Coenzyme Q distribution in HL-60 human cells depends on the endomembrane system

Coenzyme Q (Q) is an essential factor in the mitochondrial electron chain but also exerts important antioxidant functions in the rest of cell membranes of aerobic organisms. However, the mechanisms of distribution of Q among cell membranes are largely unclear. The aim of the present work is to study the mechanisms of distribution of endogenous Q(10) and exogenous Q(9) among cell membranes in human HL-60 cells. Endogenous Q(10) synthesized using the radiolabelled precursor [(14)C]-pHB was first detected in mitochondria, and it was later incorporated into mitochondria-associated membranes and endoplasmic reticulum (ER). Plasma membrane was the last location to incorporate [(14)C]-Q(10). Brefeldin A prevented Q(10) incorporation in plasma membrane. Exogenous Q(9) was preferably accumulated into the endo-lysosomal fraction but a significant amount was distributed among other cell membranes also depending on the brefeldin-A-sensitive endomembrane system. Our results indicate that mitochondria are the first location for new synthesized Q. Exogenous Q is mainly incorporated into an endo-lysosomal fraction, which is then rapidly incorporated to cell membranes mainly to MAM and mitochondria. We also demonstrate that both endogenous and dietary Q is distributed among endomembranes and plasma membrane by the brefeldin A-sensitive endo-exocytic pathway.     

Coenzyme Q distribution in HL-60 human cells depends on the endomembrane system

Coenzyme Q (Q) is an essential factor in the mitochondrial electron chain but also exerts important antioxidant functions in the rest of cell membranes of aerobic organisms. However, the mechanisms of distribution of Q among cell membranes are largely unclear. The aim of the present work is to study the mechanisms of distribution of endogenous Q₁₀ and exogenous Q₉ among cell membranes in human HL-60 cells. Endogenous Q₁₀ synthesized using the radiolabelled precursor [¹⁴C]-pHB was first detected in mitochondria, and it was later incorporated into mitochondria-associated membranes and endoplasmic reticulum (ER). Plasma membrane was the last location to incorporate [¹⁴C]-Q₁₀. Brefeldin A prevented Q₁₀ incorporation in plasma membrane. Exogenous Q₉ was preferably accumulated into the endo-lysosomal fraction but a significant amount was distributed among other cell membranes also depending on the brefeldin-A-sensitive endomembrane system. Our results indicate that mitochondria are the first location for new synthesized Q. Exogenous Q is mainly incorporated into an endo-lysosomal fraction, which is then rapidly incorporated to cell membranes mainly to MAM and mitochondria. We also demonstrate that both endogenous and dietary Q is distributed among endomembranes and plasma membrane by the brefeldin A-sensitive endo-exocytic pathway.     

Specificity of coenzyme Q10 for a balanced function of respiratory chain and endogenous ubiquinone biosynthesis in human cells

Coenzyme Q (Q) is an obligatory component of both respiratory chain and uncoupling proteins. Also, Q acts as an antioxidant in cellular membranes. Several neurodegenerative diseases are associated with modifications of Q10 levels. For these reasons, therapies based on Q supplementation in the diet are currently studied in order to mitigate the symptoms of these diseases. However, the incorporation of exogenous Q also affects aging process in nematodes probably affecting reactive oxygen species (ROS) production. The aim of the present work is to clarify if supplementation with both Q10 and Q6 isoforms affects mitochondrial Q10 content, respiratory chain activity and ROS levels in human cells. Cells incorporated exogenously added Q10 and Q6 isoforms into mitochondria that produced changes in mitochondrial activity depending on the side chain length. Supplementation with Q10, but not with Q6, increased mitochondrial Q-dependent activities. However, Q6 affected the mitochondrial membrane potential, ROS production, and increased the protein levels of both catalase and Mn-superoxide dismutase (Mn-SOD). Also, Q6 induced a transient decrease in endogenous mitochondrial Q10 levels by increasing its catabolism. These results show that human cells supplemented with Q6 undergo a mitochondrial impairment, which is not observed with Q10 supplementation.     

Effect of ubiquinone-10 on the stability of biomimetic membranes of relevance for the inner mitochondrial membrane

Ubiquinone-10 (Q10) plays a pivotal role as electron-carrier in the mitochondrial respiratory chain, and is also well known for its powerful antioxidant properties. Recent findings suggest moreover that Q10 could have an important membrane stabilizing function. In line with this, we showed in a previous study that Q10 decreases the permeability to carboxyfluorescein (CF) and increases the mechanical strength of 1-palmitoyl-2-oleyl-sn-glycero-phosphocholine (POPC) membranes. In the current study we report on the effects exerted by Q10 in membranes having a more complex lipid composition designed to mimic that of the inner mitochondrial membrane (IMM). Results from DPH fluorescence anisotropy and permeability measurements, as well as investigations probing the interaction of liposomes with silica surfaces, corroborate a membrane stabilizing effect of Q10 also in the IMM-mimicking membranes. Comparative investigations examining the effect of Q10 and the polyisoprenoid alcohol solanesol on the IMM model and on membranes composed of individual IMM components suggest, moreover, that Q10 improves the membrane barrier properties via different mechanisms depending on the lipid composition of the membrane. Thus, whereas Q10's inhibitory effect on CF release from pure POPC membranes appears to be directly and solely related to Q10's lipid ordering and condensing effect, a mechanism linked to Q10's ability to amplify intrinsic curvature elastic stress dominates in case of membranes containing high proportions of palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE).     

Effect of Sclerin on Amino Acid Incorporation into Mitochondria Isolated from Rat Liver

Though sclerin (SCL) stimulated amino acid incorporation into the protein fraction of post mitochondrial supernatant of rat liver homogenate, it had no effect on the incorporation into the isolated mitochondria at pH 7.2, despite of its stimulating effect on mitochondrial oxidative phosphorylation. SCL stimulated amino acid incorporation into the mitochondria at pH 6.1, and to some extent maintained the activity on that in mitochondria during aging in hypotonic Tris-HCl buffer (pH 7.2). Since SCL prevented leakage of amino acids from the mitochondria into these buffers, it was suggested that SCL may protect a structure of mitochondrial membrane which appeared to have a significance on transport of amino acids. In liver slices, SCL stimulated amino acid incorporation only into the extra-mitochondrial fraction for the first 3 min, but gradually turned to stimulate incorporation into mitochondria within 30 min.     

Partition of CoQ-homologs in lipid membranes

After studies on incorporation of Coenzyme-Q-homologs into mitochondrial membranes, we have undertaken the same investigation on phospholipid vesicles in order to know the behavior of different Ubiquinones in the biphasic system lipid membrane/H2O. Using concentrations of egg lecithin corresponding to the phospholipid content of 1 mg mitochondrial protein, it was found that some homologs are partitioned in lipid vesicles nearly the same as they are incorporated by mitochondria, while others behave differently. On the basis of the results it is possible to calculate approximately partition coefficients for each Q-homolog. This work shows that also membrane structure, besides the partition membrane/water, affects the incorporation of Ubiquinones.     

Hepatic mitochondrial membrane lipid environment and protein nutrition

Effect of feeding rice diet with and without lysine and threonine supplementation on hepatic mitochondria and its inner and outer membrane proteins, enzymes and phospholipids has been studied. The exchange of phosphatidylcholine and phosphatidylethanolamine between microsomes and mitochondria has also been studied under these conditions. Deficient diet lead to significant decrease in proteins as well as activities of monoamine oxidase, succinate dehydrogenase, cytochrome a + a3 and cytochrome c in mitochondria and its inner and outer membranes. Feeding of the deficient diet also significantly reduced total phospholipids and PC in mitochondria and its outer mitochondrial membrane. In the inner mitochondrial membrane, only PE and cardiolipin were reduced. The incorporation (DPM/microgram PLP) of [methyl-3H]choline and [methyl-14C]methionine into PC of mitochondria and its outer membrane and that of 32Pi into PC and PE of outer mitochondrial membrane but only into PC of inner mitochondrial membrane were significantly reduced in the deficient group. The exchange rates of PC and PE between microsomes and mitochondria were reduced in the deficient group. Supplementation of the deficient diet with lysine and threonine profoundly improved the above biochemical lesions as compared to casein fed rats.     

Uptake of exogenous coenzyme Q and transport to mitochondria is required for bc1 complex stability in yeast coq mutants.

Coenzyme Q (Q) is an essential component of the mitochondrial respiratory chain in eukaryotic cells but also is present in other cellular membranes where it acts as an antioxidant. Because Q synthesis machinery in Saccharomyces cerevisiae is located in the mitochondria, the intracellular distribution of Q indicates the existence of intracellular Q transport. In this study, the uptake of exogenous Q(6) by yeast and its transport from the plasma membrane to mitochondria was assessed in both wild-type and in Q-less coq7 mutants derived from four distinct laboratory yeast strains. Q(6) supplementation of medium containing ethanol, a non-fermentable carbon source, rescued growth in only two of the four coq7 mutant strains. Following culture in medium containing dextrose, the added Q(6) was detected in the plasma membrane of each of four coq7 mutants tested. This detection of Q(6) in the plasma membrane was corroborated by measuring ascorbate stabilization activity, as catalyzed by NADH-ascorbate free radical reductase, a transmembrane redox activity that provides a functional assay of plasma membrane Q(6). These assays indicate that each of the four coq7 mutant strains assimilate exogenous Q(6) into the plasma membrane. The two coq7 mutant strains rescued by Q(6) supplementation for growth on ethanol contained mitochondrial Q(6) levels similar to wild type. However, the content of Q(6) in mitochondria from the non-rescued strains was only 35 and 8%, respectively, of that present in the corresponding wild-type parental strains. In yeast strains rescued by exogenous Q(6), succinate-cytochrome c reductase activity was partially restored, whereas non-rescued strains contained very low levels of activity. There was a strong correlation between mitochondrial Q(6) content, succinate-cytochrome c reductase activity, and steady state levels of the cytochrome c(1) polypeptide. These studies show that transport of extracellular Q(6) to the mitochondria operates in yeast but is strain-dependent. When Q biosynthesis is disrupted in yeast strains with defects in the intracellular transport of exogenous Q, the bc(1) complex is unstable. These results indicate that delivery of exogenous Q(6) to mitochondria is required fore activity and stability of the bc(1) complex in yeast coq mutants.     

Mitochondrial glycerol-3-P acyltransferase 1 is most active in outer mitochondrial membrane but not in mitochondrial associated vesicles (MAV)

Glycerol 3-phosphate acyltransferase-1 (GPAT1), catalyzes the committed step in phospholipid and triacylglycerol synthesis. Because both GPAT1 and carnitine-palmitoyltransferase 1 are located on the outer mitochondrial membrane (OMM) it has been suggested that their reciprocal regulation controls acyl-CoA metabolism at the OMM. To determine whether GPAT1, like carnitine-palmitoyltransferase 1, is enriched in both mitochondrial contact sites and OMM, and to correlate protein location and enzymatic function, we used Percoll and sucrose gradient fractionation of rat liver to obtain submitochondrial fractions. Most GPAT1 protein was present in a vesicular membrane fraction associated with mitochondria (MAV) but GPAT specific activity in this fraction was low. In contrast, highest GPAT1 specific activity was present in purified mitochondria. Contact sites from crude mitochondria, which contained markers for both endoplasmic reticulum (ER) and mitochondria, also showed high expression of GPAT1 protein but low specific activity, whereas contact sites isolated from purified mitochondria lacked ER markers and expressed highly active GPAT1. To determine how GPAT1 is targeted to mitochondria, recombinant protein was synthesized in vitro and its incorporation into crude and purified mitochondria was assayed. GPAT1 was rapidly incorporated into mitochondria, but not into microsomes. Incorporation was ATP-driven, and lack of GPAT1 removal by alkali and a chaotropic agent showed that GPAT1 had become an integral membrane protein after incorporation. These results demonstrate that two pools of GPAT1 are present in rat liver mitochondria: an active one, located in OMM and a less active one, located in membranes (ER-contact sites and mitochondrial associated vesicles) associated with both mitochondria and ER.     

Virgin olive oil and coenzyme Q10 protect heart mitochondria from peroxidative damage during aging.

The mitochondrial theory of aging suggests that this phenomenon is the consequence of random somatic mutations in mitochondrial DNA, induced by long-term exposure to free radical attack. There are two potential dietary means of delaying the effects of free radicals on cellular aging, i.e., enrichment of mitochondrial membranes with monounsaturated fatty acids and supplementation with antioxidants. We have performed a preliminary study on male rats, 6 or 12 month old, fed with diets differing in the nature of the fat (virgin olive oil or sunflower oil) and/or with antioxidant supplementation (coenzyme Q10), analysing hydroperoxide and coenzyme Q9 and Q10 in heart mitochondria. Preliminary results allow us to conclude that the CoQ10 dietetic supplementation as well as the enrichment of the cellular membranes with monounsaturated fatty acids, successfully protect mitochondrial membranes from aged rats against the free radical insult.     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

Role of membrane contact sites in protein import into mitochondria

Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long‐standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence‐carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture.     

Mechanism of liponecrosis,a distinct mode of programmed cell death

An exposure of the yeast Saccharomyces cerevisiae to exogenous palmitoleic acid (POA) elicits “liponecrosis," a mode of programmed cell death (PCD) which differs from the currently known PCD subroutines. Here, we report the following mechanism for liponecrotic PCD. Exogenously added POA is incorporated into POA-containing phospholipids that then amass in the endoplasmic reticulum membrane, mitochondrial membranes and the plasma membrane. The buildup of the POA-containing phospholipids in the plasma membrane reduces the level of phosphatidylethanolamine in its extracellular leaflet, thereby increasing plasma membrane permeability for small molecules and committing yeast to liponecrotic PCD. The excessive accumulation of POA-containing phospholipids in mitochondrial membranes impairs mitochondrial functionality and causes the excessive production of reactive oxygen species in mitochondria. The resulting rise in cellular reactive oxygen species above a critical level contributes to the commitment of yeast to liponecrotic PCD by: (1) oxidatively damaging numerous cellular organelles, thereby triggering their massive macroautophagic degradation; and (2) oxidatively damaging various cellular proteins, thus impairing cellular proteostasis. Several cellular processes in yeast exposed to POA can protect cells from liponecrosis. They include: (1) POA oxidation in peroxisomes, which reduces the flow of POA into phospholipid synthesis pathways; (2) POA incorporation into neutral lipids, which prevents the excessive accumulation of POA-containing phospholipids in cellular membranes; (3) mitophagy, a selective macroautophagic degradation of dysfunctional mitochondria, which sustains a population of functional mitochondria needed for POA incorporation into neutral lipids; and (4) a degradation of damaged, dysfunctional and aggregated cytosolic proteins, which enables the maintenance of cellular proteostasis.     

Location and activity of ubiquinone 10 and ubiquinone analogues in model and biological membranes

Deuteriated analogues of ubiquinone 10 (Q10) have been dispersed with plasma membranes of Escherichia coli and with the inner membranes of beetroot mitochondria. Orientational order at various deuteriated sites was measured by solid-state deuterium nuclear magnetic resonance (2H NMR). Similar measurements were made, using the compounds dispersed in dimyristoylphosphatidylcholine (DMPC) and egg yolk lecithin and dispersions prepared from the lipid extracts of beetroot mitochondria. In all cases only a single unresolved 2H NMR spectrum (typically 1000-Hz full width at half-height) was observed at concentrations down to 0.02 mol % Q10 per membrane lipid. This result shows that most Q10 is in a mobile environment which is physically separate from the orientational constraints of the bilayer lipid chains. In contrast, a short-chain analogue of Q10, in which the 10 isoprene groups have been replaced by a perdeuteriated tridecyl chain, showed 2H NMR spectra with quadrupolar splittings typical of an ordered lipid that is intercalated into the bilayer. The NADH oxidase activity and O2 uptake in Escherichia coli and in mitochondria were independent of which analogue was incorporated into the membrane. Thus, despite the major difference in their physical association with membranes, or their lipid extracts, the electron transport function of the long- and short-chain ubiquinones is similar, suggesting that the bulk of the long-chain ubiquinone does not have a direct function in electron transporting activity. The physiologically active Q10 may only be a small fraction of the total ubiquinone, a fraction that is below the level of detection of the present NMR equipment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Targeting, import, and dimerization of a mammalian mitochondrial ATP binding cassette (ABC) transporter, ABCB10 (ABC-me)

ATP binding cassette (ABC) transporters are a diverse superfamily of energy-dependent membrane translocases. Although responsible for the majority of transmembrane transport in bacteria, they are relatively uncommon in eukaryotic mitochondria. Organellar trafficking and import, in addition to quaternary structure assembly, of mitochondrial ABC transporters is poorly understood and may offer explanations for the paucity of their diversity. Here we examine these processes in ABCB10 (ABC-me), a mitochondrial inner membrane erythroid transporter involved in heme biosynthesis. We report that ABCB10 possesses an unusually long 105-amino acid mitochondrial targeting presequence (mTP). The central subdomain of the mTP (amino acids (aa) 36-70) is sufficient for mitochondrial import of enhanced green fluorescent protein. The N-terminal subdomain (aa 1-35) of the mTP, although not necessary for the trafficking of ABCB10 to mitochondria, participates in the proper import of the molecule into the inner membrane. We performed a series of amino acid mutations aimed at changing specific properties of the mTP. The mTP requires neither arginine residues nor predictable alpha-helices for efficient mitochondrial targeting. Disruption of its hydrophobic character by the mutation L46Q/I47Q, however, greatly diminishes its efficacy. This mutation can be rescued by cryptic downstream (aa 106-715) mitochondrial targeting signals, highlighting the redundancy of this protein's targeting qualities. Mass spectrometry analysis of chemically cross-linked, immunoprecipitated ABCB10 indicates that ABCB10 embedded in the mitochondrial inner membrane homodimerizes and homo-oligomerizes. A deletion mutant of ABCB10 that lacks its mTP efficiently targets to the endoplasmic reticulum. Quaternary structure assembly of ABCB10 in the ER appears to be similar to that in the mitochondria.     

The biogenesis of mitochondrial membranes in the yeast Saccharomyces cerevisiae.

Membrane lipids of yeast mitochondria have been enriched by growing yeast cells in minimal medium supplemented with specific unsaturated fatty acids as the sole lipid supplement. Using the activity of marker enzymes for the outer (kynurenine hydroxylase) and inner (cytochrome c oxidase and oligomycin-sensitive ATPase) mitochondrial membranes, Arrhenius plots have been constructed using both promitochondria and mitochondria obtained from O2-adapting cells in the presence of a second unsaturated fatty acid (i.e. linoleate (N2) to elaidic (O2)). Transition temperatures which reflect the unsaturated fatty acid enrichment of the new membranes reveal interesting features involved in the mechanism of the assembly of these two mitochondrial membranes. This approach was further enforced with both lipid depletion and mitochondrial protein inhibition studies. Kynurenine hydroxylase which does not require fatty acid for its continued synthesis during aerobiosis seems to be incorporated into the preformed linoleate-anaerobic outer membrane. The newly synthesized activities of inner mitochondrial membrane enzymes on the other hand, appear to integrate their activity into newly formed aerobic-elaidic-rich inner membrane. These latter enzymes show a distinct dependence on fatty acid supplement for their continued synthesis during their aerobic phase. This suggests that O2-dependent proteo-lipid precursors are formed before these enzymes are integrated into their membrane mosaic. Two separate models are proposed to explain these results, one for the lipid-rich outer mitochondrial membrane and another for the protein-rich inner mitochondrial membrane.     

Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins

To identify the membrane regions through which yeast mitochondria import proteins from the cytoplasm, we have tagged these regions with two different partly translocated precursor proteins. One of these was bound to the mitochondrial surface of ATP-depleted mitochondria and could subsequently be chased into mitochondria upon addition of ATP. The other intermediate was irreversibly stuck across both mitochondrial membranes at protein import sites. Upon subfraction of the mitochondria, both intermediates cofractionated with membrane vesicles whose buoyant density was between that of inner and outer membranes. When these vesicles were prepared from mitochondria containing the chaseable intermediate, they internalized it upon addition of ATP. A non-hydrolyzable ATP analogue was inactive. This vesicle fraction contained closed, right-side-out inner membrane vesicles attached to leaky outer membrane vesicles. The vesicles contained the mitochondrial binding sites for cytoplasmic ribosomes and contained several mitochondrial proteins that were enriched relative to markers of inner or outer membranes. By immunoelectron microscopy, two of these proteins were concentrated at sites where mitochondrial inner and outer membranes are closely apposed. We conclude that these vesicles contain contact sites between the two mitochondrial membranes, that these sites are the entry point for proteins into mitochondria, and that the isolated vesicles are still translocation competent.     

Comparisons of coenzyme Q bound to mitochondrial membrane proteins among different mammalian species.

The objective of this study was to elucidate the mechanisms that govern the variations in the rates of mitochondrial superoxide anion radical (O2-*) generation in different species. The amounts of coenzyme Q (CoQ) associated with mitochondrial membrane proteins were compared in five different mammalian species, namely mouse, rat, rabbit, pig, and cow. Micelles of cardiac mitochondria were prepared using Triton X-100 or deoxycholate (DOC) as detergents, and the micelles containing mitochondrial proteins were sedimented by sucrose density ultracentrifugation. The amount of CoQ present in both types of micelles varied in different species, whereas alpha-tocopherol, another lipoidal molecule in mitochondrial membranes, could not be detected in the micelles of any of these species. The amounts of CoQ bound to mitochondrial proteins in DOC micelles were higher in those mammalian species where CoQ10 was the predominant CoQ homologue, and the amounts were found to be inversely correlated with the rate of mitochondrial 02-* generation among different species. Results also indicated that mitochondrial CoQ exists in at least two distinct pools, one of which is associated with the membrane proteins. The degree of association between CoQ and membrane proteins appears to be a factor determining the rate of mitochondrial O2-* generation.     

Effect of polyunsaturated fatty acids and phospholipids on [3H]-vitamin E incorporation into pulmonary artery endothelial cell membranes

Vitamin E, a dietary antioxidant, is presumed to be incorporated into the lipid bilayer of biological membranes to an extent proportional to the amount of polyunsaturated fatty acids or phospholipids in the membrane. In the present study we evaluated the distribution of incorporated polyunsaturated fatty acids (PUFA) and phosphatidylethanolamine (PE) in various membranes of pulmonary artery endothelial cells. We also studied whether incorporation of PUFA or PE is responsible for increased incorporation of [3H]-vitamin E into the membranes of these cells. Following a 24-hr incubation with linoleic acid (18:2), 18:2 was increased by 6.9-, 9.2-, and 13.2-fold in plasma, mitochondrial, and microsomal membranes, respectively. Incorporation of 18:2 caused significant increases in the unsaturation indexes of mitochondrial and microsomal polyunsaturated fatty acyl chains (P less than .01 versus control in both membranes). Incubation with arachidonic acid (20:4) for 24 hr resulted in 1.5-, 2.3-, and 2.4-fold increases in 20:4 in plasma, mitochondrial, and microsomal membranes, respectively. The unsaturation indexes of polyunsaturated fatty acyl chains of mitochondrial and microsomal membranes also increased (P less than .01 versus control in both membranes). Although incubations with 18:2 or 20:4 resulted in several-fold increases in membrane 18:2 or 20:4 fatty acids, incorporation of [3H]-vitamin E into these membranes was similar to that in controls. Following a 24-hr incubation with PE, membrane PE content was significantly increased, and [3H]-vitamin E incorporation was also increased to a comparable degree, i.e., plasma membrane greater than mitochondria greater than microsomes. Endogenous vitamin E content of the cells was not altered because of increased incorporation of PE and [3H]-vitamin E. When [3H]-vitamin E was incorporated into lipid vesicles prepared from the total lipid extracts of endothelial cells and varying amounts of exogenous PE, vitamin E content was directly related to PE content. These results demonstrate that PUFA and PE distribute in all pulmonary artery endothelial cell membranes. However, only increases in PE were associated with increased incorporation of [3H]-vitamin E in membranes of these cells.