Shape changes in human erythrocytes induced by replacement of the native phosphatidylcholine with species containing various fatty acids

Phosphatidylcholine-specific transfer protein from beef liver has been used to replace native phosphatidylcholine (PC) molecules from intact human erythrocytes by a variety of PC species differing in fatty acid composition. These replacements changed neither the total phospholipid content of the membrane, nor the composition of this fraction in terms of the various phospholipid classes. The morphology of the erythrocyte was not modified when native PC was replaced by 1-palmitoyl,2-oleoyl PC, 1-palmitoyl,2-linoleoyl PC, egg PC, or PC isolated from rat liver microsomes. Replacement with the disaturated species 1,2-dimyristoyl PC, 1,2-dipalmitoyl PC, and 1,2-distearoyl PC resulted in the formation of echinocytes and, at higher levels of replacement, in spheroechinocytes. Echinocyte-like erythrocytes were also observed after replacement with 1-palmitoyl,2-arachidonoyl PC, whereas stomatocytes were formed upon replacement with PC species containing two unsaturated fatty acids, e.g., 1,2-dioleoyl PC and 1,2-dilinoleoyl PC. The observations show that the erythrocyte membrane structure and the overall discoid cell shape of the human erythrocyte are optimally stabilized by PC species that contain one saturated and one mono- or diunsaturated fatty acid, and that the cell tolerates only limited variations in the species composition of its PC.     

Modification of LDL with human secretory phospholipase A(2) or sphingomyelinase promotes its arachidonic acid-releasing propensity

Oxidation and lipolytic remodeling of LDL are believed to stimulate LDL entrapment in the arterial wall, expanding the inflammatory response and promoting atherosclerosis. However, the cellular responses and molecular mechanisms underlying the atherogenic effects of lipolytically modified LDL are incompletely understood. Human THP-1 monocytes were prelabeled with [(3)H]arachidonic acid (AA) before incubation with LDL or LDL lipolytically modified by secretory PLA(2) (sPLA(2)) or bacterial sphingomyelinase (SMase). LDL elicited rapid and dose-dependent extracellular release of AA in monocytes. Interestingly, LDL modified by sPLA(2) or SMase displayed a marked increase in AA mobilization relative to native LDL, and this increase correlated with enhanced activity of cytosolic PLA(2) (cPLA(2)) assayed in vitro as well as increased monocyte tumor necrosis factor-alpha secretion. The AA liberation was attenuated by inhibitors toward cPLA(2) and sPLA(2), indicating that both PLA(2) enzymes participate in LDL-induced AA release. In conclusion, these results demonstrate that LDL lipolytically modified by sPLA(2) or SMase potentiates cellular AA release and cPLA(2) activation in human monocytes. From our results, we suggest novel atherogenic properties for LDL modified by sPLA(2) and SMase in AA release and signaling, which could contribute to the inflammatory gene expression observed in atherosclerosis.     

Modulation of ATPase activities of human erythrocyte membranes by free fatty acids or phospholipase A2

Summary The artificial insertion of increasing amounts of unsaturated fatty acids into human erythrocyte membranes modulated ATPase activities in a biphasic manner, depending on the number and position of double bonds, their configuration, and the chain length. Uncharged long-chain fatty acid derivatives with double bonds and short-chain fatty acids were ineffective. Stearic acid stimulated Na⁺K⁺-ATPase only. Anionic and non-ionic detergents and -lysophosphatidylcholine failed to stimulate ATPase activities at low, and inhibited them at high concentrations.Mg²⁺-ATPase activity was maximally enhanced by a factor of 2 in the presence of monoenoic fatty acids; half-maximal stimulation was achieved at a molar ratio ofcis(trans)-configurated C18 acids/membrane phopholipid of 0.16 (0.26).Na⁺K⁺-ATPase activity was maximally augmented by 20% in the presence of monoenoic C18 fatty acids at 37°C. Half-maximal effects were attained at a molar ratio oleic (elaidic) acid/phospholipid of 0.032 (0.075). Concentrations of free fatty acids which inhibited ATPase activities at 37°C were most stimulatory at reduced temperatures. AT 10°C, oleic acid increased Na⁺K⁺-ATPase activity fivefold (molar ratio 0.22).Unsaturated fatty acids simulated the effect of calmodulin on Ca²⁺-ATPase of native erythrocyte membranes (i.e., increase ofV _max from 1.6 to 5 mol PO ₄ ^3– ·phospholipid^–1·hr^–1, decrease of K _Ca from 6 m to 1.4–1.8 m). Stearic acid decreasedK _Ca (2 m) only, probably due to an increase of negative surface charges.A stimulation of Mg²⁺-ATPase, Na⁺K⁺-ATPase, and Ca²⁺-ATPase could be achieved by incubation of the membranes with phospholipase A₂.An electrostatic segregation of free fatty acids by ATPases with ensuing alterations of surface charge densities and disordering of the hydrophobic environment of the enzymes provides an explanation of the results.     

Modulation of cyclooxygenase-2 expression by phosphatidylcholine specific phospholipase C and D in macrophages stimulated with lipopolysaccharide

Lipopolysaccharide (LPS) enhances the expression of cyclooxygenase 2 (COX-2) in macrophages, and stimulates production of prostaglandins that cause endothelial dysfunction in septic shock. In an effort to identify strategies for reducing LPS-inducible expression of COX-2, inhibitors of the phospholipases involved in LPS dependent over-expression of COX-2 were studied. LPS enhances expression of COX-2 mRNA and protein by activating sequentially phosphatidylcholine-specific phospholipase C (PC-PLC), protein kinase C (PKC) and phosphatidylcholine-specific phospholipase D (PC-PLD). This stimulates production of phosphatidic acid (PA), which increases expression of COX-2 mRNA and protein. Inhibition of PC-PLC by D609 (tricyclodecanoyl xanthogenate), and of PC-PLD activity by 1-butanol, reduced LPS-dependent over-production of PA and suppressed the increase of COX-2 mRNA and protein. Activation of PKC, normally seen in LPS-treated cells, was mimicked with phorbol myristic acid (PMA), and this also increased PA production and enhanced COX-2 expression. Propranolol inhibition of phosphatidic acid phosphohydrolase (PPH) increased PA accumulation and enhanced LPS-dependent COX-2 protein synthesis. These results suggest that inhibitors of PC-PLC, PKC and PC-PLD, or activators of PPH could be useful in the management of LPS-induced overproduction of prostaglandins and of vascular dysfunction in septic shock.     

Isolation and characterization of plasma membranes from Friend erythroleukaemic cells. A study with sphingomyelinase C

Plasma membranes have been prepared from Friend erythroleukaemic cells using a Dounce homogenization technique followed by differential and sucrose gradient centrifugations. (I) A plasma membrane fraction was obtained which showed a 20- to 30-fold enrichment in 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and in 32P-labeled (poly)phosphoinositides. About 1% of the total protein, 6-7% of phospholipid, 8-9% of cholesterol and 12-15% of each of the above markers were recovered in the plasma membrane fraction with an average yield of 15-20%. The plasma membrane was characterized by a high cholesterol to phospholipid molar ratio (0.626), a 2-fold enrichment in sphingomyelin and in phosphatidylserine as compared to the whole cell and by the complete absence of diphosphatidylglycerol. (2) When compared to the phospholipid composition of the mature mouse erythrocyte membrane, the plasma membrane of the Friend cell only differs by a higher phosphatidylcholine and a lower phosphatidylethanolamine content, whereas the levels of sphingomyelin and phosphatidylinositol plus phosphatidylserine are similar. (3) Friend cells were treated with sphingomyelinase C (S. aureus) under non-lytic conditions and subsequently submitted to subcellular fractionation. The results showed that the plasma membrane accounted for 38.5% of the total phospholipid, 64.1% of the total cholesterol and about 4.4% of the total protein content of Friend cells. (4) Sphingomyelin appeared to be asymmetrically distributed in the plasma membrane of Friend cells, with about 85% of this phospholipid being present in the outer monolayer.     

Differential hydrolysis of molecular species of lipoprotein phosphatidylcholine by groups IIA, V and X secretory phospholipases A2

Human groups IIA, V and X secretory phospholipases A2 (sPLA2s) were incubated with human HDL3, total HDL and LDL over a range of enzyme and substrate concentrations and exposure times. The residual phosphatidylcholines (PtdChos) were assayed by high performance liquid chromatography with electrospray ionization mass spectrometry (LC/ESI-MS). The enzymes varied markedly in their rates of hydrolysis of the different molecular species and in the production of lysoPtdCho. The sPLA2s were compared at a concentration of 1 microg/ml and an incubation time of 4 h, when all three enzymes showed significant activity. The groups V and X sPLA2 were up to 20 times more reactive than group IIA sPLA2. Group X sPLA2 hydrolyzed arachidonate and linoleate containing species preferentially, while group V hydrolyzed the linoleates in preference to polyunsaturates. In all instances, the arachidonoyl and linoleoyl palmitates were hydrolyzed in preference to the corresponding stearates by group X sPLA2. The group IIA enzyme appeared to hydrolyze randomly all diacyl molecular species. The minor alkylacyl and alkenylacyl glycerophosphocholines (GroPChos) were poor substrates for groups V and X sPLA2s and these phospholipids tended to accumulate. The present study demonstrates a preferential release of arachidonate from plasma lipoprotein PtdCho by group X sPLA2, as well as a relative resistance of polyunsaturated PtdChos to hydrolysis by group V enzyme, which had not been previously documented. The use of lipoprotein PtdCho as substrate with LC/ESI-MS identification of hydrolyzed molecular species eliminates much of the uncertainty about sPLA2 specificity arising from past analyses of fatty acid release from unknown or ill-defined sources.     

Stimulation of CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis by incubation of rat hepatocytes with phospholipase A2

The effect of phospholipase A2 treatment of rat hepatocytes on CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis was investigated. Cytidylyltransferase is recovered from the cytosol and in a membrane-bound form with the microsomes. Digitonin treatment of cells causes rapid release into the medium of the cytosolic, but not the microsomal form of the cytidylyltransferase. Incubation of hepatocytes for 10 min with phospholipase A2 (0.9 units/dish) in the medium, resulted in a 33% decrease in the cytidylyltransferase activity released by digitonin treatment (2.5 +/- 0.15 nmol/min per mg compared to 3.9 +/- 0.10 nmol/min per mg in the control). In agreement with the digitonin experiments, incubation with 0.9 units/dish of phospholipase A2 resulted in a decrease in the cytidylyltransferase activity in the cytosol (from 4.3 +/- 0.10 nmol/min per mg to 2.6 +/- 0.14 nmol/min per mg) and a corresponding increase in the microsomal fraction (from 0.9 +/- 0.16 nmol/min per mg to 1.8 +/- 0.20 nmol/min per mg). The effect of phospholipase A2 on cytidylyltransferase translocation was concentration- and time-dependent. Incubation of hepatocytes in the presence of phospholipase A2 (0.9 units/dish) for 10 min prior to pulse-chase experiments resulted in an increase in radiolabel incorporation into phosphatidylcholine (from 2.4 +/- 0.02.10(-5) dpm/dish to 3.1 +/- 0.1.10(-5) dpm/dish) and a corresponding decrease in radiolabel associated with the choline (from 2.5 +/- 0.05.10(-5) to 1.4 +/- 0.03.10(-5) dpm) and phosphocholine fractions (from 8.5 +/- 0.07.10(-5) to 6.9 +/- 0.05.10(-5) dpm). We conclude that phospholipase A2 can cause a stimulation of CTP: phosphocholine cytidylyltransferase activity and phosphatidylcholine synthesis in cultured rat hepatocytes.     

Hydrolysis of phosphatidylcholine by phospholipase A2 does not cause dissociation of apolipoprotein C from rat plasma very low density lipoprotein.

To test the hypothesis that hydrolysis of glycerophosphatides causes displacement of apolipoprotein C from very low density lipoprotein, we have studied the effects of a snake venom phospholipase A2 on very low density lipoprotein labeled with [125I]apoC, [3H]cholesterol, [14C]palmitate and [32P]phospholipids. In spite of hydrolysis of 97% of the phosphatidylcholine, only small amounts of labeled apoC and labeled cholesterol were displaced from the very low density lipoprotein. With purified lipoprotein lipase in contrast, 80-90% of the labeled apoC and cholesterol were removed from the lipoprotein. It is concluded that hydrolysis of phosphatidylcholine does not cause an appreciable dissociation of apolipoprotein C from very low density lipoprotein.     

Modulation of human erythrocyte Ca2+/Mg2+ ATPase activity by phospholipase A2 and proteases. A comparison with calmodulin

The human erythrocyte membrane $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase responded to the presence of an acidic phospholipase A₂ and to low levels of trypsin (and chymotrypsin) in much the same way as it did to calmodulin isolated from human erythrocytes. The increased concentration of ATP hydrolyzed in 1 hour was similar to that observed when calmodulin had been added to a suspension of membranes during the assay. The observations reported here strongly suggest that activation of the $\text{Ca}{}^{\mathrm{2+}}\text{M}{}^{\mathrm{2+}}$ ATPase can proceed by introducing apparently distinct perturbations either to the protein or to phospholipid domains of the erythrocyte membrane.     

Asymmetric manipulation of the membrane lipid bilayer of intact human erythrocytes with phospholipase A, C, or D induces a change in cell shape

Changes in the membrane morphology and phospholipid content of human erythrocytes were determined after incubation of intact cells with each of various exogeneous phospholipases (PLases). PLase A2 from Naja naja or bee venom induced crenation of the cells in parallel with hydrolysis of the membrane phosphatidylcholine (PC). This crenated cell shape was reversed to a biconcave disc or cup-like form by a further treatment with lysophospholipase. In contrast, bacterial PLase C from Clostridium perfringens and Pseudomonas aureofaciens or fungal PLase D from Streptomyces chromofuscus induced invagination of the cells in parallel with hydrolysis of the PC. The action of the latter group of PLases on the membrane morphology was counteracted by PLase A2, and vice versa. Thus, participation of the membrane lipid bilayer in the induction of membrane conformational change and hence cell shape change was demonstrated.     

Selective inhibition of human platelet phospholipase A2 by buffering cytoplasmic calcium with the fluorescent indicator quin 2. Evidence for different calcium sensitivities of phospholipases A2 and C

Human platelets labelled with either [14C]arachidonic acid or [32P]orthophosphate were loaded or not with the Ca2+ fluorescent indicator quin 2. They were then incubated in the presence or in the absence of human thrombin (1 U/ml) in a medium where Ca2+ concentration was adjusted near zero or to 1 mM. Under these conditions, phospholipase A2 activity, as detected by the release of [14C]arachidonate and of its metabolites, or by the hydrolysis of [14C]phosphatidylcholine, was severely impaired in quin 2-loaded platelets upon removal of external Ca2+. However, Ca2+ was not required in non-loaded platelets, where a maximal phospholipase A2 activity was detected in the absence of external Ca2+. In contrast, phospholipase C action, as determined from the amounts of [14C]diacylglycerol, [14C]- or [32P]phosphatidic acid formed, appeared to be much less sensitive to the effects of quin 2 loading and of Ca2+ omission. By using various concentrations of quin 2, it was found that the inhibitory effect exerted against phospholipase A2 could be overcome by external Ca2+ only when the intracellular concentration of the calcium chelator did not exceed 2 mM. At higher concentrations averaging 3.5 mM of quin 2, phospholipase A2 activity was fully suppressed even in the presence of external Ca2+, whereas phospholipase C was still active, although partly inhibited. It is concluded that platelet phospholipase A2 requires higher Ca2+ concentrations than phospholipase C to display a maximal activity. By comparing platelet phospholipase A2 activity under various conditions with the values of cytoplasmic free Ca2+ as detected by quin 2 fluorescence, it is proposed that cytoplasmic free Ca2+ in control platelets stimulated with thrombin can attain concentrations above 1 microM, probably close to 5-10 microM, as recently determined with the photoprotein aequorin (Johnson, P.C., Ware, J.A., Cliveden, P.B., Smith, M., Dvorak, A.M. and Salzman, E.W. (1985) J. Biol. Chem. 260, 2069-2076).     

A new species of virus-coded low molecular weight RNA from cells infected with adenovirus type 2

A virus-coded low molecular weight RNA (5.2S), which migrates slightly faster on polyacrylamide gels than the well characterized adenovirus-specific 5.5S RNA, has been isolated from cells infected with adenovirus type 2. Hybridization-competition experiments and RNA fingerprints indicate that the two virus-associated (VA) RNAs differ in their primary structures. The gene for 5.2S RNA is located to the right of the gene for 5.5S RNA, on the I strand of a DNA segment which extends between positions 30.3 and 32.2 on the map of adenovirus type 2 DNA.Both 5.5S and 5.2S RNA can be detected early after infection and also in the presence of cytosine-arabinoside or cycloheximide. After the onset of viral DNA replication, the synthesis of 5.2S RNA levels off, whereas 5.5S RNA is synthesized in increasing amounts. Both 5.2S and 5.5S RNAs are synthesized in isolated nuclei by an enzyme which resembles RNA polymerase III in its sensitivity to α-amanitin. In isolated nuclei, both RNA species are labeled with β-³²P-labeled GTP, which suggests that they are initiated at separate promoter sites.     

Interaction of Clostridium perfringens theta-haemolysin, a contaminant of commercial phospholipase C, with erythrocyte ghost membranes and lipid dispersions. A morphological study.

Commercially available preparations of phospholipase C from Clostridium perfringens are commonly contaminated with theta haemolysin, one of a group of bacterial haemolysins called oxygen labile (O-labile) haemolysins. Treatment of erythrocyte ghosts and a mixed lipid dispersion containing cholesterol with commercially available phospholipase C in the absence of Ca-2+ and the presence of phosphate buffer and/or EDTA resulted in the formation and release of ring or arc-shaped structures. Highly purified phospholipase C, free of theta-haemolysin, produced no changes in the morphology of erythrocyte ghosts or lipid dispersions in the presence of phosphate or EDTA, but caused the formation of typical diglyceride droplets in the presence of Ca-2+ in the absence of these inhibitors. Ring structures, identical to those caused by commercial phospholipase C, were formed on addition of highly purified theta-haemolysin to erythrocyte ghost membranes, lipid dispersions containing cholesterol and cholesterol dispersions, but not on treatment of membranes from Micrococcus lysodeikticus. Heat-inactivated O-haemolysin (60 degrees C for 10 min) produced no such effects. The dimensions of rings and arcs displayed heterogeneity. The outside diameters in various preparations varied from approx. 27-58 nm with border thickness of 4.1-7.8 nm.     

Receptor-mediated activation of phospholipase D by sphingosine 1-phosphate in skeletal muscle C2C12 cells. A role for protein kinase C.

The present study showed that sphingosine 1-phosphate (SPP) induced rapid stimulation of phospholipase D (PLD) in skeletal muscle C2C12 cells. The effect was receptor-mediated since it was fully inhibited by pertussis toxin. All known SPP-specific receptors, Edg-1, Edg-3 and AGR16/H218, resulted to be expressed in C2C12 myoblasts, although at a different extent. SPP-induced PLD activation did not involve membrane translocation of PLD1 or PLD2 and appeared to be fully dependent on protein kinase C (PKC) catalytic activity. SPP increased membrane association of PKCalpha, PKCdelta and PKClambda, however, only PKCalpha and PKCdelta played a role in PLD activation since low concentrations of GF109203X and rottlerin, a selective inhibitor of PKCdelta, prevented PLD stimulation.     

Recognition of different pools of phosphatidylglycerol in intact cells and isolated membranes of Acholeplasma laidlawii by phospholipase A2.

Phospholipase A2 (EC 3.1.1.4) from pig pancreas hydrolyzes phosphatidylglycerol in intact cells and isolated membranes of Acholeplasma laidlawii. Complete degradation of phosphatidylglycerol in intact cells at 37 degrees C does not result in lysis as shown by the retention of intracellular K+ ions and the cytoplasmic glucose-6-phosphatase, as well as the inability to detect activity of membrane-bound intracellular NADH-oxidase. A. laidlawii was grown on linoleic acid. Phospholipase A2 treatment of these cells at 5 degrees C, at which temperature the lipids are still in the liquid-crystalline state, results in a rapid breakdown of 50% of the phosphatidylglycerol. The residual phosphatidylglycerol can be hydrolyzed only at elevated temperatures and at much smaller rates, depending strongly on the incubation temperature. When membranes isolated from these cells are incubated at 5 degrees C, 70% of the phosphatidylglycerol is hydrolyzed immediately. The hydrolysis of the residual 30% is again strongly temperature dependent. Cells were grown on palmitate, elaidate, or oleate to investigate possible effects of the lipid phase transition on the accessibility of phosphatidylglycerol for phospholipase A2. Under conditions in which all the lipid is in the solid state, no hydrolysis occurs. When solid and liquid-crystalline lipid phases coexist, a limited hydrolysis of phosphatidylglycerol can be observed. The results demonstrate the disposition of phosphatidylglycerol in three different pools in the membrane of A. laidlawii. Phospholipase A2 has been used to discriminate between these pools and to estimate the amount of phosphatidylglycerol which is present in the liquid-crystalline phase. The present data, however, do not allow a definite localization of the phosphatidylglycerol pools.     

Hydrolysis of a phospholipid in an inert lipid matrix by phospholipase A2: a 13C NMR study

A new approach to study phospholipase A2 mediated hydrolysis of phospholipid vesicles, using 13C NMR spectroscopy, is described. [13C]Carbonyl-enriched dipalmitoylphosphatidylcholine (DPPC) incorporated into nonhydrolyzable ether-linked phospholipid bilayers was hydrolyzed by phospholipase A2 (Crotalus adamanteus). The 13C-labeled carboxyl/carbonyl peaks from the products [lyso-1-palmitoylphosphatidylcholine (LPPC) and palmitic acid (PA)] were well separated from the substrate carbonyl peaks. The progress of the reaction was monitored from decreases in the DPPC carbonyl peak intensities and increases in the product peak intensities. DPPC peak intensity changes showed that only the sn-2 ester bond of DPPC on the outer monolayer of the vesicle was hydrolyzed. Most, but not all, of the DPPC in the outer monolayer was hydrolyzed after 18-24 h. There was no movement of phospholipid from the inner to the outer monolayer over the long time periods (18-24 h) examined. On the basis of chemical shift measurements of the product carbonyl peaks, it was determined that, at all times during the hydrolysis reaction, the LPPC was present only in the outer monolayer of the bilayer and the PA was bound to the bilayer and was approximately 50% ionized at pH approximately 7.2. Bovine serum albumin extracted most of the LPPC and PA from the product vesicles, as revealed by chemical shift changes after addition of the protein. The capability of 13C NMR spectroscopy to elucidate key structural features without the use of either shift reagents or separation procedures which may alter the reaction equilibrium makes it an attractive method to study this enzymatic process.