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Characterization of Exocytotic Events From Single PC12 Cells: Amperometric Studies in Native PC12h, DA-Loaded PC12h and Bovine Adrenal Chromaffin Cells

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Abstract

Exocytotic events from rat pheochromocytoma (PC12) cells were characterized by amperometric analysis. For single-cell amperometric recordings, PC12h cells cultured onto poly-L-lysine corted glass-base dish were incubated with 1 mM dopamine (DA) for 60 min. Amperometric recordings, with a carbon fiber microelectrode (5 μm diameter), of catecholamine release from the individual cells were conducted under an inverted microscope at 25 ∘C. To characterize a single exocytotic event that is detected as a single spike current, the spike number, spike parameters (rise time, middle width and area) and spike shape were analyzed. Exposure of DA-loaded PC12h cells to 60 mM KCl (1000 hps) for 5 min and for 4 s evoked a train of events with the event number of 114± 19 (spikes/response for 5 min) and 12± 3 (spikes/response for 15 s), respectively. We observed distinctive kinetics in the events (rise time = 0.83± 0.19 ms, middle width = 2.89± 0.62 ms, area = 62± 7.6 fC and the spikes with a “foot” = 15.4± 2.7% of total spikes). The number and mean height of the events were 3- to 4-fold higher than that in DA-unloaded cells, and the values of rise time and middle width in DA-loaded PC12h cells were approx. 5- and 10-fold less than those observed in cultured adrenal chromaffin cells. The successful application of amperometry to monitor DA released from secretory vesicles in DA-loaded PC12h cell suggest that this technique is applicable to characterize exocytotic events in neurons.

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Authors and Affiliations

  1. Laboratory of Neurochemistry and Neuropharmacology, Life Science Institute, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo, Japan

    Nobuyuki Sasakawa, Norie Murayama & Konosuke Kumakura

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  1. Nobuyuki Sasakawa
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  2. Norie Murayama
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  3. Konosuke Kumakura
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Correspondence to Konosuke Kumakura.

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Sasakawa, N., Murayama, N. & Kumakura, K. Characterization of Exocytotic Events From Single PC12 Cells: Amperometric Studies in Native PC12h, DA-Loaded PC12h and Bovine Adrenal Chromaffin Cells. Cell Mol Neurobiol 25, 777–787 (2005). https://doi.org/10.1007/s10571-005-3975-5

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  • DOI: https://doi.org/10.1007/s10571-005-3975-5

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