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

为了阐明蛋白激酶C各亚型在胞吐调控中的意义，我们利用转基因细胞检测了蛋白激酶Cα和β的过表达对单个囊泡胞吐动力学的影响。单个胞吐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。EGFP共表达证实了PKC亚型的表达。在PKCα过表达的细胞中，KCI(60 mM，5min)引起的胞吐事件的频率显著增加。这种效应明显是由于后4min的增加，而不是最初1min的增加。在pKCβ过表达的细胞中，pKCα过表达的频率在最初和以后的4min都有所增加，但在最初1min的影响比pkc DNA更明显。PKC抑制剂BIS可消除这种过度表达的影响。这可能表明PKCα和PKCβ的磷酸化靶分子不同，从而导致肾上腺嗜铬细胞胞吐功能的不同调节。此外，PKCc的激活似乎对重新安置池中的分泌囊泡重新聚集到容易释放的池中很重要。此外，微量注射肌球蛋白V抗血清、肌醇六磷酸、肌醇五焦磷酸或VAMP多肽可显著抑制KCI诱发的事件频率。这些结果表明，肌球蛋白V、高度磷酸化的肌醇多聚体和VAMP可能在嗜铬细胞胞吐的调节中起重要作用。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.