Br.J. Anaesth. (1979), 51, 619
RECENT MOLECULAR THEORIES OF GENERAL ANAESTHESIA B. WARDLEY- SMITH AND M. J. HALSEY
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CORRELATIONS WITH PHYSICAL PROPERTIES
Although almost all the early physical theories of anaesthesia involved lipid solubility, two independent workers (Miller, 1961; Pauling, 1961) proposed an aqueous site of action in the central nervous system. BRIDGET WARDLEY-SMITH,
B.SC; MICHAEL J.
HALSEY,
D.PHIL. ; Division of Anaesthesia, Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ. 0007-0912/79/070619-08 $01.00
Though different in detail, both theories attempted to relate anaesthetic potency to the stability of the gas hydrate in aqueous solution. Their original correlations for a limited range of agents were reasonable, but studies with more anaesthetics proved that the agreement between potency and hydrate dissociation pressure was much worse than that for potency and oil/gas partition coefficient. These theories of an aqueous site of action have now been mainly abandoned. It is more generally assumed that anaesthetics act by interaction with a hydrophobic phase somewhere in the nervous system. This idea is not new, however, since one of the best correlations with potency is the solubility of the anaesthetic in olive oil. This was first recognized by H. H. Meyer and by Overton, both in 1901, and the lipid solubility theory that they proposed has been expressed in modern form by K. H. Meyer in 1937, who said that "Narcosis commences when any chemically indifferent substance has attained a certain molar concentration in the lipids of the cell. This concentration depends on the nature of the animal or cell but is independent of the narcotic." The idea that a physical property of an anaesthetic was critical was further expanded by Ferguson (1939), who found a reasonable correlation between vapour pressure at 37 °C and anaesthetic potency of a series of homologous alcohols. He concluded that thermodynamic activities were important as a measure of anaesthetic potency. Mullins (1954) carried out a thermodynamic analysis of the data then available to him and suggested that anaesthetic activity was not dependent on solubility alone but also on the molar volume of the anaesthetic. He suggested that anaesthetics might "depress cell function by preventing the passage across cell membranes of any particular molecule or ion, depending only on the degree of volume occlusion occasioned by the narcotic". This was an attractive model, but inclusion of molecular size in the data analysis does not improve the Meyer-Overton correlation between potency and oil solubility. The theoretical thermodynamic approach to anaesthetic © Macmillan Journals Ltd 1979
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The mechanism of anaesthesia has attracted the interest of many eminent scientists. For example, the references in this limited review include such wellknown names as Professors Burgen, Eyring, Paton and Pauling, who have all made contributions to different aspects of this field. In view of the wide range of scientific disciplines involved, it is not surprising that, since the introduction of general anaesthesia for surgery in the 1840's, there have been numerous ideas about the mechanisms of action of anaesthetics. By 1900 more than 25 of these had been published, and by the early 1960's more than 100 different theories of the mechanism of narcosis had been proposed. Of these theories, only a few are currently accepted, and this article is intended to discuss the molecular developments in anaesthesia research since the early 1970's. At first sight much of this work, which involves various model systems, is not directly relevant to the in vivo mechanisms of anaesthesia. A complete description of the mechanisms must include an understanding of how anaesthetics affect the integrated nervous system, single neurons and synapses as well as the molecular interactions. These approaches are not entirely separate. For example, the recognition that anaesthetics acted at hydrophobic sites reduced the possibility that anaesthetic actions in aqueous areas of neurons were critical. Anaesthetic research has tended to parallel the developments in human biology, and thus one of the intensive areas of current research is concerned with molecular biology and membrane biophysics. However, it is desirable to review briefly some of the earlier ideas which were important in the formation of the currently fashionable theories.
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HIGH PRESSURE STUDIES
It is one of the most striking features of anaesthetics that many of their effects are opposed by high hydrostatic or gaseous pressures. This was first demonstrated by Johnson, Brown and Marsland (1942a, b) who showed that the light from luminous bacteria, dimmed by anaesthetics, could be restored by high pressures. Johnson and Flagler (1950, 1951) anaesthetized tadpoles with alcohol until swimming had ceased, and then applied high hydrostatic pressure until swimming had spontaneously started again. Tadpoles were used recently to show that pressure reversal occurred with a far wider range of compounds than had been expected, including narcotics and tranquillizers as well as conventional anaesthetics (Halsey and Wardley-Smith, 1975).
It was not until the early 1970's that a unifying theory was put forward to explain the interactions of anaesthetics and pressure, and to provide a hypothesis of the physical mechanism of narcosis. The critical volume hypothesis (Lever et al., 1971; Miller et al., 1973) was proposed as an extension of the work of Mulhns, and states that "anaesthesia occurs when the volume of a hydrophobic region is caused to expand beyond a critical amount by the absorption of molecules of an inert substance. If the volume of this hydrophobic region can be restored by changes of temperature or pressure then the anaesthesia will be removed." This hypothesis assumed that all anaesthetics acted at the same molecular site. It was formulated from experiments using newts. On plotting increases in anaesthetic potency against total pressure for four anaesthetics, a straight line was obtained (fig. 1). This was in accordance with the 350 300 250 200 . 150 .
50
100 He
150 200 (ATA)
FIG. 1. Original pressure reversal data in newts, x axis— Helium total pressure (Pue) m atmospheres absolute (ATA); y axis—percentage of control anaesthetic dose at 1 ATA required to reduce the rolling response of newts to 50% of control value. • = Nitrogen; • = sulphur hexafluoride; 0 = nitrous oxide; A = carbon tetrafluoride (data from Miller et al., 1973).
predictions of the hypothesis, which stated that since equi-narcotic doses of all anaesthetics caused an equal volume increase, the degree of pressure reversal for a given total pressure should be the same for all anaesthetics. The critical volume hypothesis also suggested that "temperature reversal" would occur in a manner similar to pressure reversal, and it was calculated that the effects of a normal anaesthetic dose would be removed on cooling by 10 °C. This effect was predicted on the simple basis that the expansion caused by the anaesthetic could be counteracted by the
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mechanisms is still in use today. Eyring, Woodbury and D'Arrigo (1973) proposed a molecular mechanism of anaesthesia based on thermodynamic considerations, later expanding their ideas to formulate the lipoprotein conformational change theory (Woodbury, D'Arrigo and Eyring, 1975). In 1974, Hill analysed the results of dissolving anaesthetics in membranes in terms of the "Gibbs free energy hypothesis". The correlation of physical properties of anaesthetics with anaesthetic potency is still an accepted approach to the subject. In 1969 Eger and his colleagues measured anaesthetic potency in terms of dog MAC and correlated this with both lipid solubility and hydrate dissociation pressure. Three years later Miller and colleagues (1972) correlated anaesthetic potency measured in mice with anaesthetic solubility in solvents of different solubility parameters. In a recent study Franks and Lieb (1978) measured the partition coefficient for 22 anaesthetics in several different solvents, and found that the potency/partition coefficient correlation was better for octanol than for oil, and concluded that the site of action had both polar and apolar characteristics. This concept has been developed quantitatively by Hansch and his colleagues (1975) over the past 8 years. However, there is a limit to how much information can be obtained from simple solubility characteristics and thermodynamic analysis of whole animal data. It is necessary to look at other physical changes at the molecular level. There have been two main developments, first whole animal pressure reversal studies, and second, the use of model systems of lipids and proteins.
MOLECULAR THEORIES OF ANAESTHESIA 180 160 140 120 . 100 25
50
75
100
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(ATA)
FIG. 2. Representative examples of data for pressure reversal of gaseous anaesthetics in mice, x axis—As figure 1; y axis—percentage of control anaesthetic dose at 1 ATA required to reduce righting reflex of mice to 50% of control value. V = Argon (Ar) (Smith et al., 1975); • = nitrous oxide (N2O) (White and Halsey, 1974); • = carbon tetrafluoride (CF4) (Miller, Wilson and Smith, 1978); O = nitrogen (N2) (Miller and Wilson, 1978).
compared the non-gaseous agents ethyl carbamate, a-chloralose and phenobarbitone, with argon and nitrogen and found differing degrees of pressure reversal between the two groups, but agreement within them. Some examples of the pressure reversal data for the i.v. agents are shown in figure 3. To summarize, the critical volume hypothesis stated that all anaesthetics acted at the same molecular site. It has one main predictive feature, namely that for a given increase in the total pressure, the percentage increase in anaesthetic required to maintain a constant level of anaesthesia should be the same for all agents. Theoretically this should be true to infinitely great pressures; the non-linearity at very high pressures was originally explained as being the result of the adverse physiological effects of increased pressures per se (e.g. Green, Halsey and WardleySmith, 1977). There is no doubt that our results with short acting i.v. anaesthetics do not support the concept of a single site of action for all anaesthetics. The deviations from the predicted straight line cannot be explained away as caused by pressure alone, since we have found non-linearity at pressures as small as 50 atm. We have thus developed a modification of the critical volume hypothesis in an attempt to explain the discrepancies in the data of both ourselves and others. It retains the original basic concept of the critical volume hypothesis, namely that anaesthetics expand and high pressures contract a molecular site
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contraction resulting from cooling. However, this is a more difficult concept to test, as even in cold blooded animals the predominating effect of cooling is hypothermia. Since extreme cases of hypothermia are seen as a state resembling anaesthesia, assessment of anaesthetic potency becomes unreliable. Spyropoulos (1957) removed the effect of a 3% ethanol solution on the action potential of toad nerve fibre by cooling by 15 °C, but a reduction in solubility would also occur with lowering of temperature, and thus precise analysis of his results is not easy. Preliminary experiments with nitrous oxide and nitrogen on intact animals (Halsey and Higgs, 1976) and with some of the alcohols on an in vitro nerve preparation (Richards et al., 1978) showed no evidence of "temperature reversal of anaesthesia". However, because of difficulties of interpretation this aspect of the critical volume hypothesis remains one that is not resolved. Early experiments in normothermic mammals (Halsey and Eger, 1971) provided support for the hypothesis since the pressure reversal values coincided with the predicted straight line. Subsequent mammalian experiments, however, produced less clear cut results. Smith and colleagues (1975) studied the pressure reversal of argon and nitrogen anaesthesia, while Kent and colleagues (1977) expanded the range of agents to include the volatile clinical anaesthetic, isoflurane. The original work with nitrous oxide was extended to greater pressures (White and Halsey, 1974). Recently Miller, Wilson and Smith (1978) have studied the perfluorinated anaesthetics. Examples of all these data are plotted in figure 2. The first quantitative experiments with nongaseous anaesthetics in mammals were by Winter and colleagues (1976). They used phenobarbitone in mice, and gave a single dose before compression. The anaesthetic effect of this agent was reversed by pressure, and the increase in ED50 at 103 atm was equal to that for isoflurane, but greater than that for nitrous oxide. However, the technique of preadministration of a single dose before compression makes it hard to be certain that changes in metabolism are not taking place, especially with long-acting anaesthetics such as phenobarbitone. We have developed a continuous infusion technique for studying the short acting i.v. anaesthetics at pressure. Initial experiments, with Althesin (Bailey et al., 1977), have been extended to other i.v. anaesthetics (Halsey, Wardley- Smith and Green, 1978). The degree of pressure reversal varied considerably between the different agents. Miller and Wilson (1978)
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100 FIG. 3. Representative examples of data for pressure reversal of non-inhalation anaesthetics in rats and mice. x axis—As figures 1 and 2; y axis—as figure 2. Mice data for phenobarbitone (Ph • ) and urethane (Ur v) with anaesthesia denned as loss of righting reflex (data from Miller and Wilson, 1978). Rat data for Althesin (Alth A), thiopentone (Thio • ) and ketamine (Ket ©) with anaesthesia denned as loss of response to electrical stimulus (data from Bailey et al., 1977; Halsey, Wardley-Smith and Green, 1978).
of action, but differs from it in a number of fundamental ways. We have provisionally termed it the multi-site expansion hypothesis; it is based on all the current intact animal data of both ourselves and other workers, and can be best summarized in the following tenets. (1) General anaesthesia can be produced by the expansion of more than one molecular site, and these sites may have differing physical properties. (2) The physical properties of a molecular site may themselves be influenced by the presence of anaesthetics or pressure. We believe that the most plausible receptor site characteristic which may change is the compressibility. (3) The molecular sites do not behave as if they were bulk solvents, but have a finite size and a limited degree of occupancy. (4) Pressure itself need not necessarily act at the same site as the anaesthetic. Depending on the anaesthetic, one of the sites may predominate in determining the interaction with pressure.
The correlation studies and high pressure experiments cannot provide a detailed molecular hypothesis about how general anaesthetics may modify the many membrane functions which are potentially important in the transmission of nerve impulses. There have been a number of studies on in vitro nerve preparations and isolated components of synaptic junctions, but it is difficult to interpret these data in terms of the underlying molecular events or to extrapolate the results to the in vivo integrated nervous system. It has proved more productive to study various simplified lipid models of nerve membranes. These are an oversimplification of the in vivo system, but it should be possible to separate out the several molecular changes that are produced by anaesthetics and decide which of these are likely to be critical in the production of general anaesthesia. The lipids in membranes are now thought not only to provide the basic permeability barrier, but also to influence the activity of proteins which control ionic and neurotransmitter fluxes. The important properties of lipids in controlling protein activities appear to be their ability to move and rotate within tie basic bilayer membrane structure. The fluidity or degree of order of the lipids has been shown, for example, to control the activity of the protein of the calcium pump (see review by Lee, 1975). This fluidity can be measured by either nuclear magnetic resonance studies of the lipids themselves or electron paramagnetic resonance studies of marker compounds incorporated into the bilayer. Both techniques are sensitive to the fluidity of the environment around the particular part of the molecule being studied. In pure lipid preparations it has been found that as the
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(5) The molecular sites for anaesthesia are not perturbed by a decrease in temperature in a manner analogous to an increase in pressure. A more detailed discussion of the multi-site expansion hypothesis, and the evidence on which it is based, can be found elsewhere (Halsey, WardleySmith and Green, 1978) together with the predictive features by which it can be tested. Although the multisite expansion hypothesis is more complex than the critical volume theory, it seems to us to be necessary to explain all the current pressure-anaesthetic data. However, it should not be regarded as an alternative to the molecular theories of anaesthesia based on lipid fluidity. Phase changes are accompanied by an increase in volume, and thus are susceptible to pressure reversal (Trudell et al., 1973, 1975).
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temperature is increased there is a general increase in fluidity; at a specific temperature there is a sudden step change when the fluidity increases dramatically. Below this temperature the lipids are described as being in the ordered gel state; above this temperature the fluidity of the lipids are described as being in the disordered liquid crystalline state. This "phase change" can be studied not only by magnetic resonance techniques but also by direct calorimetry, since at the phase change there is a sudden increase in the specific heat (analogous to that which occurs when any solid melts). For the past 10 years it has been known that anaesthetics alter the fluidity of the lipids and membranes. Metcalfe, Seeman and Burgen (1968) demonstrated that as the concentration of benzyl alcohol in the erythrocyte membrane (or its extracted lipids) was increased, the fluidity of the system also increased. They anticipated more recent concepts by speculating that the presence of the anaesthetic in the membrane produced more and more disorder until finally the protein was exposed. This made available more binding sites in the protein. Since 1970, there has been a series of theories as to how anaesthetic-induced changes in fluidity of the lipids might influence the activities of the proteins. In general terms, Johnson and Miller (1970) postulated that anaesthetics cause an increase in freedom of movement of the lipid parts of membranes which is associated with an increase in volume which in turn might affect conformational changes in proteins intimately associated with lipid. This paper linked lipid effects with the whole animal data on pressure reversal. Other evidence for the importance of fluidity effects was provided by the demonstration that non-anaesthetic steroids did not alter bilayer fluidity (Lawrence and Gill, 1975). Subsequently, Lee (1976) has taken the association between lipids and proteins one stage further and argued that a sodium channel protein requires an "annulus of lipid" in the more solid gel state to allow activity. Anaesthetics fluidize this lipid, causing the protein to relax into an inactive conformation. There have been a number of studies demonstrating that anaesthetics shift the phase transition to a lower temperature (Trudell, Payan and Chin, 1974) which results in a much larger increase in anaestheticinduced fluidity at the original phase transition temperature. This has led to a theory based on "lateral phase separations" (Trudell, 1977). When a lipid bilayer membrane is at the phase change temperature, both the ordered and disordered phases
are in equilibrium with each other. At this point any change in internal pressure in the system (produced, for example, by conformational changes in proteins) can be accommodated by shifting the equilibrium position—that is there is "lateral compressibility". If the position of the phase change is shifted by anaesthetics, the equilibrium between the two phases is also shifted so that one of them is no longer present. When only one phase is present the ability of the system to accommodate volume changes is much reduced and the environment of the protein becomes more rigid with respect to lateral expansion (Hanukoglu and Trudell, 1977). The hypotheses summarized so far have considered the effects of the lipid environment on the functioning of individual proteins. However, this is an oversimplification in that, in vivo, groups of a particular type of protein function together in a co-operative manner. The energetic links between these proteins are probably provided by the lipids between them, which themselves are grouped together in clusters either in the gel or in the liquid state. If these gel or liquid crystal clusters are reduced below a critical size then the co-operative linkage between the proteins is disrupted. Experiments with halothane and enflurane in simple lipid systems using differential scanning calorimetry have demonstrated that not only is the fluidity altered but also lipid co-operativity and hence cluster size is reduced (Mountcastle, Biltonen and Halsey, 1978). Two independent groups have recently put forward an entirely different concept as to how lipids may affect protein function (Ashcroft, Coster and Smith, 1977; Haydon et al., 1977; Hendry, Urban and Haydon, 1978). They proposed that the thickness of the lipid bilayer is increased by anaesthetics, which prevents the protein pore from adequately spanning the membrane. Their evidence for this phenomenon, which is based on electrical measurements of lipid films, is controversial because other techniques—such as x-ray and neutron diffraction (Franks and Lieb, 1978)—indicate that at anaesthetic concentrations there are no significant changes in membrane thickness. In addition to the discussions about the alternative hypotheses, there are at least two areas of anaesthetic actions on lipids which are controversial: the effects of varying the anaesthetic concentration and the lipid composition. For example, different groups of workers have found that "clinical concentrations" of halothane increase lipid fluidity (Vanderkooi et al., 1977), decrease fluidity (Rosenberg, Gripenberg and
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Anaesthesia may result from a direct interaction between anaesthetics and hydrophobic areas of proteins in addition to the effects on membrane lipids. Some workers have considered only the first part of this hypothesis and have argued the case for the critical site of anaesthetic action being exclusively in the proteins. However, we believe that this is a false
distinction and there is considerable indirect evidence that the molecular mechanisms of anaesthesia involve the majority of the components of the neuronal membrane. In order to prove this hypothesis it is necessary to study the membrane components separately and together. However, at the moment it is difficult to isolate and purify the various neuronal membrane proteins. For this reason the work on proteins has not advanced dramatically since Allison reviewed the subject in 1974. Woodbury, D'Arrigo and Eyring (1975) carried out a theoretical analysis of some of the general anaesthetic data and concluded that direct protein effects predominated. Their analysis was based in part on data from bacterial luciferase, which is a well-established model of a protein system for anaesthetic action. Luciferase used to be thought to be a good example of a protein unaffected by membrane lipids, but recent studies have demonstrated that this is not the case (King and White, 1976) and so Woodbury's analysis is not as convincing as it might be. The alternative approach of using model protein systems has proved useful in establishing the basis for anaesthetic-protein interactions (Brown, Halsey and Richards, 1976). Nuclear magnetic resonance studies of volatile anaesthetics altering the structure of haemoglobin (Halsey, Brown and Richards, 1978) have provided thefirstdirect evidence that anaesthetics can interact with hydrophobic pockets in proteins and the site(s) appear to behave as simple bulk solvents in terms of their solubility characteristics. The conformational changes produced by such hydrophobic interactions can be transmitted and detected in non-hydrophobic areas of the protein. The fact that conformational effects specific to individual anaesthetics are also observed in the same protein (Barker et al., 1975) is a potential explanation of the many selective actions of different anaesthetics. Haemoglobin is only a model protein, since there is no evidence that it is concerned with the mechanisms of general anaesthesia. Another protein of potentially greater neurophysiological importance is the acetylcholine receptor isolated from stingrays. The electric organs in these fish have very concentrated receptor proteins and thus it is possible to purify the material sufficiently for biochemical and biophysical experiments. Several groups of workers have started to study the anaesthetic interactions, and it has been demonstrated that the clinical volatile anaesthetics can dramatically facilitate a structural change of acetylcholine receptor protein induced by
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Jansson, 1977) or have no effect (Boggs, Young and Hsia, 1976). The confusion is probably caused by three factors. First, some of the techniques used for studying fluidity are inherently insensitive to small changes and for this reason the early workers (e.g. Trudell, Hubbell and Cohen, 1973) used high concentrations of the agent. However, more sensitive techniques, such as differential scanning calorimetry, have clearly demonstrated changes in lipid fluidity and other parameters at partial pressures well below the clinical range (Mountcastle, Biltonen and Halsey, 1978). The second factor is that Rosenberg and his colleagues have found that increasing anaesthetic concentrations can have a biphasic effect on lipid fluidity—first ordering the system and then fluidizing it (Rosenberg, Eibl and Stier, 1975; Rosenberg, Gripenberg and Jansson, 1977). Thus the problem of choosing the appropriate anaesthetic concentration is qualitative as well as quantitative. The third factor is the lipid composition. For example, pentobarbitone and the other anaesthetics can either decrease or increase the fluidity of phosphatidylcholine bilayers depending on their cholesterol content (Miller and Pang, 1976). However, when the cholesterol content exceeded 26% all the agents studied increased fluidity (Pang and Miller, 1978). There have been some recent studies with the alcohols which have shown that the short-chained compounds increase fluidity whereas those with more than 10 carbon atoms in the chain decreased fluidity (Richards et al., 1978). However, as yet these studies have not been extended to a wide range of lipid compositions and alcohol concentrations. Another study (Pringle and Miller, 1978) with two isomers of tetradecenol demonstrated that both compounds increase the fluidity of bilayers but change the phase transition temperature in opposite directions. Since the two alcohols are equipotent for tadpole anaesthesia, the evidence appears to favour a simple fluidity hypothesis. In all the alcohol studies just mentioned, the width of the transition peaks was not measured and so the potential importance of co-operativity changes has not yet been assessed.
MOLECULAR THEORIES OF ANAESTHESIA carbamoylcholine (Young et al., 1978). These experiments have been carried out with some of the membrane lipids still associated with the protein and it appears that this is a case where both components of the membrane contribute to the overall effect.
625 Eger, E. I., Lundgren, C , Miller, S. L., and Stevens, W. C. (1969). Anaesthetic potencies of SF,, CF 4 , chloroform and Ethrane in dogs: correlation with hydrate and lipid theories of anesthetic action. Anesthesiology, 30, 129. Eyring, H., Woodbury, J. W., and D'Arrigo, J. S. (1973). A molecular mechanism of general anesthesia. Anesthesiology, 38, 415.
FUTURE RESEARCH
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Haydon, D. A., Hendry, B. M., Levinson, S. R., and Requena, J. (1977). Anaesthesia by the n-Alkanes. REFERENCES A comparative study of nerve impulse blockage and the Allison, A. C. (1974). The effects of inhalational anaesproperties of black lipid bilayer membranes. Biochem. thetics on proteins; in Molecular Mechanisms in General Biophys. Acta, 470, 17. Anaesthesia (eds M. J. Halsey, R. A. Millar and J. A. Hendry, B. M., Urban, B. W., and Haydon, D. A. (1978). Sutton), p. 164. Edinburgh: Churchill Livingstone. The blockage of the electrical conductance in a poreAshcroft, R. G., Coster, H. G. L., and Smith, J. R. (1977). containing membrane by the n-Alkanes. Biochim. The molecular organisation of bimolecular lipid memBiophys. Acta, 513, 106. branes. The effect of benzyl alcohol on the structure. Hill, M. W. (1974). The Gibbs free energy hypothesis of Biochim. Biophys. Acta, 469, 13. general anaesthesia; in Molecular Mechanisms in General Bailey, C. P., Green, C. J., Halsey, M. J., and WardleyAnaesthesia (eds M. J. Halsey, R. A. Millar and J. A. Smith, B. (1977). High pressure and intravenous Sutton), p. 132. Edinburgh: Churchill Livingstone. steroid anaesthesia in rats. J. Appl. Physiol., 43, 183. Johnson, F. H., Brown, D., and Marsland, D. (1942a). Barker, R. W., Brown, F. F., Drake, R., Halsey, M. J., A basic mechanism in the biological effects of temperature, and Richards, R. E. (1975). Nuclear magnetic resonance pressure and narcotics. Science, 95, 200. studies of anaesthetic interactions with haemoglobin. (1942b). Pressure reversal of the action of Br.J. Anaesth., 47, 25. certain narcotics. J. Cell. Comp. Physiol., 20, 269. Boggs, J. M., Young, T., and Hsia, J. C. (1976). Site and Flagler, E. A. (1950). Hydrostatic pressure reversal mechanism of anesthetic action. I: Effect of anesthetics of narcosis in tadpoles. Science, 112, 91. and pressure on fluidity of spin-labeled vesicles. Molec. Pharmacol., 12, 127. (1951). Activity of narcotized amphibian larvae Brown, F. F., Halsey, M. J., and Richards, R. E. (1976). under hydrostatic pressure. J. Cell. Comp. Physiol., 37,15. Halothane interactions with haemoglobin. Proc. R. Soc. Johnson, S. M., and Miller, K. W. (1970). Antagonism of Lond. B, 193, 387. pressure and anaesthesia. Nature {Lond.), 228, 75.
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The present position is that we know a considerable amount about anaesthetic effects on lipids and it should be possible to distinguish between the alternative hypotheses on the way these changes are transmitted to functional proteins. We know considerably less about direct effects on membrane proteins and advances in this area depend on advances in the basic techniques for studying complete membranes. It is probable that interest will shift to include other aspects in synaptic transmission, such as the role of calcium and synaptic vesicle fusion processes. When the anaesthetic actions on the individual and integrated components of the synaptic functions have all been studied it may be possible to decide which are the predominant disruptions critical in general anaesthesia. The correlation studies will probably continue to be refined, but it is unlikely that any radically new concepts will emerge. The high pressure experiments have demonstrated that the in vivo molecular mechanisms are more complex than was thought 3 years ago. Future pressure work will be concerned with producing a more detailed theory that links up with the membrane lipid and protein hypotheses.
Ferguson, J. (1939). The use of chemical potentials as indices of toxicity. Proc. R. Soc. B., 127, 387. Franks, N. P., and Lieb, W. R. (1978). Where do general anaesthetics act ? Nature {Lond.), 274, 339. Green, C. J., Halsey, M. J. and Wardley-Smith, B. (1977). Possible protection against some of the physiological effects of high pressure. J. Physiol. {Lond.), 267, 46P. Halsey, M. J., Brown, F. F., and Richards, R. E. (1978). Perturbations of model protein systems as a basis for the central and peripheral mechanisms of general anaesthesia; in Molecular Interactions and Activity in Proteins, Ciba Foundation Symposium 60, Excerpta Medica, p. 123. Eger, E. I. (1971). The effect of pressure on the anesthetic potency of nitrous oxide. Fed. Proc, 30, 442. Higgs, E. G. (1976). Temperature dependence of anaesthetic potencies at high pressure. Br. J. Anaesth., 48, 265. Wardley-Smith, B. (1975). Pressure reversal of narcosis produced by anaesthetics, narcotics and tranquillizers. Nature {Lond.), 257, 811. Green, C. J. (1978). Pressure reversal of general anaesthesia—a multi-site expansion hypothesis. Br. J. Anaesth., 50, 1091. Hansch, C , Vittoria, A., Silipo, C , and Jow, P. Y. C. (1975). Partition coefficients and the structure-activity relationship of the anaesthetic gases. J. Med. Chem., 18, 546. Hanukoglu, I., and Trudell, J. F. (1977). Molecular models of anesthetic drug action—variations on a theme.
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Rosenberg, P. H., Eibl, H., and Stier, A. (1975). Biphasic effects of halothane on phospholipid and synaptic plasma membranes: a spin label study. Molec. Pharmacol., 11, 879. Gripenberg, J., and Jansson, S. E. (1977). Effects of halothane, thiopental, and lidocaine on fluidity of synaptic plasma membranes and artificial phospholipid membranes. Anesthesiology, 46, 322. Smith, R. A., Winter, P. M., Halsey, M. J., and Eger, E. I. (1975). Helium pressure produces a non-linear antagonism of argon or nitrogen anesthesia in mice. American Society of Anesthesiologists Annual Meeting Scientific Abstracts, 217. Spyropoulos, C. S. (1957). The effects of hydrostatic pressure upon the normal and narcotized nerve fibre. J. Gen. Physiol., 40, 849. Trudell, J. R. (1977). A unitary theory of anesthesia based on lateral phase separations in nerve membranes. Anesthesiology, 46, 5. Hubbell, W. L., and Cohen, E. N. (1973). The effect of two inhalation anesthetics on the order of spin-labeled phospholipid vesicles. Biochim. Biophys. Acta, 291, 321. Kendig, J. J. (1973). Pressure reversal of anesthesia: the extent of small-molecule exclusion from spin-labelled phospholipid model membranes. Anesthesiology, 38, 207. Payan, D. G., and Chin, J. H. (1974). Pressureinduced elevation of phase transition temperature in dipalmitoylphosphatidylcholine bilayers. An electron spin resonance measurement of the enthalpy of phase transition. Biochim. Biophys. Acta, 373, 436. Cohen, E. N. (1975). The antagonistic effect of an inhalational anesthetic and high pressure on the phase diagram of mixed dipalmitoyl-dimyristoylphosphatidylcholine bilayers. Proc. Nail. Acad. Sci. U.S.A., 72, 210. Vanderkooi, J. M., Landesberg, R., Selick, H. n, and McDonald, G. G. (1977). Interaction of general anesthetics with phospholipid vesicles and biological membranes. Biochim. Biophys. Acta, 464, 1. White, D. C , and Halsey, M. J. (1974). Effect of changes in temperature and pressure during experimental anaesthesia. Br. J. Anaesth., 46, 196. Winter, P. M., Smith, R. A., Smith, M., and Eger, E. I. (1976). Pressure antagonism of barbiturate anesthesia. Anesthesiology, 44, 416. Woodbury, J. W., D'Arrigo, J. S., and Eyring, H. (1975). Molecular mechanism of general anesthesia: lipoprotein conformation change theory; in Molecular Mechansims of Anesthesia (ed. B. R. Fink), Progress in Anesthesiology, Vol. 1, p. 253. New York: Raven Press. Young, A. P., Brown, F. F., Halsey, M. J., and Sigman, D. S. (1978). Volatile anesthetic facilitation of in vitro desensitization of membrane-bound acerylcholine receptor from Tropedo californica. Proc. Natl. Acad. Sci. U.S.A., 75, 4563.
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Kent, D. W., Halsey, M. J., Eger, E. I., and Kent, B. (1977). Isoflurane anesthesia and pressure antagonism in mice Anesth. Analg. (Cleve.), 56, 97. King, S M., and White, D. C. (1976). Membrane phase changes and anaesthetic action. Br.J. Anaesth., 48, 814. Lawrence, D. K., and Gill, E. W. (1975). Structurally specific effects of some steroid anesthetics on spinlabeled liposomes. Molec. Pharmacol., 11, 280. Lee, A. G. (1975). Interactions with biological membranes. Endeavour, 34, 67. (1976). Model for action of local anaesthetics. Nature (Lond.), 262, 545. Lever, M. J., Miller, K. W., Paton, W. D. M., and Smith, E. G. (1971). Pressure reversal of anaesthesia. Nature (Lond.), 231, 368. Metcalfe, J. C , Seeman, P., and Burgen, A. S. V. (1968). The proton relaxation of benzylalcohol in erythrocyte membranes. Molec. Pharmacol., 4, 87. Meyer, H. H. (1901). Zur Theorie der Alkoholnarkose. Arch. Exp. Pathol. Pharmacol., 46, 338. Meyer, K. H. (1937). Contributions to the theory of narcosis. Trans. Faraday Soc, 33, 1062. Miller, K. W., and Pang, K.-Y. Y. (1976). General anaesthetics can selectively perturb lipid bilayer membranes. Nature (Lond.), 263, 253. Paton, W. D. M., Smith, E. B., and Smith, R. A. (1972). Physicochemical approaches to the mode of action of general anesthetics. Anesthesiology, 36, 339. Smith, R. A., and Smith, E. B. (1973). The pressure reversal of anaesthesia and the critical volume hypothesis. Molec. Pharmacol., 9, 131. Wilson, M. W. (1978). The pressure reversal of a variety of anesthetic agents in mice. Anesthesiology, 48, 104. • Smith, R. A. (1978). Pressure resolves two sites of action for inert gases. Molec. Pharmacol., 14, 950. Miller, S. L. (1961). A theory of gaseous anaesthetics. Proc. Wash. Acad. Sci., 17, 481. Mountcastle, D. B., Biltonen, R. L., and Halsey, M. J. (1978). Effect of anesthetics and pressure on the thermotropic behavior of multilamellar dipalmitoylphosphatidylcholine liposomes. Proc. Natl. Acad. Sci. U.S.A., 75, 4906. Mullins, L. J. (1954). Some physical mechansims in narcosis. Chem. Rev., 54, 289. Overton, E. (1901). Studien uber die Narkose. Jena: Fischer. Pang, K.-Y Y., and Miller, K. W. (1978). Cholesterol modulates the effects of membrane perturbers in phospholipid vesicles and biomembranes. Biochim. Biophys. Ada, 511, 1. Pauling, L. (1961). A molecular theory of anaesthesia. Science, 134, 15. Pringle, M. J., and Miller, K. W. (1978). Structural isomers of tetradecenol discriminate between the lipid fluidity and phase transition theories of anesthesia. Biochem. Biophys. Res. Comm., 85, 1192. Richards, C. D., Martin, K., Gregory, S., Keightley, C. A., Hesketh, T. R., Smith, G. A., Warren, G. B., and Metcalfe, J. C. (1978). Degenerate perturbations of protein structure as the mechanism of anaesthetic action. Nature (Lond.), 276, 775.
BRITISH JOURNAL OF ANAESTHESIA
RECENT MOLECULAR THEORIES OF GENERAL ANAESTHESIA B. WARDLEY- SMITH AND M. J. HALSEY
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CORRELATIONS WITH PHYSICAL PROPERTIES
Although almost all the early physical theories of anaesthesia involved lipid solubility, two independent workers (Miller, 1961; Pauling, 1961) proposed an aqueous site of action in the central nervous system. BRIDGET WARDLEY-SMITH,
B.SC; MICHAEL J.
HALSEY,
D.PHIL. ; Division of Anaesthesia, Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ. 0007-0912/79/070619-08 $01.00
Though different in detail, both theories attempted to relate anaesthetic potency to the stability of the gas hydrate in aqueous solution. Their original correlations for a limited range of agents were reasonable, but studies with more anaesthetics proved that the agreement between potency and hydrate dissociation pressure was much worse than that for potency and oil/gas partition coefficient. These theories of an aqueous site of action have now been mainly abandoned. It is more generally assumed that anaesthetics act by interaction with a hydrophobic phase somewhere in the nervous system. This idea is not new, however, since one of the best correlations with potency is the solubility of the anaesthetic in olive oil. This was first recognized by H. H. Meyer and by Overton, both in 1901, and the lipid solubility theory that they proposed has been expressed in modern form by K. H. Meyer in 1937, who said that "Narcosis commences when any chemically indifferent substance has attained a certain molar concentration in the lipids of the cell. This concentration depends on the nature of the animal or cell but is independent of the narcotic." The idea that a physical property of an anaesthetic was critical was further expanded by Ferguson (1939), who found a reasonable correlation between vapour pressure at 37 °C and anaesthetic potency of a series of homologous alcohols. He concluded that thermodynamic activities were important as a measure of anaesthetic potency. Mullins (1954) carried out a thermodynamic analysis of the data then available to him and suggested that anaesthetic activity was not dependent on solubility alone but also on the molar volume of the anaesthetic. He suggested that anaesthetics might "depress cell function by preventing the passage across cell membranes of any particular molecule or ion, depending only on the degree of volume occlusion occasioned by the narcotic". This was an attractive model, but inclusion of molecular size in the data analysis does not improve the Meyer-Overton correlation between potency and oil solubility. The theoretical thermodynamic approach to anaesthetic © Macmillan Journals Ltd 1979
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.
The mechanism of anaesthesia has attracted the interest of many eminent scientists. For example, the references in this limited review include such wellknown names as Professors Burgen, Eyring, Paton and Pauling, who have all made contributions to different aspects of this field. In view of the wide range of scientific disciplines involved, it is not surprising that, since the introduction of general anaesthesia for surgery in the 1840's, there have been numerous ideas about the mechanisms of action of anaesthetics. By 1900 more than 25 of these had been published, and by the early 1960's more than 100 different theories of the mechanism of narcosis had been proposed. Of these theories, only a few are currently accepted, and this article is intended to discuss the molecular developments in anaesthesia research since the early 1970's. At first sight much of this work, which involves various model systems, is not directly relevant to the in vivo mechanisms of anaesthesia. A complete description of the mechanisms must include an understanding of how anaesthetics affect the integrated nervous system, single neurons and synapses as well as the molecular interactions. These approaches are not entirely separate. For example, the recognition that anaesthetics acted at hydrophobic sites reduced the possibility that anaesthetic actions in aqueous areas of neurons were critical. Anaesthetic research has tended to parallel the developments in human biology, and thus one of the intensive areas of current research is concerned with molecular biology and membrane biophysics. However, it is desirable to review briefly some of the earlier ideas which were important in the formation of the currently fashionable theories.
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HIGH PRESSURE STUDIES
It is one of the most striking features of anaesthetics that many of their effects are opposed by high hydrostatic or gaseous pressures. This was first demonstrated by Johnson, Brown and Marsland (1942a, b) who showed that the light from luminous bacteria, dimmed by anaesthetics, could be restored by high pressures. Johnson and Flagler (1950, 1951) anaesthetized tadpoles with alcohol until swimming had ceased, and then applied high hydrostatic pressure until swimming had spontaneously started again. Tadpoles were used recently to show that pressure reversal occurred with a far wider range of compounds than had been expected, including narcotics and tranquillizers as well as conventional anaesthetics (Halsey and Wardley-Smith, 1975).
It was not until the early 1970's that a unifying theory was put forward to explain the interactions of anaesthetics and pressure, and to provide a hypothesis of the physical mechanism of narcosis. The critical volume hypothesis (Lever et al., 1971; Miller et al., 1973) was proposed as an extension of the work of Mulhns, and states that "anaesthesia occurs when the volume of a hydrophobic region is caused to expand beyond a critical amount by the absorption of molecules of an inert substance. If the volume of this hydrophobic region can be restored by changes of temperature or pressure then the anaesthesia will be removed." This hypothesis assumed that all anaesthetics acted at the same molecular site. It was formulated from experiments using newts. On plotting increases in anaesthetic potency against total pressure for four anaesthetics, a straight line was obtained (fig. 1). This was in accordance with the 350 300 250 200 . 150 .
50
100 He
150 200 (ATA)
FIG. 1. Original pressure reversal data in newts, x axis— Helium total pressure (Pue) m atmospheres absolute (ATA); y axis—percentage of control anaesthetic dose at 1 ATA required to reduce the rolling response of newts to 50% of control value. • = Nitrogen; • = sulphur hexafluoride; 0 = nitrous oxide; A = carbon tetrafluoride (data from Miller et al., 1973).
predictions of the hypothesis, which stated that since equi-narcotic doses of all anaesthetics caused an equal volume increase, the degree of pressure reversal for a given total pressure should be the same for all anaesthetics. The critical volume hypothesis also suggested that "temperature reversal" would occur in a manner similar to pressure reversal, and it was calculated that the effects of a normal anaesthetic dose would be removed on cooling by 10 °C. This effect was predicted on the simple basis that the expansion caused by the anaesthetic could be counteracted by the
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mechanisms is still in use today. Eyring, Woodbury and D'Arrigo (1973) proposed a molecular mechanism of anaesthesia based on thermodynamic considerations, later expanding their ideas to formulate the lipoprotein conformational change theory (Woodbury, D'Arrigo and Eyring, 1975). In 1974, Hill analysed the results of dissolving anaesthetics in membranes in terms of the "Gibbs free energy hypothesis". The correlation of physical properties of anaesthetics with anaesthetic potency is still an accepted approach to the subject. In 1969 Eger and his colleagues measured anaesthetic potency in terms of dog MAC and correlated this with both lipid solubility and hydrate dissociation pressure. Three years later Miller and colleagues (1972) correlated anaesthetic potency measured in mice with anaesthetic solubility in solvents of different solubility parameters. In a recent study Franks and Lieb (1978) measured the partition coefficient for 22 anaesthetics in several different solvents, and found that the potency/partition coefficient correlation was better for octanol than for oil, and concluded that the site of action had both polar and apolar characteristics. This concept has been developed quantitatively by Hansch and his colleagues (1975) over the past 8 years. However, there is a limit to how much information can be obtained from simple solubility characteristics and thermodynamic analysis of whole animal data. It is necessary to look at other physical changes at the molecular level. There have been two main developments, first whole animal pressure reversal studies, and second, the use of model systems of lipids and proteins.
MOLECULAR THEORIES OF ANAESTHESIA 180 160 140 120 . 100 25
50
75
100
125
(ATA)
FIG. 2. Representative examples of data for pressure reversal of gaseous anaesthetics in mice, x axis—As figure 1; y axis—percentage of control anaesthetic dose at 1 ATA required to reduce righting reflex of mice to 50% of control value. V = Argon (Ar) (Smith et al., 1975); • = nitrous oxide (N2O) (White and Halsey, 1974); • = carbon tetrafluoride (CF4) (Miller, Wilson and Smith, 1978); O = nitrogen (N2) (Miller and Wilson, 1978).
compared the non-gaseous agents ethyl carbamate, a-chloralose and phenobarbitone, with argon and nitrogen and found differing degrees of pressure reversal between the two groups, but agreement within them. Some examples of the pressure reversal data for the i.v. agents are shown in figure 3. To summarize, the critical volume hypothesis stated that all anaesthetics acted at the same molecular site. It has one main predictive feature, namely that for a given increase in the total pressure, the percentage increase in anaesthetic required to maintain a constant level of anaesthesia should be the same for all agents. Theoretically this should be true to infinitely great pressures; the non-linearity at very high pressures was originally explained as being the result of the adverse physiological effects of increased pressures per se (e.g. Green, Halsey and WardleySmith, 1977). There is no doubt that our results with short acting i.v. anaesthetics do not support the concept of a single site of action for all anaesthetics. The deviations from the predicted straight line cannot be explained away as caused by pressure alone, since we have found non-linearity at pressures as small as 50 atm. We have thus developed a modification of the critical volume hypothesis in an attempt to explain the discrepancies in the data of both ourselves and others. It retains the original basic concept of the critical volume hypothesis, namely that anaesthetics expand and high pressures contract a molecular site
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contraction resulting from cooling. However, this is a more difficult concept to test, as even in cold blooded animals the predominating effect of cooling is hypothermia. Since extreme cases of hypothermia are seen as a state resembling anaesthesia, assessment of anaesthetic potency becomes unreliable. Spyropoulos (1957) removed the effect of a 3% ethanol solution on the action potential of toad nerve fibre by cooling by 15 °C, but a reduction in solubility would also occur with lowering of temperature, and thus precise analysis of his results is not easy. Preliminary experiments with nitrous oxide and nitrogen on intact animals (Halsey and Higgs, 1976) and with some of the alcohols on an in vitro nerve preparation (Richards et al., 1978) showed no evidence of "temperature reversal of anaesthesia". However, because of difficulties of interpretation this aspect of the critical volume hypothesis remains one that is not resolved. Early experiments in normothermic mammals (Halsey and Eger, 1971) provided support for the hypothesis since the pressure reversal values coincided with the predicted straight line. Subsequent mammalian experiments, however, produced less clear cut results. Smith and colleagues (1975) studied the pressure reversal of argon and nitrogen anaesthesia, while Kent and colleagues (1977) expanded the range of agents to include the volatile clinical anaesthetic, isoflurane. The original work with nitrous oxide was extended to greater pressures (White and Halsey, 1974). Recently Miller, Wilson and Smith (1978) have studied the perfluorinated anaesthetics. Examples of all these data are plotted in figure 2. The first quantitative experiments with nongaseous anaesthetics in mammals were by Winter and colleagues (1976). They used phenobarbitone in mice, and gave a single dose before compression. The anaesthetic effect of this agent was reversed by pressure, and the increase in ED50 at 103 atm was equal to that for isoflurane, but greater than that for nitrous oxide. However, the technique of preadministration of a single dose before compression makes it hard to be certain that changes in metabolism are not taking place, especially with long-acting anaesthetics such as phenobarbitone. We have developed a continuous infusion technique for studying the short acting i.v. anaesthetics at pressure. Initial experiments, with Althesin (Bailey et al., 1977), have been extended to other i.v. anaesthetics (Halsey, Wardley- Smith and Green, 1978). The degree of pressure reversal varied considerably between the different agents. Miller and Wilson (1978)
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100 FIG. 3. Representative examples of data for pressure reversal of non-inhalation anaesthetics in rats and mice. x axis—As figures 1 and 2; y axis—as figure 2. Mice data for phenobarbitone (Ph • ) and urethane (Ur v) with anaesthesia denned as loss of righting reflex (data from Miller and Wilson, 1978). Rat data for Althesin (Alth A), thiopentone (Thio • ) and ketamine (Ket ©) with anaesthesia denned as loss of response to electrical stimulus (data from Bailey et al., 1977; Halsey, Wardley-Smith and Green, 1978).
of action, but differs from it in a number of fundamental ways. We have provisionally termed it the multi-site expansion hypothesis; it is based on all the current intact animal data of both ourselves and other workers, and can be best summarized in the following tenets. (1) General anaesthesia can be produced by the expansion of more than one molecular site, and these sites may have differing physical properties. (2) The physical properties of a molecular site may themselves be influenced by the presence of anaesthetics or pressure. We believe that the most plausible receptor site characteristic which may change is the compressibility. (3) The molecular sites do not behave as if they were bulk solvents, but have a finite size and a limited degree of occupancy. (4) Pressure itself need not necessarily act at the same site as the anaesthetic. Depending on the anaesthetic, one of the sites may predominate in determining the interaction with pressure.
The correlation studies and high pressure experiments cannot provide a detailed molecular hypothesis about how general anaesthetics may modify the many membrane functions which are potentially important in the transmission of nerve impulses. There have been a number of studies on in vitro nerve preparations and isolated components of synaptic junctions, but it is difficult to interpret these data in terms of the underlying molecular events or to extrapolate the results to the in vivo integrated nervous system. It has proved more productive to study various simplified lipid models of nerve membranes. These are an oversimplification of the in vivo system, but it should be possible to separate out the several molecular changes that are produced by anaesthetics and decide which of these are likely to be critical in the production of general anaesthesia. The lipids in membranes are now thought not only to provide the basic permeability barrier, but also to influence the activity of proteins which control ionic and neurotransmitter fluxes. The important properties of lipids in controlling protein activities appear to be their ability to move and rotate within tie basic bilayer membrane structure. The fluidity or degree of order of the lipids has been shown, for example, to control the activity of the protein of the calcium pump (see review by Lee, 1975). This fluidity can be measured by either nuclear magnetic resonance studies of the lipids themselves or electron paramagnetic resonance studies of marker compounds incorporated into the bilayer. Both techniques are sensitive to the fluidity of the environment around the particular part of the molecule being studied. In pure lipid preparations it has been found that as the
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(5) The molecular sites for anaesthesia are not perturbed by a decrease in temperature in a manner analogous to an increase in pressure. A more detailed discussion of the multi-site expansion hypothesis, and the evidence on which it is based, can be found elsewhere (Halsey, WardleySmith and Green, 1978) together with the predictive features by which it can be tested. Although the multisite expansion hypothesis is more complex than the critical volume theory, it seems to us to be necessary to explain all the current pressure-anaesthetic data. However, it should not be regarded as an alternative to the molecular theories of anaesthesia based on lipid fluidity. Phase changes are accompanied by an increase in volume, and thus are susceptible to pressure reversal (Trudell et al., 1973, 1975).
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temperature is increased there is a general increase in fluidity; at a specific temperature there is a sudden step change when the fluidity increases dramatically. Below this temperature the lipids are described as being in the ordered gel state; above this temperature the fluidity of the lipids are described as being in the disordered liquid crystalline state. This "phase change" can be studied not only by magnetic resonance techniques but also by direct calorimetry, since at the phase change there is a sudden increase in the specific heat (analogous to that which occurs when any solid melts). For the past 10 years it has been known that anaesthetics alter the fluidity of the lipids and membranes. Metcalfe, Seeman and Burgen (1968) demonstrated that as the concentration of benzyl alcohol in the erythrocyte membrane (or its extracted lipids) was increased, the fluidity of the system also increased. They anticipated more recent concepts by speculating that the presence of the anaesthetic in the membrane produced more and more disorder until finally the protein was exposed. This made available more binding sites in the protein. Since 1970, there has been a series of theories as to how anaesthetic-induced changes in fluidity of the lipids might influence the activities of the proteins. In general terms, Johnson and Miller (1970) postulated that anaesthetics cause an increase in freedom of movement of the lipid parts of membranes which is associated with an increase in volume which in turn might affect conformational changes in proteins intimately associated with lipid. This paper linked lipid effects with the whole animal data on pressure reversal. Other evidence for the importance of fluidity effects was provided by the demonstration that non-anaesthetic steroids did not alter bilayer fluidity (Lawrence and Gill, 1975). Subsequently, Lee (1976) has taken the association between lipids and proteins one stage further and argued that a sodium channel protein requires an "annulus of lipid" in the more solid gel state to allow activity. Anaesthetics fluidize this lipid, causing the protein to relax into an inactive conformation. There have been a number of studies demonstrating that anaesthetics shift the phase transition to a lower temperature (Trudell, Payan and Chin, 1974) which results in a much larger increase in anaestheticinduced fluidity at the original phase transition temperature. This has led to a theory based on "lateral phase separations" (Trudell, 1977). When a lipid bilayer membrane is at the phase change temperature, both the ordered and disordered phases
are in equilibrium with each other. At this point any change in internal pressure in the system (produced, for example, by conformational changes in proteins) can be accommodated by shifting the equilibrium position—that is there is "lateral compressibility". If the position of the phase change is shifted by anaesthetics, the equilibrium between the two phases is also shifted so that one of them is no longer present. When only one phase is present the ability of the system to accommodate volume changes is much reduced and the environment of the protein becomes more rigid with respect to lateral expansion (Hanukoglu and Trudell, 1977). The hypotheses summarized so far have considered the effects of the lipid environment on the functioning of individual proteins. However, this is an oversimplification in that, in vivo, groups of a particular type of protein function together in a co-operative manner. The energetic links between these proteins are probably provided by the lipids between them, which themselves are grouped together in clusters either in the gel or in the liquid state. If these gel or liquid crystal clusters are reduced below a critical size then the co-operative linkage between the proteins is disrupted. Experiments with halothane and enflurane in simple lipid systems using differential scanning calorimetry have demonstrated that not only is the fluidity altered but also lipid co-operativity and hence cluster size is reduced (Mountcastle, Biltonen and Halsey, 1978). Two independent groups have recently put forward an entirely different concept as to how lipids may affect protein function (Ashcroft, Coster and Smith, 1977; Haydon et al., 1977; Hendry, Urban and Haydon, 1978). They proposed that the thickness of the lipid bilayer is increased by anaesthetics, which prevents the protein pore from adequately spanning the membrane. Their evidence for this phenomenon, which is based on electrical measurements of lipid films, is controversial because other techniques—such as x-ray and neutron diffraction (Franks and Lieb, 1978)—indicate that at anaesthetic concentrations there are no significant changes in membrane thickness. In addition to the discussions about the alternative hypotheses, there are at least two areas of anaesthetic actions on lipids which are controversial: the effects of varying the anaesthetic concentration and the lipid composition. For example, different groups of workers have found that "clinical concentrations" of halothane increase lipid fluidity (Vanderkooi et al., 1977), decrease fluidity (Rosenberg, Gripenberg and
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PROTEIN CHANGES
Anaesthesia may result from a direct interaction between anaesthetics and hydrophobic areas of proteins in addition to the effects on membrane lipids. Some workers have considered only the first part of this hypothesis and have argued the case for the critical site of anaesthetic action being exclusively in the proteins. However, we believe that this is a false
distinction and there is considerable indirect evidence that the molecular mechanisms of anaesthesia involve the majority of the components of the neuronal membrane. In order to prove this hypothesis it is necessary to study the membrane components separately and together. However, at the moment it is difficult to isolate and purify the various neuronal membrane proteins. For this reason the work on proteins has not advanced dramatically since Allison reviewed the subject in 1974. Woodbury, D'Arrigo and Eyring (1975) carried out a theoretical analysis of some of the general anaesthetic data and concluded that direct protein effects predominated. Their analysis was based in part on data from bacterial luciferase, which is a well-established model of a protein system for anaesthetic action. Luciferase used to be thought to be a good example of a protein unaffected by membrane lipids, but recent studies have demonstrated that this is not the case (King and White, 1976) and so Woodbury's analysis is not as convincing as it might be. The alternative approach of using model protein systems has proved useful in establishing the basis for anaesthetic-protein interactions (Brown, Halsey and Richards, 1976). Nuclear magnetic resonance studies of volatile anaesthetics altering the structure of haemoglobin (Halsey, Brown and Richards, 1978) have provided thefirstdirect evidence that anaesthetics can interact with hydrophobic pockets in proteins and the site(s) appear to behave as simple bulk solvents in terms of their solubility characteristics. The conformational changes produced by such hydrophobic interactions can be transmitted and detected in non-hydrophobic areas of the protein. The fact that conformational effects specific to individual anaesthetics are also observed in the same protein (Barker et al., 1975) is a potential explanation of the many selective actions of different anaesthetics. Haemoglobin is only a model protein, since there is no evidence that it is concerned with the mechanisms of general anaesthesia. Another protein of potentially greater neurophysiological importance is the acetylcholine receptor isolated from stingrays. The electric organs in these fish have very concentrated receptor proteins and thus it is possible to purify the material sufficiently for biochemical and biophysical experiments. Several groups of workers have started to study the anaesthetic interactions, and it has been demonstrated that the clinical volatile anaesthetics can dramatically facilitate a structural change of acetylcholine receptor protein induced by
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Jansson, 1977) or have no effect (Boggs, Young and Hsia, 1976). The confusion is probably caused by three factors. First, some of the techniques used for studying fluidity are inherently insensitive to small changes and for this reason the early workers (e.g. Trudell, Hubbell and Cohen, 1973) used high concentrations of the agent. However, more sensitive techniques, such as differential scanning calorimetry, have clearly demonstrated changes in lipid fluidity and other parameters at partial pressures well below the clinical range (Mountcastle, Biltonen and Halsey, 1978). The second factor is that Rosenberg and his colleagues have found that increasing anaesthetic concentrations can have a biphasic effect on lipid fluidity—first ordering the system and then fluidizing it (Rosenberg, Eibl and Stier, 1975; Rosenberg, Gripenberg and Jansson, 1977). Thus the problem of choosing the appropriate anaesthetic concentration is qualitative as well as quantitative. The third factor is the lipid composition. For example, pentobarbitone and the other anaesthetics can either decrease or increase the fluidity of phosphatidylcholine bilayers depending on their cholesterol content (Miller and Pang, 1976). However, when the cholesterol content exceeded 26% all the agents studied increased fluidity (Pang and Miller, 1978). There have been some recent studies with the alcohols which have shown that the short-chained compounds increase fluidity whereas those with more than 10 carbon atoms in the chain decreased fluidity (Richards et al., 1978). However, as yet these studies have not been extended to a wide range of lipid compositions and alcohol concentrations. Another study (Pringle and Miller, 1978) with two isomers of tetradecenol demonstrated that both compounds increase the fluidity of bilayers but change the phase transition temperature in opposite directions. Since the two alcohols are equipotent for tadpole anaesthesia, the evidence appears to favour a simple fluidity hypothesis. In all the alcohol studies just mentioned, the width of the transition peaks was not measured and so the potential importance of co-operativity changes has not yet been assessed.
MOLECULAR THEORIES OF ANAESTHESIA carbamoylcholine (Young et al., 1978). These experiments have been carried out with some of the membrane lipids still associated with the protein and it appears that this is a case where both components of the membrane contribute to the overall effect.
625 Eger, E. I., Lundgren, C , Miller, S. L., and Stevens, W. C. (1969). Anaesthetic potencies of SF,, CF 4 , chloroform and Ethrane in dogs: correlation with hydrate and lipid theories of anesthetic action. Anesthesiology, 30, 129. Eyring, H., Woodbury, J. W., and D'Arrigo, J. S. (1973). A molecular mechanism of general anesthesia. Anesthesiology, 38, 415.
FUTURE RESEARCH
Anesthesiology, 47, 532.
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The present position is that we know a considerable amount about anaesthetic effects on lipids and it should be possible to distinguish between the alternative hypotheses on the way these changes are transmitted to functional proteins. We know considerably less about direct effects on membrane proteins and advances in this area depend on advances in the basic techniques for studying complete membranes. It is probable that interest will shift to include other aspects in synaptic transmission, such as the role of calcium and synaptic vesicle fusion processes. When the anaesthetic actions on the individual and integrated components of the synaptic functions have all been studied it may be possible to decide which are the predominant disruptions critical in general anaesthesia. The correlation studies will probably continue to be refined, but it is unlikely that any radically new concepts will emerge. The high pressure experiments have demonstrated that the in vivo molecular mechanisms are more complex than was thought 3 years ago. Future pressure work will be concerned with producing a more detailed theory that links up with the membrane lipid and protein hypotheses.
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BRITISH JOURNAL OF ANAESTHESIA
