Loading of oxidizable transmitters into secretory vesicles permits carbon-fiber amperometry

Carbon-fiber amperometry detects oxidizable molecules released by exocytosi s. We extended this electrochemical technique to cells that do not normally secrete oxidizable transmitters. We incubated AtT-20 cells, pituitary gona dotropes, cultured cerebellar granule cells, and yeast with high concentrat ions of dopamine (DA) and observed spontaneous and evoked quantal release o f DA by amperometry. The rate of detectable spontaneous amperometric events was used as a measure of loading in AtT-20 cells. With 70 mM DA in the bat h, loading was complete within 40 min. Cytoplasmic accumulation preceded ve sicular loading. Loading decreased proportionally as the bath DA concentrat ion was lowered. Loading rates were similar at 37 and 25 degrees C and much slower at 15 degrees C. Loading was blocked by bafilomycin A(1), a proton pump inhibitor, but not by bupropion, an inhibitor of the plasma membrane D A transporter. Other cells were tested. Spontaneous quantal events became m ore frequent and evoked events became larger and more frequent when PC12 ce lls were loaded with DA. Fluid-phase loading of neurons by short stimulatio n in DA solutions seemed selective for the synaptic vesicles. Thus, many ce ll types can be loaded with DA to study spontaneous and evoked exocytosis. The amine molecules enter these cells passively and may become concentrated in acidic vesicles by protonation.