Stimulated Glutamate Uptake Research Articles

Rapid sodium signaling couples glutamate uptake to breakdown of ATP in perivascular astrocyte endfeet.

Perivascular endfeet of astrocytes are highly polarized compartments that ensheath blood vessels and contribute to the blood-brain barrier. They experience calcium transients with neuronal activity, a phenomenon involved in neurovascular coupling. Endfeet also mediate the uptake of glucose from the blood, a process stimulated in active brain regions. Here, we demonstrate in mouse hippocampal tissue slices that endfeet undergo sodium signaling upon stimulation of glutamatergic synaptic activity. Glutamate-induced endfeet sodium transients were diminished by TFB-TBOA, suggesting that they were generated by sodium-dependent glutamate uptake. With local agonist application, they could be restricted to endfeet and immunohistochemical analysis revealed prominent expression of glutamate transporters GLAST and GLT-1 localized towards the neuropil vs. the vascular side of endfeet. Endfeet sodium signals spread at an apparent maximum velocity of ∼120 µm/s and directly propagated from stimulated into neighboring endfeet; this spread was omitted in Cx30/Cx43 double-deficient mice. Sodium transients resulted in elevation of intracellular magnesium, indicating a decrease in intracellular ATP. In summary, our results establish that excitatory synaptic activity and stimulation of glutamate uptake in astrocytes trigger transient sodium increases in perivascular endfeet which rapidly spread through gap junctions into neighboring endfeet and cause a reduction of intracellular ATP. The newly discovered endfeet sodium signaling thereby represents a fast, long-lived and inter-cellularly acting indicator of synaptic activity at the blood-brain barrier, which likely constitutes an important component of neuro-metabolic coupling in the brain. GLIA 2017;65:293-308.

Riluzole increases glutamate uptake by cultured C6 astroglial cells

Riluzole is a drug approved for the treatment of amyotrophic lateral sclerosis (ALS) and may be effective for the treatment of other neurodegenerative and neuropsychiatric disorders. Riluzole exerts diverse actions on the central nervous system, including altering glutamate release and uptake, and therefore act diminishing glutamate extracellular levels, but the underlying mechanism of these actions is still unknown. Here, we demonstrate that riluzole stimulated glutamate uptake and augmented the expression of the glutamate EAAC1 transporter in C6 astroglial cell cultures. The effect of riluzole on glutamate uptake was reduced to below controls when it was co-administered with inhibitors of protein kinase C (PKC; bisindolylmaleimide II), phosphatidylinositol 3-kinase (PI3K; wortmannin) and fibroblast growth factor receptor 1 (FGFR1; PD173074). Riluzole also decreased reactive oxygen species load with no effect on glutathione levels. This study investigates three independent intracellular pathways and the mechanism of action of riluzole on glutamate metabolism.

High-Glucose and S100B Stimulate Glutamate Uptake in C6 Glioma Cells

Diabetes mellitus is a disease associated with several changes in the central nervous system, including oxidative stress and abnormal glutamatergic neurotransmission, and the astrocytes play an essential role in these alterations. In vitro studies of astroglial function have been performed using cultures of primary astrocytes or C6 glioma cells. Herein, we investigated glutamate uptake, glutamine synthetase and S100B secretion in C6 glioma cells cultured in a high-glucose environment, as well as some parameters of oxidative stress and damage. C6 glioma cells, cultured in 12 mM glucose medium, exhibited signals of oxidative and nitrosative stress similar to those found in diabetes mellitus and other models of diabetic disease (decrease in glutathione, elevated NO, DNA damage). Interestingly, we found an increase in glutamate uptake and S100B secretion, and a decrease in glutamine synthetase, which might be linked to the altered glutamatergic communication in diabetes mellitus. Moreover, glutamate uptake in C6 glioma cells, like primary astrocytes, was stimulated by extracellular S100B. Aminoguanidine partially prevented the glial alterations induced by the 12 mM glucose medium. Together, these data emphasize the relevance of astroglia in diabetes mellitus, as well as the importance of glial parameters in the evaluation of diabetic disease progression and treatment.

High glucose stimulates glutamate uptakes in pancreatic β-cells

Pancreatic β-cells are major cells responsible for glucose metabolism in the body. Hyperglycemia is known to be a primary factor in the induction of diabetes mellitus. Glutamate is also an excitatory neurotransmitter in diverse organs. Oxidative stress also plays a pivotal role in the development of diabetes mellitus. However, the effect of hyperglycemia in glutamate uptake in the pancreas is not clear. Furthermore, the relationship between high glucose-induced glutamate uptake and oxidative stress has not been investigated. Therefore, this study was conducted to investigate the effect of high glucose on glutamate uptake in pancreatic β-cells. In the present study, 25 mM glucose stimulated the glutamate uptake in HIT-15 cells of hamster pancreatic β-cells. The treatment of 25 mM glucose and 1 mM glutamate also decreased the cell viability in HIT-15 cells. In addition, the treatment of 25 mM glucose induced an increase of lipid peroxide formation. High glucose-induced increase of LPO formation was prevented by the treatment of antioxidants such as N-acetyl-L-cysteine and quercetin. Furthermore, high glucose-induced stimulation of glutamate uptake and decrease of cell viability were also blocked by the treatment of N-acetyl-L-cysteine and quercetin. In conclusion, high glucose stimulated glutamate uptake via oxidative stress in pancreatic β-cells.

Effects ofα-Asarone on the Glutamate Transporter EAAC1 inXenopusOocytes

The major excitatory neurotransmitter transporter EAAC1 in the mammalian central nervous system is considered a possible target for Chinese herbal medicine. Extracts of Acorus tatarinowii (Schott) were tested for their effects on EAAC1 activity. XENOPUS oocytes with heterologously expressed EAAC1 were used as the model system. Rate of glutamate uptake was determined by means of the isotopic tracer technique. Glutamate-induced current was recorded under a two-electrode voltage clamp. As a highly effective component, alpha-asarone was identified. The rate of glutamate uptake was stimulated by 200 microM of alpha-asarone by about 15 %. In contrast, the same concentration reduced the EAAC1-mediated current by about 35 % at a holding potential of - 60 mV; half maximum inhibition was obtained at about 60 microM. Our experimental data suggest that both stimulation of glutamate uptake and inhibition of EAAC1-mediated current by alpha-asarone could contribute to reduced excitatory activity.

Both B1R and B2R act as intermediate signaling molecules in high glucose‐induced stimulation of glutamate uptake in ARPE cells

Bradykinin (BK) is a potent modulator of biological processes in the retina, and retinal pigment epithelial cells (RPE) and the regulation of glutamate are believed to be important in the pathogenesis of diabetic retinopathy. However, the mechanism by which BK regulates glutamate uptake in RPE cells in diabetic retinopathy is unknown. Here, we examined the involvement of BK receptors in high glucose-induced dysfunction of glutamate uptake in human ARPE cells. High glucose stimulated glutamate uptake and the expression of excitatory amino acid transporter-4 (EAAT4) mRNA, and these were blocked by treatment with small interfering RNA (siRNA) for BK1 receptor (B1R) and BK2 receptor (B2R), but not scrambled siRNA, supporting an involvement of B1R and B2R in this process. High glucose-stimulated glutamate uptake was also blocked by the B1R antagonist [des-Arg(10)]-HOE 140 and the B2R antagonist HOE 140. High glucose increased B1R and B2R mRNA and protein expression in a time-dependent manner, increased B1R and B2R translocation from the cytosol to the nucleus, and stimulated kininogen, kallikrein, and kininase I mRNA expression. We examined whether BK receptors were involved in high glucose-induced signaling pathways. High glucose stimulated arachidonic acid release, cytosolic phospholipase A(2) and cyclooxygenase-2 proteins, nuclear factor-kappaB activation, and inhibitor-kappaB activation; these events were blocked by treatment with B1R and B2R siRNAs, but not scrambled siRNA. In addition, high glucose-induced stimulation of glutamate uptake was blocked by the cyclooxygenase-2 inhibitors arachidonyl trifluoromethyl ketone, mepacrine, 5-bromo-2-(4-fluorophenyl)-3-[4-(methyl-sulfonyl)phenyl]-thiophene, and N-[2-cyclohexyloxy-4-nitrophenyl] methane-sulfonamide, and by the nuclear factor-kappaB inhibitors pyrrolidine dithiocarbamate and SN-50.

Bradykinin stimulates glutamate uptake via both B1R and B2R activation in a human retinal pigment epithelial cells

Aims We were to examine the effect of bradykinin (BK) in the regulation of glutamate transporter and its related signaling molecules in a human retinal pigment epithelial (ARPE) cells, which are important cells to support retina. Main methods d-[2,3- 3H]-aspartate uptake, western immunoblotting, reverse transcription polymerase chain reaction, [ 3H]-arachidonic acid release, and siRNA transfection techniques were used. Key findings BK stimulated glutamate uptake as well as the mRNA expression of excitatory amino acid transporter 4 ( EAAT4) and excitatory amino acid carrier 1 ( EAAC1), which was blocked by treatment with bradykinin 1 receptor ( B1R) and bradykinin 2 receptor ( B2R) siRNA, suggesting the role of B1R and B2R in this process. The BK-induced stimulation of glutamate uptake was also blocked by [des-Arg 10]-HOE 140, a B1R antagonist, and HOE 140, a B2R antagonist, as well as by the tyrosine kinase inhibitors genistein and herbimycin A. In addition, the BK-induced stimulation of glutamate uptake was blocked by treatment with the phospholipase A 2 inhibitors mepacrine and AACOCF 3, the cyclooxygenase (COX) inhibitor indomethacin, and the COX-2 inhibitor Dup 697. Furthermore, the BK-induced increase in COX-2 expression was blocked by the PI-3 kinase inhibitors wortmannin and LY294002, Akt inhibitor, and the protein kinase C (PKC) inhibitors staurosporine and bisindolylmaleimide I, suggesting the role of PI-3 kinase and PKC in this process. BK stimulated Akt activation and the translocation of PKC activation via the activation of B1R and B2R. Significance BK stimulates glutamate uptake through a PKC–Akt–COX-2 signaling cascade in ARPE cells.

The NMDA antagonist MK-801 induces hyperalgesia and increases CSF excitatory amino acids in rats: Reversal by guanosine

Excitatory amino acids (EAAs) and their receptors play a central role in the mechanisms underlying pain transmission. NMDA-receptor antagonists such as MK-801 produce antinociceptive effects against experimental models of chronic pain, but results in acute pain models are conflicting, perhaps due to increased glutamate availability induced by the NMDA-receptor antagonists. Since guanosine and riluzole have recently been shown to stimulate glutamate uptake, the aim of this study was to examine the effects of guanosine or riluzole on changes in nociceptive signaling induced by MK-801 in an acute pain model. Rats received an i.p. injection of vehicle, morphine, guanosine, riluzole or MK-801 or a combined treatment (vehicle, morphine, guanosine or riluzole + MK-801) and were evaluated in the tail flick test, or had a CSF sample drawn after 30 min. Riluzole, guanosine, and MK-801 (0.01 or 0.1 mg/kg) did not affect basal nociceptive responses or CSF EAAs levels. However, MK-801 (0.5 mg/kg) induced hyperalgesia and increased the CSF EAAs levels; both effects were prevented by guanosine, riluzole or morphine. Hyperalgesia was correlated with CSF aspartate and glutamate levels. This study provides additional evidence for the mechanism of action of MK-801, showing that MK-801 induces hyperalgesia with parallel increase in CSF EAAs levels.

Rapid increase of glial glutamate uptake via blockade of the protein kinase A pathway

Glutamate is the main excitatory neurotransmitter in the vertebrate central nervous system. Removal of this transmitter from the synaptic cleft by glial and neuronal transporter systems plays an important role in terminating glutamatergic neurotransmission. The effects of different activators and blockers of PKA and PKC on glutamate uptake were studied in primary glial cells cultivated from the rat cortex using the patch-clamp recording technique and immunocytochemical methods. GF 109203X enhances glutamate-induced membrane currents in a concentration- and time-dependent manner. After pre-application for 40 s the maximal transport capacity was increased by 30-80%. The estimated Km-value of the transport system did not change after drug application and the enhanced glutamate uptake was reversible within a few minutes upon washout. Activators and blockers of the PKC pathway did not affect glutamate uptake, whereas H89, a selective blocker of PKA, mimicked the effects of GF 109203X, indicating involvement of the protein kinase A pathway. The GF 109203X-induced increase in transport capacity is likely to be mediated by GLAST since the GLT-1 selective blocker dihydrokainate was unable to block basal or stimulated glutamate uptake. Furthermore, the increase in transport activity may well be based on an increase in cell surface expression of the transporter protein since preincubation with cytochalasin-B, a protein that blocks actin polymerization, almost completely abolished the effect of GF 109203X and H89. These results indicate that GF 109203X and H89 enhance glial glutamate uptake via blockade of the PKA. The described effect may affect glutamatergic neurotransmission by reducing the glutamate concentration in the synaptic cleft.

Quinolinic Acid-induced Seizures Stimulate Glutamate Uptake into Synaptic Vesicles from Rat Brain: Effects Prevented by Guanine-based Purines

Glutamate uptake into synaptic vesicles is a vital step for glutamatergic neurotransmission. Quinolinic acid (QA) is an endogenous glutamate analog that may be involved in the etiology of epilepsy and is related to disturbances on glutamate release and uptake. Guanine-based purines (GBPs) guanosine 5'-monophosphate (GMP and guanosine) have been shown to exert anticonvulsant effects against QA-induced seizures. The aims of this study were to investigate the effects of in vivo administration of several convulsant agents on glutamate uptake into synaptic vesicles and investigate the role of MK-801, guanosine or GMP (anticonvulsants) on glutamate uptake into synaptic vesicles from rats presenting QA-induced seizures. Animals were treated with vehicle (saline 0.9%), QA 239.2 nmoles, kainate 30 mg/kg, picrotoxin 6 mg/kg, PTZ (pentylenetetrazole) 60 mg/kg, caffeine 150 mg/kg or MES (maximal transcorneal electroshock) 80 mA. All convulsant agents induced seizures in 80-100% of animals, but only QA stimulated glutamate uptake into synaptic vesicle. Guanosine or GMP prevented seizures induced by QA (up to 52% of protection), an effect similar to the NMDA antagonist MK-801 (60% of protection). Both GBPs and MK-801 prevented QA-induced glutamate uptake stimulation. This study provided additional evidence on the role of QA and GBPs on glutamatergic system in rat brain, and point to new perspectives on seizures treatment.

Protein Kinase C-Mediated Modulation of Glutamate Transporter Activity in Rat Retina

It has previously been shown that inhibitors of protein kinase C (PKC) attenuate retinal glutamate uptake in situ. The aim of the current study was to determine whether PKCδ -mediated inhibition differentially reduces the transport of glutamate into retinal Müller cells when compared with retinal neurons. The influence of two different types of PKC inhibitors on the uptake of [3H]D-aspartate was therefore compared in the intact retina, mixed retinal cultures, and Müller cell–enriched retinal cultures. It was found that 25 μM of the pan-isoform PKC inhibitor, chelerythrine, reduced [3H]D-aspartate uptake by 78%, 71%, and 68% in isolated retinas, mixed neuronal/glial cultures, and Müller cell–enriched cultures, respectively. Importantly, 20 μM of the PKCδ -selective inhibitor rottlerin also reduced the uptake of D-aspartate to similar extents in all three systems, and the reductions were statistically similar to those found for the pan-specific PKC inhibitor. Neither pan-isoform nor PKCδ -selective activators stimulated glutamate uptake in either culture system or the intact retina. The current results suggest that specific PKC inhibitors are quantitatively similar in reducing the uptake of glutamate into retinal neurons and Müller cells.

Glutamate uptake is stimulated by extracellular S100B in hippocampal astrocytes.

1.S100B is a calcium-binding protein expressed and secreted by astrocytes, which has been implicated in glial-neuronal communication. Extracellular S100B appears to protect hippocampal neurons against toxic concentrations of glutamate. Here we investigated a possible autocrine role of S100B in glutamate uptake activity. 2. Astrocyte cultures were prepared of hippocampi from neonate Wistar rats. [(3)H] Glutamate uptake was measured after addition of S100B protein, antibody anti-S100B or TRTK-12, a peptide that blocks S100B activity mediated by the C-terminal region. 3.Antibody anti-S100B addition decreased glutamate uptake measured 30 min after medium replacement, without affecting cell integrity or viability. Moreover, low levels of S100B (less than 0.1 ng/mL) stimulated glutamate uptake measured immediately after medium replacement. 4. This finding reinforces the importance of astrocytes in the glutamatergic transmission, particularly the role of S100B neuroprotection against excitotoxic damage.

Anticonvulsant effect of GMP depends on its conversion to guanosine

Studies on the purinergic system normally deal with adenine-based purines, namely, adenine nucleotides and adenosine. However, a guanine-based purinergic system may also have important neuromodulatory roles. Guanine-based purines exert trophic effects on neural cells, protect brain slices in a model of hypoxia and stimulate glutamate uptake. In vivo, both guanosine 5′-monophosphate (GMP) and guanosine (GUO) protected against seizures. In this study, we investigated if the anticonvulsant effect of GMP is mediated by guanosine and if guanosine or GMP treatments were able to increase adenosine levels. Intraperitoneal (i.p.) treatments with 7.5 mg/kg GMP or guanosine prevented 50% of seizures by quinolinic acid (QA) and increased guanosine cerebrospinal fluid (CSF) levels around twofold and threefold, respectively; GMP and adenosine levels remained unchanged. Intracerebroventricular treatment with 960 nmol GMP prevented 80% of seizures and the 5′-nucleotidase inhibitor α-β-methyleneadenosine 5′-diphosphate (AOPCP), when injected 3 min before, reduced this anticonvulsant effect to 30% protection as well as significantly decreased the conversion of GMP into guanosine measured in the CSF. This study shows that the previously reported effect of GMP as an anticonvulsant seems to be related to its ability to generate guanosine through the action of ecto-5′-nucleotidase.

Purification of a neuroprotective component of Parawixia bistriata spider venom that enhances glutamate uptake.

(1) In this study, we examined the effects of crude venom from the spider Parawixia bistriata on glutamate and GABA uptake into synaptosomes prepared from rat cerebral cortex. Addition of venom to cortical synaptosomes stimulated glutamate uptake and inhibited GABA uptake in a concentration-dependent manner. (2) The venom was fractionated using reverse-phase high-performance liquid chromatography on a preparative column. The fraction that retained glutamate uptake-stimulating activity was further purified on a reverse-phase analytical column followed by ion-exchange chromatography. (3) The active fraction, referred to as PbTx1.2.3, stimulated glutamate uptake in synaptosomes without changing the K(M) value, and did not affect GABA uptake. Additional experiments showed that the enhancement of glutamate uptake by PbTx1.2.3 occurs when ionotropic glutamate receptors or voltage-gated sodium and calcium channels are completely inhibited or when GABA receptors and potassium channels are activated, indicating that the compound may have a direct action on the transporters. (4) In an experimental model for glaucoma in which rat retinas are subjected to ischemia followed by reperfusion, PbTx1.2.3 protected neurons from excitotoxic death in both outer and inner nuclear layers, and ganglion cell layers. (5) This active spider venom component may serve as a basis for designing therapeutic drugs that increase glutamate clearance and limit neurodegeneration.

Extracellular conversion of guanine-based purines to guanosine specifically enhances astrocyte glutamate uptake

Guanosine (GUO) has been shown to stimulate glutamate uptake in primary astrocyte cultures. The purpose of this study was to determine the effect and specificity of guanine- or adenine-based purines on glutamate and GABA uptake in cultured astrocytes. Stimulatory effect on glutamate uptake was observed with GUO, GMP or GTP. Simultaneous exposure with these guanine-based purines did not show an additive effect. We also investigated a possible interconversion of guanine-based purines during incubation time. Action by GTP was excluded since the hydrolysis resistant GTP analog, GMP–PNP did not stimulate glutamate uptake. Addition of an ecto-5′-nucleotidase inhibitor abolished GMP-stimulatory effect on glutamate uptake, without affecting GUO action. Taken together, these results suggest that GUO is the guanine-based purines responsible for glutamate uptake activation. In addition, the stimulatory effect on glutamate uptake was not observed with adenine-based purines. Moreover, GABA uptake was not activated by GUO. These results point to specificity in the interaction between GUO and the astrocyte glutamate uptake system.

Glutamate uptake in cultured astrocytes depends on age: a study about the effect of guanosine and the sensitivity to oxidative stress induced by H 2O 2

Relatively few studies have been conducted to investigate the relationship between glutamate and development and/or aging. Rat cortical astrocyte cultures were used as a model to investigate glutamate uptake during development. The immunocontent of the markers glial fibrillary acidic protein (GFAP) and S100B increased, while basal secretion of S100B decreased, in astrocytes from 10 to 40 days in vitro (DIV). Basal glutamate uptake increased with age. Exposure to hydrogen peroxide decreased glutamate uptake more potently at 40 than 10 DIV. Moreover, 40 DIV astrocytes showed earlier loss of integrity (at 6 h) than 10 DIV astrocytes (at 24 h) after H 2O 2 exposure. Addition of guanosine stimulated glutamate uptake only in 10 DIV astrocytes. The present work shows that mature astrocytes in culture present some neurochemical alterations also observed in astrocytes of aged animals. These results can contribute to the understanding of some consequences of the excitotoxicity and oxidative stress during brain aging.

Guanosine enhances glutamate uptake in brain cortical slices at normal and excitotoxic conditions.

1. The effect of guanosine on L-[2,3-3H]glutamate uptake was investigated in brain cortical slices under normal or oxygen-glucose deprivation (OGD) conditions. 2. In slices exposed to physiological conditions, guanosine (1-100 microM) stimulated glutamate uptake (up to 100%) in a concentration-dependent manner when a high (100 microM) but not a low (1 microM) concentration of glutamate was used. 3. In slices submitted to OGD, guanosine 1 and 100 microM also increased 100 microM glutamate uptake (38 and 70%, respectively). 4. The increasing of glutamate and taurine released to the incubation medium in cortical slices submitted to OGD were significantly attenuated by the presence of guanosine in the incubation medium. 5. Guanosine prevented the increase in propidium iodide incorporation into cortical slices induced by OGD, indicating a protective role against ischemic injury. 6. These results support the hypothesis of a protective role for guanosine during brain ischemia, possibly by activating glutamate uptake into neural cells.

Stimulation of glutamate uptake and Na,K-ATPase activity in rat astrocytes exposed to ischemia-like insults.

The postsynaptic actions of glutamate are rapidly terminated by high affinity glutamate uptake into glial cells. In this study we demonstrate the stimulation of both glutamate uptake and Na,K-ATPase activity in rat astrocyte cultures in response to sublethal ischemia-like insults. Primary cultures of neonatal rat cortical astrocytes were subjected to hypoxia, or to serum- and glucose-free medium, or to both conditions (ischemia). Cell death was assessed by propidium iodide staining of cell nuclei. To measure sodium pump activity and glutamate uptake, 3H-glutamate and 86Rb were both simultaneously added to the cell culture in the presence or absence of 2 mM ouabain. Na,K-ATPase activity was defined as ouabain-sensitive 86Rb uptake. Concomitant transient increases (2-3 times above control levels) of both Na,K-ATPase and glutamate transporter activities were observed in astrocytes after 4-24 h of hypoxia, 4 h of glucose deprivation, and 2-4 h of ischemia. A 24 h ischemia caused a profound loss of both activities in parallel with significant cell death. The addition of 5 mM glucose to the cells after 4 h ischemia prevented the loss of both sodium pump activity and glutamate uptake and rescued astrocytes from death observed at the end of 24 h ischemia. Reoxygenation after the 4 h ischemic event caused the selective inhibition of Na,K-ATPase activity. The observed increases in Na,K-ATPase activity and glutamate uptake in cultured astrocytes subjected to sublethal ischemia-like insults may model an important functional response of astrocytes in vivo by which they attempt to maintain ion and glutamate homeostasis under restricted energy and oxygen supply.

Phosphocreatine-dependent Glutamate Uptake by Synaptic Vesicles: A COMPARISON WITH ATP-DEPENDENT GLUTAMATE UPTAKE

ATP-dependent uptake of glutamate into synaptic vesicles has been well documented. Stimulation of glutamate uptake into synaptic vesicles by other high-energy phosphates has not been described. In this paper, we examine the stimulation of phosphocreatine (PCr)-induced glutamate uptake and determine whether this stimulation is secondary to conversion of PCr to ATP. We found the following. 1) PCr stimulates glutamate uptake into synaptic vesicles in the absence of added ATP. 2) At a glutamate concentration of 50 microM, no concentration of added ATP could produce the degree of stimulation seen in the presence of PCr. 3) 0.5 mM iodoacetamide completely inhibits synaptic vesicle creatine kinase activity but does not inhibit PCr-stimulated glutamate uptake. 4) PCr-dependent glutamate uptake, unlike ATP-dependent uptake, is not magnesium- or chloride-dependent. 5) 0.5 mM N-ethylmaleimide, a selective H+-ATPase inhibitor, completely inhibits ATP-dependent glutamate uptake but only slightly inhibits PCr-dependent glutamate uptake. 6) PCr-dependent glutamate uptake is sensitive to valinomycin, a K+/H+ translocator, whereas the ATP-dependent uptake is not. Therefore, it appears that in addition to the well-known ATP-dependent glutamate uptake system, there is a previously unreported PCr-dependent glutamate uptake system in synaptic vesicles. The total glutamate uptake by synaptic vesicles is likely the sum of both ATP- and PCr-dependent glutamate uptake.

Nitric oxide increases cellular glutathione levels in rat lung fibroblasts.

Nitric oxide (NO) has been demonstrated to play a protective role in cell injury. In this study, we have explored the effect of NO and two NO donors (sodium nitroprusside [SNP] and isosorbide dinitrate [ISDN]) on cellular glutathione (GSH) levels in a rat lung fibroblast cell line (RFL6 cells). SNP and ISDN significantly increased cellular GSH in RFL6 cells (5 x 10(-4) M SNP: 21.9 +/- 3.6 nmol/10(6) cells and 5 x 10(-3) M ISDN: 27.6 +/- 1.7 nmol/10(6) cells versus control: 13.2 +/- 0.4 nmol/10(6) cells; P < 0.05). The stimulatory effect of SNP and ISDN on GSH was first seen at 6 h and peaked at 12 to 24 h. A similar increase in GSH was observed in RFL6 cells exposed to 400 ppm NO for 7.5 h (NO: 20.5 +/- 3.4 nmol/10(6) cells versus control: 11.9 +/- 2.4; P < 0.05). SNP and ISDN also increased cellular GSH in bovine pulmonary artery smooth muscle cells (BPSMC) and bovine pulmonary artery endothelial cells (BPAEC). Buthionine sulfoximine (BSO) (0.01 mM), an inhibitor of the GSH synthetic enzyme gamma-glutamyl cysteine synthetase, blocked the increase in GSH in RFL6 cells seen with both SNP and ISDN. In BPAEC, exposure to NO donors for 24 h stimulated glutamate uptake (SNP: 441 +/- 19 pmol/10 min/10(6) cells and ISDN: 677 +/- 48 pmol/10 min/10(6) min/10(6) cells versus control: 222 +/- 9 pmol/10 min/10(6); P < 0.05). This effect paralleled the increase in GSH. In RFL6 cells, only SNP increased glutamate uptake after 24 h of incubation. In summary, NO and NO donors increase cellular GSH in RFL6 cells, BPAEC, and BPSMC. The mechanism of this effect is unclear but may involve upregulation of the normal GSH synthetic pathways. This observation may explain in part the protective effect of NO seen in some cell culture systems and may contribute to a protective effect against oxidant injury in vivo.