Sadée W, Perry D C, Rosenbaum J S, Herz A
Eur J Pharmacol. 1982 Jul 16;81(3):431-40. doi: 10.1016/0014-2999(82)90108-x.
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.
为了研究体内阿片受体结合情况，给大鼠皮下注射[3H]二丙诺啡，在脑匀浆后采用快速过滤技术测定与膜高亲和力位点特异性结合的示踪剂。在给予示踪剂量后，结合的[3H]二丙诺啡占脑总活性的70%。体内结合位点在脑25 - 30 pmol/g时可饱和。在体内，[3H]二丙诺啡结合位点50%被占据时的剂量（10 - 15微克/千克）与二丙诺啡逆转吗啡镇痛的拮抗ED50相似。在新鲜未处理的Tris缓冲液脑匀浆中，[3H]二丙诺啡的体外结合能力也约为30 pmol/g脑；然而，大量匀浆稀释或标准膜洗涤程序会导致[3H]二丙诺啡结合位点数量减少至13 - 22 pmol/g。这些结果表明阿片受体系统在体外发生了改变。先前的研究表明，[3H]二丙诺啡示踪剂在体内数小时内保留在脑结合位点。提出了一个扩散边界模型来解释剂量依赖性示踪剂保留现象。为了研究体内结合动力学机制，在动物处死后立即测定体内标记后脑匀浆中[3H]二丙诺啡的解离情况，以尽量减少体外假象。在各种离子孵育条件下，在存在或不存在饱和二丙诺啡浓度的情况下，在“无限”匀浆稀释时，解离曲线没有差异。这一结果反对协同结合。这与以下假设一致，即[3H]二丙诺啡示踪剂在体内保留是因为结合位点（受体微区室）旁边的扩散边界在脑匀浆过程中被破坏。
