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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.

将PC 12细胞（其是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附着于微载体珠（Cytodex 1; Pharmacia），并在Dulbecco改良Eagle培养基中培养4-7天。将珠粒装入灌注微型qaurtz比色皿（体积：70穆尔）中。将比色皿置于荧光计的加热（37 ° C）样品室中。以1-1.5 ml/min的流速用洛克氏溶液灌注细胞，并通过用Fura-2 AM（2 μ M）灌注细胞15 min来加载Fura-2。通过经环注射器将刺激剂以100穆尔的体积注射到流动流中来进行细胞的脉动刺激。为了测定细胞内钙浓度（[Ca^<2+>]i），监测荧光强度（激发340和380 nm，发射500 nm）和荧光比（F_<340>/F_<380>）。将来自比色皿的灌流液直接引入电化学流动池检测器中用于检测儿茶酚胺释放。当用尼古丁、高K^+和ATP刺激PC 12细胞时，可观察到细胞内[Ca^<2+>] i和儿茶酚胺释放的一过性增加。在培养的肾上腺嗜铬细胞中，氨甲酰胆碱和KCl也以浓度依赖的方式引起[Ca^<2+>] i和儿茶酚胺释放的增加。这些结果表明，同时监测该灌流系统中[Ca^<2+>] i和儿茶酚胺的释放，有助于研究刺激-分泌偶联的调节机制。