Advertisement

Platelet-derived Growth Factor Rapidly Increases Activity and Cell Surface Expression of the EAAC1 Subtype of Glutamate Transporter through Activation of Phosphatidylinositol 3-Kinase*

Open AccessPublished:February 18, 2000DOI:https://doi.org/10.1074/jbc.275.7.5228
      Na+-dependent glutamate transporters are the primary mechanism for removal of excitatory amino acids (EAAs) from the extracellular space of the central nervous system and influence both physiologic and pathologic effects of these compounds. Recent evidence suggests that the activity and cell surface expression of a neuronal subtype of glutamate transporter, EAAC1, are rapidly increased by direct activation of protein kinase C and are decreased by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K). We hypothesized that this regulation could be analogous to insulin-induced stimulation of the GLUT4 subtype of glucose transporter, which is dependent upon activation of PI3-K. Using C6 glioma, a cell line that endogenously and selectively expresses EAAC1, we report that platelet-derived growth factor (PDGF) increased Na+-dependentl-[3H]-glutamate transport activity within 30 min. This effect of PDGF was not due to a change in total cellular EAAC1 immunoreactivity but was instead correlated with an increase cell surface expression of EAAC1, as measured using a membrane impermeant biotinylation reagent combined with Western blotting. A decrease in nonbiotinylated intracellular EAAC1 was also observed. These studies suggest that PDGF causes a redistribution of EAAC1 from an intracellular compartment to the cell surface. These effects of PDGF were accompanied by a 35-fold increase in PI3-K activity and were blocked by the PI3-K inhibitors, wortmannin and LY 294002, but not by an inhibitor of protein kinase C. Other growth factors, including insulin, nerve growth factor, and epidermal growth factor had no effect on glutamate transport nor did they increase PI3-K activity. These studies suggest that, as is observed for insulin-mediated translocation of GLUT4, EAAC1 cell surface expression can be rapidly increased by PDGF through activation of PI3-K. It is possible that this PDGF-mediated increase in EAAC1 activity may contribute to the previously demonstrated neuroprotective effects of PDGF.
      EAA
      excitatory amino acid
      PKC
      protein kinase C
      GABA
      γ-aminobutyric acid
      PI3-K
      phosphatidylinositol 3-kinase
      PMA
      phorbol 12-myristate 13-acetate
      PDGF
      platelet-derived growth factor
      DMEM
      Dulbecco's modified Eagle medium
      BSA
      bovine serum albumin
      NGF
      nerve growth factor
      EGF
      epidermal growth factor
      PLC-γ
      phospholipase C-γ
      MAP kinase
      mitogen-activated protein kinase
      Bis II
      bisindolylmaleimide II
      PKB
      protein kinase B
      The rapid clearance of glutamate from the extracellular space of the central nervous system by Na+-dependent high affinity glutamate transporters is critical to the maintenance of effective synaptic transmission and the prevention of excitotoxic injury. Increases in extracellular EAAs1 after head trauma and ischemic events have been described (
      • Faden A.I.
      • Demediuk P.
      • Panter S.S.
      • Vink R.
      ,
      • Benveniste H.
      • Drejer J.
      • Schousboe A.
      • Diemer N.H.
      ,
      • Drejer J.
      • Benveniste H.
      • Diemer N.H.
      • Schousboe A.
      ) and are presumably related to both a failure of inward transport and increased reverse operation of the carriers (
      • Attwell D.
      • Barbour B.
      • Szatkowski M.
      ,
      • Longuemare M.C.
      • Swanson R.A.
      ). A family of glutamate transporters mediates this high affinity uptake and includes five members, the glial transporters GLT-1 (human homologue EAAT2) and GLAST (EAAT1), the neuronal transporters EAAC1 (EAAT3) and EAAT4, and the retinal transporter EAAT5 (
      • Pines G.
      • Danbolt N.C.
      • Bjørås M.
      • Zhang Y.
      • Bendahan A.
      • Eide L.
      • Koepsell H.
      • Storm-Mathisen J.
      • Seeberg E.
      • Kanner B.I.
      ,
      • Storck T.
      • Schulte S.
      • Hofmann K.
      • Stoffel W.
      ,
      • Kanai Y.
      • Hediger M.A.
      ,
      • Fairman W.A.
      • Vandenberg R.J.
      • Arriza J.L.
      • Kavanaugh M.P.
      • Amara S.G.
      ,
      • Arriza J.L.
      • Eliasof S.
      • Kavanaugh M.P.
      • Amara S.G.
      ). The EAAC1 subtype of transporter is enriched in the pyramidal cells of hippocampus and cortex (
      • Rothstein J.D.
      • Martin L.
      • Levey A.I.
      • Dykes-Hoberg M.
      • Jin L.
      • Wu D.
      • Nash N.
      • Kuncl R.W.
      ,
      • Shashidharan P.
      • Huntley G.W.
      • Murray J.M.
      • Buku A.
      • Moran T.
      • Walsh M.J.
      • Morrison J.H.
      • Plaitakis A.
      ), two areas rich in glutamatergic transmission and exquisitely sensitive to excitotoxic insults (
      • Greene J.G.
      • Greenamyre J.T.
      ). Animals treated with antisense oligonucleotides to “knock down” EAAC1 expression develop a seizure phenotype, suggesting a role for EAAC1 in dampening excitability (
      • Rothstein J.D.
      • Dykes-Hoberg M.
      • Pardo C.A.
      • Bristol L.A.
      • Jin L.
      • Kuncl R.W.
      • Kanai Y.
      • Hediger M.
      • Wang Y.
      • Schielke J.P.
      • Welty D.F.
      ).
      The EAAC1 subtype is also expressed in several peripheral tissues including the kidney and intestine (
      • Rothstein J.D.
      • Martin L.
      • Levey A.I.
      • Dykes-Hoberg M.
      • Jin L.
      • Wu D.
      • Nash N.
      • Kuncl R.W.
      ,
      • Shayakul C.
      • Kanai Y.
      • Lee W.-S.
      • Brown D.
      • Rothstein J.D.
      • Hediger M.A.
      ). Although some results suggest mRNAs for the other transporters are expressed in selected peripheral tissues (for reviews see Refs.
      • Robinson M.B.
      • Dowd L.A.
      and
      • Sims K.D.
      • Robinson M.B.
      ), two studies have not observed protein expression (
      • Rothstein J.D.
      • Martin L.
      • Levey A.I.
      • Dykes-Hoberg M.
      • Jin L.
      • Wu D.
      • Nash N.
      • Kuncl R.W.
      ,
      • Furuta A.
      • Martin L.J.
      • Lin C.-L.G.
      • Dykes-Hoberg M.
      • Rothstein J.D.
      ). These studies suggest that EAAC1 may uniquely regulate extracellular acidic amino acids in the periphery. In fact, mice genetically deleted of EAAC1 excrete abnormally high levels of acidic amino acids in the urine, suggesting that this transporter mediates reabsorption of glutamate and aspartate from the glomerular filtrate (
      • Peghini P.
      • Janzen J.
      • Stoffel W.
      ). Therefore, understanding the acute regulation of this transporter may help elucidate its function during excitatory transmission and excitotoxic events, as well as in peripheral glutamate metabolism.
      Recent studies demonstrate that the activity of several neurotransmitter transporters can be rapidly regulated by direct activation of intracellular signaling molecules (PKC or cAMP-dependent protein kinase), including norepinephrine (
      • Apparsundaram S.
      • Schroeter S.
      • Giovanetti E.
      • Blakely R.D.
      ), serotonin (
      • Qian Y.
      • Galli A.
      • Ramamoorthy S.
      • Risso S.
      • DeFelice L.J.
      • Blakely R.D.
      ), dopamine (
      • Zhang L.
      • Coffey L.L.
      • Reith M.E.A.
      ,
      • Pristupa Z.B.
      • McConkey F.
      • Liu F.
      • Man H.Y.
      • Lee F.J.S.
      • Wang Y.T.
      • Niznik H.B.
      ,
      • Melikian H.E.
      • Powelka A.
      • Buckley K.M.
      ), GABA (
      • Quick M.W.
      • Corey J.L.
      • Davidson N.
      • Lester H.A.
      ,
      • Beckman M.L.
      • Bernstein E.M.
      • Quick M.W.
      ), and glutamate transporters (
      • Davis K.E.
      • Straff D.J.
      • Weinstein E.A.
      • Bannerman P.G.
      • Correale D.M.
      • Rothstein J.D.
      • Robinson M.B.
      ). In many cases, the changes in activity are correlated with a redistribution of transporter protein from the cell surface to an intracellular compartment or vice versa. There is also evidence that some of these transporters are regulated by their substrates. For example, Bernstein and Quick (
      • Bernstein E.M.
      • Quick M.W.
      ) recently demonstrated that GABA and other GABA transporter substrates increase the activity and cell surface expression of the GAT1 subtype of GABA transporter. Ramamoorthy and Blakely (
      • Ramamoorthy S.
      • Blakely R.D.
      ) provide evidence that serotonin transporter substrates decrease PKC-dependent phosphorylation and internalization of the serotonin transporter. Little is known about receptor-mediated regulation of transporter function, but there is evidence that histamine and adenosine receptor activation regulate serotonin transporter function by an unknown mechanism (
      • Launay J.M.
      • Bondoux D.
      • Oset-Gasque M.J.
      • Emami S.
      • Mutel V.
      • Haimart M.
      • Gespach C.
      ,
      • Miller K.J.
      • Hoffman B.J.
      ). Recent studies have demonstrated that activation of G protein-coupled receptors causes a decrease in cell surface expression of the GAT1 subtype of GABA transporter in neurons (
      • Beckman M.L.
      • Bernstein E.M.
      • Quick M.W.
      ). Angiotensin II and insulin may regulate norepinephrine transport in spontaneously hypertensive rats (
      • Yang H.
      • Raizada M.K.
      ) and SK-N-SH cells (
      • Apparsundaram S.
      • Blakely R.D.
      ), respectively. Both of these effects appear to be dependent on PI3-K.
      We recently demonstrated that activation of PKC with phorbol ester increases the activity and cell surface expression of the EAAC1 subtype of glutamate transporter (
      • Davis K.E.
      • Straff D.J.
      • Weinstein E.A.
      • Bannerman P.G.
      • Correale D.M.
      • Rothstein J.D.
      • Robinson M.B.
      ). Except for a PKC-induced increase in GAT1 cell surface expression observed in Xenopus oocytes (
      • Quick M.W.
      • Corey J.L.
      • Davidson N.
      • Lester H.A.
      ), this increase in EAAC1 cell surface expression following PKC activation is unique. We also found that wortmannin, an inhibitor of PI3-K, decreased EAAC1 activity and cell surface expression. These regulated changes in activity and cell surface expression qualitatively resemble translocation events observed for the insulin-sensitive glucose transporter GLUT4 (reviewed in Refs.
      • Czech M.P.
      • Corvera S.
      and
      • Pessin J.E.
      • Thurmond D.C.
      • Elmendorf J.S.
      • Coker K.J.
      • Okada S.
      ). The regulation of GLUT4 has been well studied and appears to be primarily mediated by activation of the insulin receptor tyrosine kinase cascade and stimulation of PI3-K, although some studies suggest an additional role for phorbol 12-myristate 13-acetate (PMA)-sensitive PKCs.
      In the present study, the effects of growth factors on the activity of EAAC1 were examined using C6 glioma as a model system that selectively and endogenously expresses this subtype of transporter. Of the growth factors tested, only PDGF stimulated PI3-K activity and rapidly (within minutes) increased both the activity and cell surface expression of EAAC1. These three effects of PDGF were blocked by two different inhibitors of PI3-K. Although the effects of PDGF and phorbol ester on activity and cell surface expression of EAAC1 were not additive, a PKC inhibitor did not block the effects of PDGF. These studies strongly suggest that EAAC1 cell surface expression and activity can be regulated within minutes by two independent but converging signaling pathways. This regulation may provide a novel mechanism to limit extracellular glutamate accumulation in the central nervous system.

      DISCUSSION

      The goals of this study were to determine if receptor-mediated activation of PI3-K increases the activity and cell surface expression of the neuronal glutamate transporter EAAC1 and to determine if this effect is dependent on activation of PKC. Application of PDGF BB increased EAAC1-mediated l-[3H]glutamate uptake within 30 min and also caused a comparable increase in EAAC1 cell surface expression. The PDGF AA homodimer, a ligand selective for activation of PDGF receptors containing the α subunit, had no significant effect on l-glutamate transport activity. This suggests that the effects observed in the present study are due to activation of receptors containing the β subunit. This result contrasts with an earlier preliminary study of l-aspartate transport in human fibroblasts, which reported an increase in transport activity after PDGF AA but not PDGF BB treatment (
      • Franchi-Gazzola R.
      • Visigalli R.
      • Bussolati O.
      • Gazzola G.C.
      ). The transporter subtype regulated by PDGF AA and the mechanism mediating the increase in activity were not identified in this earlier study.
      Three lines of evidence reported in the present study suggest that the effects of PDGF were mediated by PI3-K. First, several growth factors mediate a variety of cellular responses in C6 glioma, including NGF, EGF, PDGF, and insulin (
      • Wei J.-W.
      • Yeh S.-R.
      ,
      • Zhang W.
      • Nakashima T.
      • Sakai N.
      • Yamada H.
      • Okano Y.
      • Nozawa Y.
      ,
      • Goya L.
      • Feng P.T.
      • Aliabadi S.
      • Timiras P.S.
      ,
      • Hutton L.A.
      • Vellis J.D.
      • Perez-Polo J.R.
      ,
      • Abramovitch R.
      • Marikovsky M.
      • Meir G.
      • Neeman M.
      ). Each of these growth factors have been directly demonstrated to activate the same major signaling pathways, PLC-γ, PI3-K, and MAP kinase, in a number of different cellular systems (
      • Valenzuela C.F.
      • Kazlauskas A.
      • Weiner J.L.
      ,
      • Carpenter G.
      ,
      • Kapeller R.
      • Cantley L.C.
      ,
      • Goya L.
      ). However, in the present study, only PDGF stimulated PI3-K activity, and only PDGF altered EAAC1-mediated transport activity and cell surface expression. The lack of PI3-K stimulation by the other growth factors tested implies that these receptors couple to different signaling pathways in C6 glioma that do not contribute to the regulation of EAAC1 activity. Second, the effects of PDGF on EAAC1-mediated glutamate uptake activity and cell surface expression were blocked by two different PI3-K inhibitors. Finally, the effects of PDGF were not blocked by inhibitors of other signaling pathways potentially activated by PDGF. These studies suggest that PKC and PI3-K independently regulate cell surface expression of EAAC1 and provide one of the first examples of growth factor-mediated trafficking of neurotransmitter transporters.
      The C6 glioma cell line was selected as a model system for these experiments because of its endogenous and selective expression of EAAC1, allowing examination of the transporter in isolation. C6 glioma are an undifferentiated cell line of central nervous system origin that express both neuronal (glutamic acid decarboxylase) and glial (glutamine synthetase and glial fibrillary acidic protein) markers (
      • Segovia J.
      • Lawless G.M.
      • Tillakaratne N.J.K.
      • Brenner M.
      • Tobin A.J.
      ,
      • Pishak M.R.
      • Phillips A.T.
      ,
      • Bissel M.G.
      • Rubinstein L.J.
      • Bignami A.
      • Herman M.M.
      ). Although neurons in culture might more closely mimic the cellular milieu observed in vivo, we find that four transporters (EAAC1, EAAT4, GLT-1, and GLAST) are expressed in these cultures. This expression of additional transporters may be due to glial contamination, as well as changes in transporter expression properties of neurons grown in culture (
      • Brooks-Kayal A.R.
      • Munir M.
      • Jin H.
      • Robinson M.B.
      ,
      • Mennerick S.
      • Dhond R.P.
      • Benz A.
      • Xu W.
      • Rothstein J.D.
      • Danbolt N.C.
      • Isenberg K.E.
      • Zorumski C.F.
      ). Additionally, the pharmacology of transport activity in these cultures suggests that at least three of these transporters contribute to glutamate uptake activity (

      Munir, M., Correale, D. M., and Robinson, M. B. (2000) Neurochem. Int., in press

      ). Because there are no specific inhibitors of EAAC1-mediated transport activity, ongoing efforts are aimed at developing neuron-enriched cultures with minimal glial contamination for these studies. Although one might argue that the regulated trafficking of EAAC1 observed in C6 glioma is an artifact of the cell line, two studies have documented cytoplasmic as well as plasma membrane localization of EAAC1 in brain tissue (
      • Rothstein J.D.
      • Martin L.
      • Levey A.I.
      • Dykes-Hoberg M.
      • Jin L.
      • Wu D.
      • Nash N.
      • Kuncl R.W.
      ,
      • Shashidharan P.
      • Huntley G.W.
      • Murray J.M.
      • Buku A.
      • Moran T.
      • Walsh M.J.
      • Morrison J.H.
      • Plaitakis A.
      ). This cytoplasmic/intracellular localization is unique to the EAAC1 subtype of glutamate transporter (
      • Chaudhry F.A.
      • Lehre K.P.
      • Campagne M.V.L.
      • Ottersen O.P.
      • Danbolt N.C.
      • Storm-Mathisen J.
      ) and suggests that there is an intracellular pool of EAAC1 that can be redistributed to the cell surface in vivo.
      The ability of PDGF to increase EAAC1 transport activity and cell surface expression in a PI3-K-dependent fashion qualitatively resembles the regulation of GLUT4 in the periphery. In 3T3-L1 adipocytes and several other model systems, insulin increases activity and cell surface expression of the GLUT4 subtype of glucose transporter (reviewed in Refs.
      • Czech M.P.
      • Corvera S.
      and
      • Pessin J.E.
      • Thurmond D.C.
      • Elmendorf J.S.
      • Coker K.J.
      • Okada S.
      ). As is observed for the effects of PDGF on EAAC1, these effects of insulin occur within minutes and are blocked by both wortmannin and LY 294002 (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ). Although some earlier studies had not observed effects of PDGF on glucose transport, Wang et al. (
      • Wang L.
      • Hayashi H.
      • Ebina Y.
      ) recently reported that PDGF rapidly induces a 6-fold increase in glucose transport using 3T3-L1 adipocytes as a model system (for a discussion of earlier literature, see Ref.
      • Wang L.
      • Hayashi H.
      • Ebina Y.
      ). This increase in activity was correlated with an increase in cell surface expression of a myc-tagged GLUT4 transporter. As was observed in the present study, these effects of PDGF were accompanied by an increase in PI3-K activity and were blocked by wortmannin (100 nm), suggesting that the effects of PDGF on GLUT4 and EAAC1 may be comparable. Although qualitatively similar, a larger percentage of GLUT4 appears to be sequestered intracellularly, and insulin has a greater effect on glucose uptake activity and translocation (up to 20-fold) than is observed after PDGF stimulation of EAAC1 (reviewed in Refs.
      • Pessin J.E.
      • Thurmond D.C.
      • Elmendorf J.S.
      • Coker K.J.
      • Okada S.
      and
      • Rice J.E.
      • Livingstone C.
      • Gould G.W.
      ). Although it is possible that this quantitative difference in intracellular sequestration is related to differences intrinsic to EAAC1 and GLUT4, it is also possible that under different conditions EAAC1 is more effectively retained intracellularly. It has also been suggested that GLUT4 transporters are segregated into two intracellular compartments, a constitutively recycling pool and a rapidly regulated pool of transporters (
      • Zorzano A.
      • Wilkinson W.
      • Kotliar N.
      • Thoidis G.
      • Wadzinkski B.E.
      • Ruoho A.E.
      • Pilch P.F.
      ,
      • Aledo J.C.
      • Lavoie L.
      • Vollchuk A.
      • Keller S.R.
      • Klip A.
      • Hundal H.S.
      ,
      • Malide D.
      • Dwyer N.K.
      • Blanchette-Mackie E.J.
      • Cushman S.W.
      ) (reviewed in Ref.
      • Pessin J.E.
      • Thurmond D.C.
      • Elmendorf J.S.
      • Coker K.J.
      • Okada S.
      ). One argument for two compartments is the observation that transferrin receptors, a marker of the endosomal recycling compartment, and GLUT4 do not always co-localize (
      • Aledo J.C.
      • Lavoie L.
      • Vollchuk A.
      • Keller S.R.
      • Klip A.
      • Hundal H.S.
      ,
      • Hanpeter D.
      • James D.E.
      ) (for review see Ref.
      • Rice J.E.
      • Livingstone C.
      • Gould G.W.
      ). In fact, a substantial portion of cytoplasmic EAAC1 does not co-localize with transferrin receptors in control and wortmannin-treated C6 glioma, suggesting that intracellular EAAC1 may also segregate to two distinct intracellular compartments.
      K. Sims and M. Robinson, unpublished observation.
      Together, these observations suggest many similarities between the regulated trafficking of EAAC1 and GLUT4.
      The two most common PI3-K inhibitors, wortmannin and LY 294002, both blocked the effects of PDGF on EAAC1 uptake activity and cell surface expression, but in the absence of PDGF these two compounds had different effects. Wortmannin decreased basal glutamate uptake and cell surface localization and reduced the effects of PDGF to similar levels below control. In contrast, LY 294002 alone did not affect basal glutamate transport or cause a reduction of cell surface immunoreactivity. Both wortmannin and LY 294002 decreased PI3-K activity to undetectable levels and blocked PDGF stimulation of PI3-K activity. The differential effect of wortmannin on EAAC1 activity and cell surface expression might be attributable to inhibition of alternate kinase targets, but the concentrations used in the present study are below the 200–300 nm IC50 values required for inhibition of these alternate targets (myosin light chain kinase and MAP kinase) (
      • Nakanishi S.
      • Yano H.
      • Matsuda Y.
      ). Furthermore, we demonstrated in an earlier study that wortmannin inhibits basal EAAC1-mediated uptake with an IC50 value of 15 nm, which is nearly identical to the IC50 value for inhibition of PI3-K (
      • Davis K.E.
      • Straff D.J.
      • Weinstein E.A.
      • Bannerman P.G.
      • Correale D.M.
      • Rothstein J.D.
      • Robinson M.B.
      ). Wortmannin and LY 294002 both interact with the p110 subunit of PI3-K but have different mechanisms of action (wortmannin is an irreversible inhibitor and LY 294002 is reversible) (
      • Nakanishi S.
      • Yano H.
      • Matsuda Y.
      ,
      • Vlahos C.J.
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      ). Although it is possible that this difference in mechanism may account for the selective effects of wortmannin, it seems more likely that this effect on baseline activity and cell surface expression is related to inhibition of a wortmannin-sensitive, LY 294002-insensitive isoform of PI3-K. Several new isoforms of PI3-K have been identified recently, but their sensitivities to wortmannin and LY 294002 have not been systematically evaluated (for review see Ref.
      • Zvelebil M.J.
      • Macdougall L.
      • Leevers S.
      • Volinia S.
      • Vanhaesebroeck B.
      • Gout I.
      • Panayotou G.
      • Domin J.
      • Stein R.
      • Pages F.
      • Koga H.
      • Salin K.
      • Linacre J.
      • Das P.
      • Panaretou C.
      • Wetzker R.
      • Waterfield M.
      ). There are some isoforms that display differences in sensitivity to these inhibitors when compared with the “classical” mammalian PI3-K, p85/p110 PI3-K (
      • Takegawa K.
      • DeWals D.B.
      • Emr S.D.
      ,
      • Virbasius J.V.
      • Guilherme A.
      • Czech M.P.
      ,
      • Domin J.
      • Pages F.
      • Volinia S.
      • Rittenhouse S.E.
      • Zvelebil M.J.
      • Stein R.C.
      • Waterfield M.D.
      ). Therefore, it is possible that multiple isoforms of PI3-K regulate different aspects of transporter trafficking. For example, a wortmannin-sensitive, LY 294002-insensitive PI3-K may be required for recycling of EAAC1 transporters through an intracellular compartment.
      In the present study, we also found that the effects of PDGF were not blocked by the PKC antagonist Bis II, and the stimulatory effects of PDGF and PMA were not additive. This could imply that both PKC and PI3-K increase EAAC1 activity through independent but converging pathways. Activation of PKC with phorbol esters also increases activity and/or cell surface expression of the GLUT4 glucose transporter (
      • Nave B.T.
      • Siddle K.
      • Shepherd P.R.
      ,
      • Nishimura H.
      • Simpson I.A.
      ,
      • Vogt B.
      • Muschack J.
      • Seffer E.
      • Haring H.-U.
      ). The somewhat nonspecific PKC inhibitor, staurosporine, blocks the effects of phorbol esters and insulin on glucose uptake with different IC50 values, suggesting phorbol esters and insulin utilize different but possibly converging signaling pathways (
      • Nishimura H.
      • Simpson I.A.
      ). Because PKC did not activate PI3-K in our system, it seems most likely that these pathways converge downstream of PI3-K. The PKC and PDGF/PI3-K pathways may independently regulate the same limited intracellular pool of transporters. Alternatively, it is possible that a PMA-sensitive, classical PKC activates a downstream effector of PI3-K. Both atypical PKC isoforms and Akt/PKB have been implicated as downstream effectors of PI3-K during insulin-mediated regulation of GLUT4. Expression of constitutively active/dominant negative constructs of both the atypical PKC isoform, PKCζ, and Akt/PKB (
      • Kohn A.D.
      • Summers S.A.
      • Birnbaum M.J.
      • Roth R.A.
      ,
      • Tanti J.-F.
      • Grillo S.
      • Gremeaux T.
      • Coffer P.J.
      • Obberghen E.V.
      • Marchand-Brustel Y.L.
      ,
      • Cong L.-N.
      • Chen H.
      • Li Y.
      • Zhou L.
      • McGibbon M.A.
      • Taylor S.I.
      • Quon M.J.
      ) influence insulin regulation of GLUT4 transport and cell surface expression. Molecular biological approaches also suggest that PKCλ and PKCε may be downstream effectors of PI3-K signaling (
      • Kotani K.
      • Ogawa W.
      • Matsumoto M.
      • Kitamura T.
      • Sakaue H.
      • Hino Y.
      • Miyake K.
      • Sano W.
      • Akimoto K.
      • Ohno S.
      • Kasuga M.
      ,
      • Moriya S.
      • Kazlauskas A.
      • Akimoto K.
      • Hirai S.-I.
      • Mizuno K.
      • Takenawa T.
      • Fukui Y.
      • Watanabe Y.
      • Ozaki S.
      • Ohno S.
      ). At present, it is not known if these kinases can be regulated by PMA-activated PKCs nor is it known if these signaling molecules contribute to the regulation of EAAC1 trafficking.
      At present, it is not known if the effects of PDGF and phorbol ester on transport activity and cell surface expression are dependent upon direct phosphorylation of EAAC1 or indirectly mediated through phosphorylation of other proteins required for trafficking of the transporters to and from the cell surface. Recent studies have demonstrated that phorbol ester-induced decreases in GLAST-mediated transport activity are correlated with transporter phosphorylation, but this phosphorylation does not appear to be occurring at a PKC phosphorylation site consensus sequence (
      • Conradt M.
      • Stoffel W.
      ). Earlier studies have demonstrated that GLT-1 is also phosphorylated by phorbol esters (
      • Casado M.
      • Bendahan A.
      • Zafra F.
      • Danbolt N.C.
      • Aragon C.
      • Gimenez C.
      • Kanner B.I.
      ). In both of these examples of glutamate transporter phosphorylation, it has not been determined if the changes in transport activity are correlated with changes in transporter cell surface expression. More recently, it has been shown that phosphorylation of the serotonin transporter results in internalization of the transporter and a corresponding reduction in serotonin transport (
      • Ramamoorthy S.
      • Blakely R.D.
      ). Therefore, although there is currently no evidence that the effects of PDGF or phorbol ester are related to phosphorylation of the EAAC1, it is possible that the redistribution of EAAC1 could involve direct transporter phosphorylation.
      Rapid regulation of EAAC1 may be important for regulating renal reabsorption of acidic amino acids and may be critical for proper synaptic transmission and the prevention of excitotoxic injury in the brain. PDGF may represent an endogenous physiologic regulator of neuronal EAAC1 function and glutamatergic transmission, because PDGF β receptors and B-chains are expressed in neurons throughout the central nervous system and are enriched in the hippocampus, an area of high EAAC1 expression levels (
      • Smits A.
      • Kato M.
      • Westermark B.
      • Nister M.
      • Heldin C.-H.
      • Funa K.
      ,
      • Sasahara M.
      • Sato H.
      • Iihara K.
      • Wang J.
      • Chue C.-H.
      • Takayama S.
      • Hayase Y.
      • Hazama F.
      ). PDGF BB inhibits both GABAA-dependent inhibitory post-synaptic currents andN-methyl-daspartate-dependent excitatory post-synaptic currents (
      • Valenzuela C.F.
      • Kazlauskas A.
      • Brozowski S.J.
      • Weiner J.L.
      • DeMali K.A.
      • McDonald B.J.
      • Moss S.J.
      • Dunwiddie T.V.
      • Harris R.A.
      ,
      • Valenzuela C.F.
      • Xiong Z.
      • Kazkauskas A.
      • McDonald J.F.
      • Weiner J.L.
      • Frazier C.J.
      • Dunwiddie T.V.
      • Kazlauskas A.
      • Whiting P.J.
      • Harris R.A.
      ), suggesting a role in the regulation of rapid synaptic events. Both PDGF B and β receptor mRNA and immunoreactivity are increased after induction of neocortical focal ischemia (
      • Iihara K.
      • Sasahara M.
      • Hashimoto N.
      • Uemura Y.
      • Kikuchi H.
      • Hazama F.
      ,
      • Iihara K.
      • Sasahara M.
      • Hashimoto N.
      • Hazama F.
      ), and PDGF BB pretreatment reduces delayed hippocampal CA1 pyramidal neuron death in a global forebrain ischemia model (
      • Iihara K.
      • Hashimoto N.
      • Tsukahara T.
      • Sakata M.
      • Yanamoto H.
      • Taniguchi T.
      ). Although the mechanism of PDGF neuroprotection was not examined in these models, possible mechanisms have been studied in other models. In cortical and hippocampal neuronal cultures exposed to two different models of excitotoxic insult (glucose deprivation or FeSO4), PDGF AA or BB are neuroprotective (
      • Cheng B.
      • Mattson M.P.
      ). Importantly, PDGF was effective when applied before, during, or up to 4 h after the onset of the insult, implying that rapid regulatory events contribute to this protection. Prevention of neuronal death was correlated with increases in the activities of catalase, superoxide dismutase, and glutathione peroxidase, suggesting that activation of these enzymes contributes to this effect. The rapid, PDGF-mediated increase in EAAC1 activity and cell surface expression we describe in this study may represent a novel mechanism that contributes to the neuroprotective effects of this growth factor.

      Acknowledgments

      We thank Dr. J. Rothstein for providing antibodies to EAAC1, Dr. S. Summers for technical advice, and Dr. M. Birnbaum for helpful discussions and critical review of this study.

      REFERENCES

        • Faden A.I.
        • Demediuk P.
        • Panter S.S.
        • Vink R.
        Science. 1989; 244: 798-800
        • Benveniste H.
        • Drejer J.
        • Schousboe A.
        • Diemer N.H.
        J. Neurochem. 1984; 43: 1369-1374
        • Drejer J.
        • Benveniste H.
        • Diemer N.H.
        • Schousboe A.
        J. Neurochem. 1985; 45: 145-151
        • Attwell D.
        • Barbour B.
        • Szatkowski M.
        Neuron. 1993; 11: 401-407
        • Longuemare M.C.
        • Swanson R.A.
        J. Neurosci. Res. 1995; 40: 379-386
        • Pines G.
        • Danbolt N.C.
        • Bjørås M.
        • Zhang Y.
        • Bendahan A.
        • Eide L.
        • Koepsell H.
        • Storm-Mathisen J.
        • Seeberg E.
        • Kanner B.I.
        Nature. 1992; 360: 464-467
        • Storck T.
        • Schulte S.
        • Hofmann K.
        • Stoffel W.
        Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10955-10959
        • Kanai Y.
        • Hediger M.A.
        Nature. 1992; 360: 467-471
        • Fairman W.A.
        • Vandenberg R.J.
        • Arriza J.L.
        • Kavanaugh M.P.
        • Amara S.G.
        Nature. 1995; 375: 599-603
        • Arriza J.L.
        • Eliasof S.
        • Kavanaugh M.P.
        • Amara S.G.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4155-4160
        • Rothstein J.D.
        • Martin L.
        • Levey A.I.
        • Dykes-Hoberg M.
        • Jin L.
        • Wu D.
        • Nash N.
        • Kuncl R.W.
        Neuron. 1994; 13: 713-725
        • Shashidharan P.
        • Huntley G.W.
        • Murray J.M.
        • Buku A.
        • Moran T.
        • Walsh M.J.
        • Morrison J.H.
        • Plaitakis A.
        Brain Res. 1997; 773: 139-148
        • Greene J.G.
        • Greenamyre J.T.
        Prog. Neurobiol. 1996; 48: 613-634
        • Rothstein J.D.
        • Dykes-Hoberg M.
        • Pardo C.A.
        • Bristol L.A.
        • Jin L.
        • Kuncl R.W.
        • Kanai Y.
        • Hediger M.
        • Wang Y.
        • Schielke J.P.
        • Welty D.F.
        Neuron. 1996; 16: 675-686
        • Shayakul C.
        • Kanai Y.
        • Lee W.-S.
        • Brown D.
        • Rothstein J.D.
        • Hediger M.A.
        Amer. J. Physiol. 1997; 273: F1023-F1029
        • Robinson M.B.
        • Dowd L.A.
        Adv. Pharmacol. 1997; 37: 69-115
        • Sims K.D.
        • Robinson M.B.
        Crit. Rev. Neurobiol. 1999; 13: 169-197
        • Furuta A.
        • Martin L.J.
        • Lin C.-L.G.
        • Dykes-Hoberg M.
        • Rothstein J.D.
        Neuroscience. 1997; 81: 1031-1042
        • Peghini P.
        • Janzen J.
        • Stoffel W.
        EMBO J. 1997; 16: 3822-3832
        • Apparsundaram S.
        • Schroeter S.
        • Giovanetti E.
        • Blakely R.D.
        J. Pharmacol. Exp. Ther. 1998; 287: 744-751
        • Qian Y.
        • Galli A.
        • Ramamoorthy S.
        • Risso S.
        • DeFelice L.J.
        • Blakely R.D.
        J. Neurosci. 1997; 17: 45-57
        • Zhang L.
        • Coffey L.L.
        • Reith M.E.A.
        Biochem. Pharmacol. 1997; 53: 677-688
        • Pristupa Z.B.
        • McConkey F.
        • Liu F.
        • Man H.Y.
        • Lee F.J.S.
        • Wang Y.T.
        • Niznik H.B.
        Synapse. 1998; 30: 79-87
        • Melikian H.E.
        • Powelka A.
        • Buckley K.M.
        Soc. Neurosci. Abstr. 1998; 24: 609
        • Quick M.W.
        • Corey J.L.
        • Davidson N.
        • Lester H.A.
        J. Neurosci. 1997; 17: 2967-2979
        • Beckman M.L.
        • Bernstein E.M.
        • Quick M.W.
        J. Neurosci. 1998; 18: 6103-6112
        • Davis K.E.
        • Straff D.J.
        • Weinstein E.A.
        • Bannerman P.G.
        • Correale D.M.
        • Rothstein J.D.
        • Robinson M.B.
        J. Neurosci. 1998; 18: 2475-2485
        • Bernstein E.M.
        • Quick M.W.
        J. Biol. Chem. 1999; 274: 889-895
        • Ramamoorthy S.
        • Blakely R.D.
        Science. 1999; 285: 763-766
        • Launay J.M.
        • Bondoux D.
        • Oset-Gasque M.J.
        • Emami S.
        • Mutel V.
        • Haimart M.
        • Gespach C.
        Amer. J. Physiol. 1994; 266: R526-R536
        • Miller K.J.
        • Hoffman B.J.
        J. Biol. Chem. 1994; 269: 27351-27356
        • Beckman M.L.
        • Bernstein E.M.
        • Quick M.W.
        J. Neurosci. 1999; 19: RC1-RC6
        • Yang H.
        • Raizada M.K.
        J. Neurosci. 1999; 19: 2413-2423
        • Apparsundaram S.
        • Blakely R.D.
        Soc. Neurosci. Abstr. 1997; 23: 1132
        • Czech M.P.
        • Corvera S.
        J. Biol. Chem. 1999; 274: 1865-1868
        • Pessin J.E.
        • Thurmond D.C.
        • Elmendorf J.S.
        • Coker K.J.
        • Okada S.
        J. Biol. Chem. 1999; 274: 2593-2596
        • Dowd L.A.
        • Robinson M.B.
        J. Neurochem. 1996; 67: 508-516
        • Palos T.P.
        • Ramachandran B.
        • Boado R.
        • Howard B.D.
        Mol. Brain Res. 1996; 37: 297-303
        • Casado M.
        • Zafra F.
        • Aragón C.
        • Giménez C.
        J. Neurochem. 1991; 57: 1185-1190
        • Palos T.P.
        • Zheng S.
        • Howard B.D.
        J. Neurochem. 1999; 73: 1012-1023
        • Cheatham B.
        • Vlahos C.J.
        • Cheatham L.
        • Wang L.
        • Blenis J.
        • Kahn C.R.
        Mol. Cell. Biol. 1994; 14: 4902-4911
        • Clarke J.F.
        • Young P.W.
        • Yonezawa K.
        • Kasuga M.
        • Holman G.D.
        Biochem. J. 1994; 300: 631-635
        • Lowry O.H.
        • Rosebrough N.J.
        • Farr A.L.
        • Randall R.J.
        J. Biol. Chem. 1951; 193: 265-275
        • Sun X.J.
        • Rothenburg P.
        • Kahn C.R.
        • Backer J.M.
        • Araki E.
        • Wilden P.A.
        • Cahill D.A.
        • Goldstein B.J.
        • White M.F.
        Nature. 1991; 352: 73-77
        • Yeang H.Y.
        • Yusof F.
        • Abdullah L.
        Anal. Biochem. 1998; 265: 381-384
        • Haugeto Ø.
        • Ullensveng K.
        • Levy L.M.
        • Chaudhry F.A.
        • Honore T.
        • Neilsen M.
        • Lehre K.P.
        • Danbolt N.C.
        J. Biol. Chem. 1996; 271: 27715-27722
        • Wei J.-W.
        • Yeh S.-R.
        Int. J. Biochem. 1991; 23: 851-856
        • Zhang W.
        • Nakashima T.
        • Sakai N.
        • Yamada H.
        • Okano Y.
        • Nozawa Y.
        Neurol. Res. 1992; 14: 397-401
        • Goya L.
        • Feng P.T.
        • Aliabadi S.
        • Timiras P.S.
        Int. J. Dev. Neurosci. 1996; 14: 409-417
        • Hutton L.A.
        • Vellis J.D.
        • Perez-Polo J.R.
        J. Neurosci. Res. 1992; 32: 375-383
        • Abramovitch R.
        • Marikovsky M.
        • Meir G.
        • Neeman M.
        Br. J. Cancer. 1999; 79: 1392-1398
        • Valenzuela C.F.
        • Kazlauskas A.
        • Weiner J.L.
        Brain Res. Rev. 1997; 24: 77-89
        • Franke T.F.
        • Yang S.
        • Chan T.O.
        • Datta K.
        • Kazlauskas A.
        • Morrison D.K.
        • Kaplan D.R.
        • Tsichlis P.N.
        Cell. 1995; 81: 727-736
        • Burgering B.M.T.
        • Coffer P.J.
        Nature. 1995; 376: 599-602
        • Nakanishi S.
        • Yano H.
        • Matsuda Y.
        Cell. Signal. 1995; 7: 545-557
        • Vlahos C.J.
        • Matter W.F.
        • Hui K.Y.
        • Brown R.F.
        J. Biol. Chem. 1994; 269: 5241-5248
        • Carpenter G.
        FASEB J. 1992; 6: 3283-3289
        • Kapeller R.
        • Cantley L.C.
        Bioessays. 1994; 16: 565-576
        • Martiny-Baron G.
        • Kazanietz M.G.
        • Mishak H.
        • Blumberg P.M.
        • Kochs G.
        • Hug H.
        • Marme D.
        • Schachtele C.
        J. Biol. Chem. 1993; 268: 9194-9197
        • Duronio V.
        • Scheid M.P.
        • Ettinger S.
        Cell. Signal. 1998; 10: 233-239
        • Franchi-Gazzola R.
        • Visigalli R.
        • Bussolati O.
        • Gazzola G.C.
        FEBS Lett. 1994; 352 (112): 119
        • Goya L.
        Adv. Exp. Med. Biol. 1997; 429: 249-260
        • Segovia J.
        • Lawless G.M.
        • Tillakaratne N.J.K.
        • Brenner M.
        • Tobin A.J.
        J. Neurochem. 1994; 63: 1218-1225
        • Pishak M.R.
        • Phillips A.T.
        J. Neurochem. 1980; 34: 866-872
        • Bissel M.G.
        • Rubinstein L.J.
        • Bignami A.
        • Herman M.M.
        Brain Res. 1974; 82: 77-89
        • Brooks-Kayal A.R.
        • Munir M.
        • Jin H.
        • Robinson M.B.
        Neurochem. Int. 1998; 33: 95-100
        • Mennerick S.
        • Dhond R.P.
        • Benz A.
        • Xu W.
        • Rothstein J.D.
        • Danbolt N.C.
        • Isenberg K.E.
        • Zorumski C.F.
        J. Neurosci. 1998; 18: 4490-4499
      1. Munir, M., Correale, D. M., and Robinson, M. B. (2000) Neurochem. Int., in press

        • Chaudhry F.A.
        • Lehre K.P.
        • Campagne M.V.L.
        • Ottersen O.P.
        • Danbolt N.C.
        • Storm-Mathisen J.
        Neuron. 1995; 15: 711-720
        • Wang L.
        • Hayashi H.
        • Ebina Y.
        J. Biol. Chem. 1999; 274: 19246-19253
        • Rice J.E.
        • Livingstone C.
        • Gould G.W.
        Biochem. Soc. Trans. 1996; 24: 540-546
        • Zorzano A.
        • Wilkinson W.
        • Kotliar N.
        • Thoidis G.
        • Wadzinkski B.E.
        • Ruoho A.E.
        • Pilch P.F.
        J. Biol. Chem. 1989; 264: 12358-12363
        • Aledo J.C.
        • Lavoie L.
        • Vollchuk A.
        • Keller S.R.
        • Klip A.
        • Hundal H.S.
        Biochem. J. 1997; 325: 727-732
        • Malide D.
        • Dwyer N.K.
        • Blanchette-Mackie E.J.
        • Cushman S.W.
        J. Histochem. Cytochem. 1997; 45: 1083-1096
        • Hanpeter D.
        • James D.E.
        Mol. Membr. Biol. 1995; 12: 263-269
        • Zvelebil M.J.
        • Macdougall L.
        • Leevers S.
        • Volinia S.
        • Vanhaesebroeck B.
        • Gout I.
        • Panayotou G.
        • Domin J.
        • Stein R.
        • Pages F.
        • Koga H.
        • Salin K.
        • Linacre J.
        • Das P.
        • Panaretou C.
        • Wetzker R.
        • Waterfield M.
        Philos. Trans. R. Soc. Lond-Biol. Sci. 1996; 351: 217-223
        • Takegawa K.
        • DeWals D.B.
        • Emr S.D.
        J. Cell Sci. 1995; 108: 3745-3756
        • Virbasius J.V.
        • Guilherme A.
        • Czech M.P.
        J. Biol. Chem. 1996; 271: 13304-13307
        • Domin J.
        • Pages F.
        • Volinia S.
        • Rittenhouse S.E.
        • Zvelebil M.J.
        • Stein R.C.
        • Waterfield M.D.
        Biochem. J. 1997; 326: 139-147
        • Nave B.T.
        • Siddle K.
        • Shepherd P.R.
        Biochem. J. 1996; 318: 203-205
        • Nishimura H.
        • Simpson I.A.
        Biochem. J. 1994; 302: 271-277
        • Vogt B.
        • Muschack J.
        • Seffer E.
        • Haring H.-U.
        Biochem. J. 1991; 275: 597-600
        • Kohn A.D.
        • Summers S.A.
        • Birnbaum M.J.
        • Roth R.A.
        J. Biol. Chem. 1996; 271: 31372-31378
        • Tanti J.-F.
        • Grillo S.
        • Gremeaux T.
        • Coffer P.J.
        • Obberghen E.V.
        • Marchand-Brustel Y.L.
        Endocrinology. 1997; 138: 2005-2010
        • Cong L.-N.
        • Chen H.
        • Li Y.
        • Zhou L.
        • McGibbon M.A.
        • Taylor S.I.
        • Quon M.J.
        Mol. Endocrinol. 1997; 11: 1881-1890
        • Kotani K.
        • Ogawa W.
        • Matsumoto M.
        • Kitamura T.
        • Sakaue H.
        • Hino Y.
        • Miyake K.
        • Sano W.
        • Akimoto K.
        • Ohno S.
        • Kasuga M.
        Mol. Cell. Biol. 1998; 18: 6971-6982
        • Moriya S.
        • Kazlauskas A.
        • Akimoto K.
        • Hirai S.-I.
        • Mizuno K.
        • Takenawa T.
        • Fukui Y.
        • Watanabe Y.
        • Ozaki S.
        • Ohno S.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 151-155
        • Conradt M.
        • Stoffel W.
        J. Neurochem. 1997; 68: 1244-1251
        • Casado M.
        • Bendahan A.
        • Zafra F.
        • Danbolt N.C.
        • Aragon C.
        • Gimenez C.
        • Kanner B.I.
        J. Biol. Chem. 1993; 268: 27313-27317
        • Smits A.
        • Kato M.
        • Westermark B.
        • Nister M.
        • Heldin C.-H.
        • Funa K.
        Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8159-8163
        • Sasahara M.
        • Sato H.
        • Iihara K.
        • Wang J.
        • Chue C.-H.
        • Takayama S.
        • Hayase Y.
        • Hazama F.
        Mol. Brain Res. 1995; 32: 63-74
        • Valenzuela C.F.
        • Kazlauskas A.
        • Brozowski S.J.
        • Weiner J.L.
        • DeMali K.A.
        • McDonald B.J.
        • Moss S.J.
        • Dunwiddie T.V.
        • Harris R.A.
        Mol. Pharmacol. 1995; 48: 1099-1107
        • Valenzuela C.F.
        • Xiong Z.
        • Kazkauskas A.
        • McDonald J.F.
        • Weiner J.L.
        • Frazier C.J.
        • Dunwiddie T.V.
        • Kazlauskas A.
        • Whiting P.J.
        • Harris R.A.
        J. Biol. Chem. 1996; 271: 16151-16159
        • Iihara K.
        • Sasahara M.
        • Hashimoto N.
        • Uemura Y.
        • Kikuchi H.
        • Hazama F.
        J. Cereb. Blood Flow Metab. 1994; 14: 818-824
        • Iihara K.
        • Sasahara M.
        • Hashimoto N.
        • Hazama F.
        J. Cereb. Blood Flow Metab. 1996; 16: 941-949
        • Iihara K.
        • Hashimoto N.
        • Tsukahara T.
        • Sakata M.
        • Yanamoto H.
        • Taniguchi T.
        J. Cereb. Blood Flow Metab. 1997; 17: 1097-1106
        • Cheng B.
        • Mattson M.P.
        J. Neurosci. 1995; 15: 7095-7104