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Effects of General Anesthetics and Pressure on Mammalian Excitatory Receptors Expressed in Xenopus Oocytes
Abstract
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.
... Two major obstacles in earlier studies were 1) the inaccessibility of the recording microelectrode once the pressure chamber was sealed, which made it difficult to replace a broken microelectrode, and 2) mechanical disturbances that disrupted ICR when sealing the chamber and changing ambient pressure (41,42). In contrast, sharp-microelectrode ICRs have been conducted routinely in the invertebrate CNS (iCNS) (4,6,11,23,48,49), muscle cells (8,9,19), and oocytes (12,39) while ambient pressure was increased. The success of nonmammalian and nonneuronal experiments was most likely due to the robust nature of larger cells, compared with the smaller neurons in the mCNS (41,42). ...
... A major obstacle to making an ICR in an in vitro tissue preparation that is maintained inside a pressure chamber is the inaccessibility of the microelectrode once the chamber is sealed (42). Robust cell preparations, such as muscle cells (8,9,19), oocytes (12,39), and invertebrate axons and neurons (4,6,11,23,48,49), are better suited for ICR under hyperbaric conditions because of the relative ease with which they can be impaled and recorded from with the use of lower resistance microelectrodes. Because lower resistance microelectrodes break less readily, they do not have to be changed as often, and the chamber interior does not have to be accessed as frequently. ...
We developed a hyperbaric chamber for intracellular recording in rat brain stem slices during continuous compression and decompression of the tissue bath with the inert gas helium. Air, rather than helium, was also used as the compression medium in some cases to increase tissue nitrogen levels. An important feature is the chamber door, which opens or closes rapidly at 1 atmosphere absolute (ATA) for increased accessibility of the microelectrode. The door also closes and seals smoothly without disrupting the intracellular recording. Hyperbaric oxygen was administered during helium compression using a separate pressure cylinder filled with perfusate equilibrated with 2. 3-3.3 ATA oxygen. Measurements of tissue/bath PO(2) and pH confirmed that the effects of compression using helium or air could be differentiated from those due to increased PO(2). One hundred and thirteen neurons were studied during 375 compression cycles ranging from 1 to 20 ATA (mode 3.0 ATA). We conclude that it is technically feasible to record intracellularly from the same mammalian neuron while changing ambient pressure over a physiologically important range. These techniques will be useful for studying how various hyperbaric environments affect neurophysiological mechanisms.
... On the other hand, downregulation of NMDA, kainate, and quisqualate receptors is most likely to induce a state of CNS hyperexcitability that involves different neuronal pools and reverberating circuits [27]. Depression and diminished function of α 2 -autoadrenoreceptors have been implicated in the adrenergic hyperactivity that occurs during alcohol withdrawal [28]. ...
In vrijwel elk nummer van Critical Care vindt u een casus die de verschillende fases van een behandeling laat zien. Deze keer
een meisje van 14 dat bewusteloos is aangetroffen na het drinken van alcohol.
... these include high pressure neurological syndrome (hPns), which indicates a state of hyperexcitability, the phenomenon of nitrogen (n 2 ) narcosis experienced by animals breathing air during deep diving or during mining operations, anesthesia produced by chemically inert gases such as xenon (Xe), and pressure-reversal of narcosis or anesthesia for a wide variety of agents occurring among many organisms (1)(2)(3)(4)(5)(6)(7)(8). several studies have addressed neuronal conductance and the response of specific ion channels and receptor sites to hyperbaric conditions using electrophysiology methods applied to brain slice preparations and receptors expressed in Xenopus oocytes (9)(10)(11)(12)(13)(14)(15)(16). in general, it is observed that conductance velocities and action potential (AP) amplitudes are decreased under hyperbaric conditions, mainly due to slowed kinetics of na+ ion conductance at increased pressure (12). the ensemble of subtle changes within each receptor site and neuronal connection will perturb synaptic transmission and event detection among the neuronal populations that form circuits within the central nervous system (cns). ...
We used voltage-sensitive dye imaging to study the properties of transient large-scale neuronal populations under hyperbaric conditions using a newly-developed high-pressure optical cell. In our experiments we investigated propagation of neuronal voltage wave depolarization along the CA2-CA1 Schaffer collateral pathway in rat hippocampal slices. The voltage-sensitive dye responses were studied at pressures up to 20 atmospheres (atm) over a range in which changes in excitability and pressure-reversal of narcosis/anesthesia have been described to occur in animals. An electrode placed in the CA2 region was used to evoke a signal along the Schaffer collateral neuronal circuit toward CA1 using a paired pulse paradigm (PPF). Initial inspection of the data indicates that the signal amplitude of the excitation following the second PPF event is enhanced at high pressure. Data analysis using MatLab software revealed a range of conductance velocities between different layers within the Schaffer collateral and for sites at different distances from the stimulating electrode. The estimated value of the conductance velocity along the trajectory of maximum flow is in good agreement with previous determinations of axonal propagation along the Schaffer collateral.
... Ketamine, phencyclidine, ethanol and diethyl ether have been shown preferentially to depress responses of the NMDA subclass [72,73,153], whereas barbiturates depress the sensitivity of neurones to all three agonists with quisqualate/kainate activation showing the greatest sensitivity [128,153]. Oocytes injected with mRNA from rat brain express kainate receptors and the response of these cells to kainate is depressed by phenobarbitone [31]. ...
To understand the cellular and molecular basis of the anaesthetic state, it is important to remember that, in the intact CNS, synapses operate within elaborate nerve networks. From the data presented above, it is evident that block of impulse conduction in presynaptic fibres does not explain the effects of most anesthetics on synaptic activity. This is not surprising since some anaesthetics, the barbiturates in particular, may both depress excitation and enhance inhibition. General anaesthetics modulate the activity of presynaptic voltage-gated calcium channels and this appears to be sufficient to account for the reduction in transmitter secretion they produce. Transmitter operated ion channels in the postsynaptic membrane are modulated by smaller concentrations of anaesthetics than are required to modulate the presynaptic voltage-gated calcium channels. For this reason, transmitter operated channels appear to represent a major target site for anaesthetics. Finally, there are subtle effects of anaesthetics on the patterns of impulse propagation in nerve axons and on action potential generation in the cell body which result from modulation of membrane excitability. The overall effect of an anaesthetic agent depends on summation of events occurring at the many individual synapses and neurones that make up the network. The effects of anaesthetics on different neuronal pathways may therefore depend on the nature of the receptors and ion channels of the cells that comprise the network. The anaesthetic state may be the result of all these actions, but the characteristics of the state may differ somewhat from agent to agent.
... On the other hand, downregulation of NMDA, kainate, and quisqualate receptors is most likely to induce a state of CNS hyperexcitability that involves different neuronal pools and reverberating circuits [27]. Depression and diminished function of α 2 -autoadrenoreceptors have been implicated in the adrenergic hyperactivity that occurs during alcohol withdrawal [28]. ...
The modern intensive care unit (ICU) has evolved into an area where mortality and morbidity can be reduced by identification of unexpected hemodynamic and ventilatory decompensations before long-term problems result. Because intensive care physicians are caring for an increasingly heterogeneous population of patients, the indications for aggressive monitoring and close titration of care have expanded. Agitated patients are proving difficult to deal with in nonmonitored environments because of the unpredictable consequences of the agitated state on organ systems. The severe agitation state that is associated with ethanol withdrawal and delirium tremens (DT) is examined as a model for evaluating the efficacy of the ICU environment to ensure consistent stabilization of potentially life-threatening agitation and delirium.
... Glu NMDA receptors expressed in Xenopus oocytes injected with cRNA from rat cerebellum show a marked potentiation (128%) at 10MPa (Williams et al., 1996; Daniels et al., 1998). In contrast, Glu KA receptors, expressed in Xenopus oocytes following injection of cRNA from rat whole brain show little potentiation of the maximum response (<14%) and no change in the EC 50 (Daniels et al., 1991; Shelton et al., 1993). Glu NMDA receptors are composed of two subunits, R1 and one of R2A, R2B, R2C and R2D, in unknown stoichiometry. ...
The observed cellular effects of pressure are entirely compatible with the acute manifestations of CNS hyperexcitability. Inhibition of the glycine receptor will reduce post-synaptic inhibition, leading to increased excitability (cf 'Startle Disease', an hereditary disease with increased excitability arising from a genetic modification to the glycine receptor (Becker et al., 2002)). Since glycine-mediated neurotransmission is particularly associated with motor reflex circuits (Lynch, 2004) it is not surprising that many of the acute manifestations of pressure involve motor dysfunction. Potentiation by pressure of the NR1-NR2C subtype of the NMDA-sensitive glutamate receptor will lead to increased excitability within the cerebellum (where this receptor sub-type is most highly expressed (Monyer et al., 1994)). Although the cerebellum receives input from many parts of the nervous system, it projects primarily to the motor and frontal lobe cognitive areas. Thus dysfunction of the glutamate-mediated excitatory neurotransmission in this area is most likely to result in locomotor and cognitive symptoms, characteristic of acute pressure effects. Finally, the effects observed on AC/cAMP intracellular signalling, probably mediated via dopamine receptors, is also likely to produce motor dysfunction (cf Parkinson's disease). The observed cellular effects also suggest potential mechanisms that could result in long-term CNS dysfunction. Potentiation of glutamate neurotransmission is likely to lead to excessive calcium entry into those neurons. This may trigger excitotoxicity via a signal cascade in which neuronal NO synthase is activated producing the toxic free radical peroxynitrite and activation of the proapoptotic protein poly(ADP-ribose) polymerase (Aarts & Tymianski, 2005). An additional mechanism, also initially triggered by a rise in intracellular calcium through NR1-NR2C receptors, involves activation of a member of the Transient Receptor Potential (TRP) channel superfamily, the TRPM-7 channel. Activation of these channels will cause a further rise in intracellular calcium, creating a positive feedback and generating more neuronal death through the toxic signal cascade (Aarts & Tymianski, 2005). Neuronal cell death within the cerebellum might be expected to give rise to delayed motor and cognitive dysfunction the magnitude of which would tend to be related to the extent of hyperbaric exposure. There is at present no evidence that these excitotoxic mechanisms are triggered by exposure to pressure but future experimental work should investigate the extent to which pressure might activate them.
Glycine was first identified as an inhibitory neurotransmitter in a paper by Aprison and Werman (1). This role was subsequently confirmed in two papers detailing a combined neurochemical and neurophysiological approach, which led to a general acceptance of this role for glycine (2,3). Subsequently, it was established that strychnine was a selective antagonist at the glycine receptor (4). The identification and characterization of the glycine receptor as a transmembrane protein composed of two subunits, α and β, followed over a decade later (5–9).
To test the hypothesis that the anesthetic action of dexmedetomidine is similar to that of other anesthetics, dexmedetomidine was studied using two classical descriptors of anesthesia, the Meyer-Overton correlation and the pressure reversal of anesthesia. The anesthetic potency of dexmedetomidine and its isomer, levomedetomidine, were determined in Xenopus laevis tadpoles. Anesthesia was defined as loss of righting reflex. Octanol/water partition coefficients were determined using ultraviolet absorbance spectrophotometry. Pressure experiments were performed at pressures ranging from 1 to 91 atmospheres absolute (ata). A concentration-response curve was obtained with a half-maximum effective concentration (EC50) of dexmedetomidine of 7.1 +/- 1.1 micro M (mean +/- SEM), whereas levomedetomidine showed an EC50 of 59.3 +/- 5.5 micro M. The octanol/water partition coefficient was 206 +/- 49. With increasing pressure, the EC50 of dexmedetomidine increased linearly up to a value of 23.9 +/- 3.2 micro M at 91 ata. Correlation of lipid solubility and anesthetic potency demonstrates that dexmedetomidine does not adhere to the Meyer-Overton rule. However, the anesthetic action of dexmedetomidine is diminished by increased pressures, thus demonstrating a similar effect compared with other general anesthetics.
(Anesth Analg 1997;84:618-22)
This chapter discusses that high hydrostatic pressure affects equilibria and reaction rates through the molar volume changes involved in the reaction system as a whole. The chapter explains how high hydrostatic pressure affects molecules of biological interest, and their interactions; the ways in which pressure affects cellular processes; and how animals, including humans are affected by pressure. Along with high pressure, the chapter also intends to stimulate interest in a particular approach to physiology. The discussion on molecular effects of pressure and temperature includes equilibrium processes in aqueous solution, lipid bilayers under pressure and rates of chemical reactions. It is mentioned that hydrostatic pressure is not directional and that osmotic pressure is also quite separate from hydrostatic pressure. Hydrostatic pressure is a force acting in all directions, in the air one breathes, in water, or in body fluids. While high pressure, surprisingly perhaps, dissociates molecules in aqueous solution, it has the intuitively expected effect on lipid bilayers, compressing and ordering their structure. The thermodynamics of equilibrium processes in aqueous solution, and the phase state of lipid bilayers at high pressure, inevitably fall short of explaining how pressure affects rates of reaction. Muscle, a tissue whose cells are conspicuously filled with polymerized proteins, is affected by high pressure in ways determined by the contraction cycle. Simple processes, such as the diffusion of gases or water in aqueous solution, are little affected by the pressure range of interest here. Chondrocytes are cells, which synthesize the load-bearing extracellular matrixthat covers the articulating surfaces of joints. The buoyancy of aquatic animals subjected to significant pressure is interesting because buoyancy requires the creation of a void or region of low-density, working against the ambient pressure. Animals and bacteria live at high pressures in the deep sea, down to the greatest depths where the pressure is approximately 100 MPa. Several technological applications have arisen from high pressure biology—the manipulation of ploidy, and of membrane proteins; sterilization and inactivation of microorganisms; food processing; and safe human diving.
High pressure, which induces central nervous system (CNS) dysfunction (high-pressure neurological syndrome) depresses synaptic transmission at all synapses examined to date. Several lines of evidence indicate an inhibitory effect of pressure on Ca(2+) entry into the presynaptic terminal. In the present work we studied for the first time the effect of pressure on the cerebellar climbing fiber (CF) synaptic responses. Pressure modulation of cerebellar synaptic plasticity was tested in both the CF and parallel fiber (PF) pathways using paired-pulse protocols. CF synapses, which normally operate at a high baseline release probability, demonstrate paired-pulse depression (PPD). High pressure reduced CF synaptic responses at 5.1 and 10.1 MPa but did not affect its PPD. High extracellular Ca(2+) concentration ([Ca(2+)](o)) could not antagonize the effect of pressure on the CF response, whereas low [Ca(2+)](o), in contrast to pressure, decreased both the response amplitude and the observed PPD. PF synapses, which usually operate at low release probability, exhibit paired-pulse facilitation (PPF). Pressure increased PF PPF at all interstimulus intervals (ISIs) tested (20-200 ms). Several Ca(2+) channel blockers as well as low [Ca(2+)](o) could mimic the effect of pressure on the PF response but significantly increased the PPF only at the 20-ms ISI. These results, together with previous data, show that the CF synapse is relatively resistant to pressure. The lack of pressure effect on CF PPD is surprising and may suggest that the PPD is not directly linked to synaptic depletion, as generally suggested. The increase in PPF of the PF at pressure, which is mimicked by Ca(2+) channel blockers or low [Ca(2+)](o), further supports pressure involvement in synaptic release mechanism(s). These results also indicate that pressure effects may be selective for various types of synapses in the CNS.
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)
This review discusses the mechanism(s) of general anesthesia from a pharmacological viewpoint; in particular, the ability of drugs to produce many different effects is emphasised. The problems of experimental measurement of general anesthesia are discussed, and the possibilities for antagonism and potentiation of anesthesia considered. Physicochemical studies on anesthesia are described, as are the advancement of ideas beyond consideration of lipids and proteins as separate sites of action. The importance of studies on different areas of the brain is highlighted, and the review finishes with a survey of the effects of general anesthetics on synaptic transmission which emphasises the problems of extrapolation from in vitro to in vivo.
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.
To test the hypothesis that the anesthetic action of dexmedetomidine is similar to that of other anesthetics, dexmedetomidine was studied using two classical descriptors of anesthesia, the Meyer-Overton correlation and the pressure reversal of anesthesia. The anesthetic potency of dexmedetomidine and its isomer, levomedetomidine, were determined in Xenopus laevis tadpoles. Anesthesia was defined as loss of righting reflex. Octanol/water partition coefficients were determined using ultraviolet absorbance spectrophotometry. Pressure experiments were performed at pressures ranging from 1 to 91 atmospheres absolute (ata). A concentration-response curve was obtained with a half-maximum effective concentration (EC50) of dexmedetomidine of 7.1 +/- 1.1 microM (mean +/- SEM), whereas levomedetomidine showed an EC50 of 59.3 +/- 5.5 microM. The octanol/water partition coefficient was 206 +/- 49. With increasing pressure, the EC50 of dexmedetomidine increased linearly up to a value of 23.9 +/- 3.2 microM at 91 ata. Correlation of lipid solubility and anesthetic potency demonstrates that dexmedetomidine does not adhere to the Meyer-Overton rule. However, the anesthetic action of dexmedetomidine is diminished by increased pressures, thus demonstrating a similar effect compared with other general anesthetics.
(1) The effects on human homomeric alpha1 glycine receptors of 11 general anaesthetics; four barbiturates, two other intravenous anaesthetics, three volatile anaesthetics and two simple gaseous anaesthetics are described. (2) Pentobarbital and thiopental potentiate the current response to bath applied glycine (50 microM) by 200 and 300%, respectively, at clinically relevant concentrations. (3) Neither methohexital nor phenobarbital had any effect on the current response to bath applied glycine (50 microM). (4) Using maximal doses of applied glycine (1 mM) all the barbiturates acted as non-competitive antagonists. (5) Propofol and etomidate potentiate the current response to bath applied glycine (50 microM) by 200 and 10%, respectively, at clinically relevant concentrations. (6) Etomidate acts as a non-competitive antagonist for doses of glycine above the EC50 (197 microM). Propofol was without effect using maximal doses of applied glycine (1 mM). (7) Halothane, chloroform and ether potentiated the response to bath applied glycine (50 microM) by 200, 100 and 200%, respectively, at clinically relevant doses. (8) None of the volatile anaesthetics had any effect using maximal doses of applied glycine (1 mM). (9) Nitrous oxide and xenon potentiated the response to bath applied glycine (50 microM) by 75 and 50%, respectively, at clinically relevant doses. Nitrous oxide also potentiated the response using maximal doses of applied glycine (1 mM). (10) These results suggest a role for glycinergic neurotransmission in the production of anaesthesia.
1. Synapses with the brain are important components of the networks responsible for higher nervous function and current evidence suggests that general anaesthetics modulate synaptic transmission in the brain. 2. Analysis of anaesthetic action on these synapses not only defines the cellular mechanisms involved in anaesthesia but also reveals much about the molecular targets of anaesthetic action. 3. It appears that while anaesthetics affect a wide variety of processes, the most sensitive are those which are directly linked to the activity of ligand-gated ion channels. Moreover, both single channel patch clamp studies and the molecular biological investigations of the sub-unit specificity of the sensitivity to anaesthetics indicate that anaesthetics interact directly with these functional proteins.
The effects of convulsant drugs, and of thyrotropin releasing hormone (TRH), were examined on the general anaesthetic actions of ketamine, ethanol, pentobarbitone and propofol in mice. The aim was to investigate the possibility of selective antagonism, which, if seen, would provide information about the mechanism of the anaesthesia.
The general anaesthetic effects of ketamine were unaffected by bicuculline; antagonism was seen with 4-aminopyridine and significant potentiation with 300 mg kg−1 NMDLA (N-methyl-DL-aspartate). The calcium agonist, Bay K 8644, potentiated the anaesthesia produced by ketamine and antagonism of such anaesthesia was seen with TRH.
A small, but significant, antagonism of the general anaesthesia produced by ethanol was seen with bicuculline, and a small, significant, potentiation with 4-aminopyridine. There was an antagonist effect of TRH, but no effect of NMDLA.
Potentiation of the anaesthetic effects of pentobarbitone was seen with NMDLA and with 4-aminopyridine and the lower dose of bicuculline (2.7 mg kg−1) also caused potentiation. There was no significant change in the ED50 value for pentobarbitone anaesthesia with TRH.
Bicuculline did not alter the anaesthetic actions of propofol, while potentiation was seen with NMDLA and 4-aminopyridine. TRH had no significant effect on propofol anaesthetic, but Bay K 8644 at 1 mg kg−1 significantly potentiated the anaesthesia.
These results suggest that potentiation of GABAA transmission or inhibition of NMDA receptor-mediated transmission do not appear to play a major role in the production of general anaesthesia by the agents used.
British Journal of Pharmacology (2000) 129, 1755–1763; doi:10.1038/sj.bjp.0703262
The aim of this work was to study the role of pallidal GABAa and GABAb neurotransmission in the behavioral disorders induced by pressure. The effects of GABAb antagonist 5-aminovalleric acid (5-AVA) or GABAa antagonist gabazine administrations in the globus pallidus (GP) on locomotor and motor hyperactivity (LMA) and myoclonia expressions in the model of the rat submitted to 8 MPa of helium-oxygen breathing mixture were analyzed. The administration of GABAa antagonist gabazine enhances the occurrence of the epileptic seizures, slightly increases LMA but decreases myoclonia. In contrast, the administration of GABAb antagonist 5-AVA decreases both LMA and myoclonia during the compression and the beginning of the holding time at 8 MPa. These data indicate that some behavioral disorders induced by pressure are in relation with GABAergic neurotransmission and establish clearly that GABAa and GABAb receptor mediations have distinct functions in the GP of the rat.
The factors which determine the response of the in vitro luminescent
reaction of Vibrio fischeri, to the general anaesthetic diethyl ether,
have been determined. The investigations show that, as was indicated by
a study of the in vivo reaction, the levels of substrates available to
the enzyme luciferase modify its response to ether. The results indicate
that ether inhibits the binding of the aldehyde factor necessary for
luminescence. There is evidence that it also acts as a second site where
its presence appears to stimulate the binding of reduced flavin to the
enzyme.
1. Catecholamines, adenosine, gonadotrophins, vasoactive intestinal peptide (VIP) and E-series prostaglandins all elicit K+ currents in follicle-enclosed Xenopus oocytes. Evidence suggests that cyclic nucleotides act as intracellular messengers in the activation of this K+ conductance. Muscarinic agonists and some divalent cations (e.g. Co2+, Mn2+, Ni2+ and Cd2+) elicit slow oscillatory Cl- currents, which are activated through hydrolysis of inositol phospholipids and mobilization of intracellular calcium by inositol phosphates. 2. We investigated whether these membrane current responses were generated in the oocyte itself or in enveloping follicular cells which are coupled to the oocyte by gap junctions. Oocytes were defolliculated, either enzymatically using collagenase, or by manual dissection combined with rolling over poly-L-lysine-coated slides. Removal of follicular cells was checked using scanning electron microscopy. Membrane current responses of defolliculated oocytes were compared with responses seen in follicle-enclosed oocytes taken from the same ovary. 3. The K+ responses evoked by all the various hormones/neurotransmitters were either drastically reduced (greater than 90%) or abolished by defolliculation. K+ currents generated by the adenylate cyclase activator forskolin and by intraoocyte injection of adenosine 3',5'-cyclic monophosphate (cyclic AMP), or guanosine 3',5'-cyclic monophosphate were similarly reduced in defolliculated oocytes. In contrast, oscillatory Cl- currents to acetylcholine and divalent cations were selectively preserved through defolliculation. 4. Injection of cyclic AMP (1-20 pmol) into defolliculated oocytes had little or no effect on oscillatory Cl- currents elicited by ACh. However, the calcium-dependent transient Cl- current, activated by depolarization of the oocyte membrane, was consistently potentiated (100-900%) by injections of cyclic AMP (1-10 pmol). 5. These experiments suggest that cyclic nucleotide-activated K+ currents arise essentially in follicular cells and are monitored within the oocyte through electrical coupling by gap junctions. Oscillatory Cl- responses evoked by ACh and divalent cations are produced largely or wholly in the oocyte itself.
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.
Quantitative aspects of the potentiation of GABA and muscimol by benzodiazepines and barbiturates are reviewed, taking account of both electrophysiological and receptor binding data. It has been a consistent finding that barbiturates cause a greater maximal potentiation than do benzodiazepines. The steroid anaesthetic alphaxalone and some naturally occurring steroids were compared as potentiators of electrophysiological responses to muscimol. From the relative potencies, important structural features of the steroid molecule for this effect have been identified. The possibility of the barbiturates and the steroids having a common mode of action as potentiators of GABA and muscimol is discussed, together with the idea that this action may involve perturbation of membrane lipids rather than a barbiturate/steroid receptor site. The GABA-potentiating effect of ethanol may also be barbiturate-like but potentiations by chlormethiazole and ketamine appear to involve different mechanisms. It is predicted that any endogenous potentiators of GABA would be unlikely to have more than a modest effect.
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.
This chapter chapter discusses that both lipids and proteins, and their complexes, interact with general anesthetics. In both the cases, there are model systems where interactions correlate with anesthetic potency as well as other model systems where they do not. Of the lipid bilayers, those with a high ratio of cholesterol to phospholipid appear to fare best, while of the proteins, the luciferases offer the most tested and successful model. The functional changes resulting from anesthetic-lipid or anesthetic- protein interactions provides another clue to the importance of each in producing general anesthesia. The changes induced in lipid bilayers at clinical doses are small and seem unlikely per se to result in physiological effects. Most lipid theories of anesthesia have assumed that perturbation of lipid-protein interactions underlies anesthetic action. The systematic studies of average bilayer properties such as volume, order or fluidity support the lipid hypothesis in a general way, but which fail to directly address the mechanistic link with protein function. As the knowledge of membranes has increased, it has become more clear that average lipid parameters of this sort are less likely to be related to membrane protein function than are the detailed heterogeneous arrangement of lipids and proteins. In this sense, the study of these anesthetic mechanisms is in a transition between thermodynamic and molecular explanations.
The application of high hydrostatic pressure will remove, at least
partially, the effects of general anaesthetics in both newts and mice.
This feature seems to be common to all general anaesthetics and a simple
model could explain this antagonism.
The onset pressures for the tremor, myoclonus and convulsions seen in the high pressure neurological syndrome (HPNS) are increased following cis-2,3-piperidine dicarboxylic acid 1 mmol/kg in the rat. Glutamic acid diethyl ester 1-3 mmol/kg has no effect on tremor or myoclonus, but increases the convulsion pressure when 3 mmol/kg is given immediately before compression. These and earlier data with 2-amino-7-phosphonoheptanoic acid suggest that excitation at the N-methyl-D-aspartate receptor is important in HPNS tremor, and that excitation at the quisqualate receptor contributes to HPNS convulsions.
The interactions of anaesthetics and other drugs with high pressure suggest that protection against the high pressure neurological syndrome (h.p.n.s.) can no longer be considered in terms of generalized non-specific mechanisms. The evidence from our work shows that anaesthetics may either protect, have no effect, or potentiate h.p.n.s. Structural analogues of the steroid anaesthetic Althesin have a protective effect against high pressure tremors in spite of the fact that they have no anaesthetic effects. Low doses of flurazepam are effective against tremor but can be antagonized by Ro 15-1788, which implies in this case a role for the benzodiazepine receptor complex. Pressure interactions with other drugs have included the classic anticonvulsants--which, in general, were relatively ineffective--and various agents perturbing the balance of specific neurotransmitter systems. Representative examples from different studies include 6-hydroxydopamine, muscimol, and sodium valproate. Finally, the potent protection against h.p.n.s. by 2-amino-phosphonoheptanoic acid, an antagonist with preferential action against excitation produced by aspartate and N-methyl-D-aspartate, provides the first evidence that enhanced excitatory amino acid neurotransmission may have an important role in the h.p.n.s.
Important constraints on possible molecular mechanisms of general anaesthesia are derived from a quantitative reappraisal of data on the potency of general anaesthetics on whole animals. Despite their popularity, theories that invoke lipids as the prime target do not look at all promising, and available data point much more plausibly to a direct effect on particularly sensitive proteins. Structural changes of proteins on binding general anaesthetics are probably small but may be sufficient to perturb normal function; alternatively, anaesthetics may compete with an endogenous ligand. The phenomenon of pressure reversal of anaesthesia may simply be due to anaesthetics being squeezed away from their target sites.
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