Henzi V, Reichling D B, Helm S W, MacDermott A B
Department of Physiology and Cellular Biophysics, Columbia University, New York, New York 10032.
Mol Pharmacol. 1992 Apr;41(4):793-801.
The pharmacological actions of L-proline on excitatory and inhibitory amino acid receptors have been characterized under voltage-clamp conditions, using cultured dissociated neurons from the dorsal horn of the rat spinal cord. At a holding potential of -62 mV, millimolar concentrations of L-proline elicited an inward current that was partially antagonized by D-(-)-2-amino-5-phosphonopentanoic acid (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and strychnine and was virtually abolished (97% block) by a combination of all three antagonists. Currents evoked by D-proline were abolished by strychnine alone. APV-, CNQX-, and strychnine-sensitive components of L-proline-evoked currents were isolated using various combinations of the three antagonists. These currents were identical to currents elicited by N-methyl-D-aspartate (NMDA), kainate, and glycine, respectively, with respect to antagonist specificity, reversal potential, and ionic permeability. The APV- and strychnine-sensitive currents also showed a time dependence similar to that of the currents elicited by NMDA and glycine. EC50 values could not be calculated, because the response did not saturate within the tested range of L-proline concentrations (0.3-50 mM). Estimates of relative potency were obtained, however, by comparison with responses elicited by selective agonists. The APV-sensitive, CNQX-sensitive, and strychnine-sensitive currents evoked by 10 mM L-proline were comparable in size to currents elicited by 15 microM NMDA, 5 microM kainate, and 30 microM glycine, respectively. L-Proline was found to elicit an increase in intracellular [Ca2+] that was dependent upon Ca2+ entry into the cell. These Ca2+ responses were enhanced by strychnine and partially antagonized by APV, CNQX, or Mg2+. Our results using dorsal horn neurons grown in culture indicate that L-proline is a weak agonist at strychnine-sensitive glycine receptors and at both NMDA and non-NMDA glutamate receptors. These observations should help in interpreting the confusing array of L-proline actions that have been described using more intact nervous system preparations. Furthermore, the ability of L-proline to stimulate Ca2+ entry after activation of excitatory amino acid receptors implicates L-proline as a potential endogenous excitotoxin.
利用大鼠脊髓背角的原代培养解离神经元，在电压钳条件下对L-脯氨酸对兴奋性和抑制性氨基酸受体的药理作用进行了表征。在-62 mV的钳制电位下，毫摩尔浓度的L-脯氨酸引发内向电流，该电流部分被D-(-)-2-氨基-5-磷酸戊酸（APV）、6-氰基-7-硝基喹喔啉-2,3-二酮（CNQX）和士的宁拮抗，并且在三种拮抗剂联合使用时几乎完全被阻断（97%阻断）。D-脯氨酸诱发的电流仅被士的宁阻断。使用三种拮抗剂的不同组合分离出L-脯氨酸诱发电流中对APV、CNQX和士的宁敏感的成分。这些电流在拮抗剂特异性、反转电位和离子通透性方面分别与N-甲基-D-天冬氨酸（NMDA）、海人酸和甘氨酸诱发的电流相同。对APV和士的宁敏感的电流也表现出与NMDA和甘氨酸诱发的电流相似的时间依赖性。由于在测试的L-脯氨酸浓度范围（0.3 - 50 mM）内反应未达到饱和，因此无法计算EC50值。然而，通过与选择性激动剂诱发的反应进行比较获得了相对效价的估计值。10 mM L-脯氨酸诱发的对APV敏感、对CNQX敏感和对士的宁敏感的电流大小分别与15 μM NMDA、5 μM海人酸和30 μM甘氨酸诱发的电流相当。发现L-脯氨酸可引起细胞内[Ca2+]增加，这依赖于Ca2+进入细胞。这些Ca2+反应被士的宁增强，被APV、CNQX或Mg2+部分拮抗。我们使用培养的背角神经元的结果表明，L-脯氨酸是士的宁敏感的甘氨酸受体以及NMDA和非NMDA谷氨酸受体的弱激动剂。这些观察结果应有助于解释使用更完整的神经系统制剂所描述的L-脯氨酸作用的复杂情况。此外，L-脯氨酸在兴奋性氨基酸受体激活后刺激Ca2+进入的能力表明L-脯氨酸是一种潜在的内源性兴奋性毒素。
