Carmeliet P, Denef C
Laboratory of Cell Pharmacology, University of Leuven School of Medicine, Belgium.
Endocrinology. 1989 May;124(5):2218-27. doi: 10.1210/endo-124-5-2218.
Incubation of cultured rat pituitary cell aggregates with [3H]choline ([3H]Chol) yielded a derivative that was identified as [3H]acetylcholine ([3H]ACh) by several criteria: 1) the [3H]Chol derivative with the highest retention time coeluted with a [14C]ACh standard in cation exchange and reverse phase HPLC; 2) cholinesterase treatment converted this derivative to a substance with the retention time of [3H]Chol; 3) two blockers of ACh production, hemicholinium and 4-[(1-naphthylvinyl)pyridinium], eliminated 3H-labeled material in the HPLC fractions with ACh retention time. Spontaneous [3H]ACh release was increased by depolarizing potassium concentrations, and both synthesis and release of ACh were increased by the glucocorticoid hormone dexamethasone. Double immunostaining of choline acetyltransferase (CAT) and, respectively, of ACTH, GH, PRL, TSH, S100, LH, and FSH in rat pituitary cells revealed that most of the CAT-immunoreactive cells were also ACTH immunoreactive. A small proportion (less than 10%) of the PRL-immunoreactive cells also showed CAT immunoreactivity, but all other cell types were negative. The immunocytochemical evidence for colocalization of CAT within the ACTH cell was strengthened by the finding of a significantly higher rate of [3H]ACh synthesis in a corticotroph-enriched cell population obtained by separating pituitary cells on a velocity sedimentation gradient. In addition, the mouse pituitary corticotropic cell line AtT20 contained CAT immunoreactivity, converted [3H]Chol to [3H]ACh, and released bioactive ACh-like material. In conclusion, the present data provide strong evidence that pituitary corticotrophs synthesize and release ACh, and that the activity of this intrapituitary cholinergic transmission system is under regulatory control.
用[3H]胆碱（[3H]Chol）培养大鼠垂体细胞聚集体，产生了一种衍生物，通过以下几个标准鉴定为[3H]乙酰胆碱（[3H]ACh）：1）在阳离子交换和反相高效液相色谱中，保留时间最长的[3H]Chol衍生物与[14C]ACh标准品共洗脱；2）胆碱酯酶处理将该衍生物转化为保留时间与[3H]Chol相同的物质；3）两种乙酰胆碱产生阻滞剂，半胱氨酸和4-[(1-萘基乙烯基)吡啶鎓]，消除了高效液相色谱中具有乙酰胆碱保留时间的3H标记物质。去极化钾浓度可增加[3H]ACh的自发释放，糖皮质激素地塞米松可增加乙酰胆碱的合成和释放。对大鼠垂体细胞中的胆碱乙酰转移酶（CAT）以及促肾上腺皮质激素（ACTH）、生长激素（GH）、催乳素（PRL）、促甲状腺激素（TSH）、S100、促黄体生成素（LH）和促卵泡生成素（FSH）进行双重免疫染色，结果显示，大多数CAT免疫反应性细胞也具有ACTH免疫反应性。一小部分（不到10%）PRL免疫反应性细胞也显示出CAT免疫反应性，但所有其他细胞类型均为阴性。通过在速度沉降梯度上分离垂体细胞获得的富含促肾上腺皮质激素的细胞群体中，[3H]ACh合成速率显著更高，这一发现加强了CAT在ACTH细胞内共定位的免疫细胞化学证据。此外，小鼠垂体促肾上腺皮质细胞系AtT20具有CAT免疫反应性，将[3H]Chol转化为[3H]ACh，并释放生物活性ACh样物质。总之，目前的数据提供了有力证据，表明垂体促肾上腺皮质细胞合成并释放ACh，并且这种垂体内胆碱能传递系统的活性受到调节控制。
