Endogenous Phospholipids Research Articles

Phospholipid composition and organization in model beta-thalassemic erythrocytes.

The membrane phospholipid organization in human red blood cells (RBC) is rigidly maintained by a complex system of enzymes. However, several elements of this system are sensitive to oxidative damage. An important component in the destruction of beta-thalassemic RBC is the generation of reactive oxygen species and the release of redox-active iron by the unpaired alpha-hemoglobin chains. Consequently, we hypothesized that the presence of this oxidative stress to the RBC membrane could lead to alterations in membrane lipid organization. Model beta thalassemic RBC, prepared by the introduction of excess alpha-globin in the cell, have previously been shown to exhibit structural and functional changes almost identical to those observed in beta-thalassemic cells. After 24 hr at 37 degrees C, the model beta thalassemic cells exhibited a significant loss of deformability, as measured by ektacytometric analysis, indicative of extensive membrane damage. However, a normal steadystate distribution of endogenous phospholipids was found, as evidenced by the accessibility of membrane phospholipids to hydrolysis by phospholipases. Similarly, the kinetics of transbilayer movement of spin-labeled phosphatidylserine (PS) and phosphatidylethanolamine (PE) in all samples was in the normal range and was not affected by the presence of excess alpha-globin chains. In contrast, a faster rate of spin-labeled phosphatidylcholine (PC) transbilayer movement was observed in these cells. While control RBC exhibited a complete loss of their initial (2 mol%) lysophosphatidylcholine (LPC) levels following 24 hr of incubation at 37 degrees C, 1.5 mol% LPC was still present in model beta-thalassemic cells, suggesting an altered phospholipid molecular species turnover, possibly as a result of an increased repair of oxidatively damaged phospholipids.

Effects of a Low Birthweight Infant Formula Containing Human Milk Levels of Docosahexaenoic and Arachidonic Acids

Summary:Long‐chain (LC) polyunsaturated fatty acids (PUFA) (LCP) are considered conditionally essential nutrients for low birth weight infants (LBWI). Therefore, enrichment of LBWI formulae with metabolites both linoleic (ω‐6) and α‐linolenic (ω‐3) acids at levels typical for human milk has been recommended. However, previous feeding trials with LCP‐enriched formulae evaluated only a dietary supplementation with ω‐3 LCP from fish oils alone or with both ω‐3 and ω‐6 LCP at levels considerably lower than usual human milk contents. We studied the effects of an LBWI formula providing the major ω‐3 and ω‐6 LCP, docosahexaenoic and arachidonic acids, in amounts similar to those in average human milk. Twenty‐seven LBWIs were enrolled in this study when they tolerated full enteral feeding (≥ 130 ml milk/kg/day). Infants either received their own mother's milk (n = 8, birth‐weight 1218 ± 146 g, gestational age 30.2 ± 1.5 weeks, mean ± SD) fortified with protein and minerals (FM‐85, Nestle Ag, Munchen, Germany; dosage 5 g/100 ml milk) or were randomly assigned to blinded batches of an LBWI formula (Prematil, Milupa AG, Friedrichsdorf, Germany) without LCP (n = 10, 1280 ± 229 g, 31.1 ± 3.1 weeks) or with LCP (n = 9, 1253 ± 334 g, 30.4 ± 3.3 wks.). During the study period of 21 days, the three feeding groups did not differ in growth and feeding tolerances as assessed by occurrence of gastric residuals, spitting, or abdominal distention; however, firms stools were noted more frequently in the two formula groups. Compared to infants fed human milk, those on formula without LCP showed significant depletion of plasma phospholipid arachidonic acid (reduced to 74% of levels found in infants fed human milk) and, even more marked, of docosahexaenoic acid (reduced to 64%) as well as total LCP (74%), within 3 weeks of full enteral feeding. In contrast, infants receiving the LCP‐enriched formula achieved an LCP status equal to that of human milk‐fed babies. Results indicate that LBWI absorb formula LCPs and incorporate them into endogenous phospholipids. There were no adverse effects of LCP enrichment on vitamin‐E status. We conclude that the LCP enrichment of the formula used in this study was well tolerated, did not interfere with short‐term growth or vitamin‐E status, and provided sufficient amounts of essential fatty acids to match the ω‐3 and ω‐6 LCP status of infants fed human milk.

Phosphatidylethanolamine N-methyltransferase activity in isolated rod outer segments from bovine retina

Phosphatidylcholine (PC) can be synthesized in isolated rod outer segments from bovine retina by successive transfer of methyl groups from S-adenosyl-L-methionine (SAM) to phosphatidylethanolamine (PE), with the intermediate formation of phosphatidyl-N-monomethylethanolamine (PMME) and phosphatidyl-N,N-dimethylethanolamine (PDME). This reaction is time-protein-and SAM concentration-dependent. Phosphatidylethanolamine N-methyltransferase (PE N-MTase) has two pH optima, 8.5 and 10, at low (10 microM) and high (200 microM) SAM concentrations and requires magnesium ions for full activity. When ROS membranes were incubated at 5 to 200 microM SAM concentrations at pH 8.5 or pH 10, the major methylated product was PMME, followed by PC and PDME. The apparent Kms for SAM at pH 8.5 and at pH 10 were similar (37 and 38 microM, respectively). The Vmax was 13 pmol h-1 (mg protein)-1 at pH 8.5 and 12.50 pmol h-1 (mg protein)-1 at pH 10. Pulse-chase experiments demonstrated a precursor-product relationship with [3H]PC as the end product. The level of PE N-Mtase activity in the purified ROS preparation obtained from crude ROS fractions by discontinuous sucrose gradient centrifugation, was as high as 65% of the level found in the microsomal fraction obtained from the remainder of the retinas. The presence of microsomal and mitochondrial marker enzymes, however, was minimal in the ROS preparation. The radioactivity incorporated into ROS PC was measured in an upper and lower band of PC obtained by two-dimensional TLC. We found that the amount of [methyl-3H] groups incorporated into the upper PC band was 2.5-fold greater than that incorporated into the lower one. The fatty acid composition of the upper band was very different from that of the lower band, the former being enriched in very long-chain polyunsaturated fatty acids and the latter in saturated fatty acids. Phosphatidyl-ethanolamine N-methyltransferase activity increased in the presence of exogenous phospholipid substrates. PDME being augmented ten-fold and PC eight-fold when the incubations were carried out in the presence of PMME and PDME, respectively. At a 2 mM concentration, S-adenosyl-L-homocysteine (SAH) inhibited the methyl groups' incorporation into the endogenous phospholipids by 40%. When ROS membranes were selectively depleted of soluble or peripheral and soluble proteins, the PE N-MTase activity remained mainly associated to the membrane, suggesting that this enzyme (s) is an intrinsic membrane protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the ATPase activity of P-glycoprotein from multidrug-resistant Chinese hamster ovary cells

P-Glycoprotein (Pgp) was isolated from CHRC5 membranes by selective detergent extraction and further purified by lentil lectin affinity chromatography. The purified product displayed a very high basal ATPase activity (1.65 mumol/min per mg protein in the absence of added drugs or lipids) with an apparent Km for ATP of 0.4 mM. There was no evidence of cooperativity, suggesting that the two ATP sites operate independently of each other. Pgp ATPase activity was stimulated by verapamil, trifluoperazine and colchicine, and inhibited by daunomycin and vinblastine. All drugs and chemosensitizers acted as mixed activators or inhibitors, producing changes in both the Vmax of the ATPase and the Km for ATP. ADP competitively inhibited Pgp ATPase, with a Ki of 0.2 mM. The macrolide antibiotics bafilomycin A1, concanamycin A and concanamycin B, inhibited Pgp ATPase at concentrations of 0.1-10 microM, and at an inhibitor:protein stoichiometry of 0.65-1.0 mumol/mg protein, which is at the low end of the range characteristic of P-type ATPases. Pgp ATPase was relatively selective for adenine nucleotides. Several phospholipids stimulated Pgp ATPase activity in a dose-dependent manner, whereas others produced inhibition. Metabolic labelling showed that the endogenous phospholipids associated with purified Pgp consisted largely of phosphatidylethanolamine and phosphatidylserine, with only a small amount of phosphatidylcholine. 32P-Labelling studies indicated that purified Pgp was partially phosphorylated. It can be concluded that Pgp is a constitutively active, adenine nucleotide-specific ATPase whose catalytic activity can be modulated by both drugs and phospholipids.

Uptake of Exogenous sn-1-Acyl-2- lyso-phosphatidylinositol into HeLa S3 Cells: REACYLATION ON THE CELL SURFACE AND METABOLISM TO GLUCOSAMINYL(ACYL)PHOSPHATIDYLINOSITOL

A HeLa S3 subline is unusual in accumulating relatively large amounts of glucosaminyl(acyl)phosphatidylinositol (GlcN(acyl)PI), a derivative of phosphatidylinositol (PI) in which both GlcN and a fatty acid are linked to inositol hydroxyl groups (D. Sevlever, D. Humphrey, and T.L. Rosenberry, submitted for publication). This lipid is a proposed intermediate on the biosynthetic pathway for glycosyl-PI (GPI) anchors of membrane proteins. In this study we demonstrate for the first time that exogenous inositol phospholipids can enter this biosynthetic pathway and be metabolized to GlcN(acyl)PI. When HeLa S3 cells were incubated for 24 h with exogenous PI or sn-1-acyl-2-lyso-phosphatidyl-inositol (lyso-PI) labeled with 3H in the inositol group, 25-30% of the label was recovered in cell-associated lipids and most of the remaining 70-75% in hydrophilic metabolites in the medium. The predominant labeled lipid was PI, with smaller amounts of lyso-PI, phosphatidylinositol 4-phosphate (PIP), and GlcN(acyl)PI. Both exogenous lipid precursors gave the same distribution of labeled lipids, and a similar distribution was observed for endogenous inositol phospholipids metabolically labeled with [3H]inositol. Addition of excess inositol had no effect on the conversion of [3H]lyso-PI to [3H]GlcN(acyl)PI, indicating that the conversion did not result from breakdown to [3H]inositol followed by resynthesis. The cellular orientation of incorporated PI and lyso-PI was determined by incubating cells at 4 degrees C with PI-specific phospholipase C (PI-PLC). This enzyme cleaves only PI and lyso-PI on the outer leaflet of the cell membrane. After 24-h incubation with either precursor, only about 15% of cell-associated [3H]PI or [3H]lyso-PI was on the outer leaflet. However, more than 60% of the [3H]PI was on the outer leaflet after 1-h incubation with either precursor, suggesting that substantial sn-2 acylation of exogenous [3H]lyso-PI occurred in the outer leaflet. This suggestion was confirmed by examining labeled lipids in cells after uptake of [3H]lyso-PI at 4 degrees C. No transmembrane translocation of lyso-PI, PI phosphorylation, or PI glycosylation occurred at this temperature, but some sn-2 acylation was apparent and more than 90% of the [3H]PI formed was on the outer leaflet. These data indicate that sn-2 acylation can occur in the outer leaflet of the cell membrane, perhaps by transacylation from other cell surface phospholipids.

EGF and TGFα modulate structural and functional differentiation of the mammary gland from pregnant mice in vitro: Possible role of the arachidonic acid pathway

Epidermal growth factor (EGF) has been suggested to be involved in mammary gland development by mitogenic stimulation of the ductal and alveolar epithelium in virgin mice. The present studies demonstrate that also in late-pregnant mice EGF leads to proliferation of the ductal, ductular, and alveolar epithelium. The mitogenic effect is associated with structural and functional dedifferentiation of alveolar cells as revealed by analysis of morphology, expression of cytosolic and secretory proteins, and fatty acid synthesis. Using a combination of metabolic inhibitors, the dedifferentiating effect of EGF could be blocked while the mitogenic action was not influenced. This finding demonstrates that the signal transduction pathway leading to dedifferentiation and mitosis can be separated, and that the dedifferentiating effect of EGF is independent of its mitogenic properties, but is probably mediated by activation of the arachidonic acid-dependent pathways (cyclo- and lipoxygenase pathways). Release of arachidonic acid from the endogenous phospholipid pool was found to be an early response of the explants to EGF. Accordingly, arachidonic acid itself proved to be capable of inducing epithelial dedifferentiation but failed to stimulate proliferation. TGF alpha showed qualitatively similar effects as EGF but was generally a stronger agonist. It is suggested that EGF and TGF alpha also play a role in mammary gland physiology during pregnancy by final developing and maintenance of the lobulo-alveolar structure in the mammary gland and prevention of premature onset of lactation, and that this is mediated through the PLA2-arachidonic acid signalling cascade.

Phospholipid molecular species with eicosapentaenoic acid (20:5(n-3)) are less stable than species with arachidonic acid (20:4(n-6)) in isolated rat liver cells.

We have studied the incorporation of [1-14C]20:5(n-3) and [1-14C]20:4(n-6) in the molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in isolated rat liver cells. These two fatty acids are present in very different amounts in endogenous phospholipids, with 20:4 as one of the major fatty acids and 20:5 as a minor and very diet-dependent constituent. The main phospholipid species formed from 20:4(n-6) were 16:0-20:4 and 18:0-20:4. When formed, they were stable during incubations of liver cells for 2-3 h. The main species formed from 20:5(n-3) were 16:0-20:5 and 18:0-20:5. After formation, 16:0-20:5 and to a lesser degree 18:0-20:5 were, however, degraded during 1-2 h of incubation, especially in PC. Only small amounts of 22:5(n-3) and very little 22:6(n-3) were formed from 20:5(n-3) and small amounts of 22:4(n-6) were produced from 20:4(n-6). With 20:4(n-6) and 20:5(n-3) as substrates, 20:4-20:4 and 20:5-20:5 molecular species respectively were initially formed in PC and PE but both species were rapidly degraded.

A unique feature of lipid dynamics in small intestinal brush border membrane.

The lipid composition of the brush border membrane (BBM) or apical plasma membrane of enterocytes is characterized by a remarkably high glycosphingolipid content (glycosphingolipid: phospholipid:neutral lipid mole ratio of about 1:1:1). A manifestation of the high glycolipid content of the BBM is the lipid fluidity which is low compared to other mammalian plasma membranes and related to it a steep flexibility gradient: hydrocarbon chain segments close to the lipid-water interface have quasi-crystalline packing while hydrocarbon chain segments close to the centre of the lipid bilayer behave like a fluid. An important function of the BBM is the absorption of dietary lipids. The absorption of cholesterol from bile salt micelles has been shown to be protein-mediated. The integral membrane protein responsible for this activity has features similar to non-specific lipid transfer proteins. Another remarkable property of the BBM is described here: phospholipids are exchanged between the lipid bilayer of the BBM and the lipid bilayers of small unilamellar egg phosphatidylcholine (PC) vesicles. In the course of this probably 1:1 exchange, endogenous BBM phospholipids move out of the BBM and the lipid loss is compensated by the insertion of exogenous PC from the small unilamellar vesicles. This exchange activity is probably due to the same protein(s) responsible for lipid absorption in this membrane or at least related to the absorptive capacity of the BBM. The unique feature of small intestinal BBM is that the on- and off-rate of certain lipids is remarkably high: the underlying structure of this activity is still unknown.

Effect of specific phospholipid molecular species incorporated in human platelet membranes on thromboxane A2/prostaglandin H2 receptors

The incorporation of albumin-bound docosahexaenoic acid (22:6n-3), but not linoleic acid (18:2n-6), into cellular phospholipids inhibits platelet aggregation induced by the thromboxane analogue U46619. [3H]U46619 specific binding to thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors, as well as specific binding of the antagonist [3H]SQ29548 to these sites were also decreased in these modified cells (P. G., Swann et al. 1990. J. Biol. Chem. 265: 21692-21697). More than 80% of the 22:6n-3 incorporated in these cells was esterified in the various endogenous phospholipid classes and the remaining was found in neutral lipids and in the unesterified fatty acid pool. In this study, we determined whether the effects observed could be attributed to the esterification of 22:6n-3 in phospholipids and whether the 22:6n-3 biological activity might depend on its esterification in specific phospholipid classes. Therefore, pure phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecular species were transferred to platelet membranes, using lipid transfer proteins. PC and PE containing palmitate (16:0) and 22:6n-3 or 16:0 and 18:2n-6 at position sn-1 and sn-2, respectively, were incorporated into membranes only at the expense of the corresponding endogenous phospholipid class, by an apparent exchange process. When such modified membranes were tested for specific binding of U46619 and SQ29548, a significant decrease of the receptor site affinity was only observed in membranes highly enriched with 1-palmitoyl-2-docosahexaenoyl-glycerophosphocholine (16:0/22:6-GPC). Fluidity parameters measured by electron spin resonance of 5- and 16-nitroxy-stearic acids were not significantly different in membranes enriched with 16:0/22:6-GPC relative to those enriched with 16:0/18:2n-6-GPC, arguing against a generalized perturbation of the membrane due to 22:6n-3 incorporation. We conclude that molecular species of PC with 22:6n-3 at the sn-2 position can affect TXA2/PGH2 receptors. The selectivity of the inhibitory effect of PC containing 22:6n-3 is discussed.

Platelet-activating factor mediates trinitrobenzene induced colitis

Platelet-activating factor (PAF) is an endogenous phospholipid which may be an important mediator of shock and inflammation. Recent evidence suggests that PAF plays a role in the development of ischemic colitis and inflammatory bowel disease. Its effects are mediated by second messengers, including the arachidonic acid metabolites. Using an ex vivo isolated left colon rabbit perfusion model, our aims were to determine whether exogenously administered trinitrobenzene sulfonic acid (TNB), which produces experimental colitis, stimulates both PAF and eicosanoid release in the colon, and if so, whether this effect can be blocked by a PAF antagonist. Colonic inflammation was induced by the intracolonic administration of 0.25 ml of 50% ethanol containing 30 mg of TNB. Tissue and perfusate concentrations of the eicosanoids, [prostaglandin E (PGE 2), 6-keto-prostaglandin F 1α (6-keto-PGF 1α) and thromboxane B 2 (TXB 2), leukotriene B 4 (LTB 4)] and the autocoid PAF were measured by ELISA. During TNB infusion there was a significant increase in tissue levels of PAF compared to control colons. Additional studies performed pretreating the colons with the PAF receptor antagonist WEB-2170 prior to TNB infusion blocked PAF release. TNB stimulated release of luminal eicosanoids except LTB 4 and suppressed release of tissue prostanoids. Pretreatment with WEB-2170 prior to TNB inhibited luminal eicosanoids, and inhibited PGE 2 and prostacyclin, but not TX tissue suppression. Inhibition of TNB-stimulated PAF release by WEB-2170 suggests that PAF may play a role in TNB-induced colitis and this phenomenon may mediate tissue injury.

Phospholipids are necessary for calmodulin-stimulated activation of the Ca(2+)-ATPase of erythrocytes.

Treatment of red cell ghosts with increasing concentrations of the non-ionic detergent Triton X-100 caused a progressive loss of Ca(2+)-ATPase activity. Both the basal activity and the calmodulin-stimulated activity were affected and could be partially restored by acidic phospholipids. Lipid-free Ca(2+)-ATPase was prepared from solubilized ghosts by calmodulin affinity chromatography and extensive washing of the column with detergent to remove the endogenous phospholipids associated with the enzyme. The phospholipid-free, solubilized Ca(2+)-ATPase had very low activity and was not activated by calmodulin. The tryptic proteolytic pattern of the delipidated ATPase differed from the pattern of the phospholipid-associated enzyme, indicating that the delipidation had caused conformational changes. The activity was fully restored by phosphatidylserine, but was only partially restored by phosphatidylcholine. The phosphatidylcholine-activated enzyme was restored to maximal activity in the presence of calmodulin. The delipidated ATPase could be reconstituted in soybean lipid vesicles and was able to actively transport Ca2+.

Loss of phospholipid asymmetry in human platelet plasma membrane after 1-12 days of storage. An ESR study.

We used paramagnetic analogs of endogenous phospholipids to study modification of phospholipid distribution in platelet plasma membranes during aging. Asymmetrical distributions and translocation kinetics were very different for spin-labeled phosphatidylserine and spin-labeled phosphatidylcholine in fresh platelet plasma membranes. In freshly prepared platelets and up to day 7, spin-labeled phosphatidylserine very rapidly penetrated to the inner leaflet of the platelet plasma membrane. However, spin-labeled phosphatidylcholine was mainly retained on the external leaflet. From day 7 to day 9, the two translocation kinetics became identical with symmetrical distribution of both spin-labeled phospholipids at equilibrium. Inhibition of translocase activity and modification of membrane stability accounted for these transformations. The rapid re-exposition of spin-labeled phosphatidylserine after stimulation by the calcium ionophore A23187, measured in fresh platelet concentrates, persisted up to day 9 but disappeared between day 10 and day 12. From day 7 to day 9, a strong cytoskeleton proteolysis and marked decrease in intracellular ATP were observed. Moreover, complete suppression of beta-N-acetyl glucosaminidase secretion and vesicle formation after A23187 stimulation of aged platelets indicated that platelets could no longer be activated beyond day 9. Taken together, these results showed that during in vitro aging there are metabolic and membrane modifications in platelet similar to those described for platelet activation. In addition, all of the observed events occurred simultaneously between day 7 and day 9. These results highlight the importance of maintaining plasma membrane asymmetry to increase the hemostatic effectiveness of transfused platelet concentrates.

Phospholipid transverse asymmetry in trout spermatozoa plasma membrane

The phospholipid transmembrane distribution and movement in the plasma membrane of rainbow trout spermatozoa was determined with spin-labelled phospholipid analogues. After initial incorporation in the outer membrane leaflet, only the aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE) redistributed towards the inner leaflet. At equilibrium, more than 90% PS and approximately 80–85% PE were located in the cytoplasmic leaflet. The inward motion was significantly faster for PS ( t 1 2 ≈ 5 min ) than for PE ( t 1 2 ≈ 60 min ). Both the velocity and the extent of this redistribution were dependent on the cellular ATP level, arguing for the involvement of the aminophospholipid translocase. Comparison of the electron paramagnetic resonance spectra recorded from analogues located in either one of the leaflet showed an important difference of microviscosity between the cytoplasmic and the extracellular bilayer leaflets. Assuming that the analogue distribution reflects the asymmetry of the endogenous phospholipids, the higher probe mobility in the inner leaflet could be correlated to a higher degree of unsaturation of the fatty acids present in this hemi-leaflet.

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.

Phospholipid requirement of epididymal testosterone 5α-reductase and phospholipid composition of epididymal microsomes

We have investigated phospholipid requirement for testosterone 5α-reductase solubilized from microsomal and nuclear fractions of rat epididymis. The 5α-reductase from microsomal fraction was stimulated by phosphatidylcholine (PC) with long acyl-chain lengths, but inhibited by short chain PC. The nuclear enzyme activity was weakly activated by PC with various acyl-chain lengths tested. Synthetic phosphatidylserine (PS), such as dioleoylPS, most strongly stimulated the microsomal enzyme activity, but did not exhibit any activation of the nuclear enzyme activity. Endogenous phospholipids, such as PC, PS, and phosphatidylethanolamine (PE) separated from bovine epididymal microsomes were tested for their stimulatory effects on microsomal and nuclear enzymes. Among these endogenous phospholipids, PS most greatly stimulated the microsomal 5α-reductase activity, whereas both PC and PE weakly activated the enzyme activity. On the other hand, endogenous PC and PS had no ability to support the nuclear enzyme activity. The fatty acid compositions of PC and PS from bovine epididymal microsomes were determined, in order to elucidate the relationship between 5α-reductase activation by these phospholipids and the structure of their acyl chains. The relative content of fatty acids in PC, in a decreasing order, was palmitate linolate > oleate; that in PS was stearate > oleate > palmitate. Based on these observations, the roles of microsomal PS and PC in epididymal 5α-reductase reaction will be discussed.

Mobilization of acyl chains from endogenous cellular phospholipids into cholesteryl esters during foam-cell formation in mouse peritoneal macrophages

We previously showed that phosphatidylserine-containing liposomes (phosphatidylserine liposomes) are effectively metabolized and induce foam-cell formation in cultured macrophages (Nishikawa, K., Arai, H. and Inoue, K. (1990) J. Biol. Chem. 265, 5226–5231). When cultured mouse peritoneal macrophages were incubated with phosphatidylserine liposomes of different cholesterol content, cholesteryl esters were formed in the cells in proportion to the cholesterol content of the liposomes added to the culture. Cholesteryl esters were formed only when the cells took up more than a certain amount of cholesterol (approx. 160% above the basal cell level). Fatty acyl moiety derived from the exogenous phospholipids were utilized preferentially for cholesteryl ester formation. Once cholesterol as an acceptor is exhausted within cells, the acyl chain may be utilized for triacylglycerol formation. Not only exogenous lipid acyl chains but also endogenous phospholipid acyl chains were also utilized for cholesteryl ester and triacylglycerol formation under the present conditions, since the radioactivity in phospholipids was incorporated into both cholesteryl esters and triacylglycerols in macrophages whose endogenous phospholipids had been prelabeled with radioactive fatty acids. Among phospholipid classes, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol served as the acyl donor, whereas sphingomyelin and cardiolipin did not. Mobilization of acyl chains from endogenous phospholipids to the neutral lipid fraction was also observed during foam-cell formation induced by acetyl low-density lipoprotein. The substance(s) produced within cell in the process of foam-cell formation may trigger the acyl chain mobilization from endogenous phospholipids.

Aging does not affect the susceptibility to lipid peroxidation and lysosomal enzyme release of rat visual system structures and sciatic nerve.

The aim of the present study was to clarify the issue of lipid peroxidation operating in visual system structures and sciatic nerve of the rat as a contributing factor to senescence. In 4-, 14- and 28-month-old male rats, the amount of endogenous malondialdehyde, conjugated dienes and extractable phospholipids were all taken as indices of lipid peroxidation. In addition, the total free and released enzyme activities of four lysosomal hydrolases were evaluated. The susceptibility of all these parameters to in vitro iron-induced peroxidation was also taken as an age-related indicator of the endogenous peroxidative potential of the nervous tissues examined. Our data show that the content of malondialdehyde and phospholipids did not change in an age-related fashion. Furthermore, the susceptibility of rat visual system structures to lipid peroxidation, together with the release of lysosomal enzymes were unchanged as a function of aging. The results do not lend support to the hypothesis that an increase in overall lipid peroxidation is peculiar to the aging phenomenon of the central nervous system areas which delimit the rat visual pathway.

Mathematical modelling of lipid transbilayer movement in the human erythrocyte plasma membrane.

A model is presented to simulate transverse lipid movement in the human erythrocyte membrane. The model is based on a system of differential equations describing the time-dependence of phospholipid redistribution and the steady state distribution between the inner and outer membrane monolayer. It takes into account several mechanisms of translocation: (i) ATP-dependent transport via the aminophospholipid translocase; (ii) protein-mediated facilitated and (iii) carrier independent transbilayer diffusion. A reasonable modelling of the known lipid asymmetry could only be achieved by introducing mechanism (iii). We have called this pathway the compensatory flux, which is proportional to the gradient of phospholipids between both membrane leaflets. Using realistic model parameters, the model allows the calculation of the transbilayer motion and distribution of endogenous phospholipids of the human erythrocyte membrane for several biologically relevant conditions. Moreover, the model can also be applied to experiments usually performed to assess phospholipid redistribution in biological membranes. Thus, it is possible to simulate transbilayer motion of exogenously added phospholipid analogues in erythrocyte membranes. Those experiments have been carried out here in parallel using spin labeled lipid analogues. The general application of this model to other membrane systems is outlined.

Transbilayer mobility and distribution of red cell phospholipids during storage.

We studied phospholipid topology and transbilayer mobility in red cells during blood storage. The distribution of phospholipids was determined by measuring the reactivity of phosphatidylethanolamine with fluorescamine and the degradation of phospholipids by phospholipase A2 and sphingomyelinase C. Phospholipid mobility was measured by determining transbilayer movements of spin-labeled phospholipids. We were unable to detect a change in the distribution of endogenous membrane phospholipids in stored red cells even after 2-mo storage. The rate of inward movement of spin-labeled phosphatidylethanolamine and phosphatidylserine was progressively reduced, whereas that for phosphatidylcholine was increased. These changes in phospholipid translocation correlated with a fall in cellular ATP. However, following restoration of ATP, neither the rate of aminophospholipid translocation nor the transbilayer movement of phosphatidylcholine were completely corrected. Taken together, our findings demonstrate that red cell storage alters the kinetics of transbilayer mobility of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine, the activity of the aminophospholipid translocase, but not the asymmetric distribution of endogenous membrane phospholipids, at least at a level detectable with phospholipases. Thus, if phosphatidylserine appearance on the outer monolayer is a signal for red cell elimination, the amount that triggers macrophage recognition is below the level of detection upon using the phospholipase technique.

Release of platelet activation factor from activated neutrophils. Transglutaminase-dependent enhancement of transbilayer movement across the plasma membrane.

Extracellular release of platelet activating factor (PAF) following synthesis in inflammatory cells is variable and modulated by a number of as yet undefined cellular mechanisms. Using human neutrophils loaded with the tritiated, nonmetabolizable PAF analog, 1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (C-PAF), extracellular release was studied by techniques involving an albumin extraction method. Further modeling of plasma membrane events in the neutrophil was accomplished using movement across the membrane of erythrocyte ghosts. The data demonstrate that C-PAF release is dependent on cellular activation and is accompanied by alterations in the physical properties of the plasma membrane as measured by enhancement of merocyanine 540 (MC540) staining, as well as by bulk, nonspecific transbilayer movement of endogenous phospholipids as detected by the procoagulant activity of externalized phosphatidylserine (a phospholipid class usually sequestered in the plasma membrane inner leaflet). The finding that competitive inhibitors of transglutaminase significantly inhibited C-PAF release, enhancement of MC540 staining, and externalization of phosphatidylserine, strongly suggest a role for this enzyme in the enhancement of phospholipid transbilayer movement. Furthermore, the data suggest that recycling of C-PAF in the plasma membrane is likewise transglutaminase dependent and limits the net extracellular release of C-PAF which, like liberation of endogenously produced PAF, is dependent on extracellular "acceptors" and shown to be albumin concentration- and cell density-dependent.