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
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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. 1994; Tanelian et al.. 1993; Lin et al.. 1993). The focus of this thesis is the interactions of propofol and benzodiazepines on GABA,\R hnction.
1.1.1. NON-SPECIFIC AND SPECIFIC THEORTES OF ANAESTHETIC ACTION
Two general theories of anaesthetic action have evolved; the unitary theory, whereby anaesthetic dmgs mediate their effect by a common mechanism, and a theory in which different dmgs act by specific sites of action.
The great diversity in structure of anaesthetic drugs suggests a nonspecific mechanism of action. At the tum of the century, Meyer and Overton noted that the potency of an anaesthetic was correlated with its lipid solubility (Overton, 1901). This relationship can be applied to a wide variety of anaesthetic compounds and is true, whether the potency is measured in whole animais or at the cellular or subce1luIa.r level, over a wide concentration range (Urbane, 1985; Miller
1981 ; Moore et al., 1964).
Unitary models of anaesthetic action suggest that anaesthetics dissolve into the cell membrane and perturb the structure and dynamic properties of the lipid bilayer (Franks and Lieb,
1994; Tanelian et al., 1993). Thus, lipids are the primary target and membrane proteins are influenced secondarily. At clinically relevant concentrations, anaesthetics alter lipid bilayer fluidity. However, this effect was mimicked by small increases in temperature (+l OC ). These changes of temperature have no anaesthetic effect suggesting that the action of anaesthetics on lipid bilayers does not underlie the drugs clinical effects (Franks and Lieb, 1994). Further, the unitary theory of anaesthetic action does not account for cimg-specific effects observed at the molecular, cellular and whole animal level (Tanelian et al., 1993; Franks and Lieb, 1994).
Therefore, it has been increasingly difficult to support a unitary theory of general anesthesia.
Ueda and Karnaya (1973) provided the first convincing evidence that anaesthetics modulate protein function by demonstrating the inhibitory effect of volatile agents on the enzyme luciferase. Subsequently it was demonstrated, in a photoaffinity labeling study, that the binding of halothane to rat brain synaptosomes was saturable (K, = 490 FM). These data suggested anaesthetics bind to a specific number of sites in the brain (El-Maghrabi et al., 1992). These and other studies resulted in a shift of interest £?om non-specific mechanisms of anaesthetics to the study anaesthetic interactions with specific receptors (for review Franks and Lieb, 1994). 1.2. INHIBITORY NEUROTRANSMISSION AND GABA, RECEPTORS
1.2.1. GABA
Approximately 50 years ago, GABA and its synthesizing enzyme, glutamic acid decarboxylase (GAD) were discovered in the gray matter of the mammalian central nervous system (CNS) (Tanelian et. al., 1993). Not long afker, GABA was shown to be an inhibitory neurotransmitter (Bazemore et al., 1957; Tanelian et al., 1993). GABA is present in high concentrations in brain and spinal cor& and trace amounts have been round in peripheral nervous tissues (Miyata and Otsuka, 1972; Rabow et al., 1995).
GABA is synthesized fhm glutamate by GAD and stored for release in the presynaptic neuron (Figure A). During an action potential, a cascade of presynaptic events leads to an increase in intracellular calcium. The increase in cytosolic Ca- stimulates the release of GABA from the presynaptic terminal. Based on the low variance at the peak of synaptic responses and their smdl amplitudes, receptor saturation at the synapse has been postulated (Mody et al., 1994).
Further, it is thought that there are a smdl number of GABA receptors on the post synaptic membrane activated during an inhibitory post synaptic potential (IPSC). Jones and Westbrook
(1995) compared the on rates of brief pulses of GABA to those of IPSCs. They estirnated the peak concentration of GABA in the synaptic cleft was 526 FM.
GABA acts on pre- and postsynaptic GABA, recepton (GABABRs), postsynaptic
GABAARs and postsynaptic GABA, receptors (GABA&). GABA,Rs act via guanine- nucleotide-binding proteins (G-protehs) to inhibit Ca* channels or activate K' channels. The
GABA,R is selectively activated by baclofen, is insensitive to GABA analogs isoguavane or
THIP and is not inhibited by the GABAARantagonist bicuculline (Bomann, 1992; Curtis et al., Figure A
GABAergic synapses and the topology of a GABAARsubunit
A. Diagram of a GABAergic synapse, showing GABA synthesis and release from a presynaptic
terminal and GABA actions on the pre- and postsynaptic neuron. GABA is synthesized from
*glutamate by the enzyme GAD and stored in the presynaptic neuron for release. Once
released. G.4BA cm bind to post synaptic GABAARsand GABA,Rs. GABA also binds to
presynaptic GABA,Rs. GABA is removed From the synaptic clef? by diffusion and by Na--
coupled active transport into pre- and postsynaptic neurons and glial cells. Once GABA is in
the cell it is rnetabolized in the mitochondria to glutamate.
B. Genenc GABA,R protein subunit sequence and topological stmcture. The Y- and C-
terminal of the polypeptide is suggested to be extracellular. There are 4 TM domains (TM1 -
TM). Between the third and fourth transrnernbrane domain there is a hydrophilic putative
cytoplasrnic region of highly variable sequence involved in intracellular regulatory
mechanisms such as phosphorylation. GAD
GLIAL CELL
- -\c . 7, - -- -+f ,' v b -, 4- '- Na Ca" P p.-. - - . .-- - - - .- - GABA \. A A v r - GABA, . . r C 1'
POSTSYNAPTIC
.,+. -' TMtTM2 TM3 TM4 Lipid Bilayer 1970). Presynaptic GABABRs have been implicated in the regdation of Ca" dependent transmitter release (Bowery et ai., 198 1 and 1980) while activation of postsynaptic GABABRs increase K* conductance (Dutar and Nicol, 1988; Karlsson et al., 1988). Recently two isoforms of the GABA,R have been cloned (Kaupmann et al., 1997). GABABRshave been suggested to be important targets for therapy. Therefore, the cloning of the GABA,R will allow for the study of their molecular and functional diversity and lead to a greater understanding of their clinical importance.
GABA, recepton (GAB&Rs) are associated wiîh a Cl' channel but are pharmacologically distinct fiom the GABAAR (Bormann and Feigenspan, 1995). They are insensitive to bicuculline and baclofen and selectively bind Cis-4-aminopent-2-enoic acid
(Krogsgaard-Larsn et al., 1994). The p subunit present in the GAB&R is thought to confer baclofen resistance. The only place in the CNS where GAB&Rs have been found to be expressed is in the retina (Krogsgaard-Lmn et al., 1994; Bommand Feigenspan, 1995).
The major postsynaptic action of GABA is activation of GABAARs.Activation of the
GABAARresults in an increase in chloride conductance which hyperpolarizes the neuron. This creates a curent shunt that reduces the effects of any other currents on membrane voltage thereby decreasing the response to excitatory input (Mody et al., 1994). Synaptic inhibition is terminated when GABA dimises out of the cleft or is actively transport& into neurons or glial cells by a sodium-coupled active transporter (for review see Kanner, 1997; Tanelian et al., 1993).
GABA is mainly metabolized by GABA-transaminase, an enzyme which is pnmarily localized in mitochondria (Tanelian et al., 1993). 1.2.2 GABAARPROPERTIES
The GABAARbelongs to the superfamily of ligand-gated channels (Stephenson, 1995).
The GABAAR channel is formed kom five glycoprotien subunits that CO-assembleto form a functional Cl' channel. GABA bùids to the GABAARto regulate channel gating (opening and closing of the channel pore). The GABA dose response cwe(DRC) is sigrnoidal and has a Hill coefficient of approximately 2 suggesting that at Ieast two molecules of GABA bind in a cooperative manner to the GABAAR for full activation of the receptor (Macdonald and Olsen,
1994).
There are multiple high amnity (nM) and low aanity (PM) binding sites on the
GABAAR for GABA. It is not known if the low and high &nity sites are distinct or interconvertable. However, the low afiinity binding site for GABA is thought to be associated with the conformational change that results in an open channel (Macdonald and Olsen, 1994).
Single channel analysis has revealed that the GABAAR channel opens to multiple conductance levels. The main conductance level, responsible for 95% of the current, has a single channel conductance of is 27-30 pS. The two less fiequent conductance levels have a single channel conductance of 17-19 pS and 11-12 pS.
The GABAAR has agonist recognition sites for GABA, barbiturates, propofol and anaesthetic steroids, as well as antagonist recognition sites for bicuculline and flumazenil
(Stephenson, 1995; Tanelian et al., 1993). Further, the GABAAR complex is modulated by several second messenger systems. It is subject to phosphorylation by several protein kinase systems including protein kinase C, protein kinase A, and tyrosine kinases (Tanelian et al., 1993;
Moss et al., 1995). 1e2.3. KINETIC MODEL OF GABAARFCTNCTION B inding Gating
To explain the complex gating properties of the GABAAR,various kinetic modeis have been described (Orser et al., 1994; Macdonald and Olsen, 1994; Jones and Westbrook, 1995 and
1996; Lavoie and Twyman, 1996). In the model presented above two stages regulate channel isomerization fkom a closed to open state; ligand binding and channel gating. Receptor activation is regulated by the sequential binding of two agonist molecules (C to CG and C?,) followed by an isomenzation step to the open state (O). The rates of channel opening and closing are P and a and the rates of association and dissociation of agonist molecules are 2k,[,,, kZpl and k-,p,, 2k-2,,,, respectively. The rates of entry into, and recovery Ekom a nonconducting desensitized @) state are indicated by &, and K-,, respectively. Orser et al. (1994) suggested that there was a high probability the desensitized receptor will enter the open state prior to closing. However, other models suggest the desensitized state proceeds f?om a closed configuration (Macdonald and Olsen, 1994). in addition, Orser et al. (1994) proposed a third binding site for agonist which governed the transition to the desensitized state, however this model is simplified and does not include this binding state. Drugs other than GABA have been shown to alter the transition rates between the closed, open and desensitized states (Macdonald and Olsen, 1994). Benzodiazepines, propofol and barbiturates increase the affinity of the GABA,R for GABA and enhance the rate of GABA binding (Macdonald and Olsen, 1994, ûrser et al., 1994 and Lavoie and Twyman, 1997). In addition propofol and barbiturates alter the rates governing receptor desensitization (Oeer et al.,
2 994; Macdonald and Olsen, 2 994).
1.2-4- DESENSITIZATION
Ligand-gated channeis open in response to the binding of neurotransmitter but also close or "desensitize" with the agonist still bound (Jones and Westbrook, 1995). Desensitization has been viewed ~aditionally as a negative-feedback rnechanisrn to prevent the undesirable consequences of excess receptor activation such as excitotoxicity (Jones and Westbrook, 1996).
However, recent expenments suggest desensitization of GABAARsprolong, rather than curtail synaptic currents (Jones and Westbrook, 1995 and 1996).
Traditionally, it was thought that the decay of the post synaptic current (PSC) was govemed by agonist clearance and reuptake of transrnitter. However, several kinetic mechanisms contribute to the decay of the IPSC, including unbinding, desensitization and deactivation. Jones and Westbrook (1995 and 1996) suggested receptor desensitization might conaibute to the decay of the slow component of the IPSC. In expenments with pairs of closely timed responses, the second response was strongly depressed as predicted by fast desensitization of GABAAR. The second response returned to the full amplitude with a time course similar to the fast decay time course of the initial response. They interpreted these data to indicate the decay of the IPSC was related to the movement through fast desensitized states (Jones and
Westbrook, 1996). Further, they suggested the peak open probability and duration of the fast decay component of the IPSCs were lirnited by fast desensitization, whereas the slow component
was produced by channel reopening after exit fiom desensitized states. Therefore, hgsthat
increase receptor desensitization maintain the channel within a set of states that had a high probability of reopening before the agonist could dissociate. These dmgs would pmlong the decay curent of the PSC (Otis and Mody, 1992; Mody et ai., 1994). Additionally, receptor desensitization could limit the fiquency at which GABA recepton produced full amplitude
responses to GABA (Lavoie and Twyman, 1996). The slow recovery from desensitization might
also result in the attenuation of successive IPSCs during high fkequency release. Attenuation of
IPSCs might also develop in the presence of low background levels of GABA.
1.2.5. SUBUNITS OF GABAAR
To date 18 subunits of the GABAAR have been identified and are classified based
according to the conservation of their amino acid sequence. There are six GABA,R subunit types and various isofoms: al-6,B 1-4, y 1-4, pl-2, a and E (Sieghart, 1995; Davies et al.,
1997). Additionally, some of the GABAAR gene products undergo alternative splicing such as
the human P3 (Kirkness and Fraser, 1993), the rat a6 (Korpi et al., 1994) and the y2 subunit which exists in two foms, y2 short (y2S) and y2 long (y2L). The y2L splice variant has an 8
arnino acid insert (Whiting et al., 1990; Kofûji et al., 1991).
Eac h GABAARsubunit has an extended extracellular, hydrop hilic N-teminal domain of
the order of 220 arnino acids and has four putative trausmembrane domains (TM 1-TM4), which
form a wall of the chloiide ion channel (Figure A). The positively charged residues in the TM2
region are thought to form one side of the selectivity filter which permits negatively charged
chlonde ions to pass through the channel central pore (Tanelian et al., 1993). The TMI-TM3 regions are adjacent to the N-terminal domain, whereas TM4 is at the C-terminal end of the protein. This transmernbrane topology predicts that the N- and C- terminal regions are extracellular however, this remains unproved. Separating TM3 and TM4, there is a putative hydrophilic cytoplasmic region that is possibly involved in intracellular regulatory rnechanisms.
It contains several putative sequences for phosphorylation by various protein kinases. It is here that the y2L splice variant has an 8 amino acid insert which contains a putative sequence for PKC phosphorylation (Khan et al., 1994).
GABAARsubunits are unevenly distributed throughout the brain and different neuronal populations possess different compliment of subunits (Wisden et al, 1992). In situ hybridization for rnRNA of the various subunits revealed that the pl subunit was largely confined to the hippocampus (Widsen et al., 1992). Ln contrast the P2 and P3 subunits were widely disîributed within the CNS. The al, a2, a4, a5, pl, P2, y2 mRNA were strongly expressed in the hippocampus, whereas there was lower expression of the P3 and yl subunits. The a3,y3, 6 and a6 subunits were minimally or not expressed in the hippocampus.
Over 10,000 possible pentameric subunit combinations of the GABAARpotentially exist however, some combinations are more likely to occur. To detemùne which subunits co-existed in the same receptor cornplex, McKemnan and Whiting (1996) summarized studies where
GABA,Rs were immunoprecipitated using different combinations of antisera. They proposed almost half of al1 GABAARs in the brain contained the alp2y2 subunits. Two other triheteromeric combinations constituted a Mer 35% of the total GABAARs present in the brain; a2P3y2 and a3py2/y3. The following combinations are found in the hippocarnpus; alp2y2 present in intemeurons, a2P2/3y2 and aSP3y2/y3 present in pyramidal cells and u4P6 present in the dentate gyms. In spinal cord motor neurons, a2PU3-12 is the most cornmon subunit combination. 1.2.6. GABAARAND ANAESTHESIA
It is now recognized that anaesthetics influence GABAAR fûnction by several different mechanisms. However, it is not known which mechanisrn is the most important for sedation, hypnosis and anaesthesia (Tanelian et al., 1993). Agents such as propofol, barbiturates and neurosteroids directly activate the GABAAR., enhance GAB A binding to the receptor and influence desensitization (Orser et al., 1994; Rabow et al., 1995; Harris et al., 1995). In contrast sedative benzodiazepines only potentiate agonist evoked responses (Choi et al., 198 1 ).
It is thought that there are a small number of postsynaptic recepton which are saturated during synaptic transmission (Mody et al., 1994; Jones and Westbrook, 1995). This limits the number of mechanisms by which anaesthetics might exert a positive allosteric influence upon
GABAergic transmission (Mody et al., 1994). For example, in the presence of saturating concentrations of GABA it is unlikeiy that anaesthetics increase the amplitude of the IPSC since anaesthetics increase the amnity rather than efficacy of GABA for the GABAAR. However, anaesthetics could proiong the decay and thereby modify the temporal summation of inhibitory tone (Lambert et ai., 1997). Benzodiazepines, barbiturates and propofol have been show to prolong the decay of the PSC (Orser et al., 1994; Otis and Mody, 1992; Poncer et al., 1996).
This could be explained by an increase in the affinity of the receptor for GABA and a decrease in the rate of deactivation since GABA would remain bound longer. Further, prolongation of the decay could be due to an increased channel open time which would increase the probability of the channel being open during the decay.
1.3. PROPOFOL EFF'ECTS ON GABAAR
Propofol(2,6-di-isopropylphenol)is an aikylphenol recently introduced for clinical use as a general anesthetic. It is an intravenous agent which rapidly and reliably causes loss of consciousness. Propofol is an important anaesthetic because it does not cause use-dependent tolerance, as seen with the barbiturates and benzodiazepines (Fassoulaki et al., 1994). Use- dependent tolerance refers to an increase in dose requixements of drug during anaesthesia to maintain a constant same level of unconsciousness.
Propofol is a highly lipophilic molecule (octanoVwater partition coefficient = 4,300)
(Tonner et al., 1992; Veintemilla et al., 1992). In principle, propofol could disturb GABAAR channel function indirectly by pertmbing the plasma membrane. However, when propofol was applied to the extracellular domain of the GABAAR it directly activated channel opening, whereas propofol added to the pipette solution did not enhance a GABA evoked current. Thus, propofol exhibited a clear membrane asymmetry suggesting it did not difhise into the membrane to activate the GABAAR (Hales and Lambert, 1991). Additionally, bicuculline inhibited receptor activation by propofol Mersuggesting that propofol is acting at a specific site on the GABAAR
(Hara et al., 1993).
Propofol has been reported to enhance inhibitory synaptic transmission and potentiate
GABA-induced depolarizations in slices fkom the rat olfactory cortex (Collins, 1988). In addition, propofol reversibly, and dose-dependently potentiated GABA-evoked responses recorded from acutely dissociated CA1 pyramidal neurons fiom the rat hippocampus (Hales and
Lambert, 199 1). Potentiation of the GABA-evoked responses by propofol was associated with a parailel shift to the lefi of the GABA DRC, indicating an apparent increase in the finity of the receptor for GABA (Orser et al, 1994; Hara et al., 1994; Adodra and Hales, 1995). Propofol has also been shown to reduce the extent of desensitization during prolonged drug applications
(Orser et al., 1994). Single charnel analysis, indicated that low concentrations of propofol greatly increased the frequency and open probability of GABAAR channels but had little effect on the
open duration (Orser et al., 1994).
Recombinant GABAARreceptors have shown that propofol cm potentiate GABA-evoked
currents in al1 combinations of subunits tested (for review see Harris et al., 1995). The extent of
propofol potentiation of recombinant receptors did not differ between receptor combinations that
contained the al, a4 or a6 isoform. In contrast, pentobarbitone produced a much greater
potentiation of currents fiom receptors composed of the a4 and a6 subunit compared to al
containing receptors, suggesting pentobarbitone and propofol modulatory sites are distinct
(Wafford et al., 1996).
Propofol, ai concentrations generally greater than those required for allostenc modulation
of the GABAAR directly activate the GABAAR(Haies and Lambert, 1991 ; Hara et al., 1993;
Orser et al., 1994; Adodra and Hales, 1995; Sanna et al., 1995qb). Propofol-induced currents are potentiated by diazepam, and antagonized by bicuculline and zinc (Haies and Lambert, 199 1;
Hara et al 1993; Adodra and Hales, 1995). The modulation of propofol-evoked currents is similar to the modulation of GABA-evoked cments which are potentiated by benzodiazepines and antagonized by bicuculline and zinc. The direct actions of propofol and pentobarbital require the p subunit, unlike the potentiating action of propofol which does not require a specific subunit
(Sanna et al., 1995qb). Correlation analysis showed no relationship between the direct and potentiating actions of propofol. Therefore, Sanna et al. (1995b) postulated two sites of action
for propofol: one on the P subunit responsible for direct activation, and a second site that could not be placed on any single subunit, responsible for potentiating GABA-evoked currents. The P subunit is essential for direct-activation of the GABA,R by propofol. However, the extent of receptor activation is modulated by the a subunits. Recepton composed of aJy2s subunits were maximally activated by propofol when the combination contained a6, activation was reduced when the al subunit was substituted for a6. Further, when the a4 subunit was present the receptor was not activated by propofol (Waf5ord et al., 1996). Hence, the P subunit is essential however, the a subunit can modulaie the extent of propofol activation of the GABAAR.
1.4. BENZODIAZEPINE ACTION AT THE GABAAR
There are three types of benzodiazepine (BDZ)ligands: 1) agonists which increase Cl' conductance, 2) inverse agonisa which decrease CI- conductance and 3) antagonists which have little intrinsic activity but block agonist and inverse-agonist effects (Haefely, 1990 and 1991 ;
Tanelian et al., 1993; Macdonald and Olsen, 1994).
Potentiation of GABAAR-meàiated synaptic inhibition likely underlies the therapeutic efficacy of benzodiazepines as anxiolytics, anticonvulsants and sedatives. Benzodiazepines bind to an integral site on the GABAARand increase the apparent affinity of the receptor for GABA
(Pritchett et al., 1989; Choi et al., 1981; Study and Barker, 1981). Radiolabelling studies demonstrated hinctional coupling between the benzodiazepine receptor and the GABAAR;both
GABA and benzodiazepine agonists reciprocally increase the binding affinity for the other agent
(Costa and Guidotti, 1979; Braestrup and Neilson, 198 1). Fluctuation studies and single channel analysis suggested that diazepam increased GABAARcurrents by increasing opening frequency without altering channel conductance, open duration or bursting properties (Study and Barker,
1981; Twyman et al., 1989). Application of the benzodiazepine agonist, rnidazolarn, (< 0.3 PM) increased the frequency at which single charnels open, whereas higher concentrations decreased the fkequency of channel opening (Rogers et al., 1994). Detailed kinetic analysis indicated the increase in fkequency of opening reflected an increase in the binding affinity of the receptor for the first of the two GABA molecules (Rogers et al., 1994; Lavoie and Twyman et al, 1996).
1.4.1. BENZODIAZEPINE BINDING IS INFLUENCED BY SUBUNIT COMPOSITION
OF THE GABAAR
Historically, benzodiazepine agonists were classified into two groups depending on their affinity for the GABAAR;benzodiazepine Type 1 and 2 agonists (BZ1 and BZ2) bind with hi& affinity to BZ1 receptors and BZ2 recepton, respectively. More recently it has been shown that
BZ1 agonists have a high affinity for GABAARs containing the a 1 subunit, whereas BZ2 agonists have a high affinity for receptors containing a2, a3 or a5 subunits.
In order for benzodiazepines to modulate GABA-evoked currents, the y subunit must be present in the GABAAR complex (Rovira and Ben-An, 1993). The a subunit influences the efficacy, as well as the amnity of benzodiazepine ligands (Macdonald and OIsen, 1994). Site- directed mutagenesis has confirmed the sequence specificity for berwdiazepine ligand binding on the a subunit (Pritchett and Seeburg, 1991). Cornparison of sequences for a 1, a2 and a3 subunits revealed differences in the N-texminal putative extracellular domain. Using chimenc cDNAs which mixed the N-terminal domains of the a 1 and a3 subunits, lead to the identification of a single residue that detemiined the affuiity of GABAARsfor benzodiazepine agonist. There was a Gly in the al subunit at amino acid 201, whereas in the a2,a3 and a5 subunits the amino acid in homologous position was Glu. Therefore, for BZ1 agonists to have a high afEnity for
GABAARsthe a subunit must have a glycine at arnino acid 201. Recepton containing the a6 subunit have a unique pharmacology where they do not bind flunitrazeparn. Comparisons of the al and a6 subunit revealed a single residue that conferred the ability of the receptor with the isoform to bind 'H-fl~nitraze~am.The kg-100 was mutated to His-100 which was found in a 1, a2, a3 and a5, it restored the flunitrazepam binding for recombinant receptors containing the a6 subunit (Wieland et al., 1992). Therefore, rnolecular studies have revealed that the difference in agonist affinity depends on the a subunit present in the GABAARcornplex.
1.5. DRUG INTERACTIONS BETWEEN PROPOFOL AND BENZODIAZEPINES
Propofol and midazolam are comrnonly used clinically in combination. Propofol and midazolam have been shown to act synergistically for the induction of hypnosis (Short and Chui,
1991). The ED,,'s for propofol and midazolarn to induce hypnosis were reduced by 44% when used in combination compared to each agent acting individually. Additionally, midazolam reduced the ED,, for anesthesia of propofol by 52% (Short and Chui, 1991). However, a recent paper by Oxom et al. (1997) has show that midazolarn, given immediately prior to propofol did not influence the dose of propofol required for the induction of hypnosis or maintenance of anaesthesia.
The mechanism of synergism which underlies the sedative and hypnotic effects of midazolam and propofol is not certain but may be due to interactions occurring at the GABA,R.
The interactions between midazolam and propofol at the GABAARare studied in this thesis.
1.5.1. DETERMIMNG DRUG INTERACTIONS
Synergism occurs when the effect of the combination of drugs is greater than that expected Eom the additive effect of each drug alone (Berenbaurn, 1989). Drugs do not have to act at the sarne site to have a synergistic action; they can indirectly modulate each other by increasing or decreasing the dlinity or efficacy of the other hg. Alternatively, they may act in concert by activating the same recepton (Wessinger, 1986). Several methods have been used to detemine if drug interactions are additive, superadditive or subadditive, including the Fixed-
Dose Model and Isobolographic Analysis.
The Fixed-Dose Model predicts that the combination of drug 1 and dmg 2 will produce an effect equai to the sum of each effect alone. The response fkom the combination of drugs is compared to the theoretical additive response. If response of the drugs in combination is significantly lower than the theoretical response, the drugs interact in a superadditive rnanner
(Wessinger, 1986). The Fixed-Dose Model does not take into consideration that the DRCs for dmgs are not linear (Tallarida et al., 1989). Hence, for a specific effect size the potencykoncentration of a drug has to be determined. Further, to adequately explore dmg interactions, a wide variety of doses and dose combinations need to be tested (Wessinger, 1986).
A method that takes both of these concepts into consideration is Isobolographic Anaiysis
(Wessinger, 1986). For Isobolographic analysis, the potency of a cimg, or mixture of two dmgs, that produces a specified "effect level" is measured as a dose or concentration. The relative potencies of drug 1 and 2 are plotted on the isobolograph plot and the diagonal line connecting these endpoints is termed the isobol of additivity (Wessinger, 1986). If the two dmgs do not interact and the effects are additive, the concentrations of the two drugs to reach the effect level will lie on the isobol of additivity (Berenbaum, 1989; Tallarida, 1992). When agents in combination are more effective than expected fkom thek dose response curves (Le. they interact synergistically), smaller amounts of drugs are needed to produce the effect under consideration and would lie below the isobol of additivity. This mode1 of studying hginteractions will determine if the dmgs interact in a synergistic, additive of subadditive manner. 2. OBJECTIVES AND WORKRYG HYPOTHESES
2.1.1. SECTION 1
Anaesthetics, such as propofol and barbiturates, have been shown to have multiple actions at the GABA,R: they directly activate the receptor; they potentiate GABA-evoked
responses, and they influence receptor desensitization. Previous studies suggest that GABA and anaesthetics activate the GABAARby binding to distinct sites. In addition, distinct sites likely
modulate the anaesthetic-induced potentiation and the direct activation of the GABAAR.
However, it is not known if a distinct site mediates anaesthetic-induced changes in GABAAR desensitization.
Our hypothesis is that the effect of propofol on GABAARdesensitization is mediated by a site distinct fiom the two binding sites involved in potentiation of GABA-evoked responses and direct activation. Here, we will determine if the abilities of propofol to directly activate the
GABAARand decrease receptor desensitization are differentially modulated by benzodiazepines.
In Section 1, we will address the following questions:
1. Does midazolam increase the &ty of the GABAARfor GABA?
2. Does midazolam increase the fityof the GABAARfor propofol?
3. Does midazolam alter desensitization of the GABAAR?
4. What are the characteristics of the propofol modulation of GABAARdesensitization?
5. Does midazolam alter propofol modulation of GABAARdesensitization? 2.2. SECTION 2
A variety of agents are used for CO-inductionof anaesthesia. This practice is based on the principle of synergism, whereby dnigs in combination produce a greater-than-additive effect.
Propofol and midazolam are used clinically in combination, and it has been suggested that they act synergistically for the induction of hypnosis (Short and Chi, 1991). Our hypothesis is that the synergistic effects observed in the clinicd studies are due to the interactions between midazolam and propofol at the GABAAR
Ln Section 2, we will address the following questions:
1. Do midazolam and propofol potentiate GABA-evoked responses in an additive,
superadditive, or antagonistic manner?
2. 1s the interaction between propofol and midazolam dependent on the concentrations of
GABA and propofol?
3. Does ce11 type influence the interaction between midazolarn and propofol? 3. METHODS
3.1. CELL CULTURES
Cultures of ernbryonic hippocarnpal or spinal cord neurons were prepared fkom Swiss white mice as previously described (MacDonald et al., 1989). Bnefly, feu1 pups ( 17 days in utero) were removed from mice sacrificed by cervicai dislocation. The hippocampi were dissected fiom each few, then placed in an ice-cooled petri dish. Neurons were dissociated by mechanical trituration using two Pasteur pipettes (tip diameter 150-200 mm) and plated at a density of 1x106 cellu'ml on 35-mm culture dishes. The culture dishes had been coated with collagen (from cdf skin) or poly-D-lysine (Sigma Chemical Co., St. Louis, MO). For the fint 1O days in vitro, cells were maintained in Minimal Essential Media (MEM) (Life Technologies,
Grand Island, NY) supplemented with glucose (ha1 concentration 33.6 mM), NaHCO, (final concentration 31.56 mM), 10% horse se-, 10% fetal bovine serum, and 1% insulin (Life
Technologies, Grand Island, NY). The neurons were cultured at 36.S°C in a 5% CO2I 95% air environment. Once the background cells had grown to confluence (4 to 7 days), 0.1 mL of
FUDR mixture (4 mg 5-fluorodeoxyuridine and 10 mg uridine in 20 rnL MEM) was added to each dish to arrest ce11 division. The supplemented media was changed every 3-4 days. Afer 10 days in culture, the media was changed to a new media containing MEM supplemented with 10% hone senim and 1% insulin. There was a heterogeneous population of neurons in culture as there were neurons kom dl of the hippocampal regions.
The sarne procedure was followed for spinal cord neurons with the following additions.
The spinal cord was dissected nom fetal pups after 15 days in utero. Once the spinal cord was dissected it was subjected to a trypsin digestion for 30 min then dissociated by mechanical trituration. The rest of the procedure was not aitered. Rior to recording, neurons were rinsed with an extracellular recording solution containing (in mM): 140 NaCl, 1.3 CaCI,, 5.4 KCl, 25 N-2-hydroxy-ethylpiperazine-N'-2- ethanesulphonic acid (HEPES), 33 glucose, 1 MgCl, and 0.0003 tetrodotoxin (TTX) (pH 7.4,
325-335 mOsm). TTX was added to inhibit voltage-activated Na* channels. Cells were studied
12- 16 days after dissociation.
3.2. RECORDING PIPETTES
Whole-ce11 patch-pipettes (3- 10 MR) were consûucted fiom borosilicate glass capillary nibing containing an inner filament (outer diameter 1.5 mm, TW150F-4, World Precision
Instruments Inc., Sarasota, FL). Electrodes were pulled using a two-stage vertical puller
(Narishige PP-83,Narishige Scientific Lab., Setagaya-Ku, Tokyo) and tips were occasionally firepolished with a microforge (Narishige MF-83, Narishige Scientific Lab., Setagaya-Ku,
Tokyo) .
Pipettes were filled with the pipette solution (see below) and mounted in a teflon holder that contained a silvedsilver chloride-coated silver wue (Ag/AgCl). ui this system, the Ag/AgCI acted as a reversible electrode that converted ionic CI- cwent in the pipette solution into electron current in the wire. A ground electrode (AglAgCI pellet) was placed in the bath solution and the offset potential was nulled pnor to the seal formation.
3.3. PIPETTE SOLUTION
Patch-pipettes were filled with a pipette solution containing (in mM): 140 choline-CI, 10
HEPES, 11 EGTA, 1 CaCl,, 10 tetraethylarnrnonium chlonde (TEA-CI), 2 MgCl, and 4 Mg-ATP
(pH 7.4, 290-300 mOsm). Choline was used as an irnpemeant cation which has been used in previous studies to record GABA-evoked currents (Orser et al., 1994). 3.4. WOLE-CELL VOLTAGE-CLAMP RJECORDINGS
Whole-ce11 voltagetlamp recording techniques measure ion flow across the ce11 membrane and indirectly provide information regarding receptor activity. The voltage-clamp system ernploys a negative feedback amplifier which compares a signal received fiom the recording electrode to a reference or "command potential". The potential difference between the command potential and the reference potential is arnplified and transmitted to the recording electrode.
Whole-ce11 currents were recorded using the Axopatch 1D amplifier (Axon Instruments
Inc., Foster City, CA). The recording-pipette was filled with pipette solution and secured into the pipette holder. The holder was inserted into the head stage of the amplifier and the pipette tip was positioned close to the ce11 using a course rnanipulator then a fine hydraulic micromanipulator (Narishige Scientific Lab., Setagaya-Ku, Tokyo). The junction potential between the bath and patch-pipette solutions was nullified by adjusting the input offset of the amplifier. A +20 mV test pulse was used to measure the electrode resistance and to track the development of the seal between the pipette and the ce11 membrane. Following seal formation, the command potential was set and the membrane patch disrupted by applying negative pressure and a brief current pulse.
Neurons were voltage clamped at -60 mV. Once patched, we waited 10 minutes for the cell to stabilize and to reduce the effect of rundown. Responses were recorded and analyzed on a cornputer using the pClamp program (Axon Instruments Inc., Foster City, CA). Ali experiments were conducted at room temperature (20-25°C). 3.5. DRUG AND AGONIST PERFUSION SYSTEM
Drugs and agonists were applied to the neurons using a multi-banel pefision system
(Figure B; Johnson and Ascher, 1987). A senes of 2 or 3 square glass capillary tubes (400 x 400
Fm, Longreach Scientific Resources, Orr's Island, ME) were horizontally aligned and glued together with cyanoacrylate glue. The array of barrels was then placed in a holder. Lateral movement of the pefision barrels was regulated using a stepping motor (Vexta, Oriental Motor
Co.) which was attached to a Leitz manipulator (Germany). The manipulator was controlled by a custom-made, computerized switching system. The solution bathing the patched neuron was changed by laterally displacing the barrels. The speed of the fast perfusion system was previously detemined f?om the rate of omet of Mg+-induced inhibition of NMDA currents recorded at hyperpolerizing potentials and was estimated to be less than 50 msec. nie barrels were comected by silastic tubing to separate soiution reservoirs and the flow rate (approxirnately
0.5 mllrnin) was regulated by changing the height of the reservoirs above the bath. Agonists were applied only once every few minutes to allow receptors to recover f?om desensitization.
Drugs and agonists were applied for 15 or 18 seconds at a time to obtain the peak and relative steady-state current.
3.6. WOLE-CELL CURRENT ANALYSIS
Whole-ce11 data were analyzed using the CLAMPFIT program of pClamp (Axon
Instruments Inc., Foster City, CA). For a11 experiments the cells used were Eom at lest 3 different dissections.
For dose-response analysis, the current amplitudes were plotted versus the agonist concentrations and fit using a standard logistic equation (I= hax (l+CEC,,)", where 1 is the Figure B iMulti-barre1 fast-perfusion system
Three square glas capillary tubes (400 x 400 pm) were aligned and glued together as shown.
The barrels were connected by silastic tubing to separate solution reservoirs. Perfusion solutions were exchanged by laterally displacing the barrels a distance of one barre1 diameter. Solutions perfused cultured munne hippocampal neurons. Currents were recorded using the whole-ceIl patch clamp technique. The holding potential was -60 mV. current amplitude and C is the concentration of the agonist) (Graph Pad Prism, San Diego, Ca).
The concentration of agonist that produced 50% of the maximal response (EC,,) and the Hill coefficient (n) were determined fkom die equation.
The extent of receptor desensitization was quantified by detexmining the steady-state to peak current ratio (IssAp; see Figure C). An increase in the Iss/Ip ratio correlated with a decrease in receptor desensitization. The IssAp ratio was plotted versus the concentration of agonist. This relationship was best fit using a nonlinear regression for GABA-evoked currents (equation above) and a linear regression for propofol-evoked currents. The rate of receptor desensitization was estimated from the rate of current decay during prolonged agonist application. The decay of the current was best fit using a biexponential equation I(t) = A, exp(-Vrf) + A, exp(-t/r3 + C where I(t) is the current amplitude at any given time t, C is the baseline current, r, and r, are the fast and slow time constants of current decay, respectively . A, and 4 are the estimated fast and slow intercepts of the cornponents at time zero, respectively.
Deactivation refers to the current decay during agonist washout (Figure C). The rate of deactivation was best fit using the above equation. The biexponential equation above was modified, the baseline current, C, was zero.
3.7. DRUG INTERACTION ANALYSIS
Fixed-Dose Ratio mode1 and Isobolic Analysis were used to detemine if the interactions of rnidazolam and propofol, with respect to potentiation of GABA-evoked currents, were synergistic, additive or subadditive. Using the Fixed-Dose Ratio method, low, clinically relevant doses of propofol, midazolarn and GABA were chosen. The concentrations were previously noted by Reynolds et al. (1996) to have synergistic interactions for the enhancement of GABA- Figure C
Desensitization and deactivation of GABA induced currents.
The bar on top of the current represents the duration of drug application. Desensitization is the process by which receptors are inactivated in the prolonged presence of agonist. The extent of receptor desensitization was calculated from the steady-state to peak current ratio (Iss/lp).
Additionaily. the extent of desensitization was quantified by fitting the decay with a biexponential equation. AAer agonist application. the current retumed to a base line current.
This is tened deactivation and was quantified by fit~ingthe decay with a biexponential equation.
evoked currents in oocytes expressing human GABAARs. Since the degree of potentiation of
GAE3A-evoked currents dependents on the concentration of agonist, doses of propofol and
GABA were varied in different experiments (Harris et al., 1995).
Potentiation of GABA-evoked currents were quantified by estimating the increase in the peak response induced by midazolam or propofol compared to the response evoked by GABA.
The theoretical additive response was estimated by adding the enhancement produced by each dmg alone to the amplitude of the response evoked by GABA. These values were compared to the amplitude of the currents evoked by the two dmgs in combination. In addition, charge transfer was measured (using pClamp Software) by taking the integral of the area under the current response. The charge transfer was nomalized to the control GABA response. The tieoretical additive value was estimated by adding the relative enhancement by each dmg alone and these values were compared to the measured charge transfer of GABA-evoked currents in combination with the two drugs.
Isobolographic analysis was used to determine if the dmg interactions were synergistic.
Testing a pair of drugs for synergism first required the determination of the potency of each dmg alone then exarnining the potency of the two dnigs in combination. The potency of a dmg or mixture, was defined as the dose that produced a chosen effect level. Here, we chose an effect level where the dmg potentiated the GABA-evoked response by a factor of 3, that is the current was 3 times the control or 200% greater than the control.
The doses of propofol and midazolam that enhanced the GABA response by a factor of 3 were determined from individual DRCs (2,. and +, respectively; Figure D). The average dose of midazolam and propofol that produced the 3 times effect level was plotted on the X-and Y-axis of the isobologram, respectively. These points were joined to fom the isobol of ad&iviy. Figure D
Isobolographic analysis
The Y awis represents the concentration of dnig 1 and the X axis represents the concentration of drug 2. 2,. is the concentration of dnig 1 that potentiated the control response by a factor of 3.
Z,. is the concentration of dmg 2 that potentiated the control response by a factor of 3. The line connecting 2,. and z2. contains coordinates which are dose pairs that produce an additive interaction. The line is called the isobol of additivity. P represents a dose pair that interacts in a synergistic manner. Q represents a dose pair that interacts in a subadditive manner. subadditive
synergistic Subsequently, we selected a dose of midazolam that enhanced the GABA-evoked curent by less than the 3 times effect level. in the presence of this fixed concentration of midazolarn, the concentration of propofol that produced the 3 times effect was less than the propofol concentrations estimated ftom the isobol of additivity (Figure D).
The measured concentration of propofol that produced the 3 tirnes effect level in the presence of rnidazolarn was then compared to the theoretical additive concentration. To detemine the theoretical additive concentration of propofol, the following equations were used as descnbed by Tallarinda (1992). The doses of propofol and rnidazolam that each produced the
3 times effect level were denoted as z,* and q*,respectively (Figure D). The relative potency
(R) was defined as R = z,*/q*. The confidence of this ratio, or the variance of R, was calculated according to :
C = V(Z,*)/(q*)' + (2, *)2~(q*)/(~2*)4, where V was the variance of the average dose. The theoretical additive concentration of propofol was calculated by: z,ad,j = z1* - E-i where q W~Sthe fixed concentration of midazolarn. The variance of this value was caIculated b y:
V(z,,&J = V(z,*) + (ZJZC- 2qV(z,*)/q*.
If the theoretical additive concentration (z,& was significantly less than the actual concentration, the dmg interaction was not additive. If the theoretical value was significantly greater than the actual concentration, the dmg interaction was synergistic. 3.8. DRUGS AND OTHER CHEMICALS
Propofol was prepared fiom ~ipnvan' (Zeneca Pharma, Missassauga, ON). Each mL of
Diprivan contained (in mg): 10 2,6 di-isopropylphenol, 100 soybean oil, 12 egg lecithin and 22.5 glycerol. Stocks of propofol (10 mM and 1 rnM) were prepared every 3 days and stored at 4°C.
Midazolarn was prepared fiom Versedm(Hohann-LaRoche Ltd., Missassauga, ON). Each mL of Versed contained (in mg): 1 midazolam, 0.1 disodium edetate, 10.45 beruyl alcohol and 8
NaCl. In addition, rnidazolam was generously donated by Hofiann-LaRoche. There was no difference in potentiation of GABA-evoked currents when midazolam was prepared from vend compared to midazolam which was dissolved in dimethylsulfoxide (DMSO). EGTA, HEPES and KCl were purchased from Fluka Biochemika (Bucks, Switzerland), DMSO from Fischer
Scientific (Fairlawn, NJ), CNQX nom Tocns Cookson (St. Louis, MO), TTX fiom Precision
Biochernicals, hc. (Vancouver, Canada) and NaCl, CaC12, glucose and NaOH fiom BDH inc.
(Toronto, ON). Unless specified otherwise, al1 other compounds were purchased fkorn the Sigma
Chernical Co. (St. Louis, MO).
3.9. STATISTICS
Al1 data are presented as mean t S.E.M unless otherwise indicated. An analysis of variance (ANOVA) was used to compare multiple groups of data. Al1 groups of data were tested to ensure that the requirernents for a paramenic test were met. If the data were not normally distributed, a non-parametric test was substinited for the parametric test. For experirnents including only two cornparison a Student's t-test was used The EC, values and Hill slopes of the DRC were compared using a paired t-test. 4. RESULTS
4.1. SECTION 1
Sedative benzodiazepines, such as diazepam, are known to enhance GABA-evoked currents by increasing the affinity of the GABAAR for GABA (Study and Barker, 1981).
However, benzodiazepines have no inainsic GABA-mimetic properties (Choi et al., 198 1).
Propofol activates the GABAARpossibly by binding to a site distinct from the GABA binding domain (Sanna et al., 1995a,b). The purpose of these experirnents was to determine if midazolarn infiuenced propofol-evoked currents or altered the finity of the GABA,R for propofol. In addition, we investigated the effects of midazolarn on GABA-evoked responses.
4.1.1. MIDAZOLAM INCREASED THE AFFINITY OF THE GABAARFOR GABA AND
PROPOFOL
In the absence of GABA or propofol, rnidazolarn (0.5 PM) did not induce a GABA,R- mediated current. However, midazolarn (0.5 PM) potentiated cuments evoked by subsaturating concentrations of GABA (0.6-20 PM; Figure 1). The amplitudes of currents evoked by near- saturating concentrations of GABA (1 00-600 PM) were not enhanced. Midazolam (0.5 pM) shifted the GABA dose-response curve (DRC)to the lefl and significantly decreased the EC,, from 9.49 + 1.85 PM to 6-92 i 1.32 pM @ < 0.05, paired t-test, n = 11, Figure lb). However, midazolarn did not change the Hill coefficient (1 -54 + 0.19 to 1.65 t 0.25, p > 0.05, paired t-test).
As previously reported (Orser et al., 1994), propofol (1-600 PM) induced GABAAR- mediated inward currents in al1 hippocampal neurons tested (Figure 2). The thresho Id concentration for propofol-activated cments was approximately 1 pM and maximal currents were observed at 600 pM propofol. Higher concentrations of propofol (approximately 600-1 000 Figure 1
Midazolam increased the aflinity of the GABAARfor GABA.
A. Responses evoked by 0.6, 3. 6. 10. 100, and 600 pM GABA, in the absence and
presence of 0.5 FM rnidazolam. Midazolam increased the amplitude of currents
evoked by subsaturating concentrations of GABA. However, rnidazolarn did not
change the amplitude of the maximal response. The bars indicate the duration of drug
application.
B. The dose response curves for peak currents activated by GABA in the absence or
presence of 0.5 uM midazolam (n = II) are shown. iMidazolam shifted the GABA
DRC to the Ieft and shified the EC,, from 9.49 f 1.85 pM to 6.92 21.32 pM (p <
0.05, paired t-test) but did not affect the Hill coefficient (1-54 r 0.19 to 1.65 k0.25. p
> 0.05, paired t-test). I 4 sec
GABA GABA+MDZ
1 10 GABA (PM) Figure 2
Midazolam increased the affinity of the GABAARfor propofol.
A. Responses evoked by 1. 6. 10. 60. 100, and 600 pM propofol. in the absence and
presence of 0.5 pM midazolam. Midazolam increased the amplitude of currents
evoked by subsaturating concentrations of propofol. However. midazolam did not
change the amplitude of the maximal response. Note that is 3 of 6 cells. midazolam
increased the peak of the after-response.
B. Dose response cuves for peak currents activated by propofol, in the absence or
presence of 0.5 pM midazolam are shown (n = 6). Midazolam shifted the propofol
DRC to the left and decreased the EC,, from 24.3 & 3.4 pM to 16.1 + 2.3 @M (p <
0.05. paired t-test) but did not affect the Hill coefficient (124 + 0.1 1 to 1.35 +O. 16. p
> 0.05, paired t-test). Further, midazolam did not change the amplitude of the
maximal response. 3 sec
i PRO A PROt MD2
10 1O0 Propofol (PM) 42
CiM) activate progressively smaller currents (Orser et al., 1994). Therefore, the highest
concentration of propofol used in these experiments was 600 pM. Upon cessation of the
application of a high concentration of propofol (600pM), a current surge, also refexred to as an
"after-response", was observed. in contrast, there was no decrease in the peak response or after- response observed at high concentrations GABA. Propofol-activated currents were inhibited by bicuculline suggesting they were mediated by the GABAAR.The rate of onset and offset of currents evoked by propofol (1-100 FM) were slower than the rates for currents activated by
GABA (Orser et al., 1994).
Midazolam (0.5 pM) potentiated currents evoked by subsaturating concentrations of propofol (1-100 PM, Figure 2a) but did not alter the maximal amplitude of responses evoked by
600 pM propofol. As illustrated in Figure 2b, midazolam shified the propofol DRC to the left and decreased EC,, fiom 24.3 f 3.4 pM to 16.4 f 2.3 pM @ < 0.05, paired t-test, n = 6) but caused no change in the Hill coefficient (1.24 t 0.1 1 to 1.35 f 0.16, p > 0.05, paired t-test). In 3 of the 6 cells tested, midazolam increased the amplitude of the propofol-induced "after response".
Flumazenil, a selective antagonist for the benzodiazepine site, inhibited the effect of midazolam on propofol-evoked currents (Figure 3) suggesting midazolam potentiated propo fol- evoked currents by acting at the benzodiazepine site.
The Current Voltage (W) relationship of propofol (20 LM)-evokedcurrents, recorded in the absence and presence of midazolam (0.5 PM), are sumrnarized in Figure 4. Responses were noxmalized to the peak currents measured at a holding potential of -60 mV. The W relationships were nonlinear and indicated currents reversed polarity at approxirnately -6 mV. Figure 3
Midazolam potentiated currents evoked by propofol.
In the absence of propofol (PRO).rnidazolam (MDZ) did not evok ent. However.
PRO (20 FM) directly-acrivated a current. When ;MD2 was CO-appliedwith PRO the current was enhanced. When flumazenil (FMZ) was CO-appliedwith PRO and MDZ. the potentiation was depressed. --,--y.-- -,-- --- MDZ 0.5pM
200 pA 1 300 msec Figure 4
The IN relationship of propofol-induced currents recorded in the absence and presence of midazolam.
Propofol (20 piM) was applied to neurons voltage clamped at holding potentials ranging from -60 mV to +20 mV in the absence and presence of midazolam (0.5 FM). -411 currents were normalized to the current evoked at -60 mV (n = 4). The W relationship was nonlinear and currents reversed polarity at approximately -6 mV.
The reversa1 potential was close to the calculated Nernst equilibnum potential for chloride ions
(+1 mV) suggesting the currents evoked by propofol are mediated by chloride ions (Hales and
Lambert, 1991; Hales et ai., 1993).
4.1.2. MIDAZOLAM D1D NOT INFLUENCE DESENSITIZA TION OF THE GABA$
Previous reports suggest that benzodiazepines including diazeparn and chlordiazepoxide, increase desensitization of the GABAAR (Frosch et al., 1992; Mierlak and Farb, 1988).
However, the increase in GABAAR desensitization by benzodiazepines could possibly result from an increase in agonist binding rather than a direct effect on channel gating. Therefore, the purpose of these experiments was to detemiine if benzodiazepines have an intrinsic effect on
GABAARdesensitization.
Currents recorded in the presence of a prolonged agonist application peaked then decayed to an apparent steady-state. In order to examine the effects of midazolam on desensitization of the GABAAR we calculated the Iss/Ip ratio for GABA (0.1-600 PM)-evoked cwents in the absence and presence of midazolam (0.5 PM, Figure 5). Midazolam increased the peak current evoked by subsaturating concentrations of GABA (0.1 - 60 PM) and decreased the IssAp ratio as the concentration of GABA increased. Midazolam did not significantly change the IssAp ratio at any concentration of GABA tested @ > 0.05, ANOVA, n = 11). The Iss/Ip ratios for GABA (0.6-
600 FM) and GABA + MDZ (0.5 PM) were plotted versus the concentration of GABA (Figure
5b). The EC,, and Hill coefficient for this relationship were not different for currents recorded in the absence or presence of midazolam (4.15 f 0.69 to 5.27 t 1.1 3 FM, and - 1.36 f 0.25 to - 1.34 k
0.18, respectively, p > 0.05, paired t-test). Therefore, although midazolam increased the amplitude of the currents, it did not significantly alter the extent of GABAARdesensitization. Figure 5
Midazolam did not influence desensituation of the GABA,%Rfor currents evoked by
GABA.
A. Responses evoked by 3 or 10 pM GABA, in the absence and presence of 0.5 piM
midazolam. peaked and then desensitized.
B. The dose response curved for the Iss/Ip ratios of GABA-evoked currents, in the
absence and presence of midazolarn, are shown. Midazolam did not change the Iss/Ip
value at any concentration of GABA (p > 0.05. ANOVA). In Addition. there was no
difference in the absence or presence of midazolarn for the EC,, values (4.15 2 0.69
uiM versus 5.27 2 1.13 FM . p > 0.05, paired t-test) or the Hill coefficients (-1.36 IT
0.25 versus -1.34 + 0.18. p > 0.05, paired t-test) in the absence and presence of
midazolam, respectively.
The effects of midazolam on desensitization of the GABAARby propofol-evoked cments were also examined. The IssAp ratio for propofol (1-600 PM)-evoked currents recorded in the absence or presence of midazolam (0.5 pM) are shown in Figure 6. The Iss/Ip ratios for propofol
(1-600 FM) and propofol + MDZ (0.5 PM) responses were plotted venus agonist concentration
(Figure 6b). Midazolam had no significant effect on the IssAp ratios at al1 concentrations of propofoi, (p > 0.05, ANOVA, n = 6). The Iss/Ip relationship was linear for the concentrations of propofol exarnined. The Iss/Ip concentration relationship was fit with a linear regression; the dopes were not different in the absence and presence of midazolarn (-0.285 t 0.008 to -0.275 i
0.006, p > 0.05, paired t-test), nor were the correlation coefficients (? values) different (0.95 +_
0.02 to 0.93 k 0.02, p > 0.05, paired t-test). Note that the Iss/Ip relationship is strikingly different than the sigrnoidal relationship observed with GABA-evoked currents. This could be due to a decrease in receptor desensitization in the presence of propofol compared to GABA. in summary, midazolarn enhanced currents activated by subsaturating concentrations of propofol, but did not alter the IssAp ratios significantly. These data suggest, midazolarn did not directly alter receptor desensitization.
4.1.3. PROPOFOL DECREASED GABAARDESENSITIZATION
Propofol decreases receptor desensitization and increases the steady-state current observed during prolonged applications of agonist. However, the effects of propofol on
GABAAR desensitization had not been fûlly characterized (ber et al., 1994). Here we investigated the effect of various concentrations of propofol (0.0 1- 100 PM) on currents evoked by GABA (600 FM) (Figure 7). A saturating concentration of GABA (600 PM) induced currents that peaked rapidly, then decayed to an apparent steady-state. When propofol (0.01-100 pM) Figure 6
Midazolam did not alter desensitization of propofol-evoked currents.
A. Responses evoked by 6 or 100 pM propofol. in the absence and presence of 0.5 pM
midazolarn peaked. and then desensitized.
B. The dose response curves for the Iss/Ip ratios of propofol-evoked currents in the
absence and presence of midazolam are show. Midazolam did not change the Iss/Ip
value at any concentration of propofol (p > 0.05. ANOVA). The propofol DRCs for
the WIp ratios were linear in the absence and presence of midazolarn. There was no
difference in the slopes in the absence and presence of midazolarn (-0.285 k 0.008
venus -0.775 + 0.006, p > 0.05, paired t-test, n = 6) or the $ values (0.95 - 0.02
versus 0.93 f 0.02, p > 0.05, paired t-test, n = 6).
Figure 7
Propofol decreased GABA,R desensitization in a concentration dependent manner
A. Responses to 600 pM GABA and increasing concentrations of propofol (1-20 PM)
are shown.
B. The Iss/Ip ratio decreased as the concentration of propofol increased from 0.01-20
PM, suggesting that propofol decreased GABAAR desensitization. The Iss/Ip ratios
were normalized to the Iss/Ip ratio at 10 pM propofol. The nurnber of cells tested at
each concentration of propofol is noted below the mean. The EC5,]of the propofol
DRC was 1.15+ 0.33 pM and the Hill coefficient was 1.14 2 0.13. When the
concentration of propofol was increased from 70 piM to 100 PM, there was an
decreased steady-state current. Thus, the IssiIp DRC for propofol was biphasic. The
decrease in GAEIA,R current observed at higher concentrations of propofol was
possibly due to channel blockade. rlGABA+PRO 1 pM \ '- GABA +PRO 6 pM GABA +PRO 10 pM 1 / GABA + PRO 20 pM
4 sec
GABA 0.0 1 o. 1 I 600 pM Propofol (PM) was CO-appliedin the presence of GABA, the amplitude of the steady-state current increased suggesting a decrease in receptor desensitization. The threshold concentration of propo fol which induced a change in the steady-state current was approxirnately 0.1 FM (Figure 7). The concentration of propofol was plotted versus the Isdp ratio. The Iss/Ip values were normalized to the maximal IssAp value. In the presence of 0.01 to 20 pM propofol the Isdp ratio increased in a dose-dependent manner. The relationship was biphasic as the Iss/Ip ratio decreased with concentrations of propofol greater than 20 @A. The EC, for the propofol DRC representing the decrease in GABAARdesensitization was 1.15 t 0.33 pM and the Hill coefficient was 1.14 f
O. 132 (Figure 7b).
We also investigated the effects of propofol on the rate of desensitization. The decay phase of the response was fitted using a biexponential equation. For cu~~entsillustrated in Figure
7a propofol did not influence the fast time constant (TJ but increased the slow time constant (rJ of the decay (Table la).
In addition, the deactivation of currents was slowed in the presence on propofol (Figure
7a). The decay of the response, observed following temination of agonist application, was fit with a biexponential equation. As summarized in Table lb, propofol increased the fast and slow time constant of deactivation in a dose dependent manner.
4.1.4. PROPOFOL-INDUCED MODULA TION OF GABAJ2 DESENSITIZA TION WAS
NOT INFLUENCED B Y MIDAZOLAM
In the previous section, we demonstrated that rnidazolam potentiated propofol-evoked currents but did not modulate GABAAR desensitization. We then wished to determine if midazolarn influenceci propofol induced changes in GABAAR desensitization in both Table 1
A
.Desensitization Lt(sec) %O,V (sec) GABA 600 PM 1 .O3 4.30 1 GABA 600 uM + PRO 1 DM 1.04 5.49 1 GABA 600 PM + PRO 6 pM 1.O7 8.36 GABA 600 pM + PRO 10 pM 1.04 9.30 GABA 600 uM + PRO 20 MM 1 .O8 8.56 1
Deactivation Th, (sec) rdow(sec) GABA 600 pM 0.232 2.456 GABA 600 PM + PRO 1 MM 0.383 2.593 1 GABA 600 uM + PRO 6 uM 0.52 1 2.785 1 GABA 600 pM + PRO 10 ph4 0.735 2.991 GkBA 600 PM + PRO 20 pM 0.766 3.273
Propofol decreased the rate of GABAARdeseasitization and siowed receptor deactivation in the presence of saturating concentrations of GMA.
A. The rate of receptor desensitization was determined for cuments in Figure 7. The fast and slow components were calculated using a biexponential fit. As the concentration of propofol increased there was linle change in the fast time constant however, there was dose-dependent enhancement of the slow time constant. B. The rate of deactivation for currents in Figure 7. There was a dose dependent increase in both the fast and slow time constants for deactivation. hippocampal and spinal cord neurons. Further, the effect of midazolam was examined at 3 di fferent concentrations of propofol.
ïhe IssAp ratios were calculated for currents evoked by 600 pM GABA, 600 pM GABA and 0.5 pM midazolam (GABA + MDZ), 600 FM GABA and 10 pM propofol (GABA + PRO), and GABA, propofol and midazolarn (GABA + PRO + MDZ) in hippocampal neurons. As previously described, midazolam did not alter the steady-state current evoked by 600 pM GABA
(Figure 8a). The IsslIp ratios for currents evoked by GABA and GABA + MDZ were 0.14 k
0.01 and 0.13 t 0.01, respectively @ > 0.05, ANOVA, n = 8, Figure 8b). When 10 pM propofol was CO-appliedwith GABA, the IssAp ratio increased nom 0.14 t 0.01 to 0.20 i 0.02 @ c 0.05,
ANOVA). However, when midazolam was CO-appliedwith propofol and GABA, the IsslIp ratio was not significantly different fiom that observed in the presence of propofol and GABA (0.19 f
0.01 versus 0.20 + 0.02, p > 0.05, ANOVA). Hence, midazolam did not alter the effects of 10 pM propofol on GABAARdesensitization (600 pM GABA).
in spinal cord neurons, the Isdp ratios for currents activated by 600 pM GABA, GABA
+ 0.5 pM MDZ, GABA + 10 pM PRO or GABA + PRO + MDZ were 0.16 f 0.02, 0.16 + 0.02,
0.21 f 0.02 and 0.21 + 0.02, respectively (n = 7; Figure 8 c,d). The IsslIp values by GABA and
GABA + MDZ evoked responses were significantly different fiom those evoked by GABA +
PRO and GABA + PRO + MDZ @ c 0.05, ANOVA). Notably, the conclusions were not different between hippocampal and spinal cord neurons.
To ensure that the lack of effect of midazolam on propofol-modulated currents was not due to the relatively high concentrations of propofol, propofol was decreased fiom 10 FM to 1 or
6 pM and tested in hippocampal neurons. In the presence of lower concentrations of propofol(1 Figure 8
Midazolam did not effect propofol-induced modulation of GABAARdesensitization.
A,C. Responses evoked in hippocampal and spinal cord neurons by 600 FM GABA in the presence of 0.5 pM midazolarn, 10 pM propofol, or midazolam plus propofol are shown.
B,D. The bar graphs illustrate mean Iss/Ip ratios calculated for currents evoked by
GABA. GABA A MDZ. GABA + PRO and GABA + PRO + MD2 in hippocampal (n =
S) and Spinal cord neurons (n = 5). The WIp ratios for currents evoked by GABA and
GABA - MDZ were different from those of GABA + PRO and GABA -iPRO - MDZ in both hippocampal neurons and spinal cord neurons (p<0.05,repeated measures ANOVA. n= 8 and 5, respectively).. or 6 PM), midazolam did not influence the extent of GABAAR desensitization (Figure 9).
Therefore, midazolarn did not modulate GABAARdesensitization, nor did it effect the ability of propo fol to decrease GABAARdesensitization.
Despite the lack of effect that rnidazolarn has on the extent of receptor desensitization, midazolarn slowed the rate of current deactivation in the presence of propofol. The rate of deactivation for the currents in Figure 9 were fit with a biexponential equation. Midazolam increased the fast and slow time constants for both concentrations of propofol (Table 2). Figure 9
Midazolam did not influence GABAAR desensitization at lower concentrations of propofol.
A. Responses evoked by 600 FM GABA, in the presence of propofol (1 FM), or
propofol plus rnidazolarn (0.5 FM), were recorded from hippocampal neurons.
B. Responses evoked by 600 pM GABA. in the presence of propofol (6 FM). or
propofol plus midazolarn were recorded From hippocampal neurons. also indicate that
rnidazolarn does not modulate receptor desensitization regardless of the dose of
propo fol. PRO 1 pM PRO 6 pM
GABA GABA+PRO GABA+PRO+MDZ Table 2
Deac tivation Sy,t (sec) 5,hW (sec) GABA 600 pM 0.232 2.456 ------
GABA 600 PM + PRO 1 FM 0.383 2.593 1 GABA 600 MM+ PRO 1 IJM + MDZ 0.5 uM 0.475 2.773 1
1 GABA 600 pM + PRO 6 pM ( GABA 600 pM + PRO 6 pM + MDZ 0.5 MM 0.797 4.337 1
The rate of current deactivation was decreased by midazolam
The deactivation rates f?om currents in Figure 9 were fit with a bi-exponential equation. The fast and slow time constant were increased in the presence of propofol and they were further increased in the presence of midazolarn and propofol. 4.2. SECTION 2
4.2.1. EFFECTS OF MIDAZOLAM AND PROPOFOL ON CURRENT EVOKED BY
SUBSA TURA TING CONCENTRA TIONS OF GABA
The purpose of these experiments was to determine if propofol and rnidazolarn enhanced currents evoked by subsaturating concentrations of GABA in an additive, superadditive or subadditive manner. Two models were employed; a Fixed-Dose Model and Isobolographic
Analysis.
Using the Fixed-Dose Model, we examined the effit of each dmg aione and the combination of propofol and midamlam. The calculated additive response was then compared to the effect of the dmgs in combination. To determine if the interactions were influenced by ce11 type we recorded responses from hippocampal and spinal cord neurons for GABA (3 PM), propofol (10 PM) and rnidazolarn (0.5 FM). Expenments were also conducted using different concentrations of propofol (10 and 1 PM) and GABA (3 and 0.3 pM) in hippocampal neurons.
These concentrations were previously reported by Reynolds et al. (1996) to produce a superadditive enhancement of GABA-evoked responses recorded fiom oocytes expressing hurnan a 1B2y2L and a2P2y2L subunits.
In cultured hippocampal neurons, PRO (10 FM), and MDZ (0.5 PM), enhanced GABA- evoked currents (3 PM) recorded fiom hippocampal neurons (Figure Na). The currents potentiated by MD2 andor PRO were less than the maximal current generated by 600 pM
GABA. GABA-activated currents were enhanced by MDZ, PRO and PRO + MDZ by a factor of
1-45 + 0.21, 3.44 2 0.41 and 4.15 f 0.54, (n = 8; Figure lob). There were no significant differences between currents enhanced by PRO + MDZ and the theoretical additive response @ > Figure 10
Propofol and midazolam (or flurazepam) produced an additive enbancement of
GABA (3 PM)-evoked currents from hippocarnpal neurons.
A,D. Responses evoked by combinations of 3 pM GABA, 10 pM propofol, and 0.5 pM midazolarn (or flurazepam) were srnaller than the responses evoked by 600 pM GABA in hippocampal neurons.
B,E. Currents were normalized to the peak response evoked by 3 piM GABA. There was no difference between the measured response to PRO + MDZ (n = 8) (or FLü (n = 7)) and the theoretical additive values (p > 0.05, Student's t-test).
C,F. The charge transfer was calcuiated for al1 combinations of dmgs and normalized to the value calculated for 3 pM GABA. There was no difference between the measured
PRO + MDZ (or FLU) response and the theoretical additive value for charge transfer (p >
0.05. Student's t-test).
0.05, Students t-test). Similar results were obtained when another benzodiazepine, flurazepam
(FLU,0.5 FM), was substituted for midazolam. GABA-activated currents were enhanced by
FLU,PRO and PRO + FLU by a factor of 1.26 k 0.06,2.49 f 0.36, and 3.24 f 0.56, respectively
(n = 7; Figure 10 d,e). This suggested that the additive interaction was not influenced by the benzodiazepine being examined.
In addition to examining the effects of propofol and midazolam on peak currents, we also measured the total current recorded during the application of agonist. Charge transfer was measured by integrating the area under the current (see methods). Charge transfer was enhanced by MDZ, PRO or MD2 + PRO by a factor of 1.83 t 0.26, 3.99 + 1.08, and 5.04 + 1.75 (n = 8;
Figure 10c). The enhancement of charge transfer by FLU, PRO or PRO + FLU was 1.30 t 0.07,
2.20 + 0.31 and 2.83 k 0.39, respectively (n = 7; Figure [Of). Again, the theoretical additive enhancement of charge transfer was not significantly different than the enhancement for MD2
(or FLU) + PRO modulated currents @ > 0.05, Students t-test). Therefore, the interaction between propofol and midazolam or flurazepam, at these concentrations, was additive.
The W relationship for GABA-evoked cunents potentiated by MDZ and MD2 + PRO were examined (Figure 11). The reversal potential of MD2 and MDZ + PRO potentiation of
GABA-evoked currents was approximately -6 mV. The experimental reversa1 potential was close to the reversai potential caiculated according to the Nernst equation (+1 mV) suggesting the currents are mediated by Cl' ions.
The extent of potentiation of GABA-evoked currents by drugs is dependent on both the concentration of GABA and anaesthetic (Harris et al., 1995). Therefore, the interaction between propofol and rnidazolam was also examined at a lower concentration of propofol (1 FM). MDZ Figure 1 1
Midazolam and propofol did not alter the reversal potential of the G.4BA-evoked currents.
GABA (3 PM) + MRZ (0.5 PM) and GABA + iMDZ + PRO (IO PM) was applied to neurons voltage clamped at holding potentials ranging fiom -60 mV to +10 mVs. Current reversed polarity at approximateiy -6 mV. Al1 currents were normalized to currents evoked at -60 mV (n = 3).
(0.5 PM), PRO (1 pM) or PRO + MDZ enhanced peak currents evoked by 3 pM GABA in hippocampal neurons by a factor of 1.62 f 0.40, 1.89 I0.60 and 2.72 f 1.08, respectively (n = 5;
Figure 12). Enhancement of currents by propofol and rnidazolarn was additive @ > 0.05,
Students t-test).
When the concentration of GABA was reduced from 3 pM to 0.3 FM, MDZ (0.5 FM),
PRO (1 FM), or MDZ + PRO enhanced cuments by a factor of 3.54 + 0.39,4.93 t 0.80 and 14.73 k 3.03, respectively (n = 4; Figure 13). Enhancernent by propofol and rnidazolarn of currents evoked by 0.3 pM GABA was superadditive when compared to the theoretical additive response
@ c 0.05, Students t-test).
To determine if the interactions were influenced by the anatomical source of the neurons, the interactions between 10 pM propofol and 0.5 FM midazolam or 0.5 pM flurazepam for currents evoked by 3 pM GABA were also examined in spinal cord neurons. The peak curent was enhanced by MDZ, PRO or PRO + MDZ by a factor of 2.28 I0.24,4.54 t 1.01 and 6.05 k
1.24, respectively (n = 7; Figure 144b). Currents were potentiated by FLU, PRO or PRO + FLU by a factor of 1.66 ?r 0.10, 2.53 + 0.27,and 3.53 k 0.68, respectively (n = 7; Figure 14d, e). The currents evoked by PRO + MDZ (or FLU) were not different nom the theoretical additive value
@ > 0.05, Students t-test) indicating that the interaction was additive not superadditive.
Additionaily, the same trends were seen with the charge transfer (Figure 14c, f).
These data indicate interactions between propofol and benzodiazepines depend on the concentrations of GABA and propofol. At higher concentrations of propofol and GABA, an additive enhancement between propofol and a benzodiazepine was observed in hippocampal and Figure 1 2
Additive interactions when the concentration of propofol was reduced.
4- Responses evoked by 3 u5I GM3.A . 0.5 .di1 midazolam and 1 u41 propofol are
shoun.
B. There \vas no difference bew-een the measured response by propofol and rnidazolam
compared to the rheore
Figure 13
Propofol and midazolam produced a superadditive enhancement of GABA (0.3 piM)-evoked currents when the concentration of GIIBG was reduced.
A. Responses evoked by 0.3 pM GABA, GABA + 0.5 pM MDZ. GABA + 1piM PRO.
and GABA - PRO + MDZ were smaller than the currents evoked by a saturaring
concentration of GABA (600 PM).
B. The potentiated current evoked by MDZ, PRO and MDZ + PRO were normalized to
the peak GABA (0.3 FM)-evoked current. The theoretical additive potentiation was
less than the potentiation of midazolam and propofol evoked currents. indicating a
superaddi tive interaction between propo fol and midazolarn (p c 0.05. Student's t-test.
n = 4).
Figure 14
Propofol and rnidazolarn produced an additive enhancement of GABA (3 piCl)- evoked currents in spinal cord neurons.
A,D. Responses evoked by combinations of 3 FM GABA. 10 pM propofol, and 0.5 pM rnidazolarn (or flurazepam) were smaller than the responses evoked by 600 PM GABA.
B,E. The current peaks for MDZ or FLU. PRO. MDZ or FLU + PRO potentiation of
GABA (3 FM) currents were normalized to the 3 pM GABA response. There was no difference benveen the potentiation by PRO + MDZ (or FLU) and the theoretical additive values (p > 0.05. Student's t-test. n = 7 each).
C,F. The charge transfer was measured for a11 combinations of dmgs and normalized to the charge transfer measured from the current evoked by 3 pM GABA. There was no difference between the enhancement of PRO + MDZ (or FLü) and the theoretical additive value (p > 0.05, Student's t-test).
spinal cord neurons. However when the concentraiions of propofol and GABA were reduced the
interactions between propofol and midazolam were superadditive.
4.2.2. SMYERGISTIC INTElRACTION BE'lW'EEN PROPOFOL AND MIDAZOLAM IN
THE PRESENCE OF LOW CONCENTRATIONS OF GABA
Isobolographic Analysis was another mode1 used to determine if hginteractions were
synergistic. In the previous section, we reported a superadditive interaction in the presence of 1
PM propofol, 0.5 pM midazolam and 0.3 FM GABA. GABA 0.3 FM did not produce a
consistent response that could be analyzed, therefore, the concentration was increased to 1 FM.
The current produced by 1 pM GABA was used as the control response for isobolographic
analysis. Here we chose an effect level that represented enhancement of the GABA-evoked
response by a factor of 3 (200% increase). First, the concentration of rnidazolam that potentiated
the 1 pM GABA-evoked current by a factor of 3 was determined. This concentration of
midazolam was estimated as illustrated in Figure 15. We then determined the concentration of
propofol that potentiated the 1 pM GABA-evoked response by a factor of 3 (Figure 16). Finally, we selected a fixed concentration of midazolarn and determined the dose of propofol that produced the effect Ievel.
Midazolam produced a concentration-dependent increase in the amplitude of the GABA current (1 PM; Figure 15). The EC,, for the midazolarn potentiation was 0.102 k 0.034 pM and the Hill coefficient was 1.1 82 t- 0.182. Midazolam maximally potentiated the GABA-evoked response by a factor of 4.25 + 0.55 (rH;Figure 1%). The concentration of midazolam that potentiated GABA-evoked responses by a factor of 3 was 0.182 f 0.076 pM(n = 7; Figure 15b). Figure 15 hlidazolam potentiation of the GABA (1 PM)-evoked response.
A. Responses evoked by 1 pM GABA were potentiated by increasing concentrations of
midazolarn (0.1 pM to 6 PM).
B. Midazolarn dose-dependentl y potentiated GAE3 A-evoked currents. The ECI0 and Hill
coefficient For midazolarn potentiation were 0.107 ? 0.034 pM and 1.182 k 0.182.
respectively (n=9). Midazolam maxirnally potentiated GAE3A-evoked currents by a
factor of 4.75 k 0.55. The concentration of midazolam that produced the 3 times
effect level, in the presence of 1 pM GABA. was 0.182 2 0.076 piM (n = 7). The
dashed Iine on the DRC indicated the dose that produced the 3 times effect level.
Figure 16
Propofol potentiation of the GABA (lph.1)-evoked response.
A. Responses evoked by I pM GABA were potentiated by increasing concentrations of
propofol (0.1 pM to 100 PM).
B. Propofol dose-dependently potentiated GABA-evoked currents. The EC,, and Hill
coefficient for propofol potentiation were 7.21k1.34 pM and 1 2720.15. respectively
(n=9). Propofol rnaxirnally potentiated the GABA-evoked response by a factor of
12-64+ 3.41. The concentration of propofol that potentiated the GAf3A-evoked
response by a factor of 3 was 2.12 + 0.43 pM (n = 9). The dashed Iine on the DRC
indicates the dose that produced a 3 times effect Ievel. in two of the nine cells we did not observe potentiation of the GABA-evoked response greater than a factor of 3. The maximal increase in these cells was 2.25 and 2.37.
Propofol also produced a concentration-dependent increase in the response to GAE3A (1
PM). The EC, for propofol potentiation was 7.12 + 1.34 pM and the Hill coefficient was 1.27 t
0.15. Propofol (100 PM) maximally potentiated the response of 1 pM GABA by a factor of
12-64 + 3.41 (n = 9) (Figure 16b). The concentration of propofol that potentiated the GABA- evoked response by a factor of 3 was 2.42 t 0.43 pM (n = 9; Figure 16b).
The mean concentration of propofol and midazolarn that resulted in a 3 times effect level was plotted on the Y and X mis, respectively. These doses were joined to form the isobol of additivity (Figure 18).
The third dose response cwewas generated in order to complete the isobolograrn. A concentration of midazolam that produced an effect level consistently less than a factor of 3 was selected (0.06 FM). The concentration of propofol that produced the effect level in the presence of rnidazolam (0.06 PM) was determined (Figure 17a). The DRC for propofol enhancement of
GABA-evoked currents was shifted to the left in the presence of midazolam (Figure 1%).
Individual doses of propofol potentiation of the GABA-evoked response by a factor of 3 were plotted on the isobolograrn. These concentrations were below the isobol of additivity (Figure
18). The mean concentration of propofol that produced the effect level in the presence of MDZ
(0.06 FM) was 0.625 + 0.200 pM (n = 9).
The theoretical additive concentration of propofol that resulted in 3 times enhancement of the GABA-evoked respowe in the presence of 0.06 FM midazolam was 1.628 t 1.220 FM. The experimental concentration of propofol was significantly lower than the theoretical concentration Figure 17
Propofol enhancement of currents evoked by CO-application of midazolam and
GAB.4.
A. Raw traces evoked by 1 FM GABA in the presence of 0.06 pM midazolarn and
varying concentrations of propofol(0.06 pM to 2 PM) are shown.
B. Propofol potentiated GABA-evoked currents in a dose-dependent manner as
previously shown in Figure 16b (square, thick line). Addition of a fixed dose of
midazolam (0.06 p) resulted in a shifi to the lef3 of the DRC for propofol potentiation
of GABA (1 @)-evoked responses (triangle. thin line). The concentration of
propofol that potentiated the GABA-evoked response in the presence of midazolam
by a factor of 3 was 0.625 2 0.200 pM (n = 9). The dashed line on the DRC
represents the concentration of propofol that produced a 3 times effect level. GABA + MDZ 0.06 pM
GABA + MDZ +PRO 0.3 pM GABA + MDZ + PRO 0.6 pM
GABA + MDZ + PRO 1 pM
+PRO pM 200 pA GABA+ MDZ 2
I 2 sec
Propofol (PM) Figure 18
Synergistic in teractions between propofol and midazolam.
The concentration of propofol that resulted in a 3 times effect level compared to the
GABA IpiM response was plotted on the Y-êxis (diamond). The concentration of midazolarn that resulted in a 3 times effect level was ploaed on the X-auis (square). The isobol of additivity is shown as a solid line drawn between the aforementioned concentrations. The doses of propofol, in the presence of 0.06 pM midazolarn, that enhanced the GMA-evoked response by a factor of 3 al1 lie below the isobol of additivity (circles with dots in centre, n = 9). The average concentration of propofol. in the presence ofmidazolam, that produced the 3 timed effect level was 0.624 It 0.700 piM
(solid circle). indicating the interaction between propofol and rnidazolam was synergistic @ < 0.05, Students t- test).
In sumniary, the interactions between midazolam (0.06 FM) and propofol (0.625 pM) interacted synergistically to enhance GABA (1 FM)-evoked currents. 5. DISCUSSION
5.1. SECTION 1
Midazolam, 1) potentiated GABA-evoked currents, 2) potentiated propofol-evoked currents and 3) failed to influence GABAAR desensitization. Moreover, midazolam did not influence propofol-induced changes in GABAAR desensitization. These results suggest activation and desensitization of the GABAARare distinct processes that can be differentially modulated by anaesthetics.
5.1.1. MIDAZOLAM POTENTIATION OF GABA-EVOKED CURRENTS
Midazolam potentiated currents evoked by subsaturating concentrations of GABA and caused a shift to the left of the GABA DRC. These data indicate rnidazolarn increased the afKnity of the GABAARfor GABA.
Radiolabelling studies have suggested a functional coupling between the benzodiazepine and GABA binding site: GABA and benzodiazepine agonists reciprocally increase the affinity of the GABAAR for the other agent (Costa and Guidotti, 1979; Braestrup and Neilson, 1981).
Electrophysiological studies also indicated benzodiazepines, including diazepam and chlordiazepoxide, increased the afhity of the GABAARfor GABA by shifting the GABA DRC to the left (Choi et al., 198 1; Study and Barker, 198 1). Single channel studies have shown that diazepam enhanced the macroscopic current in part by increasing the £kequency of GABA, charme1 opening not by altering channel open time or butkinetics (Rogers et al., 1994).
More recently, Lavoie and Twyman (1996) investigated the effects of diazepam on
GABA binding and channel gating of a2ply2 receptors expressed in HEK 293 cells. Diazepam increased the rate of onset of currents evoked by low, but not saturating concentrations of GABA (Lavoie and Twyman, 1996). The rates of current decay afler bnef applications of agonis were not afTected by diazepam suggesting that benzodiazepines had a specific effect on ligand binding rather than channel gating (Lavoie and Twyrnan, 1996). Using cornputer modeling, it was suggested that benzodiazepines positively modulate GABA-mediated currents by accelerating the rate of GABA association to the receptor not by directly effecting charme1 gating (Lavoie and
Twyman, 1996). These results are in agreement with our data: midazolarn increased the affinity of the GABAARfor GABA but did not alter GABAARdesensitization.
5.1.2. MIDAZOLAM POTENTIATION OF PROPOFOL-EVOKED CURRENTS
Propofol directly activated the GABAAR in a dose-dependent rnanner as previously observed (Orser et al., 1994; Hales and Lambert, 1991; Hara et al., 1993; Adodra and Hales,
1995; Jones et al., 1995; Sanna et al., 1995a,b). However, the EC, for propofol-activated currents (24.3 PM) was less than that previously reported by Orser et al. (1994) (EC, = 61 FM) for cultured embryonic murine hippocampal neurons but was greater than that reported by Hara et al. (1993) for propofol activated currents (EC,, = 12 PM) recorded from acutely dissociated rat hippocampal CA1 region neurons. The differences in the EC,, values possibly result Eom the time between propofol applications or the GABAARsubunits present in different ce11 types. If the rate of propofol application was too rapid, GABAARswould not recover fiom a desensitized state resulting in a reduction in the peak amplitude if the subsequent application. Orser et al.
(1994) determined that it took approximately 2 minutes for recepton to recover from desensitization after a saturating concentration of GABA was applied. We waited at lest 2 to 4 minutes between appiications of propofol to ensure the GABA, recepton had hlly recovered
From the desensitized state. The difference between our value and that previously reported by Hara et al. (1993) could be due to a difference in subunit combination. Ham et al. (1993), studied currents recorded fiom acutely dissociated neurons fiom the hippocampal CA1 region fiom 1- to 2-week-old rats. Numerous snidies have demonstrated significant alterations in subunit expression in early development. Expression of mRNAs encoding the GABAARschange during development resulting in an alteration of the huictional properties of the channel (Griffith and Murchinsom, 1995; Zang et al., 1992; Gambarana et al., 1991). Hara et al., (1993) were using cells that had developed in the animal longer which may have resulted in a different complement of receptors. Further, it has been shown that the complement of GABAARsubunits in corncal neurons change over the when in culture (Hu and Ticku, 1994). These changes rnay parallel the changes that occur in the brain with maturation, but this has not been investigated.
Additionally, Hara et al. (1993) used neurons fkom the hippocampal CA1 region, whereas we used neurons obtained nom the entire hippocampus. Receptor composition is important because receptoa have different affinities for drugs depending on the subunits expressed (Jones et al., i 995).
The EC,, value likely underestimates the tme afinity of the receptor site for propofol since the slow onset of propofol action allows significant desensitization to occur before the peak effect is attained. Notably, desensitization affects the responses evoked by higher concentrations of agonist compared to lower agonist concentrations (Pemefather and Quastel, 1982).
Propofol is thought to directly activate the receptor by binding to a site distinct from that of GABA. Electrophysio logical studies indicated the P subunit was essential for propofol- induced activation of the GABAAR(Sanna et al., 1995a). Receptors composed alyb subunits were activated by GABA but not by propofol, which emphasized the importance of the P subunit for the direct actions of propofol on the GABAAR. pl homomenc receptors were the rnost sensitive to propofol. When other subunits (al, a2, as) were added to the recombinant receptor a reduction in the amplitude of propofol-evoked cments was observed (Sanna et al., 1995b).
Perhaps the propofol binding site on the P subunit was occluded by the a subunits or that the affinity of the propofol site was rnarkedly reduced in the presence of the a subunit.
in addition to the site for propofol on the BI subunit, the site responsible for the direct actions of barbiturates was present on the Pl subunit. Amin and Weiss (1993) determined there were two domains on the P2 subunit essential for receptor activation by GABA. Each dornain contributed a tyrosine and a threonine amino acid necessary for GABA binding. When these sites were mutated conservatively (tyrosine to phenylalanine; threonine to serine), there was a decrease in the recepton sensitivity to GABA, however, the mutations did not impair activation by pentobarbital. When the sites were mutated in a non-conservative manner (tyrosine to senne or asparagine), GABA, but not pentobarbital, failed to activate the channel. These data provided direct evidence that GABA and pentobarbital activate the GABAARby binding to ciifferent sites.
Binding and electrophysiological studies suggest that propofol and GABA bind to distinct sites on the GABAAR. Propofol enhanced 'H-GABA binding and muscimol stimutated
36Cl-uptake measured in brain membrane preparations (Concas et al., 1991). One would expect that propofol would decrease, rather than enhance 'H-GABA binding if propofol and GABA were competing for the sarne site. However, the propofol site is likely close to, or similar in stmcture to, the GABA site. Propofol-activated currents were partially inhibited by bicuculline
(Hara et al., 1993). Bicuculline also inhibited the propofol-induced reduction of "s-TBPS binding (Concas et al., 1991). These studies suggest the sites responsible for propofol and GABA activation of the GABA,R are similar but distinct.
The propofol binding site is also distinct fiom the benzodiazepine site. Flurnazenil did not effect "S-TBPS inhibition by propofol (Concas et ai., 1992), nor did propofol displace 'H- flunitrazepam from cerebral synaptosomes (Peduto et al., 1991). Further, flurnazenil did not modulate propofol-evoked currents (Sanna et al., 1995b; Hara et al., 1993). However, Prince and
Simmonds (1992) found that propofol stirnulated 'H-flunitrazepam binding to rat membranes.
Here, we also showed midazolam increased the affinity of the GABAAR for propofol. In combination with our observations, these data suggest a reciprocal interaction between propofol and midazolam binding.
In sumrnary, the propofol binding site on the GABAARis thought to be distinct fiom that of GABA and the benzodiazepine site. However, the GABA, benzodiazepine and propofol site are functionally coupled.
5.1.3. PROPOFOL BLOCKADE OF THE GABAAR
High concentrations of propofol (- 600 FM) reduced the peak current and produced a transient after-response (Orser et al., 1994; Adodra and Hales, 1995). Similar responses have been observed with hi& concentrations of other anaesthetics, including pentobarbital, alphaxalone and etornidate (Akaikie et al., 1986; Robertson 1987; Ikemoto et ai., 1988).
GABAAR inhibition by propofol was aIso observed in binding studies: low concentrations of propofol (3-10FM) enhanced "S-TBPS binding, whereas high concentrations of propofol (50-
100 FM) inhbited "S-TBPS binding (Concas et al., 1994). A blocking mechanism has been proposed to account for the b'afler-response" observed in the presence of high co~centrationsof anaesthetics (Oner et aï., 1994; Adodra and Hales, 1995).
The onset of the block and the appearance of the after response were too rapid to represent the onset and recovery from propofol-evoked desensitization suggesting that receptor desensitization is not likely the mechanism of the block (Orser et al., 1994).
Here we reported that midazolam did not enhance the peak response to maximal concentrations of propofol but potentiated the amplitude of the after response. This observation is consistent with a blocking mechanism.
5.1.4. MIDAZOLAM DID NOT INFLUENCE DESENSITIZATION OF THE GABAAR
Midazolarn did not influence the amplitude of the steady-state current evoked by saturating concentrations of propofol or GABA. Additionally, midazolam did not influence the
IssAp relationship measured at various concentrations of GABA and propofol. We interpret these results to indicate that midazolam does not influence GABAARdesensitization.
Previously, it was reported that benzodiazepines, such as midazolam and diazepam. increase the rate of receptor desensitization (Frosch et al., 1992; Mierlak and Farb, 1988; Farrant et al., 1990). However, in these studies subsahirating concentrations of GABA were employed.
Midazolarn and diazeparn increased agonist binding and the predicted increase in the peak current was not considered. When the enhancement of the peak response by diazepam was taken into account, the change in desensitization by diazepam was no longer apparent (Orser et al.,
1994).
Lavoie and Twyman (1996) also exarnined the effect of diazepam on desensitization kinetics using fast applications of GABA. They suggest that diazepam accelerated the apparent entry into a desensitized state by increasing the rate of association of GABA to the receptor. As desensitization likely proceeds from the fully ligand-bound state, the receptor is accelerated into the desensitized state in the presence of diazeparn (Lavoie and Twyman, 1996; Jones and
Westbrook, 1995). In summary, benzodiazepines do not alter the rate of desensitization which agrees with our data.
There are limitations to rneasuring the extent of desensitization. Here, the application of the agonist was for 18 seconds and for subsaturating concentrations of GABA and propofol this may have not allowed the recepton to achieve a true steady state current. However, it appean that midazolam does not influence desensitization. To Merdetermine if midazolam effects desensitization, subsaturating concentrations of GABA could be potentiated by midazolam, then a concentration of GABA that evoked the same peak current could be deterrnined and the rate and extent of desensitization compared.
5.1 -5. PROPOFOL MODULATION OF GABAARDESENSITIZATION
Orser et al. (1994) previously reported that propofol decreased GABA,R desensitization, however, this effect was not hilly characterized. Here we demonstrate that propofol dose- dependently increased the steady-state currents observed in the presence of saturating concentrations of GABA. In addition, propofol increased the slow time constant for desensitization. The propofol DRC for the Iss/Ip ratio was biphasic with an EC, of 1.15 PM.
Orser et al. (1994) also showed that propofol increases the second theconstant for the decay of currents hgprolonged agonist application.
In addition to propofol decreasing GABAARdesensitization there was also a decrease in receptor deactivation. We demonstrated that propofol increased both the slow and fast time constants associated with deactivation in a dose-dependent manner. This agrees with our results that propofol and midazolarn increase the affinity of the GABAARfor GABA.
It should be noted that the EC,, for propofol-induced changes in the steady-state current was significantly lower than for propofol-induced activation of the GABAAR(EC,, = 24.3 FM), or propofohnduced potentiation of GABA-evoked currents (EC, = 7.12 PM) By cornparhg the potentiating and direct action of propofol on numerous subunit combinations, it has been shown that there are distinct binding sites that mediate these two effects (Sanna et al., 1995b).
However, it is not known if there is a distinct site that modulates desensitization. This could be investigated by determining if activation and desensitization of the GABA,R are subunit specific.
Midazolam did not influence the peak or the steady-state currents evoked by varying concentrations of propofol in the presence of saturating concentrations of GABA. However, midazolam decreased the rate of deactivation in the presence of propofol. These observations are consistent with the hypothesis that midazolam increased the affinity of the receptor for GABA and propofol; midazolam increased the affinity of the receptor for agonist thereby increasing the probability of the agonist rebinding (or decreased unbinding) during hgwashout. Our results are consistent with those of Lavoie and Twyman (1996) who observed midazolarn prolonged deactivation of GABA evoked-currents. These results indicate that rnidazolarn influenced activation.
In summary, we demonstrated that rnidazolarn potentiated GABA- and propofol-activated currents, However, midazolarn did not alter GABAARdesensitization. In contrat, propofol dose dependently decreased GABA,R desensitization. Therefore, activation and desensitization of the 96
GABA,4Rare two distinct processes that cm be differentially modulated by anaesthetic dmgs. 5.2. SECTION 2
5.2.1. PHARMACOLOGICAL SYNERGISM BETWEEN PROPOFOL AND
MIDAZOLAM
Here we report that the interactions between benzodiazepines and propofol is influenced by the concentration of the anaesthetic or GABA. The interaction between propofol (10 or 1
FM) and midazolam (0.5 FM) for the enhancement of GABA (3 FM)-evoked responses was
additive. These data support binding studies which revealed an additive enhancement of 'H-
GABA binding and muscimol-stimulated "Cl' uptake by propofol (0.3-3 PM) in the presence of diazepam (0.3 FM)(Concas et al., 1991).
When the concentrations of GABA and propofol were reduced (GABA to 0.3 pM; propofol to 1 FM), a superadditive interaction between midazolam and propofol potentiation of
GABA-evoked responses was observed. Previously, Harris et al. (1 995) demonstrated that potentiation of GABA-evoked currents by anaesthetics was dependent on the dose of GABA. A greater potentiation was observed at Iowa concentrations of GABA. Here we demonstrated that a reduction in the concentration of GABA revealed a superadditive interaction between propofol and midazolam.
We subsequently used Isobolographic Analysis and demonstrated that midazolam (0.06
FM) and propofol (0.625 PM) interact in a synergistic rnanner to potentiate the GABA (1 FM)- evoked response by a factor of 3. Al1 the concentrations of propofol in the presence of midazolam to potentiate GABA by a factor of 3 were below the isobol of additivity. In addition, the mean dose of propofol (0.625 FM) was significantly lower than the calculated theoretically additive concentration indicating a synergistic interaction between propofol and midazolam at these concentrations. Our results agree with those of Reynolds et al. (1996) who reported that the interactions between flurazepam and propo fo 1 were synergistic when exarnined in Xenopus oocytes expressing human recombinant a 1P272L and a2P2y2L receptor constnicts.
An additive interaction can be explained as a summation of the individual effects. We speculate that at low concentrations of GABA, direct activation of the receptor by propofol contributes considerably to the current and this effect is potentiated by rnidazolam. However, at higher concentrations of GABA, direct activation by propofol is occluded and an additive effect is only observed.
Physiologically, synergism observed at lower concentrations of GABA could be important for influencing the tail current of the IPSC where the concentration of GABA is decreased. Anaesthetics have been shown to influence the second time constant of the IPSC and there would be lower concentrations of GABA present at that time.
5.2.2. CLINICAL SYMCRGISM BETWEEN PROPOFOL AND MIDAZOLAM
Midazolarn is a short-acting bernodiatepine used prior to the induction of general anaesthesia for its sedative, anxiolytic and amnestic properties (Reves, 1985), Midazolarn is also used for "CO-inductionof anesthesia" to accelerate the onset of hypnosis and to reduce the dose requirernents of other dmgs used to induce anesthesia (Vinik, 1993; Solomon et al., 1994). The therapeutic advantage of CO-induction is based on the principle of synergism: drugs interact synergistically when the effect produced in combination of the two dmgs is greater than the surn of the effects produced alone (Tallarida, 1992). By using dmgs of two different phmacological classes, the dose of each dnig can be reduced so that side effects are minimized. Clinical studies indicate that the combinations of propofol and midazolarn (Short and Chui, 1991, Short et al., 1992, McClune et al., 1992) have a synergistic interaction with respect to their ability to produce hypnosis. Ln these studies, hypnosis was defined as the inability to open eyes in response to a verbal command, or the loss of the lid reflex. In contrast to hypnotic effect, synergism between midazolam and propofol has not been demonstrated for anaesthesia which is defined as no movement in response to a surgical stimulus. Indeed, hypnosis and surgicd anaesthesia are likely two distinct clinicai effects that are mediated by different mechanisms.
Schultz and Macdonald (1991) suggested that direct activation of the GABAARis the mechanisrn by which anaesthetics induce general anaesthesia. Whereas GABA potentiation might be responsible for the "subtle" effects of anxiolytics anticonvulsants and hypnosis.
Propofol, neurosteroids and barbiturates are potent anaesthetics which can directly activate the
GABAAR whereas benzodiazepine agonists potentiate GABA-evoked current, do not directly activate the receptor and they are sedative hypnotics not analgesics.
This agrees with our results where we observed synergistic interactions at lower concentrations of the anaesthetics. In clinical studies they found synergistic interactions for the induction of hypnosis which requires lower concentrations of anaesthetics. Whereas when then concentrations of the anaesthetics were increased the interaction was additive. This agrees with the clinical observation that an additive interaction between propofol and midazolam has not been observed for the induction of anesthesia which requires higher doses of anaesthetics.
The blood concentrations of propofol that induces hypnosis in humans ranges from 1.5 to
6 pg/ml (8 - 34 PM) and recovery £kom anaesthesia occurs when serurn concentrations are less than approxirnately 6 pM (Kanto, 1988; Jensen et al., 1994). These concentrations of propofol modulate the activity of the GABAAR. Propofol is highly protein bound (greater than 98%, Kirkpatnck et al., 1988) and it is likely that the protein-fiee propofol, rather than the protein-
bound form modulates the receptor and is lower in concentration. Assuming 2% of the drug
resides in the unbound form, the fkee concentration of propofol is approxirnately 0.68 pM when
the serum concentration equals 34 PM. The high level of protein bindings suggests that the
actions of propofol associated with lower concentrations of propofol likely contribute to
propofol's clinical effects such as potentiation of GABA-evoked responses and decreased
desensitization. This analysis fails to account, however, for important experimental factors
including recording conditions in vitro, temperature and pH which influence the potency and
efficacy of propofol. In addition, the potency of propofol, similar to ketarnine, may be greater in
humans compared to rodents. For example the plasma concentration of ketamine required to produce anaesthesia in rats is greater than 50 FM while the plasma concentration in hurnans in approximately 5 - 10 pM (Cohen et al., 1973; Chang and Glazko, 1972).Therefore, a clinically relevant concentration of propofol may be anywhere from 0.5 to 5 pM (Sanna et al., 1995b). Our studies indicate that clinically relevant concentrations of propofol potentiate GABA-evoked responses, modulate GABA,R desensitization and at the upper range for clinically relevant concentrations of propofol, directly activate the receptor.
Midazolam is a water soluble 1,4 benzodiazepine denvative with a short duration of action. Midazolarn is the most potent benzodiazepine in causing anterograde amnesia (O'Boyle,
1986). It is water soluble at a pH of 4 and highly lipophilic at a physiological pH (Reves et al.,
1985). The concentration of midazolarn that was used in these studies was 0.5 pM which is close to the range obtained in humans after a 8.2 mg intravenous dose (Heizmann et al., 1983). This dose when administered to humans produced a peak serum concentration of 291 ng/ml (0.893 FM) (Heizrnann et al., 1983). However, the effective concentration is considerable lower because 96-97% of midazolarn is protein bound (Reves et al., 1995). Therefore, the concentration that we used was less than the peak serum concentration but greater than the Free midazo larn concentration after protein binding.
in summary, synergism between propofol and benzodiazepines for modulation of
GABAARfunction in vitro as shown by Isobolographic Analysis and the Fixed Dose Mode1 for low concentrations of drug might underlie the synergistic interactions observed in vivo.
Additionally, the additive interactions observed at higher concentrations of propofol and midazolarn for enhanced GABAAR function could explain the non-synergistic interaction observed when higher doses of anaesthetics are employed. 6. CONCLUSIONS
Midazolarn potentiated propofol and GABA-evoked responses. However, midazolam did not influence GABAAR desensitization. In contrast, propofol dose-dependently decreased
GABAARdesensitization at saturating concentrations of GABA. This effect was not modulated by midazolam suggesting that receptor activation and desensitization were mediated differently b y propo fol and midazolam.
Low concentrations of propofol (0.625 PM) and midazolam (0.06 FM) interacted synergistically to enhance GABA (1 FM)-evoked responses. Further, there was a superadditive interaction between propofol (1 FM) and midazolam (0.5 PM) to potentiate GABA (0.3 FM)- evoked responses. However, when the concentrations of GABA and propofol were increased there was an additive interaction between propofol and midazolarn. This suggests that the drug interactions are highly dependent on the concentration of GABA and the anaesthetic. 7. REFERENCES
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