Wick P F, Senter R A, Parsels L A, Uhler M D, Holz R W
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0626.
J Biol Chem. 1993 May 25;268(15):10983-9.
We have developed a transient transfection method to measure protein secretion from non-dividing, primary bovine chromaffin cells and from the continuous cell line, PC12. A plasmid coding human growth hormone (GH) was expressed in sufficient amounts in bovine chromaffin and PC12 cells to allow precise measurements of secretion from the small fraction (less than 1%) of transfected cells in a dish. GH was secreted in a similar proportion to endogenous catecholamine upon nicotinic stimulation, depolarization with elevated K+, and upon permeabilization with digitonin and subsequent stimulation with micromolar Ca2+. GH in homogenates from GH-transfected chromaffin cells cosedimented with catecholamine on discontinuous sucrose gradients. The data indicate that transiently expressed human GH in chromaffin and PC12 cells is localized predominantly in secretory vesicles in the regulated secretory pathway. With transient transfection there is a high probability of coexpression in the same cell of two plasmids which are cotransfected. Coexpression of a plasmid for GH and a plasmid for the non-N-methyl-D-aspartate glutamate receptor, GluR1, created chromaffin cells in which Ca(2+)-dependent GH secretion could be stimulated by the glutamatergic agonist kainate. The ability to coexpress a plasmid of interest with a plasmid for GH will allow the investigation of the role of other cloned proteins in the regulated secretory pathway in differentiated, non-dividing cells.
我们开发了一种瞬时转染方法，用于测量非分裂的原代牛嗜铬细胞和连续细胞系PC12中的蛋白质分泌。编码人生长激素（GH）的质粒在牛嗜铬细胞和PC12细胞中大量表达，从而能够精确测量培养皿中转染细胞的小部分（小于1%）的分泌情况。在烟碱刺激、用升高的K+去极化以及用洋地黄皂苷通透细胞并随后用微摩尔浓度的Ca2+刺激时，GH的分泌比例与内源性儿茶酚胺相似。来自转染GH的嗜铬细胞匀浆中的GH在不连续蔗糖梯度上与儿茶酚胺共沉降。数据表明，嗜铬细胞和PC12细胞中瞬时表达的人生长激素主要定位在调节性分泌途径的分泌小泡中。通过瞬时转染，共转染的两个质粒在同一细胞中共表达的可能性很高。GH质粒与非N-甲基-D-天冬氨酸谷氨酸受体GluR1质粒的共表达产生了嗜铬细胞，其中谷氨酸能激动剂海藻酸盐可刺激Ca(2+)依赖性GH分泌。将感兴趣的质粒与GH质粒共表达的能力将有助于研究其他克隆蛋白在分化的非分裂细胞的调节性分泌途径中的作用。
