Oxidative stress leads to inhibition of calcium transport by sarcoplasmic reticulum in skeletal muscle

Iron administration results in the development of oxidative stress in skeletal muscles, as evidenced by increases in amounts of lipid oxidation fluorescent end products, decreases in vitamin E concentration, and inhibition of calcium transport by sarcoplasmic reticulum. Exhaustive physical loading or hyperoxia, or their combination, does not lead to apparent modification in calcium transport by sarcoplasmic reticulum in skeletal muscle homogenates. However, physical loading or hyperoxia does in fact induce oxidative stress since they magnify the effect of iron loading on the inhibition of calcium transport.     

Nitrosative stress inhibits the aminophospholipid translocase resulting in phosphatidylserine externalization and macrophage engulfment: implications for the resolution of inflammation

Macrophage recognition of apoptotic cells depends on externalization of phosphatidylserine (PS), which is normally maintained within the cytosolic leaflet of the plasma membrane by aminophospholipid translocase (APLT). APLT is sensitive to redox modifications of its -SH groups. Because activated macrophages produce reactive oxygen and nitrogen species, we hypothesized that macrophages can directly participate in apoptotic cell clearance by S-nitrosylation/oxidation and inhibition of APLT causing PS externalization. Here we report that exposure of target HL-60 cells to nitrosative stress inhibited APLT, induced PS externalization, and enhanced recognition and elimination of "nitrosatively" modified cells by RAW 264.7 macrophages. Using S-nitroso-L-cysteine-ethyl ester (SNCEE) and S-nitrosoglutathione (GSNO) that cause intracellular and extracellular trans-nitrosylation of proteins, respectively, we found that SNCEE (but not GSNO) caused significant S-nitrosylation/oxidation of thiols in HL-60 cells. SNCEE also strongly inhibited APLT, activated scramblase, and caused PS externalization. However, SNCEE did not induce caspase activation or nuclear condensation/fragmentation suggesting that PS externalization was dissociated from the common apoptotic pathway. Dithiothreitol reversed SNCEE-induced S-nitrosylation, APLT inhibition, and PS externalization. SNCEE but not GSNO stimulated phagocytosis of HL-60 cells. Moreover, phagocytosis of target cells by lipopolysaccharide-stimulated macrophages was significantly suppressed by an NO. scavenger, DAF-2. Thus, macrophage-induced nitrosylation/oxidation plays an important role in cell clearance, and hence in the resolution of inflammation.     

Mitochondria targeting of non-peroxidizable triphenylphosphonium conjugated oleic acid protects mouse embryonic cells against apoptosis: role of cardiolipin remodeling

Peroxidation of cardiolipin in mitochondria is essential for the execution of apoptosis. We suggested that integration of oleic acid into cardiolipin generates non-oxidizable cardiolipin species hence protects cells against apoptosis. We synthesized mitochondria-targeted triphenylphosphonium oleic acid ester. Using lipidomics analysis we found that pretreatment of mouse embryonic cells with triphenylphosphonium oleic acid ester resulted in decreased contents of polyunsaturated cardiolipins and elevation of its species containing oleic acid residues. This caused suppression of apoptosis induced by actinomycin D. Triacsin C, an inhibitor of acyl-CoA synthase, blocked integration of oleic acid into cardiolipin and restored cell sensitivity to apoptosis.     

Genetic re-engineering of polyunsaturated phospholipid profile of Saccharomyces cerevisiae identifies a novel role for Cld1 in mitigating the effects of cardiolipin peroxidation

Cardiolipin (CL) is a unique phospholipid localized almost exclusively within the mitochondrial membranes where it is synthesized. Newly synthesized CL undergoes acyl remodeling to produce CL species enriched with unsaturated acyl groups. Cld1 is the only identified CL-specific phospholipase in yeast and is required to initiate the CL remodeling pathway. In higher eukaryotes, peroxidation of CL, yielding CL_OX, has been implicated in the cellular signaling events that initiate apoptosis. CL_OX can undergo enzymatic hydrolysis, resulting in the release of lipid mediators with signaling properties. Our previous findings suggested that CLD1 expression is upregulated in response to oxidative stress, and that one of the physiological roles of CL remodeling is to remove peroxidized CL. To exploit the powerful yeast model to study functions of CLD1 in CL peroxidation, we expressed the H. brasiliensis Δ¹²-desaturase gene in yeast, which then synthesized poly unsaturated fatty acids(PUFAs) that are incorporated into CL species. Using LC-MS based redox phospholipidomics, we identified and quantified the molecular species of CL and other phospholipids in cld1Δ vs. WT cells. Loss of CLD1 led to a dramatic decrease in chronological lifespan, mitochondrial membrane potential, and respiratory capacity; it also resulted in increased levels of mono-hydroperoxy-CLs, particularly among the highly unsaturated CL species, including tetralinoleoyl-CL. In addition, purified Cld1 exhibited a higher affinity for CL_OX, and treatment of cells with H₂O₂ increased CLD1 expression in the logarithmic growth phase. These data suggest that CLD1 expression is required to mitigate oxidative stress. The findings from this study contribute to our overall understanding of CL remodeling and its role in mitigating oxidative stress.     

Cytochrome c/cardiolipin relations in mitochondria: a kiss of death

Recently, phospholipid peroxidation products gained a reputation as key regulatory molecules and participants in oxidative signaling pathways. During apoptosis, a mitochondria-specific phospholipid, cardiolipin (CL), interacts with cytochrome c (cyt c) to form a peroxidase complex that catalyzes CL oxidation; this process plays a pivotal role in the mitochondrial stage of the execution of the cell death program. This review is focused on redox mechanisms and essential structural features of cyt c’s conversion into a CL-specific peroxidase that represent an interesting and maybe still unique example of a functionally significant ligand change in hemoproteins. Furthermore, specific characteristics of CL in mitochondria—its asymmetric transmembrane distribution and mechanisms of collapse, the regulation of its synthesis, remodeling, and fatty acid composition—are given significant consideration. Finally, new concepts in drug discovery based on the design of mitochondria-targeted inhibitors of cyt c/CL peroxidase and CL peroxidation with antiapoptotic effects are presented.     

Nitric oxide regulates the 26S proteasome in vascular smooth muscle cells

It is well established that nitric oxide (NO) inhibits vascular smooth muscle cell (VSMC) proliferation by modulating cell cycle proteins. The 26S proteasome is integral to protein degradation and tightly regulates cell cycle proteins. Therefore, we hypothesized that NO directly inhibits the activity of the 26S proteasome. The three enzymatic activities (chymotrypsin-like, trypsin-like and caspase-like) of the 26S proteasome were examined in VSMC. At baseline, caspase-like activity was approximately 3.5-fold greater than chymotrypsin- and trypsin-like activities. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly inhibited all three catalytically active sites in a time- and concentration-dependent manner (P < 0.05). Caspase-like activity was inhibited to a greater degree (77.2% P < 0.05). cGMP and cAMP analogs and inhibitors had no statistically significant effect on basal or NO-mediated inhibition of proteasome activity. Dithiothreitol, a reducing agent, prevented and reversed the NO-mediated inhibition of the 26S proteasome. Nitroso-cysteine analysis following S-nitrosoglutathione exposure revealed that the 20S catalytic core of the 26S proteasome contains 10 cysteines which were S-nitrosylated by NO. Evaluation of 26S proteasome subunit protein expression revealed differential regulation of the α and β subunits in VSMC following exposure to NO. Finally, immunohistochemical analysis of subunit expression revealed distinct intracellular localization of the 26S proteasomal subunits at baseline and confirmed upregulation of distinct subunits following NO exposure. In conclusion, NO reversibly inhibits the catalytic activity of the 26S proteasome through S-nitrosylation and differentially regulates proteasomal subunit expression. This may be one mechanism by which NO exerts its effects on the cell cycle and inhibits cellular proliferation in the vasculature.     

Iron binding to alpha-tocopherol-containing phospholipid liposomes

Tocopherols (vitamin E) located in the hydrophobic domains of biological membranes act as chain breaking antioxidants preventing the propagation of free radical reactions of lipid peroxidation. The naturally occurring form, d-alpha tocopherol is an exquisite molecule in that it is intercalated in the membrane in such a way that the hydrophobic tail anchors the molecule positioning the chromanol ring containing the hydroxyl group, which is the essence of its antioxidant function, at the polar hydrocarbon interface of phospholipid membranes. The interaction of this group with water soluble substances is not very well understood. In the present study, an investigation was made of the interaction of ascorbate and ferrous ions (Fe+2) initiators of lipid peroxidation with alpha tocopherol. The results show that tocopherol increases membrane associated iron. The formation of a tocopherol iron complex in the presence of phospholipid liposomes and ascorbate in its reduced form is indicated. These results suggest a new way in which tocopherols act to inhibit lipid peroxidation.     

Nitric oxide and dihydrolipoic acid modulate the activity of caspase 3 in HepG2 cells

Herein, we report that dihydrolipoic acid and lipoic acid (LA) plus lipoamide dehydrogenase and NADH denitrosate S-nitrosocaspase 3 (CASP-SNO). In HepG2 cells, S-nitroso-l-cysteine ethyl ester (SNCEE) impeded the activity of caspase 3 (CASP-SH), while a subsequent incubation of the cells in SNCEE-free medium resulted in endogenous denitrosation and reactivation of CASP-SH. The latter process was inhibited in thioredoxin reductase-deficient HepG2 cells, in which, however, LA markedly reactivated CASP-SH. The data obtained are discussed with focus on low molecular mass dithiols that mimic the activity of thioredoxin in reactions of protein S-denitrosation.     

Unusual peroxidase activity of polynitroxylated pegylated hemoglobin: Elimination of H2O2 coupled with intramolecular oxidation of nitroxides

Polynitroxylated hemoglobin (Hb(AcTPO)₁₂) has been developed as a hemoglobin-based oxygen carrier. While Hb(AcTPO)₁₂ has been shown to exert beneficial effects in a number of models of oxidative injury, its peroxidase activity has not been characterized thus far. In the blood stream, Hb(AcTPO)₁₂ undergoes reduction by ascorbate to its hydroxylamine form Hb(AcTPOH)₁₂. Here we report that Hb(AcTPOH)₁₂ exhibits peroxidase activity where H₂O₂ is utilized for intramolecular oxidation of its TPOH residues to TPO. This represents an unusual redox-catalytic mechanism whereby reduction of H₂O₂ is achieved at the expense of reducing equivalents of ascorbate converted into those of Hb(AcTPOH)₁₂, a new propensity that cannot be directly associated with ascorbate.     

Formation of nitroxyl and hydroxyl radical in solutions of sodium trioxodinitrate: effects of pH and cytotoxicity

Despite its negative redox potential, nitroxyl (HNO) can trigger reactions of oxidation. Mechanistically, these reactions were suggested to occur with the intermediate formation of either hydroxyl radical (.OH) or peroxynitrite (ONOO-). In this work, we present further experimental evidence that HNO can generate.OH. Sodium trioxodinitrate (Na2N2O3), a commonly used donor of HNO, oxidized phenol and Me2SO to benzene diols and.CH3, respectively. The oxidation of Me2SO was O2-independent, suggesting that this process reflected neither the intermediate formation of ONOO- nor a redox cycling of transition metal ions that could initiate Fenton-like reactions. In solutions of phenol, Na2N2O3 yielded benzene-1,2-diol and benzene-1,4-diol at a ratio of 2:1, which is consistent with the generation of free.OH. Ethanol and Me2SO, which are efficient scavengers of.OH, impeded the hydroxylation of phenol. A mechanism for the hydrolysis of Na2N2O3 is proposed that includes dimerization of HNO to cis-hyponitrous acid (HO-N=N-OH) with a concomitant azo-type homolytic fission of the latter to N2 and.OH. The HNO-dependent production of.OH was with 1 order of magnitude higher at pH 6.0 than at pH 7.4. Hence, we hypothesized that HNO can exert selective toxicity to cells subjected to acidosis. In support of this thesis, Na2N2O3 was markedly more toxic to human fibroblasts and SK-N-SH neuroblastoma cells at pH 6.2 than at pH 7.4. Scavengers of.OH impeded the cytotoxicity of Na2N2O3. These results suggest that the formation of HNO may be viewed as a toxicological event in tissues subjected to acidosis.