Olverman H J, Jones A W, Watkins J C
Department of Pharmacology, The Medical School, Bristol, U.K.
Neuroscience. 1988 Jul;26(1):1-15. doi: 10.1016/0306-4522(88)90123-6.
Tritiated D-2-amino-5-phosphonopentanoate has been prepared and evaluated as a radioligand for investigating N-methyl-D-aspartate receptors in rat brain membranes. A radioactive impurity, which was more acidic than 2-amino-5-phosphonopentanoate, interfered with the binding assay for [3H]D-2-amino-5-phosphonopentanoate in preliminary experiments and developed progressively with time of storage of the ligand, was isolated by ion-exchange purification and its binding site characterized. Binding of the 3H-impurity was increased in the presence of calcium ions, with a maximum effect at a concentration of 1-3 mM, but not by sodium, potassium or magnesium ions. It was inhibited by omega-phosphonate analogues of D-2-amino-5-phosphonopentanoate and by inorganic phosphate but not by L-glutamate or any other omega-carboxylates, omega-sulphinates or omega-sulphonates tested. The site of binding for the 3H-impurity was not identified, but from its pharmacological profile it appears to be unrelated to any excitatory amino acid receptor so far described. Binding of purified [3H]D-2-amino-5-phosphonopentanoate to rat cerebral cortical membranes was saturable (KD, 0.53 microM; Bmax, 4.3 pmol/mg protein), was maximal at pH 7.3, but was not particularly temperature sensitive. Dissociation of the receptor-ligand complex was very rapid. Magnesium ions had an inhibitory effect on the binding of [3H]D-2-amino-5-phosphonopentanoate, but the mechanism of this action was not clear. For a wide range of competitive excitatory amino acid antagonists with different potencies and receptor specificities there was a direct relationship between their Ki values as inhibitors of [3H]D-2-amino-5-phosphonopentanoate binding and their KD values for antagonism of N-methyl-D-aspartate induced depolarizations. Thus, [3H]D-2-amino-5-phosphonopentanoate binds to electrophysiological N-methyl-D-aspartate receptors. Among endogenous agonists, L-glutamate had the highest affinity (Ki 0.9 microM) for the [3H]D-2-amino-5-phosphonopentanoate binding site; L-homocysteate and S-sulpho-L-cysteine also had high affinity. However, quinolinate and N-acetylaspartylglutamate had relatively low affinity. It is considered that L-glutamate is the most likely substance to be the transmitter activating N-methyl-D-aspartate receptors physiologically. A study of the regional distribution of [3H]D-2-amino-5-phosphonopentanoate binding sites showed the hippocampus and cerebral cortex to have the highest density of these sites, while the cerebellum and spinal cord had the lowest.(ABSTRACT TRUNCATED AT 400 WORDS)
已制备出氚标记的D-2-氨基-5-膦酰戊酸，并将其作为放射性配体用于研究大鼠脑膜中的N-甲基-D-天冬氨酸受体。在初步实验中，一种比2-氨基-5-膦酰戊酸酸性更强的放射性杂质干扰了[3H]D-2-氨基-5-膦酰戊酸的结合测定，且随着配体储存时间的延长而逐渐增多。通过离子交换纯化分离出该杂质，并对其结合位点进行了表征。3H-杂质的结合在钙离子存在下增加，在1-3 mM浓度时效果最佳，但不受钠、钾或镁离子影响。它受到D-2-氨基-5-膦酰戊酸的ω-膦酸酯类似物和无机磷酸盐的抑制，但不受L-谷氨酸或所测试的任何其他ω-羧酸盐、ω-亚磺酸盐或ω-磺酸盐抑制。3H-杂质的结合位点尚未确定，但从其药理学特征来看，它似乎与迄今为止描述的任何兴奋性氨基酸受体无关。纯化的[3H]D-2-氨基-5-膦酰戊酸与大鼠大脑皮层膜的结合是可饱和的（KD，0.53 microM；Bmax，4.3 pmol/mg蛋白质），在pH 7.3时最大，但对温度不太敏感。受体-配体复合物的解离非常迅速。镁离子对[3H]D-2-氨基-5-膦酰戊酸的结合有抑制作用，但其作用机制尚不清楚。对于一系列具有不同效力和受体特异性的竞争性兴奋性氨基酸拮抗剂，它们作为[3H]D-2-氨基-5-膦酰戊酸结合抑制剂的Ki值与它们拮抗N-甲基-D-天冬氨酸诱导的去极化的KD值之间存在直接关系。因此，[3H]D-2-氨基-5-膦酰戊酸与电生理学上的N-甲基-D-天冬氨酸受体结合。在内源性激动剂中，L-谷氨酸对[3H]D-2-氨基-5-膦酰戊酸结合位点的亲和力最高（Ki 0.9 microM）；L-高半胱氨酸和S-磺基-L-半胱氨酸也有高亲和力。然而，喹啉酸和N-乙酰天冬氨酰谷氨酸的亲和力相对较低。认为L-谷氨酸是生理上最有可能激活N-甲基-D-天冬氨酸受体的递质。对[3H]D-2-氨基-5-膦酰戊酸结合位点区域分布的研究表明，海马体和大脑皮层这些位点的密度最高，而小脑和脊髓的密度最低。（摘要截短至400字）
