Molecular mechanism of general anaesthetic action?

TIPS -January 1988 [Vol. 9] favourable increase in free energy on ascending the series, but the maximum aqueous concentration achievable still decreases inexorably. Thus on ascending the series we have a situation which approximates very closely to one in which a molecule fits the site perfectly but where we systematically reduce its aqueous concentration the biological effect gets smaller and smaller and eventually cannot be perceived, a cut-off. Now let us apply that reasoning to the effects of n-alkanes on the functioning of sodium channels in the squid giant axon. One of the major contributors to the local anaesthetic action of hydrocarbons such as n-pentane is an increase in the fraction of channels which are inactivated at the resting potential 4. This increase is manifested as a hyperpolarizing shift in the relationship between the fraction of resting (or available} channels and the membrane potential, saturated n-pentane producing shifts of 15--20 mV. The actions of other series members have been reported s,6 and the effect cuts off at around n-octane, with n-pentane showing the maximum activity 5. The putative protein pocket should therefore enable n-pentane to fit snugly but part of larger alkanes ought to project out into the aqueous phase. Occupation of this pocket by an alkane chain should lock the sodium channel into the inactivated state, thus explaining the increase in resting inactivation. The decreased effectiveness of noctane should be explicable by the reduced aqueous concentration. (Sea water saturated with npentane contains --0.3raM, the transition to n-octane reduces that concentration 100-foldT.) It follows that in order to make n-octane an inhibitor, we need a way of increasing its aqueous concentration. Why not put a hydroxyl group on one end, giving noctanol? Then we can achieve an aqueous concentration well above even saturated n-pentane, five carbons could occupy the pentane-sized protein pocket and the hydroxyl group could still interact with the water. (Note that the model does not imply that the hydroxylation of n-pentane, giving n-pentanol, would cause an increased potency in terms of aqueous concentration since the -

11 presence of the hydroxyl group will reduce partition of the entire molecule into a hydrophobic phase. The molecule needs to be long enough to allow the hydroxyl group to maintain interactions with water while the hydrophobic tail fills the pocket.) In the luciferase system the extrapolated EDso for n-octane is higher than its saturated aqueous concentration but the n-octanol EDs0 is less than one-tenth saturated; thus n-octane is inactive while n-octanol is highly active 2. Such a model therefore predicts that n-octanol will cause large shifts in the voltage dependence of squid sodium channel inactivation. It does not: 0.44 mM(--,oneeighth saturated under the conditions of measurement) causes a shift of only - 2 mV (Ref. 8) and Oxford and Swenson 9 report shifts of less than - 4 mV for I mM noctanol, the highest concentration investigated in the squid. The conclusion seems clear: whatever the mechanism of squid sodium channel inactivation by n-alkanes, it is not by binding to a protein site accessible from the aqueous phase° There are, however,

interesting species differences in the effect of ~-octanol on Na + channel inactivation. Thus, in crayfish and frog 1°,11, n-octanol does produce hyperpolarizing shifts comparable to those produced by n-pentane in the squid. JIM ELLIOTT

Department of Physiology, The University, Dundee DD1 4HN, UK.

References 1 Hille, B. (1977) J. Gen. Physiol. 60, 497515 2 Franks, N. P. and Lieb, W.R. (1985) Nature 316, 349-351 3 Elliott, J. R. and McEIwee, A. A. Br. J. Anaesth. (in press) 4 Haydon, D. A. and Kimura, J. E. (1981) J. Physiol. (London) 312, 57-70 5 Elliott, J. R., Murreli, R. D. and Haydon, D.A. (1987) J. Membrane Biol. 95, 143-149 6 Haydon, D. A. and Urban, B. W. (1983) ]. Physiol. (London) 338, 435--450 7 Haydon, D. A., Hendry, B. M., Levinson, S.R. and Requena, J. (1977) Biochim. Biophys. Acta 470, 17-34 8 Haydon, D. A. and Urban, B. W. (1983) ]. Physiol. (London) 341, 411-427 9 Oxford, G. S. and Swenson, R. P. (1979) Biophys. J. 26, 585--590 10 Swensen, R. P. and Narahashi, T. (1980) Biochim. Biophys. Acta 603, 228-236 11 Hirche, G. (1985) Pfli~gers Arch. 405, 180-187

Molecular mechanism of general anaesthetic action? In the viewpoint entitled 'What is the molecular nature of general anaesthetic target sites?' Drs Franks and Lieb (TIPS8,169-174, May 1987) proposed that general anaesthetics interact with proteins. They are right to question the lipid theories of anaesthetic action. But in formulating their alternative theory of anaesthetic action, perhaps the vast number of studies involving the potent intravenous anaesthetics such as barbiturates, steroids, ketamine, urethanes, etc. should also be considered. The substances in their study are in general small, largely hydrophobic molecules many of which could not be regarded as clinically useful anaesthetics. Furthermore, many of the agents appeared to have been tested on aquatic non-mammalian species where there is difficulty in distinguishing between a condition resembling clinical anaesthesia in mammals, where vital functions are maintained, and a more general depression of vital func-

tions which might be best described as narcotization. The latter may also be replicated by local anaesthetics which clearly have a quite different effect to general anaesthetics in mammals. Franks and Lieb are likely to be correct in their assumptions that anaesthetics act at proteins. This begs the question, at which protein(s) do anaesthetics act? A clear body of evidence indicates that a wide range of general anaesthetics have a potent action at the GABA receptor-Cl- channel complex (the structure of which has recently been elucidated 1) and this action has the effect of prolonging synaptic inhibition in the CNS (e.g. Refs 2-5). Other experiments have shown some anaesthetics may interfere with the action of excitatory transmitters at the receptors (e.g. Refs 6, 7). The usefulness of any study which compares molecular properties would be greatly enhanced by using a range of clinically use-

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TIPS - January 1988 [Vol. 9]

12 ful general anaesthetic agents which have a wide range of physical and chemical properties. Although the literature abounds with data on anaesthetic potencies, any whole animal study is still fraught with interpretative difficulties because anaesthetic potency is often a reflection of pharmacokinetic parameters such as penetration of the blood-brain barrier, metabolism, etc. Data is available on the plasma or cerebrospinal fluid (c.s.f.) levels for some general anaesthetics but these measurements have been made by different authors in dif-

ferent species. So there is a desperate requirement for a systematic measurement of concentrations of a wide range of anaesthetics in c.s.f, of a single mammalian species. Such data would then be a great asset not only to the type of study described by Franks and Lieb but also to m a n y authors making cellular and neurophysiological studies who are also guilty of restricting their work to a few selected anaesthetics, mostly of the intravenous types. C. N. SCHOLFIELD

Department of Physiology, Queen's University, 97 Lisburn Road, Belfast, UK.

The statistical model has no clothes!

Multiple t-tests are appropriate in science In a recent letter 1, Kitchen criticized the way statistics are currently being used in pharmacology and advocated the use of more common sense and the 'Bloody Obvious Test'. The problem is, however, more fundamental. For many decades statistics texts have provided scientists with a classification of statistics and a model for their use that is, in fact, contrary to both appropriate scientific practice and to the scientific method. The discrepancy is illustrated clearly by a case in which most researchers already violate the statistical model. Inferential statistics, such as the t-test and the analysis of variance, are supposed to be used for making decisions about hypotheses. The statistical model for using inferential statistics states that before conducting an experiment, you first must propose a hypothesis and select a single probability level at which you will reject this hypothesis. In reality, however, most researchers employ multiple rejection levels, reporting p<0.05, p<0.01, p<0.001, etc. The reason for this practice is that the results of statistical tests are used not as inferential statistics but rather as descriptive statistics for improving communication in a scientific report. Researchers do not present their colleagues with a statement about whether they have decided the hypothesis is false or not. Instead, they try to provide their readers with the information they need to form their own opinions. The whole

idea of making such decisions, although central to the statistical model, is contrary to the scientific method. In science, we are never absolutely sure of anything. Instead we attach our own degree of confidence to every finding we make or read. We assign more confidence to a finding with p<0.001 than to one with p<0.05. The p value for a result in a scientific paper is, therefore, a descriptive statistic, like the mean and standard deviation. It is a tool for communicating, providing the reader with the information needed for assigning a confidence rating to the result. The t-test, the analysis of variance, and all of the other tests are merely formulas for generating the p values. There are no inferential statistics in science. The process of inference occurs not within these tests, but rather over a long period of time as a part of the scientific method. According to Wine~, 'if the meaningful comparisons are relatively few in n u m b e r and are planned before the data are collected', they can be tested separately in a manner equivalent to using multiple t-tests. However, a different rule has apparently passed down to scientists (see Refs 1 and 3), stating that it is always inappropriate to use several t-tests when the material could be combined and tested with an analysis of variance followed by one of the a posteriori tests (e.g. Duncan, Newman-Keuls, Tukey, Scheff~) 2. If we consider the real purpose of statistics in a scientific report,

~) 1988, Elsevier Publications, Cambridse 0165- 6147188/$02,00

References 1 Schofield, P. R., Daflison, M. G., Fujita, N., Buff, D. R., Stephenson, F. A., Rodriguez, H., Rhee, L. M., Ramachandran, ]., Glencorse, T. E., Seeburg, P. H. and Barnard, E. A. (1987) Nature 328, 221-227 2 Nicholl, R. A. (1972) J. Physiol. (London) 223, 803-814 3 Barker, J. L. and Ransom, B.R. (1978) J. Physiol. (London) 280, 355-372 4 Schoifield, C. N. (1980) Pfliigers Arch. 383, 249-255 5 Harrison, N. L. and Simmonds, M.A. (1984) Brain Res. 323, 287-292 6 Richards, C. D. and Smaje, J. C. (1976) Br. J. Pharmacol. 58, 347-357 7 Martin, D. and Lodge, D. (?~85) Neuropharmacology 24, 999-1003

however, it is clear that the a posteriori tests are themselves inappropriate. If the p value is to be useful as a descriptive statistic, it must bear a fixed relationship to the confidence the reader can have in the result and the p values generated by t-tests have this characteristic. The p values obtained with a posteriori tests, however, vary. with the particular test employed 2 and also with the n u m b e r of other results that happen to be included in the same experiment. For instance, suppose you study five experimental drugs, only one of which is effective, along with a placebo. If you forget about the four ineffective drugs, a comparison of the effective drug with the placebo might give p - 0.001. If you include, say, two of the other drugs in your analysis, the p for the effective drug may drop to 0.01, and if you include all of the drugs it m a y go to 0.05. A replication attempt with a total of ten drugs would probably not find a significant p value for the effective drug. The a posteriori tests allow the p value for the individual results to vary in this way because they attempt to keep the rate of type I errors (i.e. inappropriately concluding a result is significant) constant within an experiment. There is, however, no logical reason w h y a researcher or his or her readers should want this rate to be kept constant. It serves no purpose in science. There is nothing special about the individual experiment that makes it the unit over which the rate should be kept constant. The p values generated by a posteriori tests are, therefore, not