Alberto A. Noronha-Dutra

Reaction of nitric oxide with hydrogen peroxide to produce potentially cytotoxic singlet oxygen as a model for nitric oxide-mediated killing

Nitric oxide, as well as being a major regulator of vascular reactivity, has been shown to be one of the mediators of cytotoxicity in macrophages. This cytotoxic effect seems to be due to the interaction between nitric oxide and oxygen‐related free radicals. This study shows that, in vitro, nitric oxide reacts with hydrogen peroxide to release large amounts of chemiluminescence with the characteristics of the highly cytotoxic species, singlet oxygen. This is supported by the observation that when nitric oxide was added to a Superoxide generating system, catalase inhibited the production of singlet oxygen while Superoxide dismutase enhanced it.

Evidence for a cyclic GMP‐independent mechanism in the anti‐platelet action of S‐nitrosoglutathione

We have measured the ability of a range of NO donor compounds to stimulate cyclic GMP accumulation and inhibit collagen‐induced aggregation of human washed platelets. In addition, the rate of spontaneous release of NO from each donor has been measured spectrophotometrically by the oxidation of oxyhaemoglobin to methaemoglobin. The NO donors used were five s‐nitrosothiol compounds: S‐nitrosoglutathione (GSNO), S‐nitrosocysteine (cysNO), S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP), S‐nitroso‐N‐acetyl‐cysteine (SNAC), S‐nitrosohomocysteine (homocysNO), and two non‐nitrosothiol compounds: diethylamine NONOate (DEANO) and sodium nitroprusside (SNP). Using 10 μM of each donor compound, mean ±s.e.mean rate of NO release ranged from 0.04±0.001 nmol min−1 (for SNP) to 3.15±0.29 nmol min−1 (for cysNO); cyclic GMP accumulation ranged from 0.43±0.05 pmol per 108 platelets (for SNP) to 2.67±0.31 pmol per 108 platelets (for cysNO), and inhibition of platelet aggregation ranged from 40±6.4% (for SNP) to 90±3.8% (for SNAC). There was a significant positive correlation between the rate of NO release and the ability of the different NO donors to stimulate intra‐platelet cyclic GMP accumulation (r=0.83; P=0.02). However, no significant correlation was observed between the rate of NO release and the inhibition of platelet aggregation by the different NO donors (r=−0.17), nor was there a significant correlation between cyclic GMP accumulation and inhibition of aggregation by the different NO donor compounds (r=0.34). Comparison of the dose‐response curves obtained with GSNO, DEANO and 8‐bromo cyclic GMP showed DEANO to be the most potent stimulator of intraplatelet cyclic GMP accumulation (P<0.001 vs both GSNO and 8‐bromo cyclic GMP), but GSNO to be the most potent inhibitor of platelet aggregation (P<0.01 vs DEANO, and P<0.001 vs 8‐bromo cyclic GMP). The rate of NO release from GSNO, and its ability both to stimulate intra‐platelet cyclic GMP accumulation and to inhibit platelet aggregation, were all significantly diminished by the copper (I) (Cu+) chelating agent bathocuproine disulphonic acid (BCS). In contrast, BCS had no effect on either the rate of NO release, or the anti‐platelet action of the non‐nitrosothiol compound DEANO. Cyclic GMP accumulation in response to GSNO (10−9–10−5M) was undetectable following treatment of platelets with ODQ (100 μM), a selective inhibitor of soluble guanylate cyclase. Despite this abolition of guanylate cyclase stimulation, GSNO retained some ability to inhibit aggregation, indicating the presence of a cyclic GMP‐independent component in its anti‐platelet action. However, this component was abolished following treatment of platelets with a combination of both ODQ and BCS, suggesting that Cu+ ions were required for the cyclic GMP‐independent pathway to operate. The cyclic GMP‐independent action of GSNO, observed in ODQ‐treated platelets, could not be explained by an increase in intra‐platelet cyclic AMP. The impermeable thiol modifying agent p‐chloromercuriphenylsulphonic acid (CMPS) produced a concentration‐dependent inhibition of aggregation of ODQ‐treated platelets, accompanied by a progressive loss of detectable platelet surface thiol groups. Additional treatment with GSNO failed to increase the degree of aggregation inhibition, suggesting that a common pathway of thiol modification might be utilized by both GSNO and CMPS to elicit cyclic GMP‐independent inhibition of platelet aggregation. We conclude that NO donor compounds mediate inhibition of platelet aggregation by both cyclic GMP‐dependent and ‐independent pathways. Cyclic GMP generation is related to the rate of spontaneous release of NO from the donor compound, but transfer of the NO signal to the cyclic GMP‐independent pathway may depend upon a cellular system which involves both copper (I) (Cu+) ions and surface membrane thiol groups. The potent anti‐platelet action of GSNO results from its ability to exploit this cyclic GMP‐independent mechanism.

Copper chelation-induced reduction of the biological activity of S-nitrosothiols.

1 The effect of copper on the activity of the S‐nitrosothiol compounds S‐nitrosocysteine (cysNO) and S‐nitrosoglutathione (GSNO) was investigated, using the specific copper chelator bathocuproine sul‐phonate (BCS), and human washed platelets as target cells. 2 Chelation of trace copper with BCS (10 μm) in washed platelet suspensions reduced the inhibition of thrombin‐induced platelet aggregation by GSNO; however, BCS had no significant effect on the anti‐aggregatory action of cysNO. BCS inhibited cyclic GMP generation in response to both cysNO and GSNO. 3 The effect of BCS was rapid (within 30 s), and could be abolished by increasing the platelet concentration to 500 times 109 1−1. 4 In BCS‐treated platelet suspensions, the addition of Cu2+ ions (0.37–2.37 μm) led to a restoration of both guanylate cyclase activation and platelet aggregation inhibition by GSNO. 5 The anti‐aggregatory activity of GSNO was reduced in a concentration‐dependent manner by the copper (I)‐specific chelators BCS and neocuproine, and to a smaller extent by desferal. No effect was observed with the copper (II) specific chelator, cuprizone, the iron‐specific chelator, bathophenanthroline sulphonate, or the broader‐specificity copper chelator, d‐penicillamine. 6 In both BCS‐treated and ‐untreated platelet suspensions, cys NO was more potent than GSNO as a stimulator of guanylate cyclase. In BCS‐treated platelet suspensions there was no significant difference between the anti‐aggregatory potency of cysNO and GSNO; however, in untreated suspensions, GSNO was significantly more potent than cysNO. Thus, when copper was available, GSNO produced a greater inhibition of aggregation than cysNO, despite being a less potent activator of guanylate cyclase. 7 The breakdown of cysNO and GSNO was measured spectrophotometrically by decrease in absor‐bance at 334 nm. In Tyrode buffer, cysNO (10 μm) broke down at a rate of 3.3 μm min−1. BCS (10 μm) reduced this to 0.5 μm min−1. GSNO, however, was stable, showing no fall in absorbance over a period of 7 min even in the absence of BCS. 8 We conclude that copper is required for the activity of both cysNO and GSNO, although its influence on anti‐aggregatory activity is only evident with GSNO. The stimulatory effect of copper is unlikely to be explained solely by catalysis of S‐nitrosothiol breakdown. The enhancement by copper of th anti‐aggregatory activity of GSNO, relative to cysNO, suggests that copper may be required for biological activity of GSNO which is independent of guanylate cyclase stimulation.

Mitochondrial superoxide production during oxalate-mediated oxidative stress in renal epithelial cells

Crystals of calcium oxalate monohydrate (COM) in the renal tubule form the basis of most kidney stones. Tubular dysfunction resulting from COM-cell interactions occurs by mechanism(s) that are incompletely understood. We examined the production of reactive oxygen intermediates (ROI) by proximal (LLC-PK1) and distal (MDCK) tubular epithelial cells after treatment with COM (25-250 microg/ml) to determine whether ROI, specifically superoxide (O(2)(*-)), production was activated, and whether it was sufficient to induce oxidative stress. Employing inhibitors of cytosolic and mitochondrial systems, the source of ROI production was investigated. In addition, intracellular glutathione (total and oxidized), energy status (ATP), and NADH were measured. COM treatment for 1-24 h increased O(2)(*-) production 3-6-fold as measured by both lucigenin chemiluminescence in permeabilized cells and dihydrorhodamine fluorescence in intact cells. Using selective inhibitors we found no evidence of cytosolic production. The use of mitochondrial probes, substrates, and inhibitors indicated that increased O(2)(*-) production originated from mitochondria. Treatment with COM decreased glutathione (total and redox state), indicating a sustained oxidative insult. An increase in NADH in COM-treated cells suggested this cofactor could be responsible for elevating O(2)(*-) generation. In conclusion, COM increased mitochondrial O(2)(*-) production by epithelial cells, with a subsequent depletion of antioxidant status. These changes may contribute to the reported cellular transformations during the development of renal calculi.

Inhibition of NADPH supply by 6-aminonicotinamide: effect on glutathione, nitric oxide and superoxide in J774 cells.

We have examined the integrity of J774 cell nitric oxide (NO) production and glutathione maintenance, whilst NADPH supply was compromised by inhibition of the pentose pathway with 6‐aminonicotinamide. In resting cells 6‐phosphogluconate accumulation began after 4 h and glutathione depletion after 24 h of 6‐aminonicotinamide treatment. Cellular activation by lipopolysaccharide/interferon‐λ decreased glutathione by ∼50% and synchronous 6‐aminonicotinamide treatment exacerbated this to 31.2% of control (P<0.05). In activated cells NO− 2 production was inhibited by 60% with 6‐aminonicotinamide (P<0.01), and superoxide production by 50% (P<0.01) in zymosan‐activated cells. NADPH production via the pentose pathway is therefore important to sustain macrophage NO production whilst maintaining protective levels of glutathione.

Nitric oxide in cigarette smoke as a mediator of oxidative damage

A strong association is known to exist between cigarette smoking and the risk of atherosclerosis-related arterial disease (Doll et al. 1990; Pittilo & Woolf 1993). The mechanisms by which cigarette smoke (CS) produces its effects on the artery wall remain, for the most part, unknown. CS is an immensely complex mixture containing thousands of components many of which are potentially cytotoxic. There is, however, a considerable body of evidence showing that free radicals in CS may be the main contributing factor towards CS-related diseases (Noronha-Dutra et al. 1993; Church & Pryor 1985; Frei et al. 1991; Eiserich et al. 1994; Norman & Keith 1965). Exposure to cigarette smoke is reported to cause blebbing of the luminal plasma membranes of endothelial cells in both human and small mammal arteries (Woolf & Wilson-Holt 1981; Sieffert et al. 1981; Pittilo et al. 1985; Asmussen 1982). This blebbing has been suggested to be one of the morphological correlates of lipid peroxidation occurring as a result of oxidant stress in these membranes (Noronha-Dutra & Steen 1982). Later studies from this group (Noronha-Dutra et al. 1993) have shown that exposure of cultured human umbilical vein endothelial cells to plasma containing fresh cigarette smoke or to plasma from human volunteer smokers produces biochemical changes, such as activation of the hexose monophosphate shunt and extrusion of glutathione, which are consistent with such oxidant stress. Glutathione and its associated enzymes constitute one of the main lines of defence against peroxides. It can not only act as a chain breaker but, through the action of glutathione peroxidase, can eliminate peroxides already formed, reducing them to alcohol. Most radicals are derivatives of nitric oxide which is present at up to 500 p.p.m. in the gas phase of CS (Eiserich et al. 1994; Pryor & Stone 1993) and may represent one of the major sources of nitric oxide to which humans are exposed (Norman & Keith 1965). We have shown in a previous study that nitric oxide reacts with hydrogen peroxide to release large amounts of chemiluminescence with the characteristics of the highly cytotoxic species, singlet oxygen (Noronha-Dutra et al. 1993). In this study we have compared the cytotoxic potential and the amount of chemiluminescence generated in human plasma exposed to two gas-phase CSs, differing in their nitric oxide content but otherwise of similar chemical composition, from two types of cigarette. We have also investigated the possibility that human erythrocytes (r.b.c.) may play a role in scavenging CS-derived free radicals. Red blood cells contain large amounts of glutathione, which may protect against CS-linked oxidative stress.

Production of singlet oxygen by eosinophils activated in vitro by C5a and leukotriene B4

Using the trans‐methoxyvinylpyrene analogues of benzo[a]pyrene‐7,8‐dihydrodiol (MVP) as a singlet oxygen (1O2) chemiluminescence probe, we have demonstrated that guinea pig eosinophils release 1O2 when activated with the physiological agonists C5a and leukotriene B4. This release, which occurs at agonist concentrations as low as 10−7 M, occurs more rapidly than activation with phorbol ester (10−6 M), is similar in level, but is more transitory. In addition, the release of 1O2 occurs in the absence of added bromide ions and represents, we propose, an important feature of eosinophil‐mediated inflammatory damage.