ATP Mediated Neurotoxicity And Regulated The Expression Of GAP-43 To The Cultured Rat Hippocampal Neurons

Objective: Adenosine-5'-triphosphate (ATP) acting as neurotransmitters plays an important role during neurotransmission and hormone secretion following the activation of P2 receptors. Dramatic release of ATP occurs after cellular death, brain ischemia, trauma, necrosis, or reduced perfusion of malignant brain tumors, activating mechanisms involved in response to injury (astrogliosis, microglial activation, regeneration of damaged neuronal axons).The ATP receptor subtypes P2X₇ receptor appeared to be an important element in the mechanisms of cellular damage. In many cell types, the activation of the P2X₇ receptor leads to rapid cytoskeletal rearrangements, membrane blebbing and cell lysis. Whether activation of P2 receptors by ATP mediates toxicity for hippocampal neurons, the understanding of the roles of the P2X₇ receptor subtype in the hippocampal neurons is still rather limited. GAP-43 is a presynaptic membrane phosphoprotein whose expression increases during events of axonal growth or remodeling such as development, learning and regeneration of an injured axon. The main aim of this study is to investigate the nurotoxic effect of ATP and P2X₇ agonist BzATP in the cultured rat hippocampal neurons and observe the GAP-43 expression, and the cytoplasmic Ca2+ concentration in order to explore the related mechanism.Methods: The primary hippocampal neurons from neonatal rat were treated with ATP, P2 antagonist PPADS, P2X₇ agonist BzATP and antagonist BBG at 8 day in vitro. Nissl's staining and MTT assay were used to study various concentration of ATP induced injury in neurons. Immunofluorescent staining was used to observe ATP, PPADS, BzATP and BBG induced expression and distribution of GAP-43 in cultured hippocampal neurons. Calcium indictor Fluo-4/AM and a fluorescence microscope were used to detect intensity of fluorescence-marked calcium in intracellular neurons of different groups. Results 1.Compared to the control group, lower congcentration of ATP(100μM) had no cytotoxic effects. The OD value demonstrated by MTT assay was 0.278±0.039. Higher concentration of ATP(3mM, 5mM and 10mM) could induce marked cultured hippocampal neurons damaged. OD value were 0.202±0.04,0.194±0.018,and 0.159±0.016 respectively. Nissl's staining showed that ATP induced the morphological changes, such as cellular swelling, nuclei condensation, fragmentation and losses of nissl bodies. PPADS could block partly the cytotoxic effect of ATP, but not BBG obviously.2. GAP-43 was expressed on membrane of soma and neurites of neurons after exposure to PBS or ATP(100μM) for 4 hours. The level of GAP-43 of neurons was elevated significantly after exposure to 10mM ATP and expressed mainly on soma, and the number of neuritis strongly reduced even disappeared. P2 receptor antagonist PPADS could promot the nurites extension, but BBG had no such effect obviously.3. Fluorescence intensity of calcium loaded by Fluo-4/AM and the rate of fluorescence attenuation were detected and calculated. There was statistically decelerated of the fluorescence attenuation of calcium in 5mM ATP and BzATP treated groups, but celebrated in PPADS treated group. This reslts showed that intracellular calcium singal pathway participated in the ATP-induced neuron injury.Conclusions1. Lower concentration of ATP can not injure to the cultured rat hippocampal neurons.While higher concentration of ATP induce marked neurotoxicity to the neurons.2. Higher concentration of ATP could elevated significantly the expression level of GAP-43 on cell body, not on the neuritis, indicate that there is a supplementary neurite reconditioning at such condition. P2 receptor antagonist PPADS could attenuate the neuron damage and promote the neuritis extension which might take part in nerve regeneration following injury.3. P2X₇ receptor agonist BzATP has no obvious toxic to the neuron and the antagonist BBG has no marked promotion of neuritis extension, indicate that P2X₇ receptor might be not the major receptor subtype of P2 receptor of cytotoxicity mediated by higher concentration of ATP.4. ATP-induced calcium singal pathway might participate in the regulation of GAP-43 expression in the hippocampal neurons.