Buck L T, Bickler P E
University of Toronto, Department of Zoology, Toronto, Canada ON M5S 3G5.
J Exp Biol. 1998 Jan;201(Pt 2):289-97. doi: 10.1242/jeb.201.2.289.
During normoxia, glutamate and the glutamate family of ion channels play a key role in mediating rapid excitatory synaptic transmission in the central nervous system. However, during hypoxia, intracellular [Ca2+] increases to neurotoxic levels, mediated largely by the N-methyl-D-aspartate (NMDA) subfamily of glutamate receptors. Adenosine has been shown to decrease the magnitude of the hypoxia-induced increase in [Ca2+]i in mammalian brain slices, delaying tissue injury. Turtle brain is remarkably tolerant of anoxia, maintaining a pre-anoxic [Ca2+]i while cerebral adenosine levels increase 12-fold. Employing cell-attached single-channel patch-clamp techniques, we studied the effect of adenosine (200 micromol l-1) and anoxia on NMDA receptor open probability (Popen) and current amplitude. After 60 min of anoxic perfusion, channel Popen decreased by 65 % (from 6.8+/-1.6 to 2.4+/-0.8 %) an effect that could also be achieved with a normoxic perfusion of 200 micromol l-1 adenosine (Popen decreased from 5.8+/-1.1 to 2.3+/-1.2 %). The inclusion of 10 micromol l-1 8-phenyltheophylline, an A1 receptor blocker, prevented the adenosine- and anoxia-induced decrease in Popen. Mean single-channel current amplitude remained at approximately 2.7+/-0.23 pA under all experimental conditions. To determine whether a change in the membrane potential could be part of the mechanism by which Popen decreases, membrane and threshold potential were measured following each experiment. Membrane potential did not change significantly under any condition, ranging from -76.8 to -80.6 mV. Therefore, during anoxia, NMDA receptors cannot be regulated by Mg2+ in a manner dependent on membrane potential. Threshold potentials did decrease significantly following 60 min of anoxic or adenosine perfusion (control -33.3+/-1.9 mV, anoxia -28.4+/-1.5 mV, adenosine -23.4+/-2.8 mV). We conclude that anoxia modulates NMDA receptor activity and that adenosine plays a key role in mediating this change. This is the first direct measurement of ion channel activity in anoxic turtle brain and demonstrates that ion channel regulation is part of the naturally evolved anoxic defence mechanism of this species.
在正常氧条件下，谷氨酸及谷氨酸离子通道家族在介导中枢神经系统快速兴奋性突触传递中起关键作用。然而，在缺氧期间，细胞内[Ca2+]升高至神经毒性水平，这主要由谷氨酸受体的N-甲基-D-天冬氨酸（NMDA）亚家族介导。已表明腺苷可降低哺乳动物脑片中缺氧诱导的[Ca2+]i升高幅度，延缓组织损伤。龟脑对缺氧具有显著耐受性，在脑腺苷水平增加12倍的同时维持缺氧前的[Ca2+]i。采用细胞贴附式单通道膜片钳技术，我们研究了腺苷（200 μmol l-1）和缺氧对NMDA受体开放概率（Popen）和电流幅度的影响。缺氧灌注60分钟后，通道Popen降低了65%（从6.8±1.6%降至2.4±0.8%），这一效应在200 μmol l-1腺苷的正常氧灌注下也可实现（Popen从5.8±1.1%降至2.3±1.2%）。加入10 μmol l-1 8-苯基茶碱（一种A1受体阻滞剂）可阻止腺苷和缺氧诱导的Popen降低。在所有实验条件下，平均单通道电流幅度保持在约2.7±0.23 pA。为确定膜电位变化是否可能是Popen降低机制的一部分，在每次实验后测量膜电位和阈电位。在任何条件下膜电位均无显著变化，范围为-76.8至-80.6 mV。因此，在缺氧期间，NMDA受体不能以依赖膜电位的方式受Mg2+调节。在缺氧或腺苷灌注60分钟后，阈电位确实显著降低（对照-33.3±1.9 mV，缺氧-28.4±1.5 mV，腺苷-23.4±2.8 mV）。我们得出结论，缺氧调节NMDA受体活性，腺苷在介导这一变化中起关键作用。这是首次对缺氧龟脑离子通道活性进行直接测量，并证明离子通道调节是该物种自然进化的缺氧防御机制的一部分。
