Lin M J, Lin-Shiau S Y
Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei.
Eur J Neurosci. 1997 Apr;9(4):817-23. doi: 10.1111/j.1460-9568.1997.tb01431.x.
We measured neurotransmitter release and motor nerve terminal currents in mouse phrenic nerve-diaphragm and triangularis sterni preparations, to evaluate the role of Ca2+-channel subtypes in regulating transmitter release. Saturated concentrations of either omega-agatoxin IVA [omega-Aga-IVA (0.3 microM), a blocker of P-type Ca2+ channels] or omega-conotoxin MVIIC [omega-CTx-MVIIC (2 microM), a P- and Q-type Ca2+-channel blocker], inhibited nerve-evoked muscle contractions and the amplitude of endplate potentials respectively. In contrast, combined treatment with nifedipine (50 microM, a blocker of L-type Ca2+ channels) plus omega-conotoxin GVIA [omega-CTx-GVIA (2 microM), a blocker of N-type Ca2+ channels] did not elicit inhibitory effects on nerve-evoked muscle contractions, endplate potentials or nerve terminal waveforms. Because of the non-linear relationship between endplate potentials and Ca2+ signals, a small decrease in presynaptic Ca2+ entry can significantly reduce the amplitude of the endplate potential. Thus, we applied 3,4-diaminopyridine (3,4-DAP, a K+-channel blocker) or high Ca2+ (10 mM) to accelerate and amplify the endplate potentials and Ca2+ currents. The endplate potentials amplified by 3,4-DAP or by high Ca2+ correspondingly proved to be quite resistant to both omega-Aga-IVA and omgea-CTx-MVIIC; omega-Aga-IVA exerted only a partial inhibitory effect on endplate potentials, and the omega-Aga-IVA-resistant component was further inhibited by omega-CTx-MVIIC. The component that was resistant to the two toxins could be completely blocked by the non-selective Ca2+ channel blocker Cd2+ (300 microM). A combination of the two toxins had no significant effects on either spontaneous transmitter release or postsynaptic resting membrane potentials of the diaphragm preparation and the Na+ and K+ waveforms of the triangularis sterni preparations. This finding suggests a preferential inhibitory effect at a presynaptic site. Measuring the Ca2+ currents in the triangularis sterni also revealed partial inhibition by omega-CTx-MVIIC with further incomplete inhibition by omega-Aga-IVA. Cd2+ (300 microM) abolished the toxin-resistant component of the Ca2+ current. In contrast, a combination of nifedipine (50 microM) with omega-CTx-GVIA (2 microM) was without inhibitory effect. We conclude that multiple types of Ca2+ channels, i.e. omega-Aga-IVA-sensitive, omega-CTx-MVIIC-sensitive and toxin-resistant Ca2+ channels, coexist in mouse motor nerve terminals.
我们在小鼠膈神经 - 膈肌和胸骨三角肌标本中测量了神经递质释放和运动神经末梢电流，以评估钙离子通道亚型在调节递质释放中的作用。饱和浓度的ω - 芋螺毒素IVA [ω - Aga - IVA（0.3微摩尔），一种P型钙离子通道阻滞剂]或ω - 芋螺毒素MVIIC [ω - CTx - MVIIC（2微摩尔），一种P型和Q型钙离子通道阻滞剂]分别抑制神经诱发的肌肉收缩和终板电位的幅度。相比之下，硝苯地平（50微摩尔，一种L型钙离子通道阻滞剂）加ω - 芋螺毒素GVIA [ω - CTx - GVIA（2微摩尔），一种N型钙离子通道阻滞剂]联合处理对神经诱发的肌肉收缩、终板电位或神经末梢波形没有产生抑制作用。由于终板电位与钙离子信号之间存在非线性关系，突触前钙离子内流的小幅减少可显著降低终板电位的幅度。因此，我们应用3,4 - 二氨基吡啶（3,4 - DAP，一种钾离子通道阻滞剂）或高钙（10毫摩尔）来加速和放大终板电位及钙离子电流。经3,4 - DAP或高钙放大后的终板电位相应地被证明对ω - Aga - IVA和ω - CTx - MVIIC均具有相当的抗性；ω - Aga - IVA对终板电位仅产生部分抑制作用，且ω - Aga - IVA抗性成分被ω - CTx - MVIIC进一步抑制。对这两种毒素均有抗性的成分可被非选择性钙离子通道阻滞剂Cd2 +（300微摩尔）完全阻断。两种毒素的组合对膈肌标本的自发递质释放或突触后静息膜电位以及胸骨三角肌标本的钠离子和钾离子波形均无显著影响。这一发现表明在突触前位点存在优先抑制作用。测量胸骨三角肌中的钙离子电流还显示，ω - CTx - MVIIC可产生部分抑制作用，ω - Aga - IVA可进一步产生不完全抑制作用。Cd2 +（300微摩尔）消除了钙离子电流中的毒素抗性成分。相比之下，硝苯地平（50微摩尔）与ω - CTx - GVIA（2微摩尔）的组合没有抑制作用。我们得出结论，多种类型的钙离子通道，即对ω - Aga - IVA敏感、对ω - CTx - MVIIC敏感以及对毒素有抗性的钙离子通道，共存于小鼠运动神经末梢中。
