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

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