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R e f e r e n c e s

1 Huxtable, R. J. (1983) Trends Pharmacol. Sci. 4, 249-251

2 Quinn, R. J., Gregson, R. P., Cook, A. F. and Bartlett, R. J. (1980) Tetrahedron Lett. 21, 567-568

3 Kim, Y. H., Nachman, R. J., Pavelka, L. and Mosher, H. S. (1981) J. Nat. Prod. 44, 206-214

4 Grozinger, K., Freter, K.R., Farina, P. and Gladczuk, A. (1983) Eur. J. Med. Chem. Chim. Ther. 18, 221-226

5 Baird-Lambert, J. A., Marwood, J.F., Davies, L.P. and Taylor, K. M. (1980)

Life Sci. 26, 1069-1077 6 Buckle, P. J. and Spence, I. (1981)

Naunyn-Schmied Arch. Pharmacol. 316, 64--68

7 Davies, L. P., Baird-Lambert, J.A., Jamieson, D. D. and Spence, I. (1982) in Physiology and Pharmacology of Adeno- sine Derivatives (Daly, J. W., Kuroda, Y., Phillis, J.W., Shimizu, H. and Ui, H., eds), pp. 257-266, Raven Press, New York

8 Phillis, J. W. (1982) in Physiology and Pharmacology of Adenosine Derivatives (Daly, J. W., Kuroda, Y., Phillis, J.W., Shimizu, H. and Ui, H., eds), pp. 219-

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236, Raven Press, New York 9 Davies, L. P., Baird-Lambert, J. A. and

Jamieson, D. D. (1982) Gen. PharmacoL 13, 27--33

10 Kazlauskas, R., Murphy, P.T., Wells, R.J., Baird-Lambert, J.A. and Jamie- son, D. D. (1983) Aust. J. Chem. 36, 165- 170

11 Davies, L. P., Jamieson, D.D., Baird- Lambert, J. A. and Kazlauskas, R. (1984) Biochem. Pharmacol. 33, 347-355

12 Persson, C. G. A., Karlsson, J.-A. and Erjefalt, I. (1982) Life Sci. 30, 2181-2189

13 Bergmann, W. and Burke, D. C. (1956) J. Org. Chem. 21, 226-228

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Morpheus in Calgary

Third International Conference on Molecular and Cellular Mechanisms of Anesthesia, University of Calgary, Calgary, Alberta, Canada. June 1984.

The mechanisms by which anes- thetics cast their spell upon the nervous system are among the most elusive known to pharmaco- logy. A small but dedica ted band of scientists have struggled wi th the p rob lem since the turn of the century, when Meyer and Over ton formula ted the great ' rule ' which bears their names: The potency of general anesthet ics is propor t ional to their l ip id solubil i ty. This pro- por t ional i ty is l inear and is re- markab ly closely obeyed by anes- thetics ranging in potency over five orders of magni tude . The th i rd gather ing of the Interna- t ional Conference on Molecular and Cellular Mechanisms of Anes- thesia was recently held at the Univers i ty of Calgary in Alberta, Canada, jo int ly organized by Sheldon Roth (Calgary) and Keith Miller (Harvard).

The actions of anesthet ics on Voltage-Gated Ion Conductances were examined in the first sympo- s ium, chaired by Gary Strichartz (Harvard). That local anesthet ics prevent neuronal excitation pr im- ari ly by affecting the voltage- sensi t ive sod ium channels is no longer in dispute , but impor tan t ques t ions remain. In this context, Murdoch Ritchie (Yale) descr ibed the usefulness of the Modula ted Receptor model for local anesthe- tic action, p roposed in 1977 by Bertil HiUe. Briefly, this model postula tes that the sod ium chan- nel, wi th its local anesthet ic recep-

tor, undergoes t ransi t ions among (at least) three states: closed (rest- ing), open and inactivated. These states differ in the b ind ing affini- ties and rate constants which characterize their interact ion with local anesthetics. Each agent 's characterist ic profile of actions on the sod ium current thus emerges from specific per turba t ions of the channel ' s normal kinetic process.

A central predic t ion of the Modula ted Receptor model, that local anesthet ics act by s tabi l iz ing an inactive channel conformation, der ived apparen t suppor t from studies by Bruce Bean (Iowa) on the ant ia r rhythmic action of l ido- caine. He presented evidence from vol tage-clamped Purkinje fibers that, at concentrat ions far lower than those required to block impulse conduct ion in the nerve, l idocaine produces a frequency- dependen t block which arises from b ind ing to inactivated sod ium channels. In principle, however , f requency-dependent block could arise from drug b ind - ing ei ther to inact ivated states of the channel, as original ly postul- ated in the model, or to act ivated or open channels. Strichartz pre- sented several l ines of evidence ind ica t ing that in nerve, it is in fact unnecessary to postulate local anesthet ic b ind ing to inact ivated channels. For example, in nerve fibers treated with scorpion toxin or N-bromoacetamide , procedures which ' remove ' the normal inacti- va t ion process, f requency-depen- dent block was not affected. Thus, a l though a full account is not yet possible, it is clear that no single mechanism can account for the var ious effects exerted by local anesthetics at different t issue sites.

1985, Elsevier Science Pubiisher~l B.V., Amsterdam 0165 - 6147/85/$02.00

Jay Yeh (Northwestern) pre- sented a patch-clamp electrophy- siological analysis of local anes- thetic effects on sod ium channels in neuroblas toma cells. Quali ta- tive differences be tween the ac- t ions of 9-aminoacr id ine and QX- 314 (a l idocaine derivative) were observed. In the presence of 9- aminoacr id ine , channel openings were in ter rupted by numerous brief closures ( 'flickering'). The mean open t ime showed a propor- t ional decrease wi th increasing drug concentration, consistent wi th a sequential model in which drug molecules enter and block open sod ium channels, prevent- ing closure unti l the molecule has left the blocking site. QX-314, by contrast, caused no flickering, but s imply shortened the dura t ion of the open state. Both drugs left the uni ta ry conductance of the sodium channel unaltered.

Max Wil low (Universi ty of Washington) then repor ted bio- chemical and electrophysiological s tudies examining local anesthetic effects on sodium channels in synaptosomes and neuroblas toma cells. The sod ium channel is known to exhibi t at least three functionally dist inct neurotoxin- b ind ing sites: one which b inds tetrodotoxin and saxitoxin, inhi- b i t ing sod ium flux through the channels; a second at which vera- t r idine and batrachotoxin act to produce pers is tent channel activa- tion; and a third at which scor- p ion toxin b inds , inh ib i t ing the inact ivat ion process. Wil low pre- sented data indica t ing that thera- peutic concentrat ions of a var ie ty of local anesthetics allosterically antagonize the b ind ing of batra- chotoxin to synaptosomes, as well as its action on sod ium flux across neuronal membranes . Again, these results p resumably reflect the phenomenon of s ta te -depen- dent b ind ing postula ted in the

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Modula ted Receptor model , sug- gest ing that local anesthet ics stab- ilize a sod ium channel conforma- tion which cannot b ind batracho- toxin.

Dennis Haydon (Cambridge) summar ized work from his labor- atory on the effects of general anesthet ics and other l ipophi l ic agents, inc luding the straight- chain alkanes and alcohols in homologous series, on sod ium and po tass ium currents in the vol tage-c lamped squ id axon. The net suppress ion of ionic currents by each anesthet ic was shown to arise from a specific combina t ion of effects, varying wi th the mole- cular structure of the agent, on the classical Hodgk in -Hux ley para- meters. Haydon p roposed that these effects are exerted in part by direct act ions at the channel pro- tein and in part by an indirect mechanism, involving a thicken- ing of the axonal membrane by anesthetics. Membrane thicken- ing, assessed by measurement of electrical capacity, was shown to occur in the presence of the hy- drocarbon anesthet ics (but not in the presence of the alcohols). This p h e n o m e n o n can explain some of the observed effects of hydrocar- bons on the Hodgk in -Hux ley parameters , wi thout pos tu la t ing specific interact ions be tween drug and ion channel.

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A sympos ium on Chemically- Gated Ion Conductances examined the effects of anesthetics on chan- nels which are act ivated by neuro- t ransmit ters rather than voltage. Both an excitatory neurotransmit- ter receptor -channel complex, the acetylcholine receptor (AChR), and an inh ib i tory one, the y- aminobutyr ic acid (GABA) recep- tor were discussed, because it is not yet known whether the anes- thetic state results pr imar i ly from per turba t ions of excitatory, inhi- b i tory or both types of synaptic t ransmission.

In a lucid introduct ion, Palmer Taylor (San Diego) argued that the nicotinic acetylcholine receptor consti tutes a wel l -def ined system in which to s tudy anesthetic ac- t ions on pos tsynapt ic excitatory t ransmiss ion. Each AChR-chan- nel complex is an ol igomer of five t ransmembrane polypept ide chains which together contain two

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agonis t b ind ing sites, probably a single local anesthetic site, and a central channel through which ions may cross the membrane. The affinity of the AChR for its l igand ACh is altered by volatile and (most) local anesthetics (which enhance affinity) and by barbi tura tes (which reduce affin- ity).

Al though a specific, saturable, allosteric site produces the agonist affinity change noted with local anesthetics, less is known about the mechanism by which general anesthet ics produce enhanced af- f inity for nicotinic agonists. Using mammal ian skeletal muscle cells as a source of AChR, Taylor employs fluorescent toxins to measure the molecular distances be tween agonist b ind ing sites and var ious allosteric b ind ing sites. The method promises to reveal structural correlates of the func- t ional changes in receptor proper- ties p roduced by anesthetics. In particular, the high-aff ini ty state of the receptor is associated with a ' desens i t i zed ' ion channel. Desen- s i t izat ion of ACh-ga ted channels is in certain respects analogous to inact ivat ion of voltage-gated channels: both processes termin- ate membrane excitation; both are enhanced by anesthetics.

The mechanisms by which a variety of local anesthetics in- fluence AChR behavior were in- ves t igated by Jonathan Cohen (Washington Universi ty) , who presented radiol igand b ind ing ex- per iments carried out in parallel wi th ion flux studies. Using AChR-r ich membranes from the Torpedo (a mar ine ray) electric organ he found that different local anesthet ics vary greatly in their potency at an allosteric b ind ing site ( through which AChR are shifted to the high-aff ini ty, de- sensi t ized state). Since the allo- steric l inkage be tween drug- and A C h - b i n d i n g sites could conceiv- ably be med ia ted ei ther through the prote in structure itself or its su r round ing l ipid, the mechanism by which anesthetics desensi t ize AChR membranes may be quite complex.

Keith Miller (Harvard) summar- ized his f indings on the effects of volatile and barbi tura te anesthe- tics on both the structure and function of Torpedo AChR in their l ip id environment . At clinical con- centrat ions, volatile anesthetics

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dramatical ly increase ACh b ind- ing by s tabi l iz ing a high-aff ini ty receptor conformation, and in parallel, d i sorder the membrane l ip id bilayer. Barbiturates, on the other hand, b ind at low concentra- t ions to an allosteric site on the receptor, causing inh ib i t ion of ACh b inding . At h igher concen- trations, they increase ACh b ind- ing and d isorder the membrane in a manner reminiscent of the vola- tile anesthetics. Thus the effects of these drugs on the b ind ing of ACh to its receptor appear to be med ia ted through both direct (protein) and indirect (lipid) mechanisms. Their effect on the resul t ing ion flux is to decrease it, consis tent wi th the concept of enhanced desensi t izat ion.

A patch-clamp invest igat ion of general anesthetic actions on the ACh-act ivated channel followed, presented by Peter Gage (Austra- lian National University). His kinetic analysis, s imilar to Yeh's s tudy of local anesthetic actions on single sodium channels, favor- ed the concept that volatile and barbi tura te anesthetics decrease the average open t ime of AChR, but do not actually block the open channel. He has also examined anesthetic effects on the chloride channel act ivated by the inhi- b i tory t ransmit ter y -amino- butyr ic acid (GABA). In contrast to the ACh channel, the GABA channel remains open longer than normal in the presence of anes- thetics.

Richard Olsen (UCLA) then p r e s e n t e d b iochemica l exper i - men t s on the modu la t i on of GABAergic function by barbi tur - ates. These agents were shown to enhance both the b ind ing of GABA to its receptor and the result ing chloride ion flux. Thus, al though a precise matching of results der ived from biochemical and electrophysiological experi- ments is not yet possible, it is clear that these d ispara te ap- proaches are rapidly converging toward a common view of anes- thetic effects on receptor-channel function. From both directions, an apparent symmetry is emerging: that general anesthetics interfere wi th excitatory synapt ic trans- miss ion and facilitate inh ib i tory synapt ic t ransmission. Al though intui t ively appeal ing, such a general i ty would not necessari ly be predicted on the basis of

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conventional biophysical theory, and would itself invite explana- tion. It remains to be seen whether this concept will be further strengthened by studies of a wider variety of channel types.

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James TrudeU (Stanford) chaired a session entitled Biophysical Mechanisms which focused on the molecular interactions between anesthetics and their target sites. Within the framework of the lipid-oriented hypothesis of anesthetic mechanisms, a variety of such interactions have been studied and results have rein- forced the concept that much of the observed diversity of anes- thetic-lipid interactions reflects structural features of the drugs. Various biophysical probes have also shown that some of the diversity resides in the structure of the lipid itself (Ian Smith, Ottawa).

An interesting departure from the mainstream was presented by Nick Franks and William Lieb (London), who have long favored the concept that anesthetics act within the hydrophobic domains of proteins (see also March 1985 issue of TIPS. They have now shown that luciferase, the enzyme which catalyses a light-emitting chemical reaction in the common firefly, is competitively inhibited by a structurally diverse group of anesthetics. The purified (lipid- free) enzyme was inhibited at concentrations of the anesthetics essentially identical to those which produce anesthesia, over a 100000-fold range of potencies. Thus it appears, in principle, that protein sites cannot be ruled out as the membrane targets which account for the Meyer-Overton rule. This rule has traditionally been interpreted to mean that anesthetics must act within a lipid domain. The difficult question which remains, of course, is whether the functions of relevant proteins of the nervous system are altered independently of actions on their lipid matrix.

Whether anesthetics act at lipid or protein sites within the cell membrane, it is clear that they modify, in characteristic ways, the fundamental processes underlying neuronal excitation. The taxonomy of these processes has traditionally provided two great orders into which all channel species were separated: the voltage-gated and the chemically-gated ion conduc- tances. But in recent years many novel and exotic specimens have been added to the ion channel zoo (which at last count boasted some four dozen exhibits), and it has now become clear that a third major order exists. The size of the current which flows through voltage-de- pendent calcium channels of the cardiac sarcolemma, for example, is importantly regulated by neuro- transmitters and (ipso facto) many drugs. Similarly, certain neuronal potassium channels are subject to a complex regulation by voltage, (intracellular) calcium and (extra- cellular) neurotransmitters.

In the final symposium, Cellular and Synaptic Actions, the hypo- thesis that anesthetics enhance inhibition by altering the be- havior of such potassium channels was explored, and additional studies on calcium and chloride conductances were reported. Kresimir Krnjevic (McGill), who proposed some ten years ago that general anesthetics might activate a calcium-dependent potassium conductance by releasing calcium from intracellular sites, chaired the session and q reviewed the major lines of evidence. Mary Morris (Toronto) followed with intriguing preliminary evidence, using the recently introduced fluorescent calcium indicator quin-2, that barbiturates elevate intracellular free calcium, lending support to Krnjevic's hypothesis. Peter Carlen (Toronto) carried the argument a step further, using the in-vitro hippocampal slice prepar- ation to examine the effects of anesthetics on the afterhyper- polarization which characteristi- cally follows action potentials in many central neurons, and is involved in modulation of their input -output characteristics (e.g.,

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regulation of repetitive or 'burst- ing' activity). Barbiturates and ethanol both prolonged the after- hyperpolarization, and this was attributed to enhancement of a calcium-dependent potassium conductance. The effect was ob- served at lower anesthetic concen- trations than those at which ef- fects on GABA-activated chloride channels could be measured.

Robert MacDonald (University of Michigan) alluded to yet a third possible inhibitory mechanism for barbiturates: interference with the presynaptic release of (excita- tory) neurotransmitters. In studies on cultured spinal neurons, his group has shown an apparent reduction by barbiturates of pre- synaptic calcium entry, with an associated decrease in transmitter release. Enhancement of the inhi- bitory postsynaptic action of GABA, as well as reduction of the excitatory postsynaptic action of glutamate were also demonstrat- ed. David Owen (NIH), reporting experiments using both patch- clamp and classical voltage-clamp techniques on a brain slice prep- aration, confirmed the barbiturate potentiation of GABA action. Re- markably, it was further shown that these agents may act directly to open chloride channels in the absence of GABA.

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During the final evening, an informal session, 'Current Theories of the Molecular Basis of General Anesthetics, ' provided an enjoyable forum for debate. As onlookers cheered, lipidists and proteinophiles once again crossed their unwieldly swords. And Morpheus, the Shaper of Dreams, merely smiled, because his secret was still safe.

ROGER S. BRETT A N D

LEONARD L. FIRESTONE*

Department of Anesthesiology, S.U.N.Y. at Stony Brook, Stony Brook, NY, USA and *Departments of Anesthesia and Pharmaco- logy, Harvard Medical School, Boston, MA, USA.