[3H]diprenorphine receptor binding in vivo and in vitro.

In order to investigate opiate receptor binding in vivo, [3H]diprenorphine was given s.c. to rats, and the tracer specifically bound to membraneous high affinity sites was determined with a rapid filtration technique after brain homogenization. Bound [3H]diprenorphine accounted for 70% of the total brain activity after tracer doses. The in vivo binding sites were saturable at 25-30 pmol/g brain. Fifty percent occupancy of the [3H]diprenorphine binding sites in vivo occurred at a dose (10-15 micrograms/kg) that is similar to the antagonistic ED50 of diprenorphine for reversing morphine analgesia. The in vitro binding capacity for [3H]diprenorphine was also approximately 30 pmol/g brain in fresh untreated Tris buffer brain homogenate; however, extensive homogenate dilution or standard membrane washing procedures resulted in a reduction of the [3H]diprenorphine binding site population to 13-22 pmol/g. These results indicate that the opiate receptor system is modified in vitro. Previous studies have shown that the [3H]diprenorphine tracer is retained at cerebral binding sites over several hours in vivo. A diffusion boundary model was proposed to account for the dose dependent tracer retention. In order to investigate the mechanism of the in vivo binding kinetics, [3H]diprenorphine dissociation was measured in brain homogenates after in vivo labeling, immediately following sacrifice of the animals to minimize in vitro artefacts. No differences were found in the dissociation curves at 'infinite' homogenate dilution in the presence or absence of saturating diprenorphine concentrations under various ionic incubation conditions. This result argues against cooperative binding. It is consistent with the hypothesis that the [3H]diprenorphine tracer is retained in vivo because of a diffusion boundary next to the binding sites (receptor micro-compartment) that is destroyed during brain homogenization.