C J Weir1, A T Y Ling, D Belelli, J A W Wildsmith, J A Peters, J J Lambert. 1. Department of Anaesthesia, Division of Pathology and Neuroscience, Ninewells Hospital and Medical School, The University of Dundee, Dundee DD1 9SY, UK. c.j.weir@dundee.ac.uk
Abstract
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 alpha1 glycine and alpha1beta2gamma2L GABAA receptors. METHODS: Recombinant alpha1 glycine or alpha1beta2gamma2L 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 microM), Org20599 (EC50=1.1 microM) and alphaxalone (EC50=2.2 microM) enhanced currents mediated by GABAA receptors. The anaesthetics also enhanced currents mediated by glycine receptors, although with higher EC50 values (minaxolone 13.1 microM; Org20599=22.9 microM and alphaxalone=27.8 microM). 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, 5alpha-pregnan-3alpha-ol-20-one and 5beta-pregnan-3alpha-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 5alpha-pregnan-3beta-ol-20-one and 5beta-pregnan-3beta-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.
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 alpha1 glycine and alpha1beta2gamma2L GABAA receptors. METHODS: Recombinant alpha1 glycine or alpha1beta2gamma2L 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 microM), Org20599 (EC50=1.1 microM) and alphaxalone (EC50=2.2 microM) enhanced currents mediated by GABAA receptors. The anaesthetics also enhanced currents mediated by glycine receptors, although with higher EC50 values (minaxolone 13.1 microM; Org20599=22.9 microM and alphaxalone=27.8 microM). 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, 5alpha-pregnan-3alpha-ol-20-one and 5beta-pregnan-3alpha-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 steroids5alpha-pregnan-3beta-ol-20-one and 5beta-pregnan-3beta-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.
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