Rosalba Serù

Atorvastatin Improves the Course of Ischemic Acute Renal Failure in Aging Rats

Statins increase the production of nitric oxide (NO) and have beneficial effects on the course of acute renal failure (ARF) in young rats. The effects of a short-term treatment with atorvastatin (ATO) on ischemic ARF in old rats, characterized by a great susceptibility to ischemia, was tested. No difference was found in renal dynamics between young (Y, 3 mo old) and old (O, 18 mo old) rats in normal conditions (CON) or after ATO treatment (12 mg/kg/d for 14 d). Twenty-four hours after clamping of both renal arteries, a more pronounced decrease in GFR was observed in O rats versus Y rats after a greater renal vasoconstriction and hypoperfusion of aging animals. Pretreatment with ATO mitigated renal vasoconstriction in O rats and restored GFR values to Y rats. Nitrate excretion was enhanced in Y rats after ARF but was not further modified by ATO; in O rats, ARF did not increase nitrate excretion, which was raised after ATO treatment. This reflected the increase in endothelial NO synthase (eNOS)-mRNA expression and eNOS protein observed in old ATO-treated animals with ARF. ATO treatment had also a significant protective effect against the cell injury at tubular level in O, but not Y, rats. The Ras system was not influenced by ATO in O rats, whereas the activation of Rho proteins was partially inhibited by ATO. Low-dose treatment with ATO enhances NO availability in aging rats, improving renal dynamics and conferring a peculiar histologic protection at tubular level after ischemia.

Opposing functions of Ki- and Ha-Ras genes in the regulation of redox signals.

Ras p21 signaling is involved in multiple aspects of growth, differentiation, and stress response [1-2]. There is evidence pointing to superoxides as relays of Ras signaling messages. Chemicals with antioxidant activity suppress Ras-induced DNA synthesis. The inhibition of Ras significantly reduces the production of superoxides by the NADPH-oxidase complex [3]. Kirsten and Harvey are nonallelic Ras cellular genes that share a high degree of structural and functional homology. The sequences of Ki- and Ha-Ras proteins are almost identical. They diverge only in the 20-amino acid hypervariable domain at the COOH termini. To date, their functions remain indistinguishable [4]. We show that Ki- and Ha-Ras genes differently regulate the redox state of the cell. Ha-Ras-expressing cells produce high levels of reactive oxygen species (ROS) by inducing the NADPH-oxidase system. Ki-Ras, on the other hand, stimulates the scavenging of ROS by activating posttranscriptionally the mitochondrial antioxidant enzyme, Mn-superoxide dismutase (Mn-SOD), via an ERK1/2-dependent pathway. Glutamic acid substitution of the four lysine residues in the polybasic stretch at the COOH terminus of Ki-Ras completely abolishes the activation of Mn-SOD, although it does not inhibit ERK1/2-induced transcription. In contrast, an alanine substitution of the cysteine of the CAAX box has very little effect on Mn-SOD activity but eliminates ERK1/2- dependent transcription.

HaRas activates the NADPH oxidase complex in human neuroblastoma cells via extracellular signal-regulated kinase 1/2 pathway

In this study we have investigated the effects of the small GTP‐binding‐protein Ras on the redox signalling of the human neuroblastoma cell line, SK‐N‐BE stably transfected with HaRas(Val12). The levels of reactive oxygen species (ROS) and superoxide anions were significantly higher in HaRas(Val12) expressing (SK‐HaRas) cells than in control cells. The treatment of cells with 4‐(2‐aminoethyl) benzenesulfonylfluoride, a specific inhibitor of the membrane superoxide generating system NADPH oxidase, suppressed the rise in ROS and significantly reduced superoxide levels produced by SK‐HaRas cells. Moreover, HaRas(Val12) induced the translocation of the cytosolic components of the NADPH oxidase complex p67phox and Rac to the plasma membrane. These effects depended on the mitogen‐activated protein kinase kinase/extracellular signal‐regulated kinase (MEK/ERK1/2) pathway, as the specific MEK inhibitor, PD98059, prevented HaRas‐mediated increase in ROS and superoxide anions. In contrast, the specific phosphoinositide 3‐kinase (PI3K) inhibitors LY294002 and wortmannin were unable to reverse the effects of HaRas(Val12). Moreover, cholinergic stimulation of neuroblastoma cells by carbachol, which activated endogenous Ras/ERK1/2, induced a significant increase in ROS levels and elicited membrane translocation of p67phox and Rac. ROS generation induced by carbachol required the activation of ERK1/2 and PI3K. Hence, these data indicate that HaRas‐induced ERK1/2 signalling selectively activates NADPH oxidase system in neuroblastoma cells.

The Cu,Zn superoxide dismutase in neuroblastoma SK-N-BE cells is exported by a microvesicles dependent pathway

The antioxidant enzyme Cu,Zn superoxide dismutase has so far been considered costitutively expressed and exclusively localized into cytosol. In this paper we investigated Cu,Zn superoxide dismutase export in neuroblastoma SK-N-BE cells by flow cytometry analysis, confocal immunofluorescence analysis and enzyme-linked immunosorbed assay. Immunofluorescence analysis shows that the enzyme is exported by microvesicular granules; moreover the treatment of cells with brefeldin A and with 2-deoxy-D-glucose and sodium azide strongly decreases the amount of CuZn superoxide dismutase detected in the medium. Therefore the involvement of ATP-dependent mechanisms, likely including BFA-sensitive intracytoplasmic vesicles in Cu,Zn SOD export from SK-N-BE cells, has to be hypothesized. Microvesicular-mediated Cu,Zn SOD export in neurons could represent a relevant phenomenon able to influence cell excitability that is affected by reactive oxygen species.

Evidence of calcium‐ and SNARE‐dependent release of CuZn superoxide dismutase from rat pituitary GH3 cells and synaptosomes in response to depolarization

The antioxidant enzyme CuZn superoxide dismutase (SOD1) is secreted by many cell lines. However, it is not clear whether SOD1 secretion is only constitutive or can be regulated in an activity‐dependent fashion. Using rat pituitary GH3 cells that express voltage‐dependent calcium channels and are subjected to Ca2+ oscillations, we found that treatment with high K+‐induced SOD1 release that was significantly higher than the constitutive secretion. Evoked SOD1 release was correlated with depolarization‐dependent calcium influx and was virtually abolished by removal of extracellular calcium with EGTA or by pre‐incubation of GH3 cells with Botulinum toxin A that cleaves the SNARE protein SNAP‐25. Immunofluorescence experiments performed in GH3 cells and rat brain synaptosomes showed that K+‐depolarization induced a marked depletion of intracellular SOD1 immunoreactivity, an effect that was again abolished in the absence of extracellular calcium or after treatment with Botulinum toxin A. Subcellular fractionation analysis showed that SOD1 was present in large dense core vesicles. These data clearly show that, in addition to the constitutive SOD1 secretion, depolarization induces an additional rapid calcium‐dependent SOD1 release in GH3 cells and in rat brain synaptosomes. This likely occurs through exocytosis from SOD1‐containing vesicles operated by the SNARE complex.

Farnesyl transferase inhibitors induce neuroprotection by inhibiting Ha-Ras signalling pathway.

In previous studies we found that the GTPase p21 Harvey‐Ras (Ha‐Ras) stimulates the production of reactive oxygen species and induces apoptosis by oxidative stress; this effect was reversed by farnesyl transferase inhibitors (FTIs). In this study we investigated whether FTIs reduce rat brain damage induced by an excitotoxic stimulus, and the signalling pathway(s) underlying the neuroprotection by FTIs. In brain tissue, protein levels of Ha‐Ras and farnesylation inhibition were assayed by Western blot, and superoxide production was measured by hydroethidine. The excitotoxic lesion was induced by intrastriatal injection of N‐methyl‐d‐aspartate (NMDA). The survival of mouse neuronal cortical cells was assessed by 3‐(4,5 dimethylthialzol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT). In brain tissue, NMDA increased the protein levels of Ha‐Ras, FTIs caused the accumulation of non‐prenylated inactive Ras in the cytosolic fraction, and significantly reduced superoxide production and necrotic volume after excitotoxicity. FTIs increased the viability of mouse neuronal cortical cells following oxidative stress. In conclusion, FTIs inhibited Ha‐Ras, decreased oxidative stress and reduced necrotic volume by partly acting on neuronal cells. Thus, Ha‐Ras inhibition plays a role in the pathology of neuroprotection, suggesting a potential role of FTIs in the treatment of cerebrovascular diseases.

Free fatty acids modulate LDL receptor activity in BHK-21 cells

It has been shown that dietary fatty acids affect serum low density lipoprotein (LDL) levels, but the mechanism responsible for this effect is still under debate. Here we investigate the effect of different free fatty acids on LDL receptor activity in BHK-21 cells. These cells possess a classical LDL receptor strongly regulated by substances like 25-OH-cholesterol or lovastatin. Preincubation of cells for 24 h with both oleic (cis 18:1) and its trans counterpart, elaidic acid, enhanced 125I-LDL binding, internalization and degradation, being oleic acid more effective than elaidic acid. Among polyunsaturated fatty acids (PUFA) of the n-6 series arachidonic acid (20:4) enhanced LDL receptor activity more than linoleic acid (18:2), and among PUFA of the n-3 series docosahexaenoic (22:6) and eicosapentaenoic acids (20:5) were more effective compared to alpha-linolenic acid (18:3). Conversely, preincubation of cells with saturated fatty acids, palmitic (16:0) and stearic (18:0) acids, decreased binding, internalization and degradation of 125I-LDL. Scatchard analysis of binding data obtained with palmitic and oleic acids showed that these two fatty acids affect LDL receptor number without altering receptor affinity. The regulatory effect of free fatty acids on LDL receptor activity in BHK-21 cells is consistent with the hypothesis that the ability of fatty acids to modulate LDL-cholesterol levels in vivo is mediated, at least in part, by an action on receptor-dependent uptake of LDL.

Effect of Cu, Zn superoxide dismutase on cholesterol metabolism in human hepatocarcinoma (HepG2) cells

The microsomal enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase and the low density lipoprotein (LDL) receptor pathway carry out a key role on cholesterol homeostasis in eucaryotic cells. The HMG-CoA reductase is sensitive to oxidative inactivation and to phosphorylation by many kinases that are able to inactivate the protein and increase its susceptibility to proteolysis. We previously demonstrated that a calf thymus Cu,Zn SOD affects cholesterol metabolism. This protein binds with rat hepatocyte cell membrane by a specific surface membrane receptor. The involvement of Cu,Zn SOD in cholesterol metabolism is confirmed further by the presence of this antioxidant enzyme in circulating serum lipoproteins. We studied the effect of native human Cu,Zn SOD, metal-free SOD (apo SOD), and SOD-inactivated with hydrogen peroxide on cholesterol metabolism in human hepatocarcinoma HepG2 cells. Results showed that all forms of SODs used, at the concentration of 150 ng/ml, are able to affect cholesterol metabolism decreasing both HMG-CoA reductase activity and its protein levels; this inhibitory effect is accompanied by reduced cholesterol synthesis measured as [14C]acetate incorporation into [14C]cholesterol and by an increased [125I]LDL binding to HepG2 cells. Furthermore, the inhibitory effect of Cu,Zn SOD on cholesterol synthesis was completely abolished when the cells were incubated with Cu,Zn SOD in the presence of bisindoilmaleimide (BDM), an inhibitor of protein kinase C (PKC); moreover, we demonstrated that Cu,Zn SOD as well as apo SOD was able to increase PKC activity. Overall, data demonstrate that Cu,Zn SOD affects cholesterol metabolism independently from its dismutase activity and its metal content and that the inhibitory action on cholesterol synthesis is mediated by an activation of protein kinase C.

New Possible Role of Statins in Age‐Related Diseases

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Modulation of 3-Hydroxy-3-methylglutaryl- CoA Reductase Gene Expression by CuZn Superoxide Dismutase in Human Fibroblasts and HepG2 Cells

The homeostasis of intracellular cholesterol in animal cells is highly regulated by a complex system in which the microsomal rate-limiting enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase plays a key role in cholesterol synthesis. Substantial evidence has demonstrated that the cytosolic antioxidant enzyme CuZn superoxide dismutase (SOD1) inhibits the HMG-CoA reductase activity in rat hepatocytes and in human fibroblasts by decreasing cholesterol synthesis. Although these data suggest that SOD1 exerts a physiological role in cholesterol metabolism, it is still unclear whether the decrease of HMG-CoA reductase activity is mediated by transcriptional or by posttranscriptional events. The results of the present study, obtained by one-step RT-PCR assay, demonstrated that both SOD1 and the metal-free form of enzyme (Apo SOD1) inhibit HMG-CoA reductase gene expression in hepatocarcinoma HepG2 cells, in normal human fibroblasts, and in fibroblasts of subjects affected by familiar hypercholesterolemia. Accordingly, SOD1 could be used as a potential agent in the treatment of hypercholesterolemia, even in subjects lacking a functional LDL receptor pathway.