sq-23377 and palmitoyl-trifluoromethyl-ketone

Activation of phospholipase A2 (PLA2) and the subsequent metabolism of arachidonic acid (AA) to prostaglandins have been shown to play an important role in neuronal death in neurodegenerative disease. Here we report the effects of the prion peptide fragment HuPrP106-126 on the PLA2 cascade in primary cortical neurons and translocation of cPLA2 to neurites.. Exposure of primary cortical neurons to HuPrP106-126 increased the levels of phosphorylated cPLA2 and caused phosphorylated cPLA2 to relocate from the cell body to the cellular neurite in a PrP-dependent manner, a previously unreported observation. HuPrP106-126 also induced significant AA release, an indicator of cPLA2 activation; this preceded synapse damage and subsequent cellular death. The novel translocation of p-cPLA2 postulated the potential for exposure to HuPrP106-126 to result in a re-arrangement of the cellular cytoskeleton. However p-cPLA2 did not colocalise significantly with F-actin, intermediate filaments, or microtubule-associated proteins. Conversely, p-cPLA2 did significantly colocalise with the cytoskeletal protein beta III tubulin. Pre-treatment with the PLA2 inhibitor, palmitoyl trifluoromethyl ketone (PACOCF3) reduced cPLA2 activation, AA release and damage to the neuronal synapse. Furthermore, PACOCF3 reduced expression of p-cPLA2 in neurites and inhibited colocalisation with beta III tubulin, resulting in protection against PrP-induced cell death.. Collectively, these findings suggest that cPLA2 plays a vital role in the action of HuPrP106-126 and that the colocalisation of p-cPLA2 with beta III tubulin could be central to the progress of neurodegeneration caused by prion peptides. Further work is needed to define exactly how PLA2 inhibitors protect neurons from peptide-induced toxicity and how this relates to intracellular structural changes occurring in neurodegeneration.

Topics: Analysis of Variance; Animals; Arachidonic Acid; Cell Death; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation; Humans; Ionomycin; Ketones; Mice; Neurons; Peptide Fragments; Phospholipases A2, Cytosolic; Prions; Protein Transport; Synaptophysin; Tetradecanoylphorbol Acetate; Time Factors; Tritium; Tubulin

We have shown previously that ATP inhibits transmitter release at the neuromuscular junction through the action on metabotropic P2Y receptors coupled to specific second messenger cascades. In the present study we recorded K(+) or Ca(2+) currents in motor nerve endings or blocked K(+) or Ca(2+) channels in order to explore the nature of downstream presynaptic effectors. Endplate currents were presynaptically depressed by ATP. Blockers of Ca(2+)-activated K(+)-channels, such as iberiotoxin, apamin or tetraethylammonium, did not change the depressant action of ATP. By contrast, K(+) channel blocker 4-aminopyridine (4-AP) and raised extracellular Ca(2+) attenuated the effect of ATP. However, these effects of 4-AP and high Ca(2+) were reversed by Mg(2+), suggesting Ca(2+)-dependence of the ATP action. Ba(2+) promoted the depressant action of ATP as did glibenclamide, a blocker of ATP-sensitive K(+) channels, or mild depolarization produced by 7.5 mm K(+). None of the K(+) channel blockers affected the depressant action of adenosine. Focal recording revealed that neither ATP nor adenosine affected the fast K(+) currents of the motor nerve endings. However, unlike adenosine, ATP or UTP, an agonist of P2Y receptors, reversibly reduced the presynaptic Ca(2+)-current. This effect was abolished by suramin, an antagonist of P2 receptors. Depressant effect of ATP on the endplate and Ca(2+)-currents was mimicked by arachidonate, which precluded the action of ATP. ATP reduced acetylcholine release triggered by ionomycin or sucrose, suggesting inhibition of release machinery. Thus, the presynaptic depressant action of ATP is mediated by inhibition of Ca(2+) channels and by mechanism acting downstream of Ca(2+) entry.

Topics: Adenosine; Adenosine Triphosphate; Animals; Anura; Arachidonic Acid; Barium; Calcium; Calcium Channel Blockers; Drug Interactions; Electrophysiology; Enzyme Inhibitors; In Vitro Techniques; Ionomycin; Ionophores; Ketones; Membrane Potentials; Motor Neurons; Neural Inhibition; Neuromuscular Junction; Potassium; Potassium Channel Blockers; Sucrose; Suramin; Uridine Triphosphate