ON THE MECHANISM OF ACTION OF THE NON-OPIOID ANALGESIC FLUPIRTINE Doctoral Thesis at the Medical University of Vienna for obtaining the academic degree Doctor of Philosophy (PhD) Submitted by Felicia Klinger, MD Supervised by Prof. Dr. Stefan Böhm Center for Physiology and Pharmacology Department of Neurophysiology and Pharmacology Waehringerstr. 13a, 1090 Vienna Vienna, 12/2011 1. LIST OF ABBREVIATIONS...........................................................................................3 2. ABSTRACT .......................................................................................................................5 3. ZUSAMMENFASSUNG...................................................................................................6 4. INTRODUCTION .............................................................................................................8 4.1 Pain and nociception ..................................................................................................8 4.2 Nociceptive system ....................................................................................................8 4.3 Pain perception.........................................................................................................10 4.4 Pain sensitisation......................................................................................................12 4.5 Pain transmission .....................................................................................................15 4.6 Pain modulation .......................................................................................................18 4.7 Analgesics................................................................................................................20 4.8 Flupirtine..................................................................................................................24 4.9 Aims of the study .....................................................................................................27 5. MATERIALS AND METHODS....................................................................................28 5.1 Tissue preparation and cell culture ..........................................................................28 5.1.1 Hippocampal and dorsal horn cell cultures.............................................28 5.1.2 Superior cervical ganglia and dorsal root ganglia cell cultures.............29 5.1.3 Cell line and transfection...........................................................................30 5.2 Electrophysiological recordings...............................................................................30 5.2.1 General considerations ..............................................................................30 5.2.2 Patch clamp set up .....................................................................................31 5.2.3 Recording and analysis of autaptic currents ...........................................33 5.2.4 Recording currents through ligand-gated ion channels .........................33 5.2.5 Recording of miniature inhibitory postsynaptic currents (mIPSCs) ....34 5.2.6 Recording currents through KCNQ/Kv7 channels (IM).........................34 5.2.7 Calculations and statistics .........................................................................35 5.3 Western blot analysis ...............................................................................................35 5.3.1 Principle of Western blot analysis ............................................................35 5.3.2 Membrane protein preparation................................................................36 5.3.3 Preparation of the polyacrylamide gels and electrophoresis .................36 5.3.4 Blotting and immunostaining....................................................................37 5.4 Materials...................................................................................................................38 6. RESULTS.........................................................................................................................40 6.1 Flupirtine enhances currents through Kv7 channels................................................40 1 6.2 Flupirtine enhances inhibitory, but not excitatory, autaptic currents in hippocampal microculture........................................................................................41 6.3 GABAA receptors, but no other ligand-gated ion channels are modulated by low micromolar flupirtine concentrations.......................................................................42 6.4 Flupirtine acts as an allosteric modulator on GABAA receptors in hippocampal neurons.....................................................................................................................44 6.5 Flupirtine enhances currents through tonically-active GABAA receptors but does not alter synaptic transmission.................................................................................46 6.6 When synaptic GABAA receptors are blocked by Penicillin G 5 mM, flupirtine acts as a pure potentiator on GABAA receptors in hippocampal neurons................50 6.7 Effects of flupirtine on GABAA receptors in pain-related and non-related neurons.....................................................................................................................51 6.8 Differences in GABAA receptor subunits in pain-related and non-related neurons 53 6.9 Effects of flupirtine on Kv7 channels in pain-related and non-related neurons ......55 6.10 Concentration dependence of the effects of flupirtine on GABAA receptors in pain-related and non-related neurons; comparison with the effects on Kv7............56 7. DISCUSSION...................................................................................................................58 8. REFERENCES ................................................................................................................63 9. ACKNOWLEDGMENTS...............................................................................................81 10. CURRICULUM VITAE .................................................................................................82 2 1. LIST OF ABBREVIATIONS 5-HT ………………………………………………………..5-hydroxtryptamine (serotonin) aEPSC ………………………………………….…autaptic excitatory postsynaptic currents aIPSC ……………………………………………..autaptic inhibitory postsynaptic currents AIS ………………………………………………….………………..axonal initial segment AMPA …………………………...α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid APS ………………………………………….………………………..ammonium persulfate ATP …………………………………………….…………………....adenosine triphosphate BCA …………………………………………...…………………………bicinchoninic acid Bis-tris ……………………...……..Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methan BMI ………………………………………………………………….bicuculline methionide cAMP ……………………………………………………..cyclic adenosine monophosphate 2+ CaV channels .…………………………………………..………voltage-gated Ca channels CNQX ……………………………………...……..cyano-2,3-dihydroxy-7-nitroquinoxaline CNS ………………………………………………………………….central nervous system COX ...………………………………………………………………………cyclooxygenase DAD ……………………………………………...………………………….diacyl glycerol DH …………………………………………………...…………………………..dorsal horn DMEM ………………………………………………Dulbecco’s modified Eagle’s medium DRG ……………………………………………………………………...dorsal root ganglia EDTA ………………………………………………………ethylenediaminetetraacetic acid EPSC ……………………………………………………......excitatory postsynaptic current FCS ……………………………………………………………...…………..fetal calf serum GABA ……………………………………………………………gamma amino butiric acid GIRK channels ………………………...G-protein coupled inwardly rectifying K+ channels GPCR ……………………………………………………………G protein coupled receptor HB ……………………………………………………………………..homogenizing buffer HEK cells ………………………………………………...…human embryonic kidney cells HEPES ………………………………..4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid hiFCS ………………………………………………………heat inactivated fetal calf serum IPSP …………………………………………………...…..inhibitory postsynaptic potential IM …………………………………………………. M-current (current through M-channel) 3 IP3 ………………………………………………………...……..inositol 1,4,5-triphosphate mIPSC …………………………………………..miniature inhibitory postsynaptic currents mEPSC ……………………………………….....miniature excitatory postsynaptic currents MOPS ………………………………………………3-(N-morpholino)propanesulfonic acid mRNA ………………………………………………...………...messenger ribonucleic acid + NaV channels …………………………………………………...voltage-gated Na channels NBQX ..................2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide NGF ……………………………………………………………...….neuronal growth factor NKA …………………………………………………………………...………neurokinin A NMDA ..................................................................................................N-methyl-D-aspartate NO …………………………………………………………………………...…..nitric oxide NSAIDs …..................................................................non-steroidal anti-inflammatory drugs PBS ……………………………………………………………….phosphate buffered saline PCR …………………………………………………………...…polymerase chain reaction PLC ……………………………………………………………………...…phospholipase C PGE ……………………………………………………………………...….prostaglandin E PIP2 ……………………………………………….....phosphatidylinositol 4,5-bisphosphate PKC ………………………………………………………………...………protein kinase C PNS ………………………………………………………………peripheral nervous system PVDF .................................................................................................polyvinylidene fluoride rpm ……………………………………………………………………..rotations per minute SCG ………………………………………………………………..superior cervical ganglia SDS ..................................................................................................sodium