Clinical pharmacogenetics of methotrexate

It is well known that interindividual variability can affect the response to many drugs in relation to age, gender, diet, and organ function. Pharmacogenomic studies have also documented that genetic polymorphisms can exert clinically significant effects in terms of drug resistance, efficacy and toxicity by modifying the expression of critical gene products (drug-metabolizing enzymes, transporters, and target molecules) as well as pharmacokinetic and pharmacodynamic parameters. A growing body of in vitro and clinical evidence suggests that common polymorphisms in the folate gene pathway are associated with an altered response to methotrexate (MTX) in patients with malignancy and autoimmune disease. Such polymorphisms may also induce significant MTX toxicity requiring expensive monitoring and treatment. Although the available data are not conclusive, they suggest that in the future MTX pharmacogenetics could play a key role in clinical practice by improving and tailoring treatment. This review describes the genetic polymorphisms that significantly influence MTX resistance, efficacy, and toxicity

Physical and Functional Interaction of NCX1 and EAAC1 Transporters Leading to Glutamate-Enhanced ATP Production in Brain Mitochondria

Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na+-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production

Ruolo dello scambiatore sodio-calcio 1 nell'ipertrofia cardiaca indotta da batteri

L ipertrofia cardiaca (CH) in condizioni patologiche e un processo mal adattativo in risposta ad un aumentato stress biomeccanico e accompagna varie forme di malattie cardiache. I meccanismi alla base di questo processo patologico (argomento di studio di numerose ricerche) coinvolgono spesso perturbazioni funzionali nell omeostasi del [Ca2+]i. Uno dei principali regolatori del [Ca2+]i nel cuore e il trasportatore sodio/calcio 1 (NCX1). Le infezioni batteriche possono causare CH, alterando direttamente o indirettamente l omeostasi del [Ca2+]i. In questo studio abbiamo investigato l ipotesi che NCX1 sia determinante per l induzione e progressione dei danni cardiaci da infezioni batteriche. Risultati ottenuti nel nostro laboratorio hanno dimostrato che l LPS, la principale tossina dei Gram-negativi, induce CH che e contrastata da ciclosporina nei modelli dei cuori perfusi ex vivo, in cardiomiociti primari e in linee cellulari cardiache. In tutti i sistemi analizzati, abbiamo trovato alterazioni significative a carico di NCX1 dopo esposizione a 1 g/ml LPS per 3 e 24 h. In particolare, l LPS aumenta l espressione di NCX1 (mRNA e proteina) senza alterarne la distribuzione cellurare. L aumento della proteina NCX1 e stato osservato anche in miociti in cui l espressione dello scambiatore non era regolata dall attivita del promotore endogeno. In aggiunta, cellule esprimenti NCX1 hanno sviluppato difetti nel controllo del calcio intracellulare dopo esposizione a LPS, dato che l ingresso di calcio nella cellula via il reverse mode di NCX1 era significativamente elevato nelle cellule stressate. Abbiamo trovato anche che NCX1 e critico per la risposta ipertrofica da LPS, la quale era completamente bloccata dall inibitore specifico di NCX1 SN6. Infine, in miociti privi di NCX1 la risposta ipertrofica all LPS era significativamente attenuata. Questi dati suggeriscono che NCX1 sia un nuovo bersaglio farmacologico da considerare per la prevenzione del danno cardiaco da batteri

Gram-negative endotoxin lipopolysaccharide induces cardiac hypertrophy: Detrimental role of Na+–Ca2+ exchanger

Several molecular pathways involved in the development of cardiac hypertrophy are triggered by perturbation of intracellular Ca2+ homeostasis. Within the heart, Na+/Ca2+ exchanger 1 (NCX1) is one of the main determinant in controlling Ca2+ homeostasis. In cardiac hypertrophy and heart failure NCX1 expression and activity have been reported to be altered. It has been shown that chronic bacterial infections (sepsis, endocarditis, and myocarditis) can promote cardiac hypertrophy. Bacterial stressors, such as the Gram-negative endotoxin lipopolysaccharide (LPS), can directly or indirectly affect intracellular Ca2+ homeostasis in the heart and induce the development of cardiac hypertrophy. The present study aimed at evaluating the potential link between the signal pathways activated in LPS-exposed myocytes and NCX1. In the whole rat heart, LPS perfusion induced an early hypertrophy response during which NCX1 expression significantly increased. Notably, all these changes were completely prevented by the NCX inhibitor SN-6. We further dissect the role of NCX1 in the LPS-induced hypertrophic response in an in vitro cardiac model based on two H9c2 cardiomyoblast clones, namely H9c2-WT (lacking endogenous NCX1 expression) and H9c2-NCX1 (stably transfected with a functional NCX1). H9c2-NCX1 were more susceptible than H9c2-WT to develop a hypertrophic phenotype, and they displayed a significant increase in NCX1 expression and function after LPS treatment. SN-6 completely counteracted both hypertrophic response and exchanger alterations induced by LPS in H9c2-NCX1 cells, but it had no effects on H9c2-WT. Collectively, our results suggest that NCX1 plays a critical role in promoting myocardial hypertrophy triggered by LPS

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

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-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

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

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