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The Journal of Neuroscience, 0000, 20:RC101:1-5

RAPID COMMUNICATION
Loading of Oxidizable Transmitters into Secretory Vesicles Permits Carbon-Fiber Amperometry

Kyong-Tai Kim^{1, 2}, Duk-Su Koh¹, and Bertil Hille¹

¹ Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195-7290, and ² Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Korea

Carbon-fiber amperometry detects oxidizable molecules released by exocytosis. We extended this electrochemical technique to cells that do not normally secrete oxidizable transmitters. We incubated AtT-20 cells, pituitary gonadotropes, cultured cerebellar granule cells, and yeast with high concentrations of dopamine (DA) and observed spontaneous and evoked quantal release of 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 bath, loading was complete within 40 min. Cytoplasmic accumulation preceded vesicular loading. Loading decreased proportionally as the bath DA concentration was lowered. Loading rates were similar at 37 and 25°C and much slower at 15°C. Loading was blocked by bafilomycin A₁, a proton pump inhibitor, but not by bupropion, an inhibitor of the plasma membrane DA transporter. Other cells were tested. Spontaneous quantal events became more frequent and evoked events became larger and more frequent when PC12 cells were loaded with DA. Fluid-phase loading of neurons by short stimulation in DA solutions seemed selective for the synaptic vesicles. Thus, many cell 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.

Key words: secretion; exocytosis; amperometry; carbon fiber; vesicle; gonadotrope; granule cell; dopamine

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Copyright © 0000 Society for Neuroscience  0270-6474/00/$05.00/0

This article has been cited by other articles:

				M.-H. Kim, B.-H. Choi, S.-R. Jung, T. J. Sernka, S. Kim, K.-T. Kim, B. Hille, T. D. Nguyen, and D.-S. Koh Protease-activated Receptor-2 Increases Exocytosis via Multiple Signal Transduction Pathways in Pancreatic Duct Epithelial Cells J. Biol. Chem., July 4, 2008; 283(27): 18711 - 18720. [Abstract] [Full Text] [PDF]

				X.-K. Chen, Y.-F. Xiong, and Z. Zhou "Kiss-and-Run" Exocytosis in Astrocytes Neuroscientist, October 1, 2006; 12(5): 375 - 378. [Abstract] [PDF]

				E.-M. Hur, Y.-S. Park, B. D. Lee, I. H. Jang, H. S. Kim, T.-D. Kim, P.-G. Suh, S. H. Ryu, and K.-T. Kim Sensitization of Epidermal Growth Factor-induced Signaling by Bradykinin Is Mediated by c-Src: IMPLICATIONS FOR A ROLE OF LIPID MICRODOMAINS J. Biol. Chem., February 13, 2004; 279(7): 5852 - 5860. [Abstract] [Full Text] [PDF]

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