Simultaneous Monitoring of Intracellular Calcium Concentration and Catecholamine Release from Superfused PC 12 Cells and Adrenal Chromaffin Cells Cultured on Microcarrier Beads.

同时监测微载体珠上培养的灌流 PC 12 细胞和肾上腺嗜铬细胞的细胞内钙浓度和儿茶酚胺释放。

基本信息

批准号：
01870011
负责人：
KATO Ryuichi
金额：
$ 6.4万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B).
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

PC 12 cells, which are cloned cell line derived from rat adrenal pheochromocytoma, and purified bovine adrenal chromaffin cells were attached to microcarrier beads (Cytodex 1^<<ﾟ!>>; Pharmacia) and cultured in Dulbecco's Modified Eagle Medium for 4-7 days. The beads were packed into a perfusion mini qaurtz cuvette (volume : 70 mul). The cuvette was placed in the warmed (37 ^ﾟC) sample chamber of a fluorometer. The cells were perfused with Locke's solution at a flow rate of 1-1.5 ml/min and loaded with Fura-2 by perfusing the cells with Fura-2AM (2 muM) for 15 min. A pulsative stimulation of the cells was performed by an injection of stimulant into the flow stream in a volume of 100 mul through loopinjector. For the determination of intracellular calcium concentration ([Ca^<2+>]i), the fluorescence intensity (excitation 340 and 380 nm, emission 500 nm) and florescence ratio (F_<340>/F_<380>) were monitored. The perfusate from the cuvette was directly introduced into an electrochemical flow cell detector for the detection of catecholamine release. When the PC 12 cells were stimulated with nicotine, high K^+ and ATP, transient increase in [Ca^<2+>]_i and catecholamine release were observed. In cultured adrenal chromaffin cells, carbamylcholine and KCl also induced increase in [Ca^<2+>]_i and catecholamine release in a concentration-dependent manner. These results suggest that the simultaneous monitoring of [Ca^<2+>]_i and catecholamine release in this perfusion system is useful for studies on the regulatory mechanisms of stimulus-secretion coupling.

PC 12 细胞（源自大鼠肾上腺嗜铬细胞瘤的克隆细胞系）和纯化的牛肾上腺嗜铬细胞被附着到微载体珠（Cytodex 1^<<ﾟ!>>；Pharmacia）上，并在 Dulbecco's Modified Eagle 培养基中培养 4-7将珠子装入灌注迷你石英比色皿中（体积：70μl）。将比色杯置于荧光计的加热（37℃）样品室中，以 1-1.5 ml/min 的流速灌注细胞，并通过用 Fura-2AM 灌注细胞来加载 Fura-2。 (2μM) 15分钟通过将刺激剂注射到体积为100μM的液流中来进行细胞的脉冲刺激。为了测定细胞内钙浓度([Ca^<2+>]i)，荧光强度(激发340和380nm，发射500nm)和荧光比(F_<340>/F_<380>)。当 PC 12 细胞受到刺激时，将来自比色皿的灌注液直接引入电化学流动池检测器中以检测儿茶酚胺的释放。在培养的肾上腺嗜铬细胞中，观察到尼古丁、高K^+和ATP、[Ca^2+]_i和儿茶酚胺释放的短暂增加，氨甲酰胆碱和KCl也诱导[Ca^2+]_i和儿茶酚胺释放的增加。这些结果表明，同时监测该灌注系统中的[Ca^2+]_i和儿茶酚胺释放对于调节机制的研究是有用的。刺激-分泌耦合。