Undifferentiated PC12 cells do express EAAC1 but not NCX1.

<p>(A) EAAC1 is expressed in mitochondria from undifferentiated PC12 cells. The panel shows the results of western blot (left) and PCR (right) experiments demonstrating the presence, respectively, of EAAC1 protein and transcripts in mitochondria isolated from undifferentiated PC12 cells. Significant GLAST expression and undetectable GLT1 levels were observed in similar experiments (data not shown). The experiments are representative of a set of 3. (B) NCX1 is undetectable in undifferentiated PC12 cells. Note the absence of NCX1 immunoreactivity even though the blot was overexposed in the attempt to visualize even a faint NCX1 signal (left). Moreover, no NCX1 mRNA was detected (right). Western blot experiments with NCX1 antibody were not performed on mitochondrial extracts, since we found no evidence of NCX1 expression in this cell line. In each IP lane, the lower band at around 50 KDa represents the immunoglobulin. (C,D) Both NCX2 and NCX3 are expressed in undifferentiated PC12 cells.</p

Glutamate-stimulated ATP synthesis in isolated mitochondria from cells.

<p>(A) Effects of DL-TBOA on glutamate-stimulated ATP synthesis in mitochondria from SH-SY5Y neuroblastoma and C6 glioma cells. (B) EAAC1 expression in SH-SY5Y neuroblastoma and C6 glioma cell mitochondria. Samples enriched with anti-EAAC1 antibody by selective immunoprecipitation (IP) were also loaded onto the gel. Protein extracts from rat brain and from BHK cells stably expressing NCX1 were used as controls. In each IP lane, the lower band represents the immunoglobulin. (C) Effect of anti-EAAC1 (As-EAAC1) and anti-Citrin/AGC22 (As-AGC2) antisense (D) ODNs, on glutamate-stimulated ATP synthesis in SH-SY5Y neuroblastoma and C6 glioma cells. Data from cells treated with Lipofectamine (Ctl) and sense ODNs (S-EAAC1 and S-AGC2) are also reported. Each bar in panels A, C and D represents the mean ± SEM of 14 different determinations. * p<0.05 vs control; ** p<0.01 vs control; *** p<0.001 vs control; ## p<0.01 vs 0.5 or 1 mM glutamate; ### p<0.001 vs 0.5 or 1 mM glutamate; n.s.: not significant vs control. In ODNs experiments, *** p<0.001 vs S+glutamate.</p

EAAC1 Pre-embedding Immunoelectron Microscopy.

<p>(A–E) Specific staining (granular electron-dense reaction product) is present on the membrane of some mitochondria (arrows) contained into the dendrites (a,b), neuronal somata (b,d) and astrocyte soma (c) and perivascular processes (e) of both rat cerebral cortex (a–c) and hippocampus (d,e). Labeling is also scattered in the cytoplasm and present into cisterns of the rough endoplasmic reticulum and Golgi apparatus (arrowheads, inset of b). Insets are the enlargement of the corresponding framed area. Den, dendrite; Nuc, nucleus; G, Golgi apparatus; RER, rough endoplasmic reticulum; E, endothelium; F, filaments; Cap, capillary. Bar: a, 3 µm; b, 4 µm; c, 6 µm; d, 1.5 µm; e 2.5 µm; inset of a, 1 µm; inset of b, 2 µm; inset of c, 2.5 µm.</p

Glutamate-induced ATP synthesis in isolated heart mitochondria is dependent on EAAC1 and NCX1.

<p>(A) Mitochondria isolated from rat heart express EAAC1 and NCX1. Plasma membrane (m) and mitochondrial (mt) protein extracts from rat heart were resolved with EAAC1 and NCX1 antibodies. Blots are representative of a group of 3 different experiments. (B) Coimmunoprecipitation of EAAC1 and NCX1 in rat mitochondrial protein extracts. Crude protein extracts or proteins separated by immunoprecipitation with NCX1 antibody (IP-NCX1) were resolved with EAAC1 antibody. In A and B, proteins from whole rat brain were used as a positive control. In each IP lane, the lower band at around 50 KDa represents the immunoglobulin. (C) Glutamate stimulates ATP synthesis in isolated rat heart mitochondria. The bar graphs show ATP production in presence of 1 mM glutamate (black bars) or vehicle (white bars) for 1 h in with or without oligomycin. Each bar represents the mean ± SEM of data from at least 3 different experimental sessions. (D, E) Glutamate-induced ATP synthesis in rat heart mitochondria is inhibited by EAAT or NCX pharmacological blockade with DL-TBOA or CGP-37157, respectively. The bar graphs show ATP production in presence of 1 mM glutamate (black bars) or vehicle (white bars) for 1 h with or without different concentrations of DL-TBOA (panel D) or of CGP-37157 (panel E). Each bar represents the mean ± SEM of data from 6 different experimental sessions. ATP data were normalized to the protein content of the different mitochondrial preparations. *p<0.05 vs control; *** p<0.001 vs control; # p<0.05 vs 1 mM glutamate; ### p<0.001 vs 1 mM glutamate.</p

NCX dependence of glutamate-stimulated ATP synthesis.

<p>(A) The NHE blocker EIPA (10 µM) was not able to affect baseline (white bars) and glutamate-stimulated (black bars) ATP synthesis in mitochondria isolated from rat hippocampus or cortex. (B) The bar graphs illustrate the glutamate-dependent Na⁺_mit response in SH-SY5Y and C6 cells in the control condition and after 20 min exposure to CGP-37157 (3 µM). Na⁺_mit response to the glutamate challenge was measured as the percent increase from resting Na⁺_mit (Δ%), i.e. from the mean Na⁺_mit recorded during the 120 s preceding the challenge to the highest Na⁺_mit value reached after glutamate stimulation. The insets report representative records of glutamate induced Na⁺_mit response obtained in SH-SY5Y and C6 cells, under control condition (pink line) and after CGP-37157 treatment (blue line). Each bar represent the mean ± SEM of 48–58 cells recorded in 3 different experimental sessions. *** p<0.001 vs control. (C) Effects of rising concentrations of the mitochondrial NCX blocker CGP-37157 on glutamate-stimulated ATP synthesis in mitochondria from rat hippocampus or cortex; similar results were obtained in mitochondria isolated from SH-SY5Y and C6 cells (D). (E) The Ca²⁺ uniporter inhibitor Ru360 (10 µM) was not able to affect baseline (white bars) and glutamate-stimulated (black bars) ATP synthesis in mitochondria isolated from rat hippocampus or cortex. Each bar represents the mean ± SEM of 10 different determinations. * p<0.05 vs control; *** p<0.001 vs control; # p<0.05 vs 0.5 or 1 mM glutamate; ### p<0.001 vs 0.5 or 1 mM glutamate.</p

Glutamate-stimulated ATP synthesis in isolated mitochondria from rat tissues.

<p>(A) Purity of mitochondrial preparations: western blot for the integral protein of the endoplasmic reticulum calnexin; for the plasma membrane protein β1 integrin; for the selective mitochondrial markers porin and ANT in tissue homogenate or in Percoll gradient-purified mitochondria from hippocampus and cortex. (B) ATP production by mitochondria from rat hippocampus and cortex after 1 h incubation with glutamate (black bars) or vehicle (white bars) with or without oligomycin. (C) ATP production by mitochondria from rat hippocampus and cortex after 1 h incubation with glutamate (black bars) or vehicle (white bars) or different glucose concentrations (gray bars). (D) ATP production in rat hippocampal or cortical mitochondria exposed for 1 h to DL-TBOA in the presence of glutamate (black bars) or vehicle (white bars). (E) GLAST, GLT1, and EAAC1 glutamate transporters in mitochondrial protein extracts (mt) from rat hippocampus or cortex. Plasma membrane proteins (m) were used as a positive control. The same panel shows EAAC1 immunoreactivity in different rat tissues. Rat testis were used as negative control. (F) ATP production in rat hippocampal or cortical mitochondria exposed for 1 h to TFB-TBOA 50 nM in the presence of glutamate (black bars) or vehicle (white bars). Each bar in panels B, C, D, F represents the mean ± SEM of 18 different determinations. ** p<0.01 vs control; *** p<0.001 vs control; ## p<0.01 vs 1 mM glutamate; ### p<0.001 vs 1 mM glutamate.</p

Glutamate-stimulated ATP synthesis relies on NCX1.

<p>Selective loss of glutamate-stimulated ATP synthesis in SH-SY5Y (A) and C6 cells (B) exposed to NCX1 AsODNs and its persistence in cells treated with NCX2 or NCX3 AsODNs. Bars represent the mean ± SEM of 12 different determinations. Ctl: cells treated with Lipofectamine; S-NCX and As-NCX: cells treated respectively with specific sense and antisense ODNs. * p<0.05 vs control; *** p<0.001 vs control and vs S+glutamate; n.s.: not significant vs control.</p

Extramitochondrial sodium concentration affects glutamate-induced ATP synthesis.

<p>Effect of different Na⁺ concentrations on ATP synthesis in SH-SY5Y (A) and C6 (B) mitochondria. The glutamate stimulatory effect on ATP synthesis, observed with 5 mM Na⁺, was lost when 1 mM Na⁺ was used in the buffer solution, both in SH-SY5Y and C6 mitochondria (panel A and B, respectively). Each bar represents the mean ± SEM of data from 9 different experimental sessions. ATP data were normalized to the protein content of the different mitochondrial preparations *** p<0.001 vs all groups.</p

Real-time membrane potential analysis in intact cells.

<p>Experiments performed in SH-SY5Y (A,C) and C6 (B,D) cells using quenching concentration of the inner mitochondrial membrane potential indicator TMRE (150 nM). Glutamate perfusion induced mitochondrial depolarization (blue line). DL-TBOA (100 µM) and CGP-37157 (3 µM) both perfused from 20 min before through the end of recordings, prevented glutamate-stimulated mitochondrial depolarization (pink and yellow lines, respectively), while Ru360 (10 µM) was ineffective (green line). The resting condition inner mitochondrial membrane potential of SH-SY5Y neuroblastoma (A) or C6 glioma (B) cells was not affected by DL-TBOA (pink line), CGP-37157 (yellow line) or Ru360 (green line). The respiratory chain poison p-trifluoromethyl phenyl hydrazone (FCCP, 5 µM) was added at the end as an internal control in each experiment. For each group, more than 45 cells recorded in four different sessions were analyzed and the maximal depolarization induced after glutamate stimulation was used for the statistical analysis. *** p<0.001 vs control.</p

EAAC1 and NCX1 are assembled into a multimolecular complex in mitochondria.

<p>(A) Selective NCX1 immunoprecipitation by EAAC1 antibody in mitochondrial protein extracts from rat cortex and hippocampus. Three different western blots experiments using NCX1, NCX2 or NCX3 antibodies are reported. The gels were loaded with mitochondrial crude protein extracts (mt) or with EAAC1-immunoprecipitated proteins (mt-IP EAAC1). For western blots resolved with NCX1 antibody, brain tissue homogenate (brain) and normal mouse serum-immunoprecipitated proteins (mt-IP NM) were used as a positive and a negative control, respectively. For NCX2 and NCX3, proteins from isolated membranes (m) or EAAC1-immunoprecipitated membranes (m-IP EAAC1) were also analyzed. (B) Mitochondrial protein extracts (from the same preparations used in panel A) immunoprecipitated with NCX1 antibody and resolved with GLAST, GLT1 and EAAC1 antibodies. Brain tissue homogenate (brain) and normal mouse serum-immunoprecipitated proteins (mit-IP NM) were used as a positive and a negative control, respectively. (C) Immunoprecipitations with EAAC1 and NCX1 antibodies in SH-SY5Y and C6 cells, respectively. In the immunoblot analysis with NCX1 antibody the IP lane corresponds to EAAC1-immunoprecipitated protein extracts; in the EAAC1 immunoblots the IP lane corresponds to NCX1-immunoprecipitated protein extracts. Protein extracts from rat brain tissue homogenate and from BHK cells stably expressing NCX1 were used as controls. In each IP lane, the lower band at around 50 KDa represents the immunoglobulin. (D) Results from confocal microscopy analysis using NCX1 antibody and the mitochondrial marker MitoTracker on rat hippocampal and cortical mitochondria are shown on the left. Results on the same mitochondrial preparations probed with EAAC1 (red) and NCX1 (green) antibodies are shown on the right. Note the strong overlap of the two signals in the merged image.</p