Characterization of antagonistic activity and binding properties of SR 95531, a pyridazinl‐GABA derivative, in rat brain and cultured cerebellar neuronal cells

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Abstract
Experiments were performed to characterze the antagonistic activity and binding properties of SR 95531 [2-(3′carbethoxy-2′-propyl)-3-amino-6-paramethoxy-phenyl-piridazinium bromide] in rat brain. SR 95531 and bicuculline methiodide inhibited muscimol-stimulated 36Cl− uptake in cortical synaptoneurosomes in a concentration-dependent manner. The inhibitory potency of SR 95531 for the muscimol-stimulated 36Cl− uptake was 15 times higher than that of bicuculline methiodide. Scatchard plots of binding isotherms exhibited two apparent binding sites for [3H]SR 95531 in both the frontal cortex and cerebellum. The IC50 value of SR 95531 for muscimol-stimulated 36Cl− uptake into cortical synaptoneurosomes was in close agreement with the KD value of low-affinity binding sites of [3H]SR 95531 in the frontal cortex. Pretreatment of the membranes with phospholipase A2 invariably decreased [3H]SR 95531 binding in the frontal cortex and cerebellum. On the other hand, the treatment significantly increased [3H]γ-aminobutyric acid (GABA) binding in a concentration-dependent manner in the frontal cortex. Although lower concentrations of phospholipase A2 did not affect [3H]GABA binding in the cerebellum, treatment with higher concentrations of phospholipase A2 increased the binding in this region. Specific binding of [3H]SR 95531 was also detected in cultures rich in cerebellar granule cells. Pretreatment with phospholipase A2 affected the binding of [3H]GABA and [3H]SR 95531 in these cells, as in the case of the cerebellum. These effects of phospholipase A2 on the binding of [3H]GABA and [3H]SR 95531 were partially prevented by the addition of delipidated bovine serum albumin. Furthermore, the products generated by the catalytic activity of phospholipase A2, such as arachidonic acid and lysophosphatidylcholine, mimicked the effects of phospholipase A2 on the binding of [3H]GABA and [3H]SR 95531. These results suggest that SR 95531 exerts GABA-antagonistic action through its low-affinity binding site, and that exogenously added phospholipase A2 induces the conversion of GABAA receptors into the agonist-preferential conformation by the materials produced by phospholipase A2.