PROPOFOL AND BENZODIAZEPINE MODULATION OF GABA,R FUNCTION

Laura Catherine McAdam

A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Physiology

University of Toronto

G Copyright by Laura Catherine McAdam (1 997)

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A. ABSTRACT

PROPOFOL AND BENZODIAZEPM MODULATION OF GABAAR FUNCTION

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

GABAAR hnction.

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

B. ACLN0 WLEDGiMENTS

C. TABLE OF CONTENTS

D. LIST OF FIGURES

E. LIST OF TABLES

F. ABBREVIATION N D E X

t NTRODUCTION

.LVAESTHESIA

NON-SPECIFIC AND SPECEIC THEORJES OF ANAESTHETIC

ACTION

N I B I T O R Y NEUROTRANSMISSION AND GABA, RECEPTORS

GABA

GN3A,R PROPERTIES

KMETIC MODEL OF GABA,R FUNCTION

DESENSITIZATION

S U B W T S OF THE GABA,R

GABAAR AND ANAESTHESIA

PROPOFOL EFFECTS ON GABAAR

BENZODIAZEPNE ACTION AT THE GABA,R

-. I I

-. . 111

i v

... V l l l

i x

X

1 -4.1. BENZODIAZEPINE B W W G IS NFLüENCED BY SUBUNIT

COMPOSITION OF THE GABA,+R

1.5. DRUG INTERACTIONS BETWEEN PROPOFOL AND

BENZODIAZEPMS

1 S. 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

- 9 3 .-. 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 GAB A,R

desensitization

Midazolam did not influence GABA,R desensitization at lower concentrations

of D ~ O D O ~ 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 ~ ~ 3 4 ) -

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 G M . 4

F. .U3BREVIATION INDEX

. M P A

ATP

BDZ

BZ 1

BZ2

CDPX

CNS

DMSO

DRC

E G O

EGTA

FLU

FMZ

G-proteins

GABA

GABA,R

GABA,R

GAi3bR

GAD

HEPES

a-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid

adenosine-5-triphosphate

benzodiazepine

benzodiazepine type 1

benzodiazepine type 2

chlordiazepoxide

central nenious system

dimethyt sulfoxide

dose response curve

concentration that produces 50 % of the observed response

ehylene glycol-0,0'-bis(2-aminoethy1)-N,N,N,N',N7-te~~cetc acid

flurazepam

flumazenil

guanine nucleotide binding protein

7-aminobutync acid

a subtype of y-arninobutyric acid receptor

a subtype of y-arninobutyric acid receptor

a subtype of 7-aminobutyric acid receptor

glutamic acid decarboxylase

25 N-2-hydroxy-ethylpiperazine-N'-2-ethanesulphoic acid

IC,,

IPSC

KA

MDZ

MEM

nAChR

NMDA

'CMDAR

PA

PRO

P s

TE,4

TM

TTX

concentration that inhibits the response by 50 %

inhibitory post-synaptic current

kainate

midazo larn

minimum essential media

nicotinic acety lcholine recep tor

N-methyl-D-aspartate

NMDA receptor

p icoamperes

propo fol

picosiemens

tetraethylarnrnonium

transmem brane domain

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

198 1 ; 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 O C ). 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 GABAAR antagonist bicuculline (Bomann, 1992; Curtis et al.,

Figure A

GABAergic synapses and the topology of a GABAAR subunit

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 c m bind to post synaptic GABAARs and 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 (TM 1 -

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.

GLIAL CELL

GAD

-\c . 7, - - f , ' v - - -+ -,

b 4- '- Na Ca"

P p . -. - - . .- - - - - . - - GABA \. A

A v r

POSTSYNAPTIC

- GABA, . .

r C 1'

.,+. -'

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). GABABRs have 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, 1 995). 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; B o m m and Feigenspan, 1995).

The major postsynaptic action of GABA is activation of GABAARs. Activation of the

GABAAR results 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 GABAAR PROPERTIES

The GABAAR belongs to the superfamily of ligand-gated channels (Stephenson, 1995).

The GABAAR channel is formed kom five glycoprotien subunits that CO-assemble to form a

functional Cl' channel. GABA bùids to the GABAAR to regulate channel gating (opening and

closing of the channel pore). The GABA dose response c w e (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).

1 e2.3. KINETIC MODEL OF GABAAR FCTNCTION

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 GABAARs prolong, 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, h g s that

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 P S C (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 GABAAR subunit 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., 1 994).

GABAAR subunits 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

a 6 subunits were minimally or not expressed in the hippocampus.

Over 10,000 possible pentameric subunit combinations of the GABAAR potentially 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 M e r 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. GABAAR AND 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., 1 98 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 P S C (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 M e r suggesting 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 f i n i t y 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 GABAAR receptors 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 a 4 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 199 1 ;

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 GABAAR and 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, 1 98 1 ). Fluctuation studies and single channel

analysis suggested that diazepam increased GABAAR currents 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, a 3 or a 5 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, a 2 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 GABAARs for benzodiazepine agonist. There

was a Gly in the al subunit at amino acid 201, whereas in the a2, a3 and a 5 subunits the amino

acid in homologous position was Glu. Therefore, for BZ1 agonists to have a high afEnity for

GABAARs the 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 a 6 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, a 3 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 GABAAR cornplex.

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 s h o w 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 GABAAR are 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 h g . 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 h g interactions 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 GABAAR by 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 GABAAR desensitization 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

GABAAR and 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 GABAAR for GABA?

2. Does midazolam increase the f i t y of the GABAAR for propofol?

3. Does midazolam alter desensitization of the GABAAR?

4. What are the characteristics of the propofol modulation of GABAAR desensitization?

5 . Does midazolam alter propofol modulation of GABAAR desensitization?

2.2. SECTION 2

A variety of agents are used for CO-induction of 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 C h i , 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

GAB A 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 ( 1 7 days in

utero) were removed from mice sacrificed by cervicai dislocation. The hippocampi were

dissected fiom each f e w , 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 1 O

days in vitro, cells were maintained in Minimal Essential Media (MEM) (Life Technologies,

Grand Island, NY) supplemented with glucose ( h a 1 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% CO2 I 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. A f er 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 d l 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

1 2- 1 6 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. n i e 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 les t 3

different dissections.

For dose-response analysis, the current amplitudes were plotted versus the agonist

concentrations and fit using a standard logistic equation (I = h a x (l+CEC,,)", where 1 is the

Figure B

iMulti-barre1 fast-perfusion system

Three square g l a s 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 t e n e d deactivation and was quantified by fit~ing the 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.

synergistic

subadditive

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 ~ S the 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 (1 0 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 v e n d

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, h c . (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., 1 98 1 ).

Propofol activates the GABAAR possibly 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 f i n i t y 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 GABAAR FOR 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 p M @ < 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 GABAAR for 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 u M midazolam (n = I I ) are shown. iMidazolam shifted the GABA

DRC to the Ieft and shified the EC,, from 9.49 f 1.85 p M 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 GABAAR for 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 p M 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 (1 2 4 + 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 P R O t MD2

10 1 O0 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 p M 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 p M @ < 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

evoked currents by acting at the benzodiazepine site.

The Current Voltage (W) relationship of propofol (20

midazolam potentiated propo fol-

LM)-evoked currents, 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-applied with PRO the

current was enhanced. When flumazenil (FMZ) was CO-applied with 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

GABAAR desensitization.

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 GABAAR desensitization.

Figure 5

Midazolam did not influence desensituation of the GABA,%R for 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 GABAAR by 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 GABAAR DESENSITIZATION

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 ( b e r 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 W I p 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.

rl GABA+PRO 1 pM \

' - GABA +PRO 6 p M GABA +PRO 10 p M

1 / GABA + PRO 20 pM

4 sec

GABA 0.0 1 600 pM

o. 1 I Propofol (PM)

was CO-applied in 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 I s d p ratio. The Iss/Ip values were normalized

to the maximal IssAp value. In the presence of 0.01 to 20 pM propofol the I s d p 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 GABAAR desensitization 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~~en t s illustrated 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 T h , (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 p M + PRO 10 ph4 0.735 2.991 GkBA 600 PM + PRO 20 p M 0.766 3.273

Propofol decreased the rate of GABAAR deseasitization and siowed receptor deactivation in the presence of saturating concentrations of G M A .

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.

ï h e IssAp ratios were calculated for currents evoked by 600 pM GABA, 600 p M GABA

and 0.5 p M midazolam (GABA + MDZ), 600 FM GABA and 10 p M 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 p M 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-applied with 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-applied with 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 GABAAR desensitization (600 pM GABA).

in spinal cord neurons, the I s d p ratios for currents activated by 600 p M GABA, GABA

+ 0.5 p M 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 p M and tested in hippocampal neurons. In the presence of lower concentrations of propofol(1

Figure 8

Midazolam did not effect propofol-induced modulation of GABAAR desensitization.

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 W I p ratios for currents evoked by GABA and

GABA - MDZ were different from those of GABA + PRO and GABA -i PRO - MDZ in both hippocampal neurons and spinal cord neurons (p

or 6 PM), midazolam did not influence the extent of GABAAR desensitization (Figure 9).

Therefore, midazolarn did not modulate GABAAR desensitization, nor did it effect the ability of

propo fol to decrease GABAAR desensitization.

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 o f 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 , h W (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 p M + PRO 6 p M ( 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 eff i t 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 1 B2y2L 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 1 0

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 F L ü (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, S tudents 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-evoke