Johnson M I, Paik K, Higgins D
J Cell Biol. 1985 Jul;101(1):217-26. doi: 10.1083/jcb.101.1.217.
Sympathetic neurons taken from rat superior cervical ganglia and grown in culture acquire cholinergic function under certain conditions. These cholinergic sympathetic neurons, however, retain a number of adrenergic properties, including the enzymes involved in the synthesis of norepinephrine (NE) and the storage of measurable amounts of NE. These neurons also retain a high affinity uptake system for NE; despite this, the majority of the synaptic vesicles remain clear even after incubation in catecholamines. The present study shows, however, that if these neurons are depolarized before incubation in catecholamine, the synaptic vesicles acquire dense cores indicative of amine storage. These manipulations are successful when cholinergic function is induced with either a medium that contains human placental serum and embryo extract or with heart-conditioned medium, and when the catecholamine is either NE or 5-hydroxydopamine. In some experiments, neurons are grown at low densities and shown to have cholinergic function by electrophysiological criteria. After incubation in NE, only 6% of the synaptic vesicles have dense cores. In contrast, similar neurons depolarized (80 mM K+) before incubation in catecholamine contain 82% dense-cored vesicles. These results are confirmed in network cultures where the percentage of dense-cored vesicles is increased 2.5 to 6.5 times by depolarizing the neurons before incubation with catecholamine. In both single neurons and in network cultures, the vesicle reloading is inhibited by reducing vesicle release during depolarization with an increased Mg++/Ca++ ratio or by blocking NE uptake either at the plasma membrane (desipramine) or at the vesicle membrane (reserpine). In addition, choline appears to play a competitive role because its presence during incubation in NE or after reloading results in decreased numbers of dense-cored vesicles. We conclude that the depolarization step preceding catecholamine incubation acts to empty the vesicles of acetylcholine, thus allowing them to reload with catecholamine. These data also suggest that the same vesicles may contain both neurotransmitters simultaneously.
取自大鼠颈上神经节并在培养中生长的交感神经元在某些条件下会获得胆碱能功能。然而，这些胆碱能交感神经元保留了许多肾上腺素能特性，包括参与去甲肾上腺素（NE）合成的酶以及可测量量的NE的储存。这些神经元还保留了对NE的高亲和力摄取系统；尽管如此，即使在儿茶酚胺中孵育后，大多数突触小泡仍然清晰。然而，本研究表明，如果这些神经元在儿茶酚胺中孵育前被去极化，突触小泡会获得指示胺储存的致密核心。当用含有人类胎盘血清和胚胎提取物的培养基或心脏条件培养基诱导胆碱能功能时，以及当儿茶酚胺为NE或5-羟多巴胺时，这些操作是成功的。在一些实验中，神经元以低密度生长，并通过电生理标准显示具有胆碱能功能。在NE中孵育后，只有6%的突触小泡具有致密核心。相比之下，在儿茶酚胺中孵育前被去极化（80 mM K+）的类似神经元含有82%的致密核心小泡。这些结果在网络培养中得到证实，在儿茶酚胺孵育前对神经元进行去极化，致密核心小泡的百分比增加了2.5至6.5倍。在单个神经元和网络培养中，通过增加Mg++/Ca++比值在去极化期间减少小泡释放或通过在质膜（地昔帕明）或小泡膜（利血平）处阻断NE摄取来抑制小泡再装填。此外，胆碱似乎起到竞争作用，因为在NE孵育期间或再装填后其存在会导致致密核心小泡数量减少。我们得出结论，儿茶酚胺孵育前的去极化步骤作用于排空乙酰胆碱小泡，从而使其能够重新装填儿茶酚胺。这些数据还表明，相同的小泡可能同时包含两种神经递质。
