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 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
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Abstract |
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Methods. Recombinant 1 glycine or
1ß2
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 780950% 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-pregnan-3
-ol-20-one and 5ß-pregnan-3
-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
-pregnan-3ß-ol-20-one and 5ß-pregnan-3ß-ol-20-one modulated neither GABAA, nor glycine, receptors.
Conclusions. The data demonstrate that structureactivity 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: 70411
Keywords: anaesthetics i.v., steroid, minaxolone; glycine receptors; GABAA receptors
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Introduction |
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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 (1ß2
2L) and glycine (
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
-ol-20-one (eltanolone) and 5
-pregnan-3
-ol-20-one), two non-anaesthetic epimers (5
-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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Methods |
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Electrophysiological recordings
Oocytes were held in a recording chamber (0.5 ml) and continuously superfused (710 ml min1) 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 12 M 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. Concentrationresponse 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 concentrationresponse 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
-aminobutyric acid (GABA), glycine, 5
-pregnan-3
-ol-20-one, 5ß-pregnan-3
-ol-20-one, 5
-pregnan-3ß-ol-20-one, 5ß-pregnan-3ß-ol-20-one, 20
-dihydrocortisol,
-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
,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 vol1) 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.
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Results |
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Discussion |
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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 1-glycine receptor was comparable to that observed for GABAA receptors assembled from
1, ß2 and
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
1 receptor, their maximal effects in comparison to either minaxolone, or Org 20599, are relatively weak. A limited ability of high concentrations (i.e. 30100 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
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
-pregnan-3
-ol-20-one, but the magnitude of this effect was approximately 20-fold less than the maximal enhancement of currents mediated by the
1ß2
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
-pregnan-3
-ol-20-one at the
1 glycine receptor is only 1.5-fold higher than that reported for the
1ß2
2L GABAA receptor isoform (i.e. 177 nM).14
Structurally, minaxolone, Org 20599, alphaxalone and 5-pregnan-3
-ol-20-one share a pregnane ring backbone presenting a hydroxyl group at C3 in the
-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
-pregnan-3
-ol-20-one at the glycine
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
-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
-pregnan-3
-ol-20-one (Table 1). It would be of interest to explore the individual contributions of the 2ß-ethoxy and 11
-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 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
-pregnan-3
-ol-20-one, albeit with slightly reduced potency (Table 1). The difference in the steroid structureactivity relationship at GABAA and glycine receptors was emphasized by comparing the effects of the diasteromers 5
-pregnan-3
-ol-20-one, 5ß-pregnan-3
-ol-20-one, 5
-pregnan-3ß-ol-20-one and 5ß-pregnan-3ß-ol-20-one (Fig. 1). In contrast to the modest potentiation observed for 5
-pregnan-3
-ol-20-one, 5ß-pregnan-3
-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
1 glycine receptor, the trans- and cis-conformations favouring potentiation and inhibition respectively. The 3ß-epimers of the 5
- and 5ß-pregnanes were both inactive upon either
1 glycine receptors or GABAA receptors assembled from
1, ß2 and
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 1 receptor have reported predominantly inhibitory effects, as found here for 5ß-pregnan-3
-ol-20-one. Steroids causing inhibition include: pregnenolone (5
-pregnen-3ß-ol-20-one) hemisuccinate,20 pregnenolone sulphate,17 20 dehydroepiandrosterone (5-androsten-3ß-ol-17-one) sulphate,20 5
-androstan-3ß-ol-17-one sulphate,20 progesterone,17 17
-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
1 glycine receptor. The lower potency of C3-hydroxylated 5
-pregnan-3ß-ol-20-one found in the present study is in agreement with this suggestion. However, both 5
-androstan-3ß-ol-17-one sulphate and 5
-androstan-3
-ol-17-one sulphate inhibit the
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-dihydrocortisol,
-cortol and hydrocortisone had no detectable effect upon current responses to glycine mediated by the
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-pregnan-3
-ol-20-one and 5ß-pregnan-3
-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 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.
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Acknowledgements |
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