Search

In a previous report (FEBS Lett. 434 (1998) 231), we demonstrated for the first time that D-aspartate (D-Asp) is synthesized in rat pheochromocytoma 12 (PC12) cells. This unique amino acid is believed to act as a novel messenger in mammalian cell regulation. However, the dynamics of D-Asp homeostasis in mammalian cells is yet to be elucidated. In this communication, we demonstrate that D-Asp is also synthesized in MPT1 cells (a subclone of PC12 cells) and that the D- and L-Asp levels in cells are regulated by cell density of the culture. Our data show that D-Asp levels increase, while in contrast, L-Asp levels decrease as a function of increased cell density. Conversely, in PC12 cells, which do not express the glutamate transporter involved in the incorporation of D- and L-Asp into cells, L-Asp levels decrease upon cell density increase while D-Asp concentrations remain almost unchanged. The results indicate that the biochemical behaviors of D- and L-Asp in mammalian cells are distinct and that the cellular levels of these stereoisomers appear to be under different control mechanisms.

Abstract: We previously reported for the first time that D-aspartate (D-Asp) is biosynthesized by cultured mammalian cells such as pheochromocytoma (PC)12 cells and its subclone MPT1 (FEBS Lett. 434 (1998) 231, Arch. Biochem. Biophys. 404 (2002) 92). We speculated that D-Asp levels in the intra- and extracellular spaces of the cultured cells are maintained in a dynamic state of homeostasis. To test this here, we utilized a novel and potent L-Glu transporter inhibitor, TFB-TBOA. This inhibitor proved to be a genuine nontransportable blocker of the transporter even during long periods of culture. Use of this inhibitor with MPT1 cells confirmed that D-Asp levels are in a dynamic steady state where it is constantly released into the extracellular space by a yet undefined mechanism as well as being constantly and intensively taken up by the cells via the L-Glu transporter. We estimated the rate with which D-Asp is constitutively released from MPT1 cells is approx. 3.8 pmol/h/1 × 105 cells. [Copyright &y& Elsevier]

In previous studies [FEBS Lett. 434 (1998) 231, Arch. Biochem. Biophys. 404 (2002) 92], we demonstrated for the first time that d-aspartate (d-Asp) is synthesized in cultured mammalian cell lines, such as pheochromocytoma 12 (PC12) and its subclone, MPT1. Our current focus is analysis of the dynamics of d-Asp homeostasis in these cells. In this communication, we show that l-glutamate (Glu) and l-Glu transporter substrates in the extracellular space regulate the homeostasis of endogenous d-Asp in MPT1 cells. d-Asp is apparently in dynamic homeostasis, whereby endogenous d-Asp is constantly released into the extracellular space by an undefined mechanism, and continuously and intensively taken up into cells by an l-Glu transporter. Under these conditions, l-Glu and its transporter substrates in the medium may competitively inhibit the uptake of d-Asp via the transporter, resulting in accumulation of the amino acid in the extracellular space. We additionally demonstrate that dl-TBOA, a well-established l-Glu transporter inhibitor, is taken up by the transporter during long time intervals, but not on a short time-scale. [Copyright &y& Elsevier]