Levesque P C, Atchison W D
Department of Pharmacology and Toxicology, Michigan State University, East Lansing 48824.
Toxicol Appl Pharmacol. 1988 Jun 15;94(1):55-65. doi: 10.1016/0041-008x(88)90336-5.
Agents known to disrupt intraterminal Ca2+ buffering, N,N-dimethylamino-8-octyl-3,4,5-trimethoxybenzoate (TMB-8), 25 microM; caffeine, 7.5 mM; N,N-bis(3,4-dimethoxyphenethyl)-N-methylamine (YS035), 180 microM; ouabain, 200 microM; and dantrolene, 50 microM, were tested for the ability to alter effects of methyl mercury (MeHg) on spontaneous quantal release of acetylcholine (ACh) at the rat neuromuscular junction. In particular, we sought to determine whether any of the above agents could prevent the MeHg-induced increase of spontaneous release of ACh, an effect measured electrophysiologically as increased frequency of miniature end-plate potentials (MEPPs). MEPPs were recorded continuously from myofibers of the rat hemidiaphragm using conventional, intracellular recording techniques during pretreatment with an inhibitor of Ca2+ regulation and subsequently with the inhibitor plus MeHg (100 microM). When given alone, caffeine and ouabain, which release Ca2+ from the smooth endoplasmic reticulum and mitochondria, respectively, increased MEPP frequency in a biphasic manner. Following pretreatment, concomitant application of MeHg with caffeine or ouabain increased MEPP frequency after a brief latent period to peak values of 53 and 92 Hz, respectively. TMB-8 and dantrolene, putative inhibitors of Ca2+ release from smooth endoplasmic reticulum, differed in their effects on MEPP frequency; TMB-8 alone decreased MEPP frequency to approximately 10% of drug-free control, whereas dantrolene did not significantly alter control MEPP frequency. Subsequent concomitant application of MeHg with TMB-8 or dantrolene increased MEPP frequency to peak values of 40 and 100 Hz after 17 and 30 min, respectively. YS035, a putative inhibitor of mitochondrial uptake and release of Ca2+, decreased MEPP frequency to less than 10% of control after 15 min when given alone. Application of MeHg following YS035 pretreatment failed to increase MEPP frequency for up to 90 min. YS035 did not mask a MeHg effect by blocking postsynaptic sensitivity to ACh or preventing its release since subsequent treatment with La3+ (2 mM) after YS035 had abolished spontaneous release, increased MEPP frequency within 5 min. Thus, of the five inhibitors of nerve terminal Ca2+ regulation tested, only YS035 prevented the stimulatory action of MeHg on MEPP frequency. Results of the present study suggest that release of Ca2+ from nerve terminal mitochondria contributes to the increased MEPP frequency caused by MeHg while release of Ca2+ from smooth endoplasmic reticulum may not.
已知可破坏终末内钙离子缓冲的试剂，如N,N -二甲基氨基-8-辛基-3,4,5-三甲氧基苯甲酸酯（TMB - 8，25微摩尔）、咖啡因（7.5毫摩尔）、N,N -双(3,4-二甲氧基苯乙基)-N -甲胺（YS035，180微摩尔）、哇巴因（200微摩尔）和丹曲林（50微摩尔），被测试其改变甲基汞（MeHg）对大鼠神经肌肉接头处乙酰胆碱（ACh）自发量子释放影响的能力。特别地，我们试图确定上述试剂中是否有任何一种能够阻止MeHg诱导的ACh自发释放增加，这种效应通过电生理学方法测量为微小终板电位（MEPPs）频率增加。使用传统的细胞内记录技术，在大鼠半膈肌肌纤维上连续记录MEPPs，在预先用钙离子调节抑制剂处理后，随后用该抑制剂加MeHg（100微摩尔）处理。单独使用时，分别从光滑内质网和线粒体释放钙离子的咖啡因和哇巴因以双相方式增加MEPP频率。预处理后，将MeHg与咖啡因或哇巴因同时应用，在短暂潜伏期后，MEPP频率分别增加到峰值53赫兹和92赫兹。TMB - 8和丹曲林是推测的光滑内质网钙离子释放抑制剂，它们对MEPP频率的影响不同；单独使用TMB - 8可使MEPP频率降低至无药对照的约10%，而丹曲林未显著改变对照MEPP频率。随后将MeHg与TMB - 8或丹曲林同时应用，分别在17分钟和30分钟后，MEPP频率增加到峰值40赫兹和100赫兹。YS035是推测的线粒体摄取和释放钙离子的抑制剂，单独使用15分钟后可使MEPP频率降低至对照的不到10%。YS035预处理后应用MeHg，在长达90分钟内未能增加MEPP频率。YS035并未通过阻断突触后对ACh的敏感性或阻止其释放来掩盖MeHg的作用，因为在YS035消除自发释放后，随后用La3 +（2毫摩尔）处理，在5分钟内增加了MEPP频率。因此，在所测试的五种神经末梢钙离子调节抑制剂中，只有YS035阻止了MeHg对MEPP频率的刺激作用。本研究结果表明，神经末梢线粒体释放钙离子促成了MeHg引起的MEPP频率增加，而光滑内质网释放钙离子可能并非如此。
