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

Autor: Norie Murayama, Konosuke Kumakura, Nobuyuki Sasakawa
Rok vydání: 2005
Předmět:
Zdroj: Cellular and Molecular Neurobiology. 25:777-787
ISSN: 1573-6830
0272-4340
Popis: 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 mum diameter), of catecholamine release from the individual cells were conducted under an inverted microscope at 25 degrees 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.
Databáze: OpenAIRE
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