Apricò K, Beart P M, Crawford D, O'shea R D
Department of Human Physiology and Anatomy, La Trobe University, Bundoora, Australia.
Neurochem Int. 2007 Dec;51(8):507-16. doi: 10.1016/j.neuint.2007.05.011. Epub 2007 May 31.
While studies with [(3)H]D-aspartate ([(3)H]d-Asp) illustrate specific interactions with excitatory amino acid transporters (EAATs), new insights into the pharmacological characteristics and localization of specific EAAT subtypes depend upon the availability of novel ligands. One such ligand is [(3)H]-(2S,4R)-4-methylglutamate ([(3)H]4MG) which labels astrocytic EAATs in homogenate binding studies. This study examined the utility of [(3)H]4MG for binding and autoradiography in coronal sections of rat brain. Binding of [(3)H]4MG was optimal in 5mM HEPES buffer containing 96 mM NaCl, pH 7.5. Specific binding of [(3)H]4MG exhibited two components, but was to a single site when glutamate receptor (GluR) sites were masked with kainate (KA; 1 microM): t(1/2) approximately 5 min, K(d) 250 nM and B(max) 5.4 pmol/mg protein. Pharmacological studies revealed that [(3)H]4MG, unlike [(3)H]d-Asp, labeled both EAAT and ionotropic GluR sites. Further studies employed 6-cyano-7-nitroquinoxaline (30 microM) to block GluR sites, but selective EAAT ligands displayed lower potency than expected for binding to transporters relative to drugs possessing mixed transporter/receptor activities. Autoradiography in conjunction with densitometry with [(3)H]4MG and [(3)H]d-Asp revealed wide, but discrete distributions in forebrain; significant differences in binding levels were found in hippocampus, nucleus accumbens and cortical sub-areas. Although EAAT1 and EAAT2 components were detectable using 3-methylglutamate and serine-O-sulphate, respectively, the majority of [(3)H]4MG binding was to KA-related sites. Overall, in tissue sections [(3)H]4MG proved unsuitable for studying the autoradiographic localization of EAATs apparently due to its inability to selectively discriminate Na(+)-dependent binding to Glu transporters.
虽然用[（3）H] D - 天冬氨酸（[（3）H] d - Asp）进行的研究阐明了与兴奋性氨基酸转运体（EAATs）的特异性相互作用，但对特定EAAT亚型的药理学特性和定位的新见解取决于新型配体的可用性。一种这样的配体是[（3）H] - （2S，4R）-4 - 甲基谷氨酸（[（3）H] 4MG），其在匀浆结合研究中标记星形胶质细胞EAATs。本研究考察了[（3）H] 4MG在大鼠脑冠状切片中用于结合和放射自显影的效用。[（3）H] 4MG在含有96 mM NaCl、pH 7.5的5mM HEPES缓冲液中的结合最佳。[（3）H] 4MG的特异性结合表现出两个成分，但当用海藻酸（KA；1 microM）掩盖谷氨酸受体（GluR）位点时，其结合为单一位点：半衰期约5分钟，解离常数（K（d））250 nM，最大结合量（B（max））5.4 pmol/mg蛋白质。药理学研究表明，与[（3）H] d - Asp不同，[（3）H] 4MG标记了EAAT和离子型GluR位点。进一步的研究使用6 - 氰基 - 7 - 硝基喹喔啉（30 microM）来阻断GluR位点，但相对于具有混合转运体/受体活性的药物，选择性EAAT配体显示出比预期更低的与转运体结合的效力。用[（3）H] 4MG和[（3）H] d - Asp进行放射自显影并结合光密度测定显示，在前脑中分布广泛但离散；在海马、伏隔核和皮质亚区发现结合水平存在显著差异。尽管分别使用3 - 甲基谷氨酸和丝氨酸 - O - 硫酸盐可检测到EAAT1和EAAT2成分，但[（3）H] 4MG的大部分结合是与KA相关的位点。总体而言，在组织切片中，[（3）H] 4MG显然不适用于研究EAATs的放射自显影定位，这可能是由于其无法选择性区分与Glu转运体的Na（+）依赖性结合。
