Thioredoxin regenerates proteins inactivated by oxidative stress in endothelial cells.

The thioredoxin/thioredoxin reductase system has been studied as regenerative machinery for proteins inactivated by oxidative stress in vitro and in cultured endothelial cells. Mammalian glyceraldehyde-3-phosphate dehydrogenase was used as the main model enzyme for monitoring the oxidative damage and the regeneration. Thioredoxin and its reductase purified from bovine liver were used as the regenerating system. The physiological concentrations (2-14 microM) of reduced thioredoxin, with 0.125 microM thioredoxin reductase and 0.25 mM NADPH, regenerated H2O2-inactivated glyceraldehyde-3-phosphate dehydrogenase and other mammalian enzymes almost completely within 20 min at 37 degrees C. Although the treatment of endothelial cells with 0.2-12 mM H2O2 for 5 min resulted in a marked decrease in the activity of glyceraldehyde-3-phosphate dehydrogenase, it had no effect on the activities of thioredoxin and thioredoxin reductase. Essentially all of the thioredoxin in endothelial cells at control state was in the reduced form and 70-85% remained in the reduced form even after the H2O2 treatment. The inactivated glyceraldehyde-3-phosphate dehydrogenase in a cell lysate prepared from the H2O2-treated endothelial cells was regenerated by incubating the lysate with 3 mM NADPH at 37 degrees C and the antiserum raised against bovine liver thioredoxin inhibited the regeneration. The inhibition of thioredoxin reductase activity by 13-cis-retinoic acid resulted in a decrease in the regeneration of glyceraldehyde-3-phosphate dehydrogenase in the H2O2-treated endothelial cells. The present findings provide evidence that thioredoxin is involved in the regeneration of proteins inactivated by oxidative stress in endothelial cells.     

Hydrogen peroxide or heat shock induces resistance to hydrogen peroxide in Chinese hamster fibroblasts

Survival after H2O2 exposure or heat shock of asynchronous Chinese hamster ovary cells (HA-1) was assayed following pretreatment with mildly toxic doses of either H2O2 or hyperthermia. H2O2 cytotoxicity at 37 degrees C, expressed as a function of mM H2O2 was found to be dependent on cell density at the time of treatment. The density dependence reflected the ability of cells to reduce the effectiveness of H2O2 as a cytotoxic agent. When the survival data were plotted as a function of mumoles H2O2/cell at the beginning of the treatment, survival was independent of cell density. Cells pretreated with 0.1 mM (3-5 mumoles/cell X 10(-7)) H2O2 for 1 hr at 37 degrees C (30-50% survival) became resistant to a subsequent H2O2 treatment 16-36 hr after pretreatment [dose modifying factor (DMF) at 1% isosurvival = 4-6]. Their resistance to 43 degrees C heating, however, was only slightly increased over controls 16-36 hr following pretreatment (DMF at 1% isosurvival = 1.2). During this same interval, the synthesis of protein migrating in the 70 kD region of a one-dimensional SDS-polyacrylamide gel was enhanced twofold in the H2O2-pretreated cells. When the cells were heated for 15 min at 45 degrees C (40-60% survival), the survivors became extremely resistant to 43 degrees C heating and somewhat resistant to H2O2 (DMF at 1% isosurvival = 2). The heat-induced resistance to heat developed much more rapidly (reached a maximum between 6 and 13 hr) following pretreatment than the heat-induced resistance to H2O2 (16-36 hr). The enhanced synthesis of 70 kD protein after heat shock was greater in magnitude and occurred more rapidly following preheating than following H2O2 pretreatment. The cells that became resistant to H2O2 by either pretreatment (H2O2 or heat shock) also increased their ability to reduce the H2O2 cytotoxicity from the treatment medium beyond that of the untreated HA-1 cells. This may be one of the mechanisms involved in the increased resistance and a common adaptive mechanism induced by both stresses. These data indicate that mammalian cells develop resistance to H2O2 following mild pretreatment with H2O2 or heat shock. The cross-resistance induced by H2O2 and heat shock reinforce the hypothesis that some overlap in mechanisms exist between the cellular responses to these two stresses. However, the failure of H2O2 pretreatment to induce much resistance to heat indicates that there are also differences in the actions of the two agents.     

Oxidant-mediated inhibition of ligand uptake by the macrophage mannose receptor

We have investigated the effect of oxidants on ligand recognition and internalization by the macrophage mannose receptor. Rat bone marrow macrophages were treated with increasing concentrations of H2O2 for 30 min at 37 degrees C. Fifty percent inhibition of ligand uptake was observed at 250 microM, with only 10% of control uptake remaining following exposure to 1 mM H2O2 for 30 min. Electron micrographic analysis of macrophages following H2O2 treatment showed no morphological alterations compared to untreated cells. Ligand uptake was also inhibited by the following H2O2 generating systems: menadione, xanthine/xanthine oxidase, glucose/glucose oxidase, and phorbol 12-myristate 13-acetate-stimulated polymorphonuclear leukocytes. Inhibition could be blocked by catalase plus or minus superoxide dismutase. Treatment of macrophages at 4 degrees C with H2O2 had no effect on ligand binding, whereas treatment with H2O2 at 37 degrees C reduced binding to 15% of control levels and decreased the number of surface receptors to one-third of control cells. H2O2 treatment inhibited ligand degradation by macrophages, but did not prevent ligand movement from the surface to the interior of the cell. In addition, ligand delivery to lysosomes was blocked by oxidant treatment. These results suggest that treatment of macrophages with reagent H2O2 or H2O2-generating systems inhibits the normal ligand delivery and receptor recycling process involving the mannose receptor. Potential mechanisms might include receptor oxidation, alterations in ATP levels, or membrane lipid peroxidation.     

The effect of temperature and Ca2+ on the interaction of high-density lipoproteins with epithelial cells in the human small intestine

The effect of temperature and Ca2+ ions on the interaction of high-density lipoprotein (HDL3) with human enterocytes was studied. It was shown that Kd measured at 4 degrees C is similar to that at 37 degrees C. Maximal number of binding sites at 37 degrees C is 15-fold times higher than that at 4 degrees C. EDTA (10 mM) and CaCl2 (0.5-5 mM) did not affect binding and uptake of HDL3 by human enterocytes. The obtained results indicate that HDL3-binding with these cells depends on temperature and does not depend on Ca2+ ions.     

Effect of 3-aminobenzamide on DNA strand-break rejoining and cytotoxicity in CHO cells treated with hydrogen peroxide

The influence of the nuclear ADP-ribosyltransferase inhibitor 3-aminobenzamide on the DNA strand-break rejoining kinetics and cytotoxicity in Chinese hamster ovary cells following H2O2 treatment was investigated. For the DNA damage studies, cells were treated on ice with H2O2 (0-20 microM) for 1 h in serum-free medium, after which the H2O2 was removed and the cells were allowed to repair their damage in complete medium at 37 degrees C in the presence or absence of 3-aminobenzamide (5 mM) for periods up to 2 h. The DNA strand breaks remaining as a function of time were then estimated by alkaline elution. A linear relationship between the H2O2 concentration and the initial level of DNA single-strand breaks (zero time allowed for repair) was observed. No double-strand breaks or DNA-protein cross-links were detected at these doses. The rejoining of single-strand breaks after H2O2 (20 microM) alone was characterized by a single exponential process with a t1/2 of approx. 5 min. However, in the presence of 3-aminobenzamide, rejoining was much slower and biphasic, with t1/2 of approx. 10 and 36 min. The inhibitory action of 3-aminobenzamide was concentration-dependent and completely reversible in that, when the 3-aminobenzamide was removed from the treated cultures, the strand-break rejoining kinetics rapidly returned to the t1/2 of 5 min typical of H2O2 alone. Considerably higher concentrations of H2O2 (up to 600 microM) were required for cell killing compared to the DNA damage studies. Cell killing by H2O2 alone was characterized by a shoulderless, exponential survival curve (D0 = 880 microM). The cytotoxicity was potentiated when the cells were treated with 3-aminobenzamide (5 mM) for 1 h after the H2O2 treatment; the survival curve with 3-aminobenzamide also assumed a biphasic character (D0 of 212 microM and 520 microM). These results are consistent with the theory that OH.-induced single-strand breaks do not normally represent lethal lesions to the cell because of their rapid, efficient repair. However, interference with these repair processes (in this case by 3-aminobenzamide) can alter this relationship, possibly allowing lesion fixation.     

Construction of glucose oxidase-loaded human erythrocytes: a model of oxidative cytotoxicity

Human red blood cells were loaded with Glucose oxidase from Aspergillus niger by a standardized procedure of encapsulation involving transient hypotonic hemolysis followed by isotonic resealing. The amount of loaded enzyme activity, as evaluated by O2 consumption at 5 mM glucose, ranged from 40 to 75 mumoles O2/hr/ml of packed red cells at 37 degrees C. The red cells loaded with Glucose oxidase were found to behave as efficient glucose-consuming bioreactors. Moreover, at 5 mM glucose, no clear mechanism of H2O2-induced damage was apparent, with the exception of a significantly increased formation of methemoglobin (10% approximately) and of a several-fold stimulated intracellular rate of hexose monophosphate shunt activity, indicating glutathione peroxidase-mediated draining of reduced glutathione for removal of bursts of H2O2. The Glucose oxidase-loaded red cells represent a convenient model system for cytotoxicity studies aiming at clarifying the effects of intracellularly formed H2O2.     

Induction and repair of DNA single-strand breaks in EM9 mutant CHO cells treated with hydrogen peroxide

In this study we investigated the induction and rejoining of DNA single-strand breaks (SSBs) produced by H2O2 in the repair-deficient EM9 mutant Chinese hamster ovary (CHO) cell line. The effect of the poly(ADP-ribose)-transferase inhibitor 3-aminobenzamide (3-ABA) on SSB-rejoining and on cell killing was also evaluated. Results were compared with those obtained previously with the parent cell line (AA8). Cells were treated with H2O2 on ice for 1 h, after which they were either harvested or allowed to repair their damage at 37 degrees C either in the presence or absence of 3-ABA (5 mM). The cells were then assayed either for survival using a colony-forming assay or for their level of DNA SSBs using alkaline elution. EM9 cells were somewhat more sensitive than AA8 cells to the cytotoxic effects of H2O2. However, because the repair mutant showed slightly lower levels of DNA SSBs than did its parental cell line, this sensitivity could not be explained on the basis of alterations in initial damage. The rejoining of the H2O2-induced DNA SSBs followed exponential kinetics in both cell lines; however, EM9 cells rejoined these breaks at a slower rate (t1/2 of 10 min) than did AA8 cells (t1/2 of 5 min). The increased sensitivity of the EM9 cells therefore appears to correlate with a reduced ability to remove these lesions from their DNA. As previously demonstrated for the AA8 cells, 3-ABA treatment resulted in both a retardation of the removal of H2O2-induced DNA SSBs and potentiation of cytotoxicity in the EM9 cells. However, the degree of these effects were similar for both AA8 and EM9 cells. These data provide further evidence that the cytotoxic effects of low concentrations of H2O2 are mediated by damage to DNA, and suggest that the rate at which DNA SSBs are rejoined is important for cell survival.     

Low temperature protects mammalian cells from apoptosis initiated by various stimuli in vitro

Mild hypothermia shows protective effects on patients with brain damage and cardiac arrest. To elucidate the molecular mechanisms underlying these effects, we examined the effects of low temperature (32 degrees C) on cells exposed to a variety of stress in vitro. We found that 32 degrees C suppressed induction of apoptosis by cytotoxic stimuli such as adriamycin, etoposide, thapsigargin, NaCl, H(2)O(2), and anti-Fas antibody. In adriamycin-treated BALB/3T3 cells, the down-shift in temperature from 37 degrees C to 32 degrees C increased the Bcl-xL protein level and decreased the mRNA level of Puma and mitochondrial translocation of Bax, suppressing caspase-9-mediated apoptosis. Furthermore, the protein level and stability of p53 were decreased, and its nuclear export was increased concomitant with Mdm2 mRNA upregulation. The low temperature effect was not observed in p53(-/-)/Mdm2(-/-) mouse embryonic fibroblasts, suggesting that the effect is mediated by suppression of the p53 pathway. In contrast, while thapsigargin-induced apoptosis was suppressed by the low temperature, no effect on the p53 protein level was observed. Furthermore, the survival rate of p53(-/-)/Mdm2(-/-) cells exposed to thapsigargin was increased when cultured at 32 degrees C compared with 37 degrees C. In conclusion, mild hypothermia protects cells from a variety of stress by p53-dependent and p53-independent mechanisms.     

Iron-overload induces oxidative DNA damage in the human colon carcinoma cell line HT29 clone 19A

Dietary iron may contribute to colon cancer risk via production of reactive oxygen species (ROS). The aim of the study was to determine whether physiological ferric/ferrous iron induces oxidative DNA damage in human colon cells. Therefore, differentiated human colon tumour cells (HT29 clone 19A) were incubated with ferric-nitrilotriacetate (Fe-NTA) or with haemoglobin and DNA breaks and oxidised bases were determined by microgelelectrophoresis. The effects of Fe-NTA were measured with additional H(2)O(2) (75microM) and quercetin (25-100microM) treatment. Analytic detection of iron in cell cultures, treated with 250microM Fe-NTA for 15 min to 24h, showed that 48.02+/-5.14 to 68.31+/-2.11% were rapidly absorbed and then detectable in the cellular fraction. Fe-NTA (250-1000microM) induced DNA breaks and oxidised bases, which were enhanced by subsequent H(2)O(2) exposure. Simultaneous incubation of HT29 clone 19A cells with Fe-NTA and H(2)O(2) for 15 min, 37 degrees C did not change the effect of H(2)O(2) alone. The impact of Fe-NTA and H(2)O(2)-induced oxidative damage is reduced by the antioxidant quercetin (75-67% of H(2)O(2)-control). Haemoglobin was as effective as Fe-NTA in inducing DNA damage. From these results we can conclude that iron is taken up by human colon cells and participates in the induction of oxidative DNA damage. Thus, iron or its capacity to catalyse ROS-formation, is an important colon cancer risk factor. Inhibition of damage by quercetin reflects the potential of antioxidative compounds to influence this risk factor. Quantitative data on the genotoxic impact of ferrous iron (e.g. from red meat) relative to the concentrations of antioxidants (from plant foods) in the gut are now needed to determine the optimal balance of food intake that will reduce exposure to this type of colon cancer risk factor.     

Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism

Reactive oxygen species (ROS) inhibit sperm movement and have been implicated in male infertility. In this study, we determined the effects of specific ROS produced by activated leukocytes on human spermatozoa and investigated their metabolic site of action. We used chemiluminescence and electron paramagnetic resonance (EPR) to characterize the ROS generated by both blood and seminal leukocytes. We also determined the effects of these ROS on sperm energy metabolism using biochemical analyses and flow cytometry. Both blood and seminal leukocytes produced the same characteristic ROS which were determined to be hydrogen peroxide (H2O2) and superoxide radicals (O2*-). EPR using the spin trapping technique indicated that superoxide radical-dependent hydroxyl radicals (HO.) were also generated. ROS generated by PMA-stimulated blood leukocytes (2-5 x 10(6)/ml) caused inhibition of sperm movement in 2 h (p < .01). Using the hypoxanthine/ xanthine oxidase (0.5 U/ml) system to generate ROS, we determined that spermatozoa ATP levels, after ROS treatment, were reduced approximately eight-fold in 30 min (0.10 x 10(10) moles/10(6) sperm cells) compared to control (0.84 X 10(-10) moles/10(6) sperm cells) (p < .01). Sperm ATP reduction paralleled the inhibition of sperm forward progression. Neither superoxide dismutase (100 U/ml) nor dimethyl sulfoxide (100 mM) reversed these effects; however, protection was observed with catalase (4 X 10(3) U/ml). Flow cytometric analyses of sperm treated with various doses of H2O2 (0.3 mM-20.0 mM) showed a dose-dependent decrease in sperm mitochondrial membrane potential (MMP); however, at low concentrations of H2O2, sperm MMP was not significantly inhibited. Also, sperm MMP uncoupling with CCClP had no effect on either sperm ATP levels or forward progression. These results indicate that H2O2 is the toxic ROS produced by activated leukocytes causing the inhibition of both sperm movement and ATP production. O2*- and HO. do not play a significant role in these processes. Low concentrations of H2O2 causing complete inhibition of sperm movement and ATP levels inhibit sperm energy metabolism at a site independent of mitochondrial oxidative phosphorylation.     

Respiratory activity of yeast Yarrowia lipolytica under oxidative stress and heat shock

Heat shock (45 degrees C) and the effect of oxidants (H2O2) resulted in a decrease of the respiratory activity of yeast cells and their survival rate. Increased resistance to stress effects after mild heat treatment (37 degrees C) or treatment with a nonlethal dose of oxidants (0.5 mM H2O2 for 60 min) was accompanied by appearance of an alternative (cyanide-resistant) oxidative pathway in the mitochondria, which promotes survival due to retention of the capacity for ATP synthesis in the first coupling point at the level of endogenous NADH dehydrogenase. The alternative oxidative pathway is more resistant to the effect of stressors that disrupt electron transfer in the cytochrome site of the respiratory chain.     

The diisopropylfluorophosphate inhibitable step in antigen-induced histamine release from human leukocytes.

DFP inhibits early events in antigen-induced histamine release from human leukocytes. If added to cells 5 min or more after antigen it is ineffective. If added with antigen it can be removed at 5 min but release will still be inhibited. In contrast, ethylenediaminetetraacetate (EDTA) and 2 deoxyglucose (2DG) still inhibit the reactions when added 5 min after antigen. During incubation of leukocytes for 90 to 120 min at 0 degrees C they react with specific antigen since they subsequently release significant quantities of histamine after washing and reincubation at 37 degrees C without addition of antigen. Such priming at 0 degrees C is at least equivalent to priming for 2 to 4 min at 37 degrees C. During antigen priming at 0 degrees C the cells are not activated beyond the step in the release sequence which is inhibited by diisopropylfluorophosphate (DFP). This is apparent from the undiminished inhibitory activity of DFP on these cells. Furthermore, cells primed with antigen at 0 degrees C in the presence of DFP release as much histamine after washing and incubation at 37 degrees D as control cells primed in the absence of DFP. Incubation of leukocytes with specific antigen at 37 degrees C for 3 min resulted in significant but not quite complete priming for subsequent histamine release in the absence of antigen. Most of these primed cells were not activated beyond the step inhibitable by DFP. However, some had completed the entire sequence including the release of histamine while others had not released their histamine but were not inhibited by DFP from subsequent release. After 5 min incubation with antigen at 37 degrees C almost all leukocytes had progressed beyond the stage which is inhibited by DFP. Incubation of leukocytes at 37 degrees C with DFP but without antigen for up to 15 min followed by washing did not impair subsequent antigen-induced histamine release by these cells. Thus, DFP was inhibitory under these conditions only after antigen activation of leukocytes.     

Assay method for myeloperoxidase in human polymorphonuclear leukocytes

A simple assay method for measuring myeloperoxidase (MPO) has been developed. MPO is found in polymorphonuclear leukocytes and is important as a bactericidal agent in the presence of H2O2 and halide ions. This improved assay method is based on work of Andrews and Krinsky using tetramethylbenzidine (TMB) a noncarcinogenic substrate. By assaying MPO under optimal conditions of TMB at 1.6 mM, H2O2 concentration of 0.3 mM, pH 5.4, and incubation temperature of 37 degrees C, sensitivity of MPO measurements increased eightfold in comparison with the original TMB method. A method has been established to determine absorbance at 655 nm of the reaction mixture by incubation for 3 min and then stopping the reaction by the addition of pH 3.0 buffer. An attempt was also made to raise the sensitivity by using 3,3'-dimethyoxybenzidine (DMB), a carcinogenic substrate. The improved TMB method was 34 times more sensitive than the DMB method.     

Flow-cytometric characterization of stimulation, free radical formation, peroxidase activity and phagocytosis of human granulocytes with 2,7-dichlorofluorescein (DCF)

A standardized four-step assay for the flow cytometric determination of the oxidative activity of human polymorphonuclear leukocytes (PMNL) from normal human individuals and from septic patients was developed, using 2,7-dichlorofluorescin-diacetate (DCFH-DA) as indicator for the intracellular formation of H2O2 and free radicals. Spontaneous H2O2 and free radical formation was measured by preincubation of buffy coat PMNLs from fresh peripheral venous blood at 37 degrees C and pH 7.4 with 10 microM DCFH-DA. Intracellular peroxidase activity was determined by addition of 1 mM external H2O2 to this assay. A maximum of granulocyte oxidative burst activity was elicited by the addition of 150 nM phorbol-myristate-acetate (PMA). A physiological burst was generated by incubating buffy coat PMNLs together with E. coli bacteria. The DNA of dead cells was in all instances simultaneously counterstained with propidium iodide (PI). Quiescent or H2O2 or bacteria treated granulocytes moved as a single cell cluster to higher fluorescences. Stimulation with PMA, in contrast, generated always a bimodal distribution of granulocyte fluorescence with the high activity cell cluster being approximately sevenfold more active than the low activity cell cluster. Roughly half of the granulocytes in normal individuals had high fluorescence. An increase of the high activity granulocytes was observed in septic patients. Model experiments with the nonfluorescent DCFH-DA cleavage product DCFH (2,7-dichlorofluorescin) showed that DCFH was quickly photo-oxidized to fluorescent DCF (2,7-dichlorofluorescein) by UV-light and to a lower degree by daylight. DCFH even slowly autooxidized in the dark.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium enhances the hemolytic action of bile salts

The lysis of human erythrocytes by bile salts in buffer containing isotonic saline was dramatically enhanced by the addition of 5-10 mM calcium chloride. All bile acids tested showed this effect, with a marked increase in lysis occurring at 0.75 mM for deoxycholate, 1 mM for chenodeoxycholate, 2.5 mM for ursodeoxycholate and 5.5 mM with cholate in the presence of 10 mM calcium chloride. The effect appeared to be specific for calcium; strontium chloride and magnesium chloride gave no stimulatory effect. The increased lysis of the erythrocytes in the presence of 1 mM deoxycholate and 1-10 mM calcium chloride was not associated with increased uptake of the bile salt by the cells (measured with [14C]deoxycholate). Using erythrocytes previously labelled with [3H]cholesterol, there was no evidence of an enhanced removal of that membrane component in the presence of calcium and deoxycholate, compared to deoxycholate alone. The sensitivity of the cells to the effect of calcium in the presence of 1 mM deoxycholate increased with the length of time of their storage at 4 degrees C. The sensitivity returned to that of fresh cells after incubation at 37 degrees C with 30 mM adenosine plus 25 mM glucose, but this treatment did not further diminish the lysis. Lysis in the presence of 10 mM calcium chloride and 1 mM deoxycholate was partially blocked by increasing the KCl concentration at the expense of NaCl. The maximum effect occurred with a buffer comprising 100 mM KCl/50 mM NaCl. A more dramatic reduction in the lysis followed the incorporation of the calcium chelator, quin2, into the cells. The lysis induced by 1 mM deoxycholate in the presence of calcium was reduced by 80% in quin-2-loaded cells compared to controls. The data suggest that bile acids can promote the influx of calcium into erythrocytes, leading to lysis as a result of the efflux of intracellular potassium and/or the uptake of sodium from the incubation medium. The data further suggest that cellular effects may occur at lower bile acid concentrations than that thought to be required for detergent damage.     

Effects of gamma radiation and hyperthermia on DNA repair synthesis and the level of NAD+ in cultured human mononuclear leukocytes

DNA repair has been investigated, estimated by unscheduled DNA synthesis (UDS) and the cellular NAD+ pool, after exposing human mononuclear leukocytes to hyperthermia and gamma radiation separately and in combination. It was found that gamma radiation induced a decline in UDS with increasing temperature through the temperature region studied (37-45 degrees C). At 42.5 degrees C the gamma-ray-induced UDS was reduced to about 70% of that at 37 degrees C. Following gamma-ray damage the NAD+ pool dropped to about 20% of control values. Without hyperthermic treatment the cells completely recovered to the original level within 5 hr. Moderate hyperthermia (42.5 degrees C for 45 min) followed by gamma-ray damage altered the kinetics so that even after 8 hr the NAD+ pool had recovered to only 70% of the original level. After heat treatment at 44 degrees C for 45 min prior to gamma radiation the cells did not recover at all, presumably because of the cytotoxic effects from the combined treatment.     

Peroxynitrite Anion Stimulates Arginine Release from Cultured Rat Astrocytes

The biosynthesis of the physiological messenger nitric oxide (*NO) in neuronal cells is thought to depend on a glial-derived supply of the *NO synthase substrate arginine. To expand our knowledge of the mechanism responsible for this glial-neuronal interaction, we studied the possible roles of peroxynitrite anion (ONOO-), superoxide anion (O2*-), *NO, and H2O2 in L-[3H]arginine release in cultured rat astrocytes. After 5 min of incubation at 37 degrees C, initial concentrations of 0.05-2 mM ONOO- stimulated the release of arginine from astrocytes in a concentration-dependent way; this effect was maximum from 1 mM ONOO- and proved to be approximately 400% as compared with control cells. ONOO(-)-mediated arginine release was prevented by arginine transport inhibitors, such as L-lysine and N(G)-monomethyl-L-arginine, suggesting an involvement of the arginine transporter in the effect of ONOO-. In situ xanthine/xanthine oxidase-generated O2*- (20 nmol/min) stimulated arginine release to a similar extent to that found with 0.1 mM ONOO-, but this effect was not prevented by arginine transport inhibitors. *NO donors, such as sodium nitroprusside, S-nitroso-N-acetylpenicillamine, or 1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium+ ++-1,2-diolate, and H2O2 did not significantly modify arginine release. As limited arginine availability for neuronal *NO synthase activity may be neurotoxic due to ONOO- formation, our results suggest that ONOO(-)-mediated arginine release from astrocytes may contribute to replenishing neuronal arginine, hence avoiding further generation of ONOO- within these cells.     

Temperature-dependence of the proliferation of mouse bone marrow cells cultured in H2O- and D2O-media.

In an attempto to optimize and standardize the in vitro culture conditions of mouse bone marrow cells for assaying growth regulating factors, we studied the effects of incubation at low temperatures and of a nutrient medium containing deuteriumoxide instead of water. It was found that (1) the proliferative capacity of the cells is significantly increased by pre-incubation for 1-2 h at 0 degrees C rather than at 37 degrees C, measured by both a colony-forming and a 3H-thymidine (3H-tdr) uptake assay. A similar temperature effect on the colony-forming and 3H-tdr uptake ability is apparent after pre-incubation in D2O-medium, yet significantly lower than in H2O-medium. It was concluded that the previously observed protective effects of D2O on ascites tumor cell proliferation and viability and for hemolysis of human erythrocytes is not apparent in proliferating and colony-forming mouse bone marrow cells in vitro.     

Actin Involvement in Exocytosis from PC12 Cells: Studies on the Influence of Botulinum C2 Toxin on Stimulated Noradrenaline Release

Botulinum C2 toxin is known to ADP-ribosylate actin. The toxin effect was studied on [3H]noradrenaline secretion of PC12 cells. [3H]Noradrenaline release was stimulated five- to 15-fold by carbachol (100 microM) or K+ (50 mM) and 10-30-fold by the ionophore A23187 (5 microM). Pretreatment of PC12 cells with botulinum C2 toxin for 4-8 h at 20 degrees C, increased carbachol-, K+-, and A23187-induced, but not basal, [3H]noradrenaline release maximally 1.5-to three-fold, whereas approximately 75% of the cellular actin pool was ADP-ribosylated. Treatment of PC12 cells with botulinum C2 toxin for up to 1 h at 37 degrees C also increased stimulated [3H]noradrenaline secretion, whereas toxin treatment for greater than 1 h decreased the enhanced [3H]noradrenaline release stimulated by carbachol and K+ but not by A23187. Concomitantly with toxin-induced stimulation of secretion, 20-50% of the cellular actin was ADP-ribosylated, whereas greater than 60% of actin was modified when exocytosis was attenuated. The data indicate that ADP-ribosylation of actin by botulinum C2 toxin largely modulates stimulation of [3H]noradrenaline release. Moreover, the biphasic toxin effects suggest that distinct mechanisms are involved in the role of actin in secretion.