Molecular mechanisms of exocytosis: amperometric analysis of catecholamine release from single cells

胞吐作用的分子机制：单细胞儿茶酚胺释放的电流分析

基本信息

批准号：
14570086
负责人：
SASAKAWA Nobuyuki
金额：
$ 2.43万
依托单位：
Sophia University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

To clarify the implication of each PKC isoform in the regulation of exocytosis, we examined the effects of overexpression of PKC α and β on the kinetics of exocytosis of single vesicles using transfected cells. Amperometric detection of single exocytotic events is conducted by use of a carbon fiber electrode from a single cell. Expression of PKC isoformes was confirmed by co-expression of EGFP. In the PKCα-overexpressed cells, the frequency of exocytotic events evoked by KCI (60mM, 5min) was dramatically increased. This effect was apparently due to the increase in the later 4 min period, but not in the initial 1min. In the PKCβ-overexpressed cells, the frequency was increased both the initial and the later 4 min periods, but the effects was much evident in the initial 1 min in contrast to PKCα. The effects of overexpression was abolished by BIS, a PKC inhibitor. This might suggest that target molecules of phosphorylation by PKCα and PKCβ are different, resulting distinct regulation of exocytosis in adrenal chromaffin cells. Moreover, activation of PKCc seemd important for the recruitment of secretory vesicles to the readily releasable pool from the resetve pool. In addition, microinjection of myosinV-antiserum, inositol hexakisphosphate, inositol pentakispyrophosphate or Vamp peptide characteristically inhibited KCI-evoked event frequency. These results suggest that myosinV, highly phosphorylated inositol polyphoshates and Vamp may have important roles in the regulation of exocytosis in chromaffin cells. However, precise mechanisms remain to be elucidated.

为了阐明每种 PKC 亚型在胞吐作用调节中的含义，我们使用碳对单个胞吐事件进行安培检测，检查了 PKC α 和 β 的过表达对单个囊泡胞吐作用动力学的影响。通过 EGFP 的共表达证实了来自单细胞的纤维电极的 PKC 亚型的表达。 KCI（60mM，5 分钟）引起的胞吐事件显着增加，这种效应显然是由于后 4 分钟内的增加，但在 PKCβ 过表达的细胞中，频率与最初的 1 分钟无关。和后面的 4 分钟时间段，但与 PKCα 相比，最初 1 分钟的影响更为明显。BIS（一种 PKC 抑制剂）消除了过度表达的影响，这可能表明 PKCα 的靶分子。 PKCα 和 PKCβ 的磷酸化是不同的，导致肾上腺嗜铬细胞中胞吐作用的不同调节。此外，PKCc 的激活似乎对于将分泌囊泡从重置池中招募到易于释放的池中很重要。此外，肌球蛋白 V 抗血清的显微注射。肌醇六磷酸、肌醇五焦磷酸或 Vamp 肽受到特征性抑制KCI 诱发事件频率。这些结果表明，肌球蛋白 V、高度磷酸化的肌醇多磷酸盐和 Vamp 可能在嗜铬细胞胞吐作用的调节中发挥重要作用，但精确的机制仍有待阐明。