Matthew B. Grisham

Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations

The purpose of this position paper is to present a critical analysis of the challenges and limitations of the most widely used fluorescent probes for detecting and measuring reactive oxygen and nitrogen species. Where feasible, we have made recommendations for the use of alternate probes and appropriate analytical techniques that measure the specific products formed from the reactions between fluorescent probes and reactive oxygen and nitrogen species. We have proposed guidelines that will help present and future researchers with regard to the optimal use of selected fluorescent probes and interpretation of results.

Novel nonsterodial anti-inflammatory drug derivatives with markedly reduced ulcerogenic properties in the rat

BACKGROUND/AIMS The use of nonsteroidal anti-inflammatory drugs (NSAIDs) is limited by their ability to induce gastrointestinal injury. Two NSAIDs were modified by incorporation of an nitroxybutyl moiety. The short-term ulcerogenic and anti-inflammatory properties of these derivatives were compared with the native NSAIDs. METHODS Rats were given flurbiprofen, ketoprofen, or their respective derivatives, and the extent of gastric damage and effect on gastric prostaglandin E2 synthesis was assessed. The damage-promoting effects of these compounds were also compared following twice-daily administration for 1 week. Anti-inflammatory properties were examined using a carrageenan-induced paw edema model. RESULTS The derivatives of flurbiprofen and ketoprofen caused significantly less short-term gastric mucosal injury at all doses tested, despite producing comparable suppression of prostaglandin synthesis. The NSAID derivatives also showed comparable anti-inflammatory activity to the native compounds. The flurbiprofen derivative inhibited collagen-induced platelet aggregation significantly more than the native NSAID. Plasma nitrate/nitrite levels increased significantly following administration of the flurbiprofen derivative, consistent with release of a nitrogen oxide. CONCLUSIONS Addition of a nitroxybutyl moiety to two NSAIDs markedly reduced the ability of these agents to induce short-term gastric injury but did not interfere with their ability to suppress inflammatory processes, inhibit prostaglandin synthesis, or inhibit platelet aggregation. These NSAID derivatives may therefore represent a novel class of anti-inflammatory drugs with markedly less ulcerogenic effects on the stomach.

Mechanisms of acute and chronic intestinal inflammation induced by indomethacin

The objective of this study was to characterize the mechanisms of acute and chronic intestinal mucosal injury and inflammation induced by subcutaneously injected indomethacin (Indo). One injection of Indo (7.5 mg/kg) produced acute injury and inflammation in the distal jejunum and proximal ileum that were maximal at three days and completely resolved within one week. Two daily subcutaneous injections of Indo produced a more extensive and chronic inflammation that lasted in an active form in more than 75% of the rats for at least two weeks. Epithelial injury, as measured by enhanced mucosal permeability, was significantly elevated only at one day in the acute model (one injection) but was persistently elevated in the chronic model (two injections). Bile duct ligation completely attenuated increased mucosal permeability in the acute model, however, depletion of circulating neutrophils had no effect. Neither Indo (0–0.1 mg/ml) nor normal bile was cytotoxic to cultured rat intestinal epithelial cells; however, they synergistically promoted significant cytotoxicity. Bile collected from rats treated with Indo was cytotoxic towards the epithelial cells in a dose-dependent manner. Sulfasalazine and metronidazole (100 mg/kg/day, both) attenuated enhanced mucosal permeability in the chronic model. Massive bacterial translocation into the mesenteric lymph nodes, liver, and spleen following two injections of Indo was significantly attenuated by metronidazole. We conclude that: (1) a single injection of Indo produces acute intestinal mucosal injury and inflammation that resolve completely within three to seven days, whereas two daily injections of Indo produce both acute and chronic injury and inflammation, (2) enterohepatic circulation of Indo is important in promoting the acute phases of injury and inflammation, (3) circulating neutrophils do not play a role in the pathogenesis of this model, and (4) endogenous bacteria play an important role in exacerbating and/or perpetuating the chronic phases of injury and inflammation.

A comparative analysis of two models of colitis in rats.

Two models of colitis produced in rats that have received significant attention over the past few years are the acetic acid and trinitrobenzene sulfonic acid (TNBS) models. The objective of this study was to quantify and compare the temporal relationship among mucosal permeability, epithelial injury, and inflammation induced by acetic acid, ethanol (vehicle), ethanol plus TNBS (unbuffered, pH 1.0), and ethanol plus TNBS (pH 7.4). Data obtained show that the inflammation induced by these four irritants results from caustic injury to the colonic epithelium and interstitium as measured by the rapid and dramatic increases in mucosal permeability and tissue water content as well as by histological analysis. The injurious nature of TNBS was confirmed in a separate series of studies showing that buffered TNBS (pH 7.4), in the absence of ethanol, is toxic to cultured rat intestinal epithelial cell monolayers. Only after 1-2 days of the initial insult, were signs of classical inflammation observed, including increases in colonic myeloperoxidase activity (neutrophil infiltration) and colon weight as well as hyperemia and mucosal ulcerations. Although ethanol plus TNBS (pH 1.0 or 7.4) tended to produce higher mucosal permeabilities (epithelial cell injury) at 1-2 weeks after the enemas than acetic acid or ethanol groups, only the ethanol plus TNBS (pH 7.4) permeabilities were found to be significantly enhanced. In addition, all four groups showed significant elevations in colonic myeloperoxidase activity and colon weight at 1-2 weeks after enema. It is suggested that these models of colitis are useful to study events that occur at the time of inflammation and repair. However, these models may have significant limitations in understanding events that initiate inflammation of the intestine in human inflammatory bowel disease.

Modulation of leukocyte–endothelial interactions by reactive metabolites of oxygen and nitrogen: relevance to ischemic heart disease

Ischemia and reperfusion (I/R) are thought to play an important role in the pathophysiology of ischemic diseases of the heart. It is now well appreciated that leukocyte-endothelial cell interactions are important determinants for I/R-induced microvascular injury and dysfunction. There is a growing body of experimental data to suggest that reactive metabolites of oxygen and nitrogen are important physiological modulators of leukocyte-endothelial cell interactions. A number of investigators have demonstrated that I/R enhances oxidant production within the microcirculation resulting in increases in leukocyte adhesion and transendothelial cell migration. Several other studies have shown that exogenous nitric oxide (NO) donors may attenuate leukocyte and platelet adhesion and/or aggregation in a number of different inflammatory conditions including I/R. The objective of this review is to discuss the physiological chemistry of reactive metabolites of oxygen and nitrogen with special attention given to those interactions that may modulate leukocyte-endothelial cell interactions, provide an overview of the evidence implicating reactive metabolites of oxygen and nitrogen as modulators of leukocyte-endothelial cell interactions in vivo, and discuss how these mechanisms may be involved in the pathophysiology of ischemic heart disease.

Myocardial ischemia-reperfusion injury is exacerbated in absence of endothelial cell nitric oxide synthase.

Myocardial ischemia and reperfusion (MI/R) initiates a cascade of polymorphonuclear neutrophil (PMN)-mediated injury, the magnitude of which may be influenced by the bioavailability of nitric oxide (NO). We investigated the role of endothelial cell nitric oxide synthase (ecNOS) in MI/R injury by subjecting wild-type and ecNOS-deficient (-/-) mice to 20 min of coronary artery occlusion and 120 min of reperfusion. Myocardial infarct size represented 20.9 +/- 2.9% of the ischemic zone in wild-type mice, whereas the ecNOS -/- mice had significantly (P < 0.01) larger infarcts measuring 46.0 +/- 3.8% of the ischemic zone. Because P-selectin is thought to be involved with the pathogenesis of neutrophil-mediated I/R injury, we assessed the effects of MI/R on P-selectin expression in the myocardium of wild-type and ecNOS -/- mice. P-selectin expression measured with a radiolabeled monoclonal antibody (MAb) technique after MI/R in wild-type mice was 0.037 +/- 0.009 microgram MAb/g tissue, whereas ecNOS -/- coronary vasculature was characterized by significantly (P < 0.05) higher P-selectin expression (0.080 +/- 0.013 microgram MAb/g tissue). Histological examination of the postischemic myocardium revealed significantly (P < 0.01) more neutrophils in the ecNOS -/- (29.5 +/- 2.5 PMN/field) compared with wild-type (5.0 +/- 0.9 PMN/field) mice. A similar trend in infarct size and neutrophil accumulation was observed when wild-type and ecNOS -/- mice were subjected to 30 min of ischemia and 120 min of reperfusion. These novel in vivo findings demonstrate a cardioprotective role for ecNOS-derived NO in the ischemic-reperfused mouse heart.

Chemical biology of nitric oxide: Regulation and protective and toxic mechanisms

Publisher Summary This chapter discusses the important aspects of the solution chemistry of nitrogen oxide (NO) and reactive nitrogen oxide species (RNOS), biochemical targets of NO and intermediates in the autoxidation (NO X ), and the effect of NO in the presence of other toxic molecules, such as reactive oxygen species (ROS). There are two types of nitric-oxide synthase: constitutive (cNOS) and inducible (iNOS). Since cNOS generates low levels of NO, direct effects rather than indirect effects of NO would be particularly relevant. In case of iNOS, considerably higher concentrations of NO are formed for longer periods of time; therefore, both direct and indirect effects could be relevant. This chapter discusses, from a chemical perspective, those processes that are involved in the interactions with key cellular components as well as detoxification and control of NO in vivo . Defining the chemical, biochemical, and cellular pathways of NO quantitatively can provide insights into the role that NO plays in the etiology of various diseases that in turn can provide a basis for the development of new therapeutic agents. The chemical biology of NO will provide the understanding as to how NO can be regulatory, toxic, and protective in biological systems.

Leukocyte-endothelial cell interactions in nitric oxide synthase-deficient mice.

Nitric oxide (NO) is known to be an important endogenous modulator of leukocyte-endothelial cell interactions within the microcirculation. We examined leukocyte rolling and adhesion under baseline conditions and following thrombin (0.25 U/ml) superfusion in the mesentery of wild-type, inducible NOS (iNOS)-deficient (-/-), neuronal NOS (nNOS) -/-, and endothelial cell NOS (ecNOS) -/- mice to further our understanding of NO and leukocyte function. Baseline leukocyte rolling (cells/min) was significantly elevated in both the nNOS -/- (30.0 +/- 4.0) and ecNOS -/- mice (67.0 +/- 12.0) compared with wild-type mice (11.0 +/- 1.4). In addition, baseline leukocyte adherence (cells/100 micrometers of vessel) was also significantly elevated in the nNOS -/- (5.2 +/- 1.0) and ecNOS -/- (13.0 +/- 1.3) compared with wild-type animals (1.3 +/- 0.5). Deficiency of iNOS had no effect on baseline leukocyte rolling or adhesion in the mesentery. Baseline surface expression of P-selectin was observed in 68.0 +/- 9.0% of intestinal venules in ecNOS -/- mice compared with 10.0 +/- 2.0% in wild-type mice. Additional studies demonstrated that administration of an anti-P-selectin monoclonal antibody (RB40. 34) or the soluble P-selectin ligand, PSGL-1, completely inhibited the increased rolling and firm adhesion response in nNOS -/- and ecNOS -/- mice. Transmigration of neutrophils into the peritoneum following thioglycollate injection was also significantly augmented in nNOS -/- and ecNOS -/- mice. These studies clearly indicate the NO derived from both nNOS and ecNOS is critical in the regulation of leukocyte-endothelial cell interactions.Nitric oxide (NO) is known to be an important endogenous modulator of leukocyte-endothelial cell interactions within the microcirculation. We examined leukocyte rolling and adhesion under baseline conditions and following thrombin (0.25 U/ml) superfusion in the mesentery of wild-type, inducible NOS (iNOS)-deficient (-/-), neuronal NOS (nNOS) -/-, and endothelial cell NOS (ecNOS) -/- mice to further our understanding of NO and leukocyte function. Baseline leukocyte rolling (cells/min) was significantly elevated in both the nNOS -/- (30.0 ± 4.0) and ecNOS -/- mice (67.0 ± 12.0) compared with wild-type mice (11.0 ± 1.4). In addition, baseline leukocyte adherence (cells/100 μm of vessel) was also significantly elevated in the nNOS -/- (5.2 ± 1.0) and ecNOS -/- (13.0 ± 1.3) compared with wild-type animals (1.3 ± 0.5). Deficiency of iNOS had no effect on baseline leukocyte rolling or adhesion in the mesentery. Baseline surface expression of P-selectin was observed in 68.0 ± 9.0% of intestinal venules in ecNOS -/- mice compared with 10.0 ± 2.0% in wild-type mice. Additional studies demonstrated that administration of an anti-P-selectin monoclonal antibody (RB40.34) or the soluble P-selectin ligand, PSGL-1, completely inhibited the increased rolling and firm adhesion response in nNOS -/- and ecNOS -/- mice. Transmigration of neutrophils into the peritoneum following thioglycollate injection was also significantly augmented in nNOS -/- and ecNOS -/- mice. These studies clearly indicate the NO derived from both nNOS and ecNOS is critical in the regulation of leukocyte-endothelial cell interactions.

Superoxide Modulates the Oxidation and Nitrosation of Thiols by Nitric Oxide-derived Reactive Intermediates CHEMICAL ASPECTS INVOLVED IN THE BALANCE BETWEEN OXIDATIVE AND NITROSATIVE STRESS

Thiol-containing proteins are key to numerous cellular processes, and their functions can be modified by thiol nitrosation or oxidation. Nitrosation reactions are quenched by O2, while the oxidation chemistry mediated by peroxynitrite is quenched by excess flux of either NO or O2. A solution of glutathione (GSH), a model thiol-containing tripeptide, exclusively yielded S-nitrosoglutathione when exposed to the NO donor, Et2NN(O)NONa. However, when xanthine oxidase was added to the same mixture, the yield of S-nitrosoglutathione dramatically decreased as the activity of xanthine oxidase increased, such that there was a 95% reduction in nitrosation when the fluxes of NO and O2 were nearly equivalent. The presence of superoxide dismutase reversed O2-mediated inhibition, while catalase had no effect. Increasing the flux of O2 yielded oxidized glutathione (GSSG), peaking when the flux of NO and O2 were approximately equivalent. The results suggest that oxidation and nitrosation of thiols by superoxide and NO are determined by their relative fluxes and may have physiological significance.

Dynamic state of S-nitrosothiols in human plasma and whole blood.

In the vasculature, nitrosothiols derived from the nitric oxide (NO)-mediated S-nitrosation of thiols play an important role in the transport, storage, and metabolism of NO. The present study was designed to examine the reactions that promote the decomposition, formation, and distribution of extracellular nitrosothiols in the circulation. The disappearance of these species in plasma and whole blood was examined using a high-performance liquid chromatography method to separate low- and high-molecular weight nitrosothiols. We found that incubation of S-nitrosocysteine (CySNO) or S-nitrosoglutathione (GSNO) with human plasma resulted in a rapid decomposition of these nitrosothiols such that <10% of the initial concentration was recovered after 10-15 min. Neither metal chelators (DTPA, neocuproine), nor zinc chloride (glutathione peroxidase inhibitor), acivicin (gamma-glutamyl transpeptidase inhibitor), or allopurinol (xanthine oxidase inhibitor) inhibited the decomposition of GSNO. With both CySNO and GSNO virtually all NO was recovered as S-nitrosoalbumin (AlbSNO), suggesting the involvement of a direct transnitrosation reaction. Electrophilic attack of the albumin-associated thiols by reactive nitrogen oxides formed from the interaction of NO with O(2) was ruled out because one would have expected 50% yield of AlbSNO. Similar results were obtained in whole blood. The amount of S-nitrosohemoglobin recovered in the presence of 10 microM GSNO or CySNO was less than 100 nM taking into consideration the detection limit of the assay used. Our results suggest that serum albumin may act as a sink for low-molecular-weight nitrosothiols and as a modulator of NO(+) transfer between the vascular wall and intraerythrocytic hemoglobin.