Title

 

Authors

P. Castaldo, S. Magi, V. Lariccia, A.A. Nasti., S. Arcangeli, S. Amoroso.

Section of Pharmacology, Department of Neuroscience, University “Politecnica delle Marche”, Ancona, Italy

 

Abstract

Glutamatergic pathways seem to be involved in brain metabolism. In neuronal and glial cells Excitatory Amino Acid Transporters (EAATs) are responsible for glutamate transport coupled with Na⁺. When glutamate enters the cell through EAATs, an increase in intracellular Na⁺ occurs.  In astrocytesit has been shown that Na⁺entry via EAATs may activate the reverse mode of the Na⁺/Ca²⁺ exchanger (NCX), that couples the uphill extrusion of Ca²⁺ to the entrance of Na⁺. Three different NCX1-3 isoforms have been identified and all are expressed in CNS. Both NCX and EAATs activities can be affected by the transmembrane Na⁺ gradients to which these transporters are simultaneously exposed, so that their action may be co-modulated. In this study we characterized the expression and activity of EAATs and NCX and their possible involvement in energy production. We used two continuous cell lines, SH-SY5Y human neuroblastoma and rat C6 glioma. These cell lines express NCX1 and NCX3, and between the EAATs, they mainly express EAAC1.Exogenous glutamate promoted a significant increase in ATP production in both these systems. This effect was abolished when the cells were preincubated with DL-TBOA, an EAATs inhibitor. Moreover glutamate induced ATP synthesis was abrogated when EAAC1 was selectively knocked-out with antisense oligonucleotides. To establish the possible involvement of NCX in glutamate-induced ATP synthesis, we used KB-R7943, a selective NCX blocker. This compound reduced the glutamate-induced ATP production both in SH-SY5Y and C6. Moreover, by using specific antisense oligonucleotides against NCX1, we observed that, both in SH-SY5Y and C6, NCX1 knocking-out recapitulated the effect of KB-R7943; conversely, antisense oligonucleotides directed against either NCX2 or NCX3 were ineffective. Collectively, these results suggest that, in CNS, EAAC1 may be involved in the glutamate-induced ATP production and that glutamate may elicit ATP synthesis only if NCX1 activity is preserved.