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

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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.