E B Smith’s research while affiliated with Cardiff University and other places

The effect of hyperbaric pressure on the inhibitory glycine receptor has been investigated in voltage-clamped Xenopus oocytes microinjected with cRNA encoding the human alpha-1 glycine receptor subunit. Heterologous expression of the human alpha-1 subunit generated functional glycine-gated channels with properties typical of native receptors. Glycine elicited a concentration-dependent inward current which reversed polarity at -25 mV and was antagonised by nanomolar concentrations of strychnine. Concentration-response curves established for the homomeric alpha-1 glycine receptor at 5, 10 and 15 MPa were progressively shifted to the right with respect to the concentration response curve established at atmospheric pressure (0.1 MPa). Pressure had no effect on the maximal response. The EC(50) values at 0.1, 5, 10 and 15 MPa were 190 mu M, 222 mu M, 338 mu M and 482 mu M, respectively. The results demonstrate that a receptor comprised solely of the human alpha-subunit is sensitive to pressure in the range that affects divers and at which the native rat spinal cord receptor is affected. This finding is discussed in the context of the postulated binding sites for glycine and the implications for the design of drugs to protect divers from the effects of pressure.

The effect of high pressure on the response to glycine or kainate of voltage-clamped Xenopus oocytes micro-injected with messenger-RNA derived from either rat spinal cord or whole brain, respectively, has been investigated. Current responses were measured at 1 bar (= 10(5) Pa), 50 bar, 100 bar and 150 bar, with PO2 fixed at 1 bar and the balance helium. Glycine elicited a depolarizing current response which was antagonized by nanomolar concentrations of strychnine. The responses reversibly desensitized, with a decay constant of 0.01 s-1, when glycine concentrations greater than 250 microM were used. The decay constant was insensitive to both glycine concentration and pressure. Resensitization was complete within 4 min. Kainate elicited a depolarizing current which was non-desensitizing. The response was slightly sensitive to glutamate diethyl ester (50 microM), which increased the EC50 by 25%. The action of glycine was highly pressure sensitive. The dose-response curves established at 50 bar, 100 bar and 150 bar were shifted progressively to the right, with no effect on the maximal current. The EC50 increased from 216 microM to 296 microM at 50 bar, to 345 microM at 100 bar, and to 425 microM at 150 bar. The action of kainate was unaffected by pressure. No shift in the dose-response curves was established, nor was there any effect on the maximum current. The EC50 was 113 microM at 1 bar, and 111 microM at both 50 bar and 100 bar.(ABSTRACT TRUNCATED AT 250 WORDS)

The idea that general anaesthetics produce unconsciousness, analgesia and amnesia by interfering with communication between neurones is conceptually appealing to both scien- tists and non-scientists. Indeed, there is a long history of considering postsynaptic ligand-gated ion channels (LGICs) as molecular targets for general anaesthetics.59 Advances in experimental techniques, especially in electrophysiology and molecular biology, have fostered a reductionist approach and allowed exploration of the interactions between general anaesthetics and LGICs in increasingly greater molecular detail. Potential binding sites for anaesthetics on some LGICs have now been identified. However, concrete evidence for these sites awaits the determination of the high-resolution structure of LGICs in the absence and presence of anaesthetic. This review is organized in the following way. First, we give a summary of the structure and function of LGICs, and this is followed by an examination of the ways in which anaesthetics (or any drugs) might affect LGIC function, a critique of the methods used to measure LGIC function, and a compilation of the effects of general anaesthetics (and similar agents such as alcohols) on individual families of LGICs.

The effects of general anesthetics and pressure on receptors from the mammalian central nervous system have been investigated using oocyte expression techniques. Poly A+ mRNA extracted from rat whole brain was injected into mature Xenopus oocytes producing depolarizing responses to the fast excitatory neurotransmitters NMDA and kainate and the inhibitory neurotransmitters GABA and glycine. An apparatus was constructed to allow agonist dose-response curves to be determined at high pressures using voltage-clamped oocytes. This was used to investigate the excitatory transmitter kainate. It was found that anesthetics depress the current induced by kainate whereas pressure does not appear to affect the responses associated with this transmitter. Furthermore it was found that pressure does not reverse (or modify in any way) the changes in response brought about by application of anesthetics.

A range of compounds structurally related to the centrally acting muscle relaxant mephenesin and to the chemical convulsant strychnine were synthesized and tested for their ability to alter the threshold pressures for the onset of high pressure convulsions in mice. The ability of both groups of compounds to alter the threshold pressures for convulsions was found to be dependent on the nature of a simple molecular skeleton. Thus, compounds that possessed a negatively polarized group located both in the same plane as and some 4.5 A from an aromatic nucleus increased the thresholds whereas compounds with a positively polarized group at the same location reduced the thresholds. These findings support the suggestion that pressure elicits convulsions via a selective action on a receptor protein complex rather than via some general perturbation of the lipid regions of cellular membranes.

A series of benzazole-related, centrally acting muscle relaxants, comprising benzimidazole, chlorzoxazone, and zoxazolamine, were found to give substantial protection against the tremors and convulsions associated with the high pressure neurologic syndrome (HPNS) in the mouse. In this respect they represent a new class of nonanesthetic, anti-HPNS agents. Their anti-HPNS properties, like those previously established for the mephenesin group of centrally acting muscle relaxants, seem to be related to their ability to antagonize the convulsive action of strychnine. These findings are consistent with the suggestion that one of the principal effects of pressure, expressed as HPNS, arises from a perturbation of strychnine-sensitive mechanisms.

The effects of a variety of structural isomers of the centrally acting muscle relaxant mephenesin on the high pressure neurological syndrome have been investigated. Threshold pressures for the onset of the behavioural signs, tremors and convulsions, were established. The effects of these compounds on the response to pressure were also compared with their ability to antagonize the convulsive action of strychnine. The dose‐response relationships for strychnine and picrotoxin were investigated at fixed pressures. Additionally, the dose‐response relationship of strychnine, in the presence of mephenesin, at pressure was investigated. All the isomers of mephenesin protected against the effects of both pressure and strychnine. The relative potency was found to be identical with respect to both. Mephenesin was clearly the most effective; it raised the threshold pressure for tremors by 2.5 times, that for convulsions elicited by pressure by 1.5 and the ED 50 for strychnine convulsions by 1.6 times. Strychnine was found to be strictly additive with pressure whereas picrotoxin exhibited gross deviations from additivity. Mephenesin ameliorated the combined effects of pressure and strychnine equally. The marked dependence on structure of the anticonvulsant activity of the mephenesin isomers can be interpreted as evidence that pressure acts not by some general perturbation of the membranes of excitable cells but rather via some specific interaction. The finding that strychnine and pressure are strictly additive supports the idea of specificity and also indicates that they may share a common mechanism in the production of convulsions. By analogy with the established mechanism of action of strychnine, it is suggested that the hyperexcitability associated with pressure might arise from an action on glycine‐mediated inhibitory processes.

The mechanism of general anaesthesia has proved difficult to elucidate (see ref. 1 for a review), although the relative potencies of anaesthetic agents have been used to establish that the site at which anaesthetics act is hydrophobic in nature. One further clue to their mode of action is that the effects of anaesthetics on vertebrates can be eliminated by pressures of approximately 100 atm (refs 3, 4). However, the effects of anaesthetics are not always reversed in model systems, where there is evidence that the pattern of pressure reversal varies significantly. We now find that pressure fails to reverse the effects of anaesthetics on the freshwater shrimp (Gammarus pulex), although the sensitivity of these crustaceans to anaesthetics is comparable with that of higher animals. This is hard to reconcile with traditional bio-physical mechanisms and indicates that anaesthetics may act at a specific protein site rather than having a general effect on cell membranes. The pharmacology of pressure in mammals seems to be more similar to that of strychnine than of any other central stimulant. As glycine, whose action is blocked by strychnine, is absent as a neurotransmitter in the arthropod central nervous system, we believe that this substance may be involved in determining pressure-anaesthetic interactions in vertebrates.