Characterization of exocytotic events from single PC12 cells: amperometric studies in native PC12h, DA-loaded PC12h and bovine adrenal chromaffin cells.
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.
Sasakawa
Nobuyuki
N
Laboratory of Neurochemistry and Neuropharmacology, Life Science Institute, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo, Japan.
Murayama
Norie
N
Kumakura
Konosuke
K
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000311:Adrenal Glands / Q000166:cytology; D000818:Animals; D002417:Cattle; D019439:Chromaffin Cells / Q000166:cytology / Q000502:physiology*; D004298:Dopamine / Q000494:pharmacology*; D004594:Electrophysiology; D005089:Exocytosis / Q000187:drug effects* / Q000502:physiology*; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D016716:PC12 Cells; D051381:Rats
D004298:Dopamine