CHRONIC FLURAZEPAM TREATMENT PRODUCES DECREASED EFFICACY OF THE BENZODIAZEPINE LIGANDS AND PENTOBARBITAL WITH GAMMA-AMINOBUTYRIC ACID(A) RECEPTORS IN CORTICAL-NEURONS

The present study was designed to determine whether chronic benzodiazepine (BZ) agonist treatment alters the ''set point'' of the BZ pharmacological profile. This was achieved by investigating the modulation of gamma-aminobutyric acid (GABA)-mediated [Cl-36(-)] influx by BZ ligands, as well as pentobarbital after chronic flurazepam treatment, in well characterized mammalian cortical neurons. Chronic flurazepam treatment (5 mu M, 10 days) produced decreased efficacy of BZ agonists (diazepam and flunitrazepam) and inverse agonists (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3'-carboxylate and ethyl-beta-carbotine-3-carboxylate), as measured by GABA-induced [Cl-36(-)] influx. The chronic flurazepam treatment, although not altering their EC(50)/IC50 values, decreased the E(max)/-E(max) values. Furthermore, the decreased efficacy was reversed after a 72-hr withdrawal, and by concomitant exposure of the neurons to Ro15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5 alpha][1,4]-BZ-3-carboxylate), a BZ receptor antagonist, but not by R 5135 (3 alpha-hydroxy-16-imino-5 beta-17-androstan-11-one; a GABA receptor antagonist) and pircrotoxin (a channel blocker). The chronic flurazepam treatment also produced uncoupling between pentobarbital and BZ receptor sites, and decreased the efficacy of pentobarbital to enhance GABA-mediated [Cl-36(-)] influx, events also reversed at 72-hr withdrawal and concomitant exposure to Ro15-1788. Chronic flurazepam treatment-induced uncoupling and decreased efficacy of BZ agonists was not reversed by the GABA(A) receptor antagonist, R 5135, or channel blocker, picrotoxin. Taken together, these findings suggest that chronic flurazepam treatment produces the decreased efficacy of BZ agonists and inverse agonists and barbiturates with the GABA receptor gated Cl- channel, and these events are mediated selectively via the BZ reception site. However, the set point was not shifted toward the inverse agonistic spectrum of the pharmacological profile. The decreased efficacy may be responsible for the tolerance associated with the chronic BZ treatment. The molecular basis for the decreased efficacy could be due to an alteration in the relative levels of various subunits that form the functional GABA(A) receptors or due to post-translational modification of the receptors.