PROPOFOL AND BENZODIAZEPINE MODULATION OF GABA,R FUNCTION Laura Catherine McAdam A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Physiology University of Toronto G Copyright by Laura Catherine McAdam (1997) National Library Bibliothèque nationale m*m of Canada du Canada Acquisitions and Acquisitions et Bibliographic Senrices services bibliographiques 395 Wellington Street 395, rue Wellington Ottawa ON KIA ON4 ûttawa ON K 1 A ON4 Canada Canada Your Me Votre reIsrmœ Our Ne Noire reftirencB The author has granted a non- L'auteur a accordé une Licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, han, distribute or sell reproduire, prêter, distribuer ou copies of tbis thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. A. ABSTRACT PROPOFOL AND BENZODIAZEPM MODULATION OF GABAARFUNCTION Master of Science, 1997 Laura Catherine McAdam Department of Physiology, University of Toronto The general anaesthetic, propo fol, has multiple effects on the GABA,R: it potentiates GABA-evoked responses; it activates the receptor, and it alters the kinetics of receptor desensitization. In contrast, sedative benzodiazepines only potentiate GABA-evoked responses. It is not known if the multiple effects of propofol on the GABA,R are mediated by distinct binding sites or which effect accounts for propofol's anaesthetic properties. The whole-ceIl patch clamp method was used to study the effects of benzodiazepines and propofol on GABA,R's present in cultured, murine hippocarnpal neurons. The concentration of propofol that activated the GABA,R (EC,, = 24.3 k 3.4 PM) was significantly greaeater than that which decreased receptor desensitization (EC,, = 1.15 + 0.33 PM). Furthemore. midazolam potentiated propofol-induced currents but did not alter propofol- induced changes in receptor desensitization, suggesting that receptor activation and desensitization were mediated by distinct mechmisms. Low concentrations of propofol (1 PM) and midazolam (0.5 plu) interacted in a superadditive rnanner to enhance GABA (0.3 PM)-evoked responses and isobolographic analysis suggested a synergistic interaction. Ln contrast, higher concentrations of these dmgs produced an additive interaction. As low concentrations of propofol and midazolam interact synergistically to produce hypnosis and higher concentrations do not interact synergistically to induce anaesthesia, Our results suggest that hypnosis and anaesthesia may be mediated by distinct changes in GABAARhnction. B. ACKNOWLEDGMENTS 1 wish to express my sincere gratitude to my supervisors, Dr. B.A. Oner and Dr. J.F. MacDonald for their guidance and encouragement over the past two years. 1 would also like to thank Dr. Orser for introducing me to the clinical aspects of research and for the opportunities to visit Sunnybrook Health Science Centre. I also appreciate the advice provided by my cornmittee member, Dr. Wojtowicz. 1 would like to thank my labrnates for providing a stimulating, helphl and fnendly working atmosphere. I also gratefully acknowledge Lidia Brandes and Ella Czerwinska for overseeing the ce11 cultures. Finally, and most importantly. 1 would like to thank my parents and sisters for al1 of their love and support throughout the paçt Z years. This research was supported by an International Anesthetists Research Society Frontiers in Anesthesia Research Grant Award to Dr. Orser. C. TABLE OF CONTENTS -. A. ABSTRACT II -.. B. ACLN0WLEDGiMENTS 111 C. TABLE OF CONTENTS i v ... D. LIST OF FIGURES Vlll E. LIST OF TABLES i x F. ABBREVIATION NDEX X t NTRODUCTION .LVAESTHESIA NON-SPECIFIC AND SPECEIC THEORJES OF ANAESTHETIC ACTION NIBITORY NEUROTRANSMISSION AND GABA, RECEPTORS GABA GN3A,R PROPERTIES KMETIC MODEL OF GABA,R FUNCTION DESENSITIZATION SUBWTS OF THE GABA,R GABAARAND ANAESTHESIA PROPOFOL EFFECTS ON GABAAR BENZODIAZEPNE ACTION AT THE GABA,R 1 -4.1. BENZODIAZEPINE BWWG IS NFLüENCED BY SUBUNIT COMPOSITION OF THE GABA,+R 1.5. DRUG INTERACTIONS BETWEEN PROPOFOL AND BENZODIAZEPMS 1S. 1 DETERMNTNG DRUG NTERACTIONS -.7 OBJECTIVES AND WORKING HYPOTHESIS 3.1. SECTION 1 -.-.3 9 SECTION 3 LMETHODS CELL CULTURES RECORDNG PIPETTES PIPETTE SOLUTION WHOLE-CELL VOLTAGE-CLAMP RECORDINGS DRL'G Ai'AGONIST PERFUSION SYSTEM WHOLE-CELL CURRENT AYALYSIS DRUG INTERACTION ANALYSIS DRUGS AND OTHER CHEMICALS STATISTICS 4. RESC'LTS 4.1. SECTION 1 il.1.1. MIDAZOLAM MCREASED THE AFFNTY OF THE GABA,R FOR GABA -4ND PROPOFOL 37 3.1.2. MIDAZOLALM DOES NOT INFLUENCE DESENSITIZATION OF THE GABA,R 47 3.1.3. PROPOFOL DECREASED RECEPTOR DESENSITIZATION 50 4.1 .4. PROPOFOL-NUCED .MODULATION OF GABA,R 55 4.2. SECTION 2 64 3.1. EFFECTS OF MIDAZOLAM AND PROPOFOL ON CC'RREXTS EVOKED BY SUBSATURATNG CONCENTRATIONS OF GABA 64 1.2.2. SYNERGISTIC NI'ERACTION BETWEEN PROPOFOL AiÿD MIDAZOLAM IN THE PRESENCE OF LOW CONCENTR4TIONS OF GABA 77 5. DISCUSSION 88 5.1. SECTION 1 88 5.1.1. MIDAZOLAM POTENTUTION OF GABA-EVOKED CURRENTS 88 5.1.2. ,MIDAZOLAM POTENTlATION OF PROPOFOL-EVOKED Cb'RRENTS 89 5.1.3. PROPOFOL BLOCKA.DE OF THE GABA,,R 92 5 4 MID.LVOLAM DiD NOT I'CTLLT'YCE DESENSITIZ.4TIO-Y OF THE G.ABX,R 93 5.1 3. PROPOFOL MODLZXïION OF GrZBA,R DESENSITIZATIOX 94 3- .-.9 SECTION 2 97 5.1. PH.4K'.'ACOLOGICAL SYNERGISM BETWEEX PROPOFOL .%.ND 'tlIDAZOL-X.kl 97 5.2 CLiS1C.U SYXERGISM BETWEE'V PROPOFOL .k\D .MID=VOL.I.f 98 6. CONCLCSIONS -7. REFEREXCES D. LIST OF FIGURES GN3Aergic synapses and the topology of a G.4BA,R subunit ?/lultibarrel fast-perfusion system Desensitization of GABA induced currents Isobolographic anal ysis Midazolam increased the affinity of the GABA,R for GXBA .Midazolam increased the affinity of the GABA,R for propofol Midazolam potentiated currents evoked by propofol The W relationship of propofol-induced currents recorded in the absence and presence of midazolarn Midazolam did not influence GABA,R desensitization of the GABA,R for currents evoked by GABA .Midamlm did not aiter desensitization of propo fol-evoked currents Propo fo 1 decreased GN3A,R desensitization in a concentration-dependent mann er Midazo lam did not e ffect propo fol-induced modulation of GABA,R desensitization Midazolam did not influence GABA,R desensitization at lower concentrations of D~ODO~O~ Propofol and midazolam (or flurazepam) produced an additive enhancement of GABA (3 p.M)-evoked currents From hippocampal neurons 65 Widazolam and propofol did not alter the reversa1 potential of the GABA-evoked currents 68 Propofol and midazolam produced an additive enhancernent of GABA (3 pu)- evoked currents when the concentration of propofol was reduced 7 1 Propofol and midazolam produced a superadditive enhancement of G.4E5.4 (3 uM)-evoked currents when the concentration of GABA was reduced 73 Propofol and midazolam produced an additive enhancement of G.îB.4 (3~~34)- evoked currents in spinal cord neurons 'vlidazolam potentiation of GABA ( 1 ,uM)-evoked response Propofol potentiation of GABA ( 1 FM)-evoked response Propofol enhancement of currents evoked by co-application of midazolam and G.%l3-4 Synergisric interactions between propofol and midazolarn E. LIST OF TABLES 1. Propofoi increased the rate of GAB.4,R desensitization and deactivation at 56 saturating concentrations of GABA -7 - Widazolam increased the rate of GAB.A4R deactivation at two concentrations of 63 propofol in the presence of saturating concentrations of GM.4 F. .U3BREVIATION INDEX .MPA a-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid ATP adenosine-5-triphosphate BDZ benzodiazepine BZ 1 benzodiazepine type 1 BZ2 benzodiazepine type 2 CDPX chlordiazepoxide CNS central nenious system DMSO dimethyt sulfoxide DRC dose response curve EGO concentration that produces 50 % of the observed response EGTA ehylene glycol-0,0'-bis(2-aminoethy1)-N,N,N,N',N7-te~~cetcacid FLU flurazepam FMZ flumazenil G-proteins guanine nucleotide binding protein GABA 7-aminobutync acid GABA,R a subtype of y-arninobutyric acid receptor GABA,R a subtype of y-arninobutyric acid receptor GAi3bR a subtype of 7-aminobutyric acid receptor GAD glutamic acid decarboxylase HEPES 25 N-2-hydroxy-ethylpiperazine-N'-2-ethanesulphoic acid IC,, concentration that inhibits the response by 50 % IPSC inhibitory post-synaptic current KA kainate MDZ midazo larn MEM minimum essential media nAChR nicotinic acety lcholine recep tor NMDA N-methyl-D-aspartate 'CMDAR NMDA receptor PA p icoamperes PRO propo fol P s picosiemens TE,4 tetraethylarnrnonium TM transmembrane domain TTX tetrodotoxin 1. INTRODUCTION 1.1. ANAESTHESIA haesthesia is a complex phenornenon defined as a behavioural state associated with the loss of awareness and absence of pain (Tanelian. 1993). The mechanisms of dmg action that produce general anaesthesia are not well understood despite the use of these drugs for over 200 years (Harris et al.. 1995). It is commonly thought that general anaesthetics alter synaptic transmission rather than inhibit the propagation of impulses along the length of the nerve fiber (Franks and Lieb, 1994). Aithough some anaesthetics decrease the synthesis, uptake and release of neurotransmitters, evidence for major presynaptic target sites is minimal (Tanelian et al., 1993). Anaesthetics are thought to pnmady influence receptors present in the post-synaptic membrane (Franks and Lieb. 1994). The y-aminobutyric acid type,, receptor (GABA,R) is thought to play an important role in mediating the behavioural effects of intravenous anaesthetics including propofol. barbiturates, several neuroactive steroids and sedative benzodiazepines (Franks and Lieb.