1,25-Dihydroxyvitamin D induces the glutamate transporter SLC1A1 and alters glutamate handling in non-transformed mammary cells.

Genomic profiling of immortalized human mammary epithelial (hTERT-HME1) cells identified several metabolic genes, including the membrane glutamate transporter, SLC1A1, as 1,25-dihydroxyvitamin D3 (1,25D) regulated. In these studies we have surveyed the effects of 1,25D on known glutamate transporters and evaluated its impact on cellular glutamate handling. We confirm that expression of SLC1A1 and all of its known transcript variants are significantly upregulated in hTERT-HME1 cells following 1,25D treatment. Expression of the full-length cognate protein, EAAT3, is correspondingly increased in 1,25D treated hTERT-HME1 cells. Under the same conditions, the expression of two other glutamate transporters--SLC1A6 (EAAT4) and SLC1A2 (EAAT2 or GLT-1)--is enhanced by 1,25D while that of SLC1A3 (EAAT1 or GLAST) and SLC7A11 (xCT) is decreased. Glutamate is not essential for growth of hTERT-HME1 cells, and supplemental glutamate (up to 0.5 mM) does not abrogate the growth inhibitory effects of 1,25D. These data suggest that extracellular glutamate is not a major contributor to cellular energy metabolism in hTERT-HME1 cells under basal conditions and that the growth inhibitory effects of 1,25D are not secondary to its effects on glutamate handling. Instead, the effects of 1,25D on glutamate transporters translated to a decrease in cellular glutamate concentration and an increase in media glutamate concentration, suggesting that one or more of these transporters functions to export glutamate in response to 1,25D exposure. The reduced cellular glutamate concentration may also reflect its incorporation into the cellular glutathione (GSH) pool, which is increased upon 1,25D treatment. In support of this concept, the expression of GCLC (which codes for the rate-limiting enzyme in GSH synthesis) and genes which generate reducing equivalents in the form of NADPH (ie, G6PD, PGD, IDH2) are elevated in 1,25D-treated cells. Taken together, these data identify 1,25D as a physiological regulator of multiple membrane glutamate transporters that impacts on overall cellular glutamate handling.

L-Glutamate Enhances Barrier and Antioxidative Functions in Intestinal Porcine Epithelial Cells.

BACKGROUND: L-Glutamate (Glu) is a major amino acid in milk and postweaning diets for mammals (including pigs and human infants). However, effects of Glu on intestinal mucosal barrier and antioxidative functions are unknown. OBJECTIVE: This study tested the hypothesis that Glu may enhance the barrier function of intestinal porcine epithelial cell line 1 (IPEC-1) cells by upregulating the expression of tight junction proteins. METHODS: IPEC-1 cells were cultured with or without Glu in the presence or absence of 1 mmol/L diquat (an oxidant) for indicated time points. Cell numbers, transepithelial electrical resistance (TEER), mRNA, and protein abundance of glutamate transporter, the release of lactate dehydrogenase (LDH), and the abundance of tight junction proteins were determined. RESULTS: Compared with 0 mmol/L Glu, 0.5-, 1-, and 2 mmol/L Glu stimulated (P < 0.05) cell growth by 13-37% at 24 h and 12-34% at 48 h, respectively. In addition, 0.5 mmol/L Glu increased (P < 0.05) TEER (by 58% at 24 h and by 98% at 48 h, respectively). These effects of Glu were associated with increased mRNA abundance of Glu transporter solute carrier family 1 member 1 (SLC1A1) by 30-130% and protein abundance of excitatory amino acid transporter 3 (encoded by SLC1A1) by 19-34%, respectively. In a cell model of oxidative stress induced by 1 mmol/L diquat, 0.5 mmol/L Glu enhanced cell viability, TEER, and membrane integrity (as indicated by the reduced release of LDH) in IPEC-1 cells by increasing the abundance of the tight junction proteins occludin, claudin-3, zonula occludens (ZO)-2, and ZO-3. CONCLUSION: These findings indicate that Glu plays an important role in mucosal barrier function by enhancing cell growth and maintaining membrane integrity in response to oxidative stress.

SPAK and OSR1 Sensitivity of Excitatory Amino Acid Transporter EAAT3.

BACKGROUND/AIMS: Kinases involved in the regulation of epithelial transport include SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1). SPAK and OSR1 are both regulated by WNK (with-no-K(Lys)) kinases. The present study explored whether SPAK and/or OSR1 influence the excitatory amino acid transporter EAAT3, which accomplishes glutamate and aspartate transport in kidney, intestine and brain. METHODS: cRNA encoding EAAT3 was injected into Xenopus laevis oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK, catalytically inactive (D212A)SPAK, wild-type OSR1, constitutively active (T185E)OSR1, WNK insensitive (T185A)OSR1 and catalytically inactive (D164A)OSR1. Glutamate-induced current was taken as measure of electrogenic glutamate transport and was quantified utilizing dual electrode voltage clamp. Furthermore, Ussing chamber was employed to determine glutamate transport in the intestine from gene-targeted mice carrying WNK insensitive SPAK (spak(tg/tg)) and from corresponding wild-type mice (spak(+/+)). RESULTS: EAAT3 activity was significantly decreased by wild-type SPAK and (T233E)SPAK, but not by (T233A)SPAK and (D212A)SPAK. SPAK decreased maximal transport rate without affecting significantly affinity of the carrier. Similarly, EAAT3 activity was significantly downregulated by wild-type OSR1 and (T185E)OSR1, but not by (T185A)OSR1 and (D164A)OSR1. Again OSR1 decreased maximal transport rate without affecting significantly affinity of the carrier. Intestinal electrogenic glutamate transport was significantly lower in spak(+/+) than in spak(tg/tg) mice. CONCLUSION: Both, SPAK and OSR1 are negative regulators of EAAT3 activity.

Effects of dietary supplementation with glutamate and aspartate on diquat-induced oxidative stress in piglets.

This study aimed to investigate the protective effects of dietary glutamate and aspartate supplementations on diquat-induced oxidative stress in piglets. Diquat injection significantly reduced growth performance, including body weight, average daily weight gain, and feed intake (P<0.05). Meanwhile, diquat administration induced oxidative stress evidenced by the decreased serum nitric oxide (NO) and elevated malondialdeyhde (MDA) concentration (P<0.05). Furthermore, diquat-induced oxidative stress disrupted intestinal absorption system and decreased serum threonine, serine, and glycine levels. Dietary supplementation with glutamate improved final body weight, antioxidant system, and expressions of amino acids transporters and enhanced serum glutamate concentration compared with diquat group (P<0.05). While aspartate failed to alleviate diquat-induced oxidative stress, growth depression, and dysfunction of nutrients absorption except for liver relative weight. In conclusion, dietary supplementation with glutamate confers beneficial effects on diquat-induced oxidative stress in piglets, while aspartate exhibits little effects.

Changes in the neuronal glutamate transporter EAAT3 in rat brain after exposure to methamphetamine.

Methamphetamine (METH), an addictive psychostimulant, can induce glutamate release in several brain areas such as cerebral cortex, hippocampus and striatum. Excess glutamate is ordinarily removed from the synaptic cleft by glutamate transporters for maintaining homoeostasis. EAAT3, a subtype of glutamate transporter expressed mainly by neurons, is a major glutamate transporter in the hippocampus and cortex. Therefore, this study examined the effects of acute and sub-acute METH administration on the expression of the EAAT3 in the hippocampal formation, striatum and frontal cortex. Male Sprague-Dawley rats received vehicle injections (i.p.) for 13 days followed by one injection of METH (8 mg/kg, i.p.) on day 14 in acute group. Animals received METH (4 mg/kg, i.p.) or vehicle for 14 days in sub-acute and control groups, respectively. EAAT3 immunoreactivity was determined by western blotting followed by measurement of the integrated optical density. A significant increase in EAAT3 was found in the hippocampal formation after sub-acute, but not acute, METH administration. Conversely, a significant decrease in EAAT3 in striatum was observed in both acute and sub-acute groups. A trend towards a decrease in EAAT3 was also found in frontal cortex in the sub-acute group. Our results of decreased EAAT3 in striatum and frontal cortex suggest deficits of cortico-striatal glutamatergic synapses after METH exposure. Increased EAAT3 expression in the hippocampus may be a compensatory response to possible deficits of glutamatergic neurotransmission induced by METH. Moreover, our findings provide further support for glutamatergic dysfunction with abnormalities involving a transporter important in the regulation of neuronal glutamate.

Phenolic alkaloids from Menispermum dauricum rhizome protect against brain ischemia injury via regulation of GLT-1, EAAC1 and ROS generation.

Menispermum dauricum rhizome has been widely used in China to treat various cardiovascular and thrombosis disorders. Some studies have reported that the phenolic alkaloids of Menispermum dauricum rhizome (PAM) have protective effects against brain ischemia injury, but the mechanism of this action remains to be clarified. In the present study, we investigated the possible mechanisms of action of PAM on experimental brain ischemia injury. Oxygen and glucose deprivation (OGD) in rat primary cortical cultures and middle cerebral artery occlusion in rats were used to mimic ischemia-reperfusion injury, respectively. The results suggested that PAM protected rat primary cortical cultures against OGD-reoxygenation induced cytotoxicity. PAM decreased extracellular glutamate content and markedly prevented the effects induced by OGD on protein level of GLT-1 and EAAC1 glutamate transporters. In addition, it reduced intracellular ROS generation. In vivo, PAM significantly reduced cerebral infarct area and ameliorated neurological functional deficits at different time points. Our findings revealed that the possible mechanism of action of PAM protected against brain ischemia injury involves regulation of GLT-1, EAAC1 and ROS generation.

Critical role of s465 in protein kinase C-increased rat glutamate transporter type 3 activity.

Glutamate transporters, also called excitatory amino acid transporters (EAATs), uptake extracellular glutamate and regulate neurotransmission. Activation of protein kinase C (PKC) increases the activity of EAAT type 3 (EAAT3), the major neuronal EAAT. We designed this study to determine which amino acid residue(s) in EAAT3 may be involved in this PKC effect. Selective potential PKC phosphorylation sites were mutated. These EAAT3 mutants were expressed in the Xenopus oocytes. Phorbol 12-myristate 13-acetate, a PKC activator, significantly increased wild-type EAAT3 activity. Mutation of serine 465 to alanine or aspartic acid, but not the mutation of threonine 5 to alanine, abolished PKC-increased EAAT3 activity. Our results suggest a critical role of serine 465 in the increased EAAT3 activity by PKC activation.

Reduction of corticostriatal glutamatergic fibers in basic fibroblast growth factor deficient mice is associated with hyperactivity and enhanced dopaminergic transmission.

BACKGROUND: Basic fibroblast growth factor (FGF2) plays a crucial role during the development of the cerebral cortex. Mice with a knockout of the FGF2 gene have a reduced number of glutamatergic neurons within the deep layers of the cerebral cortex. METHODS: We used molecular and behavioral analyses to investigate possible alterations in corticostriatal function in FGF2 -/- mice. RESULTS: We found that FGF2 deficiency leads to decreased expression of presynaptic markers of integrity for glutamatergic fibers in the striatum, namely the membrane excitatory amino acid transporter 3 (EAAT3) and the vesicular glutamate transporter 1 (VGLUT1). The reduction of corticostriatal glutamatergic function in FGF2 -/- mice is associated with enhanced locomotor activity in a novel environment and increased responsiveness to dopaminergic drugs, such as cocaine or amphetamine. The behavioral alterations of FGF2 -/- can be normalized by injection of a low dose of the dopaminergic agonist apomorphine (.1 mg/kg) that reduces dopamine release by acting on presynaptic receptors. CONCLUSIONS: Our data demonstrate that FGF2 -/- mice have an increased tone and responsiveness of the dopaminergic system and suggest that these animals might represent a model to study disorders that are characterized by an imbalance between glutamatergic and dopaminergic neurotransmission.

Excitatory amino acid transporters expressed by synovial fibroblasts in rats with collagen-induced arthritis.

Although previous studies have demonstrated increased levels of the brain neurotransmitter glutamate (Glu) in the synovial fluid from patients with arthritis, not much attention has been paid to the possible role of Glu in joint synovial tissues to date. Constitutive expression of mRNA was for the first time shown with glutamate aspartate transporter, glutamate transporter-1 and excitatory amino acid carrier-1 (EAAC1), in addition to with particular ionotropic and metabotropic Glu receptors, in cultured synovial fibroblasts prepared from knee joints of male Lewis rats. Immunohistochemical analysis revealed high localization of immunoreactive EAAC1 at synovial tissues. The accumulation of [3H]Glu occurred in a temperature- and sodium-dependent manner in cultured synovial fibroblasts, with a Km of 23.1+/-1.1 microM and a Vmax of 237.1+/-31.1 pmol/(mg protein min), respectively. In rats with arthritis induced by immunization to type-II collagen, marked increases were seen in hind paw volume, cytokine mRNA expression and Glu levels in synovial tissues, in addition to histological erosion. In cultured synovial fibroblasts prepared from these arthritic rats, [3H]Glu accumulation was drastically increased with biochemical and pharmacological profiles similar to those seen in normal synovial fibroblasts. The exposure to Glu at 500 microM doubled the incorporation of 5-bromo-2'-deoxyuridine in cultured synovial fibroblasts of arthritic but not normal rats, without significantly affecting mRNA expression of different cytokines in both synovial fibroblasts. These results suggest that Glu may at least in part play a role in mechanisms associated with cellular proliferation through particular transporters functionally expressed by synovium in rheumatoid arthritis.