Vignes M, Blanc E, Récasens M
INSERM U 254, Hôpital Saint-Charles, Montpellier, France.
Eur J Neurosci. 1995 Aug 1;7(8):1791-802. doi: 10.1111/j.1460-9568.1995.tb00698.x.
The regulation of intracellular Ca2+ concentration ([Ca2+]i) by glutamate metabotropic receptors (mGluR) was studied in 8-day-old rat forebrain synaptoneurosomes using spectrofluorimetric methods. Here we demonstrate that metabotropic glutamate agonists induce in rat brain synaptoneurosomes a Ca2+ influx largely dependent upon the presence of Ca2+ in the external medium. The pharmacological profile of this influx is strongly correlated with the pharmacological profile of the activation of phosphoinositide hydrolysis, i.e. quisqualic acid >> 1S,3R-amino-1-dicarboxylate-1,3 cyclopentane approximately equal to glutamate. This metabotropic glutamate receptor-induced Ca2+ influx is insensitive to voltage-dependent Ca2+ channel antagonists and occurs through a Mn2+ impermeant pathway. The study of the rapid kinetics shows that this influx is triggered after a 300 ms delay compared with that elicited by depolarizing agents and Ca2+ ionophore A23187. In order to assess further if mGluR stimulate this influx through the recruitment of inositol triphosphate (IP3)-sensitive intracellular Ca2+ stores, we have tested the effect of thapsigargin on membrane potential and intracellular Ca2+ simultaneously. Thapsigargin induces a depolarization of the synaptoneurosomal membrane followed by a massive Ca2+ influx, occurring via a Mn2+ nonpermeant route. This depolarizing effect is sensitive to the presence of the intracellular Ca2+ chelator [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetoxymethyl ester], and partially sensitive to extracellular Na+, but insensitive to the presence of extracellular Ca2+. Taken together, our data suggest that mGluR stimulate self-maintained increases of [Ca2+]i in rat forebrain synaptoneurosomes via the activation of a multistep mechanism, sequenced in the following steps: (i) mGluR-induced IP3 synthesis; (ii) IP3-stimulated intracellular Ca2+ release; (iii) Ca(2+)-activated non-specific cation channel, leading to local depolarization and a Ca2+ influx; and (iv) activation of Ca(2+)-sensitive phospholipase C.
利用荧光分光光度法，在8日龄大鼠前脑突触神经小体中研究了代谢型谷氨酸受体（mGluR）对细胞内钙离子浓度（[Ca2+]i）的调节作用。在此我们证明，代谢型谷氨酸激动剂在大鼠脑突触神经小体中诱导钙离子内流，这在很大程度上依赖于细胞外培养基中钙离子的存在。这种内流的药理学特征与磷酸肌醇水解激活的药理学特征密切相关，即喹啉酸>>1S,3R-氨基-1,3-二羧酸-1,3-环戊烷≈谷氨酸。这种代谢型谷氨酸受体诱导的钙离子内流对电压依赖性钙离子通道拮抗剂不敏感，且通过一条锰离子不通透的途径发生。快速动力学研究表明，与去极化剂和钙离子载体A23187引发的内流相比，这种内流在延迟300毫秒后被触发。为了进一步评估mGluR是否通过募集对肌醇三磷酸（IP3）敏感的细胞内钙离子储存来刺激这种内流，我们同时测试了毒胡萝卜素对膜电位和细胞内钙离子的影响。毒胡萝卜素诱导突触神经小体膜去极化，随后大量钙离子内流，通过一条锰离子不通透的途径发生。这种去极化作用对细胞内钙离子螯合剂[1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸甲酯]的存在敏感，对细胞外钠离子部分敏感，但对细胞外钙离子的存在不敏感。综上所述，我们的数据表明，mGluR通过激活一个多步骤机制来刺激大鼠前脑突触神经小体中[Ca2+]i的自我维持性增加，该机制按以下步骤排序：（i）mGluR诱导的IP3合成；（ii）IP3刺激的细胞内钙离子释放；（iii）钙离子激活的非特异性阳离子通道，导致局部去极化和钙离子内流；以及（iv）钙离子敏感的磷脂酶C的激活。
