Curras M C, Dingledine R
Department of Pharmacology, University of North Carolina, Chapel Hill 27599.
Mol Pharmacol. 1992 Mar;41(3):520-6.
The endogenous neurotransmitter candidates L-aspartate, L-cysteine sulfinate (CSA), L-glutamate, L-homocysteate (HCA), and the endogenously occurring analogue quinolinate were compared in terms of potency, maximal activity, and selectivity for steady state activation of N-methyl-D-aspartate (NMDA) and non-NMDA [(RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)] types of glutamate receptors expressed in Xenopus oocytes injected with mRNA isolated from rat brain (minus cerebellum). Selective activation of NMDA receptors was achieved by deleting Mg2+ and including 3-10 microM glycine in the perfusion medium and by applying ligands in the presence of 30 microM quisqualate, which blocks the AMPA receptor and desensitizes the oocyte's own Ca(2+)-dependent Cl- current. Oocytes were voltage clamped, and steady state inward currents were measured in response to perfusion with agonists at known concentrations. Under the NMDA receptor-preferring condition, the potency rank order was L-glutamate (EC50 = 2.2 microM, 95% confidence interval = 1.4-3.6 microM) greater than L-aspartate (13 microM) = HCA (13 microM) greater than CSA (59 microM) greater than quinolinate (greater than or equal to 7200 microM). All amino acids tested evoked similar maximal currents, which were 120-159% that of NMDA itself. The Hill coefficient was greater than 1 for all agonists except L-HCA (0.6), which might reflect heterogeneity of NMDA receptors expressed. This was supported by the finding that glycine was more potent in combination with HCA than NMDA, in activating NMDA receptors. To study the activity of agonists at AMPA receptors, glycine and quisqualate were omitted and 1 mM Mg2+ was included to block NMDA receptors. Ca(2+)-dependent Cl- currents activated by L-glutamate were prevented by inclusion of 0.4 M ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the recording electrode. All amino acids were less potent at AMPA receptors than at NMDA receptors; the potency rank order for steady state activation of AMPA receptors was L-glutamate (EC50 = 11 microM, 95% confidence interval = 7.3-18 microM) greater than HCA (430 microM) greater than CSA (3300 microM). L-Aspartate and quinolinate produced little or no inward current even up to 10 mM, i.e., were inactive at forebrain AMPA receptors. The maximal currents activated by all amino acids at steady state were 5-10% that of kainate, presumably due to severe desensitization of the AMPA receptor by the natural agonists. These results are consistent with L-glutamate acting as a mixed agonist at both AMPA and NMDA synaptic receptors and L-aspartate being involved exclusively in NMDA receptor-mediated synapses.
就效力、最大活性以及对注射从大鼠脑（小脑除外）分离的mRNA的非洲爪蟾卵母细胞中表达的N-甲基-D-天冬氨酸（NMDA）和非NMDA [（RS）-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）]型谷氨酸受体稳态激活的选择性而言，对内源性神经递质候选物L-天冬氨酸、L-半胱亚磺酸（CSA）、L-谷氨酸、L-高半胱氨酸（HCA）以及内源性存在的类似物喹啉酸进行了比较。通过去除Mg2+并在灌流介质中加入3 - 10 μM甘氨酸，以及在30 μM喹氨酸存在下应用配体来实现对NMDA受体的选择性激活，喹氨酸可阻断AMPA受体并使卵母细胞自身的Ca(2+)依赖性Cl-电流脱敏。对卵母细胞进行电压钳制，并测量在已知浓度激动剂灌流时的稳态内向电流。在NMDA受体偏好条件下，效力排序为L-谷氨酸（EC50 = 2.2 μM，95%置信区间 = 1.4 - 3.6 μM）大于L-天冬氨酸（13 μM） = HCA（13 μM）大于CSA（59 μM）大于喹啉酸（≥7200 μM）。所有测试的氨基酸引发的最大电流相似，为NMDA自身的120 - 159%。除L-HCA（0.6）外，所有激动剂的希尔系数均大于1，这可能反映了所表达的NMDA受体的异质性。这一发现得到了支持，即在激活NMDA受体方面，甘氨酸与HCA联合比与NMDA联合更有效。为研究激动剂在AMPA受体上的活性，省略甘氨酸和喹氨酸，并加入1 mM Mg2+以阻断NMDA受体。通过在记录电极中加入0.4 M乙二醇双（β-氨基乙基醚）-N,N,N',N'-四乙酸来防止L-谷氨酸激活的Ca(2+)依赖性Cl-电流。所有氨基酸在AMPA受体上的效力均低于在NMDA受体上的效力；AMPA受体稳态激活的效力排序为L-谷氨酸（EC50 = 11 μM，95%置信区间 = 7.3 - 18 μM）大于HCA（430 μM）大于CSA（3300 μM）。L-天冬氨酸和喹啉酸即使高达10 mM也几乎不产生或不产生内向电流，即在脑前叶AMPA受体上无活性。所有氨基酸在稳态时激活的最大电流为海人藻酸的5 - 10%，这可能是由于天然激动剂使AMPA受体严重脱敏所致。这些结果与L-谷氨酸作为AMPA和NMDA突触受体的混合激动剂以及L-天冬氨酸仅参与NMDA受体介导的突触一致。
