Accumulation Of Phosphatidylserine Research Articles

Melatonin treatment delays ripening in mangoes associated with maintaining the membrane integrity of fruit exocarp during postharvest

The effects of exogenous melatonin on postharvest ripening of mango (Mangifera indica L. cv. Keitt) were investigated after the fruit were dipped in 0 (as the control), 100, or 200 μM melatonin solution for 30 min, and then stored at room temperature (25 ± 1 °C). The results showed that melatonin treatments could delay the ripening process as indicated by inhibition to softening, respiration, color change and chlorophyll degradation in fruit during storage. Notably, 200 μM melatonin treatment delayed the degradation of phosphatidylglycerol (PG) and phosphatidylinositol (PI), and the accumulation of phosphatidylserine (PS) and phosphatidic acid (PA) in membrane phospholipids, inhibited the decrease in unsaturated fatty acids (IUFA) index and also decreased the contents of H2O2 and malondialdehyde (MDA) in the exocarp of the fruit, which might collectively contribute to the integrity of the membrane associated with the delay in the ripening process of mango fruit during postharvest.

The Calotropis Gigantea Methanolic Extract Induces Apoptosis in Human Breast Carcinoma Cells.

Background: Calotropis gigantea (family: Asclepiadaceae) has been known to contain cardiac glycosides. The C. gigantea extracts have been reported as cytotoxic to a few cancer cell lines. The present study was designed to examine the effect of Calotropis gigantea methanolic extract (CGME) on the growth and apoptosis in human breast carcinoma cell line (MCF-7 cells). Methods:The study was conducted in Aurangabad (India) from 16 February to 10 June 2015. CGME treated MCF-7 cells were analyzed for growth inhibition and apoptosis. The exhibition of phosphatidylserine was analyzed with the Annexin-V Fluorescein isothiocyanate flow cytometry (FITC) method. Accumulated poly-caspases were determined with carboxyfluorescein poly-caspase assay, Apo-BrdU™ tunnel assay for DNA fragmentation and pro/anti-apoptotic gene expression with real-time polymerase chain reaction. The high-performance liquid chromatography analysis indicated the presence of two unknown cardenolides along with known cardenolides such as calactin, calatropagenin, usharin, afroside, calatoxin, and gamphoside. The Kruskal-Wallis and Wilcoxon tests (GraphPad Prism version 7.0) were used for statistical analyses. Results:Upon treatment with 40 µg/ml CGME, about 56.9% of the cell population underwent apoptosis. Compared to paclitaxel, the accumulation of active caspases in CGME treated with MCF-7 cells was found to be dose-dependent, whereas the G2/M cell cycle arrest was time-dependent. The Apo-BrdU™ tunnel assay confirmed that CGME treatment caused DNA fragmentation and RT-PCR analyses indicated elevated transcription for pro-apoptotic gene expression. Kruskal-Wallis test results were significant; Bcl-2 (P=0.00193), Bak-1 (P=0.00021), and Bax (P=0.0019). Conclusion:CGME treatment caused the accumulation of phosphatidylserine on the cell membrane, recruitment of poly-caspases, DNA fragmentation, and enhanced transcription of pro-apoptotic gene expression.

Phosphatidylserine exposure mediated by ABC transporter activates the integrin signaling pathway promoting axon regeneration

Following axon injury, a cascade of signaling events is triggered to initiate axon regeneration. However, the mechanisms regulating axon regeneration are not well understood at present. In Caenorhabditis elegans, axon regeneration utilizes many of the components involved in phagocytosis, including integrin and Rac GTPase. Here, we identify the transthyretin (TTR)-like protein TTR-11 as a component functioning in axon regeneration upstream of integrin. We show that TTR-11 binds to both the extracellular domain of integrin-α and phosphatidylserine (PS). Axon injury induces the accumulation of PS around the injured axons in a manner dependent on TTR-11, the ABC transporter CED-7, and the caspase CED-3. Furthermore, we demonstrate that CED-3 activates CED-7 during axon regeneration. Thus, TTR-11 functions to link the PS injury signal to activation of the integrin pathway, which then initiates axon regeneration.

Striatal astrocytes engulf dopaminergic debris in Parkinson's disease: A study in an animal model.

The role of astrocytes in Parkinson’s disease is still not well understood. This work studied the astrocytic response to the dopaminergic denervation. Rats were injected in the lateral ventricles with 6-hydroxydopamine (25μg), inducing a dopaminergic denervation of the striatum not accompanied by non-selective tissue damage. The dopaminergic debris were found within spheroids (free-spheroids) which retained some proteins of dopaminergic neurons (e.g., tyrosine hydroxylase, the dopamine transporter protein, and APP) but not others (e.g., α-synuclein). Free-spheroids showed the initial (LC3-autophagosomes) but not the late (Lamp1/Lamp2-lysosomes) components of autophagy (incomplete autophagy), preparing their autophagosomes for an external phagocytosis (accumulation of phosphatidylserine). Free-spheroids were penetrated by astrocyte processes (fenestrated-spheroids) which made them immunoreactive for GFAP and S100β, and which had some elements needed to continue the debris degradation (Lamp1/Lamp2). Finally, proteins normally found in neurons (TH, DAT and α-synuclein) were observed within astrocytes 2–5 days after the dopaminergic degeneration, suggesting that the intracellular contents of degenerated cells had been transferred to astrocytes. Taken together, present data suggest phagocytosis as a physiological role of striatal astrocytes, a role which could be critical for cleaning striatal debris during the initial stages of Parkinson’s disease.

Dysferlin-mediated phosphatidylserine sorting engages macrophages in sarcolemma repair.

Failure to repair the sarcolemma leads to muscle cell death, depletion of stem cells and myopathy. Hence, membrane lesions are instantly sealed by a repair patch consisting of lipids and proteins. It has remained elusive how this patch is removed to restore cell membrane integrity. Here we examine sarcolemmal repair in live zebrafish embryos by real-time imaging. Macrophages remove the patch. Phosphatidylserine (PS), an ‘eat-me' signal for macrophages, is rapidly sorted from adjacent sarcolemma to the repair patch in a Dysferlin (Dysf) dependent process in zebrafish and human cells. A previously unrecognized arginine-rich motif in Dysf is crucial for PS accumulation. It carries mutations in patients presenting with limb-girdle muscular dystrophy 2B. This underscores the relevance of this sequence and uncovers a novel pathophysiological mechanism underlying this class of myopathies. Our data show that membrane repair is a multi-tiered process involving immediate, cell-intrinsic mechanisms as well as myofiber/macrophage interactions.

Emerging roles for vascular smooth muscle cell exosomes in calcification and coagulation.

Vascular smooth muscle cell (VSMC) phenotypic conversion from a contractile to 'synthetic' state contributes to vascular pathologies including restenosis, atherosclerosis and vascular calcification. We have recently found that the secretion of exosomes is a feature of 'synthetic' VSMCs and that exosomes are novel players in vascular repair processes as well as pathological vascular thrombosis and calcification. Pro-inflammatory cytokines and growth factors as well as mineral imbalance stimulate exosome secretion by VSMCs, most likely by the activation of sphingomyelin phosphodiesterase 3 (SMPD3) and cytoskeletal remodelling. Calcium stress induces dramatic changes in VSMC exosome composition and accumulation of phosphatidylserine (PS), annexin A6 and matrix metalloproteinase-2, which converts exosomes into a nidus for calcification. In addition, by presenting PS, VSMC exosomes can also provide the catalytic surface for the activation of coagulation factors. Recent data showing that VSMC exosomes are loaded with proteins and miRNA regulating cell adhesion and migration highlight VSMC exosomes as potentially important communication messengers in vascular repair. Thus, the identification of signalling pathways regulating VSMC exosome secretion, including activation of SMPD3 and cytoskeletal rearrangements, opens up novel avenues for a deeper understanding of vascular remodelling processes.

ATP11C Facilitates Phospholipid Translocation across the Plasma Membrane of All Leukocytes.

Organization of the plasma membrane into specialized substructures in different blood lineages facilitates important biological functions including proper localization of receptors at the plasma membrane as well as the initiation of crucial intracellular signaling cascades. The eukaryotic plasma membrane is a lipid bilayer that consists of asymmetrically distributed phospholipids. This asymmetry is actively maintained by membrane-embedded lipid transporters, but there is only limited data available about the molecular identity of the predominantly active transporters and their substrate specificity in different leukocyte subsets. We demonstrate here that the P4-type ATPase ATP11C mediates significant flippase activity in all murine leukocyte subsets. Loss of ATP11C resulted in a defective internalization of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in comparison to control cells. The diminished flippase activity caused increased PS exposure on 7-aminoactinomycin D− (7-AAD−) viable pro-B cells freshly isolated from the bone marrow of ATP11C-deficient mice, which was corrected upon a 2-hour resting period in vitro. Despite the impaired flippase activity in all immune cell subsets, the only other blood cell type with an accumulation of PS on the surface were viable 7-AAD− developing T cells but this did not result in any discernable effect on their development in the thymus. These findings show that all leukocyte lineages exhibit flippase activity, and identify ATP11C as an aminophospholipid translocase in immune cells.

P6.07 - Real Time Photodynamic Therapy Analysis of ICG Loaded Modified Magnetic Nanoparticles on MCF-7 Cells

ABSTRACT Objective: We aimed to improve an ICG (Indocyanine Green) based nanoparticle, a light source and an application method that is centred to photodynamic therapy of MCF-7 cells. Methods: MCF-7 cells were obtained from ATCC, expanded in DMEM, 10% FBS and subjected to tissue culture E-Plate that was used to generate dynamic real-time data by measuring electrical impedance across interdigitated micro-electrodes on the bottom of the plate. ICG (has a high absorption at 770-810 nm) embedded and functional folic acid groups covered magnetic mesoporous nanoparticles that can be directed by magnetic field was prepared at various (50, 25, 10, 5, 2, 5 µg/ml) concantrations and applied to the E-plate. Light source (LEDs) was designed as a system that fit to 96 well-plate was irradiated 785 nm for 20 minute. To confirm the xCelligence system's data, proliferation assay was performed according to the manufacture's instructions (WST-1 reagent, ROCHE). Afterwards absorbance was measured at 450 nm using an ELISA reader. Phosphatidylserine accumulation was determined with the Apoptosis Detection Kit, Annexin-V-Alexa 568 (ROCHE). Results: According to xCelligence system analysis, the effect of optimal dose of irradiated MNP-MS-ICG-PAMAM-Folic Acid molecule was more potent at reducing proliferation of MCF-7 cells than MNP-MS-ICG-PAMAM. Also these results were confirmed by WST-1 assay was used in dark and light conditions for MCF-7 cells. The irradiation light itself did not cause any cytotoxic effect in the absence of photosensitizers. Our results showed that in-vitro application of MNP-MS-ICG-PAMAM-Folic Acid-PDT cause apoptosis in the MCF-7 cell line. Apoptotic cells were observed after 24 hours treatment under the irradiation. Conclusion: This study revealed that MNP-MS-ICG-PAMAM- Folic Acid –PDT is more cytotoxic and thereby lethal than the nanoparticle without Folic Acid integration. This indicates high efficiency for future in vivo studies aiming anti-cancer treatment strategies instead of using the latter.

Discovery of a bifunctional cardiolipin/phosphatidylethanolamine synthase in bacteria

Phosphatidylethanolamine (PE) and cardiolipin (CL) are major components of bacterial and eukaryotic membranes. In bacteria, synthesis of PE usually occurs via decarboxylation of phosphatidylserine (PS) by PS decarboxylases (Psd). CL is produced by various CL synthases (Cls). Membranes of the plant pathogen Xanthomonas campestris predominantly contain PE, phosphatidylglycerol (PG) and CL. The X. campestris genome encodes one Psd and six putative CLs. Deletion of psd resulted in loss of PE and accumulation of PS. The mutant was severely affected in growth and cell size. PE synthesis, growth and cell division were partially restored when cells were supplied with ethanolamine (EA) suggesting a previously unknown PE synthase activity. Via mutagenesis, we identified a Cls enzyme (Xc_0186) responsible for EA-dependent PE biosynthesis. Xanthomonas lacking xc_0186 not only lost its ability to utilize EA for PE synthesis but also produced less CL suggesting a bifunctional enzyme. Recombinant Xc_0186 in E. coli and in cell-free extracts uses cytidine diphosphate diacylglycerol (CDP-DAG) and PG for CL synthesis. It is also able to use CDP-DAG and EA for PE synthesis. Owing to its dual function in CL and PE production, we consider Xc_0186 the founding member of a new class of enzymes called CL/PE synthase (CL/PEs).

Modulation of enzymatic PS synthesis by liposome membrane composition

Phosphatidylserine (PS) is a phospholipid known to exert important physiological roles in humans. However, this phospholipid (PL) is poorly available as a natural source and hardly produced by the chemical route. In this work, PS was obtained by transphosphatidylation using phospholipase D (PLD) and PL self-assembled into liposomes as the substrates. The aim was to better understand how the liposome membrane composition could modulate PS yield. Three lecithins were used as PL substrates, one originated from a marine source providing a high amount of n-3 polyunsaturated fatty acids, and two issued from soya differing in their phosphatidylcholine (PC) content. Different parameters such as Ca2+ content, enzyme and l-serine concentrations modulated PS synthesis. The presence of Ca2+ increased PS conversion yield. The alcohol acceptor (l-serine) concentration positively acted on PL conversion, by governing the equilibrium between transphosphatidylation and hydrolysis. Beside these specific reaction conditions, it was demonstrated that the membrane composition of the liposomes modulated PS synthesis. A direct correlation between PS accumulation and the amount of cholesterol or α-tocopherol incorporated into the soya lecithins was observed. This result was interpreted in terms of “head” spacers promoting PLD transphosphatidylation. On the whole, this work provided key parameters for the formulation of liposomes using enzymatic PLD technology, to produce lecithins enriched in different proportions of PS and esterified with various types of fatty acids depending on the initial lecithin source.

Phosphatidylserine synthase 2: high efficiency for synthesizing phosphatidylserine containing docosahexaenoic acid

Phosphatidylserine (PS), the major anionic phospholipid in eukaryotic cell membranes, is synthesized by the integral membrane enzymes PS synthase 1 (PSS1) and 2 (PSS2). PSS2 is highly expressed in specific tissues, such as brain and testis, where docosahexaenoic acid (DHA, 22:6n-3) is also highly enriched. The purpose of this work was to characterize the hydrocarbon-chain preference of PSS2 to gain insight on the specialized role of PSS2 in PS accumulation in the DHA-abundant tissues. Flag-tagged PSS2 was expressed in HEK cells and immunopurified in a functionally active form. Purified PSS2 utilized both PE plasmalogen and diacyl PE as substrates. Nevertheless, the latter was six times better utilized, indicating the importance of an ester linkage at the sn-1 position. Although no sn-1 fatty acyl preference was noted, PSS2 exhibited significant preference toward DHA compared with 18:1 or 20:4 at the sn-2 position. Preferential production of DHA-containing PS (DHA-PS) was consistently observed with PSS2 purified from a variety of cell lines as well as with microsomes from mutant cells in which PS synthesis relies primarily on PSS2. These findings suggest that PSS2 may play a key role in PS accumulation in brain and testis through high activity toward DHA-containing substrates that are abundant in these tissues.

C. elegans Secreted Lipid-Binding Protein NRF-5 Mediates PS Appearance on Phagocytes for Cell Corpse Engulfment

During programmed cell death, apoptotic cells are rapidly removed by phagocytes. How dying cells are recognized remains poorly understood. Here we identify a secreted lipid transfer/LPS-binding family protein, NRF-5, which is required for efficient clearance of cell corpses. We observed that phosphatidylserine (PS), which is externalized to the outer leaflet of plasma membranes in apoptotic cells, is also detected on the surface of engulfing cells. Loss of NRF-5 function completely blocks PS appearance on engulfing cells but causes accumulation of PS on apoptotic cells, a phenotype observed in both ced-7(lf) and ttr-52(lf) mutants. The NRF-5 protein is expressed in and secreted from body wall muscle cells and clusters around apoptotic cells in a CED-7-dependent manner. NRF-5 associates with TTR-52, binds PS, and displays lipid transfer activity in vitro. Our data suggest that NRF-5 may act with CED-7 and TTR-52 to mediate PS transfer from apoptotic cells to engulfing cells and thus promotes engulfment by phagocytes.

Role of PS Product Inhibition in Accumulation of Phosphatidylserine (PS) in Neuronal Membranes

PS is a major membrane component in mammalian tissues. Neuronal tissues are highly enriched with docosahexaenoic acid (DHA, 22:6n‐3) and PS in comparison to non‐neuronal tissues. Such concentration of DHA and PS is known to be important for proper neuronal functions. In mammalian cells, PS synthase (PSS) 1 and 2 catalyze PS synthetic activity, which is inhibited by the product PS through the feedback mechanism. We have previously found that DHA containing PS species (DHA‐PS) highly accumulates in neuronal membranes mainly due to the substrate preference in PS synthesis towards DHA phospholipid species. To understand the biochemical mechanisms underlying enrichment of PS in neuronal membranes, we hypothesized that fatty acyl composition‐dependent product inhibition in PS synthesis also plays a role in PS accumulation. We found that DHA‐PS has weaker inhibitory effect compared to oleic acid (OA, 18:1n‐9) containing PS species (OA‐PS) when tested with mouse liver microsomes. Consistently, DHA‐PS inhibited PSS activity less effectively than OA‐PS when flag‐PSS2 were expressed in mammalian cells, successfully immuno‐purified, and subsequently used as an enzyme source. DHA‐PS as a weaker inhibitor of PSS together with the high specificity towards DHA‐PS synthesis may contribute to the high PS accumulation in neuronal membranes.Intramural Program of National Institutes of Health

Inducible expression of EVI1 in human myeloid cells causes phenotypes consistent with its role in myelodysplastic syndromes

The oncogene EVI1 has been implicated in the etiology of AML and MDS. Although AML cells are characterized by accelerated proliferation and differentiation arrest, MDS cells hyperproliferate when immature but fail to differentiate later and die instead. In agreement with its roles in AML and in immature MDS cells, EVI1 was found to stimulate cell proliferation and inhibit differentiation in several experimental systems. In contrast, the variant protein MDS1/EVI1 caused the opposite effect in some of these assays. In the present study, we expressed EVI1 and MDS1/EVI1 in a tetracycline-regulable manner in the human myeloid cell line U937. Induction of either of these proteins caused cells to accumulate in the G0/G1-phase of the cell cycle and moderately increased the rate of spontaneous apoptosis. However, when EVI1- or MDS1/EVI1-expressing cells were induced to differentiate, they massively succumbed to apoptosis, as reflected by the accumulation of phosphatidylserine in the outer leaflet of the plasma membrane and increased rates of DNA fragmentation. In summary, these data show that inducible expression of EVI1 in U937 cells causes phenotypes that may be relevant for its role in MDS and provides a basis for further investigation of its contribution to this fatal disease.

Sinorhizobium melilotiMutants Deficient in Phosphatidylserine Decarboxylase Accumulate Phosphatidylserine and Are Strongly Affected during Symbiosis with Alfalfa

Sinorhizobium meliloti contains phosphatidylglycerol, cardiolipin, phosphatidylcholine, and phosphatidylethanolamine (PE) as major membrane lipids. PE is formed in two steps. In the first step, phosphatidylserine synthase (Pss) condenses serine with CDP-diglyceride to form phosphatidylserine (PS), and in the second step, PS is decarboxylated by phosphatidylserine decarboxylase (Psd) to form PE. In this study we identified the sinorhizobial psd gene coding for Psd. A sinorhizobial mutant deficient in psd is unable to form PE but accumulates the anionic phospholipid PS. Properties of PE-deficient mutants lacking either Pss or Psd were compared with those of the S. meliloti wild type. Whereas both PE-deficient mutants grew in a wild-type-like manner on many complex media, they were unable to grow on minimal medium containing high phosphate concentrations. Surprisingly, the psd-deficient mutant could grow on minimal medium containing low concentrations of inorganic phosphate, while the pss-deficient mutant could not. Addition of choline to the minimal medium rescued growth of the pss-deficient mutant, CS111, to some extent but inhibited growth of the psd-deficient mutant, MAV01. When the two distinct PE-deficient mutants were analyzed for their ability to form a nitrogen-fixing root nodule symbiosis with their alfalfa host plant, they behaved strikingly differently. The Pss-deficient mutant, CS111, initiated nodule formation at about the same time point as the wild type but did form about 30% fewer nodules than the wild type. In contrast, the PS-accumulating mutant, MAV01, initiated nodule formation much later than the wild type and formed 90% fewer nodules than the wild type. The few nodules formed by MAV01 seemed to be almost devoid of bacteria and were unable to fix nitrogen. Leaves of alfalfa plants inoculated with the mutant MAV01 were yellowish, indicating that the plants were starved for nitrogen. Therefore, changes in lipid composition, including the accumulation of bacterial PS, prevent the establishment of a nitrogen-fixing root nodule symbiosis.

A role of phosphatidylserine externalization in clearance of erythrocytes exposed to stress but not in eliminating aging populations of erythrocyte in mice

Age dependent changes in phosphatidylserine (PS) externalization were studied in mouse erythrocytes of different age groups (range 1–55 days) by using a newly developed double in vivo biotinylation (DIB) technique. Around 3–4% of the erythrocytes freshly released in the circulation were PS + but this proportion fell rapidly to 1% or less and did not increase at later time points. Blocking erythrocyte clearance from the circulation by in vivo depletion of macrophages (by treatment with clodronate loaded liposomes) for up to 7 days did not result in accumulation of PS + erythrocytes in the circulation indicating that the low percentage of PS + cells within old erythrocytes (age >40 days) was not related to the clearance of PS + erythrocytes by macrophages. In vitro treatment with stress inducing agents like deoxyglucose or Ca ++/calcium ionophore resulted in a marked induction of PS externalization in mouse erythrocytes and this effect was most prominent in the youngest erythrocyte population (age <10 days). Kinetics of clearance of different age groups of stress exposed erythrocytes after intravenous infusion into recipient mice indicated that the young erythrocytes were cleared at fastest rate from the circulation as compared to erythrocytes of older age groups. Within young erythrocytes exposed to stress, PS + erythrocytes were preferentially cleared. Taken together our results suggest that PS externalization is unlikely to have a role in the removal of old erythrocytes from blood circulation but may have a role in the clearance of stressed and damaged young erythrocytes in blood circulation.

Neuronal Specific Increase of Phosphatidylserine by Docosahexaenoic Acid

Phosphatidylserine (PS), the major acidic phospholipid class in eukaryotic biomembranes, plays an important role in various signaling pathways. We have previously demonstrated that docosahexaenoic acid (DHA, 22:6n-3) positively modulates PS biosynthesis and accumulation in neuronal cells, promoting survival. In this paper, we demonstrate that the increase of PS levels upon DHA enrichment is not a universal mechanism, but specific to neuronal cells. When cells were enriched with 20 muM DHA, 18:0, 22:6-PS increased in both neuronal (Neuro 2A) and non-neuronal cells (Chinese hamster ovary K1 cells, NIH-3T3, and human embryonic kidney cells). However, the increase of the total PS level was observed only in Neuro 2A cells because of the fact that other PS species, such as 18:0, 18:1-PS and 18:1, 18:1-PS decreased significantly in non-neuronal cells, compensating for the increase of 18:0, 22:6-PS. DHA enrichment did not affect the messenger RNA levels of PS synthase 1 (PSS1) and PSS2. Over-expression of genes encoding PSS1 or PSS2 altered neither the PS level nor the effect of DHA on PS increase in both neuronal and non-neuronal cells. From these results, it is concluded that the PS increase by DHA, specifically observed in neuronal cells, may represent a unique mechanism for expanding the PS pool so far known in mammalian cells.

Protective effects of docosahexaenoic acid in staurosporine-induced apoptosis: involvement of phosphatidylinositol-3 kinase pathway.

Docosahexaenoic acid (22:6n-3, DHA) is highly enriched in neuronal membranes and is considered to be essential for proper brain function. We have previously demonstrated in Neuro 2A cells that DHA as a membrane component protects cells from apoptotic death induced by serum deprivation (Kim et al. 2000). In the present study we demonstrate that staurosporine (ST) induces apoptosis in Neuro 2A cells and DHA enrichment prior to the ST treatment significantly inhibits the apoptotic cell death, as evidenced by the reduction of caspase-3 activity, cleavage of pro-caspase-3 to active caspase-3, DNA strand-breaking and laddering. Enrichment of cells with other fatty acids such as oleic and arachidonic acids did not exert such an effect, indicating that the antiapoptotic effect was specific to DHA enrichment. Among the several protein kinase inhibitors, only phosphatidylinositol 3-kinase (PI3-K) inhibitors, wortmanin, and LY-294002 abolished the protective effect of DHA in ST-induced apoptosis. Concurrently, ST-treatment significantly decreased the phosphorylation status of Akt at Ser-473 and Thr-308 as well as Akt activity, and this reduction was partially prevented by DHA enrichment. The extent of the antiapoptotic effect of DHA correlated with a time-dependent increase in the phosphatidylserine (PS) content upon DHA enrichment. When cells were enriched with DHA in serine-free medium, the PS increase diminished and the DHA effect on caspase-3 activation as well as Akt phosphorylation in ST-induced apoptosis was no longer apparent, suggesting that DHA's role in accumulating membrane PS is an important component for the observed protection. In summary, DHA enrichment uniquely protects ST-induced apoptosis in a PS- and PI3-K-dependent manner. From these data, we suggest that the antiapoptotic effect of DHA is mediated at least in part through the PI3-K/Akt pathway, facilitated by DHA-induced PS accumulation.

Inhibition of Neuronal Apoptosis by Docosahexaenoic Acid (22:6n-3): ROLE OF PHOSPHATIDYLSERINE IN ANTIAPOPTOTIC EFFECT

Enrichment of Neuro 2A cells with docosahexaenoic acid (22:6n-3) decreased apoptotic cell death induced by serum starvation as evidenced by the reduced DNA fragmentation and caspase-3 activity. The protective effect of 22:6n-3 became evident only after at least 24 h of enrichment before serum starvation and was potentiated as a function of the enrichment period. During enrichment 22:6n-3 incorporated into phosphatidylserine (PS) steadily, resulting in a significant increase in the total PS content. Similar treatment with oleic acid (18:1n-9) neither altered PS content nor resulted in protective effect. Hindering PS accumulation by enriching cells in a serine-free medium diminished the protective effect of 22:6n-3. Membrane translocation of Raf-1 was significantly enhanced by 22:6n-3 enrichment in Neuro 2A cells. Consistently, in vitro biomolecular interaction between PS/phosphatidylethanolamine /phosphatidylcholine liposomes, and Raf-1 increased in a PS concentration-dependent manner. Collectively, enrichment of neuronal cells with 22:6n-3 increases the PS content and Raf-1 translocation, down-regulates caspase-3 activity, and prevents apoptotic cell death. Both the antiapoptotic effect of 22:6n-3 and Raf-1 translocation are sensitive to 22:6n-3 enrichment-induced PS accumulation, strongly suggesting that the protective effect of 22:6n-3 may be mediated at least in part through the promoted accumulation of PS in neuronal membranes.

Stimulated Nonspecific Transport of Phospholipids Results in Elevated External Appearance of Phosphatidylserine in ras-Transformed Fibroblasts

The content of phosphatidylserine (PS) was found to be increased three times in the plasma membrane outer leaflet of ras-transformed fibroblasts compared to their nontransformed counterparts. In an attempt to determine the mechanisms responsible for the enhanced external appearance of PS, we investigated the activities of aminophospholipid translocase and the nonspecific lipid scramblase. Both transport systems could separately or in combination contribute to PS accumulation in the extracellular leaflet. Aminophospholipid transfer was assessed by measuring the rate of NBD-PS internalization, and scramblase activity was estimated from the internalization of NBD-PC. The results showed that the aminophospholipid transport was inhibited and the nonspecific transport was stimulated in ras-transformed cells. To assess which of these two transport systems was related to elevation of PS external appearance, each of them was submitted to reversible alterations and the content of PS was measured simultaneously. Aminophospholipid translocase activity was inhibited by pyridyldithioethylamine treatment and reversed by reduction with dithiothreitol. Scramblase activity was modulated by a calcium repletion–depletion procedure. Calcium depletion was performed by cell incubation with BAPTA-AM and EGTA as Ca2+ intracellular and extracellular chelators. Restoration of the intracellular Ca2+ was achieved by cell incubation with Ca2+ and Ca2+-ionophore A23187. The results showed that the changes in PS outer appearance did not correlate with the uptake of NBD-PS but were closely related to NBD-PC internalization, suggesting that the nonspecific bidirectional lipid transfer was the major transport system translocating PS to the outer leaflet in ras-transformed cells.