Leucine 434 is essential for docosahexaenoic acid-induced augmentation of L-glutamate transporter current.

Astrocytic excitatory amino acid transporter 2 (EAAT2) plays a major role in removing the excitatory neurotransmitter L-glutamate (L-Glu) from synaptic clefts in the forebrain to prevent excitotoxicity. Polyunsaturated fatty acids such as docosahexaenoic acid (DHA, 22:6 n-3) enhance synaptic transmission, and their target molecules include EAATs. Here, we aimed to investigate the effect of DHA on EAAT2 and identify the key amino acid for DHA/EAAT2 interaction by electrophysiological recording of L-Glu-induced current in Xenopus oocytes transfected with EAATs, their chimeras, and single mutants. DHA transiently increased the amplitude of EAAT2 but tended to decrease that of excitatory amino acid transporter subtype 1 (EAAT1), another astrocytic EAAT. Single mutation of leucine (Leu) 434 to alanine (Ala) completely suppressed the augmentation by DHA, while mutation of EAAT1 Ala 435 (corresponding to EAAT2 Leu434) to Leu changed the effect from suppression to augmentation. Other polyunsaturated fatty acids (docosapentaenoic acid, eicosapentaenoic acid, arachidonic acid, and α-linolenic acid) similarly augmented the EAAT2 current and suppressed the EAAT1 current. Finally, our docking analysis suggested the most stable docking site is the lipid crevice of EAAT2, in close proximity to the L-Glu and sodium binding sites, suggesting that the DHA/Leu434 interaction might affect the elevator-like slide and/or the shapes of the other binding sites. Collectively, our results highlight a key molecular detail in the DHA-induced regulation of synaptic transmission involving EAATs.

Neuroprotective Potential of L-Glutamate Transporters in Human Induced Pluripotent Stem Cell-Derived Neural Cells against Excitotoxicity.

Human induced pluripotent stem cell (hiPSC)-derived neural cells have started to be used in safety/toxicity tests at the preclinical stage of drug development. As previously reported, hiPSC-derived neurons exhibit greater tolerance to excitotoxicity than those of primary cultures of rodent neurons; however, the underlying mechanisms remain unknown. We here investigated the functions of L-glutamate (L-Glu) transporters, the most important machinery to maintain low extracellular L-Glu concentrations, in hiPSC-derived neural cells. We also clarified the contribution of respective L-Glu transporter subtypes. At 63 days in vitro (DIV), we detected neuronal circuit functions in hiPSC-derived neural cells by a microelectrode array system (MEA). At 63 DIV, exposure to 100 µM L-Glu for 24 h did not affect the viability of neural cells. 100 µM L-Glu in the medium decreased to almost 0 µM in 60 min. Pharmacological inhibition of excitatory amino acid transporter 1 (EAAT1) and EAAT2 suppressed almost 100% of L-Glu decrease. In the presence of this inhibitor, 100 µM L-Glu dramatically decreased cell viability. These results suggest that in hiPSC-derived neural cells, EAAT1 and EAAT2 are the predominant L-Glu transporters, and their uptake potentials are the reasons for the tolerance of hiPSC-derived neurons to excitotoxicity.

Dexmedetomidine as a cardioprotective drug: a narrative review.

Dexmedetomidine (DEX), a highly selective alpha2-adrenoceptors agonist, is not only a sedative drug used during mechanical ventilation in the intensive care unit but also a cardio-protective drug against ischemia-reperfusion injury (IRI). Numerous preclinical in vivo and ex vivo studies, mostly evaluating the effect of DEX pretreatment in healthy rodents, have shown the efficacy of DEX in protecting the hearts from IRI. However, whether DEX can maintain its cardio-protective effect in hearts with comorbidities such as diabetes has not been fully elucidated. Multiple clinical trials have reported promising results, showing that pretreatment with DEX can attenuate cardiac damage in patients undergoing cardiac surgery. However, evidence of the post-treatment effects of DEX in clinical practice remains limited. In this narrative review, we summarize the previously reported evidence of DEX-induced cardio-protection against IRI and clarify the condition of the hearts and the timing of DEX administration that has not been tested. With further investigations evaluating these knowledge gaps, the use of DEX as a cardio-protective drug could be further facilitated in the management of patients undergoing cardiac surgery and might be considered in a broader area of clinical settings beyond cardiac surgery, including patients with acute myocardial infarction.

Long-term light and moderate exercise intervention similarly prevent both hippocampal and glycemic dysfunction in presymptomatic type 2 diabetic rats.

A prediabetic population has an increased risk of cognitive decline and type 2 diabetes mellitus (T2DM). This study investigated whether the progression of memory dysfunction and dysregulated brain glycogen metabolism is prevented with 4 mo of exercise intervention from the presymptomatic stage in a T2DM rat model. Memory function and biochemical and molecular profiles were assessed in the presymptomatic stage of Otsuka-Long-Evans-Tokushima fatty (OLETF) rats, a T2DM model, with Long-Evans Tokushima (LETO) rats as genetic control. These rats were subjected to light- or moderate-intensity treadmill running for 4 mo with repetition of the same experiments. Significant hippocampal-dependent memory dysfunction was observed in the presymptomatic stage of OLETF rats, accompanied by downregulated levels of hippocampal monocarboxylate transporter 2 (MCT2), a neuronal lactate-transporter, without alteration in hippocampal glycogen levels. Four months of light or moderate exercise from the presymptomatic stage of T2DM normalized glycemic parameters and hippocampal molecular normalization through MCT2, glycogen, and brain-derived neurotrophic factor (BDNF) levels with the improvement of memory dysfunction in OLETF rats. A 4-mo exercise regimen from the presymptomatic stage of T2DM at a light and moderate intensities contributed to the prevention of the development of T2DM and the progression of cognitive decline with hippocampal lactate-transport and BDNF improvement.NEW & NOTEWORTHY Type 2 diabetes mellitus is an independent risk factor for hippocampal memory dysfunction, which would progress since the prediabetic stage. We found that 4 mo of exercise both at the light and moderate intensity prevented the progression of memory dysfunction with an improvement of hippocampal MCT2 expression in presymptomatic diabetes, implying that light intensity exercise could be a therapeutic approach, and the alteration of hippocampal MCT2 would be a therapeutic target of memory dysfunction from presymptomatic diabetes.

Exercise-Induced Adrenocorticotropic Hormone Response Is Cooperatively Regulated by Hypothalamic Arginine Vasopressin and Corticotrophin-Releasing Hormone.

INTRODUCTION: Exercise becomes a stress when performed at an intensity above the lactate threshold (LT) because at that point the plasma adrenocorticotropic hormone (ACTH), a marker of stress response, increases. It is possible that the exercise-induced ACTH response is regulated at least by arginine vasopressin (AVP) and possibly by corticotropin-releasing hormone (CRH), but this remains unclear. To clarify the involvement of these factors, it is useful to intervene pharmacologically in the regulatory mechanisms, with a physiologically acceptable exercise model. METHODS: We used a special stress model of treadmill running (aerobic exercise) for male Wistar rats, which mimic the human physiological response, where plasma ACTH levels increase at just above the LT for 30 min. Animals were administered the AVP V1b receptor antagonist SSR149415 (SSR) and/or the CRH type 1 receptor antagonist CP154526 (CP) intraperitoneally before the exercise, which allowed the monitoring of exercise-induced ACTH response. Immunohistochemical evaluation of activated AVP and CRH neurons with exercise was performed for the animals' hypothalami. RESULTS: A single injection of either antagonist, SSR or CP, resulted in inhibited ACTH levels after exercise stress. Moreover, the combined injection of SSR and CP strongly suppressed ACTH secretion during treadmill running to a greater extent than each alone. The running-exercise-induced activation of both AVP and CRH neurons in the hypothalamus was also confirmed. CONCLUSION: These results lead us to hypothesize that AVP and CRH are cooperatively involved in exercise-induced ACTH response just above the LT. This may also reflect the stress response with moderate-intensity exercise in humans.

High-Pressure Synthesis of Transition-Metal Oxyhydrides with Double-Perovskite Structures.

We report on the high-pressure synthesis, crystal structure, and magnetic properties of four novel transition-metal oxyhydrides─Ba2NaVO3H3, Ba2NaVO2.4H3.6, Ba2NaCrO2.2H3.8, and Ba2NaTiO3H3─crystallizing in the double-perovskite structure. Notably, they have a higher hydride content in their anion sites (50%-63%) than known oxyhydrides with perovskite structures do (≤33%). Vanadium and chromium oxyhydrides exhibited Curie-Weiss magnetic susceptibilities with no magnetic ordering down to 2 K, which may be due to geometrical frustration in their face-centered lattices and weak magnetic interactions. Density functional theory calculations revealed that the transition metal-hydride bonding nature of the prepared oxyhydrides is more covalent than that observed for known perovskite oxyhydrides, as evidenced by the shorter bond lengths of the former. Remarkably, our double-perovskite oxyhydrides with a high hydride content may possess a bonding character intermediate between those of known oxyhydrides and hydrides.

Leptin in hippocampus mediates benefits of mild exercise by an antioxidant on neurogenesis and memory.

Regular exercise and dietary supplements with antioxidants each have the potential to improve cognitive function and attenuate cognitive decline, and, in some cases, they enhance each other. Our current results reveal that low-intensity exercise (mild exercise, ME) and the natural antioxidant carotenoid astaxanthin (AX) each have equivalent beneficial effects on hippocampal neurogenesis and memory function. We found that the enhancement by ME combined with AX in potentiating hippocampus-based plasticity and cognition is mediated by leptin (LEP) made and acting in the hippocampus. In assessing the combined effects upon wild-type (WT) mice undergoing ME with or without an AX diet for four weeks, we found that, when administrated alone, ME and AX separately enhanced neurogenesis and spatial memory, and when combined they were at least additive in their effects. DNA microarray and bioinformatics analyses revealed not only the up-regulation of an antioxidant gene, ABHD3, but also that the up-regulation of LEP gene expression in the hippocampus of WT mice with ME alone is further enhanced by AX. Together, they also increased hippocampal LEP (h-LEP) protein levels and enhanced spatial memory mediated through AKT/STAT3 signaling. AX treatment also has direct action on human neuroblastoma cell lines to increase cell viability associated with increased LEP expression. In LEP-deficient mice (ob/ob), chronic infusion of LEP into the lateral ventricles restored the synergy. Collectively, our findings suggest that not only h-LEP but also exogenous LEP mediates effects of ME on neural functions underlying memory, which is further enhanced by the antioxidant AX.

Usefulness and accuracy of a handheld ultrasound device for epidurssal landmark and depth assessment by anesthesiology residents.

PURPOSE: The aim of this study was to assess the usefulness and accuracy of a handheld ultrasound device (Accuro, Rivanna Medical, Charlottesville, VA, USA) for epidural landmark and depth assessment when epidural anesthesia is performed by residents. METHODS: Patients scheduled to receive epidural anesthesia were randomly assigned to the Accuro group (group A) or control group (group C). In group A, the depth to the epidural space and the appropriate place for epidural insertion according to Accuro was recorded. In group C, epidural anesthesia was performed using a conventional method. The following were recorded and compared between the groups: time from puncture of the Tuohy needle to loss of resistance, number of Tuohy needle redirects, and epidural-related complications. In group A, depth to the epidural space estimated by Accuro (Accuro Depth) and the actual depth measured with a marker on the needle (Needle Depth) were recorded and compared. RESULTS: Sixty patients were enrolled during the study period. There was no significant difference between the groups regarding the median or range of time required to locate the epidural space. The number of Tuohy needle redirects was 0 (0-3) in group A and 1.5 (0-7) in group C (P = 0.012). Accuro Depth was less than Needle Depth [mean difference, 0.85 cm (95% CI-1.10 to - 0.62), SD = 0.62]. CONCLUSIONS: Although there was no significant difference in time from Tuohy needle puncture to loss of resistance, Accuro reduced the number of Tuohy needle redirects and accurately indicated the depth to the epidural space. Accuro may be useful for identifying the needle insertion point and estimating depth to the epidural space when residents perform epidural anesthesia.

Hyaluronan synthesis supports glutamate transporter activity.

Hyaluronan is synthesized, secreted, and anchored by hyaluronan synthases (HAS) at the plasma membrane and comprises the backbone of perineuronal nets around neuronal soma and dendrites. However, the molecular targets of hyaluronan to regulate synaptic transmission in the central nervous system have not been fully identified. Here, we report that hyaluronan is a negative regulator of excitatory signals. At excitatory synapses, glutamate is removed by glutamate transporters to turn off the signal and prevent excitotoxicity. Hyaluronan synthesized by HAS supports the activity of glial glutamate transporter 1 (GLT1). GLT1 also retracted from cellular processes of cultured astrocytes after hyaluronidase treatment and hyaluronan synthesis inhibition. A serial knockout study showed that all three HAS subtypes recruit GLT1 to cellular processes. Furthermore, hyaluronidase treatment activated neurons in a dissociated rat hippocampal culture and caused neuronal damage due to excitotoxicity. Our findings reveal that hyaluronan helps to turn off excitatory signals by supporting glutamate clearance. Cover Image for this issue: doi: 10.1111/jnc.14516.

DS-8500a, an Orally Available G Protein-Coupled Receptor 119 Agonist, Upregulates Glucagon-Like Peptide-1 and Enhances Glucose-Dependent Insulin Secretion and Improves Glucose Homeostasis in Type 2 Diabetic Rats.

G protein-coupled receptor 119 (GPR119) has been shown to be highly expressed in small intestinal L-cells and pancreatic ß-cells and mediates intracellular cAMP concentration, glucagon-like peptide (GLP-1) secretion, and glucose-stimulated insulin secretion (GSIS). This study examined the pharmacological effects of 4-(5-{(1R)-1-[4-(cyclopropylcarbonyl) phenoxy]propyl}-1,2,4-oxadiazol-3-yl)-2-fluoro-N-[(2R)-1-hydroxypropan-2-yl]benzamide (DS-8500a), a novel, orally available, selective GPR119 agonist. In in vitro studies, DS-8500a increased intracellular cAMP in a concentration-dependent manner in human, rat, and mouse GPR119-expressing Chinese hamster ovary (CHO)-K1 cells, with EC50 values of 51.5, 98.4, and 108.1 nmol/l, respectively. DS-8500a had no effect on intracellular cAMP in pcDNA3.1/CHO-K1 cells. In in vivo studies, DS-8500a augmented plasma GLP-1 concentration in Zucker fatty (ZF) rats, and enhanced GSIS and did not change plasma glucose concentration in fasted Sprague-Dawley (SD) rats. A single dose of DS-8500a showed dose-dependent glucose-lowering effects at oral glucose tolerance test (OGTT) in ZF rats. In a repeat-dosing study, DS-8500a had statistically significant glucose-lowering effects at OGTT performed at the 1st day and after 2 weeks of treatment in neonatal streptozotocin-treated (nSTZ) rats, and the efficacy levels of DS-8500a in each test were greater than those of GSK1292263 or MBX-2982, which had been clinically tested previously as GPR119 agonists. Through pharmacokinetics and pharmacodynamics assessment, the high intrinsic activity of DS-8500a was suggested to be one of the reasons for the greater glucose lowering effect in the nSTZ rats. DS-8500a is a useful compound among GPR119 agonists that can maximize the potential benefit of GPR119 in type 2 diabetes.

Effect of Controlled Nucleation of Ice Crystals on the Primary Drying Stage during Lyophilization.

The freezing stage cannot be directly controlled, which leads to variation in product quality and low productivity during the lyophilization process. Our objective was to establish a robust design space for the primary drying stage using ice nucleation control based on the pressurization and depressurization technique. We evaluated the specific surface area (SSA), water content, scanning electron microscopy (SEM) images, and water vapor transfer resistance of the dried layer (Rp) of the products. The ice nucleation control resulted in a reduction of the SSA value and in an increase in water content. SEM observation suggested that the ice nucleation control enabled formation of large ice crystals, which was consistent with the reduction in the Rp value. As a result, the generation of collapsed cakes was inhibited, whereas 18% of the collapsed cakes were observed without ice nucleation control. Finally, this technique succeeded in determining a robust design space for the primary drying stage to produce uniform products of higher productivity. It was considered, from the present findings, that controlling the formation of large ice crystals impacted the product quality and productivity.

Scale-Up Procedure for Primary Drying Process in Lyophilizer by Using the Vial Heat Transfer and the Drying Resistance.

The objective of this study is to design primary drying conditions in a production lyophilizer based on a pilot lyophilizer. Although the shelf temperature and the chamber pressure need to be designed to maintain the sublimation interface temperature of the formulation below the collapse temperature, it is difficult to utilize a production lyophilizer to optimize cycle parameters for manufacturing. In this report, we assumed that the water vapor transfer resistance (Rp) in the pilot lyophilizer can be used in the commercial lyophilizer without any correction, under the condition where both lyophilizers were operated in the high efficiency particulate air (HEPA)-filtrated airflow condition. The shelf temperature and the drying time for the commercial manufacturing were designed based on the maximum Rp value calculated from the pilot lyophilizer (1008 vials) under HEPA-filtrated airflow condition and from the vial heat transfer coefficient of the production lyophilizer (6000 vials). And, the cycle parameters were verified using the production lyophilizer of 60000 vials. It was therefore concluded that the operation of lab- or pilot-scale lyophilizer under HEPA-filtrated airflow condition was one of important factors for the scale-up.

Electromagnetic wave irradiation promotes osteoblastic cell proliferation and up-regulates growth factors via activation of the ERK1/2 and p38 MAPK pathways.

BACKGROUND/AIMS: Periodontitis with bone resorption is caused by inflammatory reactions to bacterial infection. We recently reported that electromagnetic wave irradiation (EMWI) has bactericidal effects. However, the effects of EMWI on periodontal tissues remain unclear. This study was aimed to investigate the effects of EMWI on osteoblasts. METHODS: Osteoblastic cells MC3T3-E1 were treated with EMWI (500-1,000 kHz, 5 times, 1 sec/time). Cell growth and cytotoxicity were determined by cell proliferation assays and measurement of lactate dehydrogenase release, respectively. Gene expression and protein production of growth factors were analyzed using real-time PCR and ELISA, respectively. EMWI-activated cellular signal transduction pathways were investigated by immunoblotting and blocking assay with specific inhibitors. RESULTS: Osteoblasts proliferation was significantly enhanced 3 days after EMWI and no cytotoxicity was observed. EMWI up-regulated various growth factors, such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). EMWI induced ERK1/2, p38 MAPK and SAPK/JNK phosphorylation within 5 min, and the production of PDGF-ΑΑ and VEGF was partially reduced by MAPK-specific inhibitor. CONCLUSION: These findings demonstrated that EMWI increases osteoblastic cell activity and the expression of growth factors via ERK1/2 and p38 MAPK pathways and suggested that EMWI may be beneficial to bone tissue repair such as periodontitis.

Whole-exome sequencing confirmation of a novel heterozygous mutation in RUNX1 in a pregnant woman with platelet disorder.

We describe a successful pregnancy and delivery in a patient with platelet disorder. Prophylactic platelet transfusions ensured that there were no bleeding complications during and after cesarean section. Following delivery, we performed whole exome sequencing, using next generation sequencing, to analyze the DNA samples of the patient and her family, and to identify the disease-causing mutation or variant. To identify de-novo mutations systematically, we also analyzed DNA isolated from the parents of the patient and the neonate. We successfully identified a causative novel mutation c.419 G > A (p.S140N) in RUNX1 in the patient and the neonate. Mutations of RUNX1 have been reported to be associated with familial platelet disorder and with a predisposition for myelodysplasia and/or acute myeloid leukemia. The patient and the neonate require careful long-term hematological follow-up. Identification of mutations by a through whole-exome analysis using next-generation sequencing may be useful in the determination of a long-term follow-up schedule for the patient.

Effect of prone positioning on cannula function and impaired oxygenation during extracorporeal circulation.

Prone ventilation is an effective method for improving oxygenation in patients with acute respiratory failure. However, in extracorporeal circulation, there is a risk of cannula-related complications when changing the position. In this study, we investigated cannula-related complications when changing position for prone ventilation and the effect of prone ventilation on impaired oxygenation in patients who underwent extracorporeal membrane oxygenation (ECMO). The study subjects were patients who underwent prone ventilation during ECMO in the period from 2004 to 2011. Indication for prone ventilation was the presence of dorsal infiltration shown by lung computed tomography. Factors investigated were cannula insertion site, dislodgement or obstruction of the cannula, malfunction of vascular access and unplanned dislodgement of the catheters when changing position. Mean arterial pressure, PaO2/FiO2, PEEP level, blood flow and rotation speed of the pump were also determined before and after position change. Five patients were selected as study subjects. The mean duration of prone positioning was 15.3 ± 0.5 h. Strict management during position changes prevented cannula-related complications in the patients who underwent extracorporeal circulation. There were no significant changes in mean arterial pressure, PEEP level, blood flow and rotation speed of the pump when changing position. Low PaO2/FiO2 prior to prone ventilation was significantly increased after supine to prone and then prone to supine position. Prone positioning to improve impaired oxygenation is a safe procedure and not a contraindication in patients receiving extracorporeal circulation.

The Conventional and Breakthrough Tool for the Study of L-Glutamate Transporters.

In our recent report, we clarified the direct interaction between the excitatory amino acid transporter (EAAT) 1/2 and polyunsaturated fatty acids (PUFAs) by applying electrophysiological and molecular biological techniques to Xenopus oocytes. Xenopus oocytes have a long history of use in the scientific field, but they are still attractive experimental systems for neuropharmacological studies. We will therefore summarize the pharmacological significance, advantages (especially in the study of EAAT2), and experimental techniques that can be applied to Xenopus oocytes; our new findings concerning L-glutamate (L-Glu) transporters and PUFAs; and the significant outcomes of our data. The data obtained from electrophysiological and molecular biological studies of Xenopus oocytes have provided us with further important questions, such as whether or not some PUFAs can modulate EAATs as allosteric modulators and to what extent docosahexaenoic acid (DHA) affects neurotransmission and thereby affects brain functions. Xenopus oocytes have great advantages in the studies about the interactions between molecules and functional proteins, especially in the case when the expression levels of the proteins are small in cell culture systems without transfections. These are also proper to study the mechanisms underlying the interactions. Based on the data collected in Xenopus oocyte experiments, we can proceed to the next step, i.e., the physiological roles of the compounds and their significances. In the case of EAAT2, the effects on the neurotransmission should be examined by electrophysiological approach using acute brain slices. For new drug development, pharmacokinetics pharmacodynamics (PKPD) data and blood brain barrier (BBB) penetration data are also necessary. In order not to miss the promising candidate compounds at the primary stages of drug development, we should reconsider using Xenopus oocytes in the early phase of drug development.

Effect of using hypotension prediction index versus conventional goal-directed haemodynamic management to reduce intraoperative hypotension in non-cardiac surgery: A randomised controlled trial.

STUDY OBJECTIVE: It remains unclear whether it is the hypotension prediction index itself or goal-directed haemodynamic therapy that mitigates intraoperative hypotension. DESIGN: A single centre randomised controlled trial. SETTING: Sapporo Medical University Hospital. PATIENTS: A total of 64 adults patients undergoing major non-cardiac surgery under general anaesthesia. INTERVENTIONS: Patients were randomly assigned to either group receiving conventional goal-directed therapy (FloTrac group) or combination of the hypotension prediction index and conventional goal-directed therapy (HPI group). To investigate the independent utility of the index, the peak rates of arterial pressure and dynamic arterial elastance were not included in the treatment algorithm for the HPI group. MEASUREMENTS: The primary outcome was the time-weighted average of the areas under the threshold. Secondary outcomes were area under the threshold, the number of hypotension events, total duration of hypotension events, mean mean arterial pressure during the hypotension period, number of hypotension events with mean arterial pressure < 50 mmHg, amounts of fluids, blood products, blood loss, and urine output, frequency and amount of vasoactive agents, concentration of haemoglobin during the monitoring period, and 30-day mortality. MAIN RESULTS: The time-weighted average of the area below the threshold was lower in the HPI group than in the control group; 0.19 mmHg (interquartile range, 0.06-0.80 mmHg) vs. 0.66 mmHg (0.28-1.67 mmHg), with a median difference of -0.41 mmHg (95% confidence interval, -0.69 to -0.10 mmHg), p = 0.005. Norepinephrine was administered to 12 (40%) and 5 (17%) patients in the HPI and FloTrac groups, respectively (p = 0.045). No significant differences were observed in the volumes of fluid and blood products between the study groups. CONCLUSIONS: The current randomised controlled trial results suggest that using the hypotension prediction index independently lowered the cumulative amount of intraoperative hypotension during major non-cardiac surgery.

Niflumic acid activates additional currents of the human glial L-glutamate transporter EAAT1 in a substrate-dependent manner.

The astrocytic L-glutamate (L-Glu) transporter EAAT1 participates in the removal of L-Glu from the synaptic cleft and maintenance of non-toxic concentrations in the extracellular fluid. We have shown that niflumic acid (NFA), a non-steroidal anti-inflammatory drug (NSAIDs), alters L-Glu-induced EAAT1 currents in a voltage-dependent manner using the two-electrode voltage clamp technique in Xenopus oocytes expressing EAAT1. In this study, we characterised the effects of NFA on each type of ion-flux through EAAT1. NFA modulated currents induced by both L-Glu and L-aspartate (L-Asp) in a voltage-dependent manner. Ion-substitution experiments revealed that the activation of additional H(+) conductance was involved in the modulation of currents induced by L-Asp and L-Glu, but Cl(-) was involved only with the L-Asp currents. NFA activated additional currents of EAAT1 in a substrate-dependent manner.