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

为了阐明每种PKC异构体在胞吐调节中的作用，我们利用转染细胞检测了PKC α和β过表达对单个囊泡胞吐动力学的影响。单个胞外事件的安培检测是通过使用来自单个细胞的碳纤维电极进行的。通过EGFP的共表达证实了PKC异构体的表达。在pkc α-过表达的细胞中，KCI （60mM, 5min）诱发的胞外事件频率显著增加。这种效应显然是由于后4分钟的增加，而不是在最初的1分钟。在pkc β-过表达的细胞中，频率在初始和后4 min均增加，但与PKCα相比，在初始1 min的影响更为明显。PKC抑制剂BIS可消除过表达的影响。这可能表明PKCα和PKCβ磷酸化的靶分子不同，导致肾上腺染色质细胞胞吐的调节不同。此外，PKCc的激活对于将分泌囊泡从储备池中招募到容易释放的池中似乎很重要。此外，微量注射肌球蛋白抗血清、六磷酸肌醇、五磷酸肌醇或Vamp肽可显著抑制kci诱发的事件频率。这些结果提示myosinV、高度磷酸化的肌醇多磷酸酯和Vamp可能在调节嗜铬细胞的胞吐作用中起重要作用。然而，确切的机制仍有待阐明。

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.