Herkenham M, Lynn A B, Johnson M R, Melvin L S, de Costa B R, Rice K C
Section on Functional Neuroanatomy, National Institute of Mental Health, Bethesda, Maryland 20892.
J Neurosci. 1991 Feb;11(2):563-83. doi: 10.1523/JNEUROSCI.11-02-00563.1991.
A potent, synthetic cannabinoid was radiolabeled and used to characterize and precisely localize cannabinoid receptors in slide-mounted sections of rat brain and pituitary. Assay conditions for 3H-CP55,940 binding in Tris-HCl buffer with 5% BSA were optimized, association and dissociation rate constants determined, and the equilibrium dissociation constant (Kd) calculated (21 nM by liquid scintillation counting, 5.2 nM by quantitative autoradiography). The results of competition studies, using several synthetic cannabinoids, add to prior data showing enantioselectivity of binding and correlation of in vitro potencies with potencies in biological assays of cannabinoid actions. Inhibition of binding by guanine nucleotides was selective and profound: Nonhydrolyzable analogs of GTP and GDP inhibited binding by greater than 90%, and GMP and the nonhydrolyzable ATP analog showed no inhibition. Autoradiography showed great heterogeneity of binding in patterns of labeling that closely conform to cytoarchitectural and functional domains. Very dense 3H-CP55,940 binding is localized to the basal ganglia (lateral caudate-putamen, globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), cerebellar molecular layer, innermost layers of the olfactory bulb, and portions of the hippocampal formation (CA3 and dentate gyrus molecular layer). Moderately dense binding is found throughout the remaining forebrain. Sparse binding characterizes the brain stem and spinal cord. Densitometry confirmed the quantitative heterogeneity of cannabinoid receptors (10 nM 3H-CP55,940 binding ranged in density from 6.3 pmol/mg protein in the substantia nigra pars reticulata to 0.15 pmol/mg protein in the anterior lobe of the pituitary). The results suggest that the presently characterized cannabinoid receptor mediates physiological and behavioral effects of natural and synthetic cannabinoids, because it is strongly coupled to guanine nucleotide regulatory proteins and is discretely localized to cortical, basal ganglia, and cerebellar structures involved with cognition and movement.
一种强效合成大麻素被放射性标记，并用于在大鼠脑和垂体的载玻片切片中表征和精确定位大麻素受体。优化了在含有5%牛血清白蛋白的Tris-HCl缓冲液中3H-CP55,940结合的测定条件，测定了结合和解离速率常数，并计算了平衡解离常数（通过液体闪烁计数法为21 nM，通过定量放射自显影法为5.2 nM）。使用几种合成大麻素进行的竞争研究结果补充了先前的数据，显示了结合的对映选择性以及体外效力与大麻素作用生物学测定效力之间的相关性。鸟嘌呤核苷酸对结合的抑制具有选择性且很强：GTP和GDP的非水解类似物抑制结合超过90%，而GMP和非水解ATP类似物则无抑制作用。放射自显影显示结合在标记模式上具有很大的异质性，这些模式与细胞结构和功能域密切相符。非常密集的3H-CP55,940结合定位于基底神经节（外侧尾状核-壳核、苍白球、内囊核、黑质网状部）、小脑分子层、嗅球最内层以及海马结构的部分区域（CA3和齿状回分子层）。在其余前脑区域发现中等密度的结合。稀疏结合是脑干和脊髓的特征。密度测定证实了大麻素受体的定量异质性（10 nM 3H-CP55,940结合的密度范围从黑质网状部的6.3 pmol/mg蛋白质到垂体前叶的0.15 pmol/mg蛋白质）。结果表明，目前表征的大麻素受体介导天然和合成大麻素的生理和行为效应，因为它与鸟嘌呤核苷酸调节蛋白强烈偶联，并且离散地定位于与认知和运动有关的皮质、基底神经节和小脑结构。
