Modulation of synaptic GABA receptor binding by membrane phospholipids: possible role of active oxygen radicals

Pretreatment of cerebral synaptic membrane preparations with phospholipase (PLase) A2 invariably induced a significant enhancement of [3H]muscimol binding in a dose-dependent manner with a concomitant elevation of the content of total free fatty acids in the membrane. In vitro addition of various free fatty acids exhibited no profound alteration in [3H]muscimol binding, whereas a significant enhancement of the binding was induced by the pretreatment of the membrane with unsaturated free fatty acids such as arachidonic acid and linoleic acid, but not by that with saturated free fatty acids. None of the inhibitors of arachidonic acid metabolism including indomethacin (an inhibitor of cyclo-oxygenase) and nordihydroguaiaretic acid (an inhibitor of lipoxygenase), however, had a significant preventive action on the augmentation of [3H]muscimol binding. On the other hand, various scavengers for superoxide anion radical such as superoxide dismutase, tiron and nitroblue tetrazolium (NBT) not only suppressed the PLase A2-induced enhancement of [3H]muscimol binding, but also diminished the augmentation of the binding due to PLase C and arachidonic acid. It was also found that a remarkable facilitation of the formation of superoxide anion radical was induced by the treatment of synaptic membrane with PLase A2, PLase C and arachidonic acid, all of which exhibited a prominent stimulation of the binding. In addition, treatment of the membrane with xanthine and xanthine oxidase, a superoxide anion radical generating system, resulted in a profound stimulation of the binding. The PLase A2-induced enhancement of the binding was also attenuated by the scavengers for hydrogen peroxide like catalase as well as by those for hydroxyl radical such as dimethylnitrosoaniline, mannitol, methanol and ethanol, but not by those for singlet oxygen radical including alpha-tocopherol and beta-carotene. The present results suggest that membrane phospholipids may play an important role in the modulation of the association of GABA with its relevant receptor through the generation of active oxygen radicals from unsaturated free fatty acids which are yielded by the catalytic action of PLase A2 and/or PLase C.