Leishmania amazonensis fails to induce the release of reactive oxygen intermediates by CBA macrophages.

CBA mouse macrophages effectively control Leishmania major infection, yet are permissive to Leishmania amazonensis. It has been established that some Leishmania species are destroyed by reactive oxygen species (ROS). However, other species of Leishmania exhibit resistance to ROS or even down-modulate ROS production. We hypothesized that L. amazonensis-infected macrophages reduce ROS production soon after parasite-cell interaction. Employing a highly sensitive analysis technique based on chemiluminescence, the production of superoxide (O(·-)(2)) and hydrogen peroxide (H(2)O(2)) by L. major- or L. amazonensis-infected CBA macrophages were measured. L. major induces macrophages to release levels of (O(·-)(2)) 3·5 times higher than in uninfected cells. This (O(·-)(2)) production is partially dependent on NADPH oxidase (NOX) type 2. The level of accumulated H(2)O(2) is 20 times higher in L. major-than in L. amazonensis-infected cells. Furthermore, macrophages stimulated with L. amazonensis release amounts of ROS similar to uninfected cells. These findings support previous studies showing that CBA macrophages are effective in controlling L. major infection by a mechanism dependent on both (O(·-)(2)) production and H(2)O(2) generation. Furthermore, these data reinforce the notion that L. amazonensis survive inside CBA macrophages by reducing ROS production during the phagocytic process.

Expression and characterization of LTx2, a neurotoxin from Lasiodora sp. effecting on calcium channels.

Here, we described the expression and characterization of the recombinant toxin LTx2, which was previously isolated from the venomous cDNA library of a Brazilian spider, Lasiodora sp. (Mygalomorphae, Theraphosidae). The recombinant toxin found in the soluble and insoluble fractions was purified by reverse phase high-performance liquid chromatography (HPLC). Ca2+ imaging analysis revealed that the recombinant LTx2 acts on calcium channels of BC3H1 cells, blocking L-type calcium channels.

Glutathione and the redox control system trypanothione/trypanothione reductase are involved in the protection of Leishmania spp. against nitrosothiol-induced cytotoxicity

Glutathione is the major intracellular antioxidant thiol protecting mammalian cells against oxidative stress induced by oxygen- and nitrogen-derived reactive species. In trypanosomes and leishmanias, trypanothione plays a central role in parasite protection against mammalian host defence systems by recycling trypanothione disulphide by the enzyme trypanothione reductase. Although Kinetoplastida parasites lack glutathione reductase, they maintain significant levels of glutathione. The aim of this study was to use Leishmania donovani trypanothione reductase gene mutant clones and different Leishmania species to examine the role of these two individual thiol systems in the protection mechanism against S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a nitrogen-derived reactive species donor. We found that the resistance to SNAP of different species of Leishmania was inversely correlated with their glutathione concentration but not with their total low-molecular weight thiol content (about 0.18 nmol/10(7) parasites, regardless Leishmania species). The glutathione concentration in L. amazonensis, L. donovani, L. major, and L. braziliensis were 0.12, 0.10, 0.08, and 0.04 nmol/10(7) parasites, respectively. L. amazonensis, that have a higher level of glutathione, were less susceptible to SNAP (30 and 100 µM). The IC50 values of SNAP determined to L. amazonensis, L. donovani, L. major, and L. braziliensis were 207.8, 188.5, 160.9, and 83 µM, respectively. We also observed that L. donovani mutants carrying only one trypanothione reductase allele had a decreased capacity to survive (40 percent) in the presence of SNAP (30-150 µM). In conclusion, the present data suggest that both antioxidant systems, glutathione and trypanothione/trypanothione reductase, participate in protection of Leishmania against the toxic effect of nitrogen-derived reactive species.

Glutathione and the redox control system trypanothione/trypanothione reductase are involved in the protection of Leishmania spp. against nitrosothiol-induced cytotoxicity.

Glutathione is the major intracellular antioxidant thiol protecting mammalian cells against oxidative stress induced by oxygen- and nitrogen-derived reactive species. In trypanosomes and leishmanias, trypanothione plays a central role in parasite protection against mammalian host defence systems by recycling trypanothione disulphide by the enzyme trypanothione reductase. Although Kinetoplastida parasites lack glutathione reductase, they maintain significant levels of glutathione. The aim of this study was to use Leishmania donovani trypanothione reductase gene mutant clones and different Leishmania species to examine the role of these two individual thiol systems in the protection mechanism against S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a nitrogen-derived reactive species donor. We found that the resistance to SNAP of different species of Leishmania was inversely correlated with their glutathione concentration but not with their total low-molecular weight thiol content (about 0.18 nmol/10(7) parasites, regardless Leishmania species). The glutathione concentration in L. amazonensis, L. donovani, L. major, and L. braziliensis were 0.12, 0.10, 0.08, and 0.04 nmol/10(7) parasites, respectively. L. amazonensis, that have a higher level of glutathione, were less susceptible to SNAP (30 and 100 microM). The IC50 values of SNAP determined to L. amazonensis, L. donovani, L. major, and L. braziliensis were 207.8, 188.5, 160.9, and 83 microM, respectively. We also observed that L. donovani mutants carrying only one trypanothione reductase allele had a decreased capacity to survive (approximately 40%) in the presence of SNAP (30-150 microM). In conclusion, the present data suggest that both antioxidant systems, glutathione and trypanothione/trypanothione reductase, participate in protection of Leishmania against the toxic effect of nitrogen-derived reactive species.

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.

Uremic concentrations of guanidino compounds inhibit neutrophil superoxide production.

BACKGROUND: In uremia, diminished reactive oxygen intermediate (ROI) production is an important consequence of impaired neutrophil function. We have studied the effect of guanidino compounds, known uremic toxins, on neutrophil ROI production in vitro. METHODS: Neutrophils from healthy volunteers were exposed for three hours to individual or mixed guanidino compounds (GCmix) at concentrations encountered in uremic plasma. After removal of guanidino compounds, neutrophils were activated by adhesion, N-formyl-methionyl-leucyl-phenyalanine (fMLP), phorbol 12-myristate 13-acetate (PMA), or opsonized zymosan, and superoxide production was measured by lucigenin chemiluminescence (CL). The direct effect of guanidino compounds on superoxide production in activated neutrophils was also measured. The energy status (ATP and creatine phosphate), antioxidant status (total glutathione), and glycolytic flux (lactate production) were measured. RESULTS: The GCmix pretreatment decreased the superoxide production in activated neutrophils (fMLP or zymosan) by 50% (P < 0.01) and the ATP concentration by 60% (P < 0.05), and it inhibited glycolytic flux (lactate production) by 45% (P < 0.01), but did not alter glutathione concentration. Simultaneous exposure to GCmix and activation did not inhibit nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in cell lysates, but inhibited superoxide formation in zymosan-activated intact neutrophils, and this inhibition was reversed following removal of the guanidino compounds. CONCLUSION: Guanidino-succinate, -propionate, and -butyrate were individually as potent as the GCmix. Inhibition of neutrophil superoxide generation by guanidino compounds results from a depressed energy status. Uremic concentrations of guanidino compounds significantly inhibit neutrophil metabolism, and this has serious implications for their function in host defense.

Glutathione protects macrophages and Leishmania major against nitric oxide-mediated cytotoxicity.

The aim of this investigation was to examine whether macrophage and Leishmania major glutathione were involved in either host or parasite protection against NO cytotoxicity. Buthionine sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthase, caused a complete and irreversible depletion of macrophage glutathione, but only a 20% and reversible decrease in L. major glutathione. Glutathione-depleted macrophages, when activated with IFN-gamma/LPS, released less than 60% of the NO produced by untreated macrophages, resulting in a corresponding decrease in their leishmanicidal activity. BSO-treated macrophages were more susceptible to the cytotoxic effects of the NO donor SNAP. Treatment of macrophages with 1,3-bis(chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase and trypanothione reductase or with Br-Octane, a glutathione-S-transferase substrate, resulted in a transient decrease in glutathione levels and did not increase the susceptibility of the macrophages to SNAP. Treatment of the promastigote forms of L. major with BCNU resulted in an 80% decrease in total glutathione concentration with no concomitant change in viability. However, this treatment rendered the parasites more susceptible to SNAP. Finally, macrophage glutathione protected the internalized L. major from SNAP. Overall, these results demonstrate that glutathione is an essential protective component against NO cytotoxicity on both macrophages and parasites.

S-nitrosothiols are stored by platelets and released during platelet-neutrophil interactions.

Interaction between platelets and neutrophils is important in vascular injury. We have investigated the storage and release of nitric oxide (NO) by platelets interacting with neutrophils. Shear-activated platelets were added to neutrophils in suspension and both superoxide and peroxynitrite formations monitored by lucigenin- and luminol-enhanced chemiluminescence. In addition, intraplatelet S-nitrosothiols were measured by dichlorofluorescein fluorescence following displacement of NO by mercuric chloride. Addition of activated platelets to neutrophils caused free radical production and platelet-neutrophil rosette formation. Pretreatment of platelets with 20 microM S-nitrosoglutathione changed the balance between luminol and lucigenin chemiluminescence in favor of luminol, whereas S-nitrosoglutathione in platelet-free plasma did not produce these changes. This pattern was also observed both following inhibition of neutrophil NO synthase and in a neutrophil-free superoxide-generating system. Inhibition of platelet NO synthase decreased luminol and increased lucigenin chemiluminescence. These effects were reversed by L-arginine. Platelet activation increased intraplatelet S-nitrosothiols from 1.93+/-0.19 (mean +/- SD) to 4.9+/-1.10 x 10(-18) mol/platelet (P < 0.01); this increase halved following NO synthase inhibition, but was enhanced by approximately 220% following incubation with S-nitrosoglutathione. These results show that during shear stress platelets store S-nitrosothiols, which can be derived either endogenously from NO synthesis or exogenously by sequestration of S-nitrosoglutathione. Release of stored NO during platelet-neutrophil interaction alters the interaction of NO with superoxide and could modulate vascular inflammation.

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-lambda decreased glutathione by approximately 50% and synchronous 6-aminonicotinamide treatment exacerbated this to 31.2% of control (P < 0.05). In activated cells NO2- 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.

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

1. 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). 2. Using 10 microM 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 10(8) platelets (for SNP) to 2.67+/-0.31 pmol per 10(8) platelets (for cysNO), and inhibition of platelet aggregation ranged from 40+/-6.4% (for SNP) to 90+/-3.8% (for SNAC). 3. 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). 4. 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). 5. 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. 6. Cyclic GMP accumulation in response to GSNO (10(-9) 10(-5) M) was undetectable following treatment of platelets with ODQ (100 microM), 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. 7. The cyclic GMP-independent action of GSNO, observed in ODQ-treated platelets, could not be explained by an increase in intra-platelet cyclic AMP. 8. 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. 9. 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

Cell-mediated biotransformation of S-nitrosoglutathione.

Spontaneous release of nitric oxide (NO) from S-nitrosothiols cannot explain their bioactivity, suggesting a role for cellular metabolism or receptors. Using immortalised cells and human platelets, we have identified a cell-mediated mechanism for the biotransformation of the physiological S-nitrosothiol compound S-nitrosoglutathione (GSNO) into nitrite. We suggest the name "GSNO lyase" for this activity. GSNO lyase activity varied between cell types, being highest in a fibroblast cell line and lowest in platelets. In NRK 49F fibroblasts, GSNO lyase mediated a saturable, GSNO concentration-dependent accumulation of nitrite in conditioned medium, which was inhibited both by transition metal chelators, and by subjecting cells to oxidative stress using a combination of the thiol oxidant diamide and Zn2+, a glutathione reductase inhibitor. Activity was resistant, however, to both acivicin, an inhibitor of gamma-glutamyl transpeptidase (EC 2.3.2.2), and to ethacrynic acid, an inhibitor of Pi class glutathione-S-transferases (EC 2.5.1.18), thus neither of these enzymes could account for NO release. Although GSNO lyase does not explain the platelet-selective pharmacological properties of GSNO, cellular biotransformation suggests therapeutic avenues for targeted delivery of NO to other tissues.

Induction of nitric oxide synthesis in J774 cells lowers intracellular glutathione: effect of modulated glutathione redox status on nitric oxide synthase induction.

Under pathological conditions, the induction of nitric oxide synthase (NOS) in macrophages is responsible for NO production to a cytotoxic concentration. We have investigated changes to, and the role of, intracellular glutathione in NO production by the activated murine macrophage cell line J774. Total glutathione concentrations (reduced, GSH, plus the disulphide, GSSG) were decreased to 45% of the control 48 h after cells were activated with bacterial lipopolysaccharide plus interferon gamma. This was accompanied by a decrease in the GSH/GSSG ratio from 12:1 to 2:1. The intracellular decrease was not accounted for by either GSH or GSSG efflux; on the contrary, rapid export of glutathione in control cells was abrogated during activation. The loss of intra- and extracellular glutathione indicates either a decrease in synthesis de novo, or an increase in utilization, rather than competition for available NADPH. All changes in activated cells were prevented by pretreatment with the NOS inhibitor L-N-(1-iminoethyl)ornithine. Basal glutathione levels in J774 cells were manipulated by pretreatment with (1) buthionine sulphoximine (glutathione synthase inhibitor), (2) acivicin (gamma-glutamyltranspeptidase inhibitor), (3) bromo-octane (glutathione S-transferase substrate) and (4) diamide/zinc (thiol oxidant and glutathione reductase inhibitor). All treatments significantly decreased the output of NO following activation. The degree of inhibition was dependent on (i) duration of treatment prior to activation, (ii) rate of depletion or subsequent recovery and (iii) thiol end product. The level of GSH did not significantly affect the production of NO, after induction of NOS. Thus, glutathione redox status appears to plays an important role in NOS induction during macrophage activation.

Role of a copper (I)-dependent enzyme in the anti-platelet action of S-nitrosoglutathione.

1. S-nitrosoglutathione (GSNO) is a potent and selective anti-platelet agent, despite the fact that its spontaneous rate of release of nitric oxide (NO) is very slow. Our aim was to investigate the mechanism of the anti-aggregatory action of GSNO. 2. The biological action of GSNO could be mediated by NO released from S-nitrosocystylglycine, following enzymatic cleavage of GSNO by gamma-glutamyl transpeptidase. The anti-aggregatory potency of GSNO was not, however, altered by treatment of target platelets with the gamma-glutamyl transpeptidase inhibitor acivicin (1 mM). gamma-Glutamyl transpeptidase is not, therefore, involved in mediating the action of GSNO. 3. The rate of breakdown of S-nitrosoalbumin was increased from 0.19 +/- 0.086 nmol min-1 to 1.52 +/- 0.24 nmol min-1 (mean +/- s.e.mean) in the presence of cysteine (P < 0.05, n = 4). Inhibition of platelet aggregation by S-nitrosoalbumin was also significantly increased by cysteine (P < 0.05, n = 4), suggesting that the biological activity of S-nitrosoalbumin is mediated by exchange of NO from the protein carrier to form the unstable compound cysNO. Breakdown of GSNO showed a non-significant acceleration in the presence of cysteine, from 0.56 +/- 0.22 to 1.77 +/- 0.27 nmol min-1 (mean +/- s.e.mean) (P = 0.064, n = 4), and its ability to inhibit platelet aggregation was not enhanced by cysteine. This indicates that the anti-platelet action of GSNO is not dependent upon transnitrosation to form cysNO. 4. Platelets pretreated with the copper (I)-specific chelator bathocuproine disulphonic acid (BCS), then resuspended in BCS-free buffer, showed resistance to the inhibitory effect of GSNO. These findings suggest that BCS impedes the action of GSNO by binding to structures on the platelet, rather than by chelating free copper in solution. 5. Release of NO from GSNO was catalysed enzymatically by ultrasonicated platelet suspensions. This enzyme had an apparent K(m) for GSNO of 12.4 +/- 2.64 microM and a Vmax of 0.21 +/- 0.03 nmol min-1 per 10(8) platelets (mean +/- s.e.mean, n = 5). It was inhibited by BCS, but not by the iron chelator bathophenathroline disulphonic acid, nor by acivicin. 6. We conclude that the stable S-nitrosothiol compound GSNO may exert its anti-platelet action via enzymatic, rather than spontaneous release of NO. This is mediated by a copper-dependent mechanism. The potency and platelet-selectivity of GSNO may result from targeted NO release at the platelet surface.

Effect of cigarette smoking on cultured human endothelial cells.

OBJECTIVE: The aim was to investigate whether the morphological changes previously described in endothelium exposed to cigarette smoke are linked with the oxidative burden imposed on the cells. METHODS: Cultured human umbilical vein endothelial cells (HUVEC) were exposed to samples of plasma taken from volunteer smokers and to samples of plasma to which small doses of fresh cigarette smoke derived from a smoking machine had been added. Measurements of the pentose phosphate pathway and the extruded total glutathione (GSSG) were performed to assess the presence and degree of oxidative stress on cells. Angiotensin converting enzyme (ACE) release into the medium and the ATP content of the cells were used to assess early membrane damage and cytotoxicity. RESULTS: Treatment of endothelial cells with plasma exposed to cigarette smoke in vitro resulted in activation of the pentose phosphate pathway of glucose metabolism, increased extrusion of glutathione from the cells into the medium, a decrease in the ATP pool, and release of ACE from the cells into the medium. Plasma taken from volunteers immediately after smoking showed, as might be expected, similar but less marked changes in the release of glutathione and ACE. CONCLUSIONS: Plasma from human volunteer smokers or human plasma exposed to cigarette smoke in vitro produced injury in HUVEC as assessed by changes in the ATP pool and ACE release. The extrusion of glutathione from the cells and the activation of the hexose monophosphate shunt, which is necessary to keep glutathione in the reduced state, is indicative of oxidative stress. These findings support the view that cigarette smoke related endothelial injury is, at least in part, mediated by the oxidative burden imposed by the free radicals present in cigarette smoke.

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.

An antioxidant role for calcium antagonists in the prevention of adrenaline mediated myocardial and endothelial damage.

OBJECTIVE: To investigate the mechanism of calcium antagonist mediated cytoprotection against the damaging effects of adrenaline in vivo on cardiac myocytes and human endothelial cells from the umbilical vein. METHODS: Human endothelial cells cultured from the umbilical vein and isolated rat cardiac myocytes were treated with plasma from rats given adrenaline 30 minutes previously with pretreatment with calcium antagonists and without. The effect on indices of cell damage that suggest oxidation stress was determined. RESULTS: Pretreatment of rats with calcium antagonists before adrenaline administration largely inhibited the cytotoxic effects of their plasma on the two target cells used. Plasma taken from animals not pretreated with calcium antagonists caused release of oxidised glutathione from cells, a fall in intra-cellular reduced glutathione concentration, a fall in ATP production, and release of angiotensin converting enzyme from the endothelial cells. CONCLUSION: Calcium antagonists protect against the cardiotoxic effects of catecholamine by preventing the generation of plasma borne cytotoxic compounds, which are probably free radicals.

Loss of NAD(P)-reducing power and glutathione disulfide excretion at the start of induction of aerial growth in Neurospora crassa.

When exponentially growing hyphae of Neurospora crassa in aerated liquid cultures are filtered and the resulting mycelial mat is exposed to air, aerial hyphae develop and synchronous conidiation is obtained. The hyphae in direct contact with air adhere to each other within minutes and form aerial hyphae during the following 12 h; the hyphae which are not in direct contact with air do not adhere to each other and do not form aerial hyphae. Previous data indicated that oxidative stress was generated in the adhering hyphae; proteins and specific enzymes were found to be oxidatively modified and degraded. In this work, we report a dramatic fall in the reduced-to-oxidized ratio of NAD and NADP coenzymes during the first 6 min of exposure to air. This drop did not occur in a mycelial mat exposed to a N2-enriched atmosphere. Adding a carbon source to the mycelial mat did not abolish the loss of NAD(P)-reducing power. After the initial fall, the reducing levels of the coenzymes returned to the starting value in about 30 min. A peak of extracellular glutathione disulfide occurred simultaneously with the loss of NAD(P)-reducing power. The reducing power loss and the excretion of glutathione disulfide are thought to be consequences of a hyperoxidant state; the adhesion of hyphae is thought to be a response to the hyperoxidant state.

Epinephrine-induced cytotoxicity of rat plasma. Its effects on isolated cardiac myocytes.

The plasma from rats injected with epinephrine (5 mg/kg) (epinephrine plasma) was found to be cytotoxic when incubated with isolated heart cells. This cytotoxicity was apparent 60 minutes after the start of the incubation due to the appearance at this time of membrane microblebs and microvilli as seen by scanning electron microscopy and the start of a fall in intracellular concentrations of ATP. The membrane blebs increased in size with time and the cells became contracted. Incubation of the cells for 90 minutes with epinephrine plasma, caused an increase in permeability of plasma membrane to 86Rb+. Incubation with alpha-tocopherol prevent this change. After 180 minutes incubation, these cells no longer retained carboxyfluorescein indicating that extensive membrane damage had occurred. It has been suggested that the presence of membrane blebs are evidence that lipid peroxidation is occurring in the cell membrane. Accordingly, glutathione, which is used by the cell against free radical attack and membrane lipid peroxidation, was measured. Glutathione levels started to fall after 60 minutes incubation, the time when the ultrastructural cell membrane changes started to occur. Pretreatment of cells with 100 microM 1,3-bis(2-chloroethyl)-1-nitrosourea which inhibits glutathione reductase, before the addition of plasma, increased the sensitivity of these cells to the epinephrine plasma, as measured by fall in intracellular ATP. Cells treated with control plasma did not show the changes described above. The nature of the cytotoxic factor in the plasma is still unknown, although the evidence presented suggests that a free radical may be involved.

The correlation between catecholamine and lipid peroxidation induced damage in heart cells.

Cardiocytes incubated with isoprenaline plasma showed an initial rise in the intracellular ATP levels followed by a steady fall; after two hours the ATP levels of the cells treated with the plasma were less than half that of the control cells. This fall in intracellular ATP was accompanied by morphological changes, in particular the formation of both large and small membrane blebs.