Validation of suitable internal control genes for expression studies in aging.

Quantitative data from experiments of gene expression are often normalized through levels of housekeeping genes transcription by assuming that expression of these genes is highly uniform. This practice is being questioned as it becomes increasingly clear that the level of housekeeping genes expression may vary considerably in certain biological samples. To date, the validation of reference genes in aging has received little attention and suitable reference genes have not yet been defined. Our aim was to evaluate the expression stability of frequently used reference genes in human peripheral blood mononuclear cells with respect to aging. Using quantitative RT-PCR, we carried out an extensive evaluation of five housekeeping genes, i.e. 18s rRNA, ACTB, GAPDH, HPRT1 and GUSB, for stability of expression in samples from donors in the age range 35-74 years. The consistency in the expression stability was quantified on the basis of the coefficient of variation and two algorithms termed geNorm and NormFinder. Our results indicated GUSB be the most suitable transcript and 18s the least for accurate normalization in PBMCs. We also demonstrated that aging is a confounding factor with respect to stability of 18s, HPRT1 and ACTB expression, which were particularly prone to variability in aged donors.

Changes in blood lipid peroxidation markers and antioxidants after a single sprint anaerobic exercise.

It has been well demonstrated that the principal factor responsible for oxidative damage during exercise is the increase in oxygen consumption. However, other theoretical factors (acidosis, catecholamine autoxidation, ischemia-reperfusion syndrome, etc.) that are known to induce, in vitro, oxidative damage may also be operative during short-term supramaximal anaerobic exercise. Therefore, we hypothesized that short-term supramaximal anaerobic exercise (30-s Wingate test) could induce an oxidative stress. Lipid peroxidation markers [serum lipid radical production detected by electron spin resonance (ESR) spectroscopy and plasma malondialdehyde (MDA) levels detected by the thiobarbituric acid reactive substances (TBARS) method], as well as erythrocyte antioxidant enzyme activities [glutathione peroxidase (GPx), superoxide dismutase (SOD)] and erythrocyte glutathione (GSH) levels, were measured at rest, after the Wingate test and during the 40 min of recovery. The recovery of exercise was associated with a significant increase (x2.7) in lipid radical production detected by ESR spectroscopy, as well as with changes in the erythrocyte GSH level (-13.6%) and SOD activity (-11.7%). The paradoxical decrease in plasma TBARS (-23.7%) which was correlated with the peak power developed during the Wingate test ( r=-0.7), strongly suggests that such exercise stimulates the elimination of MDA. In conclusion, this study demonstrates that short-term supramaximal anaerobic exercise induces an oxidative stress and that the plasma TBARS level is not a suitable marker during this type of exercise.

A method for separation of heparin species from biological samples by ethanol precipitation of compounds solubilized in guanidine hydrochloride.

In this paper we describe a procedure to determine glycosaminoglycan and oligosaccharide composition of biological samples such as cell cultures or tissue explants. We demonstrate that heparin species of different molecular mass can be easily fractionated by sequential ethanol precipitation in 4.0 M guanidine hydrochloride. We studied by gradient polyacrylamide gel electrophoresis fractionation of standard heparin and heparin-derived oligosaccharides by anion-exchange chromatography on DEAE-Sephacel resin eluted by increasing concentration of guanidine hydrochloride. The use of guanidine salts followed by sequential precipitation by increasing ethanol concentration allowed recovery of heparin and heparin-derived oligosaccharides.

Free radical scavenging and antioxidant effects of lactate ion: an in vitro study.

Divergent literature data are found concerning the effect of lactate on free radical production during exercise. To clarify this point, we tested the pro- or antioxidant effect of lactate ion in vitro at different concentrations using three methods: 1) electron paramagnetic resonance (EPR) was used to study the scavenging ability of lactate toward the superoxide aion (O(2)(-).) and hydroxyl radical (.OH); 2) linoleic acid micelles were employed to investigate the lipid radical scavenging capacity of lactate; and 3) primary rat hepatocyte culture was used to study the inhibition of membrane lipid peroxidation by lactate. EPR experiments exhibited scavenging activities of lactate toward both O(2)(-). and.OH; lactate was also able to inhibit lipid peroxidation of hepatocyte culture. Both effects of lactate were concentration dependent. However, no inhibition of lipid peroxidation by lactate was observed in the micelle model. These results suggested that lactate ion may prevent lipid peroxidation by scavenging free radicals such as O(2)(-). and.OH but not lipid radicals. Thus lactate ion might be considered as a potential antioxidant agent.

Hepatotoxicity of tacrine: occurrence of membrane fluidity alterations without involvement of lipid peroxidation.

Tacrine (THA), used in the treatment of Alzheimer's disease, is known to induce hepatotoxicity, the mechanisms of which remain to be fully established. We have previously shown that THA reduced intracellular glutathione concentration in rat hepatocytes in primary culture, thus pointing to a possible role for oxidative stress in THA toxicity. To test this, the effects of antioxidant molecules, namely, the flavonoids silibinin, silibinin dihydrogensuccinate, and silymarin, were evaluated on the toxicity of THA in cultured rat hepatocytes. This toxicity was investigated after a 24-h treatment over a concentration range from 0 to 1 mM, in the presence or absence of antioxidant (1 and 10 microM). We found that simultaneous treatment of hepatocytes with any of the antioxidants and THA remained ineffective on the lactate dehydrogenase release induced by THA. Then, the production of lipid-derived radicals (to estimate lipid peroxidation) was measured in THA (0.05-0.50 mM)-treated cells using a spin-trapping technique coupled to electron paramagnetic resonance (EPR) spectroscopy. No increase of the EPR signal was observed over the period of 30 min to 24 h. In contrast, treatment of cells with the spin label 12-doxyl stearic acid followed by EPR spectroscopy showed that THA (0.05 and 0.25 mM) rapidly increased hepatocyte membrane fluidity. Extracellular application of GM1 ganglioside (60 microM) both reversed this increase in fluidity and partially reduced lactate dehydrogenase release on THA exposure. In conclusion, this work indicates that early alterations of membrane fluidity, not resulting from lipid peroxidation, are likely to play an important role in the development of THA toxicity.

Activated macrophages increase the susceptibility of rat hepatocytes to ethanol-induced oxidative stress: conflicting effects of nitric oxide.

The aim of this study was to examine how macrophages could act on ethanol-induced oxidative stress in rat hepatocytes during inflammatory conditions, well-known to induce nitric oxide (NO) synthase. For this purpose, RAW 264.7 macrophages were added to primary rat hepatocyte cultures. Co-cultures were then supplemented with lipopolysaccharide (LPS) and interferon gamma (IFN) for 18 h, in order to induce NO synthase before the addition of 50 mM ethanol. In cultures of hepatocytes alone, the addition of LPS and IFN protected from ethanol-induced oxidative stress. It has been shown previously that NO generated in hepatocytes was responsible for this effect. When macrophages were added to primary rat hepatocyte cultures supplemented with LPS and IFN, protection provided by NO against ethanol-induced oxidative stress in hepatocytes ceased. Using a pretreatment of macrophages with N(g)-monomethyl-l-arginine, a NO synthase inhibitor, it was concluded that NO generated by macrophages was responsible for macrophage toxicity. Taken together, our observations suggest that NO biosynthesis in hepatocytes protects them from ethanol-induced oxidative stress, whereas NO production in macrophages deprives hepatocytes of this NO protection.

Heparin binding peptides co-purify with glycosaminoglycans from human plasma.

Glycosaminoglycans (GAGs) are complexed with plasma proteins and proteolysis of plasma reduced the protein-GAG ratio about 140-fold. After dialysis, analysis by gradient PAGE revealed heparinase-1-sensitive GAGs, thus suggesting that heparin could be among the plasma GAGs. However, after dialysis most of the plasma GAGs were still not 'free'. PAGE of peptides resistant to proteolysis showed high molecular weight bands on the two sides of the dialysis membrane despite the 3.5 kDa molecular weight cut-off. Progressive dilution of the sample allowed passage of peptides appearing as high molecular weight bands in the diffusate. We interpret this phenomenon as the presence of low molecular weight peptides that aggregate when concentrated. Peptides on both sides of the membranes bound heparin.

Macrophage-induced inhibition of nitric oxide production in primary rat hepatocyte cultures via prostaglandin E2 release.

Kupffer cells and other macrophages play an important role in pathogenesis of toxicants in the liver. The aim of this study was to evaluate the effect of macrophages on hepatocyte production of nitric oxide (NO), which has been previously reported to be protective toward oxidative stress induced in primary rat hepatocytes. For this purpose, RAW 264.7 macrophages were added to primary rat hepatocytes at various ratios between macrophages and hepatocytes. These cocultures were supplemented with lipopolysaccharide (LPS) and interferon gamma (IFN-gamma) for 23 hours to induce NO synthase and trigger NO production. NO production was followed by quantification of nitrites in culture medium and dinitrosyl iron complexes (DNIC) in intact hepatocytes after separation from macrophages. In cocultured hepatocytes incubated with LPS and IFN-gamma, DNIC and nitrite levels decreased compared with those observed in hepatocytes cultured without macrophages in the same conditions. Moreover, inhibition of NO production in hepatocyte cocultures was macrophage-number-dependent. Macrophage-conditioned medium also inhibited NO production in hepatocytes, suggesting that the effect of macrophages was mediated by soluble factors. Among the soluble factors known to decrease NO levels are some cytokines, growth factors, reactive oxygen species, and prostaglandins. Ultrafiltration of macrophage-conditioned medium through a 500-d membrane to rule out higher-molecular-weight molecules, such as anti-inflammatory cytokines and growth factors, failed to restore NO production. In the same way, the use of superoxide dismutase (SOD) and catalase (CAT) to eliminate reactive oxygen species produced by macrophages did not lead to recovery of NO levels in hepatocytes. However, when NO synthesis was inhibited in macrophages by NG-monomethyl-L-arginine (L-NMMA), hepatocytes recovered the capacity to produce NO. A net decrease of prostaglandin E2 (PGE2) release by macrophages was concomitantly observed. Moreover, inhibition of PGE2 production in macrophages by indomethacin led to restoration of NO levels. Taken together, our observations suggest that NO synthesized by macrophages can decrease NO production in hepatocytes via PGE2 release. Because of the protective role of NO toward many liver injuries, it may be postulated that macrophages contribute through this mechanism to liver damage.

Urokinase-dependent angiogenesis in vitro and diacylglycerol production are blocked by antisense oligonucleotides against the urokinase receptor.

The plasminogen activator system is known to play a crucial role in the angiogenesis process by modulating the adhesive properties of endothelial cells to the extracellular matrix and cell-cell interaction. In the present study, we demonstrated that the urokinase-type plasminogen activator (u-PA) induced neovascular growth in the avascular rabbit cornea and dose-dependently promoted growth, chemotaxis, and matrix invasion of cultured endothelial cells. Interaction between u-PA and its receptor appears to be mandatory for the angiogenic effect of u-PA because monoclonal antibodies anti-u-PA and anti-u-PA receptor (u-PAR) blocked the proangiogenic effects of u-PA at the endothelial cell level. We then assessed the signaling pathway activated in endothelial cells by u-PA. u-PAR activation by u-PA produced de novo synthesis of diacylglycerol (DAG) from glucose by a cytochalasin B-inhibitable mechanism, indicating the involvement of a specific glucose transporter (GLUT). Endothelial cells expressed GLUT2, whose activation was tyrosine kinase-dependent and protein kinase C (PKC)-independent. The increase of glucose uptake led to DAG production, which resulted in PKC activation/translocation. Impairment of u-PAR availability by monoclonal antibodies and by antisense oligonucleotides (aODN) against u-PAR mRNA inhibited glucose uptake, DAG neosynthesis, and PKC activation, resulting in the blockade of endothelial cell proliferation, chemotaxis, and chemoinvasion. These data suggest that u-PAR activation consequent to the binding of u-PA can be regarded as an "angiogenic switch" and disclose the possibility that an anti-u-PAR aODN strategy may efficiently target endothelial cell function to control angiogenesis in vivo.

Premature induction of aging in sublethally H2O2-treated young MRC5 fibroblasts correlates with increased glutathione peroxidase levels and resistance to DNA breakage.

Human MRC5 fibroblasts, at different passages in cultures, were used as an in vitro model to assess variations and/or induction of aging parameters under basal conditions or following sublethal oxidative stress by H2O2. DNA sensitivities to oxidatively-induced breakage, rather than basal levels of damaged DNA, were significantly different between cultures at low and high population doubling level (PDL): old cells maintained most of their DNA integrity even at high concentrations of H2O2, while young cells showed more extensive DNA damage which developed in a dose-dependent fashion. However, young cells pretreated with low doses of H2O2 exhibited increased resistance against further oxidative damage to DNA thus reproducing a senescent-like profile of sensitivity. In turn, DNA from old cultures incubated in a NAD precursor-free medium was more prone to H2O2-induced strand breaks mimicking DNA sensitivity of young cells. The extent of oxidatively-induced DNA damage in MRC5 populations correlated inversely with the levels of glutathione peroxidase (GPx) activity that almost doubled when cells passed from the young to the senescent stage. In addition, H2O2-pretreatment of young cells induced an increase in GPx expression approaching old cell values and promoted also the premature appearance of neutral beta-galactosidase activity and decreased c-fos expression upon serum stimulation, both of which were assumed to be characteristic traits of the senescent phenotype.

Oltipraz stimulates the transcription of the manganese superoxide dismutase gene in rat hepatocytes.

Oltipraz (4-methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione) (OPZ) is recognized as a potent chemoprotective agent against chemical-induced carcinogenesis in several animal models and is thought to act mainly by inducing phase II conjugating together with inhibiting phase I detoxication enzymes. The present study was undertaken to determine whether oltipraz can also influence expression of genes encoding antioxidant enzymes. In rat hepatocytes in primary culture, this compound was found to selectively induce the transcription of the manganese superoxide dismutase (Mn-SOD) gene while it had no effect on copper/zinc-SOD and glutathione peroxidase genes. Oltipraz increased Mn-SOD gene expression in a time- and dose-dependent manner by 2- to 3-fold and enhanced the binding activity of the nuclear factor kappa B within 30 min. Moreover, the increase in Mn-SOD gene transcription was associated with a 2- to 3-fold increase of free malondialdehyde and conjugated dienes, two markers of lipid peroxidation, an index of oxidative stress. These results suggest that in rat hepatocytes, oltipraz induced a production of reactive oxygen species that probably acted as second messengers in order to trigger the transcription of many genes. Such a mechanism of action of OPZ and other dithiolethiones would account for the broad spectrum of action of these anticarcinogenic compounds.

Interaction of urokinase-type plasminogen activator with its receptor rapidly induces activation of glucose transporters.

The interaction of urokinase-type plasminogen activator (u-PA) or of u-PA amino-terminal fragment (u-PA-ATF) with the cell surface receptor (u-PAR) was found to stimulate an increase of glucose uptake in many cell lines, ranging from normal and transformed human fibroblasts, mouse fibroblasts transfected with human u-PAR, and cells of epidermal origin. Such increase of glucose uptake reached a peak within 5-10 min, depending on the cell line, and occurred through the facilitative glucose transporters (GLUTs), since it was inhibited by cytochalasin B. Each cell line showed a specific mosaic of glucose transporter isoforms, GLUT2 being the most widespread and GLUT1 the most abundant, when present. u-PAR stimulation was followed by translocation of GLUT1 from the microsomal to the membrane compartment, as shown by both immunoblotting and immunofluorescence of sonicated plasma membrane sheets and by activation of GLUT2 on the cell surface. Both translocation and activation resulted inhibitable by protein-tyrosine kinase inhibitors and independent of downregulation of protein kinase C (PKC). The increase of intracellular glucose was followed by neosynthesis of diacylglycerol (DAG) from glucose, as previously shown. Such neosynthesis was completely inhibited by impairment of facilitative GLUT transport by cytochalasin B. DAG neosynthesis was followed by activation of PKC, whose activity translocated into the intracellular compartment (PKM), where it probably phosphorylates substrates required for u-PAR-dependent chemotaxis. Our data show that u-PAR-mediated signal transduction, related with u-PA-induced chemotaxis, involves activation of tyrosine kinase-dependent glucose transporters, leading to increased de novo DAG synthesis from glucose, eventually resulting in activation of PKC.

Effect of nitric oxide on iron-mediated oxidative stress in primary rat hepatocyte culture.

An iron-mediated oxidative stress caused by an increase of the intracellular pool of low molecular weight complex of iron (LMWC) can be observed with iron overloading or ethanol metabolism. The aim of this study was to determine whether nitric oxide (NO) behaved as a pro-oxidant or an antioxidant in such an iron-mediated oxidative stress in rat hepatocytes. The cells were set up in primary cultures and incubated with lipopolysaccharide (LPS) and gamma-interferon (IFN) for 18 hours to induce NO synthase and to trigger NO production. Then 20 micromol/L iron or 50 mmol/L ethanol were added. Oxidative stress was evaluated by measuring lipoperoxidation using two markers: malondialdehyde (MDA) and conjugated dienes. Simultaneously, NO production was followed by the quantitation of nitrites in the culture medium, dinitrosyl iron complexes (DNICs) and mononitrosyl iron complexes (MNICs) in intact hepatocytes. DNIC and MNIC, evaluated by electron paramagnetic resonance (EPR), corresponded to NO bound to iron-containing molecules and to free NO, respectively. In cultures preincubated with LPS and IFN before iron or ethanol addition, a net decrease of lipid peroxidation induced by either NO, iron, or ethanol was noted. Moreover, an elevation of iron-bound NO and a decrease of free NO were observed in these cultures compared with the cultures incubated with only LPS and IFN. These data support the idea that there is a relationship between the changes of NO pool and the inhibition of oxidative stress. In addition, using N(G)-monomethyl-L-arginine (L-NMMA), a NO synthase inhibitor, NO was shown to be involved in the inhibition of oxidative stress induced by iron or ethanol. Addition of the chelator of LMWC iron, deferiprone, was followed by the inhibition of the increase of iron-bound NO and the reincrease of lipid peroxidation extent, which was as high as in cultures incubated only with LPS and IFN. Thus LMWC iron appeared to be involved also in the inhibition of oxidative stress induced by NO. All the results favor the conclusion that NO acts as an antioxidant in iron-mediated oxidative stress in rat hepatocytes. NO reacted with LMWC iron to form inactive iron complexes unable to induce oxidative stress in rat hepatocytes. Thus NO played a critical role in protecting the liver from oxidative stress.

Oxidative stress induced by ethanol in rat hepatocyte cultures.

Many controversies still exist with regard to the relationship between alcoholic intoxication and the occurrence of an oxidative stress. To attempt to resolve this question, first we investigated the induction by acute ethanol intoxication of lipid peroxidation in primary rat hepatocyte cultures using simultaneously two indices for each sample. When considering conjugated-diene indice, any lipid peroxidation elevation could be observed, whereas a net increase of extracellular free malondialdehyde was noted at 5 hours of incubation. These results led us to estimate the intracellular pool of low molecular weight iron which is known to be the iron species catalytically active in hydroperoxide degradation. An early enhancement of +20-30% of cellular low molecular weight iron was observed. Thus the discrepancy between conjugated dienes and malondialdehyde could be ascribed to an increase of hydroperoxide degradation into malondialdehyde by the transient cellular pool of low molecular weight iron. Lipid peroxidation and low molecular weight iron augmentation were linked to ethanol metabolism, since both were suppressed by the addition of 4-methylpyrazole, an alcohol dehydrogenase inhibitor. Superoxide dismutase activity was increased in the early incubation time (1 hour) and then markedly reduced. We conclude that ethanol metabolism can induce a lipid peroxidation accompanied by an elevation of intracellular pool of low molecular weight iron and a decrease of superoxide dismutase activity.

Induction, effects, and quantification of sublethal oxidative stress by hydrogen peroxide on cultured human fibroblasts.

Conditions to induce and parameters to evaluate sublethal oxidative stress of cultured human fibroblasts have been investigated in the attempt to identify markers for a more accurate quantification of cell injury. Sublethal oxidative stress was obtained by treating fibroblasts with 0.5 mM H2O2 in DMEM plus 5% FCS for times not exceeding 60 min. Under these conditions cells remained viable throughout long-term incubation, showing no appreciable release of cytosolic enzymes into the medium. On the contrary, exposures of fibroblasts to 0.5 mM H2O2 for times > 60 min induced a lethal cell injury which was fully expressed 2 days later by massive monolayer wasting and leakage of cytosolic components. Early metabolic effects of sublethal stress consisted of a rapid and significant fall of both ATP and NAD+ pools. Concomitantly, there was a moderate increase (about threefold) in both ADP-ribosyl transferase activity and free [Ca2+]i, while the specific activity of glyceraldehyde-3-phosphate dehydrogenase was partially decreased upon treatment. Oxidative injury also caused delayed effects consisting of a large depression of both protein and DNA synthesis. However, while the former was partially restored within 10 days of incubation, the latter remained severely impaired, as encountered in a growth-arrested population. Microfilaments of H2O2-treated cells appeared to be morphologically altered due to partial fragmentation of cytoskeleton actin which, however, was still maintained in the polymerized form as F-actin. Moreover, sublethally injured fibroblasts exhibited a reduced adhesiveness to plastic once they were detached and reseeded into new dishes. Relative adhesion efficiencies (number of adherent cells at 16 h as a percentage of seeded cells) were found to correlate inversely with times of exposure to H2O2. This finding allowed the identification of a biological parameter which showed itself to be very sensitive to oxidative stress and was also useful for developing an assay to grade sublethal injury to fibroblasts.

Increase in cellular pool of low-molecular-weight iron during ethanol metabolism in rat hepatocyte cultures. Relationship with lipid peroxidation.

Ethanol-induced lipid peroxidation was studied in primary rat hepatocyte cultures supplemented with ethanol at the concentration of 50 mM. Lipid peroxidation was assessed by two indices: (1) conjugated dienes by second-derivative UV spectroscopy in lipid extract of hepatocytes (intracellular content), and (2) free malondialdehyde (MDA) by HPLC-UV detection and quantitation for the incubation medium (extracellular content). In cultures supplemented with ethanol, free MDA increased significantly in culture media, whereas no elevation of conjugated diene level was observed in the corresponding hepatocytes. The cellular pool of low-mol-wt (LMW) iron was also evaluated in the hepatocytes using an electron spin resonance procedure. An early increase of intracellular LMW iron (< or = 1 hr) was observed in ethanol-supplemented cultures; it was inhibited by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, whereas alpha-tocopherol, which prevented lipid peroxidation, did not inhibit the increase of LMW iron. Therefore, the LMW iron elevation was the result of ethanol metabolism and was not secondarily induced by lipid hydroperoxides. Thus, ethanol caused lipid peroxidation in rat hepatocytes as shown by the increase of free MDA, although no conjugated diene elevation was detected. During ethanol metabolism, an increase in cellular LMW iron was observed that could enhance conjugated diene degradation.

[Kinetics of lipid peroxidation induced by UV beta rays in human keratinocyte and fibroblast cultures]. / Cinétique de la lipoperoxydation induite par les UVB dans les cultures de kératinocytes et de fibroblastes humains.

Lipid peroxidation has been implicated in skin damage by ultraviolet radiation. The aim of the study was to determine the kinetic of lipid peroxidation induced by ultraviolet beta (UVB) in adult keratinocytes and fibroblasts in culture. The keratinocytes were obtained from a single primary culture and the fibroblasts were in the same subculture (4 to 10 transfers). For UVB irradiation, the cells were maintained in a small volume of Hanks balanced salt solution and were irradiated (0.75, 1.5, 3 and 4.5 Jcm-2). Then cells were cultured for 3 to 48 hours. Lipid peroxidation was estimated by free MDA determination in both extracellular medium and cells using a size exclusion chromatography coupled to an HPLC procedure. In addition, LDH release in culture media was evaluated as in indice of cytotoxicity. An increase of total free MDA was observed 3 hours after cell irradiation which was dose-dependent from 0.75 to 3 Jcm-2 for keratinocytes and fibroblasts. MDA was detected both in cells and in culture media. As soon as 3 hours after irradiation 90% in total MDA was present in the culture media. Kinetic of lipid peroxidation: for 0.75 Jcm-2, an elevation of MDA was observed 12 hours after irradiation in both cultures. A further increase in MDA was noted 24 hours after fibroblasts irradiation but not in irradiated keratinocytes. LDH release in culture media increased with post irradiation time until 48 hours. The cytotoxic effect of UVB irradiation on keratinocytes and fibroblasts cultures was shown by an enhancement of lipid peroxidation which was detectable during 48 hours after irradiation. An increase of LDH release was observed simultaneously.

Production of second messengers following chemotactic and mitogenic urokinase-receptor interaction in human fibroblasts and mouse fibroblasts transfected with human urokinase receptor.

We studied urokinase-type plasminogen activator (u-PA)-dependent chemotaxis and DNA synthesis in both human fibroblasts and LB6 mouse fibroblasts transfected with human u-PA receptor (u-PAR) gene (LB6 clone 19). Both cell lines have receptors for the amino-terminal fragment of u-PA (u-PA-ATF). We observed that u-PA and u-PA-ATF stimulated chemotactic migration of both LB6 clone 19 cells and human fibroblasts, which could be impaired by down-regulation of protein kinase C (PKC) with phorbol myristate acetate (PMA). While LB6 clone 19 cells were unable to undergo mitosis following exposure to either u-PA or u-PA-ATF, human fibroblasts were stimulated to mitosis by exogenous addition of native u-PA, and u-PA-ATF was ineffective. The mitogenic activity of u-PA on human fibroblasts could also be impaired by down-regulation of PKC with PMA. We studied second messenger formation following u-PAR stimulation. Neither inositol lipid metabolism nor intracellular Ca2+ content were affected, while an increase of diacylglycerol (DAG) generation was observed. Such DAG formation was related to de novo synthesis from glucose and was dependent on ligand-receptor interaction. Both u-PA-ATF and the native u-PA molecule were able to stimulate DAG formation, u-PA being from three to fourfold more efficient than ATF. These data suggest that u-PAR stimulation per se is sufficient to trigger DAG formation. The native molecule confers on the cell an additional stimulus, possibly related with the activation of a u-PA-catalytic site-dependent substrate. Such stimulation allows the cell to reach the DAG threshold level required to trigger DNA synthesis.

Ultraviolet and infrared spectroscopy for microdetermination of oxidized and unoxidized fatty acyl esters in cells.

New methods based on ultraviolet and infrared spectroscopy were developed to quantify oxidized and unoxidized fatty acyl esters (FAE) in cells. For this study, rat hepatocyte cultures (2.5 x 10(6) cells) were submitted to an oxidative stress by a 5-h incubation with iron(III) chelated with nitrilotriacetic acid (100 microM). Control hepatocytes were incubated under the same conditions except in the absence of iron. After cell lipid extraction, oxidized FAE were evaluated by the second derivative of the conjugated-diene (CD) spectrum, which exhibited minima at 233 and 242 nm ascribed to trans,trans (t,t) and cis,trans (c,t) CD isomers, respectively. These minima were quantified in arbitrary units as d2 A/d lambda 2; hydroperoxide concentration was determined using a linear regression curve obtained from autoxidized linoleic acid micelles. Total (oxidized and unoxidized) FAE were measured by Fourier transform infrared spectroscopy using the absorption band at 1740 cm-1. A highly significant correlation coefficient (r = 0.992) was found for the standard curve performed with glycerol trioleate expressed as nanomoles fatty acid equivalents. The extent of lipid oxidation could be estimated by the sum of minima at 233 and 242 nm which allowed the calculation of hydroperoxide concentrations. The amount of oxidized FAE was related to the amount of total FAE in the same sample. The ratio of minima at 242 nm (c,t isomers) and 233 nm (t,t isomers) could provide an evaluation of cell antioxidant capacity. A decrease of this ratio would indicate a large depletion of radical termination antioxidants.

Simultaneous measurements of conjugated dienes and free malondialdehyde, used as a micromethod for the evaluation of lipid peroxidation in rat hepatocyte cultures.

Membrane lipid peroxidation in rat hepatocyte cultures was induced by a 5-h incubation with either ethanol (50 mM) or the chelate iron-nitrilotriacetic acid (Fe-NTA) (100 microM). To test the oxidative stress, two indices were measured simultaneously on the same sample: extracellular free malondialdehyde (MDA) measured by HPLC with a size exclusion column, and conjugated dienes (CD) determined by second derivative spectroscopy. With ethanol, both CD and MDA gave nearly the same values of lipid peroxidation, about 135% of the control value. With Fe-NTA, both indices indicated a higher lipid peroxidation, but the MDA and CD values were different. Iron lipid peroxidation evaluated by free MDA and CD was, 290 and 230%, respectively, of the control. This discrepancy could be ascribed to an increased decomposition of hydroperoxides by iron. In addition, the ratio of cis,trans and trans,trans conjugated dienes, which reflects the cellular redox status, remained unchanged after 5 h of lipid peroxidation induced either by ethanol or iron.