³H- Diazepam(83 Ci/mmol) and ³² P- GTP (3,000 Ci/mmol) were purchased from Mandel. ³² P- ATP (3,000 Ci/mmol) and ³H- cAMP were from Amersham and ICN, respectively. All other drugs and reagents were obtained from Sigma.

Animals: Male Sprague-Dawley rats weighing about 300g were maintained at a room temperature of 22o C, with food and water freely available. Animals were decapitated and striata dissected on ice for binding and functional assays.

Receptor Binding:Fresh striatal tissue was homogenized by polytron for 5s in 50 vol of 50mM Tris-HCL buffer ( pH 7,4 at 4o C ). After centifugation at 39,000g for 10 min and two washes in the same buffer, homogenates were resuspended in 50 mM Tris-HCL ( pH 7.4 at 30o C ) at a concentration of about 5mg/ml ( wet weight/ vol ). Binding assays were carried out at 30o C for 60 min with ³ H- Diazepam ( 0.25- 83 nM ), with or without 1mM GTP. Non-specific binding was determined in the presence of 10uM diazepam.

Adenlyl Cyclase Assay: Fresh striatal tissue was hand- homogenized in cold 50 mM HEPES /NaOH buffer containing 5 mM MgCl ₂ 1mM dithiothreitol, and 0.2 mM EGTA (pH 7.4 at 30o C). The homogenate was centrifuged at 39,000g for 10 min, and membranes were washed twice in the same buffer. AC activity was measured as the conversion of ³² P- ATP to ³² P- cAMP , in the presence or absence of forskolin and other drugs, as described previously (Tenn et al., 1996 ).

GTP Binding : Agonist- stimulated ³² P- GTP binding was measured as described by Friedman et al. (1993 ). Membranes ( ~200ug protein per assay tube) were incubated for 2 min at 30o C in assay buffer. After addition of 50nM ³² P- GTP with or without diazepam, the incubation continued for 5 min. Incubates were centrifuged at 10,000g for 15 min at 4o C, pellets were resuspended in sample buffer and incubated at room temperature for 1 h with gentle shaking. Aliquots containing 20ug protein were analyzed by SDS- PAGE and ³² P- GTP- labeled proteins were quantified by densitometry ( Tenn and Niles, 1997 ).

Effect of GTP on Diazepam Binding: Control binding parameters were: K_D = 19.3 + 0.8 nM ; B_MAX = 632 + 21 fmol/mg protein, n = 3. In the presence of GTP , there was a modest but significant ( p <.05 ) decrease in binding affinity, but no change in receptor density: K_D = 30.5 + 2.6 nM ; B_MAX = 688 + 77 fmol/mg protein, n = 3.

Effect of Benzodiazepines on AC Activity: The BZ agonists, diazepam and Ro5-4864, caused a concentration- dependent inhibition of forskolin-stimulated AC activity in the striatum, as shown in Fig. 1. An F- test indicated a significantly better fit to two-site and three- site models for Ro5-4864 and diazepam, respectively. The first receptor-mediated phase of inhibition by Ro5-4864 occurred with an EC ₅₀ = 1.36nM (0.64-2.08 ), whereas the second phase had an EC ₅₀ = 131uM (87.9-174 ). Diazepam was more potent, and inhibited enzyme activity with an EC ₅₀ = 0.43 nM for the first phase and 1.0 uM (0.68-1.32) and 194 uM for the other phases.

In this study, several lines of evidence support the presence of G protein- coupled BZ receptors,which mediate the inhibitory action of BZ agonists, on the AC-cAMP pathway in the rat striatum. The multiphasic dose- response curves observed suggest that more than one mechanism is involved. The first high- affinity phase is similar to that reported for other inhibitory receptors such as the dopamine D2 receptor ( Olianas and Onali, 1987), whereas the second phase involving low (micromolar) affinity sites, is consistent with evidence that high concentrations of BZs can act directly on AC to suppress its activity ( Dan'ura et al., 1988).

The failure of flumazenil to block the inhibitory effect of Ro5-4864 on AC activity, indicates that central-type BZ receptors are not involved. In contrast, the peripheral-type receptor ligand, PK11195, blocked the first phase of inhibition by Ro5-4864, suggesting involvement of a receptor with PBR-like characteristics. Although PK 11195 has been reported to act as an agonist in some biological systems, in the absence of BZs, it did not alter basal or stimulated AC activity, which indicates that it acts as an antagonist at a G protein- coupled BZ receptor ( G-BZR).

In addition to the foregoing, the presence of a G-BZR is supported by the ability of guanine nucleotides to uncouple high- affinity diazepam binding, as reflected by a decrease in receptor affinity, in the presence of GTP. Similarly, pertussis toxin which uncouples inhibitory G protein ( G _i )- associated receptors, blocked the effect of BZs and other drugs, which act on the G-BZR to inhibit AC activity. Finally, the ability of diazepam to stimulate GTP binding,to a G protein of the same size as G _i , is an important functional hallmark of a G protein- coupled receptor.

In conclusion, these findings indicate that G-BZRs mediate the inhibitory effect of BZ agonists on the AC-cAMP pathway in the striatum. Given the widespread involvement of cAMP-protein kinase A ( PKA) - dependent mechanisms in the regulation of synaptic transmission in the CNS ( Colwell and Levine, 1995), it is very likely that G-BZRs are involved in mediating the neuropsychopharmacological effects of BZs and related agents.