Tania Markovska

Ha‐ ras‐TRANSFORMATION ALTERS THE METABOLISM OF PHOSPHATIDYLETHANOLAMINE AND PHOSPHATIDYLCHOLINE IN NIH 3T3 FIBROBLASTS

Cultured NIH 3T3 fibroblasts were employed to investigate the changes in the phospholipid metabolism induced by Ha‐ ras transformation. All phospholipid fractions were reduced in ras ‐transformed fibroblasts except phosphatidylethanolamine (PE). The incorporation of labeled choline and ethanolamine into phosphatidylcholine (PC), PE and their corresponding metabolites were elevated in a similar manner in the transformed cells. The enhanced uptake of choline and ethanolamine correlated with the activation of choline kinase and ethanolamine kinase. Similarly, the uptake of arachidonic, oleic and palmitic acids by PC and PE was higher in ras ‐cells. Acyl‐CoA synthetases, which esterify fatty acid before their incorporation into lysophospholipids, were also activated. However, both CTP:phosphocholine‐cytidylyltransferase and CTP:phosphoethanolamine‐chytidyltransferase were inhibited in the transformed cells. This fact, taken together with the observed activation of choline‐ and ethanolamine kinases, led to accumulation of phosphocholine and phosphoethanolamine, which have been presumed to participate in the processes of tumor development. PC biosynthesis seemed to be carried out through the CDP‐choline pathway, which was stimulated in the oncogenic cells, whereas PE was more likely, a product of phosphatidylserine decarboxylation rather than the CDP‐ethanolamine pathway.

Phosphatidylethanolamine and phosphatidylcholine are sources of diacylglycerol in ras-transformed NIH 3T3 fibroblasts

Ras-transformation of cells is accompanied by an increase of the level of diacylglycerol (DAG), which participates in the signal transduction pathways. DAG could be generated from phospholipids either by activation of phospholipase C or by a more complex pathway involving phospholipase D and phosphatidate phosphohydrolase. To clarify which phospholipids produce DAG and which pathways are involved, we examined the DAG generating enzyme activities, using phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) as substrates. The study showed that the breakdown of PC and more markedly of PE by phospholipases C and D was stimulated in membranes from ras-transformed cells. Phosphatidate phosphohydrolase activity was also elevated in oncogene-expressing cells. The increase in glycerol uptake was most pronounced in cells given PE, followed by PC. The fatty acid analysis revealed apparent similarities between the acyl chains of PE and DAG only in the transformed cells. These findings suggest that PE is a source of DAG in ras-fibroblasts but does not rule out the role of PC in DAG production, due to the activation of the PC-specific phospholipases C and D.

Age-related changes in rat liver plasma membrane sphingomyelinase activity

The age-induced changes of some phospholipid fractions, membrane fluidity and neutral membrane-bound sphingomyelinase (EC 3.1.4.12) activity in rat liver plasma membranes have been investigated. Alterations in the percentage participation of phosphatidylcholine and sphingomyelin with aging have been established. Regression analysis indicated a positive linear correlation (r = 0.927) between the membrane-bound neutral sphingomyelinase activity and the phosphatidylcholine percent in the total plasma membrane phospholipids, as well as a negative linear correlation (r = -0.937) between the enzyme activity and the sphingomyelin/phosphatidylcholine ratio.

Resveratrol alters the lipid composition, metabolism and peroxide level in senescent rat hepatocytes

Investigations were performed on the influence of resveratrol on the lipid composition, metabolism, fatty acid and peroxide level in plasma membranes of hepatocytes, isolated from aged rats. Hepatocytes were chosen due to the central role of the liver in lipid metabolism and homeostasis. The obtained results showed that the level of sphingomyelin (SM) and phosphatidylserine (PS) was augmented in plasma membranes of resveratrol-treated senescent hepatocytes. The saturated/unsaturated fatty acids ratio of the two most abundant membrane phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), was decreased as a result of resveratrol treatment. The neutral sphingomyelinase was found to be responsible for the increase of SM and the decrease of ceramide in plasma membranes of resveratrol-treated senescent hepatocytes. Using labeled acetate as a precursor of lipid synthesis we demonstrated, that resveratrol treatment resulted in inhibition mainly of phospholipid synthesis, followed by fatty acids synthesis. Resveratrol induced reduction of specific membrane-associated markers of apoptosis such as localization of PS in the external plasma membrane monolayer and ceramide level. Finally, the content of lipid peroxides was investigated, because the unsaturated fatty acids, which were augmented as a result of resveratrol treatment, are an excellent target of oxidative attack. The results showed that the lipid peroxide level was significantly lower, ROS were slightly reduced and GSH was almost unchanged in resveratrol-treated hepatocytes. We suggest, that one possible biochemical mechanism, underlying the reported resveratrol-induced changes, is the partial inactivation of neutral sphingomyelinase, leading to increase of SM, the latter acting as a native membrane antioxidant. In conclusion, our studies indicate that resveratrol treatment induces beneficial alterations in the phospholipid and fatty acid composition, as well as in the ceramide and peroxide content in plasma membranes of senescent hepatocytes. Thus, the presented results imply that resveratrol could improve the functional activity of the membrane lipids in the aged liver by influencing specific membrane parameters, associated with the aging process.

Phospholipid-dependence of rat liver plasma membrane protein kinase activities--a new approach.

The influence of the phospholipid composition and fluidity on protein kinase A and protein kinase C activities in rat liver plasma membranes was studied. We observed that enrichment of membranes with phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine and dioleoylphosphatidylcholine caused activation of both protein kinases. Phosphatidylglycerol was found to be most effective activator. The enrichment of plasma membranes with dipalmitoylphosphatidylcholine and sphingomyelin led to decrease in protein kinase A and C activities. The stimulatory effect of phosphatidylglycerol was confirmed in plasma membranes pretreated with exogenous phospholipases A2, C and D, and subsequently enriched with phosphatidylglycerol. We suggest that besides the specific presence of definite phospholipids protein kinases A and C require a more fluid membrane lipid bilayer to display an optimal activity.

ACYL-COA:1-ACYL-GLYCERO-3-PHOSPHOETHANOLAMINE O-ACYLTRANSFERASE AND LIVER PLASMA MEMBRANE FLUIDITY

Investigations have been carried out on the influence of membrane lipid composition and physical state on acyl-CoA: 1-acyl-glycerol-3-phosphoethanolamine O-acyltransferase activity in rat liver plasma membranes. The lipid composition of the membranes was modified either by way of lipid transfer proteins or by partial delipidation with exogenous phospholipases and subsequent enrichment of the membranes with different phospholipids. The results indicated that membrane rigidification by enrichment of the membranes with DPPC or SM reduced the transfer of oleic and palmitic acid to lysophosphatidylethanolamine, whereas all phospholipids inducing membrane fluidization lead to acyltransferase activation. The eventual role of membrane fluidity in the deacylation-reacylation cycle is discussed.

Phospholipid composition of subcellular fractions and phospholipid-exchange activity in chicken liver and MC-29 hepatoma

The phospholipid composition of mitochondria, microsomes and plasma membranes from liver and MC-29 hepatoma from White Leghorn chickens has been investigated. It was established that all mitochondria and microsome phospholipid fractions obtained from MC-29 hepatoma are increased strongly compared to those from liver. The sphingomyelin augmentation was particularly great. In hepatoma plasma membranes only the sphingomyelin quantity was increased. Sphingomyelin- and phosphatidylcholine-exchange activities were observed in avian liver for the first time. These two activities were increased in MC-29 hepatoma cells. Three phospholipid-exchange proteins have been established in chicken liver 105000 X g supernatant. One of them specifically transports phosphatidylcholine, the second one is non-specific for phosphatidylcholine and sphingomyelin, and the third one is specific only for sphingomyelin. In hepatoma cells only a non-specific phosphatidylcholine- and sphingomyelin-exchange protein was found.

d-Galactosamine induced changes in rat liver plasma membranes lipid composition and some enzyme activities

The influence of D-galactosamine administration on rat liver plasma membranes lipid composition, fluidity and some enzyme activities was investigated. D-Galactosamine was found to induce an increase of the total phospholipids, the cholesterol level and membrane rigidity. In liver plasma membranes of D-galactosamine-treated rats the exogenous phospholipase A2 activity was enhanced about 2 fold, whereas the endogenous activity was slightly decreased. No alteration of the neutral sphingomyelinase activity was observed.

Phospholipid dependence of membrane-bound phospholipase A2 in ras-transformed NIH 3T3 fibroblasts

Although the activation of phospholipase A2 (PLA2) in ras-transformed cells has been well documented, the mechanisms underlying this activation are poorly understood. In this study we tried to elucidate whether the membrane phospholipid composition and physical state influence the activity of membrane-associated PLA2 in ras-transformed fibroblasts. For this purpose membranes from non-transfected and ras-transfected NIH 3T3 fibroblasts were enriched with different phospholipids by the aid of partially purified lipid transfer protein. The results showed that of all tested phospholipids only phosphatidylcholine (PC) increased PLA2 activity in the control cells, whereas in their transformed counterparts both PC and phosphatidic acid (PA) induced such effect. Further we investigated whether the activatory effect was due only to the polar head of these phospholipids, or if it was also related to their acyl chain composition. The results demonstrated that the arachidonic acid-containing PC and PA molecules induced a more pronounced increase of membrane-associated PLA2 activity in ras-transformed cells compared to the corresponding palmitate-stearate- or oleate- containing molecular species. However, we did not observe any specific effect of the phospholipid fatty acid composition in non-transformed NIH 3T3 fibroblasts. In ras-transformed cells incubated with increasing concentrations of arachidonic acid, PLA2 activity was altered in parallel with the changes of the cellular content of this fatty acid. The role of phosphatidic and arachidonic acids as specific activators of PLA2 in ras-transformed cells is discussed with respect to their possible role in the signal transduction pathways as well as in the processes of malignant transformation of cells.

Phospholipid dependence of rat liver microsomal acyl: CoA synthetase and acyl-CoA : 1-Acyl-sn-glycero-3-phosphocholine O-acyltransferase

SummaryInvestigations were performed on the influence of the phospholipid composition and physicochemical properties of the rat liver microsomal membranes on acyl-CoA synthetase and acyl-CoA : 1-acyl-sn-glycero-3-phosphocholine O-acyltransferase activities. The phospholipid composition of the membranes was modified by incubation with different phospholipids in the presence of lipid transfer proteins or by partial delipidation with exogenous phospholipase C and subsequent enrichment with phospholipids. The results indicated that the incorporation of phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine induced a marked activation of acyl-CoA synthetase for both substrates used—palmitic and oleic acids. Sphingomyelin occurred as specific inhibitor for this activity especially for palmitic acid. Palmitoyl-CoA: and oleoyl-CoA : lacyl-sn-glycero-3-phosphocholine acyltransferase activities were found to depend on the physical state of the membrane lipids. The alterations in the membrane physical state were estimated using two different fluorescent probes—1,6-diphenyl-1,3,5-hexatriene and pyrene. In all cases of membrane fluidization this activity was elevated. On the contrary, in more rigid membranes obtained by incorporation of sphingomyelin and dipalmitoylphosphatidylcholine, acyltransferase activity was reduced for both palmitoyl-CoA and oleoyl-CoA. We suggest a certain similarity in the way of regulation of membrane-bound acyltransferase and phospholipase A2 which both participate in the deacylation-reacylation cycle.