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

No abstract available.
Amino Acid Transport System X-AG* ; Anesthetics* ; Glutamic Acid*

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

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

BACKGROUND: Clonidine has been shown to be a potent neuroprotectant by acting at alpha2 receptors on glutamatergic neurons to inhibit the release of glutamate. The aim of this study is to investigate the effects of clonidine on the activity of EAAT3 that can regulate extracellular glutamate. METHODS: EAAT3 was expressed in the Xenopus oocytes. Using a two-electrode voltage clamp, membrane currents were recorded after application of 30 microM L-glutamate both in the presence and absence of various concentrations of clonidine. To determine the effects of clonidine on the Km and Vmax of EAAT3 and the reversibility of clonidine effects, membrane currents were recorded after the application of various concentrations of L-glutamate both in the presence and absence of 1.50 x 10(-7) M clonidine. RESULTS: Clonidine reduced the EAAT3 responses to L-glutamate in a concentration-dependent manner. This inhibition was statistically significant at higher concentrations than at the clinically relevant range. Clonidine at 1.50 x 10(-7) M reduced the Vmax, but did not affect the Km of EAAT3 for L-glutamate. CONCLUSIONS: These results suggest that the direct inhibition of EAAT3 activity is not related to the sedation effect of clonidine and that the clonidine-induced reduction of EAAT3 activity provides additional data for the possible involvement of glutamatergic hyperactivity in the proconvulsant effect of clonidine.
Amino Acid Transport System X-AG ; Animals ; Clonidine ; Glutamic Acid ; Membranes ; Neurons ; Oocytes ; Rats ; Xenopus

Chronic immobilization stress (CIS) induces low levels of glutamate (Glu) and glutamine (Gln) and hypoactive glutamatergic signaling in the mouse prefrontal cortex (PFC), which is closely related to the Glu-Gln cycle. A Gln-supplemented diet ameliorates CIS-induced deleterious changes. Here, we investigated the effects of CIS and Gln supplementation on Glu-Gln cycle-related proteins to characterize the underlying mechanisms. Using the CIS-induced depression mouse model, we examined the expression of 11 proteins involved in the Glu-Gln cycle in the PFC. CIS decreased levels of glutamate transporter 1 (GLT1) and sodium-coupled neutral amino acid transporter (SNAT) 1, SANT2, SNAT3, and SNAT5. Gln supplementation did not affect the non-stressed group but significantly increased GLT1 and SNATs of the stressed group. By immunohistochemical analysis, we confirmed that SNAT1 and SNAT2 were decreased in neurons and GLT1, SNAT3, and SNAT5 were decreased in astrocytes in the medial PFC of the stressed group, but Gln-supplemented diet ameliorated these decrements. Collectively, these results suggest that CIS may cause depressive-like behaviors by decreasing Glu and Gln transportation in the PFC and that a Gln-supplemented diet could prevent the deleterious effects of CIS.
Amino Acid Transport System X-AG ; Amino Acid Transport Systems ; Animals ; Astrocytes ; Depression ; Depressive Disorder ; Diet ; Glutamic Acid ; Glutamine ; Immobilization ; Mice ; Neurons ; Prefrontal Cortex ; Transportation

BACKGROUND: Remifentanil has gained wide clinical acceptance during anesthesia due to its short context-sensitive half time and organ-independent metabolism. However, its mechanism as an anesthetic remains unclear. Glutamate transporters may be important targets for anesthetic action in the central nervous system, and we tested whether remifentanil affected the activity of the primary neuronal glutamate transporter, EAAC1 (excitatory amino acid carrier 1). METHODS: EAAC1 was expressed in Xenopus oocytes by mRNA injection. By using two-electrode voltage clamping, membrane currents were recorded before, during, and after application of L-glutamate (30microM) in the presence or absence of remifentanil. Oocytes were exposed to a protein kinase C (PKC) activator and inhibitor to study the role of PKC on EAAC1 activity. RESULTS: L-Glutamate induced an inward current in EAAC1-expressing oocytes. This response increased in a bell-shaped manner in the presence of 0.1microM to 1 mM remifentanil. Remifentanil significantly increased Vmax (3.1 +/- 0.2microC for controls vs. 4.9 +/- 0.3 microC for remifentanil treatment; n = 12-15; P < 0.05). However, remifentanil did not significantly change Km. Treatment of the oocytes with phorbol-12-myristate-13-acetate (PMA), a PKC activator, caused a significant increase in transporter current (1.00 +/- 0.03 to 1.35 +/- 0.03microC; P < 0.05). Oocytes pretreated with the PKC inhibitor alone (staurosporine) abolished remifentanilenhanced EAAC1 activity. CONCLUSIONS: Our data suggests that remifentanil enhances EAAC1 activity and that PKC is involved in mediating this effect.
Amino Acid Transport System X-AG ; Anesthesia ; Central Nervous System ; Constriction ; Glutamic Acid ; Membranes ; Negotiating ; Neurons ; Oocytes ; Piperidines ; Protein Kinase C ; RNA, Messenger ; Xenopus

Spinocerebellar ataxias (SCAs) are autosomal dominant neurodegenerative disorders which disrupt the afferent and efferent pathways of the cerebellum that cause cerebellar ataxia. Spectrin beta non-erythrocytic 2 (SPTBN2) gene encodes the β-III spectrin protein with high expression in Purkinje cells that is involved in excitatory glutamate signaling through stabilization of the glutamate transporter, and its mutation is known to cause spinocerebellar ataxia type 5. Three years and 5 months old boy with delayed development showed leukodystrophy and cerebellar atrophy in brain magnetic resonance imaging (MRI). Diagnostic exome sequencing revealed that the patient has heterozygous mutation in SPTBN2 (p.Glu1251Gln) which is a causative genetic mutation for spinocerebellar ataxia type 5. With the patient's clinical findings, it seems reasonable to conclude that p.Glu1251Gln mutation of SPTBN2 gene caused spinocerebellar ataxia type 5 in this patient.
Amino Acid Transport System X-AG ; Atrophy ; Brain ; Cerebellar Ataxia ; Cerebellum ; Efferent Pathways ; Exome ; Glutamic Acid ; Humans ; Magnetic Resonance Imaging ; Male ; Neurodegenerative Diseases ; Purkinje Cells ; Spectrin ; Spinocerebellar Ataxias*

OBJECTIVETo investigate the effects of intrathecal injection of ginsenoside Rg1 at different doses on the changes of the behavior and the expressions of excitatory amino-acid transporter 1 (EAAT1), i. e., glutamate-aspartate transporter (GLAST) in the spinal dorsal horn of the arthritis rats with chronic morphine tolerance, and further to explore its mechanisms for morphine tolerance.

METHODSAfter successful intrathecal injection, an adjuvant arthritis model was established in 36 healthy male SD rats. They were randomly divided into 6 groups, 6 in each group. They were intrathecally injected with 10 microL normal saline (Group NS), 10 microg morphine (Group M), 10 microg morphine + 50 microg ginsenoside Rg1 (Group MG50), 10 microg morphine +100 microg ginsenoside Rg1 (Group MG100), 10 microg morphine + 200 microg ginsenoside Rg1 (Group MG200), and 100 microg ginsenoside Rg1 (Group G100), respectively. The normal saline and morphine were intrathecally injected twice daily, while ginsenoside Rg1 at different doses was intrathecally injected once daily, for 7 successive days. Fifty percent mechanical paw withdrawal threshold (PWT) was dynamically detected to evaluate their behaviors. The rats were sacrificed on day 7 after medication. The L3-L5 segment of the spinal cord was isolated for determining the expression of GLAST in the spinal dorsal horn using immunofluorescence staining.

RESULTSThe PWT of Group M was significantly higher than that of Group NS on the 1st and 3rd day after medication (P < 0.05). But it was gradually shortened along with the increasing days of medication. There was no statistical difference between Group M and Group NS on the 7th day (P > 0.05), indicating the formation of morphine tolerance. The PWT of Group MG100 also showed a decreasing tendency, but obviously slower than that of Group M (P < 0.05). The PWT of Group G100 was higher than that of Group NS (P < 0.05). Compared with Group NS, the expression of GLAST in the spinal dorsal horn of rats in Group M was down-regulated (P < 0.01). Compared with Group M, the expression of GLAST in the spinal dorsal horn of rats in Group MG100 and Group G100 was up-regulated (P < 0.05).

CONCLUSIONSSingle application of ginsenoside Rg1 showed mild antinociceptive effect in adjuvant-induced arthritis rats. Intrathecal injection of 100 microg ginsenoside Rg1 could attenuate the formation of morphine tolerance. Its mechanisms might be correlated with up-regulating of the expression of GLAST.

Amino Acid Transport System X-AG ; metabolism ; Animals ; Arthritis, Experimental ; metabolism ; Drug Tolerance ; Ginsenosides ; administration & dosage ; pharmacology ; Injections, Spinal ; Male ; Morphine ; pharmacology ; Pain Measurement ; Rats ; Rats, Sprague-Dawley

The neuronal activity-dependent change in the manner in which light is absorbed or scattered in brain tissue is called the intrinsic optical signal (IOS), and provides label-free, minimally invasive, and high spatial (~100 µm) resolution imaging for visualizing neuronal activity patterns. IOS imaging in isolated brain slices measured at an infrared wavelength (>700 nm) has recently been attributed to the changes in light scattering and transmittance due to aquaporin-4 (AQP4)-dependent astrocytic swelling. The complexity of functional interactions between neurons and astrocytes, however, has prevented the elucidation of the series of molecular mechanisms leading to the generation of IOS. Here, we pharmacologically dissected the IOS in the acutely prepared brain slices of the stratum radiatum of the hippocampus, induced by 1 s/20 Hz electrical stimulation of Schaffer-collateral pathway with simultaneous measurement of the activity of the neuronal population by field potential recordings. We found that 55% of IOSs peak upon stimulation and originate from postsynaptic AMPA and NMDA receptors. The remaining originated from presynaptic action potentials and vesicle fusion. Mechanistically, the elevated extracellular glutamate and K⁺ during synaptic transmission were taken up by astrocytes via a glutamate transporter and quinine-sensitive K2P channel, followed by an influx of water via AQP-4. We also found that the decay of IOS is mediated by the DCPIB- and NPPB-sensitive anion channels in astrocytes. Altogether, our results demonstrate that the functional coupling between synaptic activity and astrocytic transient volume change during excitatory synaptic transmission is the major source of IOS.
Action Potentials ; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid ; Amino Acid Transport System X-AG ; Astrocytes ; Brain ; Electric Stimulation ; Glutamic Acid ; Hippocampus ; Jupiter ; Neurons ; Receptors, N-Methyl-D-Aspartate ; Synaptic Transmission ; Water