Glutamate receptor agonists evoked Ca2+-dependent and Ca2+-independent release of [3H]d-Aspartate from cultured chick retina cells

We studied the release of [³H]d-aspartate evoked by glutamate receptor agonists from monolayer cultures of chick retina cells, and found that activation of the glutamate receptors can evoke both Ca²⁺-dependent and Ca²⁺-independent release of [³H]d-aspartate. In Ca²⁺-free (no added Ca²⁺) Na⁺ medium, the agonists of the glutamate receptors induced the release of [³H]d-aspartate with the following rank order of potency: kainate>α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)∼N-methyl-d-aspartate (NMDA). In media containing 1 mM CaCl₂ the release of [³H]d-aspartate evoked by NMDA, kainate and AMPA was increased by about 112%, 20% and 39%, respectively, as compared to the release evoked by the same agonists in Ca²⁺-free medium. NMDA was the most potent agonist in stimulating the Ca²⁺-dependent release of [³H]d-aspartate, possibly by exocytosis, and AMPA was as potent as kainate. The Ca²⁺-dependent release of [³H]d-aspartate evoked by kainate was dependent on the influx of Ca²⁺ through the receptor associated channel, as well as through the N- (ω-Conotoxin GVIA-sensitive) and L- (nitrendipine-sensitive)type voltage-sensitive Ca²⁺ channels (VSCC). The exocytotic release of [³H]d-aspartate evoked by AMPA relied exclusively on Ca²⁺ entry through the L-type VSCC, whereas the effect of NMDA was partially mediated by the influx of Ca²⁺ through the receptor-associated channel, but not through L- or N-type VSCC. Thus, activation of these different glutamate receptors under physiological conditions is expected to cause the release of cytosolic and vesicular glutamate, and the routes of Ca²⁺ entry modulating vesicular release may be selectively recruited.     

CHARACTERIZATION OF PUTATIVE AMINO ACID TRANSMITTER RELEASE FROM SLICES OF RAT DENTATE GYRUS

Abstract— Superfused slices of the rat dentate gyrus were employed to study the release of GABA, glutamate and aspartate, which are considered strong neurotransmitter candidates in this region. The introduction of Ca²⁺ to a Ca²⁺-free superfusion medium containing a depolarizing agent augmented the efflux of all three amino acids. The response to application of Ca²⁺ nearly always occurred within 30 s, the shortest interval tested in these studies. The efflux rate reached a peak within 90 s and then declined to a level slightly greater than the prestimulation baseline. The failure to maintain the maximal rate with continued exposure to Ca²⁺ and depolarizing influences appeared not to result from a reduction in Ca²⁺ permeability caused by continuous depolarization. Ca²⁺ also stimulated the efflux of exogenously loaded radiolabeled GABA, glutamate and aspartate, but not proline. Exogenously loaded GABA was more readily released than endogenous GABA. Otherwise the effects of various treatments on their efflux rates were qualitatively similar. Mg²⁺ inhibited Ca²⁺-dependent efflux. Ba²⁺, but not Mg²⁺, stimulated amino acid efflux in the absence of Ca²⁺. Extracellular Na⁺ was not required to support Ca²⁺-dependent efflux. Addition of Ca²⁺ to a Ca²⁺-free medium in the absence of a depolarizing agent released GABA from the slices, but not glutamate or aspartate. K⁺-enriched medium and the depolarizing alkaloid, veratridine, stimulated both Ca²⁺-dependent and Ca²⁺-independent release processes. Na⁺-free medium enhanced the Ca²⁺-independent releasing action of elevated K⁺. Ca²⁺-independent release was inhibited by raising the Mg²⁺ concentration by 15 or 30 mM and appeared to be inhibited by Ca²⁺ as well. Amino acid output in the absence of Ca²⁺ is probably not directly related to transmission and is considered to result partially from a general increase in membrane permeability induced by depolarization in a Ca²⁺-free medium and partially from stimulation of carrier-mediated amino acid efflux. These results support previously suggested transmitter roles for GABA, glutamate and aspartate in the rat dentate gyrus.     

The Hydroxyl Side Chain of a Highly Conserved Serine Residue Is Required for Cation Selectivity and Substrate Transport in the Glial Glutamate Transporter GLT-1/SLC1A2

Glutamate transporters maintain synaptic concentration of the excitatory neurotransmitter below neurotoxic levels. Their transport cycle consists of cotransport of glutamate with three sodium ions and one proton, followed by countertransport of potassium. Structural studies proposed that a highly conserved serine located in the binding pocket of the homologous Glt_Ph coordinates l-aspartate as well as the sodium ion Na1. To experimentally validate these findings, we generated and characterized several mutants of the corresponding serine residue, Ser-364, of human glutamate transporter SLC1A2 (solute carrier family 1 member 2), also known as glutamate transporter GLT-1 and excitatory amino acid transporter EAAT2. S364T, S364A, S364C, S364N, and S364D were expressed in HEK cells and Xenopus laevis oocytes to measure radioactive substrate transport and transport currents, respectively. All mutants exhibited similar plasma membrane expression when compared with WT SLC1A2, but substitutions of serine by aspartate or asparagine completely abolished substrate transport. On the other hand, the threonine mutant, which is a more conservative mutation, exhibited similar substrate selectivity, substrate and sodium affinities as WT but a lower selectivity for Na⁺ over Li⁺. S364A and S364C exhibited drastically reduced affinities for each substrate and enhanced selectivity for l-aspartate over d-aspartate and l-glutamate, and lost their selectivity for Na⁺ over Li⁺. Furthermore, we extended the analysis of our experimental observations using molecular dynamics simulations. Altogether, our findings confirm a pivotal role of the serine 364, and more precisely its hydroxyl group, in coupling sodium and substrate fluxes.     

Regulation of the Na+-Dependent Glutamate/Aspartate Transporter in Rodent Cerebellar Astrocytes

The regulation of the Na⁺-dependent glutamate/aspartate transporter system GLAST expressed in rat and mouse cerebellar and cortical astrocytic cultures was examined. Pretreatment of the cerebellar cells with l-glutamate and 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a known Ca²⁺/ diacylglicerol-dependent protein kinase (PKC) activator, produced a decrease in [³H]-d-aspartate uptake. This reduction was dose- and time-dependent and sensitive to PKC inhibitors. Furthermore, the l-glutamate–dependent [³H]-d-aspartate uptake decrease is a non-receptor dependent process, because neither of the agonists or antagonists were effective in mimicking or reverting the effect. Interestingly, transportable substrates could reproduce the l-glutamate effect. In sharp contrast, in cortical astrocytes, both l-glutamate and TPA pre-exposure result in an augmentation of the [³H]-d-aspartate uptake. These findings suggest that the Na⁺-dependent glutamate uptake GLAST undergoes a region-specific regulation.     

GLAST/EAAT1‐induced Glutamine release via SNAT3 in Bergmann glial cells: evidence of a functional and physical coupling

Glutamate, the major excitatory transmitter in the vertebrate brain, is removed from the synaptic cleft by a family of sodium‐dependent glutamate transporters profusely expressed in glial cells. Once internalized, it is metabolized by glutamine synthetase to glutamine and released to the synaptic space through sodium‐dependent neutral amino acid carriers of the N System (SNAT3/slc38a3/SN1, SNAT5/slc38a5/SN2). Glutamine is then taken up by neurons completing the so‐called glutamate/glutamine shuttle. Despite of the fact that this coupling was described decades ago, it is only recently that the biochemical framework of this shuttle has begun to be elucidated. Using the established model of cultured cerebellar Bergmann glia cells, we sought to characterize the functional and physical coupling of glutamate uptake and glutamine release. A time‐dependent Na⁺‐dependent glutamate/aspartate transporter/EAAT1‐induced System N‐mediated glutamine release could be demonstrated. Furthermore, D‐aspartate, a specific glutamate transporter ligand, was capable of enhancing the co‐immunoprecipitation of Na⁺‐dependent glutamate/aspartate transporter and Na⁺‐dependent neutral amino acid transporter 3, whereas glutamine tended to reduce this association. Our results suggest that glial cells surrounding glutamatergic synapses may act as sensors of neuron‐derived glutamate through their contribution to the neurotransmitter turnover.     

Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na⁺ were found to take up Ca²⁺ when incubated in K⁺ media or in sucrose media containing micromolar concentrations of free Ca²⁺. Li⁺- or choline⁺loaded ghosts did not take up Ca²⁺. The Ca²⁺ accumulated by Na⁺-loaded ghosts could be released by the Ca²⁺ ionophore A23187, but not by EGTA. Ca²⁺ uptake was inhibited by external Sr²⁺, Na ⁺, Li ⁺, or choline ⁺. All the ⁴⁵Ca²⁺ accumulated by Na⁺-dependent Ca²⁺ uptake could be released by external Na ⁺, indicating that both Ca²⁺ influx and efflux occur in a Na⁺-dependent manner. Na ⁺ -dependent Ca²⁺ uptake and release were only slightly inhibited by Mg²⁺. In the presence of the Na⁺ ionophore Monensin the Ca²⁺ uptake by Na ⁺-loaded ghosts was reduced. Ca²⁺ sequestered by the Na⁺-dependent mechanism could also be released by external Ca²⁺ or Sr²⁺ but not by Mg²⁺, indicating the presence of a Ca²⁺/Ca²⁺ exchange activity in secretory membrane vesicles. This Ca²⁺/Ca²⁺ exchange system is inhibited by Mg²⁺, but not by Sr²⁺. The Na ⁺ -dependent Ca²⁺ uptake system in the presence of Mg²⁺ is a saturable process with an apparent K_m of 0.28 μM and a V_max= 14.5 nmol min^?1 mg protein^?1. Ruthenium red inhibited neither the Na⁺/Ca²⁺ nor the Ca²⁺/Ca²⁺ exchange, even at high concentrations.     

Skeletal Muscle-specific Calpain Is an Intracellular Na+-dependent Protease

Because intracellular [Na⁺] is kept low by Na⁺/K⁺-ATPase, Na⁺ dependence is generally considered a property of extracellular enzymes. However, we found that p94/calpain 3, a skeletal-muscle-specific member of the Ca²⁺-activated intracellular “modulator proteases” that is responsible for a limb-girdle muscular dystrophy (“calpainopathy”), underwent Na⁺-dependent, but not Cs⁺-dependent, autolysis in the absence of Ca²⁺. Furthermore, Na⁺ and Ca²⁺ complementarily activated autolysis of p94 at physiological concentrations. By blocking Na⁺/K⁺-ATPase, we confirmed intracellular autolysis of p94 in cultured cells. This was further confirmed using inactive p94:C129S knock-in (p94CS-KI) mice as negative controls. Mutagenesis studies showed that much of the p94 molecule contributed to its Na⁺/Ca²⁺-dependent autolysis, which is consistent with the scattered location of calpainopathy-associated mutations, and that a conserved Ca²⁺-binding sequence in the protease acted as a Na⁺ sensor. Proteomic analyses using Cs⁺/Mg²⁺ and p94CS-KI mice as negative controls revealed that Na⁺ and Ca²⁺ direct p94 to proteolyze different substrates. We propose three roles for Na⁺ dependence of p94; 1) to increase sensitivity of p94 to changes in physiological [Ca²⁺], 2) to regulate substrate specificity of p94, and 3) to regulate contribution of p94 as a structural component in muscle cells. Finally, this is the first example of an intracellular Na⁺-dependent enzyme.     

In Vitro Release of Endogenous Amino Acids from Granule Cell-, Stellate Cell-, and Climbing Fiber-Deficient Cerebella

Abstract: The K⁺-stimulated, Ca²⁺-dependent release of glutamate, aspartate, -γ-aminobutyric acid (GABA), alanine, taurine, and glycine was measured in slices of cerebella obtained from control, and granule cell-, granule cell plus stellate cell-, or climbing fiber-deficient cerebella of the rat. The 55 mm -K⁺-stimulated release of glutamate and GABA was 10-fold greater in the presence of Ca²⁺ than in its absence. The stimulated release of aspartate was 4-fold higher when Ca²⁺ was present in the bathing media, while the value for alanine was twice as high as the amount obtained in the absence of Ca²⁺. There was no stimulated release of either taurine or glycine from the cerebellar slices. Increasing the Mg²⁺ concentration to 16 HIM inhibited the K⁺-stimulated, Ca²⁺-dependent release of glutamate, GABA, aspartate, and alanine 85% or more. The K⁺-stimulated, Ca²⁺ dependent release of glutamate, aspartate, and alanine from x-irradiated cerebella deficient in granule cells was reduced to 50–57% of control value. Additional x-irradiation treatment, which further reduced the cerebellar granule cell population and also prevented the acquisition of stellate cells, decreased the release of glutamate by 77%, aspartate by 66%, alanine by 91%, and, in addition, decreased the release of GABA by 55%. The K⁺-stimulated, Ca²⁺-dependent release of glutamate, aspartate, GABA, and alanine was not changed in climbing fiber-deficient cerebella obtained from 3-acetylpyridine-treated rats. The data support a transmitter role for GABA and glutamate in the cerebellum, but do not support a similar function for either taurine or glycine. The data also suggest that alanine and aspartate may be co-released along with glutamate from granule cells.     

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.     

The role of intracellular sodium in the regulation of NMDA-receptor-mediated channel activity and toxicity

Sodium (Na⁺) is the major cation in extracellular space and, with its entry into cells, may act as a critical intracellular second messenger that regulates many cellular functions. Through our investigations of mechanisms underlying the activity-dependent regulation of N-methyl-d-aspartate (NMDA) receptors, we recently characterized intracellular Na⁺ as a possible signaling factor common to processes underlying the upregulation of NMDA receptors by non-NMDA glutamate channels, voltage-gated Na⁺ channels, and remote NMDA receptors. Furthermore, although Ca²⁺ influx during the activation of NMDA receptors acts as a negative feedback mechanism that downregulates NMDA receptor activity, Na⁺ influx provides an essential positive feedback mechanism to overcome Ca²⁺-induced inhibition, thereby potentiating both NMDA receptor activity and inward Ca²⁺ flow. NMDA receptors may be recruited to cause excitoxicity through a Na⁺-dependent mechanism. Therefore, the further characterization of mechanisms underlying the regulation of NMDA receptors by intracellular Na⁺ is essential to understanding activity-dependent neuroplasticity in the nervous system.     

Glutamate release inhibitors: A critical assessment of their action mechanism

Summary A number of important experimental data do not support the widespread hypothesis that Na⁺-channels block is cerebroprotective, essentially because it reduces presynaptic glutamate release: (i) the inhibition of exocytosis by these compounds is not specific to glutamate; (ii) aspartate efflux produced by various stimuli was also reduced, but aspartate cannot be released by exocytosis because it is not concentrated within presynaptic vesicles; and (iii) glutamate accumulated extracellularly during ischaemic or traumatic insult to the CNS is mainly of cytosolic origin. As an alternative, we propose that use-dependent Na⁺-channel blockers enhance the resistance of nerve cells to insults, primarily by decreasing their energy demand, and that reduced efflux of glutamate and other compounds is aconsequence of attenuated cellular stress.     

Effectors ofd-[3H]aspartate release from rat cerebellum

The effect of aminooxyacetic acid (AOAA), NH₄ ⁺, phenylsuccinate (Phs), ketone bodies (KB) and glutamine (Gln), that might interfere with the biosynthesis of neurotransmitter glutamate on the K⁺-evoked Ca²⁺-dependent release ofd-[³H]aspartate from rat cerebellar slices was studied. Therefore slices were preincubated in a Krebs-Ringer-bicarbonate-glucose (KR) buffer, loaded withd-[³H]aspartate and superfused in the presence of Ca²⁺ or when Ca²⁺ was replaced by Mg²⁺ or in some cases by EGTA. AOAA, NH ₄ ⁺ and Phs increase the K⁺-evoked Ca²⁺-dependent release of radioactivity by 30%, 68% and 188% compared to the control respectively indicating that these agents are inhibitors of the K⁺-evoked Ca²⁺-dependent release of glutamate. KB and Gln had no effect on the Ca²⁺-dependent release of radioactivity. AOAA., NH ₄ ⁺ , Phs and KB but not Gln increase the total release of radioactivity by 43%, 69%, 139%, and 37% respectively. AOAA, NH ₄ ⁺ and KB but not Phs or Gln increase the Ca²⁺-independent release (Mg²⁺ replacing Ca²⁺) of radioactivity by 71%, 71% and 108% respectively. The present results indicate that in the cerebellum: 1) Neurotransmitter glutamate is mostly synthesized through the phosphate activated glutaminase (PAG) reaction 2) It is further supported that glutamate released in a Ca²⁺-dependent manner before entering its pool in the cytosol has to move into the mitochondrial matrix.     

The pH-Sensitive Dye Acridine Orange as a Tool to MonitorExocytosis/Endocytosis in Synaptosomes

Abstract : We introduce the use of the pH-sensitive dye acridine orange (AO) to monitor exo/endocytosis of acidic neurotransmitter-containing vesicles in synaptosomes. AO is accumulated exclusively in acidic v-ATPase-dependent bafilomycin (Baf)-sensitive compartments. A fraction of the accumulated AO is rapidly released (fluorescence increase) upon depolarization with KCl in the presence of Ca²⁺. The release (completed in 5-6 s) is followed by reuptake to values below the predepolarization baseline. The reuptake, but not the release, is inhibited by Baf added 5 s prior to KCl. In a similar protocol, Baf does not affect the initial fast phase of glutamate release measured enzymatically, but it abolishes the subsequent slow phase. Thus, the fast AO release corresponds to the rapid phase of glutamate release and the slow phase depends on vesicle cycling. AO reuptake depends in part on the progressive accumulation of acid-loaded vesicles during cycling. Stopping exocytosis at selected times after KCl by Ca²⁺ removal with EGTA evidences endocytosis : Its T_1/2 was 12 ± 0.6 s. The K_A⁺, channel inhibitors 4-aminopyridine (100 μM) and α-dendrotoxin (10-100 nM) are known to induce glutamate release by inducing the firing of Na⁺ channels ; their action is potentiated by the activation of protein kinase C. Also these agents promote a Ca²⁺-dependent AO release, which is prevented by the Na⁺ channel inhibitor tetrodotoxin and potentiated by 4β-phorbol 12-myristate 13-acetate (PMA). With α-dendrotoxin, endocytosis was monitored by stopping exocytosis at selected times with EGTA or alternatively with Cd²⁺ or tetrodotoxin. The T_1/2 of endocytosis, which was unaffected by PMA, was 12 ± 0.4 s with EGTA and Cd²⁺ and 9.5 ± 0.5 s with tetrodotoxin. Protein kinase C activation appeared to facilitate vesicle turnover.     

Functional Characterization of a Na+-dependent Aspartate Transporter from Pyrococcus horikoshii

Excitatory amino acid transporters (EAATs) are crucial in maintaining extracellular levels of glutamate, the most abundant excitatory neurotransmitter, below toxic levels. The recent three-dimensional crystal structure of Glt_Ph, an archaeal homolog of the EAATs, provides elegant structural details of this family of proteins, yet we know little about the mechanism of the bacterial transporter. Conflicting reports in the literature have described Glt_Ph as an aspartate transporter driven by Na⁺ or a glutamate transporter driven by either Na⁺ or H⁺. Here we use purified protein reconstituted into liposomes to thoroughly characterize the ion and substrate dependence of the Glt_Ph transport. We confirm that Glt_Ph is a Na⁺-dependent transporter that is highly selective for aspartate over other amino acids, and we show that transport is coupled to at least two Na⁺ ions. In contrast to the EAATs, transport via Glt_Ph is independent of H⁺ and K⁺. We propose a kinetic model of transport in which at least two Na⁺ ions are coupled to the cotransport of each aspartate molecule by Glt_Ph, and where an ion- and substrate-free transporter reorients to complete the transport cycle.     

Co-expression of vesicular glutamate transporters (VGLUT1 and VGLUT2) and their association with synaptic-like microvesicles in rat pinealocytes

A vesicular glutamate transporter (VGLUT) is responsible for the accumulation of l-glutamate in synaptic vesicles in glutamatergic neurons. Two isoforms, VGLUT1 and VGLUT2, have been identified, which are complementarily expressed in these neurons. Mammalian pinealocytes, endocrine cells for melatonin, are also glutamatergic in nature, accumulate l-glutamate in synaptic-like microvesicles (SLMVs), and secrete it through exocytosis. Although the storage of l-glutamate in SLMVs is mediated through a VGLUT, the molecular nature of the transporter is less understood. We recently observed that VGLUT2 is expressed in pinealocytes. In the present study, we show that pinealocytes also express VGLUT1. RT-PCR and northern blot analyses indicated expression of the VGLUT1 gene in pineal gland. Western blotting with specific antibodies against VGLUT1 indicated the presence of VGLUT1 in pineal gland. Indirect immunofluorescence microscopy with a section of pineal gland and cultured cells indicated that VGLUT1 and VGLUT2 are co-localized with process terminal regions of pinealocytes. Furthermore, immunoelectronmicroscopy as well as subcellular fractionation studies revealed that both VGLUT1 and VGLUT2 are specifically associated with SLMVs. These results indicate that both VGLUTs are responsible for storage of l-glutamate in SLMVs in pinealocytes. Pinealocytes are the first exception as to complementary expression of VGLUT1 and VGLUT2.     

Analysis of a Vesicular Glutamate Transporter (VGLUT2) Supports a Cell-leakage Mode in Addition to Vesicular Packaging

VGLUT2 is one of three vesicular glutamate transporters that play crucial roles in glutamatergic excitatory neurotransmission. We explored the functional properties of the rat VGLUT2 by heterologous expression of VGLUT2 in Xenopus oocytes. Immunocytochemical analysis indicated that most VGLUT2 protein was expressed in intracellular compartments but that some expression occurred also on the plasma membrane. Functional analysis revealed VGLUT2 to be active in two independent modes, namely, uptake into intracellular organelles and efflux at the plasma membrane. VGLUT-specific transport was identified based on the strong preference for glutamate over aspartate—in contrast to plasma-membrane or mitochondrial glutamate transporters—and sensitivity to known VGLUT blockers. VGLUT2 expression in oocytes (1) stimulated the influx of l-[³H]glutamate, but not d-[³H]aspartate, into digitonin-permeabilized oocytes and (2) stimulated efflux of l-glutamate, but not l-aspartate, from intact oocytes preinjected with ³H-labeled amino acids. In the latter assay, cellular efflux of glutamate (which was blocked by rose bengal and trypan blue) may be analogous to vesicular packaging of glutamate. Our data are consistent with VGLUT2-mediated H⁺/l-glutamate antiport, but not antiport with chloride. Expression of mammalian VGLUT1 and VGLUT3 also stimulated l-[³H]glutamate efflux from Xenopus oocytes, suggesting that this phenomenon is a general feature of vesicular glutamate transporters. Our findings support the idea that vesicular glutamate transporters, when transiently expressed on the neuronal plasma membrane, may mediate Ca²⁺-independent glutamate leakage in addition to their traditional role of packaging glutamate into synaptic vesicles for Ca²⁺-dependent exocytosis. Special issue article in honor of Dr. Frode Fonnum.     

Rapid Stimulation of EAAC1-Mediated Na+-Dependent l-Glutamate Transport Activity in C6 Glioma Cells by Phorbol Ester

Abstract: C6 glioma cells were used as a model system to study the regulation of EAAC1-mediated Na⁺-dependent l -[³H]glutamate transport. Although a 30-min preincubation with forskolin had no effect on transport activity, preincubation with phorbol 12-myristate 13-acetate (PMA) increased transport activity two- to threefold. PMA caused a time-dependent and concentration-dependent increase in EAAC1-mediated l -[³H]glutamate transport activity. A 2-min preincubation with PMA was sufficient to cause more than a twofold increase in transport activity and the protein synthesis inhibitor cycloheximide had no effect on the increase. These data suggest that this increase is independent of protein synthesis. The EC₅₀ value of PMA for stimulation of transport activity was 80 nM. Kinetic analyses demonstrated that the increase in transport activity was due to a 2.5-fold increase in V_max with no change in K_m. PMA also increased the transport of the nonmetabolizable analogue, d -[³H]aspartate to the same extent. In parallel assays, PMA did not, however, increase Na⁺-dependent glycine transport activity in C6 glioma. The inactive phorbol ester 4α-phorbol 12,13-didecanoate, did not stimulate l -[³H]glutamate transport activity, and the protein kinase C inhibitor chelerythrine blocked the stimulation caused by PMA. Okadaic acid and cyclosporin A, which are phosphatase inhibitors, had no effect on the stimulation of transport activity caused by PMA. The Ca²⁺ ionophore A23187 did not act synergistically to increase PMA stimulation. In previous studies, PMA caused a rapid increase in amiloride-sensitive Na⁺/H⁺ transport activity in C6 glioma. In the present study, pre- and coincubation with amiloride had no effect on the stimulation of transport activity caused by PMA. These studies suggest that activation of protein kinase C causes a rapid increase in EAAC1-mediated transport activity. This rapid increase in Na⁺-dependent l -[³H]-glutamate transport activity may provide a novel mechanism for protection against acute insults to the CNS.