Triggering Glutamate Excretion in Corynebacterium glutamicum by Modulating the Membrane State with Local Anesthetics and Osmotic Gradients

Corynebacterium glutamicum can be triggered to excrete glutamate by the addition of local anesthetics, particularly tetracaine. Glutamate efflux is a carrier-mediated process and not due to unspecific membrane permeabilization. The concentration of local anesthetics triggering optimum excretion depended on the type of anesthetic and varied, ranging from 0.1 (chlorpromazine), 1.3 (tetracaine), and 2.6 mM (butacaine) to 15 mM (benzocaine), in close resemblance to the order of efficiency in anesthetic effect. The onset of glutamate excretion was not correlated to a change in the viscosity or fluidity of the membrane, as measured by electron spin resonance spectroscopy, nor was it related to an action of the anesthetic as an uncoupler. Tetracaine-triggered glutamate excretion was sensitive to changes in the transmembrane osmotic gradient, although an osmotic gradient alone could not trigger glutamate excretion. Tetracaine-triggered glutamate efflux was inhibited by an external rise in osmolality and stimulated by a corresponding decrease. The effects of osmotic gradients and the addition of local anesthetics on glutamate excretion were mutually exchangeable, indicating similar modes of action. We suggest that this common principle is a change in the membrane strain. C. glutamicum cells which excrete glutamate without manipulation of the membrane, e.g., biotin-limited cells or glutamate production mutants, were not stimulated by the addition of tetracaine.     

Glutamate excretion as a major kinetic bottleneck for the thermally triggered production of glutamic acid by Corynebacterium glutamicum

The study was aimed at evaluating the extent of flux control exercised by the amino acid excretion step on the glutamate production flux in C. glutamicum 2262 strain that is induced for glutamate excretion by an upward temperature shift. Cells initially induced to excrete glutamate were cultivated at different controlled temperatures between 33 and 40 degrees C, and changes in glutamate excretion flux and intracellular concentration were determined in response to increased culture temperature. The fastest growth rate of 0.45 h(-1) and the lowest glutamate excretion rate of 1 mmole/g dw x h were observed at 33 degrees C, together with a high intracellular 0.5 mmole/g dw glutamate accumulation. On the contrary, the fastest glutamate excretion rate of 6 mmole/g dw x h was obtained at 40 degrees C, when cell growth was arrested and the internal glutamate level reduced to 0.25 mmol/g dw. The observed sixfold increase in excretion flux as a result of the temperature increase clearly suggests a specific effect of temperature on the glutamate export system which appears as the major kinetic bottleneck for the glutamate production flux. This conclusion is corroborated by the high internal accumulation of glutamate which, even under the fastest excretion conditions, severely inhibits the activity of the glutamate biosynthesis pathway.     

Blockade of NMDA-receptors or calcium-channels attenuates the ischaemia-evoked efflux of glutamate and phosphoethanolamine and depression of neuronal activity in rat organotypic hippocampal slice cultures

We have investigated the effects of various insults on extracellular glutamate and phosphoethanolamine levels as well as electrical activity alterations in the early period following these insults in organotypic hippocampal slice cultures. Cultures prepared from 7-day-old rats were maintained in vitro for 7-14 days and then metabolic inhibition was induced: cultures were briefly exposed to potassium cyanide to induce chemical anoxia, 2-deoxyglucose with glucose removal to produce hypoglycaemia, or a combination of both to simulate ischaemia. Chemical anoxia induced a small increase in glutamate and a reversible decrease in evoked field potentials and these were greatly potentiated following simulated ischaemia: high, biphasic glutamate efflux and irreversible field potential abolition as well as increase in phosphoethanolamine levels were observed. We have characterised the effects of treatments using NMDA-receptor antagonists and the L-type calcium channel blocker diltiazem. Anoxia-induced glutamate accumulation was prevented by MK-801 and diltiazem D-AP5. Following simulated ischaemia, diltiazem totally prevented glutamate and phosphoethanolamine accumulations, whereas MK-801 did not block the first phase of glutamate accumulation and D-AP5 prevented none. We demonstrated that glutamate and phosphoethanolamine ischaemic-evoked efflux as well as the recovery of electrical activity in organotypic hippocampal slice cultures are sensitive to both NMDA-receptor and calcium-channel blockade. This model thus represents a useful in vitro system for the study of ischaemic neurodegeneration paralleling results reported using in vivo models.     

The mitochondrial aspartate/glutamate and ADP/ATP carrier switch from obligate counterexchange to unidirectional transport after modification by SH-reagents

The influence of various SH-reagents on the aspartate/glutamate carrier was investigated in the reconstituted system. When liposomes carrying partially purified carrier protein were treated with 5,5'-dithiobis(2-nitrobenzoic acid) or N-ethylmaleimide, antiport activity was strongly reduced. Several mercury compounds exerted a dual effect. They completely blocked the antiport and, in addition, induced an efflux pathway for internal aspartate. The maximum rate of this unidirectional flux was comparable to the original antiport activity. Induction of efflux always was coupled to inhibition of antiport. Efflux was neither due to unspecific leakage of proteoliposomes nor to a possible contamination by porin, but depended on active carrier protein, as elucidated by the sensitivity to proteinases and protein-modifying reagents. Besides efflux of aspartate, HgCl2 and mersalyl also induced a slow efflux of ATP from liposomes carrying coreconstituted aspartate/glutamate and ADP/ATP carrier. The two efflux activities could be discriminated taking advantage of the differential effectiveness of several inhibitors and proteinases. Although basic carrier properties were changed by the applied mercurials (Dierks, T., Salentin, A. and Kr?mer, R. (1990) Biochim. Biophys. Acta 1028, 281), aspartate and ATP efflux could clearly be correlated with the aspartate/glutamate and the ADP/ATP carrier, respectively. When purifying these two translocators the respective efflux activity copurified with the antiporter, thus elucidating that the two different transport functions are mediated by the same protein. These results argue for a participation of the aspartate/glutamate and the ADP/ATP carrier in the generally observed increase of mitochondrial permeability after treatment with SH-reagents.     

Presynaptic facilitation of glutamate release from isolated hippocampal mossy fiber nerve endings by arachidonic acid

Hippocampal mossy fiber synaptosomes were used to investigate the role of arachidonic acid in the release of endogenous glutamate and the long-lasting facilitation of glutamate release associated with long-term potentiation. Exogenous arachidonate induced a dose-dependent efflux of glutamate from the hippocampal mossy fiber synaptosomes and this effect was mimicked by melittin. Neither treatment induced the release of occluded lactate dehydrogenase at the concentrations used in these experiments. In each case, removal of the biochemical stimulus allowed for glutamate efflux to return to spontaneous levels. However, there was a persistent effect of exposure to either arachidonate or melittin, since these compounds facilitated the glutamate release induced by the subsequent addition of 35 mM KCl. This facilitation of glutamate release resulted from an enhancement of both the magnitude and duration of the response to depolarization. Although exogenous prostanoids were also able to stimulate the release of glutamate, they appeared to play no direct role in secretion processes, since inhibition of eicosanoid synthesis potentiated the glutamate efflux in response to membrane depolarization or exogenous arachidonic acid. We suggest that the calcium-dependent accumulation of arachidonic acid in presynaptic membranes plays a central role in the release of endogenous glutamate and that the persistent effects of arachidonic acid may be related to the maintenance of long-term potentiation in the hippocampal mossy fiber-CA3 synapse.     

Evaluation of three techniques used to determine surfactant phytotoxicity

Three techniques were used to measure and compare the phytotoxicities of Triton X-45 and Agral 90 (two organic surfactants) with two organosilicone surfactants (Silwet L-77 and L-7607), and to compare these four non-ionic compounds with a cationic tallow amine surfactant (Hyspray). Total ion efflux and ethylene response methods were used in vivo and in vitro, while a betacyanin efflux method was an in vitro system only. The first two methods, using intact leaves, were considered to be more closely related to normal spraying conditions than the betacyanin efflux test which used explant material. However, the use of intact leaves was thought to bias the results in favour of the leaf penetration properties of the surfactants rather than their phytotoxicities. The in vitro pigment efflux method provided a simple way of ranking the surfactants in order of their potential phytotoxicities, especially with regard to effects on membrane permeability. This ranking, in increasing order of toxicity, was: Silwet L-7607, Silwet L-77, Agral 90, Triton X-45 and Hyspray.     

Effects of Glutamate Antagonists on Methamphetamine and 3,4-Methylenedioxymethamphetamine-Induced Striatal Dopamine Release In Vivo

Abstract: Several amphetamine analogues are reported to increase striatal glutamate efflux in vivo, whereas other data indicate that glutamate is capable of stimulating the efflux of dopamine (DA) in the striatum via a glutamate receptor-dependent mechanism. Based on these findings, it has been proposed that the ability of glutamate receptor-blocking drugs to antagonize the effects of amphetamine may be explained by their capacity to inhibit DA release induced by glutamate. To examine this possibility further, we investigated in vivo the ability of glutamate antagonists to inhibit DA release induced by either methamphetamine (METH) or 3,4-methylenedioxymethamphetamine (MDMA). Both METH and MDMA increased DA efflux in the rat striatum and, in animals killed 1 week later, induced persistent depletions of DA and serotonin in tissue. Pretreatment with MK-801 or CGS 19755 blocked the neurotoxic effects of METH and MDMA but, did not significantly alter striatal DA efflux induced by either stimulant. Infusion of 6-cyano-7-nitroquinoxaline-2,3-dione into the striatum likewise did not alter METH-induced DA overflow, and none of the glutamatergic antagonists affected the basal release of DA when given alone. The findings suggest that the neuroprotective effects of NMDA antagonists do not involve an inhibition of DA release, nor do the data support the proposal that glutamate tonically stimulates striatal DA efflux in vivo. Whether phasic increases in glutamate content might stimulate DA release, however, remains to be determined.     

Amphetamine and D1 Dopamine Receptor Agonists Produce Biphasic Effects on Glutamate Efflux in Rat Ventral Tegmental Area: Modification by Repeated Amphetamine Administration

Abstract: Amphetamine or selective D1 and D2 dopamine receptor agonists and antagonists were administered to the ventral tegmental area (VTA) through a microdialysis probe to determine their effects on glutamate and aspartate efflux in rats pretreated for 5 days with vehicle or 5 mg/kg (+)-amphetamine sulfate. In vehicle rats, glutamate efflux declined during 2 h of perfusion with the D1 receptor agonist SKF-82958 (10 and 100 µ M ). After SKF-82958 perfusion ended, glutamate efflux rebounded to basal levels and continued to increase gradually over the next 2 h. A similar biphasic pattern was observed with intra-VTA amphetamine (10 and 100 µ M ) and with another D1 agonist (100 µ M SKF-38393). The biphasic effects of SKF-82958 were prevented by coperfusion with a D1 antagonist (SCH-23390; 30 µ M ). Glutamate efflux was unaffected by a D2 agonist (100 µ M quinpirole) and by D1 or D2 antagonists administered alone (SCH 23390 and eticlopride; 30 µ M ). In amphetamine-pretreated rats tested 2 days after the last injection, both the decrease during SKF-82958 perfusion and the delayed increase in glutamate efflux were attenuated. In rats tested 12–14 days after the last amphetamine injection, only the decrease during SKF-82958 perfusion was attenuated. None of these drug treatments produced consistent effects on aspartate efflux. We showed previously that systemic amphetamine (5 mg/kg, i.p.) has no immediate effect on VTA glutamate efflux but produces a delayed increase in glutamate efflux that reaches statistical significance 2–3 h after injection. Because behavioral sensitization can be elicited either by repeated systemic or repeated intra-VTA administration, neurochemical effects common to both routes (such as the delayed increase in glutamate efflux) are most likely to contribute to its induction.     

Pore-like and carrier-like properties of the mitochondrial aspartate/glutamate carrier after modification by SH-reagents: evidence for a performed channel as a structural requirement of carrier-mediated transport.

Upon modification of the reconstituted aspartate/glutamate carrier by mercury reagents the antiporter was converted into a unidirectional efflux carrier (Dierks, T., Salentin, A., Heberger, C. and Kr?mer, R. (1990) Biochim. Biophys. Acta 1028, 268). In addition to this basic change in the mechanism, the mercurials, reacting with exofacial cysteines, also affected the internal binding site of the carrier leading to an unmeasurable high Km and to a drastically reduced substrate specificity. The spectrum of efflux substrates comprised small anions from chloride to glutamate, but not cationic amino acids and ATP, hence resembling pore-like properties. However, in the efflux state important carrier properties were also observed. The activation energy (86 kJ/mol) was as high as for the antiport. Furthermore, efflux was inhibited by the presence of external substrate. This trans-inhibition strongly suggests that the external binding site of the carrier, prerequisite in the antiport mechanism, also is involved in conformational transitions during efflux function. However, antiport no longer is catalyzed after switching to the efflux state. Reversion of the induced efflux carrier to the antiport state was achieved using dithioerythritol, thereby further restoring substrate specificity and saturation kinetics. A model for antiport-efflux interconversion is presented suggesting that two reactive cysteines have to be modified in order to uncouple the inward and outward directed component of antiport. The pore-type characteristics of efflux are taken as evidence that a channel-like structure determines the selectivity of unidirectional transport. This intrinsic channel of the protein then is required for substrate translocation also during antiport function.     

Functional asymmetry of the amine transporter from chromaffin granules

The studies presented in this communication describe the existence of a pH-dependent kinetic barrier in amine translocation in membrane vesicles isolated from chromaffin granules from bovine adrenal medulla. This barrier prevents efflux of amines previously accumulated in the membrane vesicles. In these preparations, once the amine is accumulated, there is no further need for ATP. This would suggest a low permeability of the membrane, both to ions and to the amine. In addition, we show that under conditions which thermodynamically favour efflux this is kinetically blocked. This block is due to a rapid protonation of the transporter in the interior of the vesicle. Net efflux can be induced by agents that bring upon an alkalinization of the internal pH such as ammonium salts or nigericin. On the other hand, the rate of exchange with extravesicular substrate is almost identical at the various pH values tested. The physiological implications of this mechanism are discussed.     

Kinetics of glutamate efflux in rat liver mitochondria

The transport of glutamate was studied in isolated rat liver mitochondria preloaded with glutamate in the presence of respiratory inhibitors. Glutamate efflux was initiated by dilution of the loaded mitochondria into a glutamate-free medium, and the rate of transport was measured by following the disappearance of glutamate from the mitochondrial matrix following rapid centrifugation through silicone oil. Glutamate efflux was inhibited extensively by bromcresol purple and partially by N-ethylmaleimide, compounds which are both known to inhibit mitochondrial glutamate uptake. The efflux process was stereospecific for L-glutamate and exhibited an activation energy of 19.2 kcal/mol. The rate of glutamate efflux was not affected by changes in the mitochondrial membrane potential. However, a good correlation was observed between the rate of glutamate efflux and the matrix pH, the efflux rate being stimulated by a decrease in matrix pH in the range from 8.0 to 7.2. In contrast, acidification of the incubation medium in the pH range 7.4 to 6.5 inhibited the rate of glutamate efflux. A kinetic analysis was made of the efflux reaction by a computer curve-fitting procedure which fits the experimental data to an integrated rate equation (Williamson, J.R., and Viale, R.O. (1979) Methods Enzymol. 56, 252-278). The results indicated that a fall in the matrix pH primarily caused a decrease in the K'm for matrix glutamate, with little change in V'max. In contrast, a low external pH had an effect on the V'max but not on the K'm for intramitochondrial glutamate. The results are in agreement with a symmetrical sequential model of glutamate transport where the glutamate anion binds to the protonated carrier.     

Mode of Action of Linenscin OC2 against Listeria innocua

Linenscin OC2 is a small hydrophobic substance produced by the orange cheese coryneform bacterium Brevibacterium linens OC2. Linenscin OC2 inhibits growth of gram-negative bacteria with an altered outer membrane permeability and gram-positive bacteria. It is also able to lyse eucaryotic cells. The mode of action of linenscin OC2 on the Listeria innocua cytoplasmic membrane and the effects of environmental parameters were investigated. Addition of low doses of linenscin OC2 resulted in an immediate perturbation of the permeability properties of the cytoplasmic membrane and of the bacterial energetic state. Linenscin OC2 induced a loss of cytoplasmic potassium, depolarization of the cytoplasmic membrane, complete hydrolysis of internal ATP, efflux of inorganic phosphate, and transient increase in oxygen consumption. Potassium loss occurred in the absence of a proton motive force and was severely reduced at low temperatures, presumably as a result of increased ordering of the lipid hydrocarbon chains of the cytoplasmic membrane. We propose that linenscin OC2 interacts with the cytoplasmic membrane and that the permeability changes observed at low doses reflect the formation of pore-like structures in this membrane.     

Glutamate Induces Glutathione Efflux Mediated by Glutamate/Aspartate Transporter in Retinal Cell Cultures

This study was undertaken in order to characterize the role of the glutamate/aspartate transporter (GLAST) in the glutathione (GSH) efflux induced by glutamate. Our results demonstrated that retinal cell cultures exhibit two mechanisms of GSH release, one Na⁺-independent and other Na⁺-dependent. Glutamate and aspartate induced GSH efflux only in presence of Na⁺. Treatment with PCD (L-trans-Pyrrolidine-2,4-dicarboxylate), a transportable glutamate uptake blocker, increased GSH release indicating that GSH can be carried by glutamate transporters in retinal cell cultures. Added to this, treatment with zinc ion cultures, a recognized inhibitor of GLAST blocked GSH efflux evoked by glutamate. Treatment with NMDA antagonist (MK-801) did not have any effect on the GSH release induced by glutamate. These results suggest that glutamate induces GLAST-mediated release of GSH from retinal cell cultures and this could represent an important mechanism of cellular protection against glutamate toxicity in the CNS.     

Glutathione Efflux Induced by NMDA and Kainate

Neurotoxicity in acute as well as chronic neurological diseases may be partly mediated by oxidative stress caused by overactivation of glutamate receptors. A key component of the cellular defense against oxidative stress is reduced glutathione. In our earlier work, we have shown that ischemia in brain induces increased efflux, elevated metabolism, and decreased tissue concentrations of glutathione. In this study, we have evaluated the effect of glutamate receptor activation on the efflux of glutathione from hippocampus in vitro. NMDA and kainate induced a delayed increase in glutathione, taurine, and phosphoethanolamine efflux. Extracellular glutathione was recovered mainly in the reduced form (85-95%); the efflux was dependent on extracellular calcium but unrelated to dantrolene-sensitive intracellular calcium release and independent of glutathione or NO synthesis. The NMDA-induced efflux of glutathione was enhanced by blockage of gamma-glutamyl transpeptidase, indicating an increased transpeptidation of glutathione after NMDA receptor activation. Our results suggest that increased efflux of glutathione could be a factor in initiating nerve cell death via a change in intracellular redox potential and/or a decrease in the intracellular capacity for inactivation of reactive oxygen species.     

Evidence for an efflux carrier system involved in the secretion of glutamate by Corynebacterium glutamicum

Corynebacterium glutamicum effectively secretes L-glutamate when growing under biotin limitation. The secretion of glutamate was studied with respect to kinetic and energetic parameters: rate of glutamate uptake and efflux, specificity of transport, dependence of efflux on the energy state of the cell, concentration gradient of glutamate and ions, and membrane potential. By comparing these parameters when measured in biotin-limited, i.e. producer cells, and biotin-supplemented, i.e. non-producer cells, respectively, the following conclusions could be drawn: 1. The efflux of L-glutamate in C. glutamicum cannot be explained by passive permeation of this amino acid through the plasma membrane, as it has been assumed in the generally accepted model of glutamate secretion in biotin-limited cells. 2. It is unlikely that the efflux of glutamate occurs via an inversion of the glutamate uptake system. 3. Based on our results concerning the specificity and the kinetics of glutamate transport as well as the observed regulation phenomena, we conclude that secretion of glutamate in C. glutamicum occurs by a special efflux carrier system.Abbreviations dw dry weight - OD optical density - TPP tetraphenyl phosphonium bromide     

Glutamate excretion by Corynebacterium glutamicum: a study of glutamate accumulation during a fermentation course

Summary Corynebacterium glutamicum was used in fed-batch fermentation for glutamate production. Both intracellular and extracellular concentrations were determined which allowed us to study the repartition of the amino acid according to the culture conditions and in the presence or absence of surfactants. A decrease in cell volume was observed after addition of surfactants during the exponential phase of growth; glutamate accumulates in the cell, whereas in standard industrial conditions the glutamate concentration in the medium during the production phase can be 30-fold higher than that found inside the cell. The level of excretion is compatible with industrial production.     

Uptake of glutamate in Corynebacterium glutamicum. 1. Kinetic properties and regulation by internal pH and potassium

The active uptake system for glutamate in Corynebacterium glutamicum is inducible by growth on glutamate as sole energy and carbon source and is also susceptible to catabolite repression by glucose. The basic level of uptake activity is low in glucose-grown cells (1.5 nmol.mg dry mass-1.min-1), it is intermediate when acetate is the carbon source (3.8 nmol.mg dry mass-1.min-1) and becomes fully induced by glutamate (15 nmol.mg dry mass-1.min-1). In all cases the uptake has, except for different Vmax values, identical kinetic and energetic properties, and is characterized by a low apparent Km value of 0.5-1.3 microM and by high substrate specificity. The transported substrate species is the deprotonated form which can also be concluded from the extremely high pH optimum of transport above pH 9. Glutamate uptake in cells grown in media with low K+ concentration is not influenced by external Na+ but is drastically stimulated by addition of K+. Stimulation by K+ could be separated into two different mechanisms. (a) Addition of K+ increases the internal pH, thereby stimulating glutamate uptake which is regulated by the internal pH in C. glutamicum. The apparent pK of the internal 'pH switch' is 6.6; below this value, uptake of glutamate is inhibited. (b) Internal K+ also directly promotes glutamate uptake. Effective uptake of glutamate can be observed only when the cytosolic K+ concentration exceeds a threshold value of about 200 mM. Stimulation of glutamate uptake by external K+ is not due to functional coupling of K+ and glutamate transport but reveals the necessity to replenish the internal K+ pool.     

Functional and energetic characterization of P-gp-mediated doxorubicin transport in rainbow trout (Oncorhynchus mykiss) hepatocytes

An assessment of energetic costs associated with P-glycoprotein (P-gp)-mediated xenobiotic efflux is important in understanding the energy budgets, tradeoffs, and fitness of organisms inhabiting contaminated environments. Here, a functional characterization and determination of the energetic costs associated with doxorubicin (DOX) efflux was examined in isolated hepatocytes of rainbow trout. The accumulation and efflux of DOX were both concentration dependent. The efflux of DOX over a 3 h incubation period resulted in a significant decrease in intracellular ATP concentrations (maximum decrease 25%) compared to control baseline levels, while significant increases in concentrations of ADP (max. 26%), AMP (max. 36%) and inorganic phosphate (max. 11%). were observed. In addition, significant reductions in the adenylate energy charge ([AEC]: max 11%), and phosphorylation potential ([PP]: max. 53%) were shown in cells incubated with DOX compared to control cells. Inhibition of DOX efflux (max. 61%) by the non-competitive P-gp inhibitor tariquidar (XR9576), demonstrated that changes in ATP, ADP, AMP, inorganic phosphate concentrations, AEC and PP in DOX-exposed hepatocytes were mainly due to P-gp activity. Overall, these results indicate that the exposure of trout hepatocytes to DOX increases energetic and metabolic costs that are associated specifically with P-gp efflux activity.     

Aluminum rapidly depolymerizes cortical microtubules and depolarizes the plasma membrane: evidence that these responses are mediated by a glutamate receptor

Efforts to understand how plants respond to aluminum have focused on describing the symptoms of toxicity and elucidating mechanisms of tolerance; however, little is known about the signal transduction steps that initiate the plant's response. Here, we image cortical microtubules and quantify plasma-membrane potential in living, root cells of intact Arabidopsis seedlings. We show that aluminum depolymerizes microtubules and depolarizes the membrane, and that these responses are prevented by calcium channel blockade. Calcium influx might involve glutamate receptors, which in animals are ligand-gated cation channels and are present in the Arabidopsis genome. We show that glutamate depolymerizes microtubules and depolarizes the plasma membrane. These responses, and also the inhibition of root elongation, occur within the first few min of treatment, but are evoked more rapidly by glutamate than by aluminum. Microtubule depolymerization and membrane depolarization, induced by either glutamate or aluminum, are blocked by a specific antagonist of ionotropic glutamate receptors, 2-amino-5-phosphonopentanoate; whereas an antagonist of an aluminum-gated anion channel blocks the two responses to aluminum but not to glutamate. For growth, microtubule integrity, and membrane potential, responses to combined glutamate and aluminum were not greater than to glutamate alone. We propose that signaling in response to aluminum is initiated by efflux of a glutamate-like ligand through an anion channel and the binding of this ligand to a glutamate receptor.