Selective Antagonism of 5-Reduced Neurosteroid Effects at GABAA Receptors

Although neurosteroids have rapid effects on GABAA receptors, study of steroid actions at GABA receptors has been hampered by a lack of pharmacological antagonists. In this study, we report the synthesis and characterization of a steroid analog, (3 ,5 )-17-phenylandrost-16-en-3-ol (17PA), that selectively antagonized neurosteroid potentiation of GABA responses. We examined 17PA using the 1 2 2 subunit combination expressed in Xenopus laevis oocytes. 17PA had little or no effect on baseline GABA responses but antagonized both the response augmentation and the direct gating of GABA receptors by 5 -reduced potentiating steroids. The effect was selective for 5 -reduced potentiating steroids; 5 -reduced potentiators were only weakly affected. Likewise, 17PA did not affect barbiturate and benzodiazepine potentiation. 17PA acted primarily by shifting the concentration response for steroid potentiation to the right, suggesting the possibility of a competitive component to the antagonism. 17PA also antagonized 5 -reduced steroid potentiation and gating in hippocampal neurons and inhibited anesthetic actions in X. laevis tadpoles. Analogous to benzodiazepine site antagonists, the development of neurosteroid antagonists may help clarify the role of GABA-potentiating neurosteroids in health and disease. GABAA receptors are important participants in central nervous system inhibition and are the target of many clinically important compounds, including benzodiazepines, barbiturates, neuroactive steroids, and other general anesthetics (Macdonald and Olsen, 1994; Belelli et al., 1999). By allosterically potentiating GABAA receptor-mediated responses, these compounds generally increase inhibition and decrease overall activity of neuronal circuits (Macdonald and Olsen, 1994). Neuroactive steroids are particularly interesting because they may be present endogenously at concentrations that modulate GABAA receptor function (Robel and Baulieu, 1994). Many important questions remain unanswered regarding steroid actions at GABAA receptors. Despite concerted efforts, a steroid binding site on the GABAA receptor has not been identified. GABA-potentiating steroids have two distinct structural classes: 5 -reduced and 5 -reduced (Covey et al., 2001). Both classes potentiate GABAA responses with similar potency and efficacy; whether the two classes work at the same site or by the same mechanisms is unclear. In addition, the role of endogenous neurosteroids in modulating GABAA receptors is obscure. New pharmacological tools for exploring neuroactive steroids, including neurosteroid antagonists, would advance efforts to understand the interaction of steroids with GABAA receptors. Several earlier studies explored potential steroid antagonists. [H]Flunitrazepam binding assays suggest that 3 -hydroxysteroids competitively antagonize the actions of the potentiating steroids (3 ,5 )-3-hydroxypregnan-20-one (3 5 P) and (3 ,5 )3-hydroxypregnan-20-one (3 5 P), two standard representatives of the 5 -reduced and 5 -reduced classes of potentiating steroids (Prince and Simmonds, 1992, 1993). In electrophysiological studies, however, 3 -hydroxypregnane steroids noncompetitively antagonize potentiating steroids through activationdependent block of GABAA receptors (Wang et al., 2002). The block by 3 -hydroxysteroids is not specific; barbiturate potentiation is reduced, as are responses to GABA itself at high This study was supported by National Institutes of Health grant MH45493, by a gift from the Bantly Foundation (to C.F.Z.), by National Institutes of Health grants GM47969 (to D.F.C., C.F.Z., and A.S.E.), NS40488 and AA12952 (to S.M.), and a Klingenstein Foundation grant (to S.M.). M.W. was supported by a postdoctoral fellowship from Umeå University-Washington University Network and EU regional fund. 1 Present address: Systems and Cognitive Neuroscience, National Institute of Neurological Disorders and Stroke, Neuroscience Center, Suite 2115, 6001 Executive Boulevard, Bethesda, MD 20892-9521. 2 Present address: Department of Clinical Science, Section of Obstetrics and Gynecology, Umeå, University, S-90187 Umeå, Sweden. ABBREVIATIONS: 3 5 P, (3 ,5 )-3-hydroxypregnan-20-one; 3 5 P, (3 ,5 )-3-hydroxypregnan-20-one; 17PA, (3 ,5 )-17-phenylandrost-16en-3-ol; DMSO, dimethyl sulfoxide; LRR, loss of righting reflex; LSR, loss of swimming reflex. 0026-895X/04/6505-1191–1197$20.00 MOLECULAR PHARMACOLOGY Vol. 65, No. 5 Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 3079/1143632 Mol Pharmacol 65:1191–1197, 2004 Printed in U.S.A. 1191 at A PE T Jornals on M ay 9, 2017 m oharm . aspeurnals.org D ow nladed from GABA concentrations (Wang et al., 2002). Therefore, 3 -hydroxysteroids, although interesting functional antagonists of steroid potentiation, lack the specificity desired of a tool for exploring steroid potentiation of GABAA receptors. In the present study, we examined the actions of a synthetic neuroactive steroid analog that was inactive as a potentiator at GABAA receptors in our functional screens, (3 ,5 )-17-phenylandrost-16-en-3-ol (17PA). 17PA represents the first described selective antagonist of neurosteroid potentiation at GABAA receptors. It is noteworthy that 17PA antagonized the physiological and anesthetic actions of 5 reduced steroids but had little effect on 5 -reduced steroids. Furthermore, 17PA inhibited direct gating of GABAA receptor channels by 5 -reduced but not 5 -reduced steroids. Inhibition of 5 -reduced steroid potentiation decreased with increasing potentiating steroid concentration, suggesting the possibility of a competitive interaction between the antagonist and potentiating steroids. 17PA did not antagonize barbiturate or benzodiazepine potentiation. Our results suggest that 5 -reduced and 5 -reduced steroids are likely to possess different sites or mechanisms of potentiation and support the hypothesis that GABAA receptors are critical participants in the behavioral anesthesia induced by neuroactive steroids. Materials and Methods 17PA Synthesis. Fig. 1A shows the reaction scheme for the