Formation Of Lysophospholipids Research Articles

Phospholipase A2 Activity and Substrate Specificity of Bothrops Asper and Crotalus Durissus Cumanensis Snake Venom Collected from the Guajira Region of Colombia

Phospholipase A2 (PLA2) is a versatile enzyme present in all organisms that hydrolyzes the sn-2 ester bond of phospholipids resulting in the formation of lysophospholipids and free fatty acids. PLA2 found in snake venom is responsible for multiple systemic effects such as neurotoxicity, myotoxicity, and hemolysis. We measure the specificities of PLA2 in whole venom samples towards different phospholipids. This study provides a first look at the biophysical characteristics of venoms isolated from Colombia. Crude venom samples were taken from Bothrops asper and Crotalus durissus cumanensis in the Guajira region of Colombia. The venom was lyophilized until used. Vesicles composed of the non-hydrolyzable 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (DEthPC) carrying calcein at a self-quenching concentration (50mM) were used as reporter vesicles, and target vesicles (substrate) made from DMPG, DMPC, POPE or sphingomyelin (SM) were used for measuring snake venom specificity. All measurements were performed in a pc1 Fluorometer (ISS, Urbana, IL) between 20°C and 60°C with and without calcium (30 μM). In the presence of calcium both venoms have similar catalytic action with C. durissus being slightly higher than B. asper. In the absence of calcium B. asper showed the highest activity indicating differences in the intrinsic calcium levels in both venoms. Overall B. asper showed the lowest lagtime for all assays. No calcein release was observed for SM. DMPG was the only one that presented activity at all temperatures suggesting that both venoms have strong affinity towards negatively charged membranes. Both snake venoms showed activity in DMPC only in the presence of extrinsic calcium and only at 24°C, where liquid-crystalline/gel phase coexistence is present, suggesting that the presence of membrane defects plays an important role in the initiation of catalysis.

Enzymatic degumming

AbstractThe oldest enzymatic degumming process (the Lurgi EnzyMax® process) was launched in 1992. It used porcine phospholipase A2, which has the disadvantages of limited availability and not being kosher/halal. To overcome these disadvantages, various microbial enzymes have been developed; they have different specificities and therefore offer different advantages. Phospholipase C for instance has the advantage that it leads to the formation of diacylglycerols that remain in the oil being degummed. This constitutes a significant yield improvement which also results from the formation of lysophospholipids that retain less oil than their precursors. In the laboratory, a fine dispersion of the aqueous enzyme solution in the oil can be maintained so that the phospholipase enzymes can be made to interact with non‐hydratable phosphatides (NHP) in the oil phase and catalyse their hydrolysis. On an industrial scale, dispersions coalesce before the enzymatic NHP hydrolysis is complete. Accordingly, enzymatic degumming processes that claim NHP‐removal and a low residual phosphorus content in the enzymatically degummed oil are invariably preceded by an acid treatment in which a degumming acid (citric acid) is finely dispersed into the oil to be degummed and made to react with the NHP present in the oil before the enzyme is added. This enzyme then only interacts with the phospholipids present in the water phase. This raises the question whether the yield increase resulting from the use of enzymes should be realised by treating the oil to be degummed or the gums that have already been isolated from the oil during a degumming treatment. Lack of experimental evidence prevents a firm answer to this question but the arguments in favour of treating the gums look more impressive than what can be said in favour of treating the oil. In short: Enzymes do not degum the oil but can be used to de‐oil the gums.

NMR study of the lipid profile of mullet raw roe and bottarga

AbstractSamples of mullet (Mugil cephalus) roes from two different fishing areas were studied by means of 1H, 13C and 31P NMR and GC techniques. The lipid fraction of unprocessed roes and that of the corresponding salted and dried commercial products “bottarga” were analyzed and the data compared. Roes of mullets from different origin showed different composition regarding mainly triacylglycerols, monounsaturated and n‐3 polyunsaturated fatty acids. The NMR spectra of processed roes, compared to those of the corresponding raw materials, showed a release of free fatty acids and the formation of other minor molecular components, such as free fatty alcohols, lysophospholipids, and diacylglycerols, thus indicating that hydrolytic processes were the main consequence of manufacturing. An increase of cholesterol ester concentration after processing was also detected. Through the analysis of the 31P NMR spectra, it was possible to follow the fate of the phospholipids and the formation of lysophospholipids induced by the salting and drying procedures.

Lipase-Catalyzed Acyl Exchange of Soybean Phosphatidylcholine in n-Hexane: A Critical Evaluation of Both Acyl Incorporation and Product Recovery

Lipase-catalyzed acidolysis was examined for the production of structured phospholipids in a hexane system. In a practical operation of the reaction system, the formation of lyso-phospholipids from hydrolysis is often a serious problem, as demonstrated from previous studies. A clear elucidation of the issue and optimization of the system are essential for the practical applications in reality. The effects of enzyme dosage, reaction temperature, solvent amount, reaction time, and substrate ratio were optimized in terms of the acyl incorporation, which led to the products, and lyso-phospholipids formed by hydrolysis, which led to the low yields. The biocatalyst used was the commercial immobilized lipase Lipozyme TL IM and substrates used were phosphatidylcholine (PC) from soybean and caprylic acid. A response surface design was used to evaluate the influence of selected parameters and their relationships on the incorporation of caprylic acid and the corresponding recovery of PC. Incorporation of fatty acids increased with increasing enzyme dosage, reaction temperature, solvent amount, reaction time, and substrate ratio. Enzyme dosage had the most significant effect on the incorporation, followed by reaction time, reaction temperature, solvent amount, and substrate ratio. However the parameters had also a negative influence on the PC recovery. Solvent amount had the most negative effect on recovery, followed by enzyme dosage, temperature, and reaction time. Individually substrate ratio had no significant effect on the PC recovery. Interactions were observed between different parameters. On the basis of the models, the reaction was optimized for the maximum incorporation and maximum PC recovery. With all of the considerations, the optimal conditions are recommended as enzyme dosage 29%, reaction time 50 h, temperature 54 degrees C, substrate ratio 15 mol/mol caprylic acid/PC, and 5 mL of hexane per 3 g substrate. No additional water is necessary. Under these conditions, an incorporation of caprylic acid up to 46% and recovery of PC up to 60% can be obtained from the prediction. The prediction was confirmed from the verification experiments.

Calcium-independent phospholipase A2 mediates store-operated calcium entry in rat cerebellar granule cells

Store-operated Ca(2+) entry (SOCE) has been extensively studied in non-neuronal cells, such as glial cells and smooth muscle cells, in which Ca(2+)-independent phospholipase A(2) (iPLA(2)) has been shown to play a key role in the regulation of SOCE channels. In the present study, we have investigated the role of iPLA(2) for store-operated Ca(2+) entry in rat cerebellar granule neurons in acute brain slices using confocal Ca(2+) imaging. Depletion of Ca(2+) stores by cyclopiazonic acid (CPA) induced a Ca(2+) influx, which could be inhibited by SOCE channel blockers 2-aminoethoxy-diphenylborate (2-APB) and 3,5-bistrifluoromethyl pyrazole derivative (BTP2), but not by the voltage-operated Ca(2+) channel blocker diltiazem and by the Na+ channel blocker tetrodotoxin. The inhibitors of iPLA(2), bromoenol lactone (BEL) and 1,1,1-trifluoro-2-heptadecanone, and the selective suppression of iPLA(2) expression by antisense oligodeoxynucleotides, inhibited CPA-induced Ca(2+) influx. Calmidazolium, which relieves the block of inhibitory calmodulin from iPLA(2), elicited a Ca(2+) influx similar to CPA-induced Ca(2+) entry. The product of iPLA(2), lysophosphatidylinositol, elicited a 2-APB- and BTP2-sensitive, but BEL-insensitive, Ca(2+) influx. Spontaneous Ca(2+) oscillations in granule cells in acute brain slices were reduced after inhibiting iPLA(2) activity or by blocking SOCE channels. The results suggest that depletion of Ca(2+) stores activates iPLA(2) to trigger Ca(2+) influx by the formation of lysophospholipids in these neurons.

Regulation of Store-Operated Calcium Entry by Calcium-Independent Phospholipase A2in Rat Cerebellar Astrocytes

We have studied store-operated Ca2+ entry (SOCE) in Bergmann glia and granule cell layer astrocytes in acute brain slices of the rat cerebellum, using the Ca2+-sensitive fluorescent dye Fluo-4 and confocal laser scanning microscopy. Astrocytes were identified by their morphology, location, and their Ca2+ response in K+-free solution. Depletion of Ca2+ stores by cyclopiazonic acid (CPA) (20 microM) induced SOCE in both types of astrocyte. A similar Ca2+ influx was elicited by the calmodulin antagonist calmidazolium (CMZ) (1 microM). The SOCE channel blocker 2-aminoethoxy-diphenylborate (2-APB) (100 microM) and the Ca2+ release-activated channel blocker 3,5-bistrifluoromethyl pyrazole derivative (BTP2) (20 microM) suppressed the CPA- and the CMZ-induced Ca2+ influx. Pretreatment of acute slices with the specific Ca2+-independent phospholipase A2 (iPLA2) inhibitor bromoenol lactone (BEL) (25 microM) blocked the CPA- and the CMZ-induced Ca2+ influx. The lysophospholipid products of iPLA2, lysophosphatidylcholine (250 nM) and lysophosphatidylinositol (250 nM), but not lysophosphatidic acid (250 nM), induced a BTP2- and 2-APB-sensitive, but BEL-insensitive, Ca2+ influx. CPA or CMZ enhanced the BEL-sensitive enzymatic activity of iPLA2 in cerebellar astrocyte culture. Inhibition of iPLA2 expression by specific antisense oligodeoxynucleotide of iPLA2 reduced the SOCE and the Ca2+ store refilling in cultured astrocytes. Spontaneous Ca2+ oscillations in astrocytes in situ were reduced after inhibiting SOCE channels or iPLA2 activity. The results suggest that the depletion of Ca2+ stores activates iPLA2 to open Ca2+ channels in the plasma membrane by the formation of lysophospholipids in astrocytes, presumably to refill the stores and allow normal Ca2+ signaling.

Leukocytic myeloperoxidase-mediated formation of bromohydrins and lysophospholipids from unsaturated phosphatidylcholines

Using MALDI-TOF mass spectrometry, we have shown that leukocytic myeloperoxidase (MPO) in the presence of its substrates (H2O2 and Br?) does not induce any changes in saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. Incubation of liposomes prepared from mono-unsaturated phosphatidylcholine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) with the (MPO + H2O2 + Br-) system resulted in formation of bromohydrins as the main products. 1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lysophosphatidylcholine) was the main product of the reaction of polyunsaturated phosphatidylcholine (1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) with the (MPO + H2O2 + Br-) system. The formation of lysophospholipids as well as of bromohydrins was not observed when the enzyme or one of its substrates (H2O2 or Br-) was absent from the incubation medium, or if an inhibitor of MPO (sodium azide) or hypobromite scavengers (taurine or methionine) were added. Thus, it can be postulated that the formation of bromohydrins as well as lysophospholipids by the (MPO + H2O2 + Br-) system results from reactions of hypobromite formed during MPO catalysis with double bonds of acyl chains of phosphatidylcholine. Such destructive processes may take place in vivo in membrane- or lipoprotein-associated unsaturated lipids in centers of inflammation.

Destabilization of the acrosome results in release of phospholipase A2 from human spermatozoa and subsequent formation of lysophospholipids

Phospholipase A(2) controls the phospholipid composition in spermatozoal membranes and is released from the acrosome of human spermatozoa. The extracellular phospholipase A(2) activity of human spermatozoa was determined by matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry after destabilization of acrosome by the calcium-ionophore calcimycin. MALDI-TOF mass spectrometry allowed the monitoring of changes in both substrate and products of spermatozoal phospholipase A(2) (PLA(2)) without the use of labelled phospholipids. The spermatozoal PLA(2) was characterized as a secretory one (sPLA(2)). Secretory PLA(2) exhibited a high substrate specificity for 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC), the most abundant spermatozoal phospholipid. A time- and cell number-dependent formation of the lysophospholipid PDPC was observed following incubation of extracellular medium of calcimycin-treated spermatozoa (CTS) with PDPC. Antibodies against sPLA(2), specific inhibitors of sPLA(2) and Ca(2+)-chelators could inhibit its generation. An antibody against lysophospholipase enhanced the lysoproduct concentration in the extracellular medium of CTS containing sPLA(2) because further metabolization of these products was blocked. The results demonstrated that destabilization of the acrosome is able to induce a release of secretory phospholipase A(2) from human spermatozoa with subsequent generation of lysophosphocholine in the surrounding of spermatozoa.

Myeloperoxidase-induced formation of chlorohydrins and lysophospholipids from unsaturated phosphatidylcholines.

The formation of lysophosphatidylcholines and chlorohydrins from unsaturated phosphatidylcholines upon the treatment with the myeloperoxidase-hydrogen peroxide-chloride system was evaluated by means of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Lyso-products were primarily found in phosphatidylcholine samples containing highly unsaturated fatty acid residues such as arachidonic or docosahexenoic acid. On the other hand, chlorohydrins dominate in mono- or bis-unsaturated phosphatidylcholines. No formation of these products was detected in the absence of one of the components of the MPO-H(2)O(2)-Cl(-) system or in the presence of MPO inhibitors (sodium azide) or scavengers of hypochlorous acid (taurine, methionine). Thus, hypochlorous acid formed by the MPO-H(2)O(2)-Cl(-) system is responsible for the observed modification in unsaturated phosphatidylcholines. In the presence of the complete MPO system, lyso-products and chlorohydrins were only formed at pH values lower than pH 6.0 with an optimum at pH 4.3. In contrast, the reagent hypochlorous acid caused the formation of these products even at neutral pH values, indicating a clear dependence of the yield of products on the presence of undissociated HOCl. We conclude that the formation of lysophospholipids and chlorohydrins from unsaturated phosphatidylcholines by myeloperoxidase can be relevant in vivo under acute inflammatory conditions.

Formation of lysophospholipids from unsaturated phosphatidylcholines under the influence of hypochlorous acid

The formation of lysophosphatidylcholines from unsaturated phosphatidylcholines upon treatment with hypochlorous acid was evaluated by means of MALDI-TOF mass spectrometry and 31P NMR spectroscopy. With an increasing number of double bonds in a fatty acid residue, the yield of lysophosphatidylcholines with a saturated fatty acid residue increased considerably in comparison to the total amount of higher molecular weight products like chlorohydrins and glycols. High amounts of lysophosphatidylcholines were formed from phospholipids containing arachidonic or docosahexaenoic acid residues. In phospholipids with monounsaturated fatty acid residues, the position of the double bond did not influence the yield of lyso-products. Besides the exclusive formation of chlorohydrin and glycol, hypochlorous acid caused the cleavage of the unsaturated fatty acid residue independent of its location at the first or second position of the glycerol backbone. In contrast, strong alkaline conditions, i.e. saponification led also to a hydrolysis of the saturated fatty acid residue from phosphatidylcholines. It is concluded that both MALDI-TOF mass spectrometry and 31P NMR spectroscopy are able to detect the formation of lysophosphatidylcholines. We conclude also that the formation of lysophospholipids from unsaturated phosphatidylcholines by hypochlorous acid can be relevant in vivo under acute inflammatory conditions.

Calcium-independent phospholipase A(2) mediates CREB phosphorylation and c-fos expression during ischemia.

In isolated, perfused adult rat hearts, global ischemia increased the phosphorylation of cAMP response element-binding protein (CREB) relative to control levels, and this phosphorylation was reversed with reperfusion. CREB phosphorylation elicited by 5 min of global ischemia was sensitive to treatments with the calcium-independent phospholipase A(2) (iPLA(2)) inhibitor bromoenol lactone (BEL) and occurred in the absence of increases in myocardial cAMP content. In contrast, CREB phosphorylation elicited by 15 min of global ischemia was likely mediated by elevated cAMP levels. The expression of c-fos, in response to brief myocardial ischemia, was also sensitive to BEL treatment. The induction of iPLA(2)-mediated CREB phosphorylation was further substantiated by the observations that lysoplasmenylcholine increased both the phosphorylation of CREB and the induction of c-fos expression in the absence and presence of BEL. CREB phosphorylation in both ischemic hearts and lysoplasmenylcholine-perfused hearts was inhibited by pretreatment of hearts with the specific cAMP-dependent protein kinase (PKA) inhibitor H-89. Taken together, these data demonstrate that iPLA(2) mediates CREB phosphorylation through a PKA-dependent pathway during brief periods of myocardial ischemia, possibly through the formation of lysophospholipids.

Activation of latent transforming growth factor beta1 by stromelysin 1 in extracts of growth plate chondrocyte-derived matrix vesicles.

Previous studies have shown that matrix vesicles isolated from cultures of costochondral growth zone chondrocytes and treated with 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] can activate recombinant human latent transforming growth factor beta1 (rhTGF-beta1). It is unknown what enzyme or other factor in the extracellular organelles is responsible for the activation. This study tested the hypothesis that enzymes present in matrix vesicles can activate latent TGF-beta1 and that this is regulated by 1alpha,25(OH)2D3. To do this, we examined the ability of matrix vesicle extracts to activate small latent rhTGF-beta1. In addition, enzymes previously determined to be present in matrix vesicles were screened for their ability to activate small latent rhTGF-beta1. Recombinant human matrix metalloproteinase 2 (rhMMP-2; 72 kDa gelatinase), rhMMP-3 (stromelysin 1), purified human plasminogen, and purified urokinase (plasminogen activator) were each tested at varying concentrations. To assess the role of cell maturation, we used a cell culture model in which chondrocytes are derived from two distinct zones of rat costochondral cartilage, the resting zone and the growth zone. Matrix vesicles were isolated from these cultures and then tested. The results showed that extracts of matrix vesicles produced by both growth zone and resting zone chondrocytes were able to activate small latent rhTGF-beta1. The effects were dose and time dependent, with greater activity being found in extracts of matrix vesicles from the growth zone chondrocyte cultures. Only rhMMP-3 was able to activate small latent rhTGF-beta1, indicating that stromelysin-1, but not MMP-2, plasminogen, or urokinase, was involved. As observed in the extracts, the effect of rhMMP-3 was time and dose dependent. When anti-MMP-3 antibody was added to matrix vesicle extracts from both cell types, activation of small latent rhTGF-beta1 was dose-dependently blocked. Neither 1alpha,25(OH)2D3 nor 24R,25(OH)2D3 had a direct effect on activation of small latent rhTGF-beta1 by the extracts. However, when intact matrix vesicles were treated with 1alpha,25(OH)2D3, their ability to activate small latent rhTGF-beta1 was increased. Inhibition of phospholipase A2 with quinacrine blocked the 1alpha,25(OH)2D3-dependent effect. These results suggest that the ability of 1alpha,25(OH)2D3-treated matrix vesicles to activate small latent TGF-beta1 is via action of the secosteroid on the matrix vesicle membrane, not on the enzymes responsible for activating latent TGF-beta1. Because matrix vesicles isolated from growth zone chondrocytes have been shown to contain increased phospholipase A2 activity after treatment with 1alpha,25(OH)2D3, it is likely that this secosteroid promotes loss of membrane integrity through phospholipase A2-dependent formation of lysophospholipids, resulting in the release of MMP-3 into the matrix, where latent TGF-beta1 is stored. Taken together, the results of the current study show that matrix vesicles produced by growth plate chondrocytes contain MMP-3, that this enzyme is at least partially responsible for activation of small latent TGF-beta1 in the matrix, and that 1alpha,25(OH)2D3 regulates MMP release from matrix vesicles.

Endothelial degradation of extracellular lyso-phosphatidylcholine

Formation of lysophospholipids, including lyso-phosphatidylcholine (lysoPC), is enhanced during oxidation of low-density lipoprotein, in ischaemic tissue and under inflammatory conditions. Besides being potentially cytotoxic, extracellular lysoPC induces changes in several properties of vascular endothelial cells. These include expression of endothelial adhesion molecules and interference with the endothelial production of nitrogen monoxide, prostacyclin and growth factors. One way of controlling the concentration of extracellular lysoPC is by the action of lysophospholipases, which degrade lysoPC into a free fatty acid and glycerophosphocholine. We therefore tested whether vascular endothelial cells have the ability to degrade extracellular lysoPC. Monolayers of primary cultures of human umbilical vein endothelial cells degraded an average of 84+/-24 nmol lysoPC/10(6) cells/2 h. By comparison, monocytes degraded 9.7+/-3.7 nmol lysoPC/10(6) cells/2 h, and erythrocytes and platelets < 1 nmol lysoPC/10(6) cells/2 h. The ability of endothelial cells to degrade extracellular phospholipids (diacylphosphatidyl choline) was found to be relatively low (9.5+/-6.4 nmol/10(6) cells/2 h). Triacylglycerol hydrolase activity was just above detection level. In conclusion, endothelial cells seem to degrade extracellular lysoPC effectively. This endothelial property may be important in controlling plasma and tissue levels of extracellular lysoPC as well as in the interaction between lysoPC and the vascular endothelium.

Preterm labor and bacterial intraamniotic infection: Arachidonic acid liberation by phospholipase A 2 of Fusobacterium nucleatum

Objective: The studies presented in this report were undertaken to evaluate whether Fusobacterium nucleatum , a common anaerobic isolate in intrauterine infection, stimulates arachidonic acid metabolism, a rate-limiting step for prostaglandin synthesis, in the human uterine endometrium. Study Design: Effects of F nucleatum on arachidonic acid liberation from human uterine endometrial cells and of F nucleatum extract on lysophosphatidylcholine production in human uterine endometrial cells were investigated. Results: When human uterine endometrial cells labeled with tritiated arachidonic acid to an isotopically steady state were exposed to an extract of F nucleatum , arachidonic acid liberation was stimulated, accompanied by lysophospholipid formation. Similar stimulatory effects on phospholipid degradation were also observed in the experiment with bacterially conditioned media. Conclusions: These results suggest that F nucleatum stimulates endometrial phospholipid metabolism, related to activity of phospholipase A 2 , which might induce the onset of labor associated with intraamniotic infection. (Am J Obstet Gynecol 1998;179:1579-82.)

Preterm Labor and Bacterial Intra-amniotic Infection: Arachidonic Acid Liberation by Phospholiphase A2ofPrevotella bivia

There is a strong association between preterm labor and infection, presumably through an increase in prostaglandin formation. The studies presented in this report were undertaken to evaluate whetherPrevotella bivia, a common anaerobic isolate of intrauterine infection, stimulates arachidonic acid metabolism, as a rate-limiting step for prostaglandin synthesis in the human uterine endometrium. When human uterine endometrial cells prelabeled with [3H]arachidonic acid to an isotopically steady state were exposed to an extract ofP. bivia, arachidonic acid liberation was stimulated, accompanied by lysophospholipid formation. Similar stimulatory effect on phospholipid degradation was also observed in the experiment with the bacterial conditioned media which was spent as culture media. These results suggests thatP. biviastimulates endometrial phospholipid metabolism, related with activity of phospholipase A2, which might induce the onset of labor associated with intra-amniotic infection.

Activation of Liver Mitochondrial Phospholipase A2 by Superoxide

Mitochondrial damage is one of the prominent features of cell injury during oxidative stress and altered mitochondrial lipids may contribute to this damage. Lipid changes were observed when liver mitochondria were exposed to superoxide generating systems. Phosphatidylcholine and phosphatidylethanolamine contents were decreased with simultaneous formation of lysophospholipids when exposed to superoxide. Among the neutral lipids there was an increase in the level of free fatty acids. This alteration in lipid composition could be prevented by the simultaneous presence of superoxide dismutase or phospholipase A2 (PLA2) inhibitors. H2O2did not have any effect on liver mitochondrial PLA2. This suggests that superoxide anion stimulates phospholipase A2 which is prevented by superoxide scavenging agents and PLA2 inhibitors. The products of phospholipase A2 are membrane-damaging agents which may be responsible for mitochondrial damage seen during oxidative stress.

Carnitine palmitoyl-transferase enzyme inhibition protects proximal tubules during hypoxia

The role of inhibition of the CPT enzymes responsible for accumulation of long chain acylcarnitines (LCAC) during hypoxia in the proximal tubule has not been previously examined. We have characterized CPT enzyme activities in mitochondrial fractions of rabbit proximal tubules. Malonyl CoA-sensitive CPT I activity (1.1 +/- 0.3 nmol/min/mg protein), and detergent-solubilized, malonyl CoA-insensitive CPT II activity (2.3 +/- 0.4 nmol/min/mg protein) were readily detected in proximal tubule mitochondrial fractions. Subjecting rabbit proximal tubules to various periods of hypoxia did not significantly change mitochondrial CPT I or CPT II activities. Thirty minutes of hypoxia resulted in an increase in lysophospholipid mass from 440 +/- 105 to 720 +/- 93 pmol/mg protein, N = 5, LCAC mass from 79 +/- 11 to 618 +/- 34 pmol/mg protein, N = 5, and lactate dehydrogenase (LDH) release from 9 +/- 1% to 46 +/- 3%, N = 8. Pretreatment of proximal tubules with two different CPT inhibitors, glybenclamide (Glyb) 400 microM and oxfenicine (Oxfe) 1 mM, resulted in reduction in the magnitude of hypoxia-induced lysophospholipid formation 490 +/- 160 (Glyb), 342 +/- 150 pmol/mg protein (Oxfe), N = 4, hypoxia-induced LCAC formation 295 +/- 27 (Glyb), 128 +/- 16 pmol/mg protein (Oxfe). N = 5, and LDH release 25 +/- 1% (Glyb) and 19 +/- 2% (Oxfe), N = 8. The protective effect of CPT inhibition was also associated with increased production of lactate suggesting the modulation of a substrate-mediated metabolic switch. Immunoblots demonstrated that hypoxia caused a time dependent hydrolysis of fodrin-alpha subunit and that CPT inhibition protected against hypoxia-induced fodrin proteolysis. These data suggest a unifying hypothesis that links phospholipase A2 (PLA2) activation, and hypoxia-mediated fodrin proteolysis to the proximal tubule mitochondrial CPT system. I propose that CPT inhibition may represent a novel mechanism to ameliorate proximal tubule cell death during hypoxia.

A requirement for membrane-associated phospholipase A2 in platelet cytotoxicity activated by receptors for immunoglobulin G and complement.

Platelets are potent antibody- and complement-dependent cytotoxic effector cells. We showed previously that a single platelet can lyse a target cell sensitized with immunoglobulin G (IgG) and complement components up to C3 (C integral of 3b denotes the target cell-bound fragment of complement up to C3; the precise nature of the bound C3 fragment has not been established), and that the complete cytotoxic system capable of specific recognition and lysis resides in platelet membranes. To define the components of platelet membranes required for cytotoxicity, a set of inhibitors of phospholipase A2 (PLA2) that act by different chemical mechanisms was tested. The lytic reaction is blocked at appropriate concentrations of bromophenacylbromide, mepacrine, and manoalide. When platelets are treated with bromophenacylbromide, inhibition of cytolytic activity and that of PLA2 enzymatic activity occur in parallel. Platelets release arachidonate when incubated with target cells bearing IgG and C integral of 3b, confirming that Fc gamma R and complement receptor trigger both PLA2 action and efficient lysis. Inhibition by thimerosal of a reverse reaction, i.e., reacylation catalyzed by acyltransferase, causes increased target cell lysis, presumably by increasing the products of PLA2 action. Platelet cytotoxicity is increased when platelets are pretreated with some products of PLA2: exogenous lysophospholipids and not free arachidonic acid increase cytotoxicity. Electron microscopy suggests that platelets and target cells may fuse, possibly as a result of the formation of lysophospholipids which are well-known membrane fusogens. Fixation with paraformaldehyde does not affect platelet cytotoxicity, suggesting that the complete cytotoxic system resides as a preformed complex in platelet membranes. The results indicate that platelet membrane-associated PLA2, together with receptors for Fc and complement, are required for platelet cytotoxicity.

IL-1α increases arachidonyl-COA: Lysophospholipid acyltransferase activity and stimulates [ 3H] arachidonate incorporation into phospholipids in rat mesangial cells

The proinflammatory cytokine interleukin-1α is a potent stimulus of prostaglandin synthesis. We have previously shown that IL-1 amplifies mesangial cell prostaglandin synthesis by inducing synthesis of a non-pancreatic phospholipase A 2. Phospholipase A 2 activation results in the formation of lysophospholipids and free fatty acids. We now investigate the effects of IL-1α on reacylation of lysophospholipids. Incubations with IL-1α for 24 hours significantly stimulated mesangial cell [ 3H] arachidonic acid incorporation but not [ 3H] oleic acid incorporation into phosphatidylinositol and phosphatidylethanolamine. Lysophospholipid acyltransferase activity was measured in vitro. Cytokine treatment increased enzyme activity when lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidylinositol were used as exogenous substrates. We conclude that IL-1 promotes cellular phospholipid remodeling by stimulating the deacylation and reacylation of phospholipids.

Regulation of arachidonic acid release in vascular endothelium. Ca2+-dependent and -independent pathways

Ca2+ metabolism and its relationship to arachidonic acid release were studied in cultured pig aortic endothelial cells. When cells were treated with bradykinin, a rapid rise in intracellular Ca2+ concentration ([Ca2+]i) occurred. Arachidonic acid release from cells prelabelled with [3H]arachidonic acid and subjected to flow-through conditions closely followed the changes in [Ca2+]i. Attenuation of the Ca2+ response by chelating extracellular and intracellular Ca2+ or by desensitization of receptors led to comparable attenuation of arachidonate release. Activation of protein kinase C inhibited Ca2+ mobilization in response to bradykinin and stimulated arachidonic acid release. Inhibition of protein kinase C had no effect on bradykinin-stimulated arachidonic acid release, suggesting that protein kinase C does not mediate the bradykinin response. The role of GTP-binding regulatory proteins (G-proteins) in mediating the bradykinin response was also investigated. Bradykinin-stimulated arachidonic acid release was not diminished by preincubation with pertussis toxin. Treatment with the G-protein activator AlF4- resulted in the release of a large pool of arachidonic acid and the formation of lysophospholipids. Combined treatment with AlF4- and bradykinin resulted in a greater than additive effect on arachidonic acid release. In contrast with bradykinin, AlF(4-)-stimulated arachidonic acid release was not dependent on the presence of extracellular Ca2+ or the mobilization of intracellular Ca2+. These results demonstrate Ca(2+)-dependent (bradykinin) and Ca(2+)-independent (AlF4-) pathways of phospholipase A2 activation.