Brain Research, 404 (1987) 355-360 355 Elsevier BRE 22052 Pregnenolone-sulfate: an endogenous antagonist of the y-aminobutyric acid receptor complex in brain? Maria Dorota Majewska and Rochelle D. Schwartz* Section on Molecular Pharmacology, Clinical Neuroscience Branch. National Institute of Mental Health, Bethesda, MD (U.S.A.) (Accepted 14 October 1986) Key words: Pregnenolone; y-Aminobutyric acid (GABA); g-Aminobutyric acid receptor The interaction of the 'neurosteroid', pregnenolone-sulfate (PS), with the GABA/benzodiazepine/chloride ionophore receptor complex was investigated in rat brain subcellular preparations. At low micromolar concentrations PS competitively inhibited the binding of the convulsant [35S]t-butylbicyclophosphorothionate (TBPS) and antagonized pentobarbital-stimulated [3H]flunitrazepam binding to synaptosomes. In addition, PS inhibited muscimol-stimulated 36C1-uptake in brain synaptoneurosomes, indicating that PS has characteristics of a relatively potent antagonist of the chloride channel coupled to the GABA receptor. Together with our previous finding that A-ring reduced metabolites of progesterone and deoxycorticosterone also interact with the GABA receptor complex hut as hypnotic barbiturates, these data suggest that the regulation of GABAergic neurotransmission by various neurosteroids may be an important mechanism for controlling neuronal excitability. Significant amounts of 3fl-hydroxy-As-steroids in- cluding pregnenolone and its sulfate ester have been recently found in brain 3'7, and since they appear to be synthesized locally, they have been termed 'neuro- steroids '3. The role of these steroids in central ner- vous system (CNS) function is yet unknown, al- though electrophysiologic studies indicate that ionto- phoretically applied pregnenolone-sulfate (PS) in- creases the firing rate of some spontaneously firing neurons 5. In exploring the possible mechanism(s) underlying the excitatory effects of PS we have pre- viously demonstrated a complex interaction of this steroid with GABA a receptors in rat brain 12. The GABA A receptor is an oligomeric protein complex, which, upon activation by agonists, leads to an increase in the neuronal membrane conductance of chloride (C1-) ions 4"8. The latter results usually in membrane hyperpolarization and a decrease in neu- ronal excitability. A number of centrally acting drugs including convulsants, anticonvulsants, anesthetics and anxiolytics have been shown to act at distinct, but interacting domains on this receptor complex to mod- ulate el- conductance 2"6m'11"~5-:°. Recently we have reported that certain endoge- nous metabolites of progesterone and deoxycortico- sterone also interact with the GABA A receptor com- plex and have pharmacological and electrophysiolog- ical actions similar to those of the anesthetic barbitu- rates 13. |n this paper we extend our previous findings that the neurosteroid PS also interacts with the GABA receptor complex 12 and we report that it has neurochemical actions similar to those of the GABA antagonist, picrotoxin, and some convulsant barbitu- rates. These findings were presented recently in a preliminary form ~4. Since we have previously shown that PS affects (both enhances and inhibits) [3H]muscimol binding to GABA A receptors az, in the present study we ex- amined whether this steroid interacts either directly or allosterically with benzodiazepine recognition sites and/or sites labeled with the convulsant t-butyl- bicyclophosphorothionate (TBPS) that are asso- * Present address: Department of Pharmacology, Box 3813, Duke University Medical Center, Durham, NC 27710, U.S.A. Correspondence." M.D. Majewska. Present address: Fidia-Georgetown Institute for Neuroscience, Georgetown University/3900 Reservoir Road N.W., Washington, DC 20007, U.S.A.