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

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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