MINIREVIEW SERIES FOR THE 50TH
VOLUME
Molecular mechanisms of anesthesia
Issaku Ueda
Anesthesia Service and Research, VA Salt Lake City Health Care System, University of Utah School of Medicine,
Salt Lake City, UT, USA(Received for publication on December 19, 2000)

Abstract. Anesthesia was a blessing to humankind. It is a miracle that simple molecules such as
chloroform (CHCl3 ), diethyl ether (CH3  CH2  O  CH2  CH3 ), or nitrous oxide (N2 O) induce a stateof unconsciousness where patients can tolerate surgery. The diversity of the structures of these molecules indicates that there are no common receptors. The action of anesthetics is nonspeci®c and physical. After the demonstration by Meyer and Overton that anesthetic potencies correlate to theirsolubility into olive oil, the nonspeci®c lipid theories monopolized anesthesia theories for almost a
century. The dominance of lipid theories invited repulsions against the nonspeci®city idea. Protein
theories that stress receptor bindings became the top mode. Nevertheless, the wide varieties of anesthetic molecules and the wide varieties of responding systems are dif®cult to reconcile with the speci®cinteraction concept. This article discusses the recent progress and controversies on the molecularmechanisms of anesthesia. Anesthetics are unique drugs in pharmacology. They affect all macromolecules. The only comparable drugs are disinfectants. Both are nonspeci®c drugs. We use alcohols
and phenols to wipe off the injection sites. We do not use penicillin or any other antibiotics for thispurpose, because they are speci®c binders. Interestingly, these two nonspeci®c drugs opened the window for the modern medicine. (Keio J Med 50 (1): 20±25, March 2001)
Key words: anesthetics, molecular mechanisms, ®re¯y luciferase, pressure effect, anesthetic-protein
interaction

Though the lipid theories of anesthesia assume lipid
membranes as the anesthetic action site, there is no
similarity between olive oil and lipid membrane. Nevertheless, the correlation that covers 5-orders of magnitude is just amazing. The potency of trichloroethylene
to anesthetize 50% of the population (ED50 ) is 0.5 volume percent when expressed by the gas phase concentration, or 0.005 atm when expressed by the partialpressure. The ED50 partial pressure of nitrous oxide is
1.01 atm. Nitrogen has anesthetic potency and the ED50
is about 50 atm. The correlation covers 0.005 to 50 atm.
The eminent contribution of Meyer and Overton was to®nd olive oil from a milliard of organic solvents.
When the Fluid-Mosaic Model of lipid membrane
was proposed by Singer and Nicolson4 in 1972, themembrane ¯uidity suddenly ran through the world like

The Meyer-Overton Rule
The year 2001 commemorates the centenary of the
publication of Charles Ernest Overton1 on the correlation between the potency of anesthetics and their olive
oil solubility. Together with the work by Hans Meyer,2
the lipid theory of anesthesia dominated the anesthesia
research for more than one hundred years. The lipid
theories are often misinterpreted to mean that the anesthetic action site is lipid membranes. This includes theoriginal authors. There is a major difference between
solvation into unstructured (isotropic) olive oil andbinding to structured lipid membranes. There are nounits for hydrophobicity. When dielectric constant isused as a measure for hydrophobicity, olive oil is about
10, water is 80, and the core of lipid bilayer is 1.8.3

Reprint requests to: Dr. Issaku Ueda, Anesthesia 112A DVA Medical Center, Salt Lake City, UT 84148, USA, e-mail: issaku.ueda@m.cc.
utah.edu
Supported by R&D Medical Research Department of VA
20

Keio J Med 2001; 50 (1): 20±25

wild ®re. Though the membrane ¯uidity has never beenclearly de®ned, the ¯uidity soon became the fad inmembrane biology. As a backlash to the dominance oflipid theories of anesthesia, protein theories became the
top fashion. Protein theories often assume speci®c
receptors.
Anesthetics and Ion Channels
Huge amounts of reports on volatile anesthetic interaction with neurotransmission systems have accumulated. The articles presented at the New York Academyof Science Symposium5 in 1991 included the nicotinic
acetylcholine receptor, GABAA receptor, GABAB
receptor, NMDA receptor, non-NMDA glutamate receptors, glycine receptor, peptide receptor, G-proteinlinked systems, a2 -adrenergic receptor, Na channel, K
channel, and Cl channel, to name a few. In addition,
volatile anesthetics affect the presynaptic transmitter
release and a number of enzymes, e.g., protein kinase
C, phospholipase C, inositide turnover, mitochondrial
electron transport, transport ATPases, luciferases, etc.
Actually, almost all channels are affected by volatile
anesthetics when the concentrations are raised. To thisauthor, this means nonspeci®c interaction.
Channels are highly organized lipoproteins. It is
impossible to isolate active channel proteins in thelipid-free form. Surfactants are necessary to isolate thechannel proteins. The isolated protein is covered bysurfactant layers. It is dif®cult, if not impossible, to distinguish anesthetic interaction with proteins from the
supporting lipid bilayer or surfactant layers.
Claude Bernard
In 1875, Claude Bernard6 published an essay on themechanisms of anesthesia. He emphasized that anesthetics are not a special poison to neuronal systems andthat all tissues are affected. Using an animal muscle
model, he reported the application of the aqueous solutions of chloroform or diethyl ether, or exposure to
their vapor, made the muscle unresponsive to electricalstimuli. At the same time, the muscle became rigid andthe transparency was decreased. When the anesthetics
were removed by washing or evaporation, the muscle
returned to the normal transparency and responded toelectrical stimuli. He interpreted these observations asa physicochemical phenomenon and discussed the similarity between this phenomenon and that of snails losing transparency and activity when placed in arid conditions, returning to normal appearance and activity
when adequate humidity is regained.
In modern terminology, Claude Bernard envisioned
anesthesia as a reversible equilibrium state of protein
colloid in which the conformation and the state of the21

hydration changed. He also emphasized the advantage
of introducing a suitable model that can be used successfully to analyze drug effects. His wisdom probably
encompasses the whole present concept of anestheticprotein interactions.
Fire¯y Luciferase
I graduated medical school in 1948 and spent about
seven years in biochemistry, studying ATP degradationintermediates. Then, the Fulbright Grant started. WhenI passed the examination, the committee advised me toapply for clinical positions. Medical schools are suffering a shortage of residents, and the positions are wide
open. I started my anesthesia residency in July 1957. In
the 1950s, the mechanisms of the ®re¯y bioluminescence and the action of Na -K ATPases were getting
established. Both are ATP-dependent enzymes. Because my background in biochemistry concerns ATP,these reports excited me. However, it was impossible tocatch ®re¯ies in Utah. I was determined to work withthis enzyme when I returned to Japan. The chancecame earlier than I expected. The United States returned
one of its occupied hospitals to Japan. The Welfare
Ministry of Japan decided to start the ®rst National
Center under direct control of the Ministry. I was calledback to Japan to manage the anesthesia department.
The National Cancer Center was equipped with top
anesthesia machines, monitors, CO2 analyzers, etc.,
plus the research building with an ultracentrifuge,
spectrophotometers, etc. To my surprise, there were®re¯y suppliers in Tokyo for entertainment. I preparedcell-free luciferase from ®re¯y tails and added anesthetics. When the anesthetics inhibited the ATPinduced light output at subclinical concentrations, I was
ecstatic.7
Fire¯y tail contains an enzyme luciferase and a substrate luciferin, and addition of ATP in the presence ofmolecular oxygen induces a ¯ash of light.8
FFL  Luciferin  ATP
 FFL-Luciferyl  AMP  PPi
FFL-Luciferyl  AMP  O2 ! FFL  Oxyluciferin
 AMP  CO2  Light
where FFL is ®re¯y luciferase and PPi is pyrophosphate.
The light intensity is stoichiometrically proportional tothe amount of added ATP.
In 1967 we9 studied the anesthetic effect on theNa -K ATPase. The enzyme was puri®ed from thecerebral microsomal fraction of rabbits. The tissue washomogenized and the enzyme was obtained by ultracentrifugation with sucrose gradient. The result was disappointing. Anesthetics inhibited ATPase activity only

22

Ueda I: Molecular mechanisms of anesthesia

when the concentration exceeded the clinical range tenfold. The enzyme also failed to respond to sedatives, suchas hydroxyzine, haloperidol, and diazepam,10 at concentrations equivalent to the clinical use. The enzyme responded to these drugs when the concentrations exceeded one-order of magnitude over the clinical doses.
Fire¯y luciferase turned out to be the most sensitive enzyme to anesthetics.7 I was extremely lucky.
Fire¯y Luciferase and Nonspeci®city ofAnesthetic Effect
The detailed anesthetic action mode on ®re¯y luciferase was evaluated by Ueda and Kamaya11 in 1973
according to the rate-process theory of Eyring. The
dose-response curves of FFL bioluminescence aretransformed into linear form according to the JohnsonEyring equation.12
I1
I2

1  KX r


I1
ln
I2


1

 r lnX  ln K  r lnX

DG
RT

where I1 and I2 are the control and the inhibited light intensities, respectively, X is the mole concentration of theinhibitor, K is the association constant of the anesthetic
and FFL, and r is the number of bound ligands. Whenthe logarithm of the inhibited portion I1 I2  and theuninhibited portion I2  is plotted against the logarithmof the inhibitor concentrations, the r value is obtained.
When the left side is plotted against the reciprocal ofabsolute temperature, DG is obtained. The r value isequivalent to the nH of the Hill plot. From the temperature-dependence of the light intensity, we estimated thefree energy changes, DG, of the enzyme by volatile anesthetics. The enthalpy change, DH, values were in therange of 90 kcal/mol. The entropy change, DS, values
were in the range of 300 cal/mol/deg. This means that theanesthetics induce a large conformational change andrelax the structure nonspeci®cally. From the entropychange, Eyring, et al.13 estimated about 50 water molecules are released from the ®re¯y luciferase by clinicalconcentrations of volatile anesthetics. Incidentally, theequation is supported by a rigorous thermodynamic
argument and a convenient procedure to analyze doseresponse curves.
Disputes on Fire¯y Luciferase
Eleven years later, Franks and Lieb14 objected to our1973 conclusion,11 that anesthetics unfold ®re¯y luciferase. They claimed that anesthetics inhibit luciferase
by competing with the substrate luciferin. However, it isinconceivable that the uncharged volatile anesthetics

compete with the negatively charged luciferin. The
charge-induced dipole interaction (luciferin binding) isstronger than the dipole-induced dipole interaction(anesthetic binding). It is dif®cult to displace stronger
binders by weaker binders, except in overtly excessconcentrations. They also claimed that the size of theluciferin binding cavity discriminates between anesthetics and nonanesthetics.
Their opposition has an underlying story. They wereoutraged about our article15 in the Molecular Pharmacology which pointed out the error in their article16 in
the Nature about anesthetic interaction with lipid
bilayer. We showed that their de®nition of the partial
molal volume of anesthetics in lipid bilayers is incorrect.
The story of luciferin-anesthetic competition was a
result of erroneous use of the Lineweaver-Burk plot.
The Lineweaver-Burk plot analyzes one-substrate oneinhibitor reaction. Even with a one-inhibitor one-substrate system, the analysis of the reaction rate is highly
complicated.17 Fire¯y luciferase is a three-substrates(luciferin, ATP, and oxygen) enzyme with two productinhibitors: one at the initial stage by pyrophosphate andthe other at the ®nal stage by oxyluciferin. The plot is
inapplicable unless supported by a proper theory.
Without rigorous derivation, the double reciprocal plot
only measures whether the inhibition is surmountable
or insurmountable by the in®nite increase of the substrate. It does not prove or disprove ligand-substratecompetition.
The bioluminescent reaction starts with the acylationof ATP to form the high-energy acyl-AMP complex,
followed by oxidation of luciferin to boost the luciferinluciferase complex to the exited state. Transition backto the ground state produces light. Anesthetics can interact in any one of these processes. It is wrong to conclude that anesthetics compete at the ®rst stage (association of luciferin and luciferase) from the ®nal
reaction (light emission).
In addition, Franks and Lieb14 used the initial ¯ashintensity to construct a Lineweaver-Burk plot. The
rapid kinetics of the transition state theory18 shows thatwhen the rate of the initial reaction to form the ®rst
product is faster than that of the ®nal reaction to form
the last product in a multiple stage reaction, the ®rst
product accumulates rapidly. The accumulation is represented by a peak. The peak is designated as the presteady-state burst. The use of the non-steady-state initial ¯ash by Franks and Lieb violates the basic rules ofinhibition kinetics.
Real Competitive Inhibitor of Fire¯y Luciferase
Ueda and Suzuki19,20 found that long-chain fatty
acids are 1000-times stronger inhibitor than anesthetics.

Keio J Med 2001; 50 (1): 20±25

23High Pressure Reversal of Anesthesia

Fig. 1 The Lineweaver-Burk plot. The broken line is halothane 1.0
mM, and the solid straight lines are, from the bottom, control without
additives, myristic acid 1.0 and 2.0 mM, respectively.

The 14-carbon fatty acid, myristate, inhibited ®re¯yluciferase at the micromolar range, compared with themillimolar range of anesthetics. The Lineweaver-Burkplot produced linear plots with luciferin, whereas thoseof anesthetics were nonlinear (Fig. 1). The curved broken line is halothane 1.0 mM, and the solid straightlines are, from the bottom, control without additives,
myristic acid 1.0 and 2.0 mM, respectively. The difference between our curved Lineweaver-Burk plot19 andthe linear plot by Franks and Lieb14 was caused by thesteady-state condition and unsteady-state condition, respectively.
It is not surprising that long-chain fatty acids arestrong luciferase inhibitors. Luciferin is a hydrophobic
carboxylic acid, and long-chain fatty acids are alsohydrophobic carboxylate. Both are negatively charged.
Luciferin acylates ATP by ®re¯y luciferase to formhigh-energy acyl-AMP. Fatty acids also acylate ATP by
acylCoA synthetases and form high-energy acyl-AMP.
Suzuki and coworkers21 reported signi®cant homology
among these enzymes.
Anesthetics inhibit bacterial luciferase,12 but myristic acid does not.22 Rather, myristic acid activates
certain bacterial luciferase.22 This proves clearly thatthe myristate action is speci®c but anesthetic actions arenot. Myristate increased the thermal transition temperature of luciferase, whereas anesthetics decreased
it.19,20 The report by DeLuca23 that 2-( p-toluidino)-6naphthalenesulfonate, TNS, and 1-anilino-8-naphthalene sulfonate compete with luciferin indicates thatnegative charges are required to compete with the luciferin binding.

High pressure antagonism of anesthesia is wellknown. However, Moss, et al.24 reported that highpressure did not show any effect on the light intensity of®re¯y luciferase in the absence or presence of anesthetics. This is highly strange because thermodynamics
dictates that all reactions are the function of temperature and pressure. Thermodynamics is the law of theuniverse. Every reaction must obey. Besides, the pressure reversal of anesthesia was ®rst discovered withbacterial luciferase by Johnson, Eyring and coworkers25±27 in 1942. Hydrostatic pressure about 10 MPa
reversed the anesthetic effects on bacterial luciferase. It
opened a new era for the understanding of the molecular mechanism of anesthesia. The criticism that bacterial light intensity has no relevance to anesthesia wascountered by Johnson and Flagler27 nine years later byshowing pressure reversal of anesthesia in tadpoles.
Tadpoles anesthetized by ethanol and staying quietly atthe bottom of the container started swimming again
when 100 atm pressure was applied.
There are sporadic negative reports on pressure reversal. Smith, et al.29 reported that high pressure did
not reverse anesthesia in freshwater shrimps. They
proposed that crustaceans lack strychnine receptors:
high pressure reverses anesthesia by stimulating thestrychnine receptor. The deviation of crustaceans fromothers originates from their anomalous response to highpressure. High pressure suppresses activity of shrimps
even in the absence of anesthetics. When both anesthetics and pressure inhibit the activity, the analysis of
the combined effect becomes complicated. Simon, etal.30 reported that pressure antagonized anesthesia inbrine shrimps. They came to this conclusion by comparing the anesthetic effect with the control in the absence of anesthetics. We31 described a method of analyzing the combined effect of pressure and anesthetics
when both are inhibitory. Above all, strychnine does
not reverse anesthesia. Most importantly, however, theshrimp studies demonstrated that the pressure effect isunrelated to the pain or discomfort associated withcompression.
The difference between the negative result with FFL
bioluminescence by Moss, et al.24 and the positive resultwith the bacterial bioluminescence by Johnson, Eyring,
et al.25±27 is caused by the difference between the continuous light intensity (steady-state condition) of luminous bacteria and initial ¯ash intensity (unsteady-state
condition) ®re¯y enzyme. By a high-pressure stopped¯ow study, Ueda, et al.32 con®rmed that pressure did
not affect the FFL light intensity when measured by the
initial ¯ash intensity. When the light output of the ®rst
10 sec was integrated, however, pressure did antagonize
the anesthetic action.32 Or, when studied at a quasi-

24

Ueda I: Molecular mechanisms of anesthesia

Fig. 2 The thermal effect on Fourier-transform infrared spectroscopy. Temperature elevation relaxes the protein structure demonstrated by
the increase of intensity. Ethanol increased the size of the peak intensity of Amide-I' and Amide-II' region whereas myristate decreased thepeak intensity. Alcohol relaxed the secondary structure of FFL, and myristate tightened it.

steady state under deceleration of the reaction rate withpyrophosphate, pressure also antagonized the anesthetic effect.33 Pressure always antagonizes anesthesia.
The pressure reversal of anesthesia means only one
thing: the volume of the anesthetized state is larger
than the awake state. It indicates that an increase of thepartial molar volume of the system is the crucial condition for anesthesia.
The Enthalpy of Anesthetic EffectDickinson, et al.34 contested our 1973 thermodynamic
study.11 They argued that the anesthetic action on ®re¯y luciferase is not nonspeci®c conformational change
and our DH values were wrong. They reached theirconclusion by measuring the initial peak light intensity
again. From the anesthetic effect on the ¯ash intensity,
they constructed the Lineweaver-Burk plot again andsupported the 1981 erroneous report by Franks andLieb.14
From the temperature dependence of the inhibitor
constant, they estimated DH and obtained negativesmall values. The averaged value for halothane,
methoxy¯urane and diethyl ether was 3.87 kcal/mol.
Our result11 was 80 kcal/mol. They argued that ourvalue is the result of using the old kinetic scheme, andinsisted on the idea that our conclusions are wrong.
They further proposed that the anesthetic interaction
with ®re¯y luciferase should be characterized by thenegative free energy. The article was accompanied withan editorial35 designating it as an enthalpic model ofanesthesia.

These authors34,35 did not recognize that the sign ispositive with ours and negative for theirs. Their values
correspond to the transfer of anesthetic molecules(molecular weight about 300) from water to the protein.
Our values represent the transfer of ®re¯y luciferase(molecular weight 62,000) from water to the anesthetic
solution. The change of the enthalpy of anesthetic molecule has little signi®cance to the mechanism of anesthesia. What is important is the change of energy levelof the enzyme when anesthetized. The large positive
DH and DS represent relaxation of the protein structureby anesthetics.
We19,20,36 demonstrated the structural relaxation of®re¯y luciferase by the effect on the thermal transition19,20 of the protein, and by Fourier transform infrared spectroscopy36 at the Amide-I' and Amide-II'region (Fig. 2). The infrared spectra showed that thetemperature elevation relaxed the secondary structureof ®re¯y luciferase. Ethanol increased the structural relaxation, whereas myristate antagonized the relaxation.
Conclusion
Biophysical studies attempt to ®nd physicochemicalmechanisms that dictate the interaction of anesthetic
molecules with proteins and lipids. I believe there is no
system that is unin¯uenced by anesthetics. The crux ofanesthetic action is the pressure reversal. Anesthetizedstate is larger than the awake state. Whatever decreases
the volume antagonizes anesthesia. Our study showedthat anesthetics unfolded and expanded ®re¯y luciferase whereas myristate tightened the structure. Because

Keio J Med 2001; 50 (1): 20±25

myristate may antagonize the anesthetic effect by
decreasing the volume, we measured the effect of myristate on the anesthesia potencies in gold®sh. Myristate
successfully antagonized anesthesia in gold®sh. The
halothane partial pressure that induces anesthesia inhalf of the population increased 300% in the presenceof 40 mM myristate.37 Myristate has no action on channels proposed to be the targets of anesthetic actions.
In this context, it is futile to pinpoint one neurotransmission system for the speci®c anesthetic target
site. We found that the Hill number of anesthetizinggold®sh averages about ®ve. The large Hill coef®cientsin intact animals indicate that multiple neurotransmission systems respond to anesthetics with high cooperativeness. There is no single button that can be pushed
to induce anesthesia.

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