BACKGROUND & AIMS:
:The involvement of phospholipase A2 (PLA2) and fatty acid release in the regulation of sodium-dependent high-affinity choline uptake in rat brain was assessed in vitro through the use of the specific binding of 3H-hemicholinium-3 (3H-HCh-3). Addition of arachidonic acid and other unsaturated fatty acids to rat striatal membranes in vitro resulted in a dose-dependent, temperature-independent activation of 3H-HCh-3 binding. Scatchard analysis revealed that these changes in binding result from a 2-fold increase in the affinity and capacity of 3H-HCh-3 binding. Saturated fatty acids, lysophospholipids, and phospholipids did not affect specific 3H-HCh-3 binding. Addition of defatted BSA to membranes, which had been treated previously with arachidonic acid, completely reversed the increase in specific 3H-HCh-3 binding. However, several inhibitors of fatty acid metabolism, including nordihydroguaiaretic acid, indomethacin, catalase, and superoxide dismutase, did not alter arachidonic acid-induced changes in 3H-HCh-3 binding, suggesting that unsaturated fatty acids, and not their metabolites, are directly responsible for the observed activation of specific 3H-HCh-3 binding. Additionally, unsaturated fatty acids dose-dependently inhibited high-affinity 3H-choline uptake in rat striatal synaptosomes, apparently due to the disruption of synaptosomal integrity. The phospholipase A2 inhibitors quinacrine hydrochloride, trifluoperazine, and 4-bromophenacylbromide dose-dependently inhibited potassium depolarization-induced activation of specific 3H-HCh-3 binding in slices of rat brain in vitro. Similarly, both quinacrine and trifluoperazine inhibited the metabolism of phospholipids and the release of fatty acids evoked by either elevated KCl or calcium ionophore A23187. These results support the involvement of PLA2 and subsequent fatty acid release in the increase of 3H-HCh-3 binding in cholinergic neurons and suggest that activation of PLA2 may be the penultimate step in regulating the velocity of sodium-dependent choline transport.
背景与目标:
: 通过使用3h-hemichholinium-3 (3h-hchh-3) 的特异性结合,在体外评估了磷脂酶A2 (PLA2) 和脂肪酸释放在调节钠依赖性高亲和力胆碱摄取中的参与。hch-3)。在体外向大鼠纹状体膜中添加花生四烯酸和其他不饱和脂肪酸会导致3H-HCh-3结合的剂量依赖性,温度依赖性激活。Scatchard分析表明,结合的这些变化是由3H-HCh-3结合的亲和力和能力增加2倍引起的。饱和脂肪酸,溶血磷脂和磷脂不影响特定的3H-HCh-3结合。将脱脂的BSA添加到先前用花生四烯酸处理过的膜中,完全逆转了特异性3H-HCh-3结合的增加。然而,脂肪酸代谢的几种抑制剂,包括去甲二氢愈创木酸,吲哚美辛,过氧化氢酶和超氧化物歧化酶,并未改变花生四烯酸诱导的3H-HCh-3结合的变化,这表明不饱和脂肪酸而不是其代谢物直接导致观察到的3H-HCh-3结合的激活。此外,不饱和脂肪酸剂量依赖性地抑制了大鼠纹状体突触小体中高亲和力的3h-胆碱摄取,这显然是由于突触小体完整性的破坏。磷脂酶A2抑制剂盐酸奎纳克林,三氟拉嗪和4-溴苯甲酰溴在体外大鼠脑切片中剂量依赖性地抑制钾去极化诱导的特异性3H-HCh-3结合的激活。同样,奎纳克林和三氟拉嗪均抑制磷脂的代谢以及升高的KCl或钙离子载体a23187引起的脂肪酸的释放。这些结果支持PLA2和随后的脂肪酸释放参与胆碱能神经元中3H-HCh-3结合的增加,并表明PLA2的激活可能是调节钠依赖性胆碱转运速度的倒数第二步。