Oxidant-induced DNA damage of target cells.

In this study we examined the leukocytic oxidant species that induce oxidant damage of DNA in whole cells. H2O2 added extracellularly in micromolar concentrations (10-100 microM) induced DNA strand breaks in various target cells. The sensitivity of a specific target cell was inversely correlated to its catalase content and the rate of removal of H2O2 by the target cell. Oxidant species produced by xanthine oxidase/purine or phorbol myristate acetate-stimulated monocytes induced DNA breakage of target cells in proportion to the amount of H2O2 generated. These DNA strand breaks were prevented by extracellular catalase, but not by superoxide dismutase. Cytotoxic doses of HOCl, added to target cells, did not induce DNA strand breakage, and myeloperoxidase added extracellularly in the presence of an H2O2-generating system, prevented the formation of DNA strand breaks in proportion to its H2O2 degrading capacity. The studies also indicated that H2O2 formed hydroxyl radical (.OH) intracellularly, which appeared to be the most likely free radical responsible for DNA damage: .OH was detected in cells exposed to H2O2; the DNA base, deoxyguanosine, was hydroxylated in cells exposed to H2O2; and intracellular iron was essential for induction of DNA strand breaks.

Oxidant injury of cells. DNA strand-breaks activate polyadenosine diphosphate-ribose polymerase and lead to depletion of nicotinamide adenine dinucleotide.

To determine the biochemical basis of the oxidant-induced injury of cells, we have studied early changes after exposure of P388D1 murine macrophages to hydrogen peroxide. Total intracellular NAD+ levels in P388D1 cells decreased with H2O2 concentrations of 40 microM or higher. Doses of H2O2 between 0.1 and 2.5 mM led to an 80% depletion of NAD within 20 min. With doses of H2O2 of 250 microM or lower, the fall in NAD and, as shown previously, ATP, was reversible. Higher doses of H2O2 that cause ultimate lysis of the cells, induced an irreversible depletion of NAD and ATP. Poly-ADP-ribose polymerase, a nuclear enzyme associated with DNA damage and repair, which catalyzes conversion of NAD to nicotinamide and protein-bound poly-ADP-ribose, was activated by exposure of the cells to concentrations of 40 microM H2O2 or higher. Activation of poly-ADP-ribose polymerase was also observed in peripheral lymphocytes incubated in the presence of phorbol myristate acetate-stimulated polymorphonuclear neutrophils. Examination of the possibility that DNA alteration was involved was performed by measurement of thymidine incorporation and determination of DNA single-strand breaks (SSB) in cells exposed to H2O2. H2O2 at 40 microM or higher inhibited DNA synthesis, and induced SSB within less than 30 s. These results suggest that DNA damage induced within seconds after addition of oxidant may lead to stimulation of poly-ADP-ribose polymerase, and a consequent fall in NAD. Excessive stimulation of poly-ADP-ribose polymerase leads to a fall in NAD sufficient to interfere with ATP synthesis.

Role of oxidants in DNA damage. Hydroxyl radical mediates the synergistic DNA damaging effects of asbestos and cigarette smoke.

The mechanism by which cigarette smoking and asbestos exposure synergistically increase the incidence of lung cancer is unknown. We hypothesized that cigarette smoke and asbestos might synergistically increase DNA damage. To test this hypothesis we exposed isolated bacteriophage PM2 DNA to cigarette smoke and/or asbestos, and assessed DNA strand breaks as an index of DNA damage. Our results supported our hypothesis. 78 +/- 12% of the DNA exposed to both cigarette smoke and asbestos developed strand breaks, while only 9.8 +/- 7.0 or 4.3 +/- 3.3% of the DNA exposed to cigarette smoke or asbestos, respectively, developed strand breaks under the conditions of the experiment. Our experimental evidence suggested that cigarette smoke and asbestos synergistically increased DNA damage by stimulating .OH formation. First, significant amounts of .OH were detected by electron paramagnetic resonance (EPR) in DNA mixtures containing both cigarette smoke and asbestos, but no .OH was detected in mixtures containing cigarette smoke alone or asbestos alone. Second, the .OH scavengers, dimethylsulfoxide (DMSO), mannitol, or Na benzoate decreased both .OH detection by EPR and strand breaks in DNA mixtures exposed to cigarette smoke and asbestos. Third, the H2O2 scavenger, catalase, and the iron chelators, 1,10-phenanthroline and desferrithiocin, decreased both .OH detection and strand breaks in DNA mixtures exposed to cigarette smoke and asbestos. These latter findings suggest that iron contained in asbestos may catalyze the formation of .OH from H2O2 generated by cigarette smoke. In summary, our study indicates that cigarette smoke and asbestos synergistically increase DNA damage and suggests that this synergism may involve .OH production.

Alterations in adenosine triphosphate and energy charge in cultured endothelial and P388D1 cells after oxidant injury.

To investigate mechanisms whereby oxidant injury of cells results in cell dysfunction and death, cultured endothelial cells or P388D1 murine macrophage-like cells were exposed to oxidants including H2O2, O2-. (generated by the enzymatic oxidation of xanthine), or to stimulated polymorphonuclear leukocytes (PMN). Although Trypan Blue exclusion was not diminished before 30 min, cellular ATP was found to fall to less than 30% of control values within 3 min of exposure to 5 mM H2O2. Stimulated PMN plus P388D1 caused a 50% fall in cellular ATP levels. During the first minutes of oxidant injury, total adenylate content of cells fell by 85%. Cellular ADP increased 170%, AMP increased 900%, and an 83% loss of ATP was accompanied by a stoichiometric increase in IMP and inosine. Calculated energy charge [(ATP + 1/2 AMP)/(ATP + ADP + AMP)] fell from 0.95 to 0.66. Exposure of P388D1 to oligomycin plus 2-deoxyglucose (which inhibit oxidative and glycolytic generation of ATP, respectively) resulted in a rate of ATP fall similar to that induced by H2O2. In addition, nucleotide alterations induced by exposure to oligomycin plus 2-deoxyglucose were qualitatively similar to those induced by the oxidant. Loss of cell adenylates could not be explained by arrest of de novo purine synthesis or increased ATP consumption by the Na+-K+ ATPase or the mitochondrial F0-ATPase. These results indicate that H2O2 causes a rapid and profound fall in cellular ATP levels similar to that seen when ATP production is arrested by metabolic inhibitors.

Damage to the bases in DNA induced by stimulated human neutrophils.

Leukocyte-induced DNA damage may partially account for the known association between chronic inflammation and malignancy. Since elucidation of the chemical nature of leukocyte-induced DNA damage may enhance our understanding of the mechanisms underlying leukocyte-induced DNA damage and the carcinogenesis associated with inflammation, the present study was undertaken to characterize the chemical modifications that occur in DNA exposed to stimulated human neutrophils. Calf thymus DNA was exposed to phorbol myristate acetate (PMA)-stimulated neutrophils in the presence or absence of exogenously added iron ions. DNA samples were subsequently hydrolyzed, derivatized and analyzed by gas chromatography-mass spectrometry with selected-ion monitoring. A variety of base modifications including cytosine glycol, thymine glycol, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine were identified. The yield of these various base products was increased by the addition of iron ions. Specifically, in the presence of physiologic quantities of iron ions, approximately 7 of every 1,000 DNA bases were modified. Addition of the superoxide anion scavenger, superoxide dismutase, the hydrogen peroxide scavenger, catalase, the hydroxyl scavenger, dimethylsulfoxide, or the iron chelator, deferoxamine, to DNA mixtures containing PMA, neutrophils, and iron ions, greatly decreased the yield of the damaged DNA base products. Our results indicate that stimulated human neutrophils can damage each of the four bases in DNA. It is likely that hydroxyl radical, generated via an iron catalyzed Haber-Weiss reaction, mediates neutrophil-induced DNA base damage, since: (a) the chemical structure of neutrophil-induced DNA base damage is consistent with a hydroxyl radical-mediated mechanism, (b) hydroxyl radical generated via ionizing radiation in aqueous solution produces DNA base modifications that are identical to neutrophil-induced DNA base modifications, (c) iron ions increase neutrophil-induced DNA base damage, and (d) iron chelators or scavengers of superoxide anion, hydrogen peroxide or hydroxyl radical decrease neutrophil-induced DNA base damage.

Mechanisms of hypochlorite injury of target cells.

HOCl, which is produced by the action of myeloperoxidase during the respiratory burst of stimulated neutrophils, was used as a cytotoxic reagent in P388D1 cells. Low concentrations of HOCl (10-20 microM) caused oxidation of plasma membrane sulfhydryls determined as decreased binding of iodoacetylated phycoerythrin. These same low concentrations of HOCl caused disturbance of various plasma membrane functions: they inactivated glucose and aminoisobutyric acid uptake, caused loss of cellular K+, and an increase in cell volume. It is likely that these changes were the consequence of plasma membrane SH-oxidation, since similar effects were observed with para-chloromercuriphenylsulfonate (pCMBS), a sulfhydryl reagent acting at the cell surface. Given in combination pCMBS and HOCl showed an additive effect. Higher doses of HOCl (greater than 50 microM) led to general oxidation of -SH, methionine and tryptophan residues, and formation of protein carbonyls. HOCl-induced loss of ATP and undegraded NAD was closely followed by cell lysis. In contrast, NAD degradation and ATP depletion caused by H2O2 preceded cell death by several hours. Formation of DNA strand breaks, a major factor of H2O2-induced injury, was not observed with HOCl. Thus targets of HOCl were distinct from those of H2O2 with the exception of glyceraldehyde-3-phosphate dehydrogenase, which was inactivated by both oxidants.

Glutathione cycle activity and pyridine nucleotide levels in oxidant-induced injury of cells.

Exposure of target cells to a bolus of H2O2 induced cell lysis after a latent period of several hours, which was prevented only when the H2O2 was removed within the first 30 min of injury by addition of catalase. This indicated that early metabolic events take place that are important in the fate of the cell exposed to oxidants. In this study, we described two early and independent events of H2O2-induced injury in P388D1 macrophagelike tumor cells: activation of the glutathione cycle and depletion of cellular NAD. Glutathione cycle and hexose monophosphate shunt (HMPS) were activated within seconds after the addition of H2O2. High HMPS activity maintained glutathione that was largely reduced. However, when HMPS activity was inhibited--by glucose depletion or by incubation at 4 degrees C--glutathione remained in the oxidized state. Total pyridine nucleotide levels were diminished when cells were exposed to H2O2, and the breakdown product, nicotinamide, was recovered in the extracellular medium. Intracellular NAD levels fell by 80% within 20 min of exposure of cells to H2O2. The loss of NADP(H) and stimulation of the HMPS could be prevented when the glutathione cycle was inhibited by either blocking glutathione synthesis with buthionine sulfoximine (BSO) or by inhibiting glutathione reductase with (1,3-bis) 2 chlorethyl-1-nitrosourea. The loss of NAD developed independently of glutathione cycle and HMPS activity, as it also occurred in BSO-treated cells.

Effects of insulin on the lipid structure of liver plasma membrane measured with fluorescence and ESR spectroscopic methods.

Insulin increased the lipid order of rat and mouse liver plasma membrane domains sampled by the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in a concentration-dependent saturable manner. The ordering is half maximal at 5.1 X 10(-11) M and fully saturated at 1.7 X 10(-10) M insulin. Membranes prepared from obese hyperglycemic (ob/ob) mice demonstrated a right-shift in the dose-dependent ordering induced by insulin, such that ordering was half maximal at 1.2 X 10(-10) M and fully saturated at 2.0 X 10(-10) M. Insulin also increased the order of rat liver plasma membranes labeled with the cis- and trans-parinaric acid methyl esters. The ordering caused by insulin as detected with cis methyl parinarate was complete within approx. 15 min. after hormone addition at 37 degrees C, and the ordering was approximately double that observed with the trans isomer. Additional ESR experiments demonstrated that the addition of insulin increased the outer hyperfine splittings of spectra recorded from membranes labeled with the steroid-like spin labels, nitroxide cholestane and nitroxide androstane, but not the fatty acid spin probe, 5-nitroxide stearate. Studies utilizing model membrane systems strongly suggest that the 5-nitroxide stearate samples a cholesterol-poor domain of the membrane, while the steroid-like probes preferentially sample cholesterol-rich regions of the membrane. Finally, insulin-induced membrane ordering was dose-dependently inhibited by cytochalasin B in the range 1-50 microM. From these results, we conclude that (1) the ordering effect of insulin addition to isolated liver plasma membrane fractions occurs within the physiological range of hormone concentration, and the dose-response is right-shifted in membranes from 'insulin resistant' animals; (2) the relative responses of the fluorescent and spin probes suggest that the effects of insulin are confined to specific domains within the membrane matrix; and (3) the direct effects of insulin on the membranes may involve protein components having cytochalasin B binding sites.

Membrane structural/functional properties of adipocytes from normal and streptozotocin-diabetic rats.

The uptake of 2-deoxy-D-[1-14C]-glucose, in the presence and absence of insulin, was measured in adipocytes from normal and streptozotocin-diabetic animals, over a wide temperature range (25-45 degrees C). Optimum temperatures for uptake in the presence of maximally stimulating insulin concentrations occurred near physiologic temperatures. Both high and low temperatures lead to progressive inhibition of insulin-sensitive uptake, whereas basal uptake was only marginally affected. It is possible that the apparent "resistance" to insulin action at high (febrile) temperatures may have pathologic significance. The magnitude of 2-deoxy-D-glucose uptake at all temperatures in the diabetic adipocytes was much reduced, both in the presence and absence of insulin, on a per cell basis, compared with cells from age-matched control animals. However, the fold stimulation of uptake caused by insulin at 35 degrees C is comparable in both normal and diabetic adipocytes (approximately 2-3-fold). Photomicrographs of adipocytes were used to estimate the cell diameters of the average normal (50 micron) and diabetic (37 micron) cells. The diabetic adipocytes are not "resistant" to insulin action in terms of 2-deoxy-D-glucose uptake since the basal and insulin-stimulated uptake magnitudes per micron2 membrane surface area are identical in both normal and diabetic cells (within experimental error). It is possible that the decreased diabetic cell size reflects the reduced antilipolytic effects of chronic hypoinsulinemia rather than any inherent resistance to insulin action in the cell itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Temperature and benzyl alcohol as probes of the antilipolytic mechanism of insulin action in adipocytes.

The temperature dependence of cAMP accumulation and glycerol release in response to epinephrine and insulin in adipocytes is examined. (1) Glycerol release in the presence of epinephrine demonstrated linear Arrhenius kinetics to 41 degrees C, and above 45 degrees C glycerol release was progressively inhibited. (2) In contrast, incubation of the cells with both epinephrine and insulin resulted in glycerol release rates that were relatively temperature insensitive. (3) Calculation of the efficacy of insulin to inhibit epinephrine-stimulated glycerol release as a function of temperature yielded a biphasic response, with a distinct optimum around 41 degrees C, in a similar manner to the effects of insulin on hexose transport activation determined previously. (4) A saturating dose of insulin (40 ng/ml) was found to have no significant effect on epinephrine-stimulated intracellular cAMP over the temperature range studied. (5) Addition of benzyl alcohol (to 40 mM) resulted in substantial inhibition of basal, epinephrine stimulated, and insulin inhibited glycerol release, without affecting the magnitude of insulin inhibition. We conclude from these studies that (a) insulin inhibition of glycerol release can not be mediated directly by intracellular cAMP modulation, (b) as in the case of hexose transport activation, the signalling mechanism by the occupied insulin receptor appears to be relatively independent of the membrane lipid environment.

The amyloidogenic peptide human amylin augments the inflammatory activities of eosinophils.

The amyloidogenic peptides, amyloid-beta (A beta) and human amylin, are the major constituents of amyloid deposits found in patients with the chronic degenerative disorders Alzheimer's disease (AD) and type 2 diabetes, respectively. Recent studies have shown that a variety of inflammatory proteins such as cytokines are associated with the amyloid deposits of AD brain tissues. Therefore, in the present study, we sought to determine whether A beta and/or human amylin could modulate the various inflammatory activities of eosinophils. We observed that human amylin but not A beta peptides inhibited the in vitro interleukin-5 (IL-5)-mediated survival of cord blood-derived eosinophils (CBEs) in a concentration-dependent manner. By contrast, rat amylin, a nonamyloidogenic peptide that is highly homologous to human amylin, failed to affect the IL-5-mediated survival of CBEs. Similar inhibitory effects of human amylin were observed for peripheral blood eosinophils. Human amylin also enhanced the release of the cytokine granulocyte-macrophage colony-stimulating factor by CBEs that were stimulated with the calcium ionophore A23187 but was incapable of directly stimulating CBEs to release cytokines. In addition, the A23187-induced release of the inflammatory lipid mediator leukotriene C4 by CBEs was augmented by human amylin. These results suggest that the amyloidogenic peptide human amylin is capable of amplifying the various inflammatory activities of eosinophils.

Hydrogen peroxide as a potent bacteriostatic antibiotic: implications for host defense.

Host defense against bacterial pathogens in higher organisms is mediated in part by the generation of reactive oxygen species (ROS) by PMN. In this study, we determined the following effects of exposure of constant concentrations of H2O2 on E. coli in a culture continuously monitored for H2O2 concentration, numbers, and viabilities of cells: (1) E. coli growth rates monitored for 1 h were profoundly affected by concentrations of H2O2, between 25-50 microM. (2) Complete bacteriostasis was observed at 100 microM. (3) Significant cell killing was not observed until the concentration of H2O2 was greater than 500 microM. (4) Bacteriostatic (25-50 microM) concentrations of H2O2 appeared not to be toxic to human skin fibroblasts for a 2-h exposure. (4) Bacteriostasis by H2O2 could not be explained by metabolic inhibition, because intracellular ATP levels were not compromised at bacteriostatic doses of H2O2. (5) Measurements of H2O2 concentrations in subcutaneous abscess fluid infected with both E. coli and S. aureus indicated prevailing concentrations of the oxidant consistent with a proposed role of H2O2 in host defense.

Evidence for N-formyl chemotactic peptide-stimulated GTPase activity in human neutrophil homogenates.

Neutrophil homogenates contained a high affinity guanosine triphosphatase (GTPase) that was stimulatable (+27%) by the addition of 100 nM N-formyl chemotactic peptide (CHO-pep), but not by 1 microgram X ml-1 phorbolmyristate acetate (PMA). Kinetic analysis of the stimulation demonstrated an apparent lagtime of 14.3 +/- 6.9 s between the addition of CHO-pep and the optimal GTPase stimulation. The GTPase activity (but not CHO-pep-stimulated GTPase activity) was preserved in a highly purified plasma membrane fraction of the homogenate. From these observations we suggest that both a high affinity guanine nucleotide binding protein and GTPase are closely associated with the plasma membrane CHO-pep receptor. The possibility that GTPase activity may influence guanine nucleotide regulation of adenylate cyclase during CHO-pep stimulation of neutrophils is discussed.

Insulin stimulation of adipocyte membrane glucose transport. A graded biologic response insensitive to bilayer lipid disordering.

Aspects of the mechanism by which insulin stimulates the membrane glucose transport system were examined by assessing the influence of the bilayer lipid structure on transport stimulation characteristics, and considering the form of the insulin dose-response curve. We tested the effects of membrane lipid perturbation on the insulin stimulation process. Benzyl alcohol, at concentrations (25 mM) that grossly fluidize lipids forming the adipocyte membrane bilayer matrix, caused 50% inhibition of intrinsic transporter activity. However, this membrane perturbation had no significant effect on either the insulin dose-response curve (conducted at 37 degrees) or the time-course of the insulin stimulation of hexose transport (conducted at 32 degrees). These data are difficult to rationalize in terms of a model in which transport stimulation involves interaction of transporters and hormone-bound receptors that is limited by lateral diffusion of these proteins in the fluid lipid bilayer. Curve-fitting experimental insulin dose-response data for stimulation of 2-deoxy-D-glucose and D-glucose uptake provided an estimate of an insulin "association constant" for transport regulation that may be compared with recent insulin receptor binding data. Similar magnitude constants were obtained whether estimated directly from plots of transport velocity versus arithmetic hormone dose, or by extrapolation from linear segments of sigmoidal velocity versus log dose plots, or from inverse (Lineweaver-Burk-type) plots of the insulin dose-response data. Insulin apparently regulates transport by associating with a binding site, having an apparent dissociation constant which is determinable through kinetic measurements of hexose uptake (KDapp approx. 17-40 pM). This is in good agreement with the dissociation constant, KD, determined from Scatchard plots of recent binding data to adipocytes, for a class of receptors representing the "high affinity" binding sites for insulin. Insulin dose-response curve simulations also indicated that the stimulation process may be classified in pharmacologic terms as a typical graded biologic response and may involve insulin association with a site that regulates transport rates in a manner kinetically analogous to allosteric modulation of a V-series enzyme by a noncompetitive ligand. From the results we suggest that a relatively close association occurs between transport and receptor proteins in the membrane, where the relative activation of transport depends on the fractional occupancy of functional high affinity receptors by insulin, and the insulin stimulation of transport involves regions of the membrane that are not influenced significantly by

Membrane structural/functional perturbations induced by gossypol. Effects on membrane order, liposome permeability, and insulin-sensitive hexose transport.

The effects of gossypol on membrane structure and membrane-associated functions were studied to explore possible reasons for the ability of gossypol to disrupt cellular processes, many of which involve intracellular and plasma membranes. The experiments reported here measured the effects of gossypol on membrane order, permeability, and hexose transport. Electron spin resonance (ESR) studies of I(12,3) nitroxide fatty acid spin-labeled unilamellar liposomes showed that exposure to 0.05 to 4 mM gossypol caused a dose-dependent increase in the polarity-corrected order parameter (S), indicating reduced motional freedom of the spin probe after exposure to gossypol. This observation is consistent with the idea that gossypol causes an ordering or "condensing" of the membrane lipid matrix. Gossypol-induced changes in order parameter in phosphatidylcholine:cholesterol liposomes varied depending on the liposome composition. Liposomes exposed to gossypol also showed increasing permeability to glycerol as the gossypol:phospholipid ratio increased up to 10 mole %. Higher concentrations of gossypol were less effective at enhancing permeability. In addition, basal and insulin-stimulated 2-deoxy-D-[3H]glucose transport were inhibited in freshly isolated rat adipocytes incubated with gossypol at 37 degrees. Half-maximal inhibition occurred at approximately 0.2 mM for uptake in both the presence and absence of 40 ng/ml insulin. Microscopic observation of the cells under low power (40 X) confirmed that diminished hexose transport was not simply due to breakage of the adipocyte plasma membrane, resulting in a decrease in intact cell population and decreased accumulation of label in the gossypol-treated cells. Gossypol produced no significant changes in numbers of intact cells or gross morphology at the concentrations tested. We suggest that ordering and increased permeability of the lipid regions of plasma and subcellular membranes may contribute to some of the toxic and pharmacologic properties of gossypol. Our results also support the idea that gossypol may exert more pronounced effects in cells that are most sensitive to variations in availability of glucose substrates for energy metabolism.

Sensitivity of adipocyte basal and insulin-stimulated hexose transport to the membrane lipid structure.

A series of anesthetic alcohols inhibited basal and insulin-stimulated 2-deoxy-D-[1-14C]glucose transport in adipocytes over total alcohol concentration ranges that cause local anesthesia of rat sciatic nerve. The relative potencies of the inhibition caused by the alcohols increased in the following order: methanol less than ethanol less than propanol less than butanol less than benzyl alcohol less than hexanol less than octanol. The inhibition was reversible and correlated well with the known partitioning of the alcohols into lipids of biological membranes. Adipocyte membranes were labeled with the 5-nitroxide stearate spin probe to investigate the effects of the alcohols on the dynamic structure of membrane lipids of the adipocyte. The alcohols increased the membrane "fluidity", and the relative concentration dependence of the effects closely paralleled that noted from methanol to octanol in transport studies. Alcohols from methanol to hexanol caused inhibition of hexose transport at molar potencies comparable to that observed for membrane disordering. This suggests that hydrophobic regions of the transporter and its lipid environment are perturbed by a comparable mechanism for each alcohol. The cholesterol-complexing polyene antibiotic filipin inhibited hexose transport and influenced the mobility of lipid domains sampled with the nitroxide cholestane, cholesterol-like spin probe. The data are consistent with the concept that the membrane structural/functional effects are mediated by formation of 1:1 cholesterol:filipin complexes. Alcohols and filipin inhibited inherent transporter activity and perturbed the membrane lipid structure without dramatically diminishing transport stimulation by insulin above basal. The specific organization of membrane lipids (particularly cholesterol) may provide an essential environment for optimal transport system activity.

Mechanism of protection of oxidant-injured endothelial cells by glutamine.

Glutamine supplementation before oxidant exposure has recently been shown to significantly enhance adenosine triphosphate (ATP) levels and viability in endothelial cells. The aim of this study was to determine if glutamine can help cells after oxidant injury has been initiated and to demonstrate the mechanism of its action. The activity of glyceraldehyde 3-phosphate dehydrogenase was measured in bovine pulmonary artery endothelial cells exposed to H2O2 (0 to 10 mmol/L). Glyceraldehyde 3-phosphate dehydrogenase activity was completely inhibited by 10 mmol/L H2O2 after 1 minute, resulting in inhibition of glycolysis. The endothelial cells were then exposed to 10 mmol/L H2O2, with glutamine (2 mmol/L) being added at different times in relation to the injury. ATP levels were monitored during a 3-hour time course, and short-term viability was measured 6 hours after addition of the oxidant. Significant improvement of endothelial cell ATP levels and short-term viability was seen with addition of glutamine as late as 15 minutes after addition of H2O2. Mitochondrial inhibition with oligomycin (650 nmol/L) abolished the protective effect of glutamine on ATP levels and short-term viability. Cellular survival at 24 hours was not enhanced by glutamine, which suggests that ATP may not be the only factor determining long-term survival after oxidant injury.

Measurement of striatal H2O2 by microdialysis following global forebrain ischemia and reperfusion in the rat: correlation with the cytotoxic potential of H2O2 in vitro.

Toxic reactive oxygen species have been implicated as important mediators of tissue injury after reperfusion of ischemic organs. When rats are subject to 30 min global forebrain ischemia, 24 h following this insult, there is substantial loss of medium-sized neurones as revealed by histological sectioning of the striatal region of the forebrain. The goal of this study was to utilize microdialysis to directly measure one of the more stable intermediates of reduced molecular oxygen, H2O2 in the rat striatum following 4-vessel occlusion and reperfusion, and to correlate these levels with H2O2 toxicity to neurones grown in culture. A significant rise in striatal H2O2 levels was observed for about 1 h during reperfusion, amounting to an increase of approximately 100 microM at the peak. In control experiments where the dialysis probe was embedded in cortical regions surrounding the striatum (where there is no neuronal loss due to the ischemic episode), there was no measurable increase in tissue H2O2 levels. H2O2 has been previously shown to be neurotoxic to PC12 cells as well as rat primary hippocampal neurones at comparable concentrations striatal neurones experience during reperfusion. We demonstrate that H2O2 is also neurotoxic to the human cortical neuronal cell line, HCN-1A. These experiments establish an important link between oxidant generation and neuronal loss in this tissue following global forebrain ischemia.

A cellular model of oxidant-mediated neuronal injury.

Oxidants derived from the partial reduction of oxygen are thought to play a significant role in neuronal injury. We present here a cellular model of neuronal injury mediated by hydrogen peroxide (H2O2) using the PC 12 rat pheochromocytoma cell line. The organization of microtubules and microfilaments within neurites of PC 12 cells differentiated by exposure to nerve growth factor was examined after H2O2 injury using fluorescence microscopy. Concentrations of H2O2 as low as 100 microM produced an initial periodic pattern of microtubule depolymerization over 3-4 h which later progressed to complete depolymerization. Neuritic microspikes containing actin filaments were relatively more resistant to injury by H2O2 than microtubules. Blebbing of PC 12 cell bodies and neurites also was seen after H2O2 injury and the blebs appeared to contain microtubules. The destructive changes affecting neuritic structure preceded but were not essential for PC 12 cell lysis. Exposure of the cells to the Ca2+ ionophore, ionomycin (25 microM) also produced the same pattern of microtubule depolymerization in PC 12 neurites as was seen after H2O2 injury suggesting that H2O2 may mediate its destructive effect on the neurites via elevation of intracellular Ca2+.

Impairment of integrin-mediated cell-matrix adhesion in oxidant-stressed PC12 cells.

Oxidants are believed to play an important and complex role in neuronal injury and death in the aging process and various neurode generative diseases. We studied the effect of oxidative stress on integrin-mediated cell-extracellular matrix (ECM) interactions using the PC12 neuronal cell line. In assays in which attachment was measured between 30 and 90 min, addition of hydrogen peroxide (H2O2) to the attachment medium resulted in a dose-dependent inhibition of initial cell attachment to collagen. Addition of H2O2 also caused previously attached cells to detach from collagen. The inhibition by H2O2 was specific for integrin-mediated adhesion, since attachment to substrata coated with non-ECM molecules was much less affected. Exposure of cells to H2O2 resulted in a rapid and profound reduction of intracellular ATP, accompanied by only a slight increase in intracellular free Ca2+ concentration ([Ca2+]i). Treatment of cells with the microfilament-disrupting agent, cytochalasin B, like that with H2O2, inhibited cell adhesion to collagen. We propose that integrin-mediated cell adhesion, which requires interactions between cytoplasmic portions of integrin subunits and cytoskeletal microfilaments, is impaired by oxidative stress as a result of the depletion of intracellular ATP and that such depletion is an early event in the process of oxidant-induced neuronal injury.