David Allan

Accumulation of 1,2-diacylglycerol in the plasma membrane may lead to echinocyte transformation of erythrocytes.

THE change in shape of erythrocytes from the normal biconcave disk to a spiculed sphere (echinocyte1) can be caused by an increased intracellular concentration of Ca2+ (refs 2 to 5). We have found that an immediate biochemical effect of raising intracellular [Ca2+] is to increase the production of 1,2-diacylglycerol and cause its accumulation in the plasma membrane. This change in membrane composition may alter the structure of the membrane and thus cause the change in shape. Energy-depleted cells also show increased 1,2-diacylglycerol and similar but less extreme morphological changes, even in the absence of Ca2+.

A calcium-activated polyphosphoinositide phosphodiesterase in the plasma membrane of human and rabbit erythrocytes

Haemoglobin-free human erythrocyte ghosts that were prepared in the presence of EDTA and were then exposed to Ca2+ showed a substantial loss of phosphatidylinositol phosphate and phosphatidylinositol diphosphate, measured either chemically or by loss of 32P from the lipids of prelabelled membranes. At the same time there was, as reported previously (Allan, D. and Michell, R.H., (1976) Biochim. Biophys. Acta 455, 824--830), and approximately equivalent rise in the diacylglycerol content of the membranes. Analysis of the 32P-labelled water-soluble material released during this process showed that the major products were inositol diphosphate and inositol triphosphate. No change was seen in the phosphatidylinositol or phosphatidate content of the membranes, and there was no Ca2+-activated loss of 32P from the phosphatidate of prelabelled membranes: this suggests that Ca2+ did not activate phosphoinositide phosphomonoesterases or phosphatidate phosphomonoesterase in human erythrocyte membranes. It is concluded that human erythrocyte membranes contain at their cytoplasmic surface a Ca2+-activated phosphodiesterase that is active against both phosphatidylinositol phosphate and phosphatidylinositol diphosphate. Rabbit erythrocytes also contained this enzyme, but in these cells there was also evidence for the presence of a Ca2+-activated phosphatidate phosphomonoesterase.

Changes in lipid metabolism and cell morphology following attack by phospholipase C (Clostridium perfringens) on red cells or lymphocytes

When intact human erythrocytes were treated with phospholipase C (Clostridium perfringens), up to 30% of the membrane phospholipids were broken down without significant cell lysis. Only phosphatidylcholine and sphingomyelin were attacked. Ceramide (derived from sphingomyelin) accumulated, but 1,2-diacylglycerol (derived from phosphatidylcholine) was largely converted into phosphatidate. Up to 12% of the cell phospholipid could be converted into phosphatidate in this way. Pig erythrocytes and lymphocytes showed a similar but smaller synthesis of phosphatidate after phospholipase C attack. Phospholipase C also caused a marked morphological change in erythrocytes, giving rise to spherical cells containing internal membrane vesicles. This change appeared to be due to ceramide and de and diacylglycerol accumulation rather than to increased phosphatidate content of the cells.

Microvesicles from Sickle Erythrocytes and their Relation to Irreversible Sickling

Summary. Incubation of sickle (HbS) erythrocytes for periods up to 96 h leads to the formation of irreversibly sickled cells (ISCs) and to the release of spectrin‐free microvesicles similar to those derived from aged or Ca2+‐ionophore‐treated normal erythrocytes. The sickle microvesicles were somewhat larger than those from normal cells and showed minor differences in their membrane polypeptide composition. Sickle microvesicles were no different from their parent cells in their content of fetal haemoglobin. Neither microvesiculation nor formation of irreversibly sickled cells required the presence of Ca2+ in the medium but Ca2+ did accelerate both processes. Although in these prolonged incubations microvesiculation appeared to occur concomitantly with the formation of ISCs, it is not clear whether or not microvesiculation is a necessary prelude to irreversible sickling.

A possible metabolic explanation for drug‐induced phospholipidosis

A large variety of amphiphilic cationic drugs which are in widespread clinical use produce a generalized phospholipidosis when administered for prolonged periods. These drugs, which vary widely in their potency in causing phospholipidosis, include chlorphentermine, fenfluramine, triparanol, trans-1,4-bis (2-chlorobenzylaminoethy1)-cyclohexane (A79944), azacosterol, 53 ’ diethylaminoethyoxyhexestrol, 1-chloroamitriptyline, iprindole, 2-N-methyl-piperazino-methyl-1,3-diazofluoroanthen 1-oxide (AC 3579), chlorcyclizine, chloroquine, chlorpromazine, thioridazine, imipramine, clomipramine, haloperidol and boxidine (Yamamoto, Adachi & others, 1971a, b; Shikata, Kanetaka &others, 1972; Hruban, Slesers & Ashenbrenner, 1973 ; Lullman, Lullman-Rauch & Wasserman, 1973; Wherrett & Huterer, 1973; De La Iglesia, Feuer & others, 1974; Kasama, Yoshida & others, 1974; Lullman-Rauch, 1974a, b, 1975; Schmien, Seiler & Wasserman, 1974). Although these drugs have a variety of therapeutic effects they are physicochemically rather similar, in that they all possess both a hydrophobic region and a primary or substituted amine group which can bear a net positive charge. This amphiphilic nature enables the drugs to interact with phospholipids, particularly the anionic phospholipids which are quantitatively minor constituents of membranes (e.g. phosphatidate, phosphatidylinositol, phosphatidylserine, cardiolipin). Their capacity both to cause phospholipidosis and to interact with lipids depends largely on the size and hydrophobicity of the apolar portions of the molecule. We recently suggested that interactions of these drugs with anionic phospholipids might cause some of the therapeutic actions or side-effects of these drugs (Brindley, Allan & Michell, 1975). The lipids which accumulate in the lysosomes of a variety of tissues during drug treatment are mainly glycerophospholipids. There are clear indications that compared with normal tissue, these tend to include increased proportions of anionic phospholipids (phosphatidate, phosphatidylinositol, phosphatidylglycerol and lysobisphosphatidate) and decreased proportions of triglyceride and of the major zwitterionic glycerophospholipids (phosphatidylcholine and phosphatidylethanolamine) (Yamamoto & others, 1971a, b; Wherrett & Huterer, 1973; Kasama, Yoshida & others, 1974; Allan & Michell, 1975; Karabelnik & Zbinden, 1975). This pattern of lipid accumulation is in marked contrast to that seen in the classical hereditary lipidoses in which sphingolipids, particularly glycosphingolipids, are the main lipids which accumulate in lysosomes. One proposed explanation of this effect is that phospholipids are normally degraded in lysosomes by phospholipases, but that when amphiphilic cationic drugs form complexes with the phospholipids this prevents phospholipase attack and the phospholipid-drug complexes therefore accumulate and engorge the lysosomes (Liillman & others, 1973 ; Lullman-Rauch, 1974a). Although this mechanism would explain many of the experimental findings it does not provide a complete explanation. For example, it does not explain

Echinocytosis and microvesiculation of human erythrocytes induced by insertion of merocyanine 540 into the outer membrane leaflet

Echinocytosis and release of microvesicles from human erythrocytes treated with the impermeant fluorescent dye merocyanine 540 (MC540) has been correlated with the extent of dye binding to intact cells and ghosts. At 20 degrees C binding appeared to saturate at about 9.3.10(6) molecules per cell (3.6 mol/100 mol phospholipid), equivalent to an expansion of the outer leaflet lipid area of about 2.7%. Stage 3 echinocytes were formed upon binding of (3-4).10(6) molecules of MC540/cell (about 1.3 mol/100 mol phospholipid), equivalent to an expansion of the outer leaflet lipid area of about 1.0%. Negligible release of microvesicles was observed with MC540 at 20 degrees C. Binding of MC540 to permeable ghosts was approximately twice that to cells suggesting that there was no selective binding to the unsaturated (more fluid) phospholipids which are concentrated in the inner lipid leaflet of the membrane. At 37 degrees C apparent maximal binding of MC540 was about 3.2 mol/100 mol phospholipid and correlated with the maximal release of microvesicles from the cells as measured by release of phospholipid and acetylcholinesterase. These results are discussed in relation to the bilayer couple hypothesis of Sheetz and Singer (Proc. Natl. Acad. Sci. USA 71 (1974) 4457-4461).

Membrane phospholipid asymmetry in Semliki Forest virus grown in BHK cells

Abstract The distribution of phospholipids across the membrane bilayer of Semliki Forest virus grown in BHK cells has been examined by treating the virus with bee venom phospholipase A 2 and sphingomyelinase C from Staphylococcus aureus . From the amounts of different phospholipids which are degraded rapidly (half-time about 1 min for phospholipase A 2 ) we calculate that in virus isolated 16 h after infection about 95% of sphingomyelin, 55% of phosphatidylcholine, 20% of phosphatidylethanolamine and less then 5% of phosphatidylserine is present on the outer leaflet of the virus envelope. Less than 5% of the virus was permeable to macromolecules before or after treatment with phospholipases as judged by accessibility of the genome to external ribonuclease. A much slower (half-time about 1 h) breakdown by phospholipase A 2 of originally inaccessible phosphatidylcholine and phosphatidylethanolamine appeared to be due to an enzyme-induced loss of lipid asymmetry since the original asymmetric distribution of phospholipids was maintained for several hours when the virus alone was incubated at 37°C. However, virus incubated for 20 h at 37°C showed a marked loss of phosphatidylethanolamine and phosphatidylserine asymmetry and a greater susceptibility to lysis by longer treatment with phospholipase A 2 .

Production of 1,2-diacylglycerol in human erythrocyte membranes exposed to low concentrations of calcium ions

A specific increase in the membrane content of 1,2-diacylglycerol occurred when erythrocytes were lysed at 20 degrees C in media which did not inclued a chelator of Ca2+ and also when Ca2+ was added to haemoglobin-free erythrocyte ghosts which had been prepared in the presence of ethyleneglycol-bis-(beta-aminoethylether)-N,N-tetraacetic acid (EGTA). The maximum increase was about 20-fold. The production of 1,2-diacylglycerol appeared to be caused by an endogenous membrane-bound phospholipase C which was half-maximally activated at less than 1 muM Ca2+ and which had access to only about 0.6-0.8% of the cells glycerolipids. This activity was optimal at pH 7.0-7.2 in the presence of 0.1 mM Ca2+; under these conditions diacylglycerol production was complete within 5-10 min. Enzyme activity was markedly decreased at low temperatures, and was abolished by heating at 100 degrees C for 1 min.

Is plasma membrane lipid composition defined in the exocytic or the endocytic pathway

Compared with intracellular membranes, the plasma membrane is rich in cholesterol and sphingomyelin. How does this distinct composition arise? Here David Allan and Karl-Josef Kallen take a critical view of the belief that these lipids arrive at the plasma membrane via vesicular traffic from the Golgi complex and propose instead that they may be accreted in the endocytic recycling pathway.

Inositol cyclic phosphate as a product of phosphatidylinositol breakdown by phospholipase C (Bacillus cereus)

Eukaryotic cells contain inositol phospholipids and also enzymes which cleave the glycerol-phosphate bond in these lipids. The activity of these enzymes within cells is in some way controlled by certain extracellular stimuli, which may thus provoke phosphatidylinositol breakdown. The enzymes which have been studied so far all catalyse breakdown of phosphatidylinositol to 1,2-diacylglycerol and a mixture of inosito1 1,2-cyclic phosphate and inositol 1 -phosphate, i.e. they act both as cyclizing phosphotransferases and as phosphodiesterases. It has been suggested, but not proved, that inositol cyclic phosphate may be released intracellularly and may function as a second messenger (for a review, see ref. [l] ). The production of inositol cyclic phosphate by the inositide-specific eukaryote ‘phospholipase C’ might be the result either of a special enzymic mechanism or of the attack of an enzyme with the usual phospholipase C mechanism upon a substrate which can yield a cyclic phosphate. This question cannot be resolved by study of the eukaryote enzymes since they do not attack those lipids which cannot lead to cyclic phosphates. However, the phospholipase C which is present in the culture filtrate of Bacillus cereus can be used for such an experiment since it attacks, inter alia, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol. The experiments reported here demonstrate that the attack of this enzyme on phosphatidylinositol leads exclusively to production of inositol cyclic phosphate. This enzyme can therefore act either as a phosphodiesterase