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.     

Appetizing rancidity of apoptotic cells for macrophages: oxidation,externalization, and recognition of phosphatidylserine

Programmed cell death (apoptosis) functions as a mechanism to eliminate unwanted or irreparably damaged cells ultimately leading to their orderly phagocytosis in the absence of calamitous inflammatory responses. Recent studies have demonstrated that the generation of free radical intermediates and subsequent oxidative stress are implicated as part of the apoptotic execution process. Oxidative stress may simply be an unavoidable yet trivial byproduct of the apoptotic machinery; alternatively, intermediates or products of oxidative stress may act as essential signals for the execution of the apoptotic program. This review is focused on the specific role of oxidative stress in apoptotic signaling, which is realized via phosphatidylserine-dependent pathways leading to recognition of apoptotic cells and their effective clearance. In particular, the mechanisms involved in selective phosphatidylserine oxidation in the plasma membrane during apoptosis and its association with disturbances of phospholipid asymmetry leading to phosphatidylserine externalization and recognition by macrophage receptors are at the center of our discussion. The putative importance of this oxidative phosphatidylserine signaling in lung physiology and disease are also discussed.     

Quantitative analysis of phospholipid peroxidation and antioxidant protection in live human epidermal keratinocytes

To characterize oxidative stress in phospholipids of normal human epidermal keratinocytes we metabolically labeled their membrane phospholipids with a natural oxidation-sensitive fluorescent fatty acid, cis-parinaric acid, and exposed the cells to two different sources of oxidants—a lipid-soluble azo-initiator of peroxyl radicals, 2,2'-azobis(2,4-dimethyl-valeronitrile), AMVN, and a superoxide generator, xanthine oxidase/xanthine. We demonstrated that both oxidants induced pronounced oxidation of four major classes of cis-parinaric acid-labeled phospholipids—phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol—in normal human epidermal keratinocytes that was not detectable as any significant change of their phospholipid composition. Vitamin E was effective in protecting the cells against phospholipid peroxidation. Since viability of normal human epidermal keratinocytes was not changed either by labeling or exposure to oxidants the labeling protocol and oxidative stress employed are compatible with the quantitative analysis of phospholipid peroxidation in viable cells.     

Inhibition of cyclic nucleotide phosphodiesterase from the rod outer segments of the frog retina by 3-hydroxypyridine

The influence of 8 analogues of 3-hydroxypyridine upon the phosphodiesterase of rod outer segments of frog retinae has been investigated. It has been shown that the analogues of 3-hydroxypyridine inhibit the enzeme reversely and noncompetitively in case of hydrolysis towards the cAMP and cGMP. The natural analogues of 3-hydroxypyridine (pyridoxol, pyridoxale, pyridoxale-phosphate) do not exert the inhibiting effect. It is suggested that the inhibition of phosphodiesterase from rod outer segments of retinae is caused by the interaction of 3-hydroxypyridines with the hydrophobic microenvironment of the active site of the enzyme.     

Changes in the cyclic nucleotide level and the inhibition of human thrombocyte aggregation in 3-hydroxypyridine exposure

The effect of six 3-oxypiridine derivatives (at a concentration of 10(-3) and 5 X 10(-3) M) on cyclic nucleotide level in human platelets and platelet aggregation was studied. Five 3-oxypiridine derivatives were shown to depress platelet aggregation, four of them causing the increase in cAMP platelet level. The correlation between antiaggregation activity of 3-oxypiridine derivatives and their ability to rise cyclic nucleotide level in human platelets is discussed.     

Accumulation of lipid peroxidation products in cataractous lenses

A study was made of the content of lipid peroxidation products (LPP) in the lenses extracted during operations for cataract as well as in transparent human lenses. In a mature cataract, the elevated content of primary, secondary and end products of lipid peroxidation (diene and triene conjugates, Schiff bases) was revealed. The content of LPP was identical in different clinical patterns of a mature cataract (senile, traumatic, complicated), which points to the universal role of lipid peroxidation in lenticular opacity.     

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.     

Impaired clearance and enhanced pulmonary inflammatory/fibrotic response to carbon nanotubes in myeloperoxidase-deficient mice

Advancement of biomedical applications of carbonaceous nanomaterials is hampered by their biopersistence and pro-inflammatory action in vivo. Here, we used myeloperoxidase knockout B6.129X1-MPO (MPO k/o) mice and showed that oxidation and clearance of single walled carbon nanotubes (SWCNT) from the lungs of these animals after pharyngeal aspiration was markedly less effective whereas the inflammatory response was more robust than in wild-type C57Bl/6 mice. Our results provide direct evidence for the participation of MPO - one of the key-orchestrators of inflammatory response - in the in vivo pulmonary oxidative biodegradation of SWCNT and suggest new ways to control the biopersistence of nanomaterials through genetic or pharmacological manipulations.     

The action of émoksipin on the basal activity of cyclic nucleotide phosphodiesterase and on the late receptor potential of the isolated retina

The influence of emoxypin (derivate of 3-hydroxypyridine) upon the late receptor potential (LRP) and activity of the cyclic 3',5'-nucleotide phosphodiesterase (PDE) have been investigated. The inhibition of PDE and increase of the amplitude of LPR have been shown. The curve (RP as a function of the stimulus light intensity) was moved towards the lesser lighting and the time of the achievement of the maximum was increased. Thus, emoxypin produces an effect on the LRP like classical inhibitors of PDE. It is suggested that increase of the functional activity of the retinae upon the influence of emoxypin in caused by the influence of the one towards the system of the cyclic nucleotides.