Trotti D, Volterra A, Lehre K P, Rossi D, Gjesdal O, Racagni G, Danbolt N C
Department of Anatomy, University of Oslo, Norway.
J Biol Chem. 1995 Apr 28;270(17):9890-5. doi: 10.1074/jbc.270.17.9890.
Glutamate is believed to be the major excitatory transmitter in the mammalian central nervous system. Keeping the extracellular concentration of glutamate low, the glutamate transporters are required for normal brain function. Arachidonic acid (AA) inhibits glutamate uptake in relatively intact preparations (cells, tissue slices, and synaptosomes (Rhoads, D.E., Ockner, R. K., Peterson, N. A., and Raghupathy, E. (1983) Biochemistry 22, 1965-1970 and Volterra, A., Trotti, D., Cassutti, P., Tromba, C., Salvaggio, A., Melcangi, R. C., and Racagni, G. (1992b) J. Neurochem. 59, 600-606). The present study demonstrates that the effect of AA occurs also in a reconstituted system, consisting of a purified glutamate transporter protein incorporated into artificial cell membranes (liposomes). The characteristics of the AA effect in this system and in intact cells are similar with regard to specificity, sensitivity, time course, changes in Vmax, and affinity. AA-ethyl ester is inactive, suggesting that the free carboxylic group is required for inhibitory activity. When incubated with proteoliposomes, AA (300 microM, 15 min) mostly partitions to the lipid phase (lipid/water about 95:5). However, uptake inhibition is abolished by rapid dilution (6.5-fold) of the incubation medium (water phase), a procedure that does not modify the amount of AA associated with lipids. On the contrary, inhibition remains sustained if the same dilution volume contains as little as 5 microM AA, a concentration inactive before saturation of liposome lipids with 300 microM AA. The same degree of inhibition (60%) is obtained by 5 microM AA following preincubation with the inactive AA-ethyl ester (300 microM) instead of AA. The lipids apparently inactivate AA by extracting it from the water phase. The results suggest that AA acts on the transporter from the water phase rather than via the membrane. This could be true for other proteins as well since gamma-aminobutyric acid uptake is similarly affected by AA.
谷氨酸被认为是哺乳动物中枢神经系统中的主要兴奋性神经递质。为保持细胞外谷氨酸浓度较低，谷氨酸转运体对于正常脑功能是必需的。花生四烯酸（AA）在相对完整的标本（细胞、组织切片和突触体）中可抑制谷氨酸摄取（Rhoads, D.E., Ockner, R. K., Peterson, N. A., and Raghupathy, E. (1983) Biochemistry 22, 1965 - 1970；Volterra, A., Trotti, D., Cassutti, P., Tromba, C., Salvaggio, A., Melcangi, R. C., and Racagni, G. (1992b) J. Neurochem. 59, 600 - 606）。本研究表明，AA的这种作用在一个重构系统中也会发生，该系统由掺入人工细胞膜（脂质体）的纯化谷氨酸转运体蛋白组成。在该系统和完整细胞中，AA作用的特性在特异性、敏感性、时间进程、Vmax变化和亲和力方面是相似的。AA - 乙酯无活性，这表明游离羧基是抑制活性所必需的。当与蛋白脂质体一起孵育时，AA（300微摩尔，15分钟）大多分配到脂质相（脂质/水约为95:5）。然而，通过快速稀释（6.5倍）孵育介质（水相）可消除摄取抑制，该操作不会改变与脂质相关的AA量。相反，如果相同的稀释体积中仅含有低至5微摩尔的AA（该浓度在脂质体脂质被300微摩尔AA饱和之前无活性），抑制作用仍会持续。在用无活性的AA - 乙酯（300微摩尔）而非AA预孵育后，5微摩尔的AA可产生相同程度的抑制（60%）。脂质显然通过从水相中提取AA使其失活。结果表明，AA从水相作用于转运体而非通过膜。由于γ - 氨基丁酸摄取同样受到AA的类似影响，这对于其他蛋白质可能也是如此。
