Verdoorn T A, Kleckner N W, Dingledine R
Department of Pharmacology, University of North Carolina, Chapel Hill 27599.
Mol Pharmacol. 1989 Mar;35(3):360-8.
Quantitative pharmacological studies were done to determine the properties of excitatory amino acid receptors expressed in Xenopus oocytes injected with rat brain mRNA. Smooth currents with properties indicative of N-methyl-D-aspartate (NMDA) and quisqualate/kainate receptors were observed in mRNA-injected oocytes. Schild analysis of currents evoked by NMDA indicated that the EAA receptor antagonist D-2-amino-5-phosphonovalerate (D-APV) exerted a competitive block of the oocyte NMDA receptor, because the Schild regression was linear with a slope not significantly different from unity (1.03 +/- 0.025) up to 100 microM D-APV. The pA2 estimated for D-APV antagonism of NMDA currents (5.87 +/- 0.043) was nearly identical to that for D-APV as an L-aspartate antagonist (pA2 = 5.86 +/- 0.073, slope = 0.97 +/- 0.036), suggesting that these two agonists are selective for NMDA receptors in oocytes up to concentrations well above 1 mM. 6-Nitro-7-cyano-quinoxaline-2,3-dione (CNQX) reduced the maximum NMDA response significantly (70% reduction by 15 microM CNQX) but had no effect on the NMDA EC50. CNQX exerted a mixed competitive-noncompetitive block of the glycine site on NMDA receptors; 15 microM CNQX increased the glycine EC50 by 5-fold and reduced the maximum glycine response by 35%. In addition, CNQX exerted a potent and competitive antagonism of currents evoked by kainate. The Schild regression was linear up to 30 microM CNQX with a slope of 1.02 +/- 0.014 and a pA2 of 6.53 +/- 0.029. The block of kainate or NMDA currents by 2 microM CNQX was not voltage dependent. D-APV exerted a weak antagonism of kainate-evoked currents, with a pA2 of 3.39 +/- 0.044, but the slope of the Schild regression was slightly less than 1 (0.90 +/- 0.03). These data demonstrate a clear pharmacological distinction between receptors that mediate the kainate- and NMDA-induced currents and quantify the potency of CNQX and D-APV acting at NMDA/glycine and quisqualate/kainate receptors. The implications of these data for the identification of EAA receptors in oocytes and the classification of neuronal EAA receptors are discussed.
进行了定量药理学研究，以确定在注射了大鼠脑信使核糖核酸（mRNA）的非洲爪蟾卵母细胞中表达的兴奋性氨基酸受体的特性。在注射了mRNA的卵母细胞中观察到了具有N-甲基-D-天冬氨酸（NMDA）和quisqualate/海人藻酸受体特性的平滑电流。对NMDA诱发的电流进行的希尔德分析表明，兴奋性氨基酸受体拮抗剂D-2-氨基-5-膦酰基戊酸（D-APV）对卵母细胞NMDA受体产生竞争性阻断，因为在高达100微摩尔D-APV时，希尔德回归呈线性，斜率与1无显著差异（1.03±0.025）。估计D-APV对NMDA电流拮抗作用的pA2（5.87±0.043）与D-APV作为L-天冬氨酸拮抗剂时的pA2几乎相同（pA2 = 5.86±0.073，斜率 = 0.97±0.036），这表明这两种激动剂在浓度远高于1毫摩尔时对卵母细胞中的NMDA受体具有选择性。6-硝基-7-氰基喹喔啉-2,3-二酮（CNQX）显著降低了最大NMDA反应（15微摩尔CNQX降低了70%），但对NMDA的半数有效浓度（EC50）没有影响。CNQX对NMDA受体上的甘氨酸位点产生了竞争性-非竞争性混合阻断；15微摩尔CNQX使甘氨酸的EC50增加了5倍，并使最大甘氨酸反应降低了35%。此外，CNQX对海人藻酸诱发的电流产生了强效且竞争性的拮抗作用。在高达30微摩尔CNQX时，希尔德回归呈线性，斜率为1.02±0.014，pA2为6.53±0.029。2微摩尔CNQX对海人藻酸或NMDA电流的阻断不依赖电压。D-APV对海人藻酸诱发的电流产生了微弱的拮抗作用，pA2为3.39±0.044，但希尔德回归的斜率略小于1（0.90±0.03）。这些数据证明了介导海人藻酸和NMDA诱发电流的受体之间存在明显的药理学差异，并量化了CNQX和D-APV作用于NMDA/甘氨酸和quisqualate/海人藻酸受体的效力。讨论了这些数据对鉴定卵母细胞中的兴奋性氨基酸受体以及对神经元兴奋性氨基酸受体进行分类的意义。
