Enhanced proteolysis and changes in membrane-associated calpain following phenylhydrazine insult to human red cells

Phenylhydrazine-mediated protein damage in human red cells has been assessed using HPLC, one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblot analysis of major membrane proteins. The association of the Ca(2+)-activated neutral protease, calpain, with membrane proteins following hydrazine insult was also examined using immunoblot analysis. HPLC amino acid analysis of red cell suspensions was employed to quantify proteolysis. Phenylhydrazine (4 mM) increased the rate of leucine, lysine, and histidine release by approximately 12-, 7-, and 5-fold, respectively. N-acetylcysteine (20 mM), dithiothreitol (50 mM), and dimethylthiourea (50 mM) decreased the rate of phenylhydrazine-stimulated amino acid release by approximately 30-50%; in contrast, the free radical scavengers and antioxidants dimethylfuran (50 mM) and dimethyl sulfoxide (50 mM) were without significant effect. The calcium chelator, EGTA (10 mM) inhibited phenylhydrazine-stimulated proteolysis by approximately 30%. Phenylhydrazine (4 mM) caused attenuation of the major membrane protein bands present in the SDS-PAGE pattern and extensive smearing of a band in the region of approximately 28 kDa. Free radical scavengers and antioxidants failed to ameliorate significantly membrane protein damage in phenylhydrazine-treated cells as judged by SDS-PAGE. Immunoblot analysis of spectrin confirmed these results. Two-dimensional SDS-PAGE of membrane proteins following phenylhydrazine treatment, however, revealed the appearance of new protein spots and a loss of existing protein spots as compared to control. Western blot analysis of membrane-associated calpain (79 kDa (proenzyme), 77- and 75-kDa forms) was also performed. Phenylhydrazine-treated red blood cells exhibited concentration- and time-dependent changes in the level of membrane-associated procalpain relative to control. The inhibitors N-acetylcysteine, dithiothreitol, dimethylthiourea, and dimethyl sulfoxide in the presence of phenylhydrazine appeared to preserve the level of procalpain in association with the membrane proteins, but only N-acetylcysteine and dithiothreitol protected the 77- and 75-kDa forms. In contrast, dimethylfuran in the presence of phenylhydrazine caused a substantial decrease in all three forms of membrane-associated calpain. In phenylhydrazine-treated hemolysate, the level of the 77- and 75-kDa forms of membrane-associated calpain was decreased relative to control. These forms were absent when EGTA (10 mM) was included in the incubation and the level of proenzyme was decreased. These data suggest that calpain is recruited to the membrane following hydrazine insult, undergoes a Ca(2+)-dependent conversion to the active forms, and may be involved in the degradation of damaged cytosolic and membrane protein(s).     

Effect of calcium and phosphate ions on hemolysis induced by Pyrularia thionin and Naja naja kaouthia cardiotoxin.

Pyrularia thionin is a strongly basic bioactive peptide of 47 amino acids isolated from nuts of Pyrularia pubera. It is hemolytic, cytotoxic and activates an endogenous phospholipase A2 in 3T3 cells. Earlier studies have shown that the cardiotoxin from Naja naja kaouthia has similar activities and binds to the same site as Pyrularia thionin. Since the peptides appear to bind to the phospholipids of cell membranes to elicit their cellular responses, the effect of modifying the electrostatic environment was studied by separately adding phosphate ion and Ca2+, and by removing Ca2+ from the membrane by treatment with EGTA. Analysis of erythrocyte hemolysis for both Pyrularia thionin and cardiotoxin shows that the reactions follow Michaelis-Menten kinetics, with the peptides serving as the substrate. The basal rate of hemolysis in physiological saline is markedly increased by the addition of phosphate in the 5-10 mM range and also by removing membrane-bound Ca2+ by incubation of the cells with 10 mM EGTA. These treatments do not change the apparent K(m) values, but increase the V(max), indicating that more binding sites are made available by these treatments. On the other hand, added Ca2+ in the 5-10 mM range competitively inhibits the reaction by inhibiting the binding of the peptide to the membrane.     

Binding properties of Pyrularia thionin and Naja naja kaouthia cardiotoxin to human and animal erythrocytes and to murine P388 cells.

Pyrularia thionin and snake venom cardiotoxin are strongly basic peptides which induce hemolysis, depolarization of muscle cells and activation of endogenous phospholipase A2. An earlier study of the hemolysis reaction indicated that the two peptides bind to and compete for the same site on human erythrocytes. A recent study examined the hemolysis induced by both peptides as the phosphate and Ca2+ content of the reaction mixture was varied. The results of the recent study (VERNON, L. P. and ROGERS, A., Toxicon 30, 701-709) agree with this companion study on the binding of 125I-labeled pyrularia thionin and cardiotoxin to erythrocytes under the same conditions. Added phosphate ion at 5 mM and removal of membrane-bound Ca2+ by treatment with 10 mM EGTA make more binding sites of the same affinity available to both peptides, which are shown to bind in a competitive fashion to the same site. Addition of 10 mM Ca2+ to the medium decreases peptide binding due to competitive binding of Ca2+ to the same site on the membrane. For human erythrocytes the number of binding sites/cell for the thionin ranged from 0.7 to 1.7 x 10(5) and for cardiotoxin from 0.82 to 1.6 x 10(5). The calculated dissociation constants (Kd) from the Scatchard plots ranged from 0.43 to 1.1 microM for the thionin and from 0.40 to 0.98 microM for the cardiotoxin. The binding sites for thionin and cardiotoxin with sheep erythrocytes were 1.7 and 2.0 x 10(4) sites/cell, respectively, and both cow and horse erythrocytes demonstrated 2.7 x 10(4) sites/cell for the thionin. Binding studies with murine P388 cells showed 7.0 and 9.5 x 10(6) sites per cell for Pyrularia thionin and cardiotoxin, respectively.     

Dissociation of the PAF-receptor from NADPH oxidase and adenylate cyclase in human neutrophils results in accelerated influx and delayed clearance of cytosolic calcium

The magnitude and duration of the abruptly occurring increases in cytosolic Ca2+ in human neutrophils following activation with PAF (20 and 200 nM) and FMLP (1 microM), have been compared and related to alterations in NADPH oxidase activity, membrane potential and intracellular cyclic AMP. Cytosolic Ca2+ and membrane potential were measured by spectrofluorimetry, transmembrane fluxes of Ca2+ by radiometric procedures, and NADPH oxidase activity and cyclic AMP by chemiluminescence and radioimmunoassay respectively. Activation of neutrophils with both PAF (200 nM) and FMLP (1 microM) was accompanied by an abrupt increase in cytosolic Ca2+, which was of similar magnitude for each activator (393+/-9 and 378+/-17 nM respectively). Unlike FMLP-activated cells in which Ca2+ was rapidly removed from the cytosol, peak levels of cytosolic Ca2+ were sustained for longer (0.14+/-0.02 vs 1.16+/-0.04 min, P相似文献   

Studies on the contracture of the mouse diaphragm induced by sodium selenite

In this study, we found that sodium selenite was potent in inducing contracture of the mouse diaphragm. The possible mechanism of action of selenite was investigated. Contracture was induced by a direct action of selenite on the muscle membrane rather than that selenite enhanced transmitter release from the motor nerve terminals, since denervation, d-tubocurarine and tetrodotoxin did not inhibit the selenite-induced contracture. Although selenite decreased both the membrane potential and the amplitude of the muscle action potential, neither high K+ nor glycerol treatment, which closed the transverse tubule, reduced the selenite-induced contracture, suggesting that depolarization of the muscle membrane was not essential for the induction of the contracture. EGTA (1-50 mM) inhibited the selenite-induced contracture in a concentration-dependent manner. In contrast, varying the external Ca2+ concentrations from 10(-3) to 10 mM or raising Mg2+ concentration to 10 mM did not affect the contracture. Similarly, the contracture induced by caffeine was not affected by lowering the external Ca2+ concentration to 10(-3) mM but was completely inhibited by 30 mM EGTA. Selenite pretreatment markedly potentiated the caffeine contracture and prolonged treatment with caffeine inhibited the selenite contracture. All of these findings suggest that the selenite contracture was not dependent on external Ca2+ but was induced by the release of Ca2+ from internal membranes such as the sarcoplasmic reticulum. Pretreatment with trypsin, glutathione or cyanide blocked the selenite-evoked contracture. Therefore, we postulate that the selenite-induced contracture was induced by the initial binding of selenite to the sulfhydryl groups of the muscle membrane, which then triggered the release of Ca2+ from internal membranes such as the sarcoplasmic reticulum.     

Effects of calyculin A on tension and myosin phosphorylation in skinned smooth muscle of the rabbit mesenteric artery.

1. Using beta-escin and ionomycin-treated skinned smooth muscle strips of the rabbit mesenteric artery, the effects of calyculin A (CL-A, an inhibitor of type 1 and 2A phosphatases) on mechanical activities, phosphorylation of myosin light chain (MLC) and the relationship between the two were studied in Ca(2+)-free solution containing 4 mM EGTA and these effects were compared with those evoked by Ca2+. 2. The threshold concentration of Ca2+ required to increase either tension or MLC-phosphorylation was 0.1 microM and maximum effects were obtained at 10 microM. MLC was mainly monophosphorylated, rather than diphosphorylated, in the presence of Ca2+. ED50 value for Ca2+ was 0.54 microM for either tension or MLC-phosphorylation. The relationship between tension and MLC-phosphorylation is linear in the pCa range 7-5.5. 3. In Ca(2+)-free solution (containing either 20 mM EGTA or 4 mM EGTA with or without 4 mM BAPTA), 3 microM CL-A produced a contraction, the maximum amplitude of which was similar to that evoked by 10 microM Ca2+. CL-A (0.03-3 microM) concentration-dependently increased both tension and MLC-phosphorylation in Ca(2+)-free solution containing 4 mM EGTA. The threshold concentration of CL-A required for the increase in either tension or MLC-phosphorylation was 0.03 microM and maximum effects were obtained at 3 microM. In the presence of CL-A, MLC was not only monophosphorylated but also diphosphorylated. ED50 values for CL-A were 0.39 microM for tension, 0.44 microM for the monophosphorylated form of MLC and 0.54 microM for all phosphorylated (mono + di) forms. The relationship between tension and the monophosphorylated form of MLC was linear over the concentration range studied and was similar to that for Ca2+. 4. H-7 (3 microM, an inhibitor of protein kinase C) inhibited neither the tension nor phosphorylation of MLC induced by 10 microM Ca2+ or 3 microM CL-A. At a high concentration (30 microM), H-7 slightly inhibited both the tension and phosphorylation of MLC induced by either stimulant without a change in the tension-MLC-phosphorylation relationship. KN-62, an inhibitor of Ca(2+)-calmodulin-dependent protein kinase II, did not modify either the tension or the phosphorylation of MLC induced by 10 microM Ca2+ or 3 microM CL-A. CK-II, another inhibitor of Ca(2+)-calmodulin-dependent protein kinase II, did not inhibit the contraction induced by 3 microM CL-A.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of pentoxifylline on the plasma membrane Ca2+ ATPase in age-separated rat and human erythrocytes.

Pentoxifylline, a dimethyl xanthine derivative given to patients with peripheral vascular disorders, increases erythrocyte deformability, diminishes Ca2+ entry, inhibits Ca(2+)-dependent transglutaminase activity and elevates ATP levels. The present study examined the effects of pentoxifylline on the Ca2+ pump ATPase, an enzyme which regulates intracellular Ca2+ levels. Studies were carried out with inside-out vesicles (IOVs) prepared from young (Ey) and old (Eo) human and rat erythrocytes. The pumping of Ca2+ depends on the concomitant hydrolysis of ATP; the two processes were measured using radiolabeled substrates. The catalytic properties of IOVs from young and old erythrocytes were stimulated by pentoxifylline when added directly to the assay medium. Pentoxifylline (0.5 to 5.0 mM) significantly activated the rates of Ca2+ dependent ATP hydrolysis in Ey and Eo IOVs of rat erythrocytes. The percent of activation was greater in the IOVs from older erythrocytes. The Ca2+ translocation was also affected by pentoxifylline. The early burst of Ca2+ uptake into IOVs decreased in the presence of pentoxifylline in Ey IOVs prepared from either species whereas steady state rates of Ca2+ transport only declined at 5.0 mM pentoxifylline. This pattern was not evident in the corresponding Eo IOVs. The response of Ey and Eo IOVs to pentoxifylline may be the basis of the difference in sensitivity of young and old erythrocytes to the drug in regulating intracellular Ca2+ concentrations.     

Differential effects of organic hydroperoxides and hydrogen peroxide on proteolysis in human erythrocytes

The effects of tert-butyl hydroperoxide, cumene hydroperoxide, and hydrogen peroxide on proteolysis in human red blood cells have been examined. The organic hydroperoxides effectively stimulated the rate of protein degradation in red cells and in hemolysate; in contrast, H2O2 addition was without significant effect in either system. tert-Butyl hydroperoxide or cumene hydroperoxide (8 mM) increased the rate of protein degradation in red cells 2.3- and 4-fold, respectively, relative to control as monitored by tyrosine release. In hemolysate, tert-butyl hydroperoxide and cumene hydroperoxide, present at 8 mM, produced a 2- and 3-fold increase in the rate of protein degradation, respectively, as compared to controls. Hydroperoxide-stimulated proteolysis in red cells or in hemolysate was concentration-dependent and reached saturation at 8 mM hydroperoxide. The reaction was linear for 2 h after which a plateau was reached. In contrast to the results observed for the organic hydroperoxides, H2O2 (100 or 200 mM) addition either alone or in the presence of the catalase inhibitor 3-amino-1,2,4-triazole (50-200 mM), failed to stimulate proteolysis. N-Acetylcysteine (20 mM) and dimethylthiourea (50 mM) inhibited the rate of hydroperoxide-stimulated proteolysis in red cells by approximately 50 and approximately 35%, respectively, and in hemolysate by 25 and 40%, respectively. The hydroxyl radical scavengers methyl sulfoxide (50 mM) or dimethylfuran (50 mM), metal ion chelators, or spin traps failed to decrease significantly the rate of organic hydroperoxide stimulated proteolysis. In addition, inhibitors of the calpain/procalpain system in red cell or hemolysate incubations challenged by organic hydroperoxide were without significant effect on the rate of proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mode of action of contractile effects induced by external calcium and its related bivalent cations in the KCl-depolarized rat uterus.

The cumulative log dose-response curve (DRC) isometrically produced by CaCl2 on depolarized uteri of rats in Ca2+-free KCl Ringer's solution (pH 7.8) showed a plateau-shaped type, when responses were plotted as a percent of the maximal tension of 50 micrometer acetylcholine, whereas those produced by BaCl2 or SrCl2 were a simple sigmoid type with the maximal response near the height of the plateau induced by Ca2+. The plateau phase of Ca2+ was inhibited competitively by Mg2+ (0.5--50 mM) and non-competitively by Mn2+ (30 micrometer--1 mM), whereas the maximal contraction of Ca2+ was not inhibited by either ion. Dantrolene (0.1 mM) inhibited both the plateau and the maximal contraction of Ca 2+ and retained the plateau-shaped type. EGTA (2mM) potentiated the plateau to the maximal response level and changed the DRC induced by Ca2+ to the simple sigmoid type. From these results, the plateau of DRC induced by Ca2+ seems to be mainly due to actions of external Ca2+ on the calcium binding of internal surface of cell membrane, suggesting that the internal surface is the site of the bivalent cations tested.     

Decrease in the Ca2+-activated K+ current of pulmonary arterial smooth muscle in pulmonary hypertension rats

Pulmonary hypertension exhibits acute elevation of vascular tone and hyperreactivity of pulmonary vasculature, which are closely related to patient mortality. In the present study, we investigated the characteristics of membrane currents of isolated pulmonary artery smooth muscle cells taken from rats with monocrotaline-induced pulmonary hypertension. Male Wistar rats were given a single subcutaneous injection of monocrotaline or saline, and then sacrificed between 18 to 21 days after the injection. The membrane currents in the smooth muscle cells from both groups of rats were compared using the whole-cell patch clamp technique. With 0.1 mM EGTA in the pipette, the densities of outward currents in monocrotaline-injected rats were smaller than those in control rats. When EGTA in patch pipettes was increased to 10 mM, the densities of the outward currents in monocrotaline-injected rats were equal to those of control rats. The Ca2+-activated K+ channel blockers (TEA, iberiotoxin) and nisoldipine were less effective on the outward currents of monocrotaline-injected rats. In the current clamp mode, a depolarization of membrane potential induced by 4-aminopyridine was greater in monocrotaline-injected rats than in control rats because of the reduced activity of the Ca2+-activated K+ channels. The Ca2+-activated K+ channels were decreased in pulmonary hypertension. The reduced activity of the currents may be related to the vascular hyperreactivity in pulmonary hypertension.     

Mechanism of barium-induced contraction in the vascular smooth muscle of rabbit aorta.

In a solution containing 1.5 mM Ca2+, cumulative application of 0.3-10.0 mM Ba2+ induced a concentration-dependent contraction of the rabbit aorta. This contraction was reduced by the Ca2+ channel inhibitors, verapamil (10(-6) M), nifedipine (10(-7) M) and lanthanum (2.0 mM), and was potentiated by the Ca2+ channel facilitator, Bay K8644 (10(-7) M). In a Ca2+-free solution containing EGTA (1.0 mM), cumulative application of Ba2+ still induced a concentration-dependent contraction, the maximum contractile tension of which was comparable to that in the presence of 1.5 mM Ca2+. The Ba2+-induced contraction which was not dependent on the external Ca2+ was also inhibited by verapamil, nifedipine and lanthanum and was potentiated by Bay K8644. A high concentration (65.4 mM) of K+ potentiated this Ba2+-induced contraction whereas noradrenaline (10(-6) M) did not have such an effect. In order to deplete the releasable Ca2+ store in the cell, the muscle strip was treated with noradrenaline (10(-6) M) and/or caffeine (20.0 mM) in a Ca2+-free solution. In such a Ca2+-depleted muscle, Ba2+ still induced a contraction of a similar magnitude to that without such treatment. Further, the second application of Ba2+ in a Ca2+-free solution induced a similar contraction to that induced by the first application of Ba2+. These results suggest that Ba2+ depolarizes the cell membrane and opens the voltage-dependent Ca2+ channels resulting in a Ca2+ influx in the presence of Ca2+. In the absence of external Ca2+, Ba2+ may enter the cell through the voltage-dependent Ca2+ channels and induce contraction without mobilizing the Ca2+ store which is sensitive to noradrenaline and caffeine.     

A comparison of the actions of cobra cardiotoxin and scorpion toxin II on the guinea-pig taenia coli

Both cobra cardiotoxin (CTX) and scorpion toxin II induce contracture of the guinea-pig taenia coli, the latter being more potent than the former. Tachyphylaxis was observed with each toxin. Acetylcholine (ACh) contracture was enhanced by toxin II but not by CTX. High K+ (152 mM) abolished toxin II contracture but only partially inhibited CTX and ACh contractures. Ca2+-free medium, especially in the presence of 1 mM EGTA, inhibited CTX- and toxin II-contracture by more than 60%, but abolished ACh contracture. On the other hand, high Ca2+ (12 mM) was without effect on ACh contracture but inhibited both CTX- and toxin II-contracture, and enhanced K+ contracture. Contracture was produced after washout of Ca2+-free medium and toxins but not after washout of high Ca2+ medium, suggesting that the binding of toxins was inhibited by high Ca2+. Tetrodotoxin (TTX) abolished toxin II contracture, but only partially inhibited CTX contracture. Atropine markedly inhibited toxin II contracture but did not significantly affect CTX contracture. Procaine potentiated CTX contracture but abolished toxin II contracture. The spontaneous contraction induced by toxin II was also inhibited by TTX, atropine and procaine. ACh contracture was abolished by atropine and potentiated by procaine, but unaffected by TTX. The rate of decline of K+ (60 mM) contracture in Ca2+-free medium was enhanced by CTX but unaffected by toxin II. 45Ca2+ efflux was increased by CTX more than by toxin II. Both toxins also significantly increased 45Ca2+ uptake. These findings indicate that CTX induces contracture of taenia coli mainly by releasing the Ca2+ pool of the muscle membrane, whereas toxin II does so mainly by releasing acetylcholine from nerve terminals and partially by acting on the muscle membrane as a result of an increase of the membrane Na+ permeability.     

Halothane inhibits hyperpolarization and potassium channels in human red blood cells

The effect of halothane on the Ca2+-sensitive K+ channel in human erythrocytes has been investigated. The red cells were suspended in buffer-free salt solutions containing Ca2+ or 45Ca2+. The protonophore CCCP was added to bring about a rapid equilibration of protons across the plasma membrane. After addition of the divalent cation ionophore A23187, the cells took up Ca2+ and this caused the K+ channels to open. When the medium contained 1 mM K+, the addition of A23187 induced a transient hyperpolarization of the cells, as monitored by measurement of the pH of the medium. The cellular pH, being buffered by haemoglobin, was virtually constant. Halothane reversibly inhibited hyperpolarization and limited the release of cellular K+ in a dose-dependent way, but did not inhibit the Ca2+-transporting properties of A23187. No stimulatory effects of halothane were observed even at low halothane concentrations. In conclusion, halothane reversibly inhibits the Ca2+-sensitive K+ channel in human erythrocytes with an ED50 of about 0.5 mM.     

Calcium plays a key role in the effects induced by a snake venom Lys49 phospholipase A2 homologue on a lymphoblastoid cell line.

A catalytically-inactive Lys49 phospholipase A2 homologue from the venom of the snake Bothrops asper induces diverse effects (necrosis, apoptosis and proliferation) in a lymphoblastoid cell line, depending on the toxin concentration. The increments in cytosolic Ca2+ levels induced by this toxin in this cell line were assessed. At high toxin concentration (100 microg/mL) the toxin induces drastic disruption of the plasma membrane, associated with a prominent Ca2+ influx and necrosis. Previous incubation of the cells with the chelating agent EGTA or with ruthenium red, an inhibitor of the uniporter mitochondrial Ca2+ transport, greatly reduced necrosis. At a toxin concentration of 12.5 microg/mL, apoptosis is the predominant response, being associated with lower increments in cytosolic Ca2+. This effect was inhibited by preincubation with ruthenium red and the cytosolic Ca2+ chelator BAPTA-AM. The proliferative response, which occurs at a low toxin concentration (0.5 microg/mL), is associated with a small and oscillatory increment in cytosolic Ca2+. It was inhibited by EGTA, ruthenium red and BAPTA-AM, by inhibitors of the endoplasmic reticulum Ca2+ -ATPase (SERCA) and by blockade of the ryanodine receptor. It is concluded that necrosis and apoptosis induced by this toxin are associated with increments in cytosolic Ca2+ levels following plasma membrane perturbation, together with the involvement of mitochondria. The cellular proliferative response depends on a limited Ca2+ influx through the plasma membrane, being associated with a concerted functional unit constituted by SERCA, the ryanodine receptor and mitochondria, which regulate the observed oscillations in cytosolic Ca2+ concentration.     

Pharmacological interventions of cyanide-induced cytotoxicity and DNA damage in isolated rat thymocytes and their protective efficacy in vivo

Cyanide inhibits the mitochondrial respiratory chain enzyme cytochrome oxidase causing histotoxic hypoxia. It is primarily considered as a neurotoxin but its other toxic manifestations are also well documented. Cyanide-induced apoptosis in neuronal cells has also been demonstrated recently. At the same time we also reported that potassium cyanide (KCN) produces extensive cytotoxicity and DNA fragmentation in rat thymocytes. The DNA damage was sensitive to elevated levels of extracellular Ca2+ and was attenuated by Zn2+ (modulator of Ca2+ dependent endonuclease), N-acetylcysteine (free radical scavenger) and diltiazem (Ca2+ channel blocker). In a continuation of this work, in the present study we have shown that the cytotoxicity and DNA fragmentation induced by 5 mM KCN was preceded by loss of mitochondrial integrity (MTT assay and rhodamine-123 staining) and nuclear viability (propidium iodide uptake) which were mediated by generation of reactive oxygen species (DCHF-DA staining). The DNA damage was also accompanied by nuclear fragmentation (Hoechst 33342 staining), a phenomenon that characterises the 'apoptotic' type of cell death. The in vitro toxic insult of KCN was challenged by pre-treatment (0.5 h), simultaneous treatment or post-treatment (0.5-3 h) of various pharmacological agents viz., Trolox (antioxidant), EGTA (Ca2+ modulator) and aurintricarboxylic acid (ATA; Ca2+/Mg2+ dependent endonuclease inhibitor). In addition, Quercetin (antioxidant) was tested as simultaneous treatment alone and was found to be ineffective. On the basis of various biochemical indices and DNA fragmentation (quantitative and qualitative), simultaneous treatment of Trolox was found to be the most effective in attenuating cyanide toxicity in vitro. This protection can be attributed to interventions in oxidative stress-mediated cell injury which is an early event preceding DNA damage. Both EGTA and ATA could not prevent this damage. Trolox also increased the LD(50) of KCN in mice 2.5-fold as compared to 1.8- and 1.6-fold for EGTA and ATA, respectively.     

Structural component changes of erythrocytes caused by oxidative stress generated by indoxyl sulfate

Indoxyl sulfate (IS) belongs to groups of uremic toxins binding to proteins. This compound may contribute to the generation of oxidative stress in chronic kidney disease (CKD) patients. We hypothesized that a high concentration of IS in the blood may induce structural changes of erythrocyte components and thus may contribute to CKD progression.In the present study, we evaluated the influence of IS on hemolysate and membrane proteins' conformational state, lipid membrane fluidity, and internal viscosity in erythrocytes. We examined thiols, carbonyl groups, peroxides, and TBARS levels in erythrocyte incubated with IS.The treatment of erythrocytes with IS led to increase in lipid membrane fluidity, decrease in the internal viscosity of the cells and the motion of the spin labels attached to hemolysate proteins. We did not observe conformational changes in plasma membrane proteins; however, in the plasma membranes of erythrocytes incubated with IS, a decrease in the content of thiol groups and increase in the carbonyls levels and peroxides and TBARS in comparison with the control was observed.The obtained results indicate that IS induces the oxidative damage of erythrocyte components. This may be an important factor that affects the functional properties of erythrocytes in CKD patients.     

Potentiation of stretch-induced myogenic tone of dog cerebral artery by hemolysate and the inhibitory action of calcium antagonists

Quick stretching of a helical strip of dog basilar artery at a rate of 10 cm/s to 140% of the slack length of the muscle evoked a delayed contraction. A small amount of hemolysate (0.01-0.2 mg/ml as hemoglobin), which increased the basal tone by 10-15% of the maximum contracture produced by 80 mM K+, potentiated the stretch-induced contraction 2- to 3-fold over the control. The enhanced response to stretch was attenuated by removal of the endothelium and was readily suppressed by Ca2+ antagonists such as nisoldipine and other dihydropyridine drugs as well as diltiazem. The hemolysate had no apparent effect on the contraction due to a quick stretch in Ca2+-free medium or on the contractile response of a chemically skinned arterial strip in the relaxing solution. The results suggest that the hemolysate potentiates the contractile response to stretch by promoting the transmembrane supply of Ca2+. The presence of endothelium seems to amplify the vasoconstrictor action of the hemolysate.     

Inhibition of erythrocyte Ca2(+)-pump by Ca2+ antagonists

Inside-out vesicularized membrane fragments from human erythrocytes were prepared to study the effects of various Ca2+ channel entry blockers of plasma membrane Ca2+ transport and (Ca2+ + Mg2+)-ATPase activity concomitantly. Verapamil and diltiazem (0.01 to 5 mM) inhibited both (Ca2+ + Mg2+)-ATPase activity and initial rates of 45Ca2+ net uptake analogously. In general, the parameter affected most by these drugs, using either Ca2+ transport or (Ca2+ + Mg2+)-5'-adenosine-triphospho-hydrolase (EC 3.6.1.3) ([Ca2+ + Mg2+]-ATPase) measurements, was the stimulation by calmodulin. However, the specificity and selectivity of inhibition appeared to be highly concentration and membrane preparation dependent. Verapamil and diltiazem inhibited the calmodulin-Ca2+ transport concentration-effect relationship by changing its apparent affinity as well as the maximal velocity of the process. In a "white ghost" membrane preparation, bepridil inhibited calmodulin activation with a high degree of selectivity as opposed to its effects on calmodulin activation in the vesicular preparation. Nifedipine failed to exhibit any specificity and modestly inhibited basal and calmodulin-activated inside-out vesicular Ca2+ transport and (Ca2+ + Mg2+)-ATPase alike. Our results suggest that verapamil, diltiazem and bepridil (0.01 to 0.3 mM), but not nifedipine (1 nM to 0.01 mM), in relatively high concentrations can antagonize the calmodulin-stimulated Ca2(+)-pump, i.e. the ATPase as well as the transport process. The inhibitors differed with regard to potency, selectivity, and the type of inhibition they produced.     

Ca2+-induced aortic contraction in normal K+ after exposure to Ca2+- and Mg2+ -free solution

Guinea pig and rabbit aortic rings which have been incubated in a Ca2+- and Mg2+ -free solution containing ethylene glycol bis-(beta-aminoethyl ether) N,N,N'N'-tetraacetic acid (EGTA) responded to addition of Ca2+ in a similar Ca2+ - and Mg2+ -free solution without EGTA by increasing tension gradually at first and then more sharply at Ca2+ concentrations above 2 mM. Prior addition of D 600 (10 or 50 microM) inhibited contractions to low Ca2+ concentrations, but only partially attenuated those to high Ca2+ concentrations. The resulting contraction is thought to represent Ca2+ entry from both voltage-dependent (D 600-sensitive) Ca2+ channels and Na+ -Ca2+ exchange (D 600-insensitive).     

Ketamine-induced relaxation in intact and skinned smooth muscles of the rabbit ear artery.

1. The effects of ketamine, an intravenous anaesthetic, on the rabbit ear artery were investigated by measuring the tension in intact and saponin-treated skinned smooth-muscle fibres. 2. Ketamine dose-dependently inhibited contractions of intact smooth-muscle fibres induced by high K+ solution and by noradrenaline (NA) or histamine in Krebs solution. This drug similarly attenuated both phasic and tonic contractions induced by high K+ solution. 3. Ketamine also inhibited NA- or histamine-induced contractions in Ca2+-free solution containing 2mM EGTA, but it did not affect the caffeine-induced contraction in this solution. 4. Because the pCa-tension relationship of saponin-treated skinned smooth-muscle fibres was not affected, it can be proposed that ketamine does not have an effect on the contractile proteins. 5. In the presence of 5mM NaN3, 20 microM inositol 1,4,5-trisphosphate (InsP3) or 25mM caffeine produced a contraction in skinned smooth-muscle fibres after accumulation of Ca2+ by intracellular stores. Analysis of the InsP3- or caffeine-induced contractions indicates that ketamine does not have an effect on the Ca2+ accumulation into and Ca2+ release from the intracellular stores. 6. These results indicate that the relaxant effects produced by ketamine in the rabbit ear artery are not likely to be due to an intracellular action. The inhibitory effects of ketamine could be caused by a decrease of the Ca2+ influx through the plasma membrane or interference with the process of signal transduction between receptors on the plasma membrane and intracellular stores.