dioleoylphosphatidic-acid and lysophosphatidic-acid

Previously, we suggested that dioleoyl phosphatidic acid (PA) and lysophosphatidic acid (LPA) increased [Ca(2+)](i) through endogenous LPA receptors coupled to pertussis toxin-sensitive G proteins in rat C6 glioma cells. In the present report, we investigated morphological changes and cytotoxicity induced by PA and LPA in C6 glioma cells. Isoproterenol treatment led to changes in the cell morphology of rat C6 glioma cells, which were reverted by the addition of PA and LPA. PA-and LPA-induced morphological reversions were inhibited by treatment with Ki16425, an LPA(1)/LPA(3) receptor antagonist. VPC32183, another LPA(1)/LPA(3) receptor antagonist with a different structure, only inhibited PA-induced morphological reversion but not LPA-induced reversion. However, the reversions were not inhibited by treatment with pertussis toxin, a specific inhibitor of G(i/o) proteins. In addition, cytotoxicity was only induced by LPA but not by PA in C6 glioma cells. Our results suggest that PA may act as a partial agonist at endogenous LPA receptors, which are sensitive to Ki16425 and coupled to PTX-insensitive G proteins, to evoke morphological changes in C6 glioma cells.

Topics: Animals; Cell Line, Tumor; Cell Survival; Cytotoxins; Drug Partial Agonism; Glioma; Isoproterenol; Isoxazoles; Lysophospholipids; Organophosphates; Pertussis Toxin; Phosphatidic Acids; Propionates; Pyridines; Rats; Receptors, Lysophosphatidic Acid

The formation of phosphatidic acid (PA) from lysophosphatidic acid (LPA), diacylglycerol, or phosphatidylcholine plays a key role in the regulation of intracellular membrane fission events, but the underlying molecular mechanism has not been resolved. A likely possibility is that PA affects local membrane curvature facilitating membrane bending and fission. To examine this possibility, we determined the spontaneous radius of curvature (R(0p)) of PA and LPA, carrying oleoyl fatty acids, using well-established X-ray diffraction methods. We found that, under physiological conditions of pH and salt concentration (pH 7.0, 150 mM NaCl), the R(0p) values of PA and LPA were -46 A and +20 A, respectively. Thus PA has considerable negative spontaneous curvature while LPA has the most positive spontaneous curvature of any membrane lipid measured to date. The further addition of Ca(2+) did not significantly affect lipid spontaneous curvature; however, omitting NaCl from the hydration buffer greatly reduced the spontaneous curvature of PA, turning it into a cylindrically shaped lipid molecule (R(0p) of -1.3 x 10(2) A). Our quantitative data on the spontaneous radius of curvature of PA and LPA at a physiological pH and salt concentration will be instrumental in developing future models of biomembrane fission.

Topics: Buffers; Calcium Chloride; Hydrogen-Ion Concentration; Intracellular Membranes; Linear Models; Lysophospholipids; Membrane Fusion; Membrane Lipids; Models, Chemical; Phosphatidic Acids; Salts; Sodium Chloride; X-Ray Diffraction

Phosphatidic acid and lysophosphatidic acid are minor but important anionic bioactive lipids involved in a number of key cellular processes, yet these molecules have a simple phosphate headgroup. To find out what is so special about these lipids, we determined the ionization behavior of phosphatidic acid (PA) and lysophosphatidic acid (LPA) in extended (flat) mixed lipid bilayers using magic angle spinning 31P NMR. Our data show two surprising results. First, despite identical phosphomonoester headgroups, LPA carries more negative charge than PA when present in a phosphatidylcholine bilayer. Dehydroxy-LPA [1-oleoyl-3-(phosphoryl)propanediol] behaves in a manner identical to that of PA, indicating that the difference in negative charge between LPA and PA is caused by the hydroxyl on the glycerol backbone of LPA and its interaction with the phosphomonoester headgroup. Second, deprotonation of phosphatidic acid and lysophosphatidic acid was found to be strongly stimulated by the inclusion of phosphatidylethanolamine in the bilayer, indicating that lipid headgroup charge depends on local lipid composition and will vary between the different subcellular locations of (L)PA. Our findings can be understood in terms of a hydrogen bond formed within the phosphomonoester headgroup of (L)PA and its destabilization by competing intra- or intermolecular hydrogen bonds. We propose that this hydrogen bonding property of (L)PA is involved in the various cellular functions of these lipids.

Topics: Cell Membrane; Endoplasmic Reticulum; Hydrogen Bonding; Hydrogen-Ion Concentration; Intracellular Membranes; Ions; Least-Squares Analysis; Lipid Bilayers; Lysophospholipids; Magnetic Resonance Spectroscopy; Membranes, Artificial; Models, Molecular; Molecular Structure; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylethanolamines; Protons; Titrimetry

External application of dioleoyl-phosphatidic acid and oleoyl-lysophosphatidic acid stimulated Ca(2+)-dependent chloride currents in voltage-clamped Xenopus laevis oocytes. The responses were observed in oocytes from which follicular cells had been removed, indicating they were intrinsic to the oocyte itself. The lipid-induced Ca(2+)-dependent chloride currents were observed in the absence of extracellular calcium, were blocked by intracellular injection of the calcium chelator, bis(O-aminophenoxy)-ethane N,N,N'N'-tetraacetic acid, and could not be elicited by direct intracellular injection of the active lipids. The thresholds for dose-dependent current responses to dioleoyl-phosphatidic acid (100 nM) and for oleoyl-lysophosphatidic acid (10 nM) indicated that the lipid activities on oocytes were potent. With repeated or prolonged administration of either active lipid, responses exhibited desensitization. These results demonstrate that the Xenopus oocyte expresses endogenous functional responses for the mitogenic lipids phosphatidic acid and lysophosphatidic acid and thus provides a powerful model for characterization of the pharmacology and transduction pathways of these responses.

Topics: Animals; Chloride Channels; Egtazic Acid; Female; In Vitro Techniques; Ion Channels; Kinetics; Lysophospholipids; Membrane Potentials; Membrane Proteins; Oocytes; Phosphatidic Acids; Receptors, Cell Surface; Signal Transduction; Tetradecanoylphorbol Acetate; Xenopus laevis