British Journal of Anaesthesia 1997; 78: 290–295
LABORATORY INVESTIGATIONS
Anaesthetic potency of inhalation agents is independent of membrane
microviscosity
R. I. NORMAN, R. HIRST, B. L. APPADU, M. MCKAY, P. BRADLEY, R. GRIFFITHS AND
D. J. ROWBOTHAM
Summary
The decrease in membrane microviscosity of
erythrocyte ghosts in the presence of clinically
relevant concentrations of seven inhalation anaesthetic agents was studied using fluorescence
polarization
anisotropy
of
the
membrane
incorporated fluorescent probes 1,6-diphenyl-1,3,5hexatriene and 1-[4-trimethylammoniumphenyl]-6phenyl-1,3,5-hexatriene. All anaesthetic agents
produced a dose-dependent decrease in anisotropy
of both probes, indicating decreased membrane
microviscosity. The reduction in anisotropy
measured at the minimum alveolar concentration
(ED50) for anaesthesia was related inversely to the
anaesthetic potency of the agent and was directly
proportional to the hypothetical concentration of
agent in the membrane calculated from lipid-water
partition coefficients. These findings do not
support the hypothesis that volatile anaesthetic
agents act by increasing membrane microviscosity
of the bulk lipid bilayer to produce anaesthesia.
(Br. J. Anaesth. 1997; 78: 290–295).
Key words
Measurement techniques, anisotropy. Potency, anaesthetic,
MAC. Anaesthetics volatile. Theories of anaesthetic action,
molecular.
The mechanism of action of inhalation anaesthetics
remains unknown even though these agents are used
widely in clinical practice. The anaesthetic potency
of the volatile agents is correlated most closely with
their solubility in non-polar solvents and lipid.1–5 It
has been implied from this relationship that volatile
anaesthetic agents may act to alter neuronal membrane function.3–5 This could occur either through a
generalized perturbation of neuronal membrane
bilayer structure, by non-specific interaction of
membrane dissolved agents with bilayer lipids or by
direct modification of neuronal membrane protein
activity by interaction with specific hydrophobic
binding sites on sensitive proteins. A variety of
changes in membrane dynamic properties in the
presence of volatile agents have been demonstrated
by various biophysical techniques and include
increased rotational motion, decreased phase
transition temperature, decreased surface viscosity,
weakened binding of interfacial water, volume
expansion and competition of membrane hydrogen
bonds (reviewed by Aloia, Curtain and Gordon6).
Many of these phenomena have been observed at
supra-clinical anaesthetic concentrations and it
remains to be determined if any of these perturbations in membrane dynamic properties are relevant
to the process of anaesthesia.
The activity and kinetics of membrane-bound
enzymes and carrier proteins can be markedly affected
by the fluidity of the lipid phase of the membrane.7
Several neuronal protein activities have been reported
to be sensitive to the presence of volatile agents8–13
and some of these could be involved in the mechanism of anaesthesia. Modification of membrane
dynamic properties by volatile anaesthetic agents may
be sufficient to transduce the anaesthetic effect of
agents in the nervous system. For example, a link
between membrane lipid order and sodium channel
activity has been proposed from a strong linear relationship between voltage-dependent uptake of 24Na;
into isolated mouse brain synaptosomes and the
decrease in fluorescence polarization of membrane
incorporated probes, 1,6-diphenyl-1,3,5-hexatriene
(DPH) and 1-[4-trimethylammoniumphenyl]-6phenyl-1,3,5-hexatriene (TMA-DPH).14 By analogy,
it has been suggested that changes in protein function
induced by membrane dynamic changes, such as the
sodium channel, perhaps in conjunction with alterations in other synaptic processes, could result in
modified membrane conductance or synaptic transmission and transduce the anaesthetic effect of
membrane dissolved agents.3–5
If the primary action of the inhalation anaesthetics
involves alteration of a property of the lipid bilayer to
transduce the effect of agent in the membrane at a
functional level, it would be predicted that when
tested at equipotent concentrations for anaesthesia
the volatile agents may be expected to produce the
ROBERT I. NORMAN*, BSC, PHD, MARY MCKAY, HND, PATRICK
BRADLEY (Department of Medicine and Therapeutics); ROBERT
HIRST, BSC, BALRAJ L. APPADU, MD, FRCA, RICHARD GRIFFITHS,
MD, FRCA, DAVID J. ROWBOTHAM, MD, MRCP, FRCA (Department
of Anaesthesia); University of Leicester, Leicester Royal
Infirmary, Leicester LE2 7LX. Accepted for publication:
November 7, 1996.
*Address for correspondence: Department of Medicine and
Therapeutics, Robert Kilpatrick Building, Leicester Royal
Infirmary, Leicester LE2 7LX.
Membrane perturbations and anaesthetic potency
same degree of perturbation in that variable.
Although not all biological membranes would be
involved in transducing the anaesthetic effect, anaesthetic perturbation of membrane dynamics would be
expected to be universal. To test this hypothesis,
anaesthetic-induced increases in erythrocyte membrane microviscosity produced by clinically relevant
concentrations of seven volatile anaesthetic agents
were investigated to allow the degree of membrane
perturbation at the ED50 for anaesthesia for the
agents to be determined. Probes studied were DPH,
which incorporates into the core hydrophobic region
of the lipid bilayer, and TMA-DPH, which localizes
in the outer regions of the lipid bilayer by virtue of
the cationic trimethylammornium group.15 These
probes intercalate between membrane lipid molecules and the fluorescence polarization anisotropy
reports on the restriction of movement of the probe
molecule and thus reflects largely the rotational
movement of the probe in the membrane. The
greater the polarization of the fluorescent emission in
response to a polarized excitation the more restricted
is the probe in the membrane. Thus fluorescent
polarization anisotropy reports on the membrane
microviscosity in the environs of the probe.
Materials and methods
DPH and TMA-DPH were Obtained from
Molecular Probes Inc. (Eugene, Oregon, USA).
Anaesthetic agents were obtained as follows:
desflurane was a gift of Pharmacia (St Albans,
Hertfordshire, UK), halothane and trichloroethylene
were from ICI Pharmaceuticals (Macclesfield,
Cheshire, UK), isoflurane and enflurane were from
Abbott Laboratories Ltd (Maidenhead, Berkshire,
UK), methoxyflurane was from C-Vet (Bury St
Edmunds, Suffolk, UK) and nitrous oxide from
BOC Ltd (Derby, Derbyshire, UK). Air and oxygen
were from BOC Ltd. All other reagents used were of
the highest grade available.
ERYTHROCYTE GHOST MEMBRANES
Venous blood samples were collected from 10
healthy volunteers into tubes containing ethylenediaminetetraacetic acid as anticoagulant. Plasma
and the buffy coat were removed after centrifugation
at 1900 x g for 10 min at 4 ⬚C. Erythrocyte ghost
membranes were prepared as reported previously16
and stored at 920 ⬚C until use.
MEMBRANE MICROVISCOSITY MEASUREMENTS
Erythrocyte ghost membranes, 100 ␮g of protein
ml91 in 1 ml of Tris-HCl 10 mmol litre91 (pH 7.4),
were incubated in a thermistor-controlled water
bath at 37⫾0.1 ⬚C and volatile anaesthetic agents
delivered at the required concentration from laser
refractometer calibrated vaporizers (Ohmeda TEC
(0–2% trichloroethylene; 1–3% methoxyflurane;
0–5% halothane; 0–4% isoflurane; 0–4% enflurane) or TEC 6 (0–20% desflurane)) via a manifold of 10 outlets, each delivering humidified air
60 ml min91 to each sample. The deadspace over
291
each sample was 11 ml. Carrier gas flow through
the vaporizers was 2 litre min91 and any excess gas
not delivered to experimental tubes was exhausted
to the atmosphere. The time taken for each agent
to reach stable equilibrium in the aqueous phase
was determined in preliminary experiments by following the decrease in DPH and TMA anisotropy
with gas delivery at the lowest concentration of
each agent to be studied until steady state
anisotropy was attained (not shown). Equilibration
times were confirmed at approximate MAC concentrations of agent delivered by gas chromatographic analysis using a Perkin-Elmer 8410 gas
chromatograph17 (table 1). Volatile agents (0.2%
trichloroethylene, 0.5% methoxyflurane, 0.75%
halothane, 1.25% isoflurane and 1.7% enflurane)
were delivered for various times (0–60 min) to
samples containing 100 ␮g of erythrocyte ghost
membrane protein in 1 ml of Tris-HCl 10 mmol
litre91 (pH 7.4). Aliquots (100 ␮l) were mixed
with n-heptane 100 ␮l (halothane, isoflurane,
enflurane) or toluene 100 ␮l (trichloroethylene,
methoxyflurane) in glass vials on ice. The vials
were sealed to prevent evaporation before analysis.
The non-aqueous phase was injected onto a 30-m
DB17 megabore column under the following conditions: injection temperature 120 ⬚C; oven temperature 90 ⬚C; flame ionization detector at 120 ⬚C.
The amount of anaesthetic in each sample was
determined from the peak height against a standard curve for the agent. All experiments in which
polarization anisotropy was measured were performed under steady state conditions to ensure
equilibration of the anaesthetics. Pre-equilibration
was for 15 min for enflurane, halothane, isoflurane
and desflurane, and 60 min for methoxyflurane
and
trichloroethylene.
Nitrous
oxide
was
delivered with oxygen from a Boyles anaesthetic
machine for 15 min. Control samples were
exposed to air or oxygen only, as appropriate, for
the same period.
DPH (in tetrahydrafuran) and TMA-DPH (in
dimethylformamide) were added to erythrocyte
ghost membranes to final concentrations of 0.5 and
5 ␮mol litre91, respectively, and incubated for 30
min at 37 ⬚C with gas delivery as before. Addition of
solvent from the stock solution never exceeded
0.05%. Fluorescence polarization anisotropy, as a
measure of the degree of depolarization of the
emitted signal, was measured in duplicate at 37 ⬚C
using a Perkin-Elmer LS50 luminescence spectrofluorimeter. Excitation and emission wavelengths
were 365 nm and 456 nm. Background fluorescence
from unlabelled membrane samples, which was
always less than 3% of the total signal, was subtracted from measurements in which probes were
incorporated. All measurements were made in
duplicate. Under vertical polarized light excitation
(subscript V), fluorescence polarization anisotropy
was defined by the equation:
(I|| − I||S )V − G(I Ⲛ − I ⲚS )V
r=
(I|| − I|| S )V + 2G(I Ⲛ − I ⲚS)V
with a correcting grating factor, to correct for
differences in instrument sensitivity for emitted
292
polarized light in the parallel and perpendicular
planes, measured on each sample of:
(I||)H
G=
(I Ⲛ) H
where subscript H:horizontal polarized light excitation, and subscripts # and 芯:components of the
emitted light, parallel and perpendicular to the direction of polarization of the excitation beam.
Superscript S:contribution of scattered light
determined for an unlabelled reference sample of
the same composition. Experiments were performed
on erythrocytes from 4–10 subjects for each
concentration of anaesthetic studied.
PROTEIN DETERMINATION
Protein concentration was measured by the method
of Lowry and colleagues.18
British Journal of Anaesthesia
Results
Maximum concentrations of agents used in this
study did not alter the wavelength of emission
maxima or affect the intensity of emission of membrane incorporated probes in the analyses of fluorescence spectra. We conclude that depolarization
measured in the presence of anaesthetic agents was a
result of membrane disruption and not alteration of
fluorescence life-time by quenching.
A dose-dependent decrease in the anisotropy of
both DPH and TMA-DPH was observed over the
clinically relevant range of concentrations studied
for each agent (fig. 1). The shapes of the
dose–response curves varied with each agent and
with the distribution of the fluorophore in the
membrane.
The ability of equipotent concentrations of agents
Figure 2 Relationship between the change in fluorescence
polarization anisotropy of erythrocyte ghost incorporated DPH
(A) and TMA-DPH (B), measured at the minimum alveolar
concentration (MAC) of the delivered agent, and anaesthetic
potency (MAC). Anaesthetic agents: 1:methoxyflurane;
2:trichloroethylene; 3:halothane; 4:isoflurane; 5:enflurane;
6:desflurane and 7:nitrous oxide (anisotropy at 100% taken to
approximate to MAC of 104%24). MAC values used are those
given in figure 1.
Figure 1 Dose–response relationships between the fluorescence
polarization anisotropy of membrane incorporated DPH (!) and
TMA-DPH (●) and the concentration of volatile anaesthetic. A:
Methoxyflurane; B: trichloroethylene; C: halothane; D: isoflurane;
E: enflurane; F: desflurane; and G: nitrous oxide. Values of
anisotropy are mean (SD). Experiments were performed on
erythrocytes from 4–10 subjects for each concentration of
anaesthetic. The minimum alveolar concentration (MAC) of
each agent is shown by the arrow head. MAC values used were:
methoxyflurane, 0.16%,19 trichloroethylene, 0.17%
(calculated),20 halothane, 0.74%,19 isoflurane, 1.15%,21
enflurane, 1.68%,22 desflurane, 7.25%23 and nitrous oxide,
104%.24
Table 1 Equilibration of volatile anaesthetic agents in 1 ml of
Tris-HCl 10 mmol litre91 (pH 7.4) containing 100 µg of
erythrocyte ghost protein ml91. The concentration of each agent
delivered is given in parentheses. Data are mean (SEM) of three
individual experiments
Volatile anaesthetic
Half-life (min)
Trichloroethylene (0.2)
Methoxyflurane (0.5)
Halothane (0.75)
Isoflurane (1.25)
Enflurane (1.7)
1.8 (0.3)
3.0 (0.2)
1.8 (0.3)
5.5 (1.8)
4.9 (0.2)
Membrane perturbations and anaesthetic potency
293
approached
P:0.06).
statistical
significance
(r:90.79,
Discussion
Figure 3 Relationship between the change in fluorescence
polarization anisotropy of erythrocyte ghost incorporated DPH
(A) and TMA-DPH (B), measured at the Minimum alveolar
concentration (MAC) of the delivered agent, and the calculated
theoretical concentration of agent in the membrane at MAC.
Anaesthetic agents: 1:methoxyflurane; 2:trichloroethylene;
3:halothane; 4:isoflurane; 5:enflurane; 6:nitrous oxide
(anisotropy at 100% taken to approximate to MAC of 104%24).
MAC values used are those given in figure 1.
to perturb membrane microviscosity was assessed by
comparing the change in anisotropy of the two membrane incorporated probes at the concentration of
agents that produces anaesthesia in 50% of a population, ED50 (minimum alveolar concentration,
MAC). Values for the change in anisotropy at MAC
were derived from the data in figure 1. An inverse
relationship between the change in anisotropy of
both membrane probes at MAC and anaesthetic
potency was observed (fig. 2: DPH, correlation
coefficient r:90.91, P:0.005; TMA-DPH,
r:90.85, P:0.02). No other relationship between
the potency and degree of membrane perturbation,
for example the concentration of agent required
to produce a defined change in anisotropy, was
identified with either fluorescent probe.
Stable equilibrium between gaseous and aqueous
phases at delivered gaseous concentrations of approximately 1 MAC was confirmed for five of the agents
(table 1). A theoretical concentration of agent in the
erythrocyte membrane at the MAC concentration of
delivered gases was calculated,12 using olive oil-gas
partition coefficients where known (unknown for
desflurane), and related to the change in anisotropy
at MAC for each of the agents (fig. 3). A linear relationship between the change in anisotropy and theoretical membrane concentration was observed for
anisotropy determined with the DPH probe (correlation coefficient r:90.89, P:0.02) but when TMADPH was used as probe this relationship only
The identity of the primary site(s) of action of
volatile anaesthetic agents remains to be determined.
There has been much discussion on whether or not
an interaction in the membrane bilayer or a direct
interaction with hydrophobic sites on target proteins
is responsible for anaesthesia (reviewed in Aloia,
Curtain and Gordon,6 Miller,8 Koblin9 and Franks
and Lieb12 13). Polarization anisotropy of the membrane incorporated fluorescent probes DPH and
TMA- DPH in the presence of clinically relevant
concentrations of volatile agents suggested that
decreased membrane microviscosity is linked to
changes in functional properties of the membrane.14
25
To test if there is a relationship between anaesthetic potency and the ability of volatile anaesthetic
agents to produce membrane perturbation, reflected
by polarization anisotropy of membrane incorporated probes, the change in anisotropy of the probes
DPH and TMA-DPH was investigated in this study
for a range of volatile anaesthetic agents at clinically
relevant concentration ranges. The erythrocyte
membrane was used as a simple model biological
membrane system with a defined composition and
devoid of organelle membranes of varying composition that could have complicated interpretation.
Although the magnitude of changes produced in the
erythrocyte membrane may be quantitatively different, this membrane system would be expected to
mirror changes that occur in other membranes that
may have a more direct involvement in the
mechanism of anaesthesia.
Using DPH and TMA-DPH to probe the core
and outer regions of the bilayer, respectively,15 we
found that clinical concentrations of all agents produced a decrease in anisotropy consistent with
decreased membrane microviscosity. The different
shapes of the dose-response curves for alteration of
membrane microviscosity obtained both for different
agents and with respect to localization of the probe
in the bilayer suggested complex and non-uniform
interactions of the agents within the membrane.
Contrary to the proposed hypothesis that equipotent
clinical concentrations of volatile anaesthetics would
produce a similar degree of membrane perturbation,
the most potent anaesthetic agents produced the
least change in anisotropy of probes at MAC concentrations. This series of observations indicates that
there is no direct relationship between the ability of
an agent to alter the anisotropy of membrane probes
and anaesthetic potency and argues against a role for
disruption of bulk phase membrane microviscosity
in the transduction of the effect(s) of volatile anaesthetic agents to membrane mechanisms. These
observations further suggest that links between
membrane microviscosity and membrane function
identified previously14 25 are unrelated to anaesthetic
action.
Dissociation of membrane perturbing effects and
anaesthetic mechanism has been suggested previously in a study comparing the actions of
294
2-[2-methoxyethoxy]-ethyl
8-[cis-2-n-octylcyclopropyl]-octanoate (A2C) and benzyl alcohol.26 In
common with benzyl alcohol, i.v. administration of
A2C was found to perturb brain membranes but,
unlike benzyl alcohol, A2C did not produce either
intoxication or anaesthesia in mice. Both A2C and
benzyl alcohol were found to inhibit [35S]t-butylbicyclophosphorothionate (TBPS) binding and reduce
muscimol- stimulated 36Cl9 influx into brain vesicles
at the same concentrations that reduced membrane
order. However, at lower concentrations benzyl
alcohol, but not A2C, stimulated muscimolactivated 36Cl9 influx. A two-site model was
proposed in which the “perturbant” site responsible
for decreased 36Cl9 uptake was proposed to be
distinct from an “anaesthetic” site responsible for
augmentation of 36Cl9 flux and anaesthesia.
From the inability to detect membrane perturbations at clinically relevant concentrations of volatile
agents using several biophysical techniques,27–30 it
has been argued that the mechanism of anaesthesia
of these agents does not involve acting as hydrophobic solutes to produce non-specific perturbation
of bulk phase membrane properties and, hence,
membrane protein function. Such conclusions presuppose that the chosen biophysical variable
investigated reflects any membrane action of an
anaesthetic, that a change in properties is observed
within the time scale of the measurement and that
the variable is sufficiently sensitive to demonstrate an
interaction. As important as showing absence of
anaesthetic effect at clinical concentrations is investigation of the significance of anaesthetic-induced
membrane perturbations that have been reported at
clinically relevant concentrations of volatile agents.14
25
The inverse relationship between the ability to
perturb membrane microviscosity and anaesthetic
potency observed in this study suggests that links
between membrane perturbation and membrane
protein function reported previously14 25 may be
secondary phenomena, reflecting the presence of the
anaesthetic molecules in the membrane rather than
perturbations of relevance to the mechanism of
anaesthesia.
As measurements were made at steady state
between the gaseous and aqueous phases we were
able to calculate a theoretical concentration of each
agent in the lipid phase using olive oil-gas partition
coefficients. Partition coefficients into lipid systems
related to a phospholipid bilayer were not known for
enough of the agents to allow this relationship to be
calculated for a more representative lipid phase.
Although partitioning of gases into olive oil and the
erythrocyte membrane is unlikely to be identical, a
proportionality in solubility between the two systems
would be expected. Despite the differences in
chemical structure between the anaesthetics, the
change in anisotropy at the MAC concentration was
proportional to the calculated theoretical concentration of agent in the lipid phase and, hence, the relative number of molecules of anaesthetic in the lipid
bilayer. This correlation was highly significant with
the DPH probe but only approached statistical
significance for the TMA-DPH probe. Although
several assumptions have been made in these
British Journal of Anaesthesia
calculations, it would appear that membrane
microviscosity changes in the presence of volatile
anaesthetic agents simply reflect the presence of
anaesthetic molecules in the lipid membrane and are
unrelated to the mechanism of anaesthesia.
In conclusion, our results are inconsistent with a
role for perturbation in membrane microviscosity in
the mechanism of anaesthesia of volatile agents.
While perturbation of the dynamic properties of the
bulk phase of the membrane may be unimportant,
the ability of anaesthetic agents to dissolve in
membrane bilayers may still prove to be important
in their action.
Acknowledgements
R.G. was a British Journal of Anaesthesia Research Fellow.
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