Studies on GABA_A receptor complexes in cultured cerebellar neuronal cells and synaptoneurosomes

本研究对SR 95531[2-（3‘碳氧基-2’-丙基）-3-氨基-6-参氧基-苯基-吡嗪溴化]的拮抗活性和结合特性进行了实验表征。sr95531和双管碱（BMI）以浓度依赖的方式抑制肌肉醇刺激的^<36>Cl^-摄取到皮质突触体。sr95531对<36>Cl -摄取的肌肉抑制效能比BMI高15倍。SR 95531的IC_<50>值与[^3H]SR 95531在FC低亲和力结合位点的K_D值密切一致。sr95531和BMI可拮抗gaba诱导的皮层膜[^<35>S]TBPS结合抑制。SR 95531比BMI更有效。用磷脂酶A_2 （PLase A_2）预处理膜，一定程度上降低了sr95531在大脑皮层和小脑中的结合。另一方面，pase - A_2预孵育显著增加了[^3H]GABA在皮质膜上的结合，并呈浓度依赖性。虽然低浓度的PLase A_2对[^3H]GABA在小脑的结合没有影响，但高浓度的PLase A_2处理增加了该区域的结合。此外，plaa_2诱导的大脑皮层[^3H]GABA结合的增加大于小脑。[^3H]SR 95531在培养的小脑神经元细胞中也有特异性结合。PLase A_2预处理影响[^3H]GABA和[^3H] sr95531在培养的小脑神经元细胞中的结合。上述结果表明，SR 95531通过SR 95531的低亲和力结合位点发挥GABA的拮抗作用，外源添加的PLase A_2通过PLase A_2的催化活性产物诱导GABA_A受体转化为激动剂优先构象。此外，培养的小脑神经元细胞可能是研究各种药物对体外GABA_A受体复合物的长期影响的有用工具。少

In the present study, experiments were performed to characterize antagonistic activity and binding properties of SR 95531 [2-(3'carbethoxy-2'-propyl)-3-amino-6-Paramethoxy-phenyl-pyidaziniun bromide]. SR 95531 and bicuculline methiodide (BMI) inhibited muscimol-stimulated ^<36>Cl^- uptake into cortical synaptoneurosomes in a concentration-dependent manner. Inhibitory potency of SR 95531 for the muscimlstimlated ^<36>Cl^- uptake was 15 times higher than that of BMI. The IC_<50> value of SR 95531 for muscimol-stimulated ^<36>Cl^- uptake into cortical synaptoneurosomes was in close agreement with the K_D value of low-affinity binding sites of [ ^3H]SR 95531 in the FC. SR 95531 and BMI antagwonized GABA-induced inhibition of [ ^<35>S]TBPS binding in cortical membranes. SR 95531 was more potent than BMI. Pretreatment of the membranes with phospholipase A_2 (PLase A_2) invariably decreased [ ^3H]SR 95531 binding in the cerebral cortex and the cerebellum. On the other hand, preincubation with … More PLase A_2 significantly, increased the binding of [ ^3H]GABA in a concentration-dependent manner in cortical membranes. Although lower concentrations of PLase A_2 did not affect the binding of [ ^3H]GABA in the cerebellum, treatment with higher concentrations of PLase A_2 increased the binding in this region. In addition, PLase A_2-induced increase in the binding of [ ^3H]GABA in the cerebral cortex was greater than that in the cerebellum. Specific binding of [ ^3H]SR 95531 was also detected in cultured cerebellar neuronal cells. Pretreatment with PLase A_2 affected [ ^3H]GABA and [ ^3H]SR 95531 binding in the cultured cerebellar neuronal cells as in the case of the cerebellum. These results suggest that SR 95531 exerts GABA antagonistic action through the low-affinity binding site of SR 95531 and that exogenously added PLase A_2 induces the conversion of GABA_A receptors into the agonist preferential conformation by the products of catalytic activity of PLase A_2. In addition, cultured cerebellar neuronal cells may be a useful tool to investigate the long term effects of various drugs on GABA_A receptor complexes in vitro. Less