Mulkey R M, Zucker R S
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
J Neurosci. 1992 Nov;12(11):4327-36. doi: 10.1523/JNEUROSCI.12-11-04327.1992.
The fluorescent indicator fura-2 was used to measure cytoplasmic calcium in presynaptic terminals in the crayfish Procambarus clarkii under conditions that raise intracellular sodium to examine whether sodium can elevate intracellular calcium concentration ([Ca2+]i) or prolong its efflux and thus influence the magnitude and duration of posttetanic potentiation (PTP). Sodium was elevated in presynaptic terminals at rest by either (1) injection of sodium into the excitatory axon, (2) application of veratridine to open sodium channels, or (3) addition of ouabain to block Na/K exchange, with [Ca2+]i increasing by either 430, 400, or 180 nM, respectively. Intracellular calcium concentration increased only when external calcium was present, indicating that calcium influx occurred through Na/Ca exchange. Additionally, ouabain enhanced excitatory junctional potentials (EJPs) eightfold. Elevation of sodium using a high-frequency stimulation in zero-calcium Ringer's did not elevate [Ca2+]i during the train or immediately afterward when calcium-containing Ringer's was re-introduced. This indicates that a physiological sodium load does not release calcium from internal stores or reverse Na/Ca exchange to levels where [Ca2+]i accumulation is detectable. We examined the ability of sodium to interfere with calcium efflux from presynaptic terminals by loading boutons with both sodium and calcium or calcium alone using high-potassium depolarization. Elevation of internal sodium slowed calcium efflux from the terminal (12.3 min) compared to calcium removal without a sodium load (4.0 min). When sodium loading was increased during a tetanus by application of ouabain, the time constants for decay of EJP potentiation, 17.3 min, and for [Ca2+]i, 35 min, were longer than control values, 4.4 min and 5.8 min, respectively. In addition, using lithium to inhibit the efflux of calcium by Na/Ca exchange following a PTP-inducing train also lengthened the decay of [Ca2+]i to 15.7 min. Intracellular sodium accumulation in presynaptic terminals slows the efflux of calcium through Na/Ca exchange, and may therefore augment and prolong PTP.
荧光指示剂fura-2用于在提高细胞内钠离子浓度的条件下测量克氏原螯虾突触前终末的细胞质钙，以研究钠离子是否能升高细胞内钙浓度（[Ca2+]i）或延长其外流，从而影响强直后增强（PTP）的幅度和持续时间。在静息状态下，通过以下方式升高突触前终末的钠离子浓度：（1）向兴奋性轴突注射钠离子；（2）应用藜芦碱打开钠离子通道；（3）添加哇巴因阻断Na/K交换，[Ca2+]i分别升高430、400或180 nM。细胞内钙浓度仅在存在细胞外钙时升高，表明钙内流是通过Na/Ca交换发生的。此外，哇巴因使兴奋性突触后电位（EJP）增强了八倍。在零钙林格氏液中使用高频刺激升高钠离子浓度，在刺激期间或随后重新引入含钙林格氏液时，[Ca2+]i并未升高。这表明生理性钠离子负荷不会从内部储存库释放钙，也不会使Na/Ca交换逆转到可检测到[Ca2+]i积累的水平。我们通过使用高钾去极化使轴突终末同时加载钠离子和钙离子或仅加载钙离子，研究了钠离子干扰突触前终末钙外流的能力。与未加载钠离子时去除钙相比（4.0分钟），细胞内钠离子升高使终末钙外流减慢（12.3分钟）。当在强直刺激期间通过应用哇巴因增加钠离子加载时，EJP增强衰减的时间常数为17.3分钟，[Ca2+]i衰减的时间常数为35分钟，均长于对照值，分别为4.4分钟和5.8分钟。此外，在诱导PTP的刺激序列后，使用锂抑制通过Na/Ca交换的钙外流也使[Ca2+]i衰减延长至15.7分钟。突触前终末内细胞内钠离子积累会减慢通过Na/Ca交换的钙外流，因此可能增强并延长PTP。
