Effects of ionic compositions of the medium on monosodium glutamate binding to taste epithelial cells.
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Monosodium glutamate and nucleotides are umami taste substances in animals and have a synergistic effect on each other. We studied the ligand-binding properties of the glutamate receptors in taste epithelial cells isolated from bovine tongue. Specific glutamate binding was observed in an enriched suspension of taste receptor cells in Hanks' balanced salt solution, while no specific glutamate binding was apparent in the absence of divalent ions or when the cells had been depolarized by a high content of potassium in Hanks' balanced salt solution. There was no significant difference between the release of glutamate under depolarized or divalent ion-free conditions and under normal conditions. However, glutamate was easily released from the depolarized cells in the absence of divalent ions. These data suggest that the binding of glutamate to receptors depends on divalent ions, which also have an effect on maintaining binding between glutamate and receptors. (+info)
Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease.
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The mature form of the cathepsin B-like protease of Leishmania major (LmajcatB) is a 243 amino acid protein belonging to the papain family of cysteine proteases and is 54% identical to human-liver cathepsin B. Despite the high identity and structural similarity with cathepsin B, LmajcatB does not readily hydrolyse benzyloxycarbonyl-Arg-Arg-7-amino-4-methyl coumarin (Z-Arg-Arg-AMC), which is cleaved by cathepsin B enzymes. It does, however, hydrolyse Z-Phe-Arg-AMC, a substrate typically cleaved by cathepsin L and B enzymes. Based upon computer generated protein models of LmajcatB and mammalian cathepsin B, it was predicted that this variation in substrate specificity was attributed to Gly234 at the S2 subsite of LmajcatB, which forms a larger, more hydrophobic pocket compared with mammalian cathepsin B. To test this hypothesis, recombinant LmajcatB was expressed in the Pichia pastoris yeast expression system. The quality of the recombinant enzyme was confirmed by kinetic characterization, N-terminal sequencing, and Western blot analysis. Alteration of Gly234 to Glu, which is found at the corresponding site in mammalian cathepsin B, increased recombinant LmajcatB (rLmajcatB) activity toward Z-Arg-Arg-AMC 8-fold over the wild-type recombinant enzyme (kcat/Km=3740+/-413 M-1.s-1 versus 472+/-72.4 M-1.s-1). The results of inhibition assays of rLmajcatB with an inhibitor of cathepsin L enzymes, K11002 (morpholine urea-Phe-homoPhe-vinylsulphonylphenyl, kinact/Ki=208200+/-36000 M-1. s-1), and a cathepsin B specific inhibitor, CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l- prolin e, kinact/Ki=199200+/-32900 M-1.s-1], support the findings that this protozoan protease has the P2 specificity of cathepsin L-like enzymes while retaining structural homology to mammalian cathepsin B. (+info)
Regulation of CBP-mediated transcription by neuronal calcium signaling.
(67/9130)
The transcription factor CREB is involved in mediating many of the long-term effects of activity-dependent plasticity at glutamatergic synapses. Here, we show that activation of NMDA receptors and voltage-sensitive calcium channels leads to CREB-mediated transcription in cortical neurons via a mechanism regulated by CREB-binding protein (CBP). Recruitment of CBP to the promoter is not sufficient for transactivation, but calcium influx can induce CBP-mediated transcription via two distinct transactivation domains. CBP-mediated transcription is stimulus strength-dependent and can be induced by activation of CaM kinase II, CaM kinase IV, and protein kinase A, but not by activation of the Ras-MAP kinase pathway. These observations indicate that CBP can function as a calcium-sensitive transcriptional coactivator that may act as a regulatory switch for glutamate-induced CREB-mediated transcription. (+info)
Glutamate uptake limits synaptic excitation of retinal ganglion cells.
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EPSCs of retinal ganglion cells decay more slowly than do those of most other CNS neurons, in part because of the long time course of glutamate release from bipolar cells. Here we investigated how glutamate clearance and AMPA receptor desensitization affect ganglion cell EPSCs in the salamander retinal slice preparation. Inhibition of glutamate uptake greatly prolonged ganglion cell EPSCs evoked by light or monosynaptic electrical stimuli but had little effect on spontaneous miniature EPSCs (mEPSCs). This suggests that single quanta of glutamate are cleared rapidly by diffusion but multiple quanta can interact to lengthen the postsynaptic response. Some interaction between quanta is likely to occur even when glutamate uptake is not inhibited. This seems to depend on quantal content, because reducing glutamate release with low Ca2+, paired-pulse depression, or weak stimuli shortened the EPSC decay. High quantal content glutamate release may lead to desensitization of postsynaptic receptors. We reduced the extent of AMPA receptor desensitization by holding ganglion cells at positive potentials. This increased the amplitude of the late phase of evoked EPSCs but did not affect the decay rate after the first 50 msec of the response. In contrast, the holding potential had little effect on mEPSC kinetics. Our results suggest that desensitization limits the late phase of AMPA receptor-mediated EPSCs, whereas glutamate uptake controls the duration of both AMPA and NMDA receptor-mediated responses. (+info)
Protection by eliprodil against excitotoxicity in cultured rat retinal ganglion cells.
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PURPOSE: To test whether eliprodil (SL 82.0715), a unique antagonist for the N-methyl-D-aspartate (NMDA) receptor, is protective in the glutamate-induced cytotoxicity model in cultured rat retinal ganglion cells (RGCs). METHODS: Two to four days after a fluorescent dye, Di-I, was injected near the superior colliculi, neonatal rats were killed, and retinal cells were dissociated and cultured. Survival of RGCs after drug treatment was assayed by counting Di-I fluorescent cells. RESULTS: In rat RGCs, glutamate-induced toxicity with a mean EC50 of 10.7 microM. Only 47% of RGCs survived after a 3-day treatment with 100 microM glutamate. Studies using selective agonists and antagonists indicated that the glutamate-induced toxicity was mediated largely by the NMDA receptor. Pretreatment with eliprodil protected against such toxicity. Eliprodil exhibited a mean IC50 of 1.0 nM (log [IC50] = -9.00 +/- 0.01, mean +/- SEM, n = 3; against cell death produced by 100 microM glutamate). At 1 microM, eliprodil was maximally protective; cell survival in the presence of 100 microM glutamate challenge was 100% +/- 5% (n = 3). This protective effect of eliprodil may be related to its reduction (by 78%) of NMDA-induced currents recorded under patch-clamp recording in these cells. CONCLUSIONS: Eliprodil is protective against glutamate cytotoxicity in retinal neurons. It may be a useful novel compound for the treatment of retinopathies including glaucoma in which excitotoxicity has been implicated. (+info)
Propofol prevents peroxide-induced inhibition of glutamate transport in cultured astrocytes.
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BACKGROUND: Glutamate transporters located in the plasma membrane of cerebral astrocytes take up excitatory neurotransmitters from the synaptic cleft. In diseases characterized by oxidative stress, the extracellular glutamate concentration increases and contributes to neuronal death. The authors wanted to determine whether propofol defends brain cells against oxidant-induced changes in their transport of glutamate. METHODS: Primary cultures of rat cerebral astrocytes were exposed to tert-butyl hydroperoxide (1 mM) to serve as an in vitro model of oxidative stress. Astrocytes were incubated with propofol for 2 h and tert-butyl hydroperoxide was added for the final hour. Alternatively, astrocytes were incubated with tert-butyl hydroperoxide for 30 min and then with propofol for another 30 min. Control cells received drug vehicle rather than propofol. The rate of uptake of glutamate, the efflux of the nonmetabolizable analog D-aspartate, and the intracellular concentration of the endogenous antioxidant glutathione were measured. RESULTS: Tert-butyl hydroperoxide decreased the glutathione concentration and inhibited glutamate uptake but had a negligible effect on D-aspartate efflux. At clinically relevant concentrations, propofol did not affect the glutathione concentration but did prevent the effect of tert-butyl hydroperoxide on glutamate transport. Furthermore, the addition of propofol after tert-butyl hydroperoxide reversed the inhibition of glutamate uptake. CONCLUSIONS: Propofol prevents and reverses the inhibition of excitatory amino acid uptake in astrocytes exposed to tert-butyl hydroperoxide. The ability of propofol to defend against peroxide-induced inhibition of glutamate clearance may prevent the pathologic increase in extracellular glutamate at synapses, and thus delay or prevent the onset of excitotoxic neuronal death. (+info)
Sodium-dependent glutamate uptake by an alkaliphilic, thermophilic Bacillus strain, TA2.A1.
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A strain of Bacillus designated TA2.A1, isolated from a thermal spring in Te Aroha, New Zealand, grew optimally at pH 9.2 and 70 degrees C. Bacillus strain TA2.A1 utilized glutamate as a sole carbon and energy source for growth, and sodium chloride (>5 mM) was an obligate requirement for growth. Growth on glutamate was inhibited by monensin and amiloride, both inhibitors that collapse the sodium gradient (DeltapNa) across the cell membrane. N, N-Dicyclohexylcarbodiimide inhibited the growth of Bacillus strain TA2.A1, suggesting that an F1F0-ATPase (H type) was being used to generate cellular ATP needed for anabolic reactions. Vanadate, an inhibitor of V-type ATPases, did not affect the growth of Bacillus strain TA2.A1. Glutamate transport by Bacillus strain TA2.A1 could be driven by an artificial membrane potential (DeltaPsi), but only when sodium was present. In the absence of sodium, the rate of DeltaPsi-driven glutamate uptake was fourfold lower. No glutamate transport was observed in the presence of DeltapNa alone (i.e., no DeltaPsi). Glutamate uptake was specifically inhibited by monensin, and the Km for sodium was 5.6 mM. The Hill plot had a slope of approximately 1, suggesting that sodium binding was noncooperative and that the glutamate transporter had a single binding site for sodium. Glutamate transport was not affected by the protonophore carbonyl cyanide m-chlorophenylhydrazone, suggesting that the transmembrane pH gradient was not required for glutamate transport. The rate of glutamate transport increased with increasing glutamate concentration; the Km for glutamate was 2.90 microM, and the Vmax was 0.7 nmol. min-1 mg of protein. Glutamate transport was specifically inhibited by glutamate analogues. (+info)
Excitatory but not inhibitory synaptic transmission is reduced in lethargic (Cacnb4(lh)) and tottering (Cacna1atg) mouse thalami.
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Excitatory but not inhibitory synaptic transmission is reduced in lethargic (Cacnb4(lh)) and tottering (Cacna1atg) mouse thalami. Recent studies of the homozygous tottering (Cacna1atg) and lethargic mouse (Cacnb4(lh)) models of absence seizures have identified mutations in the genes encoding the alpha1A and beta4 subunits, respectively, of voltage-gated Ca2+ channels (VGCCs). beta subunits normally regulate Ca2+ currents via a direct interaction with alpha1 (pore-forming) subunits of VGCCs, and VGCCs are known to play a significant role in controlling the release of transmitter from presynaptic nerve terminals in the CNS. Because the gene mutation in Cacnb4(lh) homozygotes results in loss of the beta4 subunit's binding site for alpha1 subunits, we hypothesized that synaptic transmission would be altered in the CNS of Cacnb4(lh) homozygotes. We tested this hypothesis by using whole cell recordings of single cells in an in vitro slice preparation to investigate synaptic transmission in one of the critical neuronal populations that generate seizure activity in this strain, the somatosensory thalamus. The primary finding reported here is the observation of a significant decrease in glutamatergic synaptic transmission mediated by both N-methyl-D-aspartate (NMDA) and non-NMDA receptors in somatosensory thalamic neurons of Cacnb4(lh) homozygotes compared with matched, nonepileptic mice. In contrast, there was no significant decrease in GABAergic transmission in Cacnb4(lh) homozygotes nor was there any difference in effects mediated by presynaptic GABAB receptors. We found a similar decrease in glutamatergic but not GABAergic responses in Cacna1atg homozygotes, suggesting that the independent mutations in the two strains each affected P/Q channel function by causing defective neurotransmitter release specific to glutamatergic synapses in the somatosensory thalamus. This may be an important factor underlying the generation of seizures in these models. (+info)