Cytoplasmic localization and efflux of endogenous d-aspartate in pheochromocytoma 12 cells

In our previous reports [Z. Long, H. Homma, J.-A. Lee, T. Fukushima, T. Santa, T. Iwatsubo, R. Yamada, K. Imai, FEBS Lett. 434 (1998) 231–235; Z. Long, M. Sekine, M. Adachi, T. Furuchi, K. Imai, N. Nimura, H. Homma, Arch. Biochem. Biophys. 404 (2002) 92–97], we demonstrated for the first time that d-aspartate (d-Asp) is actually synthesized in cultured mammalian cells such as PC12, MPT1, and GH3 cells. After its synthesis, this unique amino acid is spontaneously and continuously released into the extracellular space during cell culture. In the current study, we characterized two different types of d-Asp efflux in PC12 cells. One is a spontaneous and continuous form of release of cytoplasmic origin that does not involve exocytotic efflux of vesicular origin. Endogenous d-Asp is predominantly localized to the cytoplasm of cells, and this form of d-Asp release presents a striking contrast to exocytotic, quantal discharge of vesicular dopamine. The other form of efflux is also of cytoplasmic origin and occurs through volume-sensitive organic anion channels that are opened upon hyposmotic stimuli. Interestingly, this latter form of efflux is potentiated by acetylcholine stimulation.