In the present study, the modulatory effect of AMPA receptors on gamma-aminobutyric acid (GABA) transporter current was investigated on enzymatically isolated horizontal cells of carp retina. The GABA transporter current elicited by 1 mmol/L GABA was decreased immediately after pre-application of AMPA (30 mumol/L or 3 mmol/L) for 50 s. Application of 10 mmol/L BAPTA in intracellular solution inhibited the suppression effect of AMPA on GABA transporter current. The suppression effect induced by co-application of 3 mmol/L AMPA and 3 mmol/L NMDA was similar to that of 3 mmol/L AMPA or 3 mmol/L NMDA alone. These results suggest that the activation of AMPA receptors inhibits GABA transporter-mediated current by affecting intracellular Ca(2+) processes in the retinal horizontal cells, which is identical with the modulatory effect of NMDA receptors on GABA transporters.
Animals ; Carps ; Egtazic Acid ; analogs & derivatives ; pharmacology ; GABA Plasma Membrane Transport Proteins ; metabolism ; Receptors, Ionotropic Glutamate ; metabolism ; Retinal Horizontal Cells ; metabolism ; gamma-Aminobutyric Acid ; pharmacology

Glutamate is a kind of excitatory transmitter in the central nervous system of mammals; at the same time, being with excitatory toxicity, its extracellular concentration is mainly modulated by the glutamate transporters. In the case of hypoxic-ischemia, the energy failure would lead to the generation of lactate and free radical, and to the crash of [Na+] gradient, thus exerting influence on the activity and the expression of glutamate transporters. As a result, the glutamate accumulates in the synaptic cleft, activates the glutamate receptors, and causes the death of neurons.
Amino Acid Transport System X-AG ; antagonists & inhibitors ; metabolism ; Animals ; Glutamate Plasma Membrane Transport Proteins ; metabolism ; Glutamic Acid ; metabolism ; Humans ; Hypoxia-Ischemia, Brain ; metabolism ; Synapses ; physiology ; Synaptic Transmission ; physiology

OBJECTIVETo investigate the expressions of gamma aminobutyric acid transporter 1 (GAT-1) and glutamate decarboxylase 65 (GAD65) mRNA in different brain regions at brain propofol uptake equilibrium in dogs.

METHODSEighteen 12- to 18-month-old healthy hybrid dogs were randomized equally into control group (group C), low dose group (group L), and high dose group (group H). In groups L and H, anesthesia was administered by intravenous injection of 5.5 and 7.0 mg/kg propofol followed by propofol infusion at a constant rate of 55 and 70 mg·kg(-1)·h(-1) for 50 min, respectively. Blood samples were taken from the internal carotid artery and jugular vein to measure plasma propofol concentrations, and the brain tissues of the hypothalamus, sub thalamus, dorsal thalamus, hippocampus, pons, parietal lobe and frontal lobe were examined for GAT-1 and GAD65 mRNA expressions using quantitative real-time PCR.

RESULTSIn groups L and H, propofol infusion at a constant rate for 50 min resulted in comparable plasma propofol concentrations between the internal carotid artery and jugular vein (P>0.05), but the concentrations differed significantly between the two groups (P<0.01). GAT-1 mRNA levels in the hypothalamus and hippocampus were significantly higher in groups L and H than in group C (P<0.05 and P<0.01), but comparable between the former two groups. The variations of GAT-1 mRNA levels between the hypothalamus and hippocampus were similar in both group L [(61.26∓7.17)% and (79.34∓39.95)%, P>0.05] and group H [(74.64∓19.63)% and (97.12∓32.31)%, P>0.05]. GAT-1 mRNA levels in other brain regions showed no significant difference among the 3 groups. GAD65 mRNA levels were similar between group L and group H, but both significantly higher than that in group C (P<0.01). GAD65 mRNA in other brain regions had no significant difference among the 3 groups.

CONCLUSIONGAT-1 mRNA in the hypothalamus and hippocampus and GAD65 mRNA in the dorsal thalamus are upregulated when propofol uptake reaches an equilibrium in the brain of dogs.

Animals ; Brain ; drug effects ; metabolism ; Dogs ; GABA Plasma Membrane Transport Proteins ; genetics ; metabolism ; Glutamate Decarboxylase ; genetics ; metabolism ; Hippocampus ; drug effects ; metabolism ; Hypothalamus ; drug effects ; metabolism ; Propofol ; pharmacology ; RNA, Messenger ; genetics ; Thalamus ; drug effects ; metabolism

Repeated exposure to morphine leads to the addiction, which influences its clinical application seriously. The glutamatergic projection from prefrontal cortex (PFC) to the nucleus accumbens (NAc) plays an important role in rewarding effects. It is still unknown whether morphine exposure changes PFC-NAc synaptic transmission. To address this question, in vivo field excitatory postsynaptic potentials (fEPSPs) induced by electric stimulating PFC-NAc projection fibers were recorded to evaluate the effect of acute morphine exposure (10 mg/kg, s.c.) on glutamatergic synaptic transmission in NAc shell of repeated saline/morphine pretreated rats. It was showed that acute morphine exposure enhanced fEPSP amplitude and reduced paired-pulse ratio (PPR) in saline pretreated rats, which could be reversed by following naloxone injection (1 mg/kg, i.p.), an opiate receptor antagonist. However, repeated morphine pretreatment significantly inhibited both the enhancement of fEPSP amplitude and reduction of PPR induced by acute morphine exposure. Those results indicate that the initial morphine exposure enhances PFC-NAc synaptic transmission by pre-synaptic mechanisms, whereas morphine pretreatment occludes this effect.
Animals ; Excitatory Postsynaptic Potentials ; drug effects ; physiology ; Female ; Glutamate Plasma Membrane Transport Proteins ; metabolism ; Glutamates ; metabolism ; Morphine ; administration & dosage ; Morphine Dependence ; physiopathology ; Nucleus Accumbens ; physiopathology ; Prefrontal Cortex ; physiopathology ; Rats ; Rats, Sprague-Dawley

BACKGROUND: Propofol (2, 6-diisopropylphenol) has been known to have neuroprotective effects. Excitatory amino acid transporter 4 (EAAT4) is a glutamate transporter predominantly expressed in the cerebellar Purkinje cells, which is vulnerable to ischemic injury. Thus, we hypothesized that propofol reverses reduced EAAT4 activity which was induced by oxidative stress and investigated the effects of propofol on EAAT4 under oxidative stress induced by tert-butyl hydroperoside (t-BHP). METHODS: EAAT4 was expressed in Xenopus oocytes by injection of its mRNA. By using two-electrode voltage clamping, membrane currents were recorded before, during, and after application of L-aspartate (3 microM) in the presence or absence of t-BHP and propofol. RESULTS: L-aspartate induced an inward current in EAAT4 expressing oocytes. Exposure of these oocytes to t-BHP (1-20 mM) for 10 min dose-dependently decreased EAAT4 activity (1 +/- 0.01 microC for control; 0.88 +/- 0.05 microC for 1 mM; 0.83 +/- 0.03 microC for 2mM; 0.65 +/- 0.04 microC for 3 mM; 0.51 +/- 0.07 microC for 5 mM; 0.45 +/- 0.03 f microC for 10 mM and 0.24 +/- 0.06 microC for 20 mM). IC50 for t-BTH was 6.05 mM and further study was performed with 10 mM t-BTH. Propofol (3-10 microM) dose-dependently reversed this t-BHP-attenuated EAAT4 activity. CONCLUSIONS: Oxidative stress by t-BHP decreased EAAT4 activity and 3-10 microM propofol restored oxidative stress-reduced EAAT4 activity.
Amino Acid Transport System X-AG ; Aspartic Acid ; Constriction ; Excitatory Amino Acid Transporter 4 ; Glutamic Acid ; Inhibitory Concentration 50 ; Membranes ; Neuroprotective Agents ; Oocytes ; Oxidative Stress ; Propofol ; Purkinje Cells ; RNA, Messenger ; Xenopus

To study the relationship between tau hyperphosphorylation and the function of glutamate transporter okadaic acid (OA), a protein phosphatase inhibitor, 20 ng in a 0.5 microl volume, was injected into the frontal cortex of rat brain and immunostaining was used to observe the phosphorylation of tau protein and the expression of excitatory amino acid transporter 1 (EAAT1) in the brain following the injection. The results showed that (1) the neurons in the center of the injection region displayed cytoplasmic shrinkage, swelling, nuclear pyknosis, and dislocation at the early stage, and necrosis appeared 3 d after the injection. However, most neurons in the peri-injected areas showed normal morphological characters with immuno positive reaction for AT8, a tau phosphorylated marker; (2) morphological analysis showed that tau hyperphosphorylation caused by OA treatment was mainly observed in the axons and dendrites of neuronal cells at 6 h in the cell body at 1 d, which brought about dystrophic neurites and neurofibrillary tangle (NFT)-like pathological changes; (3) the induction of glutamate transporter EAAT1 was observed in the involved areas corresponding to that with AT8 immunopositive staining, and the number of EAAT1-positive staining cells markedly increased at 12 h (P<0.01), peaked at 1 d (P<0.001), then decreased at 3 d following the injection. Combined with a confocal laser scanning microscopic analysis, double fluorescent immunostaining showed that EAAT1 positive staining appeared in neurons as well as astrocytes in the peri-injected areas of the frontal cortex. These results demonstrate that OA increases glutamate transporter EAAT1 expression in neurons while it induces tau hyperphosphorylation. However, the mechanism and significance of the induction of glutamate transporter EAAT1 expression remain to be further elucidated.
Animals ; Astrocytes ; drug effects ; metabolism ; Axons ; drug effects ; metabolism ; Brain ; cytology ; Dendrites ; drug effects ; metabolism ; Excitatory Amino Acid Transporter 1 ; metabolism ; Neurofibrillary Tangles ; pathology ; Neurons ; drug effects ; metabolism ; Okadaic Acid ; pharmacology ; Phosphorylation ; Rats ; tau Proteins ; metabolism

OBJECTIVETo investigate and compare the effects of Compound Chaihu Shugan Decoction (CHSGD, "treatment from Gan") and Dingxian Pill (DXP, "treatment from the sputum") on the metabolism path of glutamate in the pentylenetetrazol-kindled seizure rats' hippocampus, thus exploring the molecular mechanism of "heterotherapy for homopathy".

METHODSA chronic kindling seizures rat model was established by intraperitoneal injecting pentylenetetrazol (PTZ). Totally 24 fully kindled seizure rats were randomized into four groups, i.e., the model control group, the Sodium Valproate (VPA) group, the DXP group, and the CHSGD group. They were respectively treated with normal saline, VPA, CHSGD, and DXP, respectively. Rats in the control group were treated with normal saline by peritoneal injection and by gastrogavage. After intragastric administration for 4 successive weeks, the glutamate (Glu) levels in the hippocampus were detected by high performance liquid chromatography (HPLC). The expressions of glutamate transporter-1 (GLT-1) proteins were detected by Western blot. The activity of glutamine synthetase (GS) was detected by using GS detection kit.

RESULTSCompared with the control group, the content of Glu in the model group significantly increased, and the expression of GLT-1 and the activity of GS significantly decreased (P < 0.01). Compared with the model group, the content of Glu in each medication group significantly decreased, and the protein expression of GLT-1 as well as the activity of GS significantly increased (P < 0.01). But when compared between the CHSGD group and the DXP group, the content of Glu was lower and the activity of GS was higher in the CHSGD group than in the DXP group (P < 0.01), while there was no statistical difference in the expression of GLT-1 between the two groups (P > 0.05).

CONCLUSIONSCHSGD ("treatment from Gan") and DXP ("treatment from the sputum") could both decrease the level of Glu and raise the expression of GLT-1 and the activity of GS, indicating that CHSGD and DXP both could regulate the metabolism path of Glu to affect the level of the Glu in the brain. But the effects of CHSGD were superior to those of DXP in decreasing the content of Glu and up-regulating the activity of GS, suggesting that there were some different effects targets between the two compounds on the metabolism path of Glu, which may be one of possible molecular mechanisms for treating epilepsy by heterotherapy for homopathy.

Animals ; Excitatory Amino Acid Transporter 2 ; metabolism ; Glutamic Acid ; metabolism ; Hippocampus ; metabolism ; Kindling, Neurologic ; Male ; Medicine, Chinese Traditional ; methods ; Pentylenetetrazole ; adverse effects ; Rats ; Rats, Wistar ; Seizures ; metabolism ; therapy

OBJECTIVEThe present study was undertaken to design antisense oligodeoxynucleotides (AS-ODNs) of glial glutamate transporter-la (GLT-1a) and to evaluate the effectiveness of the designed AS-ODNs on the expression of GLT-1a.

METHODSFive sequences of GLT-1a AS-ODNs were designed according to the C terminus specific sequences of GLT-1a mRNA using antisense design software of IDT Com- pany. Western blot analysis was used to evaluate the inhibition effects of the five GLT-1a AS-ODNs on the expression of GLT-la.

RESULTSThe sequence of GLT-1a AS-ODNs with sequence of 5'-GGTTCTTCCTCAACACTGCA-3' could specifically inhibit the expression of GLT-1a in the hippocampal CA1 subfield of rats, while it had no effect on the expression of GLT-1b. This sequence showed similar inhibition on the expression of GLT-la in sham and ceftriaxone (Cef)-treated rats. It could also significantly inhibit the cerebral ischemic preconditioning (CIP)-induced up-regulation in the expression of GLT-1a. The magnitude of the inhibition in sham, Cef- or CIP-treated rats was similar by more than 60%.

CONCLUSIONFrom the designed five sequences of GLT-1a AS-ODNs, we obtained an effective sequence which can specifically inhibit the expression of GLT-1a.

Animals ; CA1 Region, Hippocampal ; metabolism ; Excitatory Amino Acid Transporter 2 ; antagonists & inhibitors ; metabolism ; Ischemic Preconditioning ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; Rats ; Up-Regulation

BACKGROUND AND PURPOSE: The progression of migraine into chronic daily headache involves multiple risk factors, but the main contributor is not known. Glutamate is the major excitatory neurotransmitter in central sensitization, which is an important process in the pathogenesis of migraine transformation. The glutamate transporter protein excitatory amino acid transporter 2 (EAAT2) is the primary modulator of glutamatergic neurotransmission, and genetic polymorphisms of its gene, EEAT2, have been identified. The aim of this study was to determine the effect of EAAT2 polymorphisms on migraine transformation into chronic daily headache. METHODS: We included 74 migraine patients with episodic attack (M-E) and 59 migraine patients with chronic daily headache (M-CDH). After amplifying EAAT2 by polymerase chain reaction, we assessed its genotype frequencies based on restriction fragment length polymorphisms. We reclassified all migraine patients into two groups according to their EAAT2 genotype, either with the A allele (n=62) or without it (n=71), and compared the clinical variables between the two groups. RESULTS: The genotype frequencies of EAAT2 polymorphisms did not differ between the M-E and M-CDH groups. Comparison between EEAT2 genotypes revealed that the frequency of analgesic usage was significantly higher among migraine patients with the A allele (12.9+/-1.6 days/month) than in those without the A allele (8.1+/-1.2 days/month; p=0.019). The other clinical variables of migraine did not differ between the two groups. CONCLUSIONS: The results suggest that EEAT2 polymorphism contributes to the tendency toward frequent analgesic usage in migraine patients. This implies a potential genetic influence on the progression of migraine into chronic daily headache through the development of medication-overuse headache.
Alleles ; Amino Acid Transport System X-AG ; Central Nervous System Sensitization ; Excitatory Amino Acid Transporter 2 ; Genotype ; Glutamic Acid ; Headache ; Headache Disorders ; Humans ; Migraine Disorders ; Neurotransmitter Agents ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Risk Factors ; Synaptic Transmission

PURPOSE: The aim of this study was to investigate the differences of expression in glycolysis-related proteins such as Glut-1, carbonic anhydrase (CA) IX, and monocarboxylate transporter (MCT) 4 according to the myoepithelial cell (MEC) and basement membrane (BM) status in solid papillary carcinoma (SPC) of the breast. MATERIALS AND METHODS: Immunohistochemical evaluation of Glut-1, CAIX, and MCT4, as well as p63 and type IV collagen, were performed on 23 SPC cases. RESULTS: Six and nine cases of SPC showed the presence and absence of myoepithelial cells, respectively, and eight cases belonged to the borderline status (p63-positive MEC on some areas of the outer tumor surface but not in others). BM was partially or completely absent in 14 cases and present in nine cases. SPC lacking BM more frequently showed high expression of CAIX than SPC with BM (p=0.037). CONCLUSION: In SPC of the breast, a strong expression of CAIX seems to be associated with an increasing degree of loss of BM, which can be interpreted as BM degradation due to the induction of extracellular acidity with increasing expression of CAIX.
Adult ; Aged ; Basement Membrane/*metabolism ; Breast Neoplasms/*metabolism ; Carcinoma, Papillary/*metabolism ; Excitatory Amino Acid Transporter 2/metabolism ; Female ; Glycolysis ; Humans ; Immunohistochemistry ; Middle Aged ; Monocarboxylic Acid Transporters/metabolism ; Muscle Proteins/metabolism ; Tumor Markers, Biological/*metabolism