Stimulation of arachidonic acid release by vasopressin in A7r5 vascular smooth muscle cells mediated by Ca2+-stimulated phospholipase A2

Arachidonic acid (AA) regulates many aspects of vascular smooth muscle behaviour, but the mechanisms linking receptors to AA release are unclear. In A7r5 vascular smooth muscle cells pre-labelled with (3)H-AA, vasopressin caused a concentration-dependent stimulation of 3H-AA release that required phospholipase C and an increase in cytosolic [Ca2+]. Ca2+ release from intracellular stores and Ca2+ entry via L-type channels or the capacitative Ca2+ entry pathway were each effective to varying degrees. Selective inhibitors of PLA2 inhibited the 3H-AA release evoked by vasopressin, though not the underlying Ca2+ signals, and established that cPLA2 mediates the release of AA. We conclude that in A7r5 cells vasopressin stimulates AA release via a Ca2+-dependent activation of cPLA2.     

Decreased iPLA2gamma expression induces lipid peroxidation and cell death and sensitizes cells to oxidant-induced apoptosis

Our previous studies showed that renal proximal tubular cells (RPTC) express Ca(2+)-independent phospholipase A(2)gamma (iPLA(2)gamma) in endoplasmic reticulum (ER) and mitochondria and that iPLA(2)gamma prevents and/or repairs lipid peroxidation induced by oxidative stress. Our present studies determined the importance of iPLA(2)gamma in mitochondrial and cell function using an iPLA(2)gamma-specific small hairpin ribonucleic acid (shRNA) adenovirus. iPLA(2)gamma expression and activity were decreased in the ER by 24 h and in the mitochondria by 48 h compared with scrambled shRNA adenovirus-treated cells. Lipid peroxidation was elevated by 2-fold at 24 h and remained elevated through 72 h in cells with decreased iPLA(2)gamma. Using electrospray ionization-mass spectrometry, primarily phosphatidylcholines and phosphatidylethanolamines were increased in iPLA(2)gamma-shRNA-treated cells. At 48 h after exposure to the iPLA(2)gamma shRNA, uncoupled oxygen consumption was inhibited by 25% and apoptosis was observed at 72 and 96 h. RPTC with decreased iPLA(2)gamma expression underwent apoptosis when exposed to a nonlethal concentration of the oxidant tert-butyl hydroperoxide (TBHP). Exposure of control cells to a nonlethal concentration of TBHP induced iPLA(2)gamma expression in RPTC. These results suggest that iPLA(2)gamma is required for the prevention and repair of basal lipid peroxidation and the maintenance of mitochondrial function and viability, providing further evidence for a cytoprotective role for iPLA(2)gamma from oxidative stress.     

Inhibition of phospholipase A2 reduces neurite outgrowth and neuronal viability

Phospholipase A2 (PLA(2)) has been implicated in neurodevelopmental processes and in the early development of the nervous system. We investigated the effects of the inhibition of calcium-dependent and calcium-independent subtypes of cytosolic PLA2 (cPLA2 and iPLA2) on the development and viability of primary cultures of cortical and hippocampal neurons. PLA2 in these cultures was continuously inhibited with methylarachidonyl-fluorophosphonate (MAFP), an irreversible inhibitor of cPLA2 and iPLA2, or with bromoenol lactone (BEL), an irreversible selective iPLA2 inhibitor. The effect of PLA2 inhibitors on the development of neuronal cultures was ascertained by total cell count and morphological characterisation. Neuronal viability was quantified with MTT assays. Inhibition of PLA2 resulted in reduction of neuritogenesis and neuronal viability, disrupting neuronal homeostasis and leading to neuronal death. We conclude that the functional integrity of both calcium-dependent and calcium-independent cytosolic PLA2 is necessary for the in vitro development of cortical and hippocampal neurons.     

Catalytic residues of group VIB calcium-independent phospholipase A2 (iPLA2gamma)

Although human group VIB calcium-independent phospholipase A(2) (iPLA(2)gamma) contains the lipase-consensus sequence Gly-Xaa-Ser-Xaa-Gly in the C-terminal half, its overall sequence exhibits a week similarity to those of other PLA(2)s, and thus no information on the catalytic site has been available. Here we show that the C-terminal region of human iPLA(2)gamma is responsible for the enzymatic activity. Comparison of this catalytic domain with those of the mouse homologue, human cytosolic PLA(2) (cPLA(2)), and the plant PLA(2) patatin reveals that an amino acid sequence of a short segment around Asp-627 of iPLA(2)gamma is conserved among these PLA(2)s, in addition to the Ser-483-containing lipase motif; the corresponding serine and aspartate in cPLA(2) and patatin are known to form a catalytic dyad. Since substitution of alanine for either Ser-483 or Asp-627 results in a loss of the PLA(2) activity, we propose that Ser-483 and Asp-627 of human iPLA(2)gamma constitute an active site similar to the Ser-Asp dyad in cPLA(2) and patatin.     

The expression of brain cyclooxygenase-2 is down-regulated in the cytosolic phospholipase A2 knockout mouse

We examined brain phospholipase A2 (PLA2) activity and the expression of enzymes metabolizing arachidonic acid (AA) in cytosolic PLA2 knockout () mice to see if other brain PLA2 can compensate for the absence of cPLA2 alpha and if cPLA2 couples with specific downstream enzymes in the eicosanoid biosynthetic pathway. We found that the rate of formation of prostaglandin E2 (PGE2), an index of net cyclooxygenase (COX) activity, was decreased by 62% in the compared with the control mouse brain. The decrease was accompanied by a 50-60% decrease in mRNA and protein levels of COX-2, but no change in these levels in COX-1 or in PGE synthase. Brain 5-lipoxygenase (5-LO) and cytochrome P450 epoxygenase (cyp2C11) protein levels were also unaltered. Total and Ca2+-dependent PLA2 activities did not differ significantly between and control mice, and protein levels of type VI iPLA2 and type V sPLA2, normalized to actin, were unchanged. These results show that type V sPLA2 and type VI iPLA2 do not compensate for the loss of brain cPLA2 alpha, and that this loss has significant downstream effects on COX-2 expression and PGE2 formation, sparing other AA oxidative enzymes. This suggests that cPLA2 is critical for COX-2-derived eicosanoid production in mouse brain.     

Phospholipase A(2) of peroxiredoxin 6 has a critical role in tumor necrosis factor-induced apoptosis

Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with peroxidase and phospholipase A(2) (PLA(2)) activities. Although the cellular function of the peroxidase of Prdx6 has been well elucidated, the function of the PLA(2) of Prdx6 is largely unknown. Here, we report a novel function for the PLA(2) in regulating TNF-induced apoptosis through arachidonic acid (AA) release and interleukin-1β (IL-1β) production. Prdx6 knockdown (Prdx6(KD)) in human bronchial epithelial cells (BEAS2B) shows severe decreases of peroxidase and PLA(2) activities. Surprisingly, Prdx6(KD) cells are markedly resistant to apoptosis induced by TNF-α in the presence of cycloheximide, but are highly sensitive to hydrogen peroxide-induced apoptosis. Furthermore, the release of AA and the production of IL-1β induced by proinflammatory stimuli, such as TNF-α, LPS, and poly I/C, are severely decreased in Prdx6(KD) cells. More interestingly, the restoration of Prdx6 expression with wild-type Prdx6, but not PLA(2)-mutant Prdx6 (S32A), in Prdx6(KD) cells dramatically induces the recovery of TNF-induced apoptosis, AA release, and IL-1β production, indicating specific roles for the PLA(2) activity of Prdx6. Our results provide new insights into the distinct roles of bifunctional Prdx6 with peroxidase and PLA(2) activities in oxidative stress-induced and TNF-induced apoptosis, respectively.     

Antioxidant activity of isocoumarins isolated from Paepalanthus bromelioides on mitochondria

The isocoumarins (1-50 microM) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin isolated from Paepalanthus bromelioides, were assessed for antioxidant activity using isolated rat liver mitochondria and non-mitochondrial systems, and compared with the flavonoid quercetin. The paepalantine and paepalantine dimers, but not vioxanthin, were effective at scavenging both 1,1-diphenyl-2-picrylhydrazyl (DPPH(*)) and superoxide (O(2)(-)) radicals in non-mitochondrial systems, and protected mitochondria from tert-butylhydroperoxide-induced H(2)O(2) accumulation and Fe(2+)-citrate-mediated mitochondrial membrane lipid peroxidation, with almost the same potency as quercetin. These results point towards paepalantine, followed by paepalantine dimer, as being a powerful agent affording protection, apparently via O(2)(-) scavenging, from oxidative stress conditions imposed on mitochondria, the main intracellular source and target of those reactive oxygen species. This strong antioxidant action of paepalantine was reproduced in HepG2 cells exposed to oxidative stress condition induced by H(2)O(2).     

Cytosolic phospholipase A2 alpha inhibition prevents neuronal NMDA receptor-stimulated arachidonic acid mobilization and prostaglandin production but not subsequent cell death

Phospholipase A(2) (PLA(2)) enzymes encompass a superfamily of at least 13 extracellular and intracellular esterases that hydrolyze the sn-2 fatty acyl bonds of phospholipids to yield fatty acids and lysophospholipids. The purpose of this study was to characterize which phospholipase paralog regulates NMDA receptor-mediated arachidonic acid (AA) release. Using mixed cortical cell cultures containing both neurons and astrocytes, we found that [(3)H]-AA released into the extracellular medium following NMDA receptor stimulation (100 microM) increased with time and was completely prevented by the addition of the NMDA receptor antagonist MK-801 (10 microM) or by removal of extracellular Ca(2+). Neither diacylglycerol lipase inhibition (RHC-80267; 10 microM) nor selective inhibition of Ca(2+)-independent PLA(2) [bromoenol lactone (BEL); 10 microM] alone had an effect on NMDA receptor-stimulated release of [(3)H]-AA. Release was prevented by methyl arachidonyl fluorophosphonate (MAFP) (5 microM) and AACOCF(3) (1 microM), inhibitors of both cytosolic PLA(2) (cPLA(2)) and Ca(2+)-independent PLA(2) isozymes. This inhibition effectively translated to block of NMDA-induced prostaglandin (PG) production. An inhibitor of p38MAPK, SB 203580 (7.5 microM), also significantly reduced NMDA-induced PG production providing suggestive evidence for the role of cPLA(2)alpha. Its involvement in release was confirmed using cultures derived from mice deficient in cPLA(2)alpha, which failed to produce PGs in response to NMDA receptor stimulation. Interestingly, neither MAFP, AACOCF(3) nor cultures derived from cPLA(2)alpha null mutant animals showed any protection against NMDA-mediated neurotoxicity, indicating that inhibition of this enzyme may not be a viable protective strategy in disorders of the cortex involving over-activation of the NMDA receptor.     

Calcium-independent phospholipase A2 through arachidonic acid mobilization is involved in Caco-2 cell growth

Several studies indicate that phospholipase A(2) (PLA(2)) expression and/or activation account for the high levels of arachidonic acid (AA) detected in cancer and, together with the elevated expression of cyclooxygenase-2, lead to cell proliferation and tumor formation. Using Caco-2 cells, a human colorectal carcinoma cell, we studied the role of high-molecular-weight PLA(2)s, cytosolic PLA(2) (cPLA(2)), and calcium-independent PLA(2) (iPLA(2)) in the AA cascade and in cell growth. Treatment with an antisense oligonucleotide against cPLA(2)alpha decreased [(3)H]AA release induced by ionophore A23187 or by a phorbol ester but did not affect the release of [(3)H]AA, [(3)H]thymidine incorporation, or Caco-2 growth induced by fetal calf serum (FCS). However, these parameters were significantly modified by iPLA(2) inhibitors and by an antisense oligonucleotide against iPLA(2)beta. Our results show that iPLA(2) was involved in AA release and the subsequent prostaglandin production induced by serum. Moreover, these data indicate that iPLA(2) may be involved in the signaling pathways involved in the control of Caco-2 proliferation.     

Resistance to TNF-induced cytotoxicity correlates with an abnormal cleavage of cytosolic phospholipase A2

To investigate the mechanism underlying the absence of arachidonic acid (AA) release by TNF in TNF-resistant cells, we first performed comparative analysis of phospholipid pools in both TNF-sensitive (MCF7) and their equivalent resistant cells (C1001). Quantification and incorporation studies of [(3)H]AA indicated that TNF-resistant cells were not depleted in AA. Furthermore, distribution of this fatty acid in different phospholipid pools was similar in both sensitive cells and their resistant counterparts, ruling out a defect in phospholipid pools. Since phospholipase A(2) (PLA(2)) are the main enzymes releasing free AA, we investigated their relative contribution in the acquisition of cell resistance to TNF-induced cell death and AA release. For this purpose, we used two PLA(2) inhibitors, methylarachidonyl fluorophosphate (MAFP) and bromoenol lactone (BEL), which selectively and irreversibly inhibit the cytosolic PLA(2) (cPLA(2)) and the Ca(2+)-independent PLA(2), respectively. Although a significant inhibitory effect of MAFP on both TNF-induced AA release and PLA(2) activity in MCF7 was observed, BEL had no effect. The inhibitory effect of MAFP on cPLA(2) activity correlated with an inhibition of TNF-induced cell death. Western blot analysis revealed that TNF induced a differential cleavage of cPLA(2) in TNF-sensitive vs TNF-resistant cells. Although the p70 (70-kDa) form of cPLA(2) was specifically increased in TNF-sensitive cells, a cleaved form, p50 (50 kDa), was selectively observed in TNF-resistant C1001 cells in the presence or absence of TNF. These findings suggest that the acquisition of cell resistance to this cytokine may involve an abnormal cPLA(2) cleavage.     

Phospolipase A2 and apoptosis

Phospolipase A(2) (PLA(2)) is the esterase activity that cleaves the sn-2 ester bond in glycerophospholipids, releasing free fatty acids and lysophospholipids. The PLA(2) activity is found in a variety of enzymes which can be divided in several types based on their Ca(2+) dependence for their activity; Ca(2+)-dependent secretory phosholipases (sPLA(2)s) and cytosolic phospholipases (cPLA(2)s), and Ca(2+)-independent phospholipase A(2)s (iPLA(2)s). These enzymes also show diverse size and substrate specificity (i.e., in the fatty acid chain length and extent of saturation). Among the fatty acids released by PLA(2), arachidonic acid (AA) is of particular biological importance, because it is subsequently converted to prostanoids and leukotrienes by cyclooxygenases (COX) and lipoxygenases (LOX), respectively. Free AA may also stimulate apoptosis through activation of sphingomyelinase. Alternatively, it is suggested that oxidized metabolites generated from AA by LOX induce apoptosis. Although the precise mechanisms remain to be elucidated, changes are observed in glycerolipid metabolism during apoptotic processes. In some cells induced to undergo apoptosis, AA is released concomitant with loss of cell viability, caspase activation and DNA fragmentation. Such AA releases appear to be mediated by activation of cPLA(2) and/or iPLA(2). For example, tumor necrosis factor-alpha (TNF-alpha)-induced cell death is mediated by cPLA(2), whereas Fas-induced apoptosis appears to be mediated by iPLA(2). Some discrepancies among early experimental results were probably caused by differences in the experimental conditions such as the serum concentration, inhibitors used that are not necessarily specific to a single-type enzyme, or differential expression of each PLA(2) in cells employed in the experiments. Recent studies eliminated such problems, by carefully defining the experimental conditions, and using multiple inhibitors that show different specificities. Accordingly, more convincing data are available that demonstrate involvement of some PLA(2)s in the apoptotic processes. In addition to cPLA(2) and iPLA(2), sPLA(2)s were recently found to play roles in apoptosis. Moreover, new proteins that appear to control PLA(2)s are being discovered. Here, the roles of PLA(2)s in apoptosis are discussed by reviewing recent reports.     

Role of caspases in TNF-mediated regulation of cPLA(2)

A major part of the proinflammatory activity of tumor necrosis factor (TNF) is brought about by cytosolic phospholipase A(2) (cPLA(2)) that generates arachidonic acid, the precursor for the production of leukotrienes and prostaglandins. The activation of cPLA(2) and induction of proinflammatory lipid mediators is in striking contrast to the teleologic meaning of apoptosis, which is to avoid an inflammatory reaction. In this review we highlight the evidence for a caspase-mediated cleavage and inactivation of cPLA(2), which seems to be an important mechanism by which TNF downregulates cPLA(2) activity in cells undergoing apoptosis.     

Analysis of two major intracellular phospholipases A(2) (PLA(2)) in mast cells reveals crucial contribution of cytosolic PLA(2)α, not Ca(2+)-independent PLA(2)β, to lipid mobilization in proximal mast cells and distal fibroblasts

Mast cells release a variety of mediators, including arachidonic acid (AA) metabolites, to regulate allergy, inflammation, and host defense, and their differentiation and maturation within extravascular microenvironments depend on the stromal cytokine stem cell factor. Mouse mast cells express two major intracellular phospholipases A(2) (PLA(2)s), namely group IVA cytosolic PLA(2) (cPLA(2)α) and group VIA Ca(2+)-independent PLA(2) (iPLA(2)β), and the role of cPLA(2)α in eicosanoid synthesis by mast cells has been well documented. Lipidomic analyses of mouse bone marrow-derived mast cells (BMMCs) lacking cPLA(2)α (Pla2g4a(-/-)) or iPLA(2)β (Pla2g6(-/-)) revealed that phospholipids with AA were selectively hydrolyzed by cPLA(2)α, not by iPLA(2)β, during FcεRI-mediated activation and even during fibroblast-dependent maturation. Neither FcεRI-dependent effector functions nor maturation-driven phospholipid remodeling was impaired in Pla2g6(-/-) BMMCs. Although BMMCs did not produce prostaglandin E(2) (PGE(2)), the AA released by cPLA(2)α from BMMCs during maturation was converted to PGE(2) by microsomal PGE synthase-1 (mPGES-1) in cocultured fibroblasts, and accordingly, Pla2g4a(-/-) BMMCs promoted microenvironmental PGE(2) synthesis less efficiently than wild-type BMMCs both in vitro and in vivo. Mice deficient in mPGES-1 (Ptges(-/-)) had an augmented local anaphylactic response. These results suggest that cPLA(2)α in mast cells is functionally coupled, through the AA transfer mechanism, with stromal mPGES-1 to provide anti-anaphylactic PGE(2). Although iPLA(2)β is partially responsible for PGE(2) production by macrophages and dendritic cells, it is dispensable for mast cell maturation and function.     

Role of iPLA(2) in the regulation of Src trafficking and microglia chemotaxis

Microglia are immune effector cells in the central nervous system (CNS) and their activation, migration and proliferation play crucial roles in brain injuries and diseases. We examined the role of intracellular Ca(2+) -independent phospholipase A(2) (iPLA(2)) in the regulation of microglia chemotaxis toward ADP. Inhibition of iPLA(2) by 4-bromoenol lactone (BEL) or iPLA(2) knockdown exerted a significant inhibition on phosphatidylinositol-3-kinase (PI3K) activation and chemotaxis. Further examination revealed that iPLA(2) knockdown abrogated Src activation, which is required for PI3K activation and chemotaxis. Colocalization studies showed that cSrc-GFP was retained in the endosomal recycling compartment (ERC) in iPLA(2) knockdown cells, but the addition of arachidonic acid (AA) could restore cSrc trafficking to the plasma membrane by allowing the formation/release of recycling endosomes associated with cSrc-GFP. Using BODIPY-AA, we showed that AA is selectively enriched in recycling endosomes. These results suggest that AA is required for the cSrc trafficking to the plasma membrane by controlling the formation/release of recycling endosomes from the ERC.     

Localization and functional interrelationships among cytosolic Group IV, secreted Group V, and Ca2+-independent Group VI phospholipase A2s in P388D1 macrophages using GFP/RFP constructs

P388D(1) cells exposed to bacterial lipopolysaccharide (LPS) mobilize arachidonic acid (AA) for prostaglandin synthesis in two temporally distinct pathways. The "immediate pathway" is triggered within minutes by receptor agonists such as platelet-activating factor (PAF) but only if the cells have previously been primed with LPS for 1 h. The "delayed pathway" occurs in response to LPS alone over the course of several hours. We have now investigated the subcellular localization of both the Group IV cytosolic phospholipase A(2) (cPLA(2)) and the Group V secreted PLA(2) (sPLA(2)) during these two temporally distinct routes of AA release. We have prepared cells overexpressing fusion proteins of sPLA(2)-GFP and cPLA(2)-RFP. In the resting cells, cPLA(2)-RFP was uniformly located throughout the cytoplasm, and short-term treatment with LPS did not induce translocation to perinuclear and/or Golgi membranes. However, such a translocation occurred almost immediately after the addition of PAF to the cells. Long-term exposure of the cells to LPS led to the translocation of cPLA(2)-RFP to intracellular membranes after 3 h, and correlates with a significant release of AA in a cPLA(2)-dependent manner. At the same time period that the delayed association of cPLA(2) with perinuclear membranes is detected, an intense fluorescence arising from the sPLA(2)-GFP was found around the nucleus in the sPLA(2)-GFP stably transfected cells. In parallel with these changes, significant AA release was detected from the sPLA(2)-GFP transfectants in a cPLA(2)-dependent manner, which may reflect cross-talk between sPLA(2) and cPLA(2). The subcellular localization of the Group VIA Ca(2+)-independent PLA(2) (iPLA(2)) was also investigated. Cells overexpressing iPLA(2)-GFP showed no fluorescence changes under any activation condition. However, the iPLA(2)-GFP-expressing cells showed relatively high basal AA release, confirming a role for iPLA(2) in basal deacylation reactions. These new data illustrate the subcellular localization changes that accompany the distinct roles that each of the three kinds of PLA(2) present in P388D(1) macrophages play in AA mobilization.     

Lipid mediators and vector infection: Trypanosoma rangeli inhibits Rhodnius prolixus hemocyte phagocytosis by modulation of phospholipase A2 and PAF-acetylhydrolase activities

In this work we investigated the effects of Trypanosoma rangeli infection through a blood meal on the hemocyte phagocytosis in experiments using the 5th instar larvae of Rhodnius prolixus. Hemocyte phagocytic activity was strongly blocked by oral infection with the parasites. In contrast, hemocyte phagocytosis inhibition caused by T. rangeli infection was rescued by exogenous arachidonic acid (20 microg/insect) or platelet activating factor (PAF; 1 microg/insect) applied by hemocelic injection. Following the oral infection with the protozoan we observed significant attenuation of phospholipase A2 (PLA2) activities in R. prolixus hemocytes (cytosolic PLA2: cPLA2, secreted PLA2: sPLA2 and Ca+2-independent PLA2: iPLA2) and enhancement of sPLA2 activities in cell-free hemolymph. At the same time, the PAF-acetyl hydrolase (PAF-AH) activity in the cell-free hemolymph increased considerably. Our results suggest that T. rangeli infection depresses eicosanoid and insect PAF analogous (iPAF) pathways giving support to the role of PLA2 in the regulation of arachidonic acid and iPAF biosynthesis and of PAF-AH by reducing the concentration of iPAF in R. prolixus. This illustrates the ability of T. rangeli to modulate the immune responses of R. prolixus to favor its own multiplication in the hemolymph.     

The transfer of VLDL-associated phospholipids to activated platelets depends upon cytosolic phospholipase A2 activity

We previously reported that VLDL could transfer phospholipids (PLs) to activated platelets. To identify the metabolic pathway involved in this process, the transfer of radiolabeled PLs from VLDL (200 microM PL) to platelets (2 x 10(8)/ml) was measured after incubations of 1 h at 37 degrees C, with or without thrombin (0.1 U/ml) or LPL (500 ng/ml), in the presence of various inhibitors, including aspirin, a cyclooxygenase inhibitor (300 microM); esculetin, a 12-lipoxygenase inhibitor (20 microM); methyl-arachidonyl-fluorophosphonate (MAFP), a phospholipase A(2) (PLA(2)) inhibitor (100 microM); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM), a Ca(2+) chelator (20 microM); bromoenol lactone (BEL), a Ca(2+)- independent phospholipase A(2) (iPLA(2)) inhibitor (100 nM); and 1-[6-[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl-]amino]hexyl]1H-pyrrole-2,5-dione (U73122), a phospholipase C (PLC) inhibitor (20 microM). Aspirin and esculetin had no effect, showing that PL transfer was not dependent upon cyclooxygenase or lipoxygenase pathways. The transfer of PL was inhibited by MAFP, U73122, and BAPTA-AM. Although MAFP inhibited both cytosolic phospholipase A(2) (cPLA(2)) and iPLA(2), only cPLA(2) is a calcium-dependent enzyme. Because calcium mobilization is favored by PLC and inhibited by BAPTA-AM, the transfer of PL from VLDL to platelets appeared to result from a cPLA(2)-dependent process. The inhibition of iPLA(2) by BEL had no effect on PL transfers.     

Amyloid beta(1-42) and its beta(25-35) fragment induce activation and membrane translocation of cytosolic phospholipase A2 in bovine retina capillary pericytes

We investigated changes in cytosolic phospholipase A(2) (cPLA(2)) and calcium-independent PLA(2) (iPLA(2)) activities in bovine retina capillary pericytes after stimulation with 50 microM amyloid-beta (Abeta) (1-42) and its (25-35) fragment, over 24 h (mild, sublethal model of cell damage). In the presence of Abeta peptides, we found that cPLA(2) activity was increased and translocated from the cytosolic fraction to the membrane system, particularly in the nuclear region. Reversed-sequence Abeta(35-25) peptide did not stimulate or induce cPLA(2) translocation. Exposure to both Abeta peptides had no significant effect on cPLA(2) protein content as tested by Western immunoblot analysis. The addition of Abetas to quiescent pericytes was followed by phosphorylation of cPLA(2) and arachidonic acid release. Treatment with inhibitors (AACOCF(3), staurosporine and cycloheximide) resulted in a sharp decrease in basal and stimulated cPLA(2) activity. Inactivating effects of bromoenol lactone (BEL), inhibitor of iPLA(2), demonstrated that the stimulation of total PLA(2) activity by Abetas was mediated by both PLA(2) enzymes. Taken together with our previous observations that both Abeta peptides may induce hydrolysis of phosphatidylcholine, the present results provide evidence that this process is cooperatively mediated by cPLA(2) activation/translocation and iPLA(2) activation. The effect is very likely triggered by a mild prooxidant mechanism which was not able to divert the cell to degeneration. The data confirm the hypothesis that pericytes could be a target of potential vascular damage and reactivity during processes involving amyloid accumulation.     

Protective effect of fosfomycin on gentamicin-induced lipid peroxidation of rat renal tissue

Fosfomycin is clinically recognized to reduce the aminoglycoside antibiotics-induced nephrotoxicity. However, little has been clarified why fosfomycin protects the kidney from the aminoglycosides-induced nephrotoxicity. Gentamicin, a typical aminoglycoside, is reported to cause lipid peroxidation. We focused on lipid peroxidation induced by gentamicin as a mechanism for the aminoglycosides-induced nephrotoxicity. The aim of this study is to investigate the effect of fosfomycin on the gentamicin-induced lipid peroxidation. In rat renal cortex mitochondria, fosfomycin was shown to depress the gentamicin-induced lipid peroxidation, which was evaluated by formation of thiobarbituric acid reactive substances (TBARS). Interestingly, this effect was observed in rat renal cortex mitochondria, but not in rat liver microsomes. However, fosfomycin did not affect lipid peroxidation of arachidonic acid caused by gentamicin with iron. Fosfomycin inhibited the gentamicin-induced iron release from rat renal cortex mitochondria. These results indicated that fosfomycin inhibited the gentamicin-induced lipid peroxidation by depressing the iron release from mitochondria. This may possibly be one mechanism for the protection of fosfomycin against the gentamicin-induced nephrotoxicity.