Shanghai Fever: Not Only an Asian Disease.

Objectives: To describe a case of Shanghai fever disease and to analyze other published reports in non-Asiatic countries, defining clinical characteristics and highlighting that this is not only an Asian disease. Study design: A computerized search without language restriction was conducted using PubMed and Scopus; all references listed were hand-searched to identify any other relevant literature. An article was considered eligible for inclusion in the systematic review if it reported cases with Shanghai fever described in non-Asiatic countries. Our case was also included in the analysis. Results: Ten articles reporting 10 cases of Shanghai fever disease were considered. Fever, diarrhea and ecthyma gangrenosum were the most frequent symptoms observed. Blood was the most common site of isolation for Pseudomonas aeruginosa. Three patients underwent surgery due to necrotizing enteritis and intestinal perforation. Meningitis was documented in one case. None of the patients received antipseudomonal antibiotics within 24 h of admission. The outcome was good in nine cases; only one patient died due to multiple organ failure from Pseudomonas sepsis. No common primary immune deficiency was identified in these patients. Extremely young age (<1 year) was the only host factor predisposing to Shanghai fever. Conclusions: It is important to shed light on this disease in non-Asiatic countries and take into account that it can also affect healthy children. Pediatricians, therefore, should consider Shanghai fever among diagnoses in children with community-onset diarrhea, fever and skin lesions suggestive of ecthyma gangrenosum to start an appropriate treatment sooner and to reduce the mortality in these children.

The Neuroprotective Effect of L-Carnitine against Glyceraldehyde-Induced Metabolic Impairment: Possible Implications in Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive regression and memory loss. Dysfunctions of both glucose metabolism and mitochondrial dynamics have been recognized as the main upstream events of the degenerative processes leading to AD. It has been recently found that correcting cell metabolism by providing alternative substrates can prevent neuronal injury by retaining mitochondrial function and reducing AD marker levels. Here, we induced an AD-like phenotype by using the glycolysis inhibitor glyceraldehyde (GA) and explored whether L-carnitine (4-N-trimethylamino-3-hydroxybutyric acid, LC) could mitigate neuronal damage, both in SH-SY5Y neuroblastoma cells and in rat primary cortical neurons. We have already reported that GA significantly modified AD marker levels; here we demonstrated that GA dramatically compromised cellular bioenergetic status, as revealed by glycolysis and oxygen consumption rate (OCR) evaluation. We found that LC ameliorated cell survival, improved OCR and ATP synthesis, prevented the loss of the mitochondrial membrane potential (Δψm) and reduced the formation of reactive oxygen species (ROS). Of note, the beneficial effect of LC did not rely on the glycolytic pathway rescue. Finally, we noticed that LC significantly reduced the increase in pTau levels induced by GA. Overall, these findings suggest that the use of LC can promote cell survival in the setting of the metabolic impairments commonly observed in AD. Our data suggest that LC may act by maintaining mitochondrial function and by reducing the pTau level.

Gateways for Glutamate Neuroprotection in Parkinson's Disease (PD): Essential Role of EAAT3 and NCX1 Revealed in an In Vitro Model of PD.

Increasing evidence suggests that metabolic alterations may be etiologically linked to neurodegenerative disorders such as Parkinson's disease (PD) and in particular empathizes the possibility of targeting mitochondrial dysfunctions to improve PD progression. Under different pathological conditions (i.e., cardiac and neuronal ischemia/reperfusion injury), we showed that supplementation of energetic substrates like glutamate exerts a protective role by preserving mitochondrial functions and enhancing ATP synthesis through a mechanism involving the Na+-dependent excitatory amino acid transporters (EAATs) and the Na+/Ca2+ exchanger (NCX). In this study, we investigated whether a similar approach aimed at promoting glutamate metabolism would be also beneficial against cell damage in an in vitro PD-like model. In retinoic acid (RA)-differentiated SH-SY5Y cells challenged with α-synuclein (α-syn) plus rotenone (Rot), glutamate significantly improved cell viability by increasing ATP levels, reducing oxidative damage and cytosolic and mitochondrial Ca2+ overload. Glutamate benefits were strikingly lost when either EAAT3 or NCX1 expression was knocked down by RNA silencing. Overall, our results open the possibility of targeting EAAT3/NCX1 functions to limit PD pathology by simultaneously favoring glutamate uptake and metabolic use in dopaminergic neurons.

Renal angiomyolipomatosis and bleeding aneurysms in a tuberous sclerosis context: selective artery embolization in a girl with end-stage renal failure.

Recent developments in endovascular radiological techniques and devices have rendered embolization a major therapeutic option prior to surgery in many renal vascular or neoplastic diseases. A 19-yearold female patient, with a diagnosis of tuberous sclerosis complex (TSC) in childhood, was admitted with severe anemia. Polycystic kidney disease in end-stage renal failure appeared four years before and the patient has been undergoing peritoneal dialysis. The patient's medical history also included bilateral renal angiomyolipomas (AMLs). One year earlier, a unilateral endovascular embolization was performed to repair a bleeding aneurysm at the right renal upper pole. A second bilateral ruptured renal aneurysm was diagnosed at admission. To continue with peritoneal dialysis and prevent intrarenal hemorrhage and intraperitonal bleeding, an urgent bilateral renal AE was performed. Two months later she underwent a bilateral retroperitoneal nephrectomy. The posterior surgical approach, preserved the peritoneal surface area and adequate conditions to continue dialysis. At histology, bilateral AMLs were confirmed and a renal cell carcinoma of the right kidney was concurrently discovered. She undergoes continuous peritoneal dialysis. Urgent selective renal AE represents a feasible treatment for bilateral AML bleeding. It is safe and feasible before performing nephrectomy in patients with end-stage renal failure.

NCX1 and EAAC1 transporters are involved in the protective action of glutamate in an in vitro Alzheimer's disease-like model.

Increasing evidence suggests that metabolic dysfunctions are at the roots of neurodegenerative disorders such as Alzheimer's disease (AD). In particular, defects in cerebral glucose metabolism, which have been often noted even before the occurrence of clinical symptoms and histopathological lesions, are now regarded as critical contributors to the pathogenesis of AD. Hence, the stimulation of energy metabolism, by enhancing the availability of specific metabolites, might be an alternative way to improve ATP synthesis and to positively affect AD progression. For instance, glutamate may serve as an intermediary metabolite for ATP synthesis through the tricarboxylic acid (TCA) cycle and the oxidative phosphorylation. We have recently shown that two transporters are critical for the anaplerotic use of glutamate: the Na+-dependent Excitatory Amino Acids Carrier 1 (EAAC1) and the Na+-Ca2+ exchanger 1 (NCX1). Therefore, in the present study, we established an AD-like phenotype by perturbing glucose metabolism in both primary rat cortical neurons and retinoic acid (RA)-differentiated SH-SY5Y cells, and we explored the potential of glutamate to halt cell damage by monitoring neurotoxicity, AD markers, ATP synthesis, cytosolic Ca2+ levels and EAAC1/NCX1 functional activities. We found that glutamate significantly increased ATP production and cell survival, reduced the increase of AD biomarkers (amyloid ß protein and the hyperphosphorylated form of tau protein), and recovered the increase of NCX reverse-mode activity. The RNA silencing of either EAAC1 or NCX1 caused the loss of the beneficial effects of glutamate, suggesting the requirement of a functional interplay between these transporters for glutamate-induced protection. Remarkably, our results indicate, as proof-of-principle, that facilitating the use of alternative fuels, like glutamate, may be an effective approach to overcome deficits in glucose utilization and significantly slow down neuronal degenerative process in AD.

Cracking the code of sodium/calcium exchanger (NCX) gating: Old and new complexities surfacing from the deep web of secondary regulations.

Cell membranes spatially define gradients that drive the complexity of biological signals. To guarantee movements and exchanges of solutes between compartments, membrane transporters negotiate the passages of ions and other important molecules through lipid bilayers. The Na+/Ca2+ exchangers (NCXs) in particular play central roles in balancing Na+ and Ca2+ fluxes across diverse proteolipid borders in all eukaryotic cells, influencing cellular functions and fate by multiple means. To prevent progression from balance to disease, redundant regulatory mechanisms cooperate at multiple levels (transcriptional, translational, and post-translational) and guarantee that the activities of NCXs are finely-tuned to cell homeostatic requirements. When this regulatory network is disturbed by pathological forces, cells may approach the end of life. In this review, we will discuss the main findings, controversies and open questions about regulatory mechanisms that control NCX functions in health and disease.

NCX and EAAT transporters in ischemia: At the crossroad between glutamate metabolism and cell survival.

Energy metabolism impairment is a central event in the pathophysiology of ischemia. The limited availability of glucose and oxygen strongly affects mitochondrial activity, thus leading to ATP depletion. In this setting, the switch to alternative energy sources could ameliorate cells survival by enhancing ATP production, thus representing an attractive strategy for ischemic treatment. In this regard, some studies have recently re-evaluated the metabolic role of glutamate and its potential to promote cell survival under pathological conditions. In the present review, we discuss the ability of glutamate to exert an "energizing role" in cardiac and neuronal models of hypoxia/reoxygenation (H/R) injury, focusing on the Na+/Ca2+ exchanger (NCX) and the Na+-dependent excitatory amino acid transporters (EAATs) as key players in this metabolic pathway.

Excitatory Amino Acid Transporters (EAATs): Glutamate Transport and Beyond.

Na+-dependent excitatory amino acid transporters (EAATs) are the major transport mechanisms for extracellular glutamate removal in the central nervous system (CNS). The primary function assigned to EAATs is the maintenance of low extracellular glutamate levels, thus allowing glutamate to be used as a signaling molecule in the brain and to avoid excitotoxicity. However, glutamate has other recognized functions. For instance, it is a key anaplerotic substrate for the tricarboxylic acid (TCA) cycle, as it can be converted to α-ketoglutarate by transaminases or glutamate dehydrogenase. Furthermore, glutamate is a precursor of the main antioxidant glutathione, which plays a pivotal role in preventing oxidative cell death. Therefore, glutamate signaling/use is at the crossroad of multiple metabolic pathways and accordingly, it can influence a plethora of cell functions, both in health and disease. Here, we provide an overview of the main functions of glutamate and its transport systems, analyzing its role as a neurotransmitter and at the same time, the possible metabolic fates it can undergo in the intracellular milieu. Specifically, the metabolic role of glutamate and the molecular machinery proposed to metabolically support its transport will be further analyzed.

Inorganic Polyphosphate Regulates AMPA and NMDA Receptors and Protects Against Glutamate Excitotoxicity via Activation of P2Y Receptors.

Glutamate is one of the most important neurotransmitters in the process of signal transduction in the CNS. Excessive amounts of this neurotransmitter lead to glutamate excitotoxicity, which is accountable for neuronal death in acute neurological disorders, including stroke and trauma, and in neurodegenerative diseases. Inorganic polyphosphate (PolyP) plays multiple roles in the mammalian brain, including function as a calcium-dependent gliotransmitter mediating communication between astrocytes, while its role in the regulation of neuronal activity is unknown. Here we studied the effect of PolyP on glutamate-induced calcium signal in primary rat neurons in both physiological and pathological conditions. We found that preincubation of primary neurons with PolyP reduced glutamate-induced and AMPA-induced but not the NMDA-induced calcium signal. However, in rat hippocampal acute slices, PolyP reduced ion flux through NMDA and AMPA receptors in native neurons. The effect of PolyP on glutamate and specifically on the AMPA receptors was dependent on the presence of P2Y1 but not of P2X receptor inhibitors and also could be mimicked by P2Y1 agonist 2MeSADP. Preincubation of cortical neurons with PolyP significantly reduced the initial calcium peak as well as the number of neurons with delayed calcium deregulation in response to high concentrations of glutamate and resulted in protection of neurons against glutamate-induced cell death. As a result, activation of P2Y1 receptors by PolyP reduced calcium signal acting through AMPA receptors, thus protecting neurons against glutamate excitotoxicity by reduction of the calcium overload and restoration of mitochondrial function.SIGNIFICANCE STATEMENT One of the oldest polymers in the evolution of living matter is the inorganic polyphosphate (PolyP). It is shown to play a role of gliotransmitter in the brain; however, the role of polyphosphate in neuronal signaling is not clear. Here we demonstrate that inorganic polyphosphate is able to reduce calcium signaling induced by physiological or high concentrations of glutamate. The effect of polyphosphate on glutamate-induced calcium signal in neurons is due to the effect of this polymer on the AMPA receptors. The effect of PolyP on glutamate-induced and AMPA-induced calcium signal is dependent on P2Y receptor antagonist. The ability of PolyP to restrict the glutamate-induced calcium signal lies in the basis of its protection of neurons against glutamate excitotoxicity.

Low incidence of nephrotoxicity following intravenous administration of iodinated contrast media: a prospective study.

OBJECTIVES: To estimate the incidence of contrast-induced acute kidney injury (CI-AKI) after intravenous (iv) iodinated contrast material (ICM) exposure. METHODS: This prospective cohort study included all consecutive patients who underwent radiological investigations using low-osmolar iopamidol 370 mg/ml in a regional hospital over a period of 36 months, without any exclusion criteria. The estimated glomerular filtration rate (eGFR) was evaluated using the MRDR equation before (2-10 days) and after (24-36 h) radiological investigations. CI-AKI was defined as a ≥ 25% decrease in eGFR from baseline. CI-AKI incidence was estimated using a binomial distribution. The association between CI-AKI and demographic and clinical characteristics was modeled using logistic regression. RESULTS: The study included 1541 patients with a median age of 68 (1st-3rd quartiles 58-76) years with various comorbidities, 30% of whom had pre-existing CKD. Patients affected by stage III or IV chronic kidney disease (CKD) received an infusion of 0.9% normal saline (1.0-1.5 ml/kg/h) before and after iso-osmolar iodixanol administration. CI-AKI was observed in 33 patients (2.1%, 95% CI 1.5-3.0). The logistic regression analysis showed that antibiotic and statin therapies were significantly associated with CI-AKI. The probability of developing CI-AKI decreased by 80% in patients taking statins (OR = 0.20, 95% CI 0.03; 0.68) and increased approximately three times in patients with antibiotic therapy compared with those who did not take statins and antibiotics (OR = 2.92, 95% CI 1.21; 6.36). CONCLUSIONS: Our data suggest that low-osmolar iopamidol carries a low incidence of nephrotoxicity, even in subjects with various comorbid conditions or reduced renal function. KEY POINTS: • IV administration of ICM carries a low incidence of nephrotoxicity, which was transient in observed patients. • Statin therapy is negatively associated with AKI in patients exposed to ICM. • Pre-existing impairment of renal function is not associated with AKI in patients exposed to ICM.

Emphysematous Pyelonephritis Following Ureterovesical Reimplantation for Congenital Obstructive Megaureter. Pediatric Case Report and Review of the Literature.

Introduction: Emphysematous pyelonephritis (EPN) is a rare, life-threatening necrotizing infection of the kidney. To date, very few cases of EPN have been described in the pediatric age. The first case of EPN in a toddler occurring after ureterovesical reimplantation for congenital obstructive megaureter is reported with a literature review. Case Report: A 23-month-old male, with a prenatal diagnosis of obstructive megaureter and incomplete duplication of the left urinary tract, was admitted to our Unit where he underwent surgery to treat increased dilatation of the renal pelvis and appearance of an obstructive curve. The latter was revealed at renal scintigraphy, the exam highlighted the radiographic aspect of the cortical renal parenchymal sufferance. At admission preoperative exams were normal, and no recurrent urinary tract infections were documented. Surgical removal of the left stenotic ureteral common tract of the incomplete duplex collecting system was accomplished; ureterovesical reimplantation was performed without ureteral recalibration. No intraoperative complications were recorded. In the immediate postoperative period, urosepsis and the patient's lethargic condition led to life-threatening conditions requiring urgent admission to the intensive care unit. Biochemical analysis showed leukocytosis, anemia, increased C-reactive protein, prolonged prothrombin time, pancytopenia, hyponatremia. Abdominal sonographic evaluation revealed the presence of gas in the left kidney. Unilateral EPN (Class 2) was confirmed by CT- scan. Escherichia coli was cultured from peripheral blood and antimicrobial therapy was started. No additional interventions were required. The child was discharged 14 days postoperatively with normal renal function. Conclusion: EPN is a serious condition that can occur after surgical treatment for urinary tract obstruction. Early detection of air in the kidney should be considered a sign of complicated urinary tract infection. Immediate aggressive resuscitation and antimicrobial therapy are effective and curative with a positive outcome.

Tumor necrosis factor-related apoptosis-inducing ligand reduces the expression of the neuroprotective Na+ /Ca2+ exchanger isoform NCX3 in human neuroblastoma SH-SY5Y cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a cytokine belonging to the TNF superfamily, is regarded as a mediator of neurotoxicity. The constitutively expressed ion exchanger Na+ /Ca2+ exchanger isoform-3 (NCX3) has been shown to protect neurons from injury. Its expression is induced by nerve growth factor (NGF) through activation of its tyrosine kinase receptor trkA. The latter, in turn, activates downstream kinases, such as extracellular signal-regulated kinase (ERK) and the survival-related kinase protein kinase B (AKT). Here, we verified whether TRAIL could influence the expression of NCX3 via modulation of the NGF/trkA system. Differentiated human neuroblastoma SH-SY5Y cells were incubated with TRAIL and, subsequently, the expression of the NCX3 protein was studied at different times by means of western blot analysis. Then, the expression of the phosphorylated forms of either trkA, ERK or AKT was analyzed at identical intervals. Western blot analysis revealed that the expression of NCX3 protein decreased in a time-dependent fashion in SH-SY5Y cells treated with TRAIL, to reach its minimum at 48 h. On the other hand, p-trkA, p-ERK, and p-AKT expression was increased in cells treated with TRAIL after 6 and 16 h; then it declined to nearly undetectable levels after 48 h. Results indicate that the increase in TRAIL expression occurring during neuronal damage may be responsible of NCX3 down-regulation and weakens its neuroprotective effects. The TRAIL system could thus represent a potential target for treatment of neuronal damage characterized by NCX3 function impairment.

Glutamate as a potential "survival factor" in an in vitro model of neuronal hypoxia/reoxygenation injury: leading role of the Na+/Ca2+ exchanger.

In brain ischemia, reduction in oxygen and substrates affects mitochondrial respiratory chain and aerobic metabolism, culminating in ATP production impairment, ionic imbalance, and cell death. The restoration of blood flow and reoxygenation are frequently associated with exacerbation of tissue injury, giving rise to ischemia/reperfusion (I/R) injury. In this setting, the imbalance of brain bioenergetics induces important metabolic adaptations, including utilization of alternative energy sources, such as glutamate. Although glutamate has long been considered as a neurotoxin, it can also be used as intermediary metabolite for ATP synthesis, and both the Na+/Ca2+ exchanger (NCX) and the Na+-dependent excitatory amino-acid transporters (EAATs) are essential in this pathway. Here we analyzed the role of NCX in the potential of glutamate to improve metabolism and survival of neuronal cells subjected to hypoxia/reoxygenation (H/R). In SH-SY5Y neuroblastoma cells differentiated into a neuron-like state, H/R produced a significant cell damage, a decrease in ATP cellular content, and intracellular Ca2+ alterations. Exposure to glutamate at the onset of the reoxygenation phase attenuated H/R-induced cell damage and evoked a significant raise in intracellular ATP levels. Furthermore, we found that in H/R cells NCX reverse-mode activity was reduced, and that glutamate limited such reduction. All the effects induced by glutamate supplementation were lost when cells were transfected with small interfering RNA against NCX1 and EAAT3, suggesting the need of a specific functional interplay between these proteins for glutamate-induced protection. Collectively, our results revealed the potential beneficial effect of glutamate in an in vitro model of H/R injury and focused on the essential role exerted by NCX1. Although preliminary, these findings could be a starting point to further investigate in in vivo systems such protective effect in ischemic settings, shedding a new light on the classical view of glutamate as detrimental factor.

Calcium- and ATP-dependent regulation of Na/Ca exchange function in BHK cells: Comparison of NCX1 and NCX3 exchangers.

Na+/Ca2+ exchangers (NCX) mediate bidirectional Ca2+ fluxes across cell membranes and contribute to Ca2+ homeostasis in many cell types. Exchangers are regulated by gating reactions that depend on Na+ and Ca2+ binding to transport and regulatory sites. A Na+i-dependent inactivation is prominent in all isoforms, whereas Ca2+i-dependent regulation varies among isoforms. Here we characterize new details of NCX operation and describe differences and similarities between NCX3 and NCX1 regulation by intracellular Ca2+ and ATP. To compare isoforms, we employed BHK cells expressing NCX3 or NCX1 constitutively and exchange activity was analysed in whole-cell and excised patch recordings under "zero-trans" conditions (i.e., with only one transported ion species on each side). Using BHK cells with low cytoplasmic Ca2+ buffering, outward (reverse) currents, reflecting Ca2+ influx, are activated by applying extracellular Ca2+ (Cao) in the presence of Na+ on the cytoplasmic side. When firstly activated, peak outward NCX3 currents rapidly decay over seconds and then typically develop a secondary transient peak with slower kinetics, until Cao removal abolishes all outward current. The delayed rise of outward current is the signature of an activating process since peak outward NCX3 currents elicited at subsequent Cao bouts remain stimulated for minutes and slower decline towards a non-zero level during continued Cao application. Secondary transient peaks and current stimulation are suppressed by increasing the intracellular Ca2+ buffer capacity or by replacing cytoplasmic ATP with the analogues AMP-PNP or ATPγS. In BHK cells expressing NCX1, outward currents activated under identical settings decay to a steady-state level during single Cao application and are significantly larger, causing strong and long-lived run down of subsequent outward currents. NCX1 current run down is not prevented by increasing cytoplasmic Ca2+ buffering but secondary transient peaks in the outward current profile can be resolved in the presence of ATP. Finally, inward currents recorded in patches excised from NCX3-expressing cells reveal a proteolysis-sensitive, Ca-dependent inactivation process that is unusual for NCX1 forward activity. Together, our results suggest that NCX function is regulated more richly than appreciated heretofore, possibly including processes that are lost in excised membrane patches.

CHF5074 protects SH-SY5Y human neuronal-like cells from amyloidbeta 25-35 and tumor necrosis factor related apoptosis inducing ligand toxicity in vitro.

Alzheimer's disease (AD) is contributed by multiple pathogenic causes. The anomalous protein amyloid-ß (Aß) is regarded as a pivotal factor in AD, and originates from enzymatic cleavage of a precursor protein by the secretase family. 1-(3',4'-Dichloro-2-fluoro[1,1'-biphenyl]-4-yl)-cyclopropanecarboxylic acid (CHF5074) is a non-steroidal antiinflammatory derivative able to inhibit Aß deposition in the brain of transgenic mouse models of AD. The proapoptotic cytokine TRAIL has been reported to mediate Aß-dependent neurotoxicity. Here, the effects of CHF5074 on Aß25-35- triggered TRAIL toxicity were evaluated in the differentiated human neuroblastoma cell line SH-SY5Y in vitro. Cells were pre-treated 1h with CHF5074 at graded concentrations (range: 1 nM-1 uM) and then challenged for 72 h with either Aß25-35 or TRAIL. Results show that CHF5074 treatment prevented apoptotic death in SH-SY5Y cell line in a concentration- dependent fashion. Its maximally active concentration was 10 nM. Then, investigation of related molecular mechanisms underlying such protective effect of CHF5074 suggested that the levels of caspases, as well as of various kinases, including stress and MAP kinases, are modulated by CHF5074. Finally, treatment of injured human neuroblastoma cell line SH-SY5Y with CHF5074 resulted in prominent protection from apoptotic death. The bulk of these data suggest that CHF5074 represents a potential candidate for pharmacological neuroprotective treatment in neurodegenerative disorders.

Glutamate-induced ATP synthesis: relationship between plasma membrane Na+/Ca2+ exchanger and excitatory amino acid transporters in brain and heart cell models.

It is known that glutamate (Glu), the major excitatory amino acid in the central nervous system, can be an essential source for cell energy metabolism. Here we investigated the role of the plasma membrane Na(+)/Ca(2+) exchanger (NCX) and the excitatory amino acid transporters (EAATs) in Glu uptake and recycling mechanisms leading to ATP synthesis. We used different cell lines, such as SH-SY5Y neuroblastoma, C6 glioma and H9c2 as neuronal, glial, and cardiac models, respectively. We first observed that Glu increased ATP production in SH-SY5Y and C6 cells. Pharmacological inhibition of either EAAT or NCX counteracted the Glu-induced ATP synthesis. Furthermore, Glu induced a plasma membrane depolarization and an intracellular Ca(2+) increase, and both responses were again abolished by EAAT and NCX blockers. In line with the hypothesis of a mutual interplay between the activities of EAAT and NCX, coimmunoprecipitation studies showed a physical interaction between them. We expanded our studies on EAAT/NCX interplay in the H9c2 cells. H9c2 expresses EAATs but lacks endogenous NCX1 expression. Glu failed to elicit any significant response in terms of ATP synthesis, cell depolarization, and Ca(2+) increase unless a functional NCX1 was introduced in H9c2 cells by stable transfection. Moreover, these responses were counteracted by EAAT and NCX blockers, as observed in SH-SY5Y and C6 cells. Collectively, these data suggest that plasma membrane EAAT and NCX are both involved in Glu-induced ATP synthesis, with NCX playing a pivotal role.

Ischemic-LTP in striatal spiny neurons of both direct and indirect pathway requires the activation of D1-like receptors and NO/soluble guanylate cyclase/cGMP transmission.

Striatal medium-sized spiny neurons (MSNs) are highly vulnerable to ischemia. A brief ischemic insult, produced by oxygen and glucose deprivation (OGD), can induce ischemic long-term potentiation (i-LTP) of corticostriatal excitatory postsynaptic response. Since nitric oxide (NO) is involved in the pathophysiology of brain ischemia and the dopamine D1/D5-receptors (D1-like-R) are expressed in striatal NOS-positive interneurons, we hypothesized a relation between NOS-positive interneurons and striatal i-LTP, involving D1R activation and NO production. We investigated the mechanisms involved in i-LTP induced by OGD in corticostriatal slices and found that the D1-like-R antagonist SCH-23390 prevented i-LTP in all recorded MSNs. Immunofluorescence analysis confirmed the induction of i-LTP in both substance P-positive, (putative D1R-expressing) and adenosine A2A-receptor-positive (putative D2R-expressing) MSNs. Furthermore, i-LTP was dependent on a NOS/cGMP pathway since pharmacological blockade of NOS, guanylate-cyclase, or PKG prevented i-LTP. However, these compounds failed to prevent i-LTP in the presence of a NO donor or cGMP analog, respectively. Interestingly, the D1-like-R antagonism failed to prevent i-LTP when intracellular cGMP was pharmacologically increased. We propose that NO, produced by striatal NOS-positive interneurons via the stimulation of D1-like-R located on these cells, is critical for i-LTP induction in the entire population of MSNs involving a cGMP-dependent pathway.

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.

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.