Transport and decarboxylation of liposomal phosphatidylserine: effect of cations.

Decarboxylation of liposomal phosphatidylserine by rat liver and Ehrlich ascites tumor mitochondria was taken as a measure of phospholipid transfer. The process was found to be greatly enhanced by the cytoplasmic fraction of rat liver containing nonspecific lipid transfer protein, but not by the cytoplasmic fraction from tumor cells. Divalent cations, like rat liver cytoplasmic fraction, also stimulated phosphatidylserine decarboxylation by facilitating the lipid association with mitochondria. In contrast, these cations, at 0.5-3 mM concentration, inhibited the cytoplasmic fraction-mediated phosphatidylserine transport. Monovalent cations were also inhibitory but at 20-150 mM concentration. However, they had no effect on phosphatidylserine decarboxylation in the absence of the cytoplasmic fraction. Further experiments with purified rat liver nonspecific lipid transfer protein and pyrene-labeled phosphatidylcholine and phosphatidylserine have shown that cations by neutralizing net negative charge on phospholipid donor vesicles decrease the interaction of protein with them and, in consequence, lower the rate of release of molecules to the water phase.

Intramitochondrial distribution and transport of phosphatidylserine and its decarboxylation product, phosphatidylethanolamine. Application of pyrene-labeled species.

To investigate the mechanism of intramitochondrial translocation of phosphatidylserine and its decarboxylation product, phosphatidylethanolamine, the distribution of these lipids between the outer (OM) and inner (IM) mitochondrial membranes, as well as their transversal and lateral distribution in OM were studied. Fluorescent, pyrenyl derivatives of phosphatidylserine (PyrxPS) and phosphatidylethanolamine (PyrxPE) species were employed because they allow: (i), direct monitoring of PS (and PE) loading to the mitochondria; (ii) assay of PS decarboxylation by high-performance liquid chromatography with fluorescence detection and (iii), determination of the lateral distributions of PS and PE within the mitochondrial membranes. All PyrxPS species tested were efficiently decarboxylated by the solubilized decarboxylase and thus the distribution of the endogenous PE could be also studied. When the PyrxPS species were loaded to isolated mitochondria very little, if any, of the loaded PyrxPS or of the PyrxPE product was found in IM independent of the time and temperature of incubation, strongly suggesting that these lipids either never enter IM or their residence there is only transient. When mitochondria preloaded with Pyr4PS were incubated with an excess of acceptor vesicles in the presence of the lipid transfer protein, 80% of Pyr4PS and 30-40% of the Pyr4PE product were transported to the acceptor vesicles, indicating that at least corresponding fractions of these lipid were located in, or were in rapid equilibrium with the outer leaflet of OM. Since the decarboxylase is located in the inner membrane, these results signify that both PS and PE must be able to move readily across OM. Determination of the excimer to monomer ratio as the function of pyrenyl lipid concentration in mitochondria (i.e., OM) gave parallel results for PyrxPS and -PE species suggesting the lateral distribution of PS and PE in OM is similar and thus there is no specific enrichment of PS to the contact sites. To investigate the mechanism of PS transport from the outer leaflet to the decarboxylation site, the influence of PyrxPS hydrophobicity, i.e., pyrenylacyl chain length, on the rate of decarboxylation was determined. The variation of the length of the pyrenyl acyl chain from 4 to 12 carbons did not significantly affect the rate of PyrxPS decarboxylation in intact mitochondria, indicating that the transport of PS from the outer leaflet of OM to the site of decarboxylation takes place by lateral diffusion rather than by spontaneous or protein-mediated transport. The implications of these findings on the mechanism of intramitochondrial transport of PS and PE are discussed in terms of alternative models.

Structural organization of mammalian lipid phosphate phosphatases: implications for signal transduction.

This article describes the regulation of cell signaling by lipid phosphate phosphatases (LPPs) that control the conversion of bioactive lipid phosphates to their dephosphorylated counterparts. A structural model of the LPPs, that were previously called Type 2 phosphatidate phosphatases, is described. LPPs are characterized by having no Mg(2+) requirement and their insensitivity to inhibition by N-ethylmaleimide. The LPPs have six putative transmembrane domains and three highly conserved domains that define a phosphatase superfamily. The conserved domains are juxtaposed to the proposed membrane spanning domains such that they probably form the active sites of the phosphatases. It is predicted that the active sites of the LPPs are exposed at the cell surface or on the luminal surface of intracellular organelles, such as Golgi or the endoplasmic reticulum, depending where various LPPs are expressed. LPPs could attenuate cell activation by dephosphorylating bioactive lipid phosphate esters such as phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate. In so doing, the LPPs could generate alternative signals from diacylglycerol, sphingosine and ceramide. The LPPs might help to modulate cell signaling by the phospholipase D pathway. For example, phosphatidate generated within the cell by phospholipase D could be converted by an LPP to diacylglycerol. This should change the relative balance of signaling by these two lipids. Another possible function of the LPPs relates to the secretion of lysophosphatidate and sphingosine 1-phosphate by activated platelets and other cells. These exogenous lipids activate phospholipid growth factor receptors on the surface of cells. LPP activities could attenuate cell activation by lysophosphatidate and sphingosine 1-phosphate through their respective receptors.

An antagonist of ATP-regulated potassium channels, the guanidine derivative U-37883A, stimulates the synthesis of phosphatidylserine in rat liver endoplasmic reticulum membranes.

The guanidine derivative U-37883A has been found to stimulate in vitro synthesis of phosphatidylserine in endoplasmic reticulum membranes, catalyzed exclusively by a serine-specific base exchange enzyme. The stimulation of the enzyme activity by the drug was concentration-dependent, with EC50 of 54 microM, while the biologically inactive analog of U-37883A, U-42069, was without effect. The stimulation caused by U-37883A was enhanced under the conditions when active transport of Ca2+ into the lumen of microsomal vesicles was induced, whereas it was inhibited by a calcium ionophore, A23187, and by a specific inhibitor of Ca2+-ATPase, thapsigargin. On the other hand, a potassium ionophore, valinomycin, had no effect on phosphatidylserine synthesis. U-37883A did not affect the Km of the base exchange enzyme for serine, but greatly reduced the EC50 value of the enzyme for calcium. Furthermore, Ca2+ uptake by endoplasmic reticulum vesicles has been found to increase in the presence of U-37883A. These observations suggest that U-37883A enhances phosphatidylserine synthesis indirectly by acting on calcium transport, thus affecting calcium concentration within the lumen of endoplasmic reticulum membranes. Alternatively, the effect of the drug could be propagated via the mechanism by which phospholipid flip-flop movement, known to regulate the serine-specific base exchange reaction, is modulated.

Positive feedback between ethanolamine-specific phospholipid base exchange and cytochrome P450 activities in rat liver microsomes. The effect of clofibric acid.

The results of the present investigation relate the effects of the nutritional state and administration of clofibric acid (CLA), a hypolipidaemic drug and peroxisomal proliferator, on phosphatidylethanolamine (PE) synthesis in rat liver and fatty acid metabolism. Fasting and CLA treatment of animals causes an increase in the amount of PE in endoplasmic reticulum (ER) membranes and mitochondria, as well as in the PE/phosphatidylcholine (PC) ratio. Moreover, the activity of the ethanolamine-specific phospholipid base exchange (PLBE) enzyme in liver ER membranes of fasted animals was enhanced by 75% in comparison to that of animals fed ad libitum. The effect of CLA treatment was additive to that of starvation; PE synthesis tested in vitro via the Ca2+-sensitive PLBE reaction increased 3-fold in comparison to rats fed ad libitum. This is confirmed by an increased Vmax for the reaction, but the affinity of the enzyme for ethanolamine was not significantly changed. These effects were accompanied by an enhanced expression of cytochrome P450 CYP4A1 isoform and elevated activity of the enzyme upon CLA administration. The stimulatory effect of CLA administration on the efficiency of the ethanolamine-specific PLBE reaction can be explained by elimination of lauric acid, a known inhibitor of de novo PE synthesis, during the course of omega-hydroxylation catalysed by CYP4A1, and by increased expression of the PLBE enzyme. The products of omega-hydroxylation of lauric acid, which are then converted by dehydrogenase to 1,12-dodecanedioic acid, did not significantly affect the in vitro synthesis of PE.

Membrane lateral pressure as a modulator of glycerol-3-phosphate dehydrogenase activity.

Michaelis-Menten kinetics of glycerol-3-phosphate dehydrogenase activity in proteoliposomes from brown adipose tissue mitochondria with exogenously added phospholipids or cholesterol was measured. It was shown that changes in membrane lipid composition affected the membrane lateral pressure and therefore modulated the enzyme activity, namely Vmax value. Contrarily, changes in surface charge caused by minute amounts of phosphatidylserine or charged organic substances influenced only the apparent Km value. The role of bulk phospholipids in regulation of glycerol-3-phosphate dehydrogenase is discussed.

Intracellular localization of labelling of tocopherols with [U-14C]tyrosine in Calendula officinalis leaves.

The content of tocopherols in the cellular subfractions of C. officinalis leaves from plants kept in the light and dark was investigated. In addition to the previously detected alpha-, gamma- and delta-tocopherols a small amount of 7-monomethyltocol was found in the chloroplasts, mitochondria and microsomes of plants kept in the dark. Studies on the dynamics of labelling of tocopherols in the dark and in the light with [U-14C]tyrosine in the chloroplast, mitochondrial and microsomal fractions have shown that two isomers of monomethyltocol, i.e. 7- and 8-methyltocols are formed. gamma-Tocopherol originates from 7-MeT rather than from 8-MeT and is partly methylated to alpha-tocopherol and partly undergoes some unknown transformation. The light is not indispensable for condensation of aromatic ring with the phytyl chain, whereas it is indispensable for methylation of the aromatic ring of tocopherols.

The induction of cytochrome P450 isoform, CYP4A1, by clofibrate coincides with activation of ethanolamine-specific phospholipid base exchange reaction in rat liver microsomes.

Administration of a hypolipidaemic drug, clofibrate, to rats resulted, 24 h after a single intraperitoneal injection (250 mg/kg body weight), in pronounced enhancement of the rate of phosphatidylethanolamine (PE) synthesis via the PE-specific base exchange (PEBE) reaction in liver microsomes. This was accompanied by 3.4-fold activation of microsomal omega-hydroxylation of lauric acid by cytochrome P450 4A1 isoform (CYP4A1) and an increase in the protein content of this isoform in endoplasmic reticulum (ER) membranes. Since PE represents a class of phospholipids (PL) prerequisite for proper functioning of CYP4A1, and the PEBE reaction is an inducible pathway of PL synthesis in hepatocytes under metabolic stress, one may speculate that this reaction is switched on when extensive remodelling of PL molecular species or/and massive synthesis of lipid bilayer components for membrane assembly is required.

[Extracellular components of broncho-alveolar lavage fluid (BALF) as markers of interstitial pulmonary disease activity. II. Phospholipid concentration]. / Pozakomórkowe skladniki plynu z plukania oskrzelowo-pecherzykowego (BALF) jako marker aktywnosci sródmiazszowych chorób pluc. Czesc II: Stezenie fosfolipidów.

Phospholipids found in the alveolar space come from the surfactant which is produced by type II pneumocytes and whose surplus is eliminated from lungs by macrophages. In 188 patients with interstitial pulmonary diseases bronchoalveolar lavage was performed. Phospholipids from the supernatant of BALF were extracted using of Folch method and their concentration was measured colorimetrically assaying inorganic phosphorus after prior mineralization by Fiske-Subbarov method. The persons were divided into 7 groups: active sarcoidosis, inactive sarcoidosis, avian fancier's lung in the period of contact with the antigen, and after stopping the contact with the antigen, advanced idiopathic pulmonary fibrosis, moderate idiopathic pulmonary fibrosis, and a control group of healthy persons. Apart from that, the patients were divided into untreated and treated with corticosteroids. In every group of patients we have noticed increased concentration and total amount of phospholipids in BALF. Particularly distinct increase of the total number of phospholipids in BALF in pulmonary fibrosis was observed. However, this parameter does not permit to estimate disease activity in interstitial lung disease. Corticotherapy increases phospholipids concentration in BALF in patients with idiopathic pulmonary fibrosis and avian fancier's lung but decreases in sarcoidosis patients.

[Extracellular components of bronchoalveolar lavage fluid (BALF) as a marker of the activity of interstitial pulmonary diseases. III: Phospholipids to protein concentration ratio]. / Pozakomórkowe skladniki plynu z plukania oskrzelowo-pçherzykowego (BALF) jako marker aktywnosci sródmiazszowych chorób pluc. Czesc III: Stosunek stezenia fosfolipidów do stezenia bialka.

In interstitial pulmonary diseases investigations are conducted to find markers of the activity of the interstitial processes so that noninvasive monitoring of the disease might be possible. In 188 patients divided into 9 groups: 42 with active sarcoidosis, 24 with inactive sarcoidosis, 16 with active sarcoidosis treated with steroids and 22 with inactive sarcoidosis after corticotherapy, 17 with avian fanciers' lung exposed to the antigen, 16 with avian fanciers' lung after a year interval in exposure to the antigen, 20 with advanced and 13 with moderate idiopathic pulmonary fibrosis, and 18 healthy persons the BAL was performed. In the BALF concentrations of protein and phospholipids were assayed by colorimetric method. The results indicate usefulness of the studied biochemical parameters in BALF in evaluation of the activity of interstitial pulmonary diseases. Significant differences were found between the results in the active group of patients compared to the control group and to the inactive forms of interstitial pulmonary diseases. Particularly valuable is phospholipids to protein concentration ratio in BALF.

Membrane integrity and phospholipid movement influence the base exchange reaction in rat liver microsomes.

Properties of Ca(2+)-stimulated incorporation of amincalcohols, serine and ethanolamine, into phospholipids, and factors regulating the reaction were studied in endoplasmic reticulum membranes isolated from rat liver. In contrast to apparent K(m) values for either aminoalcohol, maximal velocities of the reaction were significantly affected by Ca2+ concentration. No competition between these two soluble substrates used at equimolar concentrations close to their K(m) values was observed, suggesting the existence of two distinct phospholipid base exchange activities. The enzyme utilizing the electrically neutral serine was not sensitive to changes of membrane potential evoked by valinomycin in the presence of KCl. On the other hand, when positively charged ethanolamine served as a substrate, the enzyme activity was inhibited by 140 mM KCl and this effect was reversed by valinomycin. The rates of inhibition of phospholipid base exchange reactions by various thiol group modifying reagents were also found to differ. Cd2+ and lipophylic p-chloromercuribenzoic acid at micromolar concentrations were most effective. It can be suggested that -SH groups located within the hydrophobic core of the enzymes molecules are essential for the recognition of membrane substrates. However, the influence of the -SH group modifying reagents on the protein-facilitated phospholipid motion across endoplasmic reticulum membranes can not be excluded, since an integral protein-mediated transverse movement of phospholipids within the membrane bilayer and Ca(2+)-mediated changes in configuration of the phospholipid polar head groups seem to be a regulatory step of the reaction. Indeed, when the membrane integrity was disordered by detergents or an organic solvent, the reaction was inhibited, although not due to the transport of its water-soluble substrates is affected, but due to modulation of physical state of the membrane bilayer and, in consequence, the accessibility of phospholipid molecules.

Lipid phosphate phosphohydrolase-1 degrades exogenous glycerolipid and sphingolipid phosphate esters.

Lipid phosphate phosphohydrolase (LPP)-1 cDNA was cloned from a rat liver cDNA library. It codes for a 32-kDa protein that shares 87 and 82% amino acid sequence identities with putative products of murine and human LPP-1 cDNAs, respectively. Membrane fractions of rat2 fibroblasts that stably expressed mouse or rat LPP-1 exhibited 3.1-3. 6-fold higher specific activities for phosphatidate dephosphorylation compared with vector controls. Increases in the dephosphorylation of lysophosphatidate, ceramide 1-phosphate, sphingosine 1-phosphate and diacylglycerol pyrophosphate were similar to those for phosphatidate. Rat2 fibroblasts expressing mouse LPP-1 cDNA showed 1.6-2.3-fold increases in the hydrolysis of exogenous lysophosphatidate, phosphatidate and ceramide 1-phosphate compared with vector control cells. Recombinant LPP-1 was located partially in plasma membranes with its C-terminus on the cytosolic surface. Lysophosphatidate dephosphorylation was inhibited by extracellular Ca2+ and this inhibition was diminished by extracellular Mg2+. Changing intracellular Ca2+ concentrations did not alter exogenous lysophosphatidate dephosphorylation significantly. Permeabilized fibroblasts showed relatively little latency for the dephosphorylation of exogenous lysophosphatidate. LPP-1 expression decreased the activation of mitogen-activated protein kinase and DNA synthesis by exogenous lysophosphatidate. The product of LPP-1 cDNA is concluded to act partly to degrade exogenous lysophosphatidate and thereby regulate its effects on cell signalling.