Oxidative stress can activate the epidermal platelet-activating factor receptor.
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Platelet-activating factor (1-alkyl-2-acetyl-glycero-phosphocholine) is a lipid mediator that has been implicated in keratinocyte function and cutaneous inflammation. Keratinocytes both synthesize platelet-activating factor and express functional platelet-activating factor receptors linked to calcium mobilization. Oxidative stress to various cells including keratinocytes can also result in the mobilization of intracellular Ca2+, a known stimulus for platelet-activating factor biosynthesis. The ability of the epidermal platelet-activating factor receptors to modulate oxidant-induced signaling was investigated using a unique model system created by retroviral-mediated transduction of the platelet-activating factor receptor-negative epithelial cell line KB with the platelet-activating factor receptor. Treatment of KB cells with the lipid pro-oxidant tert-butyl hydroperoxide induced transient increases in intracellular Ca2+ in a concentration-dependent fashion. Expression of the platelet-activating factor receptor in KB cells lowered the threshold for tert-butyl hydroperoxide-induced Ca2+ flux by an order of magnitude (10 microM in control KB versus 1 microM in KB cells expressing the platelet-activating factor receptors) and increased the peak change in intracellular Ca2+ concentration in response to this lipid hydroperoxide. This augmentation of tert-butyl hydroperoxide-induced Ca2+ mobilization was inhibited by pretreatment with the two competitive platelet-activating factor receptor antagonists CV-6209 and WEB 2086, as well as by the antioxidants vitamin E and 1,1,3,3-tetramethyl-2-thiourea. KB cells synthesized platelet-activating factor and the platelet-activating factor receptor agonist 1-palmitoyl-2-acetyl-glycero-phosphocholine in response to tert-butyl hydroperoxide treatment, suggesting the augmentation of oxidative stress-induced signaling seen in platelet-activating factor receptor-expressing cells was due in part to endogenous platelet-activating factor biosynthesis. These studies suggest involvement of the epidermal platelet-activating factor receptors in oxidant-mediated signaling. (+info)
Cellular responses to excess phospholipid.
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Phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells, and its synthesis is controlled by the activity of CDP:phosphocholine cytidylyltransferase (CCT). Enforced CCT expression accelerated the rate of PtdCho synthesis. However, the amount of cellular PtdCho did not increase as a result of the turnover of both the choline and glycerol components of PtdCho. Metabolic labeling experiments demonstrated that cells compensated for elevated CCT activity by the degradation of PtdCho to glycerophosphocholine (GPC). Phospholipase D-mediated PtdCho hydrolysis and phosphocholine formation were unaffected. Most of the GPC produced in response to excess phospholipid production was secreted into the medium. Cells also degraded the excess membrane PtdCho to GPC when phospholipid formation was increased by exposure to exogenous lysophosphatidylcholine or lysophosphatidylethanolamine. The replacement of the acyl moiety at the 1-position of PtdCho with a non-hydrolyzable alkyl moiety prevented degradation to GPC. Accumulation of alkylacyl-PtdCho was associated with the inhibition of cell proliferation, demonstrating that alternative pathways of degradation will not substitute. GPC formation was blocked by bromoenol lactone, implicating the calcium-independent phospholipase A2 as a key participant in the response to excess phospholipid. Owing to the fact that PtdCho is biosynthetically converted to PtdEtn, excess PtdCho resulted in overproduction and exit of GPE as well as GPC. Thus, general membrane phospholipid homeostasis is achieved by a balance between the opposing activities of CCT and phospholipase A2. (+info)
Hypertonicity-induced accumulation of organic osmolytes in papillary interstitial cells.
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BACKGROUND: Medullary cells of the concentrating kidney are exposed to high extracellular solute concentrations. It is well established that epithelial cells in this kidney region adapt osmotically to hypertonic stress by accumulating organic osmolytes. Little is known, however, of the adaptive mechanisms of a further medullary cell type, the papillary interstitial cell [renal papillary fibroblast (RPF)]. We therefore compared the responses of primary cultures of RPFs and papillary collecting duct (PCD) cells exposed to hypertonic medium. METHODS: In RPFs and PCD cells, organic osmolytes were determined by high-performance liquid chromatography; mRNA expression for organic osmolyte transporters [Na+/Cl(-)-dependent betaine transporter (BGT), Na(+)-dependent myo-inositol transporter (SMIT)], and the sorbitol synthetic and degrading enzymes [aldose reductase (AR) and sorbitol dehydrogenase (SDH), respectively] was determined by Northern blot analysis. RESULTS: Exposure to hypertonic medium (600 mOsm/kg by NaCl addition) caused intracellular contents of glycerophosphorylcholine, betaine, myo-inositol, and sorbitol, but not free amino acids, to increase significantly in both RPFs and PCD cells. The rise in intracellular contents of these organic osmolytes was accompanied by enhanced expression of mRNAs coding for BGT, SMIT, and AR in both RPFs and PCD cells. SDH mRNA abundance, however, was unchanged. Nonradioactive in situ hybridization studies on sections from formalin-fixed and paraffin-embedded, normally concentrating kidneys showed strong expression of BGT, SMIT, and AR mRNAs in interstitial and collecting duct cells of the papilla, whereas expression of SDH mRNA was much weaker in both cell types. CONCLUSIONS: These results suggest that both RPFs and PCD cells use similar strategies to adapt osmotically to the high interstitial NaCl concentrations characteristic for the inner medulla and papilla of the concentrating kidney. (+info)
Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position.
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Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252-6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0.23 micromol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis. (+info)
Formation of the aldehydic choline glycerophospholipids in human red blood cell membrane peroxidized with an azo initiator.
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The production of phospholipid hydroperoxide and aldehydic phospholipid was examined in human red blood cell (RBC) membranes after peroxidation with 2,2-azobis(2-amidinopropane)dihydrochloride (AAPH) or xanthine/xanthine oxidase (XO/XOD/Fe3+). Both radical-generation systems caused a profound decrease in the amount of polyunsaturated fatty acid (PUFA) in choline glycerophospholipid (CGP) and induced formation of peroxidized CGP in RBC membranes to different extents. No consistent generation of peroxidized lipids from CGP was evident after peroxidation with XO/XOD/Fe3+, which caused the apparent decomposition of phospholipids and the formation of large amounts of thiobarbituric acid-reactive substance (TBARS). On the other hand, CGP hydroperoxide was formed as a primary product of peroxidation with AAPH. Aldehydic CGP was also detected as a secondary product of hydroperoxide decomposition in AAPH-peroxidized RBC membranes. Aldehydic CGP was preferentially generated from arachidonoyl CGP rather than from linoleoyl CGP in AAPH-peroxidized membranes. AAPH mainly oxidized CGP to hydroperoxide and aldehydic phospholipids. The sum of hydroperoxide and aldehyde of CGP corresponded to the loss of CGP due to peroxidation by AAPH. This result indicates that CGP was mainly converted into these two oxidized phospholipids in AAPH-peroxidized RBC membranes. (+info)
Regulation of phosphatidylcholine homeostasis by calcium-independent phospholipase A2.
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Phosphatidylcholine (PtdCho) is the most abundant phospholipid in mammalian cell membranes and is essential for cell viability. The levels of this lipid must be tightly controlled to maintain homeostasis. Therefore, changes in the rate of PtdCho synthesis are generally balanced by changes in PtdCho catabolism and vice versa. It is commonly accepted that the rate of PtdCho synthesis is regulated by CTP:phosphocholine cytidylyltransferase (CT). However, it is not certain if PtdCho mass is regulated by specific catabolic enzyme(s). Our goal is to determine if PtdCho homeostasis is regulated by a phospholipase A2 (PLA2). To this end, we have prepared Chinese hamster ovary (CHO) cell lines that overexpress CT. CT activity is 7-10-fold higher in the transfected cells than in parental CHO cells. This increase in CT activity is associated with increases in both PtdCho synthesis and PtdCho catabolism. Glycerophosphocholine is the PtdCho catabolite that accumulates in the transfected cells, which suggests that PtdCho turnover is mediated by a phospholipase A2 (PLA2). Indeed, higher levels of calcium-independent PLA2 activity are measured in the cytosols of the CHO cells that overexpress CT, compared to parental CHO cells. The elevated calcium-independent PLA2 activity is associated with increases in the expression of the 80-kDa calcium-independent PLA2 (iPLA2). Together, these data suggest that the 80-kDa iPLA2 may be modulated in response to changes in PtdCho levels and therefore is involved in the regulation of PtdCho homeostasis in CHO cells. (+info)
Biosynthesis of 1,2-dieicosapentaenoyl-sn-glycero-3-phosphocholine in Caenorhabditis elegans.
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Previously, we showed that lowering the growth temperature increased the level of eicosapentaenoic acid (EPA) in the phosphatidylcholine (PtdCho) of Caenorhabditis elegans. In this study, we investigated the molecular species composition of PtdCho of C. elegans, with an emphasis on EPA-containing species. C. elegans contained a substantial amount of 1,2-dipolyunsaturated fatty acid-containing PtdCho (1,2-diPUFA-PtdCho) species, such as arachidonic acid/EPA and EPA/EPA, which are unusual phospholipids in higher animals. The EPA/EPA-PtdCho content was significantly increased in C. elegans grown at a low temperature. To examine the possibility that the acyltransferase activity involved in the remodeling of phospholipids accounts for the production of 1,2-diPUFA-PtdCho, we investigated the substrate specificity of this enzyme in C. elegans and found that it did not exhibit a preference for saturated fatty acid for acylation to the sn-1 position of PtdCho. The efficacy of the esterification of EPA to the sn-1 position was almost equal to that of stearic acid. The lack of preference for a saturated fatty acid for acylation to the sn-1 position of PtdCho is thought to result in the existence of the unusual 1,2-diEPA-PtdCho in C. elegans. (+info)
Cortical and medullary betaine-GPC modulated by osmolality independently of oxygen in the intact kidney.
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Renal osmolyte concentrations are reduced during reflow following ischemia. Osmolyte decreases may follow oxygen depletion or loss of extracellular osmolality in the medulla. Image-guided volume-localized magnetic resonance (MR) microspectroscopy was used to monitor regional osmolytes during hyposmotic shock and hypoxia in the intact rat kidney. Alternate spectra were acquired from 24-microl voxels in cortex and medulla of the isolated perfused kidney. There was a progressive decrease in the combined betaine-glycerophosphorylcholine (GPC) peak intensity of 21% in cortex and 35% in medulla of normoxic kidneys between 60 and 160 min after commencing perfusion. Hypoxia had no significant effect on the betaine-GPC peak intensity in cortex or medulla, despite a dramatic reduction in tubular sodium, potassium, and water reabsorption. The results suggest that cortical and medullary intracellular osmolyte concentrations depend on osmotically regulated channels that are insensitive to oxygen and dissociated from the oxygen-dependent parameters of renal function, the fractional excretion of sodium, the fractional excretion of potassium, and urine-to-plasma inulin concentration ratio. (+info)