Human recombinant Sema 3A and mouse recombinant Sema 3A were purchased from R&D Systems

ence studies performed on isolated mitochondria spotted on glass ” micro slides. NCX1 dependence of glutamate-stimulated ATP synthesis SH-SY5Y cells express NCX1 and NCX3 , while C6 cells express all three NCX. To establish whether the privileged association of EAAC1 and NCX1 emerging from the immunoprecipitation experiments also corre- 12 Mitochondrial NCX1/EAAC1 Sustain Brain 2883-98-9 metabolism sponded to a predominant role of NCX1 in mediating the effect of glutamate on mitochondrial metabolism we used an AsODN approach . NCX1 knock-down induced the same effect as CGP-37157, ” abrogating glutamate-induced ATP synthesis both in SH-SY5Y neuroblastoma, and in C6 glioma cells, whereas NCX2 and NCX3 knock-down was wholly ineffective. Finally, it is noteworthy that results obtained in isolated mitochondria were strengthened by experiments performed in hippocampal and cortical slices, a well-known integrated system which largely preserves the tissue architecture and physiology of brain regions. In this system, DL-TBOA and CGP-37157 completely counteracted glutamate-stimulated ATP synthesis. Glutamate-induced ATP synthesis in isolated heart mitochondria is dependent on EAAC1 and NCX1 To further assess the possible physiological importance of the EAAC1-NCX1 interplay in sustaining ATP production, we sought for this phenomenon in other tissues. We started by exploring the Mitochondrial NCX1/EAAC1 Sustain Brain Metabolism 14 Mitochondrial NCX1/EAAC1 Sustain Brain Metabolism heart. In preliminary experiments we detected a strong immunoreactivity in mitochondria obtained from rat heart for both EAAC1 and NCX1 proteins. Notably we found that, as in brain, NCX1 and EAAC1 proteins were assembled into a multimolecular complex and that glutamate was able to stimulate ex novo ATP synthesis in a DL-TBOA and CGP-37157 sensitive manner. Glutamate-induced ATP synthesis in undifferentiated PC12 cells and in liver does not rely on EAAT and NCX1 Undifferentiated PC12 cells express EAAC1 but not NCX1. Conversely, NCX2 and NCX3 were detected. When mitochondria obtained from PC12 cells were exposed to glutamate, an increase in ATP synthesis occurred. However this phenomenon was independent from EAAC1 and NCX, since neither DL- TBOA nor CGP-37157 were able to inhibit glutamate-stimulated ATP synthesis. Equivalent results were obtained in liver mitochondria, a system that does not express exchangers at all , but does express EAAC1 protein. We note that, at the concentrations used here, these two blockers were unable to block the ATP response to glutamate in mitochondria from both undifferentiated PC12 cells and rat hepatocytes. Since in these cells NCX1 is not expressed and, consequentely, the proposed EAAC1/NCX1 mechanism for the glutamate response is not working, these results strongly suggest that the pharmacological inhibition of the ATP response is not due to any side actions of DL-TBOA or CGP37157 on other mitochondrial carriers. Conclusion Mitochondrial NCX1/EAAC1 Sustain Brain Metabolism forming the so-called “transporting microdomains”. Since it has been shown that in energized mitochondria the matrix Na concentration is lower than extra-mitochondrial one, we propose that this gradient might be the driving force that allows glutamate entry through EAAC1 and then the interaction with NCX1 might be crucial to re-establish the Na gradient across the mitochondrial membrane, that likely has been lost after glutamate entry. However, the mechanism underpinning glutamate-st