Funicello Marcella, Conti Paola, De Amici Marco, De Micheli Carlo, Mennini Tiziana, Gobbi Marco
Istituto di Ricerche Farmacologiche "Mario Negri", 20157 Milano, Italy.
Mol Pharmacol. 2004 Sep;66(3):522-9. doi: 10.1124/mol.66.3..
We characterized the interaction of two conformationally constrained aspartate and glutamate analogs, 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-4-carboxylic acid (HIP-A) and 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B), with excitatory amino acid transporters (EAATs) in rat brain cortex synaptosomes. HIP-A and HIP-B were potent and noncompetitive inhibitors of [(3)H]L-glutamate uptake, with IC(50) values (17-18 microM) very similar to that of the potent EAAT inhibitor dl-threo-beta-benzyloxyaspartic acid (TBOA). The two compounds had little effect in inducing [(3)H]D-aspartate release from superfused synaptosomes but they potently inhibited l-glutamate-induced [(3)H]D-aspartate release, thus behaving as EAAT blockers, not substrates, in a manner similar to those of TBOA and dihydrokainate (DHK). HIP-A and HIP-B, but not TBOA and DHK, unexpectedly inhibited L-glutamate-induced [(3)H]D-aspartate release with IC(50) values (1.2-1.6 microM) 10 times lower than those required to inhibit [(3)H]L-glutamate uptake. There is therefore a concentration window (1-3 microM) in which the two compounds significantly inhibited l-glutamate-induced release with very little effect on L-glutamate uptake. This selective inhibitory effect required quite long preincubation (>5 min) of synaptosomes with the drugs. At these low concentrations, however, HIP-A and HIP-B had no effect on the EAAT-mediated [(3)H]d-aspartate release induced by altering the ion gradients, indicating that they specifically affect some L-glutamate-triggered process(es)--different from L-glutamate translocation itself--responsible for the induction of reverse transport. These data are inconsistent with the classic model of facilitated exchange-diffusion and provide the first evidence that EAAT-mediated substrate uptake and substrate-induced EAAT-mediated reverse transport are independent. Compounds such as HIP-A and HIP-B could be useful to further clarify the mechanisms underlying these operating modes of transporters.
我们研究了两种构象受限的天冬氨酸和谷氨酸类似物，即3-羟基-4,5,6,6a-四氢-3aH-吡咯并[3,4-d]异恶唑-4-羧酸（HIP-A）和3-羟基-4,5,6,6a-四氢-3aH-吡咯并[3,4-d]异恶唑-6-羧酸（HIP-B）与大鼠脑皮质突触体中兴奋性氨基酸转运体（EAATs）的相互作用。HIP-A和HIP-B是[(3)H]L-谷氨酸摄取的强效非竞争性抑制剂，其IC(50)值（17 - 18 microM）与强效EAAT抑制剂dl-苏式-β-苄氧基天冬氨酸（TBOA）非常相似。这两种化合物在诱导[(3)H]D-天冬氨酸从灌注的突触体中释放方面几乎没有作用，但它们能有效抑制L-谷氨酸诱导的[(3)H]D-天冬氨酸释放，因此表现为EAAT阻滞剂而非底物，其作用方式与TBOA和二氢海因酸（DHK）相似。HIP-A和HIP-B而非TBOA和DHK意外地抑制L-谷氨酸诱导的[(3)H]D-天冬氨酸释放，其IC(50)值（1.2 - 1.6 microM）比抑制[(3)H]L-谷氨酸摄取所需的值低10倍。因此存在一个浓度窗口（1 - 3 microM），在此窗口中这两种化合物显著抑制L-谷氨酸诱导的释放，而对L-谷氨酸摄取影响很小。这种选择性抑制作用需要突触体与药物进行相当长时间的预孵育（>5分钟）。然而，在这些低浓度下，HIP-A和HIP-B对通过改变离子梯度诱导的EAAT介导的[(3)H]d-天冬氨酸释放没有影响，这表明它们特异性地影响一些L-谷氨酸触发的过程——不同于L-谷氨酸转运本身——这些过程负责反向转运的诱导。这些数据与经典的易化交换扩散模型不一致，并首次证明EAAT介导的底物摄取和底物诱导的EAAT介导的反向转运是独立的。诸如HIP-A和HIP-B这样的化合物可能有助于进一步阐明这些转运体运作模式背后的机制。
