Cancer destruction in vivo through disrupted energy metabolism. Part II. Lipid peroxidation and cell death; drug resistance as a consequence of reversible cellular injury.

In the second part of this review of autoxidative cellular injury and death in tumor cells, the presence of saturated or polyunsaturated fatty acids, vitamin E, and free or esterified cholesterol in whole cells and organelles is discussed in the context of enhancing or attenuating lipid peroxidation. The disposition of unsaturation within polyunsaturated fatty acid molecules is critical for tumor promotion, but the situation appears ambivalent with regard to inflicting cellular injury. Increases in lipid peroxidation and phospholipase A2 activity following on from the administration of hormones or non-cytotoxic drugs are considered from the viewpoint of generating hydroperoxyfatty acids and lysophosphatides, both of which disrupt mitochondrial energy production by uncoupling oxidative phosphorylation. The metabolic fate of lysophosphatides is thought to be a crucial factor both in determining whether cancer cells survive or not, and in furnishing protection for surviving cells against subsequent attack. Both energy-dependent and energy-independent mechanisms for acylating lysophosphatides are reviewed. The emergence of an unstable form of drug resistance during the recovery phase is interpreted in terms of the chemical identity of the new acyl groups on the acylated lysophospholipid. Resistance to further free radical challenge can be conferred by the regeneration of phospholipids bearing saturated or monoenoic 2-substituents which are unable to undergo peroxidation.     

Characterization of the interactions between lysophosphatides, Triton X-100, and sarcoplasmic reticulum.

The interaction of lysophosphatidylcholines and lysophosphatidylethanolamines with lobster abdominal muscle sarcoplasmic reticulum was studied. Only lysophosphatidylcholines with 16 and 18 carbon acyl chains were effective solubilizing agents. The rate of membrane solubilization was most rapid with the palmitoyl and oleoyl derivatives. All lysophosphatides partially inhibited calcium-dependent ATPase activity between 0.0 and 2.0 μmol of lysophosphatide mg^?1 of membrane protein. Lysophosphatides that were active in solubilizing membranes exhibited a reactivating effect on ATPase activity between 2.0 and 6.0 μmol of lysophosphatide mg^?1 of membrane protein. Arrhenius plots of temperature-dependent ATPase activity showed high activation energies and loss of discontinuities in Arrhenius plots when inhibiting concentrations of the lysophosphatides were present. These results suggest that the inhibiting effect of lysophosphatides on membrane enzyme activity is due to intrusion of the lysophosphatide into the membrane, which results in a less fluid lipid environment around the enzyme. Subsequent membrane solubilization at higher lysophosphatide concentrations may release the enzyme from the inhibiting effects of the lysophosphatide by increasing lipid fluidity neighboring the enzyme. The effects of lysophosphatides on a membrane enzyme system were also examined in the presence of 10 mm Triton X-100. Under these conditions, little effect on membrane enzyme activity was generated by increasing concentrations of the lysophosphatidylcholines (lauryl, palmitoyl, and steroyl), while the unsaturated lysophosphatidylcholine and the lysophosphatidylethanolamines caused a two- to threefold increase in enzyme activity. Temperature-dependent enzyme activity studies showed that discontinuities in the Arrhenius plots of enzyme activity occurred at varying temperatures, depending on the lysophosphatide used. Lowest transition temperatures occurred for lysophosphatidylcholine (oleoyl) and the lysophosphatidylethanolamines. These results suggest that, in the presence of 10 mm Triton X-100, lipid exchange occurs around the sarcoplasmic reticulum ATPase enzyme and the fluidity of this lipid-protein complex is increased by lysophosphatides with unsaturated acyl chains or ethanolamine head groups.     

Inhibition of phospholipid hydrolysis by phospholipase A2 in microsomal and mitochondrial membranes subjected to lipid peroxidation

The rate of phospholipid hydrolysis in rat liver microsomal and mitochondrial membranes catalyzed by phospholipase A2 was shown to decrease after ascorbate + Fe2+-induced lipid peroxidation. The degree of inhibition was linearly dependent on the amount of lipid peroxidation products (malonyl dialdehyde) accumulated in the membrane. The decreased phospholipid hydrolysis rate in membranes after lipid peroxidation was registered using phospholipases A2 from two sources: porcine pancreas and bee venom. It was established that the inhibitory action of phospholipid peroxidation products was not linked with a direct effect on the enzyme and was not caused by depletion of phospholipase reaction substrates (as a result of lipid peroxidation). A possible role of lateral separation of oxidized and non-oxidized lipid phases in the mechanisms of inhibition of phospholipid hydrolysis by phospholipase A2 is discussed.     

Changes in Ca2+ affinity upon activation of Agkistrodon piscivorus piscivorus phospholipase A2

Changes in the affinity of calcium for phospholipase A2 from Agkistrodon piscivorus piscivorus during activation of the enzyme on the surface of phosphatidylcholine vesicles have been investigated by site-directed mutagenesis and fluorescence spectroscopy. Changes in fluorescence that occur during lipid binding and subsequent activation have been ascribed to each of the three individual Trp residues in the protein. This was accomplished by generating a panel of mutant proteins, each of which lacks one or more Trp residues. Both Trp21, which is found in the interfacial binding region, and Trp119 show changes in fluorescence upon protein binding to small unilamellar zwitterionic vesicles or large unilamellar vesicles containing sufficient anionic lipid. Trp31, which is near the Ca2+ binding loop, exhibits little change in fluorescence upon lipid bilayer binding. A change in the fluorescence of the protein also occurs during activation of the enzyme. These changes arise from residue Trp31 as well as residues Trp21 and Trp119. The calcium dependence of the fluorescence change of Trp31 indicates that the affinity of the enzyme for calcium increases at least 3 orders of magnitude upon activation. These studies suggest either that a change in conformation of the enzyme occurs upon activation or that the increase in calcium affinity reflects formation of a ternary complex of calcium, enzyme, and substrate.     

Lipid peroxidation and phospholipase A2 activity in liposomes composed of unsaturated phospholipids: a structural basis for enzyme activation

The effect of lipid peroxidation on membrane structure and phospholipase A2 activity was studied using liposomes composed of bovine liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The phospholipids were mixed at set ratios and sonicated to yield small unilamellar vesicles. The liposome preparations were subjected to lipid peroxidation as induced by cumene hydroperoxide and hematin. Under these conditions, a sharp increase in lipid peroxidation was noted over a 30 min incubation period and was accompanied by loss of polyunsaturated fatty acids (PUFA). Liposomes enriched in PE were most extensively peroxidized with a preferred oxidation of this phospholipid. The extent of PC oxidation was also greater in liposomes containing the largest proportions of PE. Analysis of liposome anisotropy, via steady-state fluorescence polarization of diphenylhexatriene indicated that progressive increases in either PE content or the level of lipid peroxidation increased the apparent microviscosity of the vesicles. Moreover, lipid peroxidation increased anisotropy more effectively than variations in the ratios of PE vs. PC. Thus, peroxidation of 5-10% of the phospholipids produced the same anisotropy increase as a 20% increase in the ratio of PE vs. PC. Analysis of vesicle turbidity suggested that fusion was also more readily achieved through lipid peroxidation. When liposomes were incubated with 0.4 U/ml of snake venom phospholipase A2, a direct correlation was found between the degree of lipid peroxidation and the extent of phospholipid hydrolysis. The more unsaturated phospholipid, PE, was most extensively hydrolyzed following peroxidation. Increasing the proportion of PE also resulted in more extensive phospholipid hydrolysis. These findings indicate that lipid peroxidation produces a general increase in membrane viscosity which is associated with vesicle instability and enhanced phospholipase A2 attack. A structural basis for membrane phospholipase A2 activation as a consequence of lipid peroxidation is discussed in light of these findings.     

Mechanisms governing the level of susceptibility of erythrocyte membranes to secretory phospholipase A2

Although cell membranes normally resist the hydrolytic action of secretory phospholipase A(2) (sPLA(2)), they become susceptible during apoptosis or after cellular trauma. Experimentally, susceptibility to the enzyme can be induced by loading cells with calcium. In human erythrocytes, the ability of the calcium ionophore to cause susceptibility depends on temperature, occurring best above approximately 35 degrees C. Considerable evidence from experiments with artificial bilayers suggests that hydrolysis of membrane lipids requires two steps. First, the enzyme adsorbs to the membrane surface, and second, a phospholipid diffuses from the membrane into the active site of the adsorbed enzyme. Analysis of kinetic experiments suggested that this mechanism can explain the action of sPLA(2) on erythrocyte membranes and that temperature and calcium loading promote the second step. This conclusion was further supported by binding experiments and assessment of membrane lipid packing. The adsorption of fluorescent-labeled sPLA(2) was insensitive to either temperature or ionophore treatment. In contrast, the fluorescence of merocyanine 540, a probe sensitive to lipid packing, was affected by both. Lipid packing decreased modestly as temperature was raised from 20 to 60 degrees C. Calcium loading enhanced packing at temperatures in the low end of this range, but greatly reduced packing at higher temperatures. This result was corroborated by measurements of the rate of extraction of a fluorescent phosphatidylcholine analog from erythrocyte membranes. Furthermore, drugs known to inhibit susceptibility in erythrocytes also prevented the increase in phospholipid extraction rate. These results argue that the two-step model applies to biological as well as artificial membranes and that a limiting step in the hydrolysis of erythrocyte membranes is the ability of phospholipids to migrate into the active site of adsorbed enzyme.     

Role of intracellular calcium and phospholipase A2 in arachidonic acid-induced toxicity in liver cells overexpressing CYP2E1

Liver cells (HepG2 and primary hepatocytes) overexpressing CYP2E1 and exposed to arachidonic acid (AA) were previously shown to lose viability together with enhanced lipid peroxidation. These events were blocked in cells pre-incubated with antioxidants (alpha-tocopherol, glutathione ethyl ester), or in HepG2 cells not expressing CYP2E1. The goal of the current study was to evaluate the role of calcium and calcium-activated hydrolases in these CYP2E1-AA interactions. CYP2E1-expressing HepG2 cells treated with AA showed an early increase in cytosolic calcium and partial depletion of ionomycin-sensitive calcium stores. These changes in calcium were blocked by alpha-tocopherol. AA activated phospholipase A2 (PLA2) in CYP2E1-expressing liver cells, and this was inhibited by PLA2 inhibitors or alpha-tocopherol. PLA2 inhibitors prevented the cell death caused by AA, without affecting CYP2E1 activity or lipid peroxidation. AA toxicity and PLA2 activation were inhibited in calcium-depleted cells, but not by removal of extracellular calcium alone. Removal of extracellular calcium inhibited the early increase in cytosolic calcium caused by AA. CYP2E1 overexpressing HepG2 cells exposed to AA showed a decrease in mitochondrial membrane potential, which was prevented by the PLA2 inhibitors. These results suggest that AA-induced toxicity to CYPE1-expressing cells: (i) is associated with release of Ca2+ from intracellular stores that depends mainly on oxidative membrane damage; (ii) is associated with activation of PLA2 that depends on intracellular calcium and lipid peroxidation; (iii) does not depend on increased influx of extracellular calcium, and (iv) depends on the effect of converging events (lipid peroxidation, intracellular calcium, activation of PLA2) on mitochondria to induce bioenergetic failure and necrosis. These interactions may play a role in alcohol liver toxicity, which requires polyunsaturated fatty acids, and involves induction of CYP2E1.     

Gonadotropin receptors in plasma membranes of bovine corpus luteum. II. Role of membrane phospholipids.

The role of phospholipids in the binding of 125I-choriogonadotropin to bovine corpus luteum plasma membranes has been investigated with the use of purified phospholipase A and phospholipase C to alter membrane phospholipids. The phospholipase C-digested plasma membrane preparation showed 85 to 90% inhibition of 125I-choriogonadotropin binding activity when 70% of the membrane phospholipid was hydrolyzed. Similarly treatment of plasma membranes with phospholipase A resulted in 45 to 55% hydrolysis of membrane phospholipid and almost 75% inhibition of receptor activity. Both these enzymes hydrolyzed membrane-associated phosphatidylcholine to a greater extent than phosphatidylethanolamine and phosphatidylserine. Phosphorylaminoalcohols of phospholiphase C end products were completely released into the medium, while phospholipase A by-products remained associated with plasma membranes. Addition of a phospholipids suspension or liposomes to plasma membranes pretreated with phospholipase A and C did not restore gonadotropin binding activity. Soluble phosphorylcholine, phosphorylethanolamine, and phosphorylserine and insoluble diglyceride products of phospholipase C action had no effect on receptor activity. In contrast, end products of the phospholipase A action, such as lysophosphatides and fatty acids, inhibited both on the membrane-associated and solubilized receptor activity. Lysophosphatidylcholine was the most effective end product inhibiting the binding of gonadotropin to the receptor, followed by lysophosphatidylethanolamine and lysophosphatidylserine. The inhibitory effects of phospholipase A or lysophosphatides were completely reversed upon removal of membrane-bound phospholipid end products by washing the membranes with defatted bovine serum albumin. However, phospholipase C inhibition could not be overcome by defatted albumin washings. Solubilization of plasma membranes with detergents which had been pretreated with phospholipase C partially restored the inhibited activity. It is concluded that the phospholipase-mediated inhibition of gonadotropin binding activity was due to hydrolysis and alterations of the phospholipid environment in the case of phospholipase C and by direct inhibition by end products in the case of phospholipase A.     

Reactive Oxygen Species Produced by a Photodynamic Effect Induced Calcium Signal in Neurons and Astrocytes

Photodynamic therapy (PDT) leads to production of reactive oxygen species (ROS) and cell destruction due to oxidative stress. We used photodynamic effect of photosensitizer radachlorin to unravel the effect of photo-induced oxidative stress on the calcium signal and lipid peroxidation in primary culture of cortical neurons and astrocytes using live cell imaging. We have found that irradiation in presence of 200 nM of radachlorin induces calcium signal in primary neurons and astrocytes. Photo-induced neuronal calcium signal depends on internal calcium stores as it was still observed in calcium-free medium and could be blocked by depletion of endoplasmic reticulum (ER) stores with inhibitor of sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA) thapsigargin. Both inhibitors of phospholipase C activity U73122 and water-soluble analogue of vitamin E Trolox suppressed calcium response activated by PDT. We have also observed that the photodynamic effect of radachlorin induces lipid peroxidation in neurons and astrocytes. This data demonstrate that lipid peroxidation induced by PDT in neurons and astrocytes leads to activation of phospholipase C that results in production of inositol 1,4,5-trisphosphate (IP3).     

Ordered and cooperative binding of opposing globular domains of calmodulin to the plasma membrane Ca-ATPase

We have investigated the mechanisms of activation of the plasma membrane (PM) Ca-ATPase by calmodulin (CaM), which result in enhanced calcium transport rates and the maintenance of low intracellular calcium levels. We have isolated the amino- or carboxyl-terminal domains of CaM (i.e. CaMN or CaMC), permitting an identification of their relative specificity for binding to sites on either the PM Ca-ATPase or a peptide (C28W) corresponding to the CaM-binding sequence. We find that either CaMN or CaMC alone is capable of productive interactions with the PM Ca-ATPase that induces enzyme activation. There are, however, large differences in the affinity and specificity of binding between CaMN and CaMC and either C28W or the PM Ca-ATPase. The initial binding interaction between CaMC and the PM Ca-ATPase is highly specific, having approximately 10,000-fold greater affinity in comparison with CaMN. However, following the initial association of either CaMC or CaMN, there is a 300-fold enhancement in the affinity of CaMN for the secondary binding site. Thus, while CaMC binds with a high affinity to the two CaM-binding sites within the PM Ca-ATPase in a sequential manner, CaMN binds cooperatively with a lower affinity to both binding sites. These large differences in the binding affinities and specificities of the amino- and carboxyl-terminal domains ensure that CaM binding to the PM Ca-ATPase normally involves the formation of a specific complex in which the initial high affinity association of the carboxyl-terminal domain promotes the association of the amino-terminal domain necessary for enzyme activation.     

Lipid peroxidation inactivates rat liver microsomal glycerol-3-phosphate acyl transferase. Effect of iron and copper salts and carbon tetrachloride

Lipid peroxidation is known to affect the activity of several enzymes including microsomal enzymes such as glucose-6-phosphatase; but its effect on the enzymes of lipid biosynthesis has not been investigated. Glycerol-3-phosphate acyltransferase (GPAT) represents the first committed step and probably the rate limiting step in glycerolipid synthesis and thus may be a good candidate for study. Rat liver microsomal GPAT was assayed after preincubating the microsomes under conditions known to induce peroxidation. In 30 min, 10 microM Fe2+ can diminish the activity by as much as 80%. The inactivating effect can be blocked to different extents by several antioxidants, while ascorbic acid enhances it. These effects, along with the concomitant measurement of lipid peroxidation, indicate that microsomal GPAT activity is inactivated by lipid peroxidation in a sensitive and rapid fashion. This is further confirmed by the inactivating effect of carbon tetrachloride, which is known to induce lipid peroxidation in microsomes. Fe3+ also inactivates the enzyme, but at a higher concentration. Copper salts inactivate GPAT by a mechanism apparently different from that of iron. The mechanism might involve a direct sulfhydryl modification by copper and lipid peroxidation apparently different from that induced by iron. It is suggested that the inactivation of GPAT by lipid peroxidation could accelerate the process of membrane disintegration caused by lipid peroxidation in pathological conditions involving free radical-mediated tissue injury.     

4-Hydroxynonenal, an Aldehydic Product of Lipid Peroxidation, Impairs Signal Transduction Associated with Muscarinic Acetylcholine and Metabotropic Glutamate Receptors: Possible Action on Gαq/11

Abstract: Considerable data indicate that oxidative stress and membrane lipid peroxidation contribute to neuronal degeneration in an array of age-related neurodegenerative disorders. In contrast, the impact of subtoxic levels of membrane lipid peroxidation on neuronal function is largely unknown. We now report that 4-hydroxy-nonenal (HNE), an aldehydic product of lipid peroxidation, disrupts coupling of muscarinic cholinergic receptors and metabotropic glutamate receptors to phospholipase C-linked GTP-binding proteins in cultured rat cerebrocortical neurons. At subtoxic concentrations, HNE markedly inhibited GTPase activity, inositol phosphate release, and elevation of intracellular calcium levels induced by carbachol (muscarinic agonist) and ( RS )-3,5-dihydroxyphenyl glycine (metabotropic glutamate receptor agonist). Maximal impairment of agonist-induced responses occurred within 30 min of exposure to HNE. Other aldehydes, including malondialdehyde, had little effect on agonist-induced responses. Antioxidants that suppress lipid peroxidation did not prevent impairment of agonist-induced responses by HNE, whereas glutathione, which is known to bind and detoxify HNE, did prevent impairment of agonist-induced responses. HNE itself did not induce oxidative stress. Immunoprecipitation-western blot analysis using an antibody to HNE-protein conjugates showed that HNE can bind to Gα_q/11. HNE also significantly suppressed inositol phosphate release induced by aluminum fluoride. Collectively, our data suggest that HNE plays a role in altering receptor-G protein coupling in neurons under conditions of oxidative stress that may occur both normally, and before cell degeneration and death in pathological settings.     

Initiator-promoter coupling of phospholipases D and A2 in platelets upon cholesterol incorporation.

Since phospholipases exist within a membrane lipid environment, it is not unreasonable to assume that cholesterol capable of changing the lipid environment can effect the coupling relationship among the signal transducing components. Our previous study showed that the 'molecular switch' through which membrane cholesterol modulates cyclic nucleotide levels and Na+/H+ exchange within human platelets is phospholipase A2. We demonstrate here that membrane cholesterol initiates the activation of phosphatidyl choline phospholipase D and phosphatidic acid thus generated promotes the activation of its phospholipase A2 in the presence of extraplatelet calcium. More important, inhibition of phospholipase D by zinc blocks the activation of phosphatidic acid phospholipase A2 in platelets upon cholesterol incorporation. Our result led us to postulate that membrane cholesterol induced initiation promotion coupling of phospholipases D and A2 in human platelets may be responsible for the hypersensitized state of platelets in hypercholesterolemic patients.     

Inhibitory effects of calcium antagonists on mitochondrial swelling induced by lipid peroxidation or amchidonic acid in the rat brain in vitro

Inhibitory effects of calcium antagonists, efonidipine (NZ-105), nicardipine, nifedipine, nimodipine and flunarizine, on mitochondrial swelling induced by lipid peroxidation or arachidonic acid in the rat brain in vitro were investigated. Mitochondrial swelling and lipid peroxidation induced by FeSO₄ and ascorbic acid system showed a close and significant relationship. Mitochondrial swelling and lipid peroxidation induced by FeSO₄ and ascorbic acid were inhibited by all of calcium antagonists tested. The order of inhibition was: flunarizine>nicardipine>efonidipine>nimodipine>nifedipine. This result suggests that calcium antagonists tested have antiperoxidant activities resulting in protection of mitochondrial membrane damage and that each moiety of these structures would play an important role in appearance of anti-peroxidant activities. Furthermore, flunarizine and efonidipine inhibited mitochondrial swelling induced by arachidonic acid, which is not associated with lipid peroxidation. In contrast, nicardipine, nifedipine, and nimodipine did not inhibited this swelling. It is possible that flunarizine and efonidipine could directly interact with mitochondrial membrane. In conclusion, it is capable that calcium antagonists tested may protect from the membrane damage induced by lipid peroxidation and that flunarizine and efonidipine could stabilize the membrane, which is attributed to a direct interaction with the membrane.     

The role of membrane structure in activation of mitochondrial phospholipases. 2. Mechanism of crambin action on the activities of mitochondrial membrane phospholipases and microviscosity

Effects of crambin on the phospholipase activity, lipid peroxidation and structure of mitochondrial membranes have been investigated. Crambin has been shown to inhibit lipid peroxidation and phospholipase activity induced by Ca2+ and freezing-thawing of mitochondria. As shown by ESR studies, these effects are based on the ability of crambin to cause changes in mitochondrial membrane structure.     

Role of a guanine nucleotide regulatory protein in the activation of phospholipase C by different chemoattractants

It is well established that formyl peptide chemoattractants can activate a phospholipase C in leukocytes via a pertussis toxin (PT)-sensitive guanine nucleotide regulatory (G) protein. Whether this pathway is similarly used by chemoattractant receptors as a class has been unclear. We now report that lipid and peptide chemoattractants in direct comparative studies induced similar amounts of initial (less than or equal to 15 sec) inositol trisphosphate (IP3) release in human polymorphonuclear leukocytes, but the response to lipid chemoattractants was more transient. Production of IP3 by all chemotactic factors was inhibited by treatment of the cells with PT, indicating that chemotactic factor receptors as a class are coupled to phospholipase C via a G protein that is a substrate for ADP ribosylation by PT. The peptide and lipid factors had comparable chemotactic activity, which was also inhibitable by PT. However, transient activation of phospholipase C is apparently an insufficient signal for full cellular activation, since the lipid chemotactic factor leukotriene B4 and platelet-activating factor were poor stimuli for O2- production and lysosomal enzyme secretion compared with N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe). Nonetheless, treatment with PT inhibited O2- production and enzyme secretion in response to all chemoattractants, but as previously noted, did not affect Ca2+ ionophores, lectins, or phorbol myristate acetate. Formyl peptide and lipid chemotactic factors induced similar levels of Ca2+ mobilization when monitored by Quin 2 or chlortetracycline (CTC) fluorescence. Although these responses to fMet-Leu-Phe were blocked by PT, the Quin 2 and initial CTC response to the lipid factors were only partially susceptible. Thus, the lipid factors apparently utilize an additional PT-resistant mechanism for redistributing intracellular Ca2+. This latter process requires extracellular Ca2+ and may be independent of the PT-sensitive G protein.     

Loss of latent activity of liver microsomal membrane enzymes evoked by lipid peroxidation. Studies of nucleoside diphosphatase, glucose-6-phosphatase, and UDP glucuronyltransferase

The effects of lipid peroxidation on latent microsomal enzyme activities were examined in NADPH-reduced microsomes from phenobarbital-pretreated male rats. Lipid peroxidation, stimulated by iron or carbon tetrachloride, was assayed as malondialdehyde formation. Independent of the stimulating agent of lipid peroxidation, latency of microsomal nucleoside diphosphatase activity remained unaffected up to microsomal peroxidation equivalent to the formation of about 12 nmol malondialdehyde/mg microsomal protein. However, above this threshold a close correlation was found between lipid peroxidation and loss of latent enzyme activity. The loss of latency evoked by lipid peroxidation was comparable to the loss of latency attainable by disrupting the microsomal membrane by detergent. Loss of latent enzyme activity produced by lipid peroxidation was also observed for microsomal glucose-6-phosphatase and UDPglucuronyltransferase. In contrast to nucleoside diphosphatase, however, both enzymes were inactivated by lipid peroxidation, as indicated by pronounced decreases of their activities in detergent-treated microsomes. According to the respective optimal oxygen partial pressure (po2) for lipid peroxidation, the iron-mediated effects on enzyme activities were maximal at a po2 of 80 mmHg and the one mediated by carbon tetrachloride at a po2 of 5 mmHg. Under anaerobic conditions no alterations of enzyme activities were detected. These results demonstrate that loss of microsomal latency only occurs when peroxidation of the microsomal membrane has reached a certain extent, and that beyond this threshold lipid peroxidation leads to severe disintegration of the microsomal membrane resulting in a loss of its selective permeability, a damage which should be of pathological consequences for the liver cell. Because of its resistance against lipid peroxidation nucleoside diphosphatase is a well-suited intrinsic microsomal parameter to estimate this effect of lipid peroxidation on the microsomal membrane.     

Oxidized LDL induces an oxidative stress and activates the tumor suppressor p53 in MRC5 human fibroblasts

It is now well established that oxidized LDL (OxLDL) is involved in the progression of the atheromatous plaque via several mechanisms, including its cytotoxicity toward the arterial wall. Our study demonstrates that a 4-h incubation of cultured human fibroblasts with 25-75 microg/ml OxLDL induced a dose-dependent increase in the intracellular levels of reactive oxygen species (ROS) and lipid peroxidation end products (TBARS). This effect was markedly prevented by the antioxidant vitamin E. The lipid extract of OxLDL partially reproduced the action of the LDL particle itself. Concomitantly, OxLDL enhanced the DNA binding activity of p53 measured by electrophoretic mobility shift assay, and the intracellular protein level of p53 determined by immunoblot analysis. Cycloheximide prevented the OxLDL-induced augmentation in both p53 binding activity and intracellular level. Again, the lipid extract of OxLDL reproduced the effect of OxLDL on p53 binding activity, whereas vitamin E prevented it. These results indicate that OxLDL initiates an intracellular oxidative stress by means of its lipid peroxidation products, leading to the activation of the tumour suppressor p53 by enhancement of p53 protein synthesis. This effect might be related to the cytotoxic effect of OxLDL since the activation of p53 is known to lead to cell cycle arrest, necrosis or apoptosis.     

The role of lipid-protein interactions in NADH-cytochrome c reductase (rotenone-insensitive) of rat liver mitochondria

The phospholipid depletion of rat liver mitochondria, induced by acetone-extraction or by digestion with phospholipase A₂ or phospholipase C, greatly inhibited the activity of NADH-cytochrome c reductase (rotenone-insensitive). A great decrease of the reductase activity also occurred in isolated outer mitochondrial membranes after incubation with phospholipase A₂. The enzyme activity was almost completely restored by the addition of a mixture of mitochondrial phospholipids to either lipid-deficient mitochondria, or lipid-deficient outer membranes. The individual phospholipids present in the outer mitochondrial membrane induced little or no stimulation of the reductase activity. Egg phosphatidylcholine was the most active phospholipid, but dipalmitoyl phosphatidylcholine was almost ineffective. The lipid depletion of mitochondria resulted in the disappearance of the non-linear Arrhenius plot which characterized the native reductase activity. A non-linear plot almost identical to that of the native enzyme was shown by the enzyme reconstituted with mitochondrial phospholipids. Triton X-100, Tween 80 or sodium deoxycholate induced only a small activation of NADH-cytochrome c reductase (rotenone-insensitive) in lipiddeficient mitochondria. The addition of cholesterol to extracted mitochondrial phospholipids at a 1 : 1 molar ratio inhibited the reactivation of NADH-cytochrome c reductase (rotenone-insensitive) but not the binding of phospholipids to lipid-deficient mitochondria or lipid-deficient outer membranes.These results show that NADH-cytochrome c reductase (rotenone-insensitive) of the outer mitochondrial membrane requires phospholipids for its activity. A mixture of phospholipids accomplishes this requirement better than individual phospholipids or detergents. It also seems that the membrane fluidity may influence the reductase activity.