Mag-fura-2 fluorescence microscopy and whole-cell patch-clamp recordings were used to measure glutamate-induced changes in the intracellular free Mg2+ concentration ([Mg2+]i) and Mg2+ currents, respectively, in cultured forebrain neurones from fetal rats in the absence of extracellular Na+ (Nao+) and Ca2+ (Cao2+). 2. Increasing the extracellular Mg2+ concentration ([Mg2+]o) from 9 to 70 mM significantly enhanced the maximum [Mg2+]i induced by a 5 min 100 microM glutamate plus 1 microM glycine stimulation ([Mg2+]i,5 min) from 2.04 +/- 0.07 to 2.98 +/- 0.20 mM. Increasing [Mg2+]o from 9 to 70 mM also significantly enhanced the initial rate of rise in [Mg2+]i upon glutamate stimulation from 0.41 +/- 0.02 to 0.81 +/- 0.08 mM min-1. 3. The glutamate-stimulated increase in [Mg2+]i was not altered by prior depletion of intracellular free Na+ (Nai+). For paired stimulations in single neurones, the mean [Mg2+]i,5 min was 1.95 +/- 0.17 mM under Na(+)-depleted conditions and 1.94 +/- 0.16 mM under control conditions. 4. The glutamate-stimulated increase in [Mg2+]i was significantly reduced when NMDA channel-permeant Cs+ or K+ ions were used as the Na+ substitute instead of the presumably NMDA channel-impermeant ions N-methyl-D-glucamine (NMDG), Tris or sucrose. The mean [Mg2+]i,5 min was 0.56 +/- 0.06 and 0.74 +/- 0.08 mM in the presence of Cs+ or K+, respectively, compared with 2.13 +/- 0.10, 1.93 +/- 0.11 and 2.07 +/- 0.22 mM in the presence of NMDG, Tris or sucrose, respectively. 5. In whole-cell recordings performed with Cs+ as the primary intracellular cation, application of 100 microM NMDA plus 10 microM glycine induced inward currents that reversed around -55 mV in an extracellular solution containing 70 mM Mg2+ and 31 mM NMDG as the only cations. The currents were reversibly inhibited by DL-2-amino-5-phosphonovaleric acid (APV). In an extracellular solution containing 2 mM Mg2+ and 140 mM NMDG, NMDA plus glycine activated outward currents at potentials more depolarized than -90 mV. 6. In whole-cell recordings made with NMDG as the principal cation in the patch pipette, application of NMDA plus glycine in the 70 mM Mg2+ extracellular solution induced inward currents at voltages more negative than +15 mV. The ratio of the current measured under these conditions to the current measured in an extracellular solution containing Na+ as the principal cation (0.073:1) was nearly constant from cell to cell. 7. Following a 5 min glutamate stimulation in the presence of 9 mM Mg2+, [Mg2+]i returned to basal levels at a mean rate of 58.1 +/- 2.1 microM min-1. Complete removal of Nao+ significantly inhibited the rate of recovery to 31% of control. Raising [Mg2+]o to 30 mM in combination with removal of Nao+ did not inhibit recovery significantly more than either manipulation alone (28% of control). 8. These results suggest that glutamate-stimulated increases in [Mg2+]i that occur in the absence of Nao+ and Cao2+ result from Mg2+ entry through NMDA-activated ion channels. Furthermore, recovery from a glutamate-induced Mg2+ load appears to be primarily due to Mg2+ efflux via a mechanism whose characteristics are consistent with Na(+)-Mg2+ exchange.
摘要
采用镁荧光素-2荧光显微镜和全细胞膜片钳记录技术，分别在无细胞外钠离子（Nao+）和钙离子（Cao2+）的情况下，测量胎鼠培养前脑神经元中谷氨酸诱导的细胞内游离镁离子浓度（[Mg2+]i）变化和镁离子电流。2. 将细胞外镁离子浓度（[Mg2+]o）从9 mM增加到70 mM，显著增强了5分钟100 μM谷氨酸加1 μM甘氨酸刺激诱导的最大[Mg2+]i（[Mg2+]i,5 min），从2.04±0.07 mM增加到2.98±0.20 mM。将[Mg2+]o从9 mM增加到70 mM，也显著增强了谷氨酸刺激后[Mg2+]i的初始上升速率，从0.41±0.02 mM min-1增加到0.81±0.08 mM min-1。3. 细胞内游离钠离子（Nai+）预先耗尽，并不改变谷氨酸刺激引起的[Mg2+]i增加。对于单个神经元的配对刺激实验，在钠离子耗尽条件下，平均[Mg2+]i,5 min为1.95±0.17 mM，对照条件下为1.94±0.16 mM。4. 当使用NMDA通道可通透的铯离子（Cs+）或钾离子（K+）作为钠离子替代物，而不是可能NMDA通道不可通透的离子N-甲基-D-葡萄糖胺（NMDG）、三羟甲基氨基甲烷（Tris）或蔗糖时，谷氨酸刺激引起的[Mg2+]i增加显著减少。在存在Cs+或K+时，平均[Mg2+]i,5 min分别为0.56±0.06 mM和0.74±0.08 mM，而在存在NMDG、Tris或蔗糖时，分别为2.13±0.10 mM、1.93±0.11 mM和2.07±0.22 mM。5. 在以Cs+作为主要细胞内阳离子进行的全细胞记录中，应用1００ μM NMDA加10 μM甘氨酸在含有70 mM Mg2+和31 mM NMDG作为唯一阳离子的细胞外溶液中诱导出内向电流，该电流在-55 mV左右反转。该电流被DL-2-氨基-5-磷酸缬氨酸（APV）可逆性抑制。在含有2 mM Mg2+和140 mM NMDG的细胞外溶液中，NMDA加甘氨酸在比-90 mV更正的电位下激活外向电流。6. 在以NMDG作为膜片钳吸管中的主要阳离子进行的全细胞记录中，在70 mM Mg2+细胞外溶液中应用NMDA加甘氨酸在比+15 mV更负的电压下诱导出内向电流。在这些条件下测量的电流与在以钠离子作为主要阳离子的细胞外溶液中测量的电流之比（0.073:1）在不同细胞之间几乎恒定。7. 在9 mM Mg2+存在下进行5分钟谷氨酸刺激后，[Mg2+]i以平均速率58.1±2.1 μM min-1恢复到基础水平。完全去除Nao+显著抑制恢复速率至对照的31%。将[Mg2+]o提高到30 mM并同时去除Nao+，对恢复的抑制作用并不比单独任何一种操作显著更强（对照的28%）。8. 这些结果表明，在无Nao+和Cao2+的情况下，谷氨酸刺激引起的[Mg2+]i增加是由于镁离子通过NMDA激活的离子通道进入细胞。此外，从谷氨酸诱导的镁离子负荷中恢复似乎主要是由于通过一种其特征与钠-镁离子交换一致的机制使镁离子外流。
