Shafer T J, Contreras M L, Atchison W D
Department of Pharmacology, Michigan State University, East Lansing 48824.
Mol Pharmacol. 1990 Jul;38(1):102-13.
The interaction of methylmercury (MeHg) with neuronal Ca2+ channels in rat forebrain synaptosomes and dihydropyridine (DHP)-sensitive Ca2+ channels in rat pheochromocytoma (PC12) cells was examined using radiotracer flux assays and radioligand binding analyses. In synaptosomes, the influx of 45Ca2+ was used to examine the voltage and state dependence of block of Ca2+ channels by MeHg, as well as the effects of MeHg on apparent inactivation of 45Ca2+ influx. In addition, the differential influx of 45Ca2+, 85Sr2+, and 133Ba2+ was used to examine the effect of MeHg on the ionic selectivity of synaptosomal Ca2+ channels. The ability of MeHg to block 45Ca2+ influx via a DHP-sensitive Ca2+ channel was examined in PC12 cells. Effects of MeHg on binding of [3H]nitrendipine in synaptosomes and 125I-omega-conotoxin GVIA (CgTx) in synaptosomes and PC12 cells were measured. In synaptosomes, MeHg blocked 45Ca2+ influx in a voltage-dependent manner, inasmuch as increasing the extracellular K+ concentration increased the magnitude of block by 100 microM MeHg. When synaptosomes were incubated for 10 sec in either a nondepolarizing or a depolarizing solution before measurement of 1 sec of depolarization-induced 45Ca2+ influx, the potency and efficacy of the block of 45Ca2+ influx by MeHg were similar. Thus, block of Ca2+ channels by MeHg does not appear to be state dependent. To determine the kinetics of apparent inactivation of 45Ca2+ influx, synaptosomes were predepolarized in Ca2(+)-free high [K+] solution, for intervals varying from 1 to 10 sec, before measurement of 1 sec of K(+)-induced 45Ca2+ influx. When compared with control, MeHg (100 microM) altered the rate constant for apparent inactivation and decreased the fraction of 45Ca2+ influx that does not inactivate. Influx of 45Ca2+, 85Sr2+, and 133Ba2+ during 1 sec of depolarization was blocked in a dose-dependent manner by MeHg, with estimated IC50 values of 125, 150, and greater than 150 microM for 45Ca2+, 85Sr2+, and 133Ba2+, respectively. In triple-label experiments, the relative flux of radiolabeled Ca2+:Sr2+:Ba2+ was altered from approximately 6:2:3 to 6:1:3 in the presence of 100 microM MeHg. In undifferentiated and nerve growth factor-differentiated PC12 cells, K(+)-induced 45Ca2+ influx was blocked by the DHP nifedipine, with an approximate IC50 value of 5 nM. MeHg reduced 45Ca2+ influx in PC12 cells with an estimated IC50 value of 50 microM, and 125 microM MeHg reduced uptake by greater than 90%. [3H]Nitrendipine bound to synaptosomes with high affinity in normal and elevated [K+] solutions.(ABSTRACT TRUNCATED AT 400 WORDS)
利用放射性示踪剂通量测定法和放射性配体结合分析法，研究了甲基汞（MeHg）与大鼠前脑突触体中神经元Ca2+通道以及大鼠嗜铬细胞瘤（PC12）细胞中二氢吡啶（DHP）敏感的Ca2+通道之间的相互作用。在突触体中，利用45Ca2+的内流来检测MeHg对Ca2+通道阻滞的电压和状态依赖性，以及MeHg对45Ca2+内流表观失活的影响。此外，利用45Ca2+、85Sr2+和133Ba2+的差异内流来检测MeHg对突触体Ca2+通道离子选择性的影响。在PC12细胞中检测了MeHg通过DHP敏感的Ca2+通道阻滞45Ca2+内流的能力。测定了MeHg对突触体中[3H]尼群地平结合以及突触体和PC12细胞中125I-ω-芋螺毒素GVIA（CgTx）结合的影响。在突触体中，MeHg以电压依赖性方式阻滞45Ca2+内流，因为增加细胞外K+浓度会增加100μM MeHg的阻滞程度。当在测量1秒去极化诱导的45Ca2+内流之前，将突触体在非去极化或去极化溶液中孵育10秒时，MeHg对45Ca2+内流的阻滞效力和效果相似。因此，MeHg对Ca2+通道的阻滞似乎不依赖于状态。为了确定45Ca2+内流表观失活的动力学，在测量1秒K+诱导的45Ca2+内流之前，将突触体在无Ca2+的高[K+]溶液中预去极化1至10秒不等的时间间隔。与对照相比，MeHg（100μM）改变了表观失活的速率常数，并降低了未失活的45Ca2+内流的比例。在1秒去极化期间，45Ca2+、85Sr2+和133Ba2+的内流被MeHg以剂量依赖性方式阻滞，45Ca2+、85Sr2+和133Ba2+的估计IC50值分别为125、150和大于150μM。在三重标记实验中，在100μM MeHg存在下，放射性标记的Ca2+:Sr2+:Ba2+的相对通量从约6:2:3变为6:1:3。在未分化和神经生长因子分化的PC12细胞中，K+诱导的45Ca2+内流被DHP硝苯地平阻滞，估计IC50值约为5 nM。MeHg降低了PC12细胞中45Ca2+内流，估计IC50值为50μM，125μM MeHg使摄取减少超过90%。[3H]尼群地平在正常和升高的[K+]溶液中以高亲和力与突触体结合。（摘要截短于400字）
