Evidence that free radical generation occurs during scorpion envenomation.

Although it is well established that symptomatology, morbidity and death following scorpion envenomation are due to increases in neurotransmitter release secondary to toxins binding to voltage-sensitive sodium channels, the mechanism by which venom action is involved in damaging heart, liver, lungs and kidneys remains unclear. We hypothesized that scorpion toxins could induce the generation of high levels of free radicals responsible for membrane damage in organs targeted by venom action. We have investigated lipid peroxidation in different organs, through the evaluation of thiobarbituric acid reactive substances (TBARS), after experimental envenomation of rats by toxic fractions of Androctonus australis Hector venom. We have shown that scorpion toxins cause considerable lipid peroxidation in most vital organs. We also evaluated the protective effects of antioxidants in mice injected with lethal doses of toxins. Among the drugs tested, N-acetylcysteine (NAC) was effective in protecting the mice when injected prior to toxin application. However, the free radical scavenging properties of NAC seem less implicated in these protective effects than its ability to increase the fluidity of bronchial secretions. We therefore conclude that free radical generation only plays a minor role in the toxicity of scorpion venom.

Small conductance calcium-activated K+ channels, SkCa, but not voltage-gated K+ (Kv) channels, are implicated in the antinociception induced by CGS21680, a A2A adenosine receptor agonist.

It has been shown that A2A adenosine receptors are implicated in pain modulation. The precise mechanism by which activation of A2A receptors produces analgesic effects, however, remains unclear. The aim of this study was to investigate the possible involvement of apamin-sensitive calcium-activated potassium channels (SKCa) and voltage-gated potassium (Kv) channels in A2A receptor activation-induced analgesic effects. Using mice, we evaluated the influence of apamin, a non specific blocker of SKCa channels, Lei-Dab7 (an analog of scorpion Leiurotoxin), a selective blocker of SKCa2 channels, and kaliotoxin (KTX) a Kv channel blocker, on the CGS 21680 (A2A adenosine receptor agonist)-induced increases in hot plate and tail pinch latencies. All drugs were injected in mice via the intracerebroventricular route. We found that apamin and Lei-Dab7, but not KTX, reduced antinociception produced by CGS21680 on the hot plate and tail pinch tests in a dose dependent manner. Lei-Dab 7 was more potent than apamin in this regard. We conclude that SKCa but not Kv channels are implicated in CGS 21680-induced antinociception.

Parameters affecting in vitro oxidation/folding of maurotoxin, a four-disulphide-bridged scorpion toxin.

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K(+) channel subtypes. MTX adopts a disulphide bridge organization of the type C1-C5, C2-C6, C3-C4 and C7-C8, and folds according to the common alpha/beta scaffold reported for other known scorpion toxins. Here we have investigated the process and kinetics of the in vitro oxidation/folding of reduced synthetic L-MTX (L-sMTX, where L-MTX contains only L-amino acid residues). During the oxidation/folding of reduced L-sMTX, the oxidation intermediates were blocked by iodoacetamide alkylation of free cysteine residues, and analysed by MS. The L-sMTX intermediates appeared sequentially over time from the least (intermediates with one disulphide bridge) to the most oxidized species (native-like, four-disulphide-bridged L-sMTX). The mathematical formulation of the diffusion-collision model being inadequate to accurately describe the kinetics of oxidation/folding of L-sMTX, we have formulated a derived mathematical description that better fits the experimental data. Using this mathematical description, we have compared for the first time the oxidation/folding of L-sMTX with that of D-sMTX, its stereoisomer that contains only D-amino acid residues. Several experimental parameters, likely to affect the oxidation/folding process, were studied further; these included temperature, pH, ionic strength, redox potential and concentration of reduced toxin. We also assessed the effects of some cellular enzymes, peptidylprolyl cis-trans isomerase (PPIase) and protein disulphide isomerase (PDI), on the folding pathways of reduced L-sMTX and D-sMTX. All the parameters tested affect the oxidative folding of sMTX, and the kinetics of this process were indistinguishable for L-sMTX and D-sMTX, except when stereospecific enzymes were used. The most efficient conditions were found to be: 50 mM Tris/HCl/1.4 mM EDTA, pH 7.5, supplemented by 0.5 mM PPIase and 50 units/ml PDI for 0.1 mM reduced compound. These data represent the first report of potent stereoselective effects of cellular enzymes on the oxidation/folding of a scorpion toxin.

Adenosine and hemodialysis in humans.

BACKGROUND: Infections and hypotension are serious complications that develop during hemodialysis (HD) treatment. Adenosine (ADO), a strong hypotensive and immunosuppressive agent, may participate in these two HD complications, because high concentrations of ADO metabolites are found in dialyzed human plasma. ADO, which is released by endothelial cells, is quickly transformed into inosine (INO) by plasmatic ADO deaminase (ADA) and mononuclear cell ADO deaminase (MCADA). In plasma, the degradation of ADO into INO and its uptake by red blood cells (RBC) are both very rapid, resulting in the short half-life of ADO in blood. METHODS: Using liquid chromatography, we evaluated ADO and INO plasma concentrations before and after HD session. RESULTS: Before the HD session, ADO and INO plasma concentrations were higher in hemodialyzed patients than in controls and in peritoneally dialyzed patients. At the end of the HD session, ADO plasma concentration was increased. ADO plasma concentration for the undialyzed patients was in the same range as that of the controls. Before HD, ADA activity was higher in hemodialyzed patients (559 +/- 349 IU) than in controls (219 +/- 48 IU), and the activity rose during the session (665 +/- 135 IU). ADA activity in the undialyzed patients (222 +/- 80 IU) was in the same range as that of the controls (219 +/- 48 IU). Before the HD session, the MCADA activity (247 +/- 144 IU) was lower than in controls (624 +/- 99 IU). HD did not modify ADO RBC uptake. ADO inhibited mononuclear cell proliferation and interferon-gamma production in humans. Finally, as much as 50 microM INO does not inhibit ADO uptake by RBC and does not modify ADA and MCADA activities. CONCLUSIONS: These data indicate that chronic HD inhibited MCADA activity and increased ADO plasma concentration. Both high ADO plasma concentration and low MCADA activity may be involved in dialysis-induced immune system failure and thereby favor infectious diseases.