用大鼠脑切片进行β—受体放射配体结合实验的方法学研究

本项研究探素了用氚标的β-受体拮抗剂~3H-双氢心得舒与大鼠中脑上丘平面冰冻切片进行结合实验的条件。实验得出配体与脑切片的最大特异性结合量为17.3±4.5 fmol/mg,组织干重,表观平衡解离常数为5.8±0.6nM,Hill系数为0.93±0.3,结合速率常数为0.112±0.025nM~(-1)·min~(-1),解离速率常数为0.69±0.07min~(-1),心得安与配体竞争结合的IC_(50)为1.00±0.15×10~(-8)M,心得宁的IC~(50)为167±0.22×10~(-8)M,异丙肾上慷素的IC_(50)为1.32±0.3×10~(-6)M。这些结果与用组织匀浆进行结合实验所得结果近似,表明在合适的条件下用冰冻组织切片进行β-受体结合实验是可行的。

THE METHODOLOGICAL STUDY ON RADIOLIGAND BINDING ASSAY OF β-ADRENERGIC RECEPTORS WITH RAT BRAIN SECTIONS

It is generally considered that the experiment of radioligand binding assay ( RBA) of receptors with frozen tissue sections is difficult. In this study the conditions involved in the RBA of β-adre-nergic receptors with rat brain sections on the superior colliculus level was investigated. 3H-Dihydroalprenolol (3H-DHA), a β-adrener-gic receptor antagonist, was used as the labeled ligand. The data obtained from this experment are as follows: Bmax=17.3±4.5fmol/mg of dry tissue, Kd=5.8±0.6nM, association rate constant=0.112±0.025 nM-1 · min-1,dissociation rate constant=0.69±0.07min-1, the IC50 of propranolol=1.00±0.15×10-8M, IC50 of practolol=1.67±0.22×10-5M, IC50 of isopreterenol=1.32±0.31 × 10-6M.These results are in agreement with those obtained from RBA with homogenate, which indicates that the frozen tissue sections are useful preparations in the RBA of β-adrenergic receptors.