163ZHENG Zhao-Qing et al: Dual Action of LPA in Cultured Cortical Neurons: Survival and ApoptogenicReceived 2003-06-06 Accepted 2003-08-18*Corresponding author. Tel: +86-351-4135052; Fax: +86-351-4083014; E-mail: qiaojtpublic.ty.sx.cnResearch PaperActa Physiologica Sinica, April 25, 2004, 56(2): 163-171http:/www.actaps.com.cnDual action of lysophosphatidic acid in cultured cortical neurons: survival and apoptogenicZHENG Zhao-Qing1, FANG Xian-Jun2, QIAO Jian-Tian1*1Department of Neurobiology, Shanxi Medical University, Taiyuan, Shanxi 030001, China2M. D. Anderson Cancer Center, Department of Molecular Therapeutic, 1515 Holcombe Blvd., Houston, TX 77030, USAAbstract: The effect of lysophosphatidic acid (LPA), with a wide range of its different concentrations, upon cultured mouse cortical neurons was assessed by electrophoresis of DNA fragments, HO33342 and TUNEL stainings, and also by ultrastructural examination attimes. The results showed that administration of LPA at lower concentrations (0.130 mol/L) dose-dependently protected cortical neurons from apoptosis that was induced by deprivation of serum from the cultural medium, while 50 mol/L or higher concentrations ofLPA failed to show this effect; and moreover, the concentrations higher than 50 mol/L induced apoptosis in neurons cultured in serum- containing complete medium. These results suggest that a moderate concentration of LPA may play as a survival factor in apoptoticcortical neurons, while an excessive level of LPA induces apoptosis in neurons cultured in complete medium.Key words: lysophosphatidic acid; apoptosis; survival factor; serum-free medium; complete medium; cortical neurons; mice121,*1, 030001; 2, 77030, : DNAHO33342TUNEL, (lysophosphatidic acid, LPA), LPA (0.130 mol/L), LPA ( 50 mol/L), , LPA, LPA: ; ; ; ; ; ; : Q421Apoptosis, or programmed cell death, is an active cell response to physiological or damaging signals1. Morpho- logically , apoptosis is characterized by condensation of nuclear chromatin, DNA fragmentation, compaction of cy- toplasmic organelles, cell shrinkage, and ruffling and blebbing of the plasma membrane1,2. Cell death via apoptosis is a component of normal neuronal develop- ment3, 4 and is also observed in response to acute and chronic neuronal insults2 5. Recently, apoptosis is found to play a key role in neurodegenerative disorders4,5.Lysophosphatidic acid (LPA) is the smallest and struc- turally simplest of all glycerophospholipids presenting in serum6, 7, and is normally released by activated plate- lets during blood clotting7. LPA binds with high affinity to serum albumin while retaining its biological activity8. LPA can mediate cell proliferation9 and promote plate- let aggregation10. LPA and LPA receptors are highly ex- pressed in the brain and so LPA might participate in both normal and pathological processes in the central ner- vous system11,12.Acta Physiologica Sinica, February 25, 2004, 56(2): 163-171164Recently, many investigators have observed that LPA acts as a survival factor blocking the serum deprivation- induced apoptosis in cultured Schwann cells13, macroph- ages14, intestinal epithelial cells15, and renal proximal tu- bular cells16. In contrast, Holtsberg et al.17 reported that LPA is cytotoxic and induces apoptosis and necrosis in rat hippocampal neurons, and Steiner et al.18 reported that PLA induces apoptosis in PC12 cells. These controversial results concerning roles of LPA at the cellular level pro- moted us to explore if the different functional effects of LPA in vitro were dependent upon the different concen- trations of LPA being used other than the different cell types being used in the experiments. Thus, we tested the effects of a wide range of concentrations of LPA on mu- rine cortical neurons cultured in serum-deprived media or in complete media containing serum.1 MATERIALS AND METHODS1.1 Materials. LPA (1-acyl-2-hydroxy-sn-glycero-3- phosphate, oleoyl, 18:1) was purchased from Avanti Po- lar-Lipids (Alabaster, AL). Fatty acid-free bovine serum albumin (BSA), HO33342, HEPES, RNase A, agarose, and proteinase K were purchased from Sigma; Dubeccos modified Eagle medium (DMEM) and fetal bovine serum were obtained from GIBCO; Apop Tag peroxidase in situ apoptosis detection kit was purchased from Intergen Com- pany (Oncor, Inc.).1.2 Primary cell culture. Primary cortical neurons were cultured as described previously19. In brief, cortical neu- rons derived from newborn mice were dissociated in DMEM supplemented with 10% heat-inactivated fetal bo- vine serum (v/v), 26 mmol/L bicarbonate, 25 mmol/L D- glucose, 25 mmol/L HEPES, 10 U/ml penicillin, and 10 g/ ml streptomycin, and then plated on 35 mm dishes coated with general type I collagen from rat tail and poly-L-lysine (2105 cells/cm2). Cultures were kept in a humidified CO2 incubator at 37C. The non-neuronal cell division was halted by exposure to 10 mol/L cytosine arabinoside