Evidence for a novel glutamate-mediated signaling pathway in keratinocytes.
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Phenotypic alterations in keratinocyte behavior are essential for maintaining epidermal integrity during growth and wound repair and rely on co-ordinated cell signaling events. Numerous growth factors and cytokines have been shown to be instrumental in guiding such changes in keratinocyte activity; here we provide evidence which proposes a novel epidermal signaling pathway mediated by the excitatory amino acid glutamate. Glutamate is the major excitatory neurotransmitter at synaptic junctions within the central nervous system; however, we have identified expression in vivo of several regulatory molecules associated with glutamate signaling in keratinocytes. In resting rat skin epidermis, different classes of glutamate receptors, transporters, and a recently described clustering protein were shown to display distinct distribution patterns, supportive of a multifunctional cellular communication pathway. Immunoreactive N-methyl-D-aspartate-type, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type, and metabotropic-type glutamate receptors were colocalized with the specific glutamate transporter EAAC1 in basal layer keratinocytes, and GLT-1, a related transporter, was expressed suprabasally. In full-thickness rat skin wounds, marked modifications in the distribution of N-methyl-D-aspartate receptors and EAAC1 were observed during re-epithelialization, and alterations in N-methyl-D-aspartate receptor expression accompanied embryonic epidermal development, implicating glutamate signaling in these important biologic events. Furthermore, we provide evidence that these receptors are functional in vitro. These data provide strong evidence to support a role for glutamate in the control of epidermal renewal, and therefore suggest potentially novel therapeutic targets for the treatment of skin disease and enhancement of wound healing. (+info)
Optical detection of synaptically induced glutamate transport in hippocampal slices.
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Although it has long been believed that glial cells play a major role in transmitter uptake at synapses in the CNS, the relative contribution of glial and neuronal cells to reuptake of synaptically released glutamate has been unclear. Recent identification of the diverse glutamate transporter subtypes provides an opportunity to examine this issue. To monitor glutamate transporter activity, we optically detected synaptically induced changes of membrane potential from hippocampal CA1 field in slice preparations using a voltage-sensitive dye, RH155. In the presence of ionotropic glutamate-receptor blockers, synaptic inputs gave rise to a slow depolarizing response (SDR) in the dendritic field. The amplitude of SDR correlated well with presynaptic activities, suggesting that it was related to transmitter release. The SDR was found to be caused by the activities of glutamate transporters because it was not affected by blockers for GABAA, nACh, 5-HT3, P2X, or metabotropic glutamate receptors but was greatly reduced by dihydrokainate (DHK), a specific blocker for GLT-1 transporter, and by D, L-threo-beta-hydroxyaspartate (THA), a blocker for EAAC, GLAST, and GLT-1 transporters. When SDR was detected with RH482 dye, which stains both glial and neuronal cells, 1 mM DHK and 1 mM THA were equally effective in suppressing SDR. The SDR was very small in GLT-1 knockout mice but was maintained in gerbil hippocampi in which postsynaptic neurons were absent because of ischemia. Because GLT-1 transporters are exclusively expressed in astrocytes, our results provide direct evidence that astrocytes play the dominant role in sequestering synaptically released glutamate. (+info)
Identification of truncated human glutamate transporter.
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Excitatory amino-acid carrier 1 (EAAC1) is a high affinity Na+-dependent L-glutamate/D, L-Aspartate transporter protein. A truncated form of EAAC1 (tEAAC1) was identified by reverse transcription and polymerase chain reaction in the human cell line, ACHN, in which EAAC1 mRNA was highly expressed. The deduced amino acid sequence of tEAAC1 lacks 31-77 amino acids including the first extracellular domain. The mRNA encoding tEAAC1 was detected in various cells of human origin but not in cells of rat or mouse origin. The expression of tEAAC1 mRNA was proportional to that of full-length EAAC1 (fEAAC1) mRNA, suggesting common transcriptional regulation between tEAAC1 and fEAAC1. In addition, the expression of EAAC1 mRNA was relatively low or non-existent in non-adherent cells. (+info)
Expression of the GLT-1 subtype of Na+-dependent glutamate transporter: pharmacological characterization and lack of regulation by protein kinase C.
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Several subtypes of Na+-dependent glutamate transporters have been pharmacologically differentiated in brain tissues. Five distinct cDNA clones that express Na+-dependent glutamate transport activity have been isolated. One goal of the current study was to compare the pharmacological properties of the rat GLT-1 subtype of transporter to those identified previously using rat brain tissues. To accomplish this goal, GLT-1 was stably transfected into two different cell lines that express low levels of endogenous transport activity (MCB and L-M (TK-)). Several clones stably transfected with GLT-1 were isolated. In each cell line, Na+-dependent glutamate transport activity was saturable with similar Km values (19 and 37 microM). The pharmacological properties of GLT-1-mediated transport in these cell lines paralleled those observed for the predominant pharmacology observed in cortical crude synaptosomes. These data are consistent with other lines of evidence that suggest that GLT-1 may be sufficient to explain most of the Na+-dependent glutamate transport activity in cortical synaptosomes. Although recent studies using HeLa cells have suggested that GLT-1 can be rapidly up-regulated by activation of protein kinase C (PKC), modulation of PKC or phosphatase activity had no effect on GLT-1-mediated activity in these transfected cell lines. To determine if GLT-1 regulation by PKC is cell-specific, HeLa cells, which endogenously express the EAAC1 subtype of transporter, were stably transfected with GLT-1. Although EAAC1-mediated activity was increased by activation of PKC, we found no evidence for regulation of GLT-1. Despite the present findings, GLT-1 activity may be regulated by PKC under certain conditions. (+info)
Nontransportable inhibitors attenuate reversal of glutamate uptake in synaptosomes following a metabolic insult.
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Na+-dependent, high-affinity glutamate transporters in the central nervous system are generally credited with regulating extracellular levels of L-glutamate and maintaining concentrations below those that would induce excitotoxic injury. Under pathological conditions, however, it has been suggested that these same transporters may contribute to excitotoxic injury by serving as sites of efflux for cellular L-glutamate. In this study, we examine the efflux of [3H]D-aspartate from synaptosomes in response to both alternative substrates (i.e., heteroexchange), such as L-glutamate, and a metabolic insult (5 mM potassium cyanide and 1 mM iodoacetate). Exposure of synaptosomes containing [3H]D-aspartate to either L-glutamate or metabolic inhibitors increased the efflux of the radiolabeled substrate to over 200% of control values. Two previously identified competitive transport inhibitors (L-trans-2, 3-pyrrolidine dicarboxylate and dihydrokainate) failed to stimulate [3H]D-aspartate efflux but did inhibit glutamate-mediated heteroexchange, consistent with the action of nontransportable inhibitors. These compounds also attenuated the efflux of [3H]D-aspartate from synaptosomes exposed to the metabolic inhibitors. These results add further strength to the model of central nervous system injury-induced efflux of L-glutamate through its high-affinity transporters and identify a novel strategy to attenuate this process. (+info)
Structural features of the glutamate transporter family.
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Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large and widespread family of secondary transporters, including bacterial glutamate, serine, and C4-dicarboxylate transporters; mammalian neutral-amino-acid transporters; and an increasing number of bacterial, archaeal, and eukaryotic proteins that have not yet been functionally characterized. Sixty members of the glutamate transporter family were found in the databases on the basis of sequence homology. The amino acid sequences of the carriers have diverged enormously. Homology between the members of the family is most apparent in a stretch of approximately 150 residues in the C-terminal part of the proteins. This region contains four reasonably well-conserved sequence motifs, all of which have been suggested to be part of the translocation pore or substrate binding site. Phylogenetic analysis of the C-terminal stretch revealed the presence of five subfamilies with characterized members: (i) the eukaryotic glutamate transporters, (ii) the bacterial glutamate transporters, (iii) the eukaryotic neutral-amino-acid transporters, (iv) the bacterial C4-dicarboxylate transporters, and (v) the bacterial serine transporters. A number of other subfamilies that do not contain characterized members have been defined. In contrast to their amino acid sequences, the hydropathy profiles of the members of the family are extremely well conserved. Analysis of the hydropathy profiles has suggested that the glutamate transporters have a global structure that is unique among secondary transporters. Experimentally, the unique structure of the transporters was recently confirmed by membrane topology studies. Although there is still controversy about part of the topology, the most likely model predicts the presence of eight membrane-spanning alpha-helices and a loop-pore structure which is unique among secondary transporters but may resemble loop-pores found in ion channels. A second distinctive structural feature is the presence of a highly amphipathic membrane-spanning helix that provides a hydrophilic path through the membrane. Recent data from analysis of site-directed mutants and studies on the mechanism and pharmacology of the transporters are discussed in relation to the structural model. (+info)
Differential expressions of glycine transporter 1 and three glutamate transporter mRNA in the hippocampus of gerbils with transient forebrain ischemia.
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The extracellular concentrations of glutamate and its co-agonist for the N-methyl-d-aspartate (NMDA) receptor, glycine, may be under the control of amino acid transporters in the ischemic brain. However, there is little information on changes in glycine and glutamate transporters in the hippocampal CA1 field of gerbils with transient forebrain ischemia. This study investigated the spatial and temporal expressions of glycine transporter 1 (GLYT1) and three glutamate transporter (excitatory amino acid carrier 1, EAAC1; glutamate/aspartate transporter, GLAST; glutamate transporter 1, GLT1) mRNA in the gerbil hippocampus after 3 minutes of ischemia. The GLYT1 mRNA was transiently upregulated by the second day after ischemia in astrocytelike cells in close vicinity to hippocampal CA1 pyramidal neurons, possibly to reduce glycine concentration in the local extracellular spaces. The EAAC1 mRNA was abundantly expressed in almost all pyramidal neurons and dentate granule cells in the control gerbil hippocampus, whereas the expression level in CA1 pyramidal neurons started to decrease by the fourth day after ischemia in synchrony with degeneration of the CA1 neurons. The GLAST and GLT1 mRNA were rather intensely expressed in the dentate gyrus and CA3 field of the control hippocampus, respectively, but they were weakly expressed in the CA1 field before and after ischemia. As GLAST and GLT1 play a major role in the control of extracellular glutamate concentration, the paucity of these transporters in the CA1 field may account for the vulnerability of CA1 neurons to ischemia, provided that the functional GLAST and GLT1 proteins are also less in the CA1 field than in the CA3 field. This study suggests that the amino acid transporters play pivotal roles in the process of delayed neuronal death in the hippocampal CA1 field. (+info)
Effects of inhibiting glutamine synthetase and blocking glutamate uptake on b-wave generation in the isolated rat retina.
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The purpose of the present experiments was to evaluate the contribution of the glutamate-glutamine cycle in retinal glial (Muller) cells to photoreceptor cell synaptic transmission. Dark-adapted isolated rat retinas were superfused with oxygenated bicarbonate-buffered media. Recordings were made of the b-wave of the electroretinogram as a measure of light-induced photoreceptor to ON-bipolar neuron transmission. L-methionine sulfoximine (1-10 mM) was added to superfusion media to inhibit glutamine synthetase, a Muller cell specific enzyme, by more than 99% within 5-10 min, thereby disrupting the conversion of glutamate to glutamine in the Muller cells. Threo-hydroxyaspartic acid and D-aspartate were used to block glutamate transporters. The amplitude of the b-wave was well maintained for 1-2 h provided 0.25 mM glutamate or 0.25 mM glutamine was included in the media. Without exogenous glutamate or glutamine the amplitude of the b-wave declined by about 70% within 1 h. Inhibition of glutamate transporters led to a rapid (2-5 min) reversible loss of the b-wave in the presence and absence of the amino acids. In contrast, inhibition of glutamine synthetase did not alter significantly either the amplitude of the b-wave in the presence of glutamate or glutamine or the rate of decline of the b-wave found in the absence of these amino acids. Excellent recovery of the b-wave was found when 0.25 mM glutamate was resupplied to L-methionine sulfoximine-treated retinas. The results suggest that in the isolated rat retina uptake of released glutamate into photoreceptors plays a more important role in transmitter recycling than does uptake of glutamate into Muller cells and its subsequent conversion to glutamine. (+info)