The interaction of anaesthetic steroids with recombinant glycine and GABAA receptors{dagger}

C. J. Weir*,1,2, A. T. Y. Ling2, D. Belelli2, J. A. W. Wildsmith1, J. A. Peters2 and J. J. Lambert2

Departments of 1 Anaesthesia and 2 Pharmacology & Neuroscience, Division of Pathology and Neuroscience, Ninewells Hospital and Medical School, The University of Dundee, Dundee DD1 9SY, UK

*Corresponding author. E-mail: c.j.weir{at}dundee.ac.uk
{dagger}This work was presented, in part, at a meeting of the ARS in Aberdeen, but publication was not requested.

Accepted for publication: December 23, 2003


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. Anaesthetic steroids are established positive allosteric modulators of GABAA receptors, but little is known concerning steroid modulation of strychnine-sensitive glycine receptors, the principal mediators of fast, inhibitory neurotransmission in the brain stem and spinal cord. This study compared the modulatory actions of five anaesthetic pregnane steroids and two non-anaesthetic isomers at human recombinant {alpha}1 glycine and {alpha}1ß2{gamma}2L GABAA receptors.

Methods. Recombinant {alpha}1 glycine or {alpha}1ß2{gamma}2L GABAA receptors were expressed in Xenopus laevis oocytes and agonist-evoked currents recorded under voltage-clamp. Steroid modulation of currents evoked by GABA, or glycine, was quantified by determining the potency (EC50) and maximal effect of the compounds.

Results. The anaesthetics minaxolone (EC50=1.3 µM), Org20599 (EC50=1.1 µM) and alphaxalone (EC50=2.2 µM) enhanced currents mediated by GABAA receptors. The anaesthetics also enhanced currents mediated by glycine receptors, although with higher EC50 values (minaxolone 13.1 µM; Org20599=22.9 µM and alphaxalone=27.8 µM). The maximal enhancement (to 780–950% of control) produced by the three steroids acting at the GABAA receptor was similar, but currents evoked by glycine were potentiated with increasing effectiveness by alphaxalone (199%) <Org20599 (525%) <minaxolone (1197%). The anaesthetic isomers, 5{alpha}-pregnan-3{alpha}-ol-20-one and 5ß-pregnan-3{alpha}-ol-20-one (eltanolone) enhanced GABAA receptor-mediated currents with similar potency and efficacy, but only the former enhanced glycine, the latter causing inhibition. The non-anaesthetic steroids 5{alpha}-pregnan-3ß-ol-20-one and 5ß-pregnan-3ß-ol-20-one modulated neither GABAA, nor glycine, receptors.

Conclusions. The data demonstrate that structure–activity relationships for steroid modulation at glycine and GABAA receptors differ. Comparing the EC50 values reported here with free plasma concentrations during steroid-induced anaesthesia indicates that a selective modulation of GABAA receptor activity is likely to occur in vivo.

Br J Anaesth 2004; 92: 704–11

Keywords: anaesthetics i.v., steroid, minaxolone; glycine receptors; GABAA receptors


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Fast inhibitory synaptic transmission within the mammalian central nervous system is mediated by {gamma}-aminobutyric acid (GABA) and glycine acting at anion-selective GABAA and strychnine-sensitive glycine receptors, respectively. Such transmitter-gated ion channels are assembled as a pentameric complex of subunits which, for the GABAA receptor, can be drawn from families that include {alpha}1–6, ß1–3, and {gamma}1–3,1 and for the glycine receptor {alpha}1–4 and ß1.2 In vitro investigations have shown that the activity of the GABAA receptor is potentiated by many general anaesthetics at clinically relevant concentrations including; thiopentone, propofol, etomidate and numerous inhalational agents.35 Moreover, recent studies using mice genetically engineered to express select GABAA receptor isoforms insensitive to the intravenous agent etomidate have demonstrated that suppression of the nocifensive reflex, or sedation, in response to this anaesthetic are associated with the modulation of ß3- and ß2-subunit-containing GABAA receptors, respectively.6 7

In common with the GABAA receptor, in vitro studies have demonstrated that the glycine receptor is subject to positive allosteric regulation by select anaesthetic agents including; propofol, trichloroethanol and volatile halogenated hydrocarbons.25 However, there is no in vivo evidence that firmly establishes the glycine receptor as a target of intravenous anaesthetic action, although evidence exists that immobility induced by halothane is, at least in part, mediated by glycine receptors.8 To further evaluate a potential role of the glycine receptor in anaesthesia, the present study has examined the activity of anaesthetic and non-anaesthetic steroids at recombinant GABAA ({alpha}1ß2{gamma}2L) and glycine ({alpha}1) receptors. Steroidal agents are of particular interest due both to their remarkable potency as modulators of GABAA receptor activity and exquisite stereoselectivity of action in vitro and in vivo.3 In addition, the amidine steroid, RU 5135, is a potent antagonist of GABAA and glycine receptors,9 10 suggesting that potential steroid binding sites exist on both receptor types. Specifically, we have examined the activity of five anaesthetic pregnane steroids (alphaxalone, minaxolone, ORG 20599, 5ß-pregnan-3{alpha}-ol-20-one (eltanolone) and 5{alpha}-pregnan-3{alpha}-ol-20-one), two non-anaesthetic epimers (5{alpha}-pregnan-3ß-ol-20-one and 5ß-pregnan-3ß-ol-20-one) and three glucocorticosteroids previously suggested to be positive allosteric modulators of glycine receptor activity11 (see Fig. 1 for chemical structures).



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Fig 1 Chemical structures of the steroid compounds examined in this study. By convention, substituents projecting below (broken wedges) and above (solid wedges) the plane of the steroid ring system are in the {alpha} and ß configurations, respectively. The orientation of the C5-hydrogen in the saturated ring systems determines the configuration of the A and B ring fusion (5{alpha}-pregnane series, trans; 5ß-pregnane series, cis). Steroids with anaesthetic activity are: alphaxalone, Org 20599, minaxolone, 5{alpha}-pregnan-3{alpha}-ol-20-one and 5ß-pregnan-3{alpha}-ol-20-one (eltanolone).

 

    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Expression of GABAA and glycine receptor subunits in Xenopus laevis oocytes
Xenopus laevis oocytes (Stage V–VI) were defolliculated by pre-treatment with 2 mg ml–1 collagenase type I (Sigma-Aldrich) for 2–3 h at room temperature (20–23°C) in Barth’s saline with Ca2+ salts omitted. cDNAs encoding the glycine receptor {alpha}1 subunit, were injected intranuclearly (20 nl of 2.0 mg ml–1) whereas cRNAs encoding GABAA receptor {alpha}1, ß2 and {gamma}2L subunits were injected into the cytoplasm (30–50 nl of 1 mg ml–1 for each subunit). Oocytes were subsequently maintained at 19–20°C for 2 to 12 days in 96-well plates containing 200 µl of standard Barth’s solution (composition in mM: NaCl 88, KCl 1, NaHCO3 2.4, Hepes (N-[2-hydroxyethyl]piperazine-N'-[2-ethane-sulphonic acid]) 15, Ca(NO3)2 0.5, CaCl2 0.5 and MgSO4 1.0; adjusted to pH 7.6 with NaOH). The solution was supplemented with 0.1 mg ml–1 gentamicin.

Electrophysiological recordings
Oocytes were held in a recording chamber (0.5 ml) and continuously superfused (7–10 ml min–1) with frog Ringer solution (composition in mM: NaCl 120, KCl 2, CaCl2 1.8, Hepes 5; adjusted to pH 7.4 with NaOH). Oocytes were voltage-clamped at a holding potential of –60 mV using a Gene Clamp 500 amplifier (Axon Instruments, Foster City, USA) in the two-electrode voltage clamp mode. Voltage-sensing and current-passing electrodes were filled with 3 M KCl and had resistances of 1–2 M{Omega} when measured in frog Ringer solution. Current and voltage signals were both acquired (at the digitization rate of 100 Hz) and analysed with the Win WCP program (courtesy of Dr J. Dempster, University of Strathclyde, UK; http://www.strath.ac.uk/Departments/PhysPharm/), utilizing a Dell Dimension Pentium PC and an Axon Instruments 1200 Digidata interface A to D converter. Timed pulses of drugs dissolved in Ringer solution were applied to oocytes via a BPS-4 bath perfusion system (Adams and List Associates, New York, USA) with a four-way manifold. For each oocyte a maximally effective concentration of GABA (3 mM) or glycine (1 mM) was applied once every 20 min until the peak inward current response produced was stable.12 In the majority of experiments investigating potentiation by steroids, a concentration of GABA, or glycine, which produced a response 10% of the maximal agonist-evoked response (EC10) was utilized. The EC10 was determined for each oocyte. The steroid was pre-applied for 30 to 60 s before co-application with the appropriate concentration of GABA, or glycine. Concentration–response data for the enhancement of agonist-evoked responses by steroids were fitted iteratively by use of SigmaPlot version 8.01 (SPSS Inc., Chicago, IL, USA), with the four-parameter Hill equation:

Im/Im(max)=Ic+([A]nH/([A]nH+EC50nH))

where Im is the amplitude of the agonist-evoked current in the presence of the steroid at concentration [A], Im(max) is the amplitude of the response in the presence of a maximally effective concentration of the steroid, Ic is the amplitude of the agonist-evoked response in the absence of the steroid, EC50 is the concentration of the steroid producing half-maximal enhancement and nH is the Hill coefficient. In all instances, the amplitude of the modulated current is expressed as a percentage of the control agonist-evoked current (Ic) recorded in the absence of steroid.

In instances where the concentration–response relationship for agonist modulation was clearly bell-shaped, curve fitting was restricted to the ascending limb and apparent maximum. Quantitative results are expressed as the arithmetic mean (SEM) (standard error of the mean).

Reagents
{gamma}-aminobutyric acid (GABA), glycine, 5{alpha}-pregnan-3{alpha}-ol-20-one, 5ß-pregnan-3{alpha}-ol-20-one, 5{alpha}-pregnan-3ß-ol-20-one, 5ß-pregnan-3ß-ol-20-one, 20{alpha}-dihydrocortisol, {alpha}-cortol and hydrocortisone were purchased from Sigma-Aldrich (Poole, Dorset, UK). Alphaxalone and minaxolone were donated by Glaxo-Smith-Kline (Stevenage, Hertfordshire, UK). Org 20599 [(2ß,3{alpha},5ß)-21-chloro-3-hydroxy-2-(4-morpholinyl)-pregnan-20-one] was provided by Organon Laboratories (Newhouse, Lanarkshire, UK). Steroidal agents (with the exception of Org 20599 and minaxolone) were initially dissolved as concentrated stock solutions in dimethylsulphoxide (DMSO) and subsequently diluted to the desired concentration in frog Ringer solution. In vehicle controls, the highest final concentration of DMSO employed (0.1% vol vol–1) had no effect upon current responses to either GABA, or glycine. All other agents were dissolved as concentrated stock solutions in double distilled deionized water. Stock solutions of all compounds were prepared daily.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Modulation of glycine and GABAA receptor activity by alphaxalone, Org 20599 and minaxolone
Within the concentration range 1–100 µM, alphaxalone, minaxolone and Org 20599 all produced a concentration-dependent enhancement of the peak inward current evoked by glycine at EC10 acting upon homo-oligomeric, strychnine-sensitive, glycine receptors expressed in Xenopus laevis oocytes. Over the range of concentrations examined, the anaesthetics did not elicit a detectable current response when applied in the absence of glycine. The EC50 values for potentiation of the response to glycine by alphaxalone (Fig. 2A) and Org 20599 (Fig. 2B) were 27.8 (5.8) µM (n=4) and 22.9 (1.5) µM (n=3) respectively, whereas that for minaxolone was 13.1 (0.9) µM (n=4, Table 1, Fig. 2C). At the highest concentration tested (100 µM), the rank order in which the steroids increased the amplitude of the glycine-evoked current (expressed as a percentage of control) was minaxolone (1197 (134)%, n=4) >Org 20599 (535 (81)%, n=3) >alphaxalone (199 (14)%, n=4, Table 1). Both the potency and maximal effect of alphaxalone and Org 20599 at the glycine receptor were substantially less than previously reported for positive allosteric modulation of GABAA receptors assembled from the {alpha}1ß2{gamma}2L subunit combination.13 In contrast, in the present study, although minaxolone was approximately 10-fold more potent (EC50=1.3 (0.2) µM, n=3) in potentiating currents evoked by GABA at EC10 acting upon the GABAA receptor, the maximal potentiation (to 901 (142)% of control) was less than that found for the glycine receptor (Fig. 2C). The modulatory effects of the steroids at glycine and GABAA receptors are summarized in Table 1.



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Fig 2 Modulation of recombinant glycine ({alpha}1) and GABAA ({alpha}1ß2{gamma}2L) receptors expressed in Xenopus laevis oocytes by steroid anaesthetics. (AC) Graphical depictions of the relationship between the concentration (logarithmic scale) of bath applied anaesthetic and the current produced by GABA, or glycine, plotted on a linear scale and expressed as a percentage of the control response in the absence of anaesthetic. Each data point is the mean (SEM) of the results obtained from 3–4 oocytes (see Table 1 for fitted parameters). All recordings and data were obtained from oocytes voltage-clamped at –60 mV. The data for the modulation of GABAA {alpha}1ß2{gamma}2L receptors by Org 20599 and alphaxalone are derived from Hill-Venning et al. (1996)13 and are shown for comparison.

 

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Table 1 Modulation of {alpha}1 glycine receptors and {alpha}1ß2{gamma}2L GABAA receptors by steroid anaesthetics. Data are quantified as the mean (SEM) of the data from 3–7 experiments. Abbreviations: 5{alpha}3{alpha}, 5{alpha}-pregnan-3{alpha}-ol-20-one; 5ß3{alpha}, 5ß-pregnan-3{alpha}-ol-20-one; ND=not determined
 
Modulation of glycine and GABAA receptor activity by 5ß-pregnan-3{alpha}-ol-20-one (eltanolone) and its stereoisomers
In preliminary experiments 5ß-pregnan-3{alpha}-ol-20-one produced a concentration-dependent inhibition of the response to glycine at EC10. To quantify this effect with greater precision, all subsequent experiments using pregnane steroid isomers were conducted using an EC50 concentration of glycine. 5ß-pregnan-3{alpha}-ol-20-one blocked the response to glycine in a concentration-dependent manner (Fig. 3). The threshold for inhibition was approximately 300 nM and at the highest concentration of the steroid tested (10 µM), the glycine-evoked response was reduced to 57 (4)% (n=6) of control (Fig. 3). Higher concentrations of the steroid, that would allow the calculation of an unambiguous IC50 value, could not be tested due to the limited solubility of the compound. This inhibitory action contrasts with a modest potentiation observed for the 5{alpha}-isomer, 5{alpha}-pregnan-3{alpha}-ol-20-one which, at a concentration of 10 µM, enhanced the glycine-evoked current to 133 (7)% (n=7) of control with an EC50 of 250 (190) nM (Fig. 3). In contrast to compounds with the 3{alpha}-ol configuration, neither 5{alpha}-pregan-3ß-ol-20-one, nor 5ß-pregnan-3ß-ol-20-one (10 nM–10 µM, each n=3), had any effect upon glycine-evoked currents (Fig. 3).



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Fig 3 Modulation of recombinant glycine ({alpha}1) receptors expressed in Xenopus laevis oocytes by pregnane steroid diastereomers. Graph illustrating the relationship between the concentration (logarithmic scale) of bath applied 5{alpha}-pregnan-3{alpha}-ol-20-one, 5ß-pregnan-3{alpha}-ol-20-one, 5{alpha}-pregnan-3ß-ol-20-one and 5ß-pregnan-3ß-ol-20-one and the current produced by glycine plotted on a linear scale and expressed as a percentage of the control response in the absence of steroid. Each data point is the mean (SEM) of the results obtained from 3–7 oocytes (see Table 1 for fitted parameters). All recordings and data were obtained from oocytes voltage-clamped at –60 mV.

 
For comparative purposes, we examined the effect of 5ß-pregnan-3{alpha}-ol-20-one, 5{alpha}-pregnan-3ß-ol-20-one and 5ß-pregnan-3ß-ol-20-one upon {alpha}1ß2{gamma}2L GABAA receptors, having reported upon 5{alpha}-pregnan-3{alpha}-ol-20-one previously.14 5ß-pregnan-3{alpha}-ol-20-one (10 nM–10 µM) increased the response to GABA at EC10 with an EC50 of 262 (74) nM and a maximal enhancement to 722 (54)% of control in the presence of the steroid at a concentration of 1 µM. Concentrations of 5ß-pregnan-3{alpha}-ol-20-one in excess of 1 µM were associated with a reduced potentiation, producing a bell-shaped concentration–response curve. Over the same concentration range, neither 5{alpha}-pregnan-3ß-ol-20-one, nor 5ß-pregnan-3ß-ol-20-one, had any significant effect upon the response to GABA (Fig. 4), consistent with data previously obtained for native GABAA receptors.1517



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Fig 4 Modulation of recombinant GABA ({alpha}1ß2{gamma}2L) receptors expressed in Xenopus laevis oocytes by pregnane steroid diastereomers. The graph illustrates the relationship between the concentration (logarithmic scale) of bath applied 5{alpha}-pregnan-3{alpha}-ol-20-one, 5ß-pregnan-3{alpha}-ol-20-one, 5{alpha}-pregnan-3ß-ol-20-one and 5ß-pregnan-3ß-ol-20-one and the current produced by GABA plotted on a linear scale and expressed as a percentage of the control response in the absence of steroid. Each data point is the mean (SEM) of the results obtained from 3–4 oocytes (see Table 1 for fitted parameters). All recordings and data were obtained from oocytes voltage-clamped at –60 mV. The data for 5{alpha}-pregnan-3{alpha}-ol-20-one are derived from Belelli et al. (2002)14 and are shown for comparative purposes.

 
The influence of glucocorticosteroids on the glycine receptor
It has been reported that 20{alpha}-dihydrocortisol, {alpha}-cortol and hydrocortisone potentiate the depolarizing response to glycine recorded extracellularly from the rat optic nerve in vitro.11 In the present study, each of these steroids was examined over the concentration range 300 nM–30 µM for their effect upon homo-oligomeric glycine receptors activated by glycine at EC10. No significant effect was detected for any of the compounds examined (n=3 in each case, data not shown).


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The major findings of the present study are firstly that minaxolone is an effective positive allosteric modulator of the {alpha}1 glycine receptor and secondly that modulation of glycine receptor activity by 5{alpha}-pregnan-3{alpha}-ol-20-one and 5ß-pregnan-3{alpha}-ol-20-one demonstrates an inverse relationship, the former eliciting modest potentiation and the latter producing inhibition.

Minaxolone has previously been shown to enhance the displacement by glycine of [3H]strychnine bound to glycine receptors in synaptosomal membranes of rat spinal cord,18 a finding congruent with the positive allosteric regulation demonstrated in the present study. The activity of minaxolone is notable because the maximal effect of the steroid to potentiate currents mediated by the {alpha}1-glycine receptor was comparable to that observed for GABAA receptors assembled from {alpha}1, ß2 and {gamma}2L subunits. Similarly, the second water-soluble steroid examined, Org 20599, also exhibited considerable activity at the glycine receptor. Although some steroids, including alphaxalone,19 pregnenolone20 and pregnenolone acetate,20 have previously been shown to potentiate responses to glycine mediated by the {alpha}1 receptor, their maximal effects in comparison to either minaxolone, or Org 20599, are relatively weak. A limited ability of high concentrations (i.e. 30–100 µM) of alphaxalone to potentiate responses to glycine was confirmed in the present study, although previous investigations performed on the rat cuneate slice21 and optic nerve preparations,11 mouse spinal neurones in culture22 and Xenopus laevis oocytes23 expressing hetero-oligomeric {alpha}1ß glycine receptors did not detect such an action. This apparent discrepancy could be explained by the fact that the present study used a maximal concentration of alphaxalone higher than that previously examined, or by differences in glycine receptor subunit composition between studies. The latter is known to influence the actions of certain steroids upon glycine receptors.20 In this study, we also detected limited enhancement of responses to glycine by 5{alpha}-pregnan-3{alpha}-ol-20-one, but the magnitude of this effect was approximately 20-fold less than the maximal enhancement of currents mediated by the {alpha}1ß2{gamma}2L GABAA receptor isoform,14 perhaps contributing to the perception that this steroid is essentially ‘inactive’ at the glycine receptor.17 24 Nonetheless, the EC50 value (250 nM) for 5{alpha}-pregnan-3{alpha}-ol-20-one at the {alpha}1 glycine receptor is only 1.5-fold higher than that reported for the {alpha}1ß2{gamma}2L GABAA receptor isoform (i.e. 177 nM).14

Structurally, minaxolone, Org 20599, alphaxalone and 5{alpha}-pregnan-3{alpha}-ol-20-one share a pregnane ring backbone presenting a hydroxyl group at C3 in the {alpha}-configuration (i.e. projecting below the surface of the steroid ring), an A and B ring fusion in the trans orientation and a keto group at C20 of the acetyl side chain, Fig. 1). Alphaxalone, containing an 11-keto substituent (Fig. 1), appears 100-fold less potent (EC50 = 27.8 µM) than 5{alpha}-pregnan-3{alpha}-ol-20-one at the glycine {alpha}1 receptor, but displays an enhanced maximal effect (Table 1). Additional C21-chloro- and 2ß-morpholinyl- substituents in the structure of alphaxalone (i.e. Org 20599) have no effect upon potency (EC50=22.9 µM), but increase maximal enhancement of the glycine evoked current (Table 1). Minaxolone, containing 2ß-ethoxy and 11{alpha}-dimethyl amino substituents (Fig. 1), is both more potent (EC50=13.1 µM) and efficacious than any of the other substituted steroids derived from 5{alpha}-pregnan-3{alpha}-ol-20-one (Table 1). It would be of interest to explore the individual contributions of the 2ß-ethoxy and 11{alpha}-dimethyl groups upon activity at the glycine receptor, particularly because the 2ß-ethoxy substituent confers superior anaesthetic potency over alphaxalone,25 but unfortunately such compounds are not currently available.

The effect of substituents at C2 and C11 upon activity at the {alpha}1 glycine receptor is distinct from that observed for GABAA receptor isoforms because at the latter alphaxalone, ORG 20599 and minaxolone exert maximal effects comparable to 5{alpha}-pregnan-3{alpha}-ol-20-one, albeit with slightly reduced potency (Table 1). The difference in the steroid structure–activity relationship at GABAA and glycine receptors was emphasized by comparing the effects of the diasteromers 5{alpha}-pregnan-3{alpha}-ol-20-one, 5ß-pregnan-3{alpha}-ol-20-one, 5{alpha}-pregnan-3ß-ol-20-one and 5ß-pregnan-3ß-ol-20-one (Fig. 1). In contrast to the modest potentiation observed for 5{alpha}-pregnan-3{alpha}-ol-20-one, 5ß-pregnan-3{alpha}-ol-20-one inhibited the response to glycine in a concentration-dependent manner. At native15 16 and recombinant GABAA receptor isoforms, this pair of diastereomers exhibits approximately equal potency and efficacy in potentiating the actions of GABA. These observations indicate that the configuration of the pregnane steroid A and B ring fusion is a determinant of activity at the {alpha}1 glycine receptor, the trans- and cis-conformations favouring potentiation and inhibition respectively. The 3ß-epimers of the 5{alpha}- and 5ß-pregnanes were both inactive upon either {alpha}1 glycine receptors or GABAA receptors assembled from {alpha}1, ß2 and {gamma}2L subunits, demonstrating that, for either inhibitory receptor, activity is suppressed if the C3-hydroxyl projects above the plane of the pregnane ring system.

Previous studies of steroidal regulation of the glycine receptors native to spinal neurones and the recombinant {alpha}1 receptor have reported predominantly inhibitory effects, as found here for 5ß-pregnan-3{alpha}-ol-20-one. Steroids causing inhibition include: pregnenolone (5{alpha}-pregnen-3ß-ol-20-one) hemisuccinate,20 pregnenolone sulphate,17 20 dehydroepiandrosterone (5-androsten-3ß-ol-17-one) sulphate,20 5{alpha}-androstan-3ß-ol-17-one sulphate,20 progesterone,17 17{alpha}-OH-progesterone,17 deoxycorticosterone17 and corticosterone.17 The more potent of these compounds (Ki=1.9 to 9.8 µM) all possess a negative charge at C3, provided by a substituted sulphate or hemisuccinate group, which Maksay and collegues20 postulate to be important for inhibition of the {alpha}1 glycine receptor. The lower potency of C3-hydroxylated 5{alpha}-pregnan-3ß-ol-20-one found in the present study is in agreement with this suggestion. However, both 5{alpha}-androstan-3ß-ol-17-one sulphate and 5{alpha}-androstan-3{alpha}-ol-17-one sulphate inhibit the {alpha}1 glycine receptor with similar potency and effectiveness,20 indicating that the orientation of the androstane A and B rings is not a determinant of action of the sulphated steroids.

In contrast to the potentiation of the extracellularly recorded response to glycine obtained with the rat optic nerve preparation,11 the glucocorticosteroids 20{alpha}-dihydrocortisol, {alpha}-cortol and hydrocortisone had no detectable effect upon current responses to glycine mediated by the {alpha}1 glycine receptor. It is possible that differences in glycine receptor subunit composition may contribute to such a discrepancy, as may the difference in the electrophysiological techniques employed. Nonetheless, the present results are in agreement with data obtained from chick spinal neurones, where responses to glycine were similarly insensitive to hydrocortisone and weakly depressed, rather than potentiated, by corticosterone and deoxycorticosterone.17

There is abundant evidence to support the notion that the GABAA receptor is a major molecular target of general anaesthetic action,37 26 but a similar role for the glycine receptor is less certain. Many classes of experimental and clinical anaesthetics including gases, alcohols, some barbiturates, propofol, etomidate and volatile agents in particular, potentiate responses to glycine, but this is not a universal feature of anaesthetic action at the molecular level.25 8 26 27 In addition, the concentrations of anaesthetic required to positively modulate the glycine receptor are, for intravenous agents at least, considerably higher than those required for an equivalent effect at the GABAA receptor and frequently lie outside the clinically relevant range.24 The present study demonstrates that modulation of glycine receptor activity by steroids is not crucial to their anaesthetic activity because the isomers 5{alpha}-pregnan-3{alpha}-ol-20-one and 5ß-pregnan-3{alpha}-ol-20-one, both of which are general anaesthetics, produced opposing effects upon glycine receptor function. This, however, does not preclude a contribution to anaesthesia that may be agent specific. Amongst intravenous anaesthetics, minaxolone emerges as a relatively potent and efficacious positive allosteric modulator of glycine receptor function. The free-plasma concentration of minaxolone that abolishes movement in response to a noxious stimulus has been estimated to be approximately 200 nM, after correction for extensive protein binding of 95%.28 Although difficult to interpret due the disposition and biophase concentrations of the anaesthetic being unknown, such a value provides an approximate guide to the concentrations of minaxolone that might be clinically relevant. The EC50 values for potentiation of GABA and glycine receptor activity by minaxolone of 1.3 and 13 µM respectively would thus suggest the potential for modulation of synaptic transmission mediated by GABA, but not glycine, in vivo. However, as a caveat, it should be noted that receptors expressed heterologously in Xenopus oocytes might not be subject to the same post-translational modifications, such as phosphorylation, as occur in their native, neuronal, environment. Phosphorylation of GABAA receptor isoforms by protein kinase C, for example, is known to enhance sensitivity to neurosteroids in the hippocampal dentate gyrus.29 Hence, extrapolations from relatively simple in vitro systems to the situation in vivo must be made with caution.

Agents that potentiate glycinergic neurotransmission are of interest because glycine has a pivotal role in the processing of sensory input at the spinal level and is known to suppress nociceptive signals.2 The importance of glycerinergic neurotransmission is vividly illustrated by numerous mutations of the glycine receptor {alpha}1 subunit that are associated with hyperekplexia, an autosomal dominant disorder in which reduced postsynaptic sensitivity to glycine underlies an exaggerated startle reflex and neonatal hypertonia.30 Although currently best managed by the benzodiazepine, clonazepam, presumably via compensatory facilitation of GABA-ergic neurotransmission, agents that enhance the actions of glycine would be a logical treatment for this condition. More generally, potentiation of glycine receptor function within the spinal cord can be anticipated to produce analgesia.2 Anaesthetics that combine the ability to potentiate both GABA-ergic and glycinergic transmission might thus be of considerable utility.


    Acknowledgements
 
C. J. Weir was supported by a Research Fellowship from the British Journal of Anaesthesia and the Royal College of Anaesthetists. D. Belelli is a MRC Non-Clinical Fellow. Some of the work reported here was supported by the Commision of the European Communities RTD programme ‘Quality of Life and Management of Living Resources’, QLK1-CT-2000-00179, Tenovus Tayside and Scottish Hospital Endowments Research Trust. We are grateful to Dr Paul Whiting, Neuroscience Research Centre, Merck Sharp and Dohme, Harlow, UK for the gift of cDNAs encoding the GABAA receptor subunits and Professor Heinrich Betz, Max Planck Institute for Brain Research, Frankfurt-Main, Germany, for the cDNA encoding the glycine {alpha}1 subunit. We thank Organon Laboratories and Glaxo SmithKline for donating Org 20599 and minaxolone, respectively.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
1 Barnard EA, Skolnick P, Olsen RW et al. International Union of Pharmacology. XV. Subtypes of gamma-aminobutyric acidA receptors: classification on the basis of subunit structure and receptor function. Pharmacol Rev 1998; 50: 291–313[Abstract/Free Full Text]

2 Laube B, Maksay G, Schemm R, Betz H. Modulation of glycine receptor function: a novel approach for therapeutic intervention at inhibitory synapses? Trends Pharmacol Sci 2002; 23: 519–27[CrossRef][ISI][Medline]

3 Belelli D, Pistis M, Peters JA, Lambert JJ. General anaesthetic action at transmitter-gated inhibitory amino acid receptors. Trends Pharmacol Sci 1999; 20: 496–502[CrossRef][ISI][Medline]

4 Krasowski MD, Harrison NL. General anaesthetic actions on ligand-gated ion channels. Cell Mol Life Sci 1999; 55: 1278–1303[CrossRef][ISI][Medline]

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