L-arginine and sodium nitroprusside caused a significant amelioration of the decrease in pO_2ti after reperfusion compared to IR animals (p < 0.05). Histological damage was also reduced in the NO donor groups (p < 0.05). After reperfusion, plasma lipase in the L-arginine-treated animals was significantly lower compared to IR and sodium nitroprusside (p < 0.05). Conclusions: The administration of both endogenous and exogenous NO donors is protective in IRI of the rat pancreas which can be seen by an improvement in post-ischemic tissue oxygenation which indicates better nutritive tissue perfusion, amelioration of the histological tissue injury and, in L-arginine animals, lower lipase levels. NO donors could be useful in the prevention and reduction of the pancreatic IRI.

The influence of nitric oxide donors on the responses to nitrergic nerve stimulation in the mouse duodenum

In the vasculature it is well established that cGMP is involved in the relaxant response to nitric oxide (NO) and NO donors. However, there is an increasing evidence that alternative/additional pathways that are cGMP-independent may also exist. A key criterion for a response to NO or a NO donor drug to be classified as cGMP-independent is lack of (or incomplete) inhibition by the selective inhibitor of soluble guanylate cyclase, ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one). In many blood vessels cGMP-independent mechanisms contribute to the vasorelaxation, and in certain vascular beds cGMP-independent relaxation may be the predominant mechanism of action of NO and NO donors. NO donor drugs that generate NO "spontaneously", like authentic NO (i.e. solutions of NO gas), appear to exhibit a larger component of cGMP-independent vasorelaxation than do those drugs that require bioactivation in the tissue. The long lasting inhibition of responses to vasoconstrictors by S-nitrosothiols, persisting after removal of these NO donors, may be a cGMP-independent process, at least in some vessels. The mechanisms involved in the inhibition of vascular growth by NO and NO donors are predominantly cGMP-independent, as are the mechanisms responsible for the effects of NO donors on apoptosis in vascular smooth muscle and endothelial cells. The ability of NO and NO donors to inhibit platelet aggregation has a significant cGMP-independent component. cGMP-independent pathways are most often, though not exclusively, seen at high concentrations (microM - mM) of NO and NO donors. Hence, in relation to the actions of endogenous NO, these pathways may be particularly important in settings when the inducible isoform of NO-synthase is expressed. Furthermore, cGMP-independent pathways are enhanced in animal models of atherosclerosis and ischaemia. This suggests that it may be possible to target cGMP-independent mechanisms with selected NO donors in disease states.

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

Nitric oxide (NO) signaling is inextricably linked to both its physical and chemical properties. Due to its preferentially hydrophobic solubility, NO molecules tend to partition from the aqueous milieu into biological membranes. We hypothesized that plasma membrane ordering provided by cholesterol further couples the physics of NO diffusion with cellular signaling. Fluorescence lifetime quenching studies with pyrene liposome preparations showed that the presence of cholesterol decreased apparent diffusion coefficients of NO approximately 20-40%, depending on the phospholipid composition. Electrochemical measurements indicated that the diffusion rate of NO across artificial bilayer membranes were inversely related to cholesterol content. Sterol transport-defective Niemann-Pick type C1 (NPC1) fibroblasts exhibited increased plasma membrane cholesterol content but decreased activation of both intracellular soluble guanylyl cyclase and vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser(239) induced by exogenous NO exposure relative to their normal human fibroblast (NHF) counterparts. Augmentation of plasma membrane cholesterol in NHF diminished production of both cGMP and VASP phosphorylation elicited by NO to NPC1-comparable levels. Conversely, decreasing membrane cholesterol in NPC1 resulted in the augmentation in both cGMP and VASP phosphorylation to a level similar to those observed in NHF. Increasing plasma membrane cholesterol contents in NHF, platelets, erythrocytes and tumor cells also resulted in an increased level of extracellular diaminofluorescein nitrosation following NO exposure. These findings suggest that the impact of cholesterol on membrane fluidity and microdomain structure contributes to the spatial heterogeneity of NO diffusion and signaling.

In this issue of the Journal, Xie and co-workers (1) demonstrate that introducing nitric oxide (NO) synthase type II (NOSII, iNOS) into tumor cells produces cytotoxicity not only in the transfected cells but also in bystander tumor cells. Here, we discuss the potential implications of these experiments in the light of present knowledge on the complex role of NO in tumor biology. The growth of solid tumors is regulated by interactions between endothelial cells of the tumor vasculature, tumorinfiltrating immune cells (such as T lymphocytes and macrophages), and the tumor cells themselves. In these cellular interactions, the unusual biologic messenger molecule and cytotoxin, NO, may play important pathobiologic roles in addition to its many physiologic functions (2). Endogenous NO production from L-arginine has been directly or indirectly demonstrated in all of these cell types. In most cases, the inducible NO synthase gene (Nos2)—one of three known human Nos genes (3)— was switched on (presumably by NF-kB-dependent mechanisms). Its gene product, the high-output NOS-II isoform, unlike its constitutively expressed low-output counterparts (NOS-I and -III), is not regulated by the intracellular concentration of free calcium (3) and is chronically active. Such continuously high exposure of cells to endogenous NO as well as exogenous NO donors will inhibit proliferation and induce cell death (4). High NO levels inhibit mitochondrial respiration, the citric acid cycle, glycolysis, and DNA replication. Locally high levels of reactive oxygen species (ROS), stemming, for example, from host immune cells or from an insufficient oxygen supply to the tumor tissue, may exacerbate these toxic effects by generating even more reactive compounds, such as peroxynitrite (ONOO). The latter compound arises from the diffusion-limited interaction of NO and O2 − and is even more reactive than NO, but it is stable enough to diffuse to and thus harm tumor cells. Apart from these tumoricidal effects, NO has facilitated tumor growth and vascularization in a few experimental models, and, in certain human carcinomas, endogenous NO production was positively associated with tumor grade (5). Thus, NO has a complex, at least dual, action on tumor growth that may depend on the local concentrations of NO, additional factors such as the presence of ROS, and the type of tumor and its susceptibility to NO. In those models where NO had a permissive effect on tumor growth, NOS activity was up to two orders of magnitude lower than that associated with NO-dependent tumor toxicity and apoptosis (5). The dual action of NO on tumor cell proliferation is reminiscent of what has been repeatedly demonstrated for ROS (6,7). Maximal growth promotion by ROS is observed when cells maintain a low but sufficient oxidant signal for the induction of growth-competence genes (8,9), as may be the case with moderate NO concentrations. At high ROS concentrations, the equilibrium balance between the cellular antioxidant defense and oxidant levels is shifted, i.e., toward lipid peroxidation and DNA fragmentation. Besides this concentration dependence, the mechanisms of action of NO may also diverge considerably (Fig. 1). Signaling functions at comparatively low NO concentrations involve soluble guanylyl cyclases as the principal molecular target and subsequent increases in the intracellular level of the second messenger molecule, cyclic guanosine monophosphate (cGMP) (10). The tumor-relevant consequences of these increases in cGMP may be the stimulation of (neo)vascularization and angiogenesis. Moreover, endogenous NO may not only promote the growth of existing tumors, but it may also be tumorigenic itself. NO can cause mutations by mediating the deamination of S-methylcytosine to thymine and also by increasing the formation of DNA strand breaks (11). Increased NO formation has been implicated in several tumor types associated with chronic infections and inflammations, such as gastric, duodenal, esophageal, bladder, and liver cancers (12,13). What are the therapeutic implications of this dual role of NO? One apparent goal may be to enhance NO synthesis to a maximum level only in tumor tissue so that cell proliferation is impaired and cell death is induced. This may be achieved in principle by simply substituting NO in the form of so-called NO donors. Indeed, NO is an effective hypoxic radiosensitizer of tumor tissue (14), while a nonselective NOS inhibitor increases tumor survival (15). Alternatively, endogenous NO formation may be induced in the tumor cell by different cytokines (tumor necrosis factor-a and interleukin 2). Tumor necrosis factor-a has been shown to induce both NO and superoxide (O2 ) production, providing an effective means of local ONOO formation (16). However, cytokines induce NOS-II expression systemically in the blood vessel wall, resulting in massive NOand cGMPmediated vasodilatation and hypotension, which has been recognized as the primary limiting factor in high-dose cytokine therapy (17). A more direct approach is to transfer cytokine or NO synthase

Comparison of the mechanisms underlying the relaxation induced by two nitric oxide donors: Sodium nitroprusside and a new ruthenium complex

Nitric Oxide

Nitric oxide: physiology and pharmacology.

We investigated the role of nitric oxide (NO) in inflammatory hyperalgesia. Coinjection of prostaglandin E2 (PGE2) with the nitric oxide synthase (NOS) inhibitor NG-methyl-L-arginine (L-NMA) inhibited PGE2-induced hyperalgesia. L-NMA was also able to reverse that hyperalgesia. This suggests that NO contributes to the maintenance of, as well as to the induction of, PGE2-induced hyperalgesia. Consistent with the hypothesis that the NO that contributes to PGE2-induced sensitization of primary afferents is generated in the dorsal root ganglion (DRG) neurons themselves, L-NMA also inhibited the PGE2-induced increase in tetrodotoxin-resistant sodium current in patch-clamp electrophysiological studies of small diameter DRG neurons in vitro. Although NO, the product of NOS, often activates guanylyl cyclase, we found that PGE2-induced hyperalgesia was not inhibited by coinjection of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylyl cyclase inhibitor. We then tested whether the effect of NO depended on interaction with the adenylyl cyclase-protein kinase A (PKA) pathway, which is known to mediate PGE2-induced hyperalgesia. L-NMA inhibited hyperalgesia produced by 8-bromo-cAMP (a stable membrane permeable analog of cAMP) or by forskolin (an adenylyl cyclase activator). However, L-NMA did not inhibit hyperalgesia produced by injection of the catalytic subunit of PKA. Therefore, the contribution of NO to PGE2-induced hyperalgesia may occur in the cAMP second messenger pathway at a point before the action of PKA. We next performed experiments to test whether administration of exogenous NO precursor or donor could mimic the hyperalgesic effect of endogenous NO. Intradermal injection of either the NOS substrate L-arginine or the NO donor 3-(4-morphinolinyl)-sydnonimine hydrochloride (SIN-1) produced hyperalgesia. However, this hyperalgesia differed from PGE2-induced hyperalgesia, because it was independent of the cAMP second messenger system and blocked by the guanylyl cyclase inhibitor ODQ. Therefore, although exogenous NO induces hyperalgesia, it acts by a mechanism different from that by which endogenous NO facilitates PGE2-induced hyperalgesia. Consistent with the hypothesis that these mechanisms are distinct, we found that inhibition of PGE2-induced hyperalgesia caused by L-NMA could be reversed by a low dose of the NO donor SIN-1. The following facts suggest that this dose of SIN-1 mimics a permissive effect of basal levels of NO with regard to PGE2-induced hyperalgesia: (1) this dose of SIN-1 does not produce hyperalgesia when administered alone, and (2) the effect was not blocked by ODQ. In conclusion, we have shown that low levels of NO facilitate cAMP-dependent PGE2-induced hyperalgesia, whereas higher levels of NO produce a cGMP-dependent hyperalgesia.