VOLTAMMETRIC AND PHARMACOLOGICAL CHARACTERIZATION OF DOPAMINE RELEASEFROM SINGLE EXOCYTOTIC EVENTS AT RAT PHEOCHROMOCYTOMA (PC12) CELLS

Although rat pheochromocytoma (PC12) neurotransmitter storage vesicles are known to contain a variety of neurotransmitters including catecho lamines, there is little evidence that the molecular species detected during amperometric monitoring of exocytosis is a catecholamine. Rathe r, as these are catecholamine-containing cells, one assumes catecholam ines are released, Additionally, although the total amount of transmit ter released can be quantified, it has been extremely difficult to eva luate the conentration at the point of release for each exocytosis eve nt. Interpreting voltammograms obtained in the attoliter volume affect ed between the electrode and the cell and defined by the size of the e xocytosis pore during exocytosis is an extreme analytical challenge. H ere we use voltammetry of similar to 10(-19) mol released from individ ual exocytosis events to identify, along with pharmacological evidence , the released compound at PC12 cells as a catecholamine, most likely dopamine. The area of the electrode at which oxidation occurs followin g an exocytosis event is proportional to the temporal delay prior to a cquisition of a voltammogram, This model allows determination of relat ive concentrations from individual release events and has been used to examine events at control cells and cells incubated with the dopamine precursor, L-3,4-dihydroxyphenylalanine (L-DOPA), Exposure to L-DOPA (100 mu M for 1 h) results in 145 detectable events for 11 cells compa red to 77 events for 29 control cells, clearly indicating that vesicle s can be ''loaded'' with dopamine. However, the concentrations measure d at the electrode surface provide similar distributions for both L-DO PA-treated and control cells. Cyclic voltammetric measurements of rela tive concentration for zeptomole levels of transmitter in attoliter vo lumes provide evidence that loading vesicles by increased transmitter synthesis does not lead to elevated concentrations at individual relea se sites.