EFFECT OF NEUROACTIVE STEROIDS ON [H-3] FLUMAZENIL BINDING TO THE GABA(A) RECEPTOR COMPLEX IN-VITRO

Modulation of benzodiazepine receptor ligand binding to the GABA(A) re ceptor complex by the neuroactive steroids 3 alpha-hydroxy-dihydroprog esterone (3 alpha-OH-DHP) and 3 alpha-hydroxycorticosterone (3 alpha-T HDOC) was assessed in an in vitro binding assay with the benzodiazepin e antagonist [H-3]flumazenil using rat cortical membranes. Neuroactive steroids, pentobarbital, GABA and bicuculline did not significantly a ffect flumazenil binding. However, the addition of neuroactive steroid s significantly decreased the K-i of benzodiazepine agonists, includin g alprazolam, diazepam and clonazepam, indicating an increase in agoni st affinity. Only the addition of 3 beta-OH-DHP, an inactive stereoiso mer had no effect on the K-i of these agonists. The binding of the ben zodiazepine inverse agonist FG 7142 was not significantly affected by these steroids, but the addition of GABA significantly increased the K -i of FG 7142 indicating a decrease in inverse agonist affinity. High concentrations of GABA or bicuculline were able to occlude the 3 alpha -THDOC mediated decrease in alprazolam K-i, indicating a GABA dependen t mechanism of binding enhancement. An advantage of using [H-3]flumaze nil is that neither the K-d nor the B-max change in the presence of al losteric site modulators, permitting the simple and direct assessment of alterations in benzodiazepine ligand affinity for the GABA(A) recep tor complex by neuroactive steroids.