Accumulation Of Lysophospholipids Research Articles

Phospholipase A2 group IVD mediates the transacylation of glycerophospholipids and acylglycerols

In mammalian cells, glycerolipids are mainly synthesized using acyl-CoA–dependent mechanisms. The acyl-CoA–independent transfer of fatty acids between lipids, designated as transacylation reaction, represents an additional mechanism for lipid remodeling and synthesis pathways. Here, we demonstrate that human and mouse phospholipase A2 group IVD (PLA2G4D) catalyzes transacylase reactions using both phospholipids and acylglycerols as substrates. In the presence of monoglycerol and diacylglycerol (MAG and DAG), purified PLA2G4D generates DAG and triacylglycerol, respectively. The enzyme also transfers fatty acids between phospholipids and from phospholipids to acylglycerols. Overexpression of PLA2G4D in COS7 cells enhances the incorporation of polyunsaturated fatty acids into triacylglycerol stores and induces the accumulation of lysophospholipids. In the presence of exogenously added MAG, the enzyme strongly increases cellular DAG formation, while MAG levels are decreased. PLA2G4D is not or poorly detectable in commonly used cell lines. It is expressed in keratinocytes, where it is strongly upregulated by proinflammatory cytokines. Pla2g4d-deficient mouse keratinocytes exhibit complex lipidomic changes in response to cytokine treatment, indicating that PLA2G4D is involved in the remodeling of the lipidome under inflammatory conditions. Transcriptomic analysis revealed that PLA2G4D modulates fundamental biological processes including cell proliferation, differentiation, and signaling. Together, our observations demonstrate that PLA2G4D has broad substrate specificity for fatty acid donor and acceptor lipids, allowing the acyl-CoA-independent synthesis of both phospholipids and acylglycerols. Loss-of-function studies indicate that PLA2G4D affects metabolic and signaling pathways in keratinocytes, which is associated with complex lipidomic and transcriptomic alterations.

Discovery of coordinately regulated pathways that provide innate protection against interbacterial antagonism.

Bacterial survival is fraught with antagonism, including that deriving from viruses and competing bacterial cells. It is now appreciated that bacteria mount complex antiviral responses; however, whether a coordinated defense against bacterial threats is undertaken is not well understood. Previously, we showed that Pseudomonas aeruginosa possess a danger-sensing pathway that is a critical fitness determinant during competition against other bacteria. Here, we conducted genome-wide screens in P. aeruginosa that reveal three conserved and widespread interbacterial antagonism resistance clusters (arc1-3). We find that although arc1-3 are coordinately activated by the Gac/Rsm danger-sensing system, they function independently and provide idiosyncratic defense capabilities, distinguishing them from general stress response pathways. Our findings demonstrate that Arc3 family proteins provide specific protection against phospholipase toxins by preventing the accumulation of lysophospholipids in a manner distinct from previously characterized membrane repair systems. These findings liken the response of P. aeruginosa to bacterial threats to that of eukaryotic innate immunity, wherein threat detection leads to the activation of specialized defense systems.

The phospholipid-repair system LplT/Aas in Gram-negative bacteria protects the bacterial membrane envelope from host phospholipase A2 attack

Secretory phospholipases A2 (sPLA2s) are potent components of mammalian innate-immunity antibacterial mechanisms. sPLA2 enzymes attack bacteria by hydrolyzing bacterial membrane phospholipids, causing membrane disorganization and cell lysis. However, most Gram-negative bacteria are naturally resistant to sPLA2 Here we report a novel resistance mechanism to mammalian sPLA2 in Escherichia coli, mediated by a phospholipid repair system consisting of the lysophospholipid transporter LplT and the acyltransferase Aas in the cytoplasmic membrane. Mutation of the lplT or aas gene abolished bacterial lysophospholipid acylation activity and drastically increased bacterial susceptibility to the combined actions of inflammatory fluid components and sPLA2, resulting in bulk phospholipid degradation and loss of colony-forming ability. sPLA2-mediated hydrolysis of the three major bacterial phospholipids exhibited distinctive kinetics and deacylation of cardiolipin to its monoacyl-derivative closely paralleled bacterial death. Characterization of the membrane envelope in lplT- or aas-knockout mutant bacteria revealed reduced membrane packing and disruption of lipid asymmetry with more phosphatidylethanolamine present in the outer leaflet of the outer membrane. Moreover, modest accumulation of lysophospholipids in these mutant bacteria destabilized the inner membrane and rendered outer membrane-depleted spheroplasts much more sensitive to sPLA2 These findings indicated that LplT/Aas inactivation perturbs both the outer and inner membranes by bypassing bacterial membrane maintenance mechanisms to trigger specific interfacial activation of sPLA2 We conclude that the LplT/Aas system is important for maintaining the integrity of the membrane envelope in Gram-negative bacteria. Our insights may help inform new therapeutic strategies to enhance host sPLA2 antimicrobial activity.

Analysis of Lysophospholipid Content in Low Phytate Rice Mutants.

As a fundamental component of nucleic acids, phospholipids, and adenosine triphosphate, phosphorus (P) is critical to all life forms, however, the molecular mechanism of P translocation and distribution in rice grains are still not understood. Here, with the use of five different low phytic acid (lpa) rice mutants, the redistribution in the main P-containing compounds in rice grain, phytic acid (PA), lysophospholipid (LPL), and inorganic P (Pi), was investigated. The lpa mutants showed a significant decrease in PA and phytate-phosphorus (PA-P) concentration with a concomitant increase in Pi concentration. Moreover, defects in the OsST and OsMIK genes result in a great reduction of specific LPL components and LPL-phosphorus (LPL-P) contents in rice grain. In contrast, defective OsMRP5 and Os2-PGK genes led to a significant increase in individual LPL components. The effect of the Os2-PGK gene on the LPL accumulation was validated using breeding lines derived from a cross between KBNT-lpa (Os2-PGK mutation) and Jiahe218. This study demonstrates that these rice lpa mutants lead to the redistribution of Pi in endosperm and modify LPL biosynthesis. Increase LPLs in the endosperm in the lpa mutants may have practical applications in rice breeding to produce "healthier" rice.

Bioactive lysophospholipids generated by hepatic lipase degradation of lipoproteins lead to complement activation via the classical pathway.

We determined bioactivity of lysophospholipids generated by degradation of the low-density (LDL), very low-density (VLDL), and high-density (HDL) lipoproteins with hepatic lipase (HL), cholesterol esterase (CE), and lipoprotein-associated phospholipase A2 (Lp-PLA2). The LDL, VLDL, and HDL were treated with HL, CE, and Lp-PLA2 after immobilization on plates, and complement activation studies were performed with diluted human serum. Complement component 3 (C3) fixation, a marker for complement activation, was determined with a monoclonal anti-human C3d antibody. Enzymatic properties of HL and CE were assayed with triglyceride and phosphatidylcholine substrates for triglyceride hydrolase and phospholipase A activities. The ARPE-19 cells were used for viability studies. The HL degradation of human lipoproteins LDL, VLDL, or HDL results in the formation of modified lipoproteins that can activate the complement pathway. Complement activation is dose- and time-dependent upon HL and occurs via the classical pathway. Enzymatic studies suggest that the phospholipase A1 activity of HL generates complement-activating lysophospholipids. C-reactive protein (CRP), known to simultaneously interact with complement C1 and complement factor H (CFH), further enhances HL-induced complement activation. The lysophospholipids, 1-Palmitoyl-sn-glycero-3-phosphocholine and 1-Oleoyl-sn-glycero-3-phosphocholine, can be directly cytotoxic to ARPE-19 cells. The HL degradation of lipoproteins, known to accumulate in the outer retina and in drusen, can lead to the formation of bioactive lysophospholipids that can trigger complement activation and induce RPE cellular dysfunction. Given the known risk associations for age-related macular degeneration (AMD) with HL, CRP, and CFH, this study elucidates a possible damage pathway for age-related macular degeneration (AMD) in genetically predisposed individuals, that HL activity may lead to accumulation of lysophospholipids to initiate complement activation, with CFH dysregulation exacerbating the effects of this process.

CPLA2α and EHD1 interact and regulate the vesiculation of cholesterol-rich, GPI-anchored, protein-containing endosomes

The lipid modifier phospholipase A2 catalyzes the hydrolysis of phospholipids to inverted-cone-shaped lysophospholipids that contribute to membrane curvature and/or tubulation. Conflicting findings exist regarding the function of cytosolic phospholipase A2 (cPLA2) and its role in membrane regulation at the Golgi and early endosomes. However, no studies addressed the role of cPLA2 in the regulation of cholesterol-rich membranes that contain glycosylphosphatidylinositol-anchored proteins (GPI-APs). Our studies support a role for cPLA2α in the vesiculation of GPI-AP-containing membranes, using endogenous CD59 as a model for GPI-APs. On cPLA2α depletion, CD59-containing endosomes became hypertubular. Moreover, accumulation of lysophospholipids induced by a lysophospholipid acyltransferase inhibitor extensively vesiculated CD59-containing endosomes. However, overexpression of cPLA2α did not increase the endosomal vesiculation, implying a requirement for additional factors. Indeed, depletion of the "pinchase" EHD1, a C-terminal Eps15 homology domain (EHD) ATPase, also induced hypertubulation of CD59-containing endosomes. Furthermore, EHD1 and cPLA2α demonstrated in situ proximity (<40 nm) and interacted in vivo. The results presented here provide evidence that the lipid modifier cPLA2α and EHD1 are involved in the vesiculation of CD59-containing endosomes. We speculate that cPLA2α induces membrane curvature and allows EHD1, possibly in the context of a complex, to sever the curved membranes into vesicles.

Deletion of the metabolic transcriptional coactivator PGC1β induces cardiac arrhythmia.

Peroxisome proliferator-activated receptor-γ coactivators PGC1α and PGC1β modulate mitochondrial biogenesis and energy homeostasis. The function of these transcriptional coactivators is impaired in obesity, insulin resistance, and type 2 diabetes. We searched for transcriptomic, lipidomic, and electrophysiological alterations in PGC1β(-/-) hearts potentially associated with increased arrhythmic risk in metabolic diseases. Microarray analysis in mouse PGC1β(-/-) hearts confirmed down-regulation of genes related to oxidative phosphorylation and the electron transport chain and up-regulation of hypertrophy- and hypoxia-related genes. Lipidomic analysis showed increased levels of the pro-arrhythmic and pro-inflammatory lipid, lysophosphatidylcholine. PGC1β(-/-) mouse electrocardiograms showed irregular heartbeats and an increased incidence of polymorphic ventricular tachycardia following isoprenaline infusion. Langendorff-perfused PGC1β(-/-) hearts showed action potential alternans, early after-depolarizations, and ventricular tachycardia. PGC1β(-/-) ventricular myocytes showed oscillatory resting potentials, action potentials with early and delayed after-depolarizations, and burst firing during sustained current injection. They showed abnormal diastolic Ca(2+) transients, whose amplitude and frequency were increased by isoprenaline, and Ca(2+) currents with negatively shifted inactivation characteristics, with increased window currents despite unaltered levels of CACNA1C RNA transcripts. Inwardly and outward rectifying K(+) currents were all increased. Quantitiative RT-PCR demonstrated increased SCN5A, KCNA5, RYR2, and Ca(2+)-calmodulin dependent protein kinase II expression. PGC1β(-/-) hearts showed a lysophospholipid-induced cardiac lipotoxicity and impaired bioenergetics accompanied by an ion channel remodelling and altered Ca(2+) homeostasis, converging to produce a ventricular arrhythmic phenotype particularly during adrenergic stress. This could contribute to the increased cardiac mortality associated with both metabolic and cardiac disease attributable to lysophospholipid accumulation.

Influence of lysophospholipid hydrolysis by the catalytic domain of neuropathy target esterase on the fluidity of bilayer lipid membranes

Neuropathy target esterase (NTE) is an integral membrane protein localized in the endoplasmic reticulum in neurons. Irreversible inhibition of NTE by certain organophosphorus compounds produces a paralysis known as organophosphorus compound-induced delayed neuropathy. In vitro, NTE has phospholipase/lysophospholipase activity that hydrolyses exogenously added single-chain lysophospholipids in preference to dual-chain phospholipids, and NTE mutations have been associated with motor neuron disease. NTE's physiological role is not well understood, although recent studies suggest that it may control the cytotoxic accumulation of lysophospholipids in membranes. We used the NTE catalytic domain (NEST) to hydrolyze palmitoyl-2-hydroxy- sn-glycero-3-phosphocholine (p-lysoPC) to palmitic acid in bilayer membranes comprising 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) and the fluorophore 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]- sn-glycero-3-phosphocholine (NBD-PC). Translational diffusion coefficients ( D L ) in supported bilayer membranes were measured by fluorescence recovery after pattern photobleaching (FRAPP). The average D L for DOPC/p-lysoPC membranes without NEST was 2.44 µm 2s -1 ± 0.09; the D L for DOPC/p-lysoPC membranes containing NEST and diisopropylphosphorofluoridate, an inhibitor, was nearly identical at 2.45 ± 0.08. By contrast, the D L for membranes comprising NEST, DOPC, and p-lysoPC was 2.28 ± 0.07, significantly different from the system with inhibited NEST, due to NEST hydrolysis. Likewise, a system without NEST containing the amount of palmitic acid that would have been produced by NEST hydrolysis of p-lysoPC was identical at 2.26 ± 0.06. These results indicate that NTE's catalytic activity can alter membrane fluidity.

A Decrease in Remodeling Accounts for the Accumulation of Arachidonic Acid in Murine Mast Cells Undergoing Apoptosis

The goal of this study was to examine arachidonic acid (AA) metabolism by murine bone marrow-derived mast cells (BMMC) during apoptosis induced by cytokine depletion. BMMC deprived of cytokines for 12-48 h displayed apoptotic characteristics. During apoptosis, levels of AA, but not other unsaturated fatty acids, correlated with the percentage of apoptotic cells. A decrease in both cytosolic phospholipase A(2) expression and activity indicated that cytosolic phospholipase A(2) did not account for AA mobilization during apoptosis. Free AA accumulation is also unlikely to be due to decreases in 5-lipoxygenase and/or cyclooxygenase activities, since BMMC undergoing apoptosis produced similar amounts of leukotriene B(4) and significantly greater amounts of PGD(2) than control cells. Arachidonoyl-CoA synthetase and CoA-dependent transferase activities responsible for incorporating AA into phospholipids were not altered during apoptosis. However, there was an increase in arachidonate in phosphatidylcholine (PC) and neutral lipids concomitant with a 40.7 +/- 8.1% decrease in arachidonate content in phosphatidylethanolamine (PE), suggesting a diminished capacity of mast cells to remodel arachidonate from PC to PE pools. Further evidence of a decrease in AA remodeling was shown by a significant decrease in microsomal CoA-independent transacylase activity. Levels of lyso-PC and lyso-PE were not altered in cells undergoing apoptosis, suggesting that the accumulation of lysophospholipids did not account for the decrease in CoA-independent transacylase activity or the induction of apoptosis. Together, these data suggest that the mole quantities of free AA closely correlated with apoptosis and that the accumulation of AA in BMMC during apoptosis was mediated by a decreased capacity of these cells to remodel AA from PC to PE.

Myocardial calcium-independent phospholipase A2 activity during global ischemia in isolated rabbit hearts

To study calcium-independent phospholipase A2 activity during global ischemia in isolated rabbit hearts by measuring the hydrolysis of the endogenous choline phospholipids. Langendorff perfused rabbit hearts were exposed to global ischemia for 15 or 60 min, or control perfusion for the same length of time. The hearts were then rapidly frozen in liquid nitrogen and lyophilized. Calcium-independent phospholipase A2 activity in the lyophilized tissue was studied by measuring accumulation of lysophospholipids resulting from hydrolysis of both the choline diacylphospholipid and the choline plasmalogen pool. The calcium-independent phospholipase A2 activity showed the same pH, temperature and calcium sensitivity in control and ischemic (15 min of ischemia) lyophilized myocardial tissue. Incubation of control and ischemic tissue showed no difference in the rate of accumulation of lysophospholipids when the ischemic tissue was obtained from hearts exposed to 15 min of ischemia (107 +/- 4 vs 111 +/- 7 nmol/g dry wt x min, ischemia versus control, mean +/- s.e.m., n = 8), but a significant decrease was noticed in tissue from hearts that had been exposed to 60 min of ischemia (31 +/- 9 vs 86 +/- 18 nmol/g dry wt x min, P < 0.05, n = 4). The decreased phospholipase A2 activity in tissue exposed to 60 min of ischemia was not due to enhanced metabolism of the lysophospholipids (84 +/- 15 vs 79 +/- 8 nmol/g dry wt x min, n = 4). The calcium-independent phospholipase A2 activity was considerably lower in fresh myocardial tissue compared with lyophilized tissue, but comparison of control and ischemic fresh tissue gave results comparable to those found using lyophilized tissue. The myocardial calcium-independent phospholipase A2 activity showed no plasmalogen selectivity in either control or ischemic myocardium. In isolated perfused rabbit hearts we found no evidence for activation of calcium-independent phospholipase A2 activity during global ischemia. With prolonged time of ischemia there was a significant decrease in calcium-independent phospholipase A2 activity.

Phospholipid Degradation in Rat Calcium Ionophore-Activated Platelets Is Catalyzed Mainly by Two Discrete Secretory Phospholipase As

An "A1 type" phospholipase activity with serine-phospholipid preference was released by rat activated platelets. It was distinct from the secretory type II phospholipase A2 [Horigome, K., Hayakawa, M., Inoue, K., and Nojima, S. (1987) J. Biochem. 101, 625-631] and co-purified with the secretory lysophosphatidylserine-selective lysophospholipase activity [Higashi, S., Kobayashi, T., Kudo, I., and Inoue, K. (1988) J. Biochem. 103, 442-447]. Several lines of evidence indicated that a single protein was responsible for the phospholipase A1 and lysophospholipase activities. Marked accumulation of lysophospholipids was observed in rat calcium ionophore-activated washed platelets and both phospholipase A1/lysophospholipase and type II phospholipase A2 were shown to contribute to this phospholipid degradation. A selective inhibitor of type II phospholipase A2 reduced the phospholipid degradation and enhanced the clotting time and prothrombinase activity. These results indicate that secretory platelet phospholipases may play a role in regulation of blood clotting.

Characterization of Rabbit Myocardial Phospholipase A2 Activity Using Endogenous Phospholipid Substrates

We have developed an assay for studying myocardial phospholipase A2 activity by measuring accumulation of lysophospholipids resulting from hydrolysis of the endogenous choline glycerophospholipid pool. This assay was used to characterize phospholipase A2 activity in rabbit myocardium. Lyophilized rabbit myocardium was incubated at 37°C in Tris-HCl buffer containing either ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid (EGTA)/EDTA or calcium, and palmitoyl-lysophosphatidylcholine (P-LPC), oleoyl-LPC, stearoyl-LPC, and 16:0-lysoplasmenylcholine were measured using a recently developed HPLC method. The identity of the individual species was confirmed by ion-spray LC-MS-MS. In the presence of EGTA/EDTA, incubation for up to 30 min caused a linear increase in all lysophospholipids. The main increases were found in P-LPC and 16:0-lysoplasmenylcholine, which increased by 37 ± 3 (mean ± SE, N = 8) and 48 ± 3 nmol/g dry wt × min, respectively. The apparent phospholipase A2 activity was found to be calcium, temperature, and pH sensitive. The pH optimum was between 6.5 and 8.0, and incubation at room temperature and 45°C decreased the activity by 80 and 40%, respectively. Studies of the metabolism of the formed lysophospholipids showed a substantial metabolism of the lysophospholipids that accounted for about 40% of the total phospholipase A2 activity. This method offers a novel approach to study phospholipase A2 activities by measuring accumulation of products resulting from hydrolysis of endogenous phospholipid pools.

Glycerophospholipid:cholesterol acyltransferase complexed with lipopolysaccharide (GCAT‐LPS) of Aeromonas salmonicida produces lysophospholipids in salmonid red cell membranes: a probable haemolytic mechanism

Abstract. The enzymic activity of GCAT‐LPS was studied using whole blood (in vivo), citrated blood or washed red blood cells as substrate. Results demonstrated in vitro and in vivo haemolysis of Atlantic salmon, Salmo salar L., red blood cells, and in vitro haemolysis of rainbow trout, Oncorhynchus mykiss (Walbaum), red blood cells, by purified GCAT‐LPS. Haemolysis coincided with 10% or more lysophospholipids, as a per cent of total phospholipids, in the red cell membranes. Addition of soybean lysophosphatidylcholine to citrated whole blood from salmon caused haemolysis. This suggests that it is the observed accumulation of lysophospholipids, caused by the enzymic activity of GCAT‐LPS, which leads to lysis of salmonid red blood cells. When membranes were used as substrate, GCAT‐LPS seemed to express acyltransferase and possibly some phospholipase activity, while accumulation of lysophospholipids suggests that the lysophospholipase activity is inhibited or severely suppressed. Deacylation of both phosphatidylcholine and phosphatidylethanolamine was observed, indicating that both phospholipids are substrates for the enzyme.

Possible implication of lysophosphatidylcholine in cell fusion accompanying implantation in rabbits.

Implantation in rabbits involves the cellular fusion of trophoblastic and uterine epithelial cells resulting in embryo penetration of the uterine endometrium. Since lysophospholipids, known to have fusigenic properties, could be responsible for this cell fusion, the metabolism of lysophospholipids was studied throughout gestation in blastocyst/yolk sac and extracoelic amnioallantoic fluids. Analysis of phospholipid composition revealed that lysophospholipids are present in blastocyst/yolk sac fluid. Their concentrations and haemolytic activity change during pregnancy. They increase and reach their highest values during days 7 to 9, the implantation days in rabbits. A clear correlation was observed between lysophosphatidylcholine concentrations in blastocyst/yolk sac fluid and haemolysis induced by this fluid. Phosphatidylcholine concentrations, phospholipase A2 activity, which generates lysophospholipids, and lysophospholipase A activity which hydrolyses lysophosphatidylcholine into fatty acid, were at their highest value at day 12. These data suggest that a transient accumulation of lysophospholipids could ensure local cell fusion. Moreover, we propose that the lysophospholipid concentrations in blastocyst/yolk sac fluid are dependent upon activities of phospholipase A2 and lysophospholipase.

The effects of a PAF antagonist on ischemic myocardial damage and arrhythmia in the dog

Myocardial ischemia is associated with accumulation of lyso-phospholipids, including lyso-platelet activating factor, the degradation product and precursor of platelet activating factor. These compounds produce cellular and microvascular damage and, in the myocardium, depression of contractility and arrhythmia. The potent platelet activating factor antagonist, WEB 2086, or placebo, was infused (IV) 10 min before constriction of the proximal left anterior descending coronary artery in open-chest dogs. Two protocols were followed: the dose of WEB 2086 was 0.5 mg/kg in those subjected to 20 min ischemia with 10 min reperfusion ( n = 40) and 5 mg/kg preceding 60 min ischemia alone ( n = 24). There was no significant difference in the number of ventricular premature complexes between WEB 2086 and placebo treated dogs during either period of ischemia. On reperfusion in those surviving 20 min of ischemia, 5 of the 18 WEB 2086 and 9 of the 18 placebo treated dogs developed ventricular fibrillation ( ns). After 60 min of myocardial ischemia, there was no statistical difference in histological changes (nuclear swelling, aggregation of chromatin, myofibrillar separation) between groups. Hence, no substantial effect of relatively large doses of WEB 2086 on ischemia-induced histological change or arrhythmia was found in this preparation.

Mobilization of cellular Ca 2+ by lysophospholipids in rat islets of Langerhans

To determine whether lysophospholipids mobilize cellular Ca 2+, intact rat islets were prelabelled with 45Ca 2+ and subjected to three maneuvers designed to simulate the physiologic accumulation of lysophospholipids: (1) exogenous provision; (2) addition of porcine pancreatic phospholipase A 2; and (3) provision of p-hydroxymercuribenzoic acid, which impedes both the reacylation and hydrolysis of endogenous lysophospholipids, leading to their accumulation in islets. Each maneuver provoked 45Ca 2+ efflux at concentrations nearly identical to those previously reported to induce insulin release in the absence of toxic efffects on the islets. Lysophosphatidylcholine (lysoPC) and lysophosphatidylinositol were active, whereas the ethanolamine and serine derivatives, and lysophosphatidic acid, were much less effective. The effects of lysoPC were reversible; they also were reduced by lanthanum or gentamicin (which are probes of superficial, plasma membrane-bound stores of Ca 2+) or by prior depletion of membrane-bound cellular Ca 2+ stores using ionomycin, but not by removal of extracellular Ca 2+ or Na +. The effects of lysoPC, phospholipase A 2 and p-hydroxymercuribenzoic acid were largely independent of any hydrolysis to, or accumulation of, free fatty acids as assessed by resistance to dantrolene or trifluoperazine (which selectively reduce arachidonic acid-induced 45Ca 2+ efflux and insulin release). Thus, lysophospholipids are a newly recognized class of lipid mediators which may promote insulin release at least in part via mobilization of a pool(s) of Ca 2+ (‘trigger Ca 2+’) bound in the plasma membrane and possibly in other cellular membranes.

Disturbance of Ca++-transport function and lipid component of sarcoplasmic reticulum membranes during total ischemia of the rat myocardium

In myocardial ischemia the contractile function of the heart is quickly disturbed. This is due to several factors, including injury to membranes of the sarcoplasmic reticulum (SR) [13]. Among the basic mechanisms of membrane damage an essential role is ascribed to activation of lipid peroxidation (LPO) and of phospholipolysis. Experiments in vitro have shown that phospholipase A2 [I0] and the xanthine--xanthine oxidase oxygen radical generation system [12] inhibit Calcium uptake by SR. Some investigators consider that the dominant role in damage to the Ca++-transport function of SR in ischemia is played by activation of LPO [4]. Activation of LPO in the liver microsomes during ischemia was observed much earlier than accumulation of lysophospholipids [2]. The determinant role of phospholipases in ischemic damage to the liver mitochondria [3] and an increase in the content of lysophospholipids even in the initial period of cardiac ischemia [8] have been demonstrated.

Lysophospholipase activity in rat brain subcellular fractions.

Lysophospholipase activity in brain subcellular fractions was measured by the release of myristic acid from 1-myristoylglycerophosphocholine or through the formation of [32P]glycerophosphocholine from [32P]lysophosphatidylcholine. Although the lysophospholipase activity was highest in microsomes, considerable enzyme activity was also found in other subcellular membrane fractions. The pH optimum for the microsomal enzyme was around 7, whereas the synaptosomes and non-synaptic plasma membranes exhibited a pH maximum around 8. Although the enzyme did not require divalent cations for activity, divalent cations (1 mM) such as Hg2+, Cu2+, and Zn2+ inhibited potently the enzyme activity. Enzyme activity was also partially inhibited by both saturated and polyunsaturated fatty acids (25-200 microM), and the inhibition seemed to be greater in the membrane than in the cytosolic fractions. Ionic detergents such as deoxycholate and taurocholate inhibited the lysophospholipase. On the other hand, the effect of Triton X-100 was biphasic, i.e., stimulation at concentrations below 100 micrograms/mg protein and inhibition at higher concentrations. Addition of cholesterol (50-250 micrograms/ml), but not cholesteryl esters, also potently inhibited enzyme activity. The presence of active lysophospholipase(s) in brain is probably an important mechanism for preventing unnecessary accumulation of lysophospholipids which may exert a deleterious effect on the membranes because of their detergent properties.

Alterations in the metabolism of lipids in ischemia of the liver and kidney.

The metabolism of lipids in the ischemic liver has been examined in the attempt to define the cause of the previously described loss of phospholipid and to determine whether additional alterations occur that may be related to the disturbances in membrane function. With 3 hr of ischemia, 30% of the cellular phospholipid was lost when measured either as phosphate in a lipid extract of the whole liver or as fatty acyl esters after separation by thin-layer chromatography of the major lipid classes in the same extracts. All phospholipid species were equally affected, and there was no accumulation of lysophospholipids. The loss of phospholipid acyl chains was not accompanied by an increased number of acyl esters as mono-, di-, or triglycerides. There was no increase in the size of the free fatty acid pool, and the content of long chain acyl CoA esters decreased by 50%. The acyl chain composition of the free fatty acid and neutral lipid pools changed, however, to resemble more closely that of the phospholipids. There was no change in the fatty acid composition of the phospholipids. The incorporation of intraportally injected [3H]arachidonic acid into total phospholipids was decreased upon reperfusion of liver that had been ischemic for only 20 min. These data are consistent with a loss of fatty acyl chains from the phospholipids into the free fatty acid pool. A few of these chains are incorporated into neutral lipids, but most are lost from the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural alterations of the inner mitochondrial membrane in ischemic liver cell injury

Mitoplasts were prepared from 3-h ischemic livers in an attempt to define the structural alterations in the inner membrane that may account for the functional deficiencies of ischemic mitochondria. Mitoplasts from both control and ischemic livers had similar specific activities of cytochrome oxidase and succinate-cytochrome c reductase. With both preparations, the specific activity of rotenone-insensitive NADH-cytochrome c reductase was 10-fold lower than in the mitochondria from which they were prepared. Ischemic mitoplasts had no respiratory control with ADP, and had a slightly reduced phospholipid to protein ratio and an increased cholesterol to protein ratio. As a result, the cholesterol to phospholipid molar ratio was increased from the control of 0.04 to 0.08. There were also differences in the content of individual phospholipid species. Phosphatidylcholine increased by 15%, while cardiolipin decreased by 60%. There were increases in sphingomyelin and in the lysophospholipids of phosphatidylcholine, ethanolamine, and cardiolipin. Pretreatment with chlorpromazine did not prevent these changes. Linoleic acid was decreased by 35% in ischemic phospholipids, and the content of free fatty acids was increased 4-fold. Electron spin resonance spectroscopy of mitoplasts spin labeled with either 5- or 12-doxyl stearic acid revealed an increased molecular order (decreased fluidity) of ischemic inner mitochondrial membranes consistent with the increased cholesterol to phospholipid ratio. The data indicate activation of a phospholipase A in ischemic mitochondria with the resulting accumulation of products of lipid hydrolysis. This conclusion further emphasizes the close similarity between the structural and functional consequences of ischemia in the intact animal and the effect on isolated mitochondria of the activation of the endogenous phospholipase A. In both cases the major functional alterations are attributable to changes in the permeability of the inner mitochondrial membrane induced by the accumulation of lysophospholipids.