Effects of phospholipid base analogues on the subcellular membrane ether composition of suspension cultured LM cells

doi:10.1016/0005-2760(76)90167-3

Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism

Volume 441, Issue 2, 23 August 1976, Pages 239-254

Biochimica et Biophy...

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Abstract

A strain of mouse fibroblasts, LM cells, was cultured in suspension with a chemically defined medium in the absence of lipid or serum. The subcellular distribution of ether-linked lipids in these cells was determined. O-alkyl and O-alk-1-enyl glycerolipids were found in plasma membranes, microsomes, and mitochondria. The glyceryl ether diesters from all three membrane fractions were devoid of O-alk-1-enyl moieties. Phospholipids contained both O-alkyl and O-alk-1-enyl ether chains. The distribution of these lipids among the subcellular fractions was not uniform. Microsomes contained the largest quantities of O-alkyldiacylglycerols, O-alkylacylphosphatides, and O-alk-1-enylacylphosphatides. Mitochondria were essentially devoid of O-alk-1-enyl phospholipids.

The in vivo incorporation of new polar head groups such as N,N-dimethyl-ethanolamine, N-monomethylethanolamine, or ethanolamine instead of N,N,N-trimethylethanolamine (choline) into LM suspension cell membrane phospholipids resulted in increased levels of ether-linked glycerolipids. These increases were inversely proportional to the number of CH₃ groups on the nitrogen atom of the base supplement. Both neutral lipids and phospholipids contained increased (2-fold or greater) quantities of O-alkyl moieties, but the levels of O-alk-1-enyl ether moieties in the phospholipids were slightly decreased or unchanged. These alterations in ether lipid content occurred primarily in the microsomes and plasma membranes while mitochondrial ether content or composition was not altered significantly. The chain length composition and degree of unsaturation of the O-alkyl and O-alk-1-enyl side chains from neutral lipids and phospholipids of these membranes were determined. Small (less than 10%) increases in unsaturation of O-alkyl side chains and larger (2-fold) increases in unsaturation of O-alk-1-enyl side chains of plasma membranes and microsomes were noted. These changes were not consistent with developmental alterations during the growth phase since the degree of unsaturation of ether-linked lipids from control LM cells decreased with age of the culture, an affect opposite to that noted in the presence of base analogues. In addition, the increases in levels of ether lipids after base analogue supplementation were much greater than could be accounted for simply by alterations in growth phase of LM suspension cells.

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Glycerophospholipids with ether linkages are found in nearly all animal and bacterial cells with the exception of most aerobic bacteria. In contrast to the diacyl types of glycerophospholipids, the plasmalogens contain a hydrocarbon chain attached to glycerol through a dehydrated hemiacetal (vinyl ether) linkage and the alkylacyl glycerophospholipids contain a hydrocarbon chain attached through an ether linkage. The oxidation levels of the ether-linked side-chains are aldehyde and alcohol for plasmalogens and alkylacyl glycerophospholipids, respectively. Plasmalogens are glycerophospholipids containing a potential aldehyde that were first discovered in the plasma of cells. Thus the name was derived from plasm + al + ogen. A correct common name for 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine is “ethanolamine plasmalogen.” Purified preparations of plasmalogens have been made by removal of the diacyl glycerophospholipids by mild alkaline hydrolysis, hydrolysis by phospholipase A₂ from snake venoms and by hydrolysis by phospholipase D from cabbage. These methods depend on the lesser reactivity of plasmalogens.
Altered phospholipid composition affects endocytosis in cultured LM fibroblasts
1981, BBA - Biomembranes
The phospholipid polar head group composition of LM fibroblast membranes was altered by growing the cells in a chemically defined, serum-free medium containing choline, $N,N′- dimethylethanolamine$ , $N- monomethylethanolamine$ , or ethanolamine. The cells incorporated these bases into their membrane phospholipid such that 29–40% of the total plasma membrane phospholipids contained these polar head groups. Alteration of the phospholipid composition correlated with a depression of polystyrene bead phagocytosis by 36, 55 and 85% when the cells had been supplemented with $N,N′- dimethylethanolamine$ , $N- monoethylethanolamine$ , or ethanolamine, respectively. Pinocytotic uptake of horseradish peroxidase was depressed 44, 39, and 32%, respectively. The phagosomal membrane phospholipid composition qualitatively resembled that of the primary plasma membrane from which it was derived. However, enrichment of phosphatidylcholine, and other quantitative differences were noted in the phagosomal membranes as compared to the parent primary plasma membrane. Approx. 50% of the phagosomal membrane's phosphatidylethanolamine was accessible to the chemical labelling reagent trinitrobenzenesulfonate at 4°C. The asymmetric distribution of phosphatidylethanolamine across the phagosomal membrane did not appear to be altered by base analogues except in the case of phagosomes from cells supplemented with ethanolamine. The data were consistend with a nonrandom site for endocytosis with regard to phospholipid composition.
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1980, BBA - Biomembranes
The regulation of the asymmetric distribution of aminophospholipids in mammalian cell plasma membranes is not understood at this time. One approach to determine the nature of such regulatory mechanisms is to attempt alteration of the plasma membrane phospholipid composition. Choline analogues such as $N,N′- dimethylethanolamine$ and $N- monomethylethanolamine$ lowered the quantity of phosphatidylethanolamine in the plasma membrane of LM fibroblasts grown in defined medium without serum. Ethanolamine supplementation increased the phosphatidylethanolamine content while ethanolamine analogues such as 2-amino-2-methyl-1-propanol, 2-amino-1-butanol, 1-aminopropanol, and 3-aminopropanol did not alter the aminophospholipid content significantly. The transverse distribution of aminophospholipids in the plasma membrane was determined by use of a chemical labelling reagent trinitrobenzenesulfonic acid. The percent phosphatidylethanolamine trinitrophenylated by trinitrobenzenesulfonate in the outer plasma membrane monolayer of LM cells supplemented with choline analogues was not altered. In contrast, ethanolamine analogue supplementation increased the percentage of aminophospholipid in the outer monolayer 2–3-fold. Ethanolamine analogue-containing phospholipids were distributed asymmetrically across the plasma membrane with 85 to 91% being located in the inner monolayer of the plasma membrane, a distribution similar to that of phosphatidylethanolamine. The fatty acyl composition of aminophospholipids in the outer monolayer was in all cases more saturated than in the corresponding phospholipids of the inner monolayer. However, choline analogues and especially the ethanolamine analogues reduced this difference. Thus, base analogues of choline and ethanolamine may alter the aminophospholipid asymmetry, the surface charge, and the acyl chain asymmetry of LM cell plasma membranes
A potential role for phospholipids in the regulation of 3-hydroxy-3-methylglutaryl-coenzyme a reductase in cultured C-6 glial cells effects of N,N-dimethylethanolamine
1979, Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism
The relation of the activity of the microsomal enzyme, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, to cellular phospholipid composition was studied in C-6 glial cells. Phospholipid composition was perturbed by growth of cells in the naturally occurring amino alcohol, N, N-dimethylethanolamine. After growth of C-6 glia in 5 mM N, N-dimethylethanolamine for 24 h, reductase activity was diminished by 50%. A similar diminution in cholesterol synthesis was observed. This effect was not accompanied by any parallel change in cell growth, DNA synthesis, protein synthesis, fatty acid synthetase activity, or microsomal NADPH-cytochrome c reductase activity. The inhibition of reductase activity by N, N-dimethylethanolamine was prevented by the addition of equimolar concentrations of choline to the culture medium and, also, could be reversed completely by removal of N, N-dimethylethanolamine from the culture medium. The effect of N, N-dimethylethanolamine on reductase was associated with the formation of phosphatidyl-N, N-dimethylethanolamine which accumulated primarily at the expense of phosphatidylcholine and, after 24 h, accounted for 27% of total phospholipid phosphorus.
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^∗: Present address: Department of Pharmacology, University of Missouri Medical School, Columbia, Mo. 65201 U.S.A.

^∗∗: Present address: Merck Sharp and Dohme Research Laboratories, Division of Merck and Co., In., Rahway N.J. 07065 U.S.A.

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Effects of phospholipid base analogues on the subcellular membrane ether composition of suspension cultured LM cells

Abstract

Prog. Chem. Fats Other Lipids

J. Lipid Res.

FEBS Lett.

Comp. Biochem. Physiol.

Biochim. Biophys. Acta

Arch. Biochem. Biophys.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

FEBS Lett.

J. Lip. Res.