Polar head groups are important for barrier-protective effects of oxidized phospholipids on pulmonary endothelium

We have previously described protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on pulmonary endothelial cell (EC) barrier function and demonstrated the critical role of cyclopentenone-containing modifications of arachidonoyl moiety in OxPAPC protective effects. In this study we used oxidized phosphocholine (OxPAPC), phosphoserine (OxPAPS), and glycerophosphate (OxPAPA) to investigate the role of polar head groups in EC barrier-protective responses to oxidized phospholipids (OxPLs). OxPAPC and OxPAPS induced sustained barrier enhancement in pulmonary EC, whereas OxPAPA caused a transient protective response as judged by measurements of transendothelial electrical resistance (TER). Non-OxPLs showed no effects on TER levels. All three OxPLs caused enhancement of peripheral EC actin cytoskeleton. OxPAPC and OxPAPS completely abolished LPS-induced EC hyperpermeability in vitro, whereas OxPAPA showed only a partial protective effect. In vivo, intravenous injection of OxPAPS or OxPAPC (1.5 mg/kg) markedly attenuated increases in the protein content, cell counts, and myeloperoxidase activities detected in bronchoalveolar lavage fluid upon intratracheal LPS instillation in mice, although OxPAPC showed less potency. All three OxPLs partially attenuated EC barrier dysfunction induced by IL-6 and thrombin. Their protective effects against thrombin-induced EC barrier dysfunction were linked to the attenuation of the thrombin-induced Rho pathway of EC hyperpermeability and stimulation of Rac-mediated mechanisms of EC barrier recovery. These results demonstrate for the first time the essential role of polar OxPL groups in blunting the LPS-induced EC dysfunction in vitro and in vivo and suggest the mechanism of agonist-induced hyperpermeability attenuation by OxPLs via reduction of Rho and stimulation of Rac signaling.     

Studies on the turnover of endogenous choline-containing phospholipids of cultured neuroblastoma cells

The effects of two polypeptide antibiotics, polymixin B and gramicidin S, on the intracellular pool size and turnover of guanosine tetraphosphate (ppGpp) were analyzed in stringent (relA+) and relaxed (relA) strains of Escherichia coli. When either one of these two drugs was added to stringent bacteria cultures at a final concentration that blocked protein and RNA synthesis, ppGpp was found to accumulate. Under similar conditions of inhibition of macromolecular synthesis, ppGpp also appeared to accumulate in relaxed bacteria. Moreover, in either type of strain, no significant accumulation of guanosine pentaphosphate (pppGpp) could be detected upon drug treatment. It was, therefore, concluded that polymixin and gramicidin elicit ppGpp accumulation through a mechanism independent of the relA gene product and, consequently, quite distinct from the stringent control system triggered by amino acid starvation. Further experiments performed by using tetracycline as an inhibitor of ppGpp synthesis, showed that the increase in the level of this nucleotide induced by drug action was due, in fact, to a strong restriction of its degradation rate.     

Phospholipase D-catalyzed synthesis of new phospholipids with polar head groups

A series of new phospholipids with polar head groups have been synthesized by enzymatic transphosphatidylation of 1,2-dioleoyl-sn-glycerophosphocholine and identified by 1H NMR and MALDI-TOF-MS. The acceptor alcohols were N- or C2-substituted derivatives of ethanolamine (diethanolamine, triethanolamine, serinol, Tris, BisTris). Phospholipases D from cabbage (PLDcab) and Streptomyces sp. (PLDStr) were compared with respect to product yield and purity as well as the initial rates in transphosphatidylation and competing hydrolysis. In all reactions, PLDStr showed a remarkably higher transphosphatidylation activity than PLDcab. However, higher yields of the phospholipids with diethanolamine, triethanolamine, and serinol were obtained by PLDcab because PLDStr resulted in the additional formation of diphosphatidyl derivatives. In the synthesis of the Tris and BisTris derivatives, PLD(Str) was much more appropriate because voluminous head group alcohols (>129A3) are poorly converted by PLDcab. With BisTris as acceptor alcohol two regioisomeric forms of phosphatidyl-BisTris were obtained.     

Phosphatase activity in cultured glioma and neuroblastoma cells

Acid phosphatase activity in human glioma cells (138 MG) and mouse neuroblastoma cells (C 1300) was associated with structures accumulating neutral red and acridine orange. Only neuroblastoma cells gave a significant positive histochemical reaction for alkaline phosphatase. Glioma and neuroblastoma cell homogenates exhibited maximal phosphatase activity at pH 5 as measured by spectrophotometer. The specific activity; μmoles phosphate released per hour/mg protein was 1.1 in glioma and 0.9 in neuroblastoma. At pH 8, glioma cells lacked activity whereas neuroblastoma cells showed another maximum. The acid phosphatase activity of both cell types was strongly inhibited by CuCl₂ (0.3 mM) and NaF (10 mM) and moderately by -tartaric acid (10 mM). cGMP (1 mM) stimulated the phosphatase activity of both cell lines. db-cAMP, in serum-free medium, induced characteristic morphological changes of the cells studied. This process was unaffected by CuCl₂, c-GMP and -tartaric acid. db-cAMP (1 mM) inhibited proliferation in both glioma and neuroblastoma cells during a 48 h incubation in serum-containing medium. This growth inhibition was associated with an increase in acid phosphatase activity of the glioma but not of the neuroblastoma cells.     

Biogenic amines in cultured neuroblastoma and astrocytoma cells

The presence of biogenic amines in cultured cells of mouse neuroblastoma C-1300 (clone NB-2a) was suggested by fluorescence-microscope histochemistry. Incubation in media containing L-[¹⁴C]tyrosine and L-[¹⁴C]tryptophan for 24 h, followed by high-voltage electrophoresis, radiochromatogram scanning, and scintillation counting, confirmed the presence of [¹⁴C]dopamine, [¹⁴C]norepinephrine, [¹⁴C]epinephrine, [¹⁴C]serotonin, [¹⁴C]tyramine, and [¹⁴C]octopamine. Dopamine, norepinephrine, epinephrine, and serotonin were demonstrated spectrophotofluorometrically in concentrations, expressed as micrograms amine per milligram protein, of 1.19, 0.027, 0.038, and 0.148, respectively, for cells in a stationary growth phase. Fluorescence-microscope histochemistry also suggested the presence of biogenic amines in cultured astrocytoma cells (cell line C6). Spectrophotofluorometric assay of cells in a stationary growth phase demonstrated intracellular dopamine, norepinephrine, epinephrine, and serotonin in concentrations significantly lower than those of neuroblastoma cells.     

Introduction of phospholipids to cultured cells with cyclodextrin

Previous studies indicate that methyl-β-cyclodextrin (meβ-CD) can greatly enhance translocation of long-chain phospholipids from vesicles to cells in culture, which is very useful when studying, e.g., phospholipid metabolism and trafficking. However, the parameters affecting the transfer have not been systematically studied. Therefore, we studied the relevant parameters including meβ-CD and vesicle concentration, incubation time, phospholipid structure, and cell type. Because meβ-CD can extract cholesterol and other lipids from cells, thereby potentially altering cell growth or viability, these issues were studied as well. The results show that efficient incorporation of phospholipid species with hydrophobicity similar to that of natural species can be obtained without significantly compromising cell growth or viability. Cellular content of phosphatidyl-serine, -ethanolamine, and -choline could be increased dramatically, i.e., 400, 125, and 25%, respectively. Depletion of cellular cholesterol could be prevented or alleviated by inclusion of the proper amount of cholesterol in the donor vesicles. In summary, meβ-CD mediates efficient transfer of long-chain (phospho) lipids from vesicles to cells without significantly compromising their growth or viability. This lays a basis for detailed studies of phospholipid metabolism and trafficking as well as enables extensive manipulation of cellular phospholipid composition, which is particularly useful when investigating mechanisms underlying phospholipid homeostasis.     

Degradation of membrane phospholipids in plant cells cultured in sucrose-free medium

It has been generally accepted that autophagy contributes to the degradation of cellular components under nutrient starvation conditions. In a previous study, however, we showed that the degradation of membrane phospholipids occurs mainly by mechanisms distinct from autophagy in suspension-cultured tobacco (Nicotiana tabacum) BY-2 cells. In response to deprivation of sucrose, the amounts of total phospholipids and a major phospholipid, phosphatidylcholine (PC), decreased. 3-Methyladenine, which inhibits autophagy, did not affect the degradation of total phospholipids or PC. On the other hand, glycerol inhibited PC degradation although it did not block autophagy. In the present study, we labeled intracellular phospholipids by loading cells with a fluorochrome-labeled fatty acid and observed cellular morphology by fluorescence microscopy. Most cellular membrane structures were stained at the start of starvation; but 12 h after starvation treatment, concomitant with PC degradation, fluorescence on membranes disappeared and instead the central vacuole became fluorescent. 3-Methyladenine did not inhibit this process, whereas glycerol did. These results suggest that the degradation of membrane phospholipids can be traced by light microscopy and support the notion that autophagy is not a main contributor to the degradation of membrane phospholipids in tobacco cells cultured in sucrose-free medium.     

Polar secretion of endothelin-1 by cultured endothelial cells.

The aim of this study was to determine the permeability of endothelial monolayers for endothelin-1 and a possible directionality of the endothelin-1 secretion process. Human umbilical vein endothelial cells were cultured on acellular amniotic membranes, dividing the tissue culture wells into an apical (luminal) and a basolateral (abluminal) compartment. Whereas in the absence of endothelial monolayers 44.9 +/- 2.3 and 43.5 +/- 2.0% of the unilaterally added endothelin-1 permeated from the apical to the basolateral side and from the basolateral to the apical side, respectively, only 6.5 +/- 0.6 and 6.6 +/- 0.4% diffused in the presence of endothelial cells. Analyzing endothelin-1 secretion, approximately 80% of the total amount of synthesized endothelin-1 was found in the basolateral compartment; thrombin (10 units/ml) stimulated the production of endothelin-1 approximately 2-fold, but did not change the relative distribution of endothelin-1 between the apical and basolateral compartments. In the presence of dexamethasone (10(-7) M), a decrease in the level of endothelin-1 was found in the apical compartment, whereas the total amount of endothelin-1 produced was not affected. Dexamethasone did not influence the permeability of human umbilical vein endothelial cell monolayers for endothelin-1. These results strongly support the hypothesis that endothelin-1 is a local paracrine regulator of vasotone.     

Cytopathogenicity of Naegleria fowleri for cultured rat neuroblastoma cells

The cytopathogenicity of Naegleria fowleri strain LEE (ATCC-30894) for cultured rat neuroblastoma cells (B-103) has been investigated. Both live N. fowleri amoebae and Naegleria lysates added to 51Cr-labeled B-103 cells caused release of radiolabel, which was dependent upon the ratio of amoebae to target cells or to the lysate concentration. Lysates of N. fowleri strains LEE, NF-66, NF-69, and HB-4 were equally injurious to B-103 target cells whereas lysates of strains 6088 and KUL were less cytotoxic. Highly pathogenic mouse-passaged strain LEE were less cytotoxic than axenically grown amoebae. Maximum cytotoxicity was observed in lysates from amoebae in late exponential or early stationary phase of growth. Cytopathogenicity of lysates was reduced after heating at 44 degrees C for 60 min or at 60 degrees C for 30 min. Cytotoxicity was stable during storage at 4 degrees C or at -20 degrees C for 26 h. Neither live amoebae nor lysates injured B-103 target cells at 4 degrees C. Live amoebae and lysates injured B-103 by a time, temperature, and concentration dependent process.     

The manipulation of polar head group composition of phospholipids in the wheat Miranovskaja 808 affects frost tolerance

Caryopses of the frost-resistant cultivar of the wheat Triticum aestivum L., Miranovskaja 808, were germinated and grown in the presence of various concentrations of choline chloride. Changes in the composition of leaf total phospholipids and leaf total fatty acids at two extreme temperatures (25°C and 2°C) as well as changes in frost resistance were followed. A choline chloride concentration-dependent accumulation of phosphatidyl choline was observed in the leaves. Seedlings grown at 2°C accumulated more phosphatidyl choline at each choline chloride concentration than those grown at 25°C. There was an inverse relationship between the contents of phosphatidyl choline and phosphatidic acid in the leaves. Neither the temperature nor choline chloride seemed to affect fatty-acid composition. Modification of polar-head group composition of phospholipids affected frost tolerance: Seedlings grown in the presence of 15 mM choline chloride at 25°C exhibited a freezing resistance equal to that of hardened controls. The data indicate that the polar-head group composition of membrane phospholipids in plants can be easily manipulated and point to the importance of phosphatidyl choline in cold adaptation processes.     

Structural changes induced by diethyl ether in cultured neuroblastoma cells

It has been found that the more differentiated growing cells of a neuroblastoma culture retract their neurites under the action of diethyl ether (for anesthesia) in a dose of 1.0 ml per 15 ml of culture medium for 2 h; as a result, their cytoplasm is gradually drawn into the cell bodies. Under these conditions the coefficient K, which reflects the ratio of the number of neuroblastoma cell bodies to the number of their processes and is 9.94±2.12 in the initial state, gradually increases to 19.66±1.93 (P<0.002). There is also a substantial change in the shape of the cells and a decrease in their volume. This reaction is characteristic both of relatively undifferentiated neuroblastoma cells and for more differentiated cells in various phases of individual cellular development and morphological differentiation. The data obtained create the prerequisites for a search for drugs capable of preventing the destruction of neurons under anesthesia.A. A. Bogomolets Institute of Physiology, Ukrainian Academy of Sciences, Kiev. Kiev Institute for the Advanced Training of Physicians, Ukrainian Ministry of Health. Translated from Neirofiziologiya, Vol. 24, No. 3, pp. 279–285, May–June, 1992.     

Separation and analysis of P-labeled phospholipids by a simple and rapid thin-layer chromatographic procedure and its application to cultured neuroblastoma cells

A two-directional thin-layer chromatographic method for the rapid analysis of phospholipids from cultured cells is described. The procedure permits reliable separation of the common and minor phospholipid species using regular silica gel G chromatoplates. It is based primarily on the shortening of the running distances of the developing solvents and the use of suitable solvent systems. The method has been used to study changes in the patterns of ³²P-labeled phospholipids in cultured cells under a variety of growth conditions. It is shown that, in the presence of -propranolol, incorporation of ³²P into choline and ethanolamine phospholipids is markedly reduced, whereas an increase of label in phosphatidylglycerol is observed. The latter may serve as a precursor for lysobisphosphatidic acid formation. Following treatment of cells with dimethylaminoethanol, a high proportion of label is incorporated into dimethylethanolamine phosphoglycerides.     

Compartmentation of phosphorylated precursors of phospholipid biosynthesis in cultured neuroblastoma cells

The continuous turnover of membrane phospholipids requires a steady supply of biosynthetic precursors. We evaluated the effects of decreasing extracellular Na+ concentration on phospholipid metabolism in cultured neuroblastoma (N1E 115) cells. Incubating cultures with 145 to 0 mM NaCl caused a concentration-dependent inhibition of [32P]phosphate uptake into the water-soluble intracellular pool and incorporation into phospholipid. Phospholipid classes were differentially affected; [32P]phosphate incorporated into phosphati-dylethanolamine (PE) and phosphatidylcholine (PC) was consistently less than into phosphatidylinositol (PI) and phosphatidylserine (PS). This could not be attributed to decreased phospholipid synthesis since under identical conditions, there was no effect on arachidonic acid or ethanolamine incorporation, and choline utilization for PC synthesis was increased. The effect of Na+ was highly specific since reducing phosphate uptake to a similar extent by incubating cultures in a phosphate-deficient medium containing Na+ did not alter the relative distribution of [32P]phosphate in phospholipid. Of several cations tested only Li+ could partially (50%) replace Na+. Incubation in the presence of ouabain or amiloride had no effect on [32P]phosphate incorporation into phospholipid. The differential effects of low Na+ on [32P]phosphate incorporation into PI relative to PC and PE suggests preferential compartmentation of [32P]phosphate into ATP in pools used for phosphatidic acid synthesis and relatively less in ATP pools used for synthesis of phosphocholine and phosphoethanolamine, precursors of PC and PE, respectively. This suggestion of heterogeneous and distinct pools of ATP for phospholipid biosynthesis, and of potential modulation by Na+ ion, has important implications for understanding intracellular regulation of metabolism.     

Effect of riboflavin on monoamine oxidase activity in cultured neuroblastoma cells

Monoamine oxidase (MAO) depends on a covalently attached FAD cofactor for activity. Activity is depressed in mouse neuroblastoma cells (NIE-115) grown in synthetic N2 medium lacking riboflavin. MAO activity in depleted cells is stimulated by added riboflavin, and this recovery is blocked by inhibitors of RNA and protein synthesis, and not by an inhibitor of protein glycosylation. Recovery from riboflavin depletion appears to depend upon new RNA and protein synthesis, and not on the addition of FAD cofactor to an inactive MAO precursor.     

Turnover of histone acetyl groups in cultured mammalian cells

The relative metobolic turnover rates of labeled histone fractions and of their labeled acetyl groups were determined in random cultures of exponentially-growing mammalian cells. Both the parent histones and their acetyl groups were conserved, neither exhibiting detectable metabolic turnover. The significance of these observations is discussed.     

Levels and distributions of phospholipids and cholesterol in the plasma membrane of neuroblastoma cells.

Murine neuroblastoma cells (clone N-2A) grown in suspension (spinner cells) or attached on a plastic surface (monolayer cells) were used in studies of the phospholipid and cholesterol composition of whole cells, primary plasma membranes, plasma membranes internalized during phagocytosis of polystyrene latex beads, mitochondria and microsomes. Monolayer cells contained higher concentrations of total phospholipid, phosphatidylserine and phosphatidylcholine, and lower concentration of phosphatidylethanolamine than spinner cells. The cholesterol levels and the relative proportions of the various phospholipids were similar in both cell types except phosphatidylethanolamine and sphingomyelin whose proportions were lower in monolayer cells. The primary plasma membranes of the two cell types differed significantly in the relative proportions of all phospholipids, except sphingomyelin, and the phospholipid to protein and the cholesterol to protein ratios were all higher in the membranes of spinner cells. In contrast to these results, all the phospholipid to protein and the cholesterol to protein ratios of the internalized plasma membranes were higher in monolayer than in spinner cells, and the proportions of all phospholipids, except phosphatidylethanolamine, were similar in both cell types. The membrane distributions of individual phospholipids and cholesterol were inferred from comparison of the phospholipid and cholesterol compositions of primary plasma membranes and plasma membranes internalized during phagocytosis of polystyrene beads. The results are consistent with a non-random distribution of most phospholipids in both spinner and monolayer cells, but the patterns of these distributions were different in the two cell types. With regard to cholesterol the results are compatible with a random or a heterogeneous distribution. All the phospholipid to protein ratios of the mitochondrial fraction of both cell types were lower than those of the plasma membranes. However, these ratios of the microsomal fraction were higher than those of the plasma membranes of monolayer cells, whereas they were comparable, with a few exceptions, to those of spinner cell membranes. The cholesterol to phospholipid molar ratios of plasma membranes were 6.4 and 4.3 fold greater than those of the mitochondrial and microsomal fractions, respectively.     

Sodium channels in cultured neuroblastoma cells grown in high glucose or l-Fucose

Patch clamp techniques were used to record whole cell and single channel Na⁺ currents from NB41A3 neuroblastoma cells grown in culture. Cells were grown for two weeks in control medium or medium supplemented with 30 mm d-glucose of 30 mm l-fucose.Cells exposed to glucose or l-fucose had smaller whole cell Na⁺ currents than cells grown in unsupplemented medium, consistent with earlier studies (Yorek, Stefani & Wachtel, 1994). Whole cell macroscopic currents showed no change in activation or inactivation kinetics. Single channel current properties and opening probability were also unchanged.The number of [³H]saxitoxin binding sites, and therefore the total number of Na⁺ channels, was not reduced in cells grown in glucose or l-fucose (Yorek et al., 1994). Therefore, we conclude that some of the channels must have been rendered nonfunctional by the conditioning media. The finding that single channel properties are not altered suggests that channels become nonfunctional in an all-or-none manner.This work was supported by Merit Review Awards to M.A. Yorek and R.E. Wachtel from the Department of Veterans Affairs and by National Institutes of Health grant DK45453 to M.A. Yorek.