Pozzan T, Di Virgilio F, Vicentini L M, Meldolesi J
Biochem J. 1986 Mar 15;234(3):547-53. doi: 10.1042/bj2340547.
Ca2+ homoeostasis was investigated in pheochromocytoma neurosecretory (PC12) cells both before and after treatment with nerve growth factor, which induces a neuronal-like differentiation accompanied by a large increase in the number of muscarinic receptors. The resting concentration of free cytosolic Ca2+, [Ca2+]i, measured by the quin2 technique, was found to be higher and more variable in differentiated cells. Moreover, the [Ca2+]i rises induced by the Ca2+ ionophore ionomycin and by depolarizing concentrations of KC1 were greater and more transient. Exposure to carbachol induced modest, but long-lasting, [Ca2+]i rises, which were faster and greater in differentiated than in non-differentiated cells. These effects were due to the activation of the muscarinic receptor, because they were unaffected by nicotinic blockers (hexamethonium and D-tubocurarine) and completely eliminated by low concentrations of the muscarinic antagonists atropine and pirenzepine [IC50 (concn. causing 50% inhibition) = 2 and 60 nM respectively]. The muscarinic-receptor-dependent [Ca2+]i rises were the result of two concomitant processes: (1) redistribution of Ca2+ from cytoplasmic stores to the cytosol, possibly mediated by generation of inositol 1,4,5-trisphosphate as a consequence of the muscarinic-receptor-coupled hydrolysis of polyphosphoinositides, and (2) increased Ca2+ influx through a pathway of the plasmalemma insensitive to verapamil and thus different from the voltage-dependent Ca2+ channel. The existence of this second process was documented: (a) by the difference of the [Ca2+]i responses brought about by carbachol in Ca2+-containing and Ca2+-free media; (b) by the occurrence of [Ca2+]i rise and increased 45Ca accumulation in cells exposed to 1 mM-CaCl2 after having been treated for 2 min with carbachol in Ca2+-free medium; (c) by typical differences in the quin2 signal kinetics observed in parallel samples of PC12 cells loaded with different concentrations of the dye.
在使用神经生长因子处理嗜铬细胞瘤神经分泌（PC12）细胞前后，对其Ca2 + 稳态进行了研究。神经生长因子可诱导神经元样分化，并伴随毒蕈碱受体数量大幅增加。通过quin2技术测量发现，分化细胞中游离胞质Ca2 +（[Ca2 + ]i）的静息浓度更高且更具变异性。此外，Ca2 + 离子载体离子霉素和去极化浓度的KCl诱导的[Ca2 + ]i升高幅度更大且更短暂。暴露于卡巴胆碱会诱导适度但持久的[Ca2 + ]i升高，分化细胞中的升高速度更快且幅度更大。这些效应是由于毒蕈碱受体的激活，因为它们不受烟碱阻断剂（六甲铵和筒箭毒碱）的影响，并被低浓度的毒蕈碱拮抗剂阿托品和哌仑西平完全消除[IC50（引起50%抑制的浓度）分别为2和60 nM]。毒蕈碱受体依赖性[Ca2 + ]i升高是两个伴随过程的结果：（1）Ca2 + 从细胞质储存库重新分布到细胞质溶胶，可能是由于毒蕈碱受体偶联的多磷酸肌醇水解产生肌醇1,4,5 - 三磷酸介导的；（2）通过质膜上一条对维拉帕米不敏感且因此不同于电压依赖性Ca2 + 通道的途径，Ca2 + 内流增加。第二个过程的存在通过以下方式得到证明：（a）卡巴胆碱在含Ca2 + 和无Ca2 + 培养基中引起的[Ca2 + ]i反应的差异；（b）在无Ca2 + 培养基中用卡巴胆碱处理2分钟后，暴露于1 mM - CaCl2 的细胞中出现[Ca2 + ]i升高和45Ca积累增加；（c）在加载不同浓度染料的PC12细胞平行样本中观察到的quin2信号动力学的典型差异。
