Phorbol ester-induced inhibition of GABA uptake by synaptosomes and by Xenopus oocytes expressing GABA transporter (GAT1)

Osawa, Ichiro; Saito, Naoaki; Koga, Tomohide; Tanaka, Chikako

doi:10.1016/0168-0102(94)90041-8

Cited by 42 publications

(17 citation statements)

References 25 publications

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“…The observation that removal of all consensus PKC sites did not affect the modulation of GAT1 by PMA [10] implies that PKC modulation in PMA injected oocytes involves an indirect mechanism. In contrast, the data of Osawa et al [9] may reflect direct phosphorylation and consequently a distinct regulatory mechanism.…”

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confidence: 76%

“…In case of the major GABA transporter in mammalian brain, GAT1, the data reported sofar are controversial. Upon treatment of Xenopus oocytes expressing GAT1 with phorbol ester known to activate protein kinase C (PKC), Osawa and colleagues [9] observed a downregulation in GABA transport resulting from a decrease in substrate affinity. In contrast, when using the same heterologous expression *Corresponding author.…”

Section: Introductionmentioning

confidence: 99%

“…However, the results obtained were contradictory, since one study reported a PMA-induced GAT1 downregulation resulting from a decrease of transporter affinthe other found enhanced [ H]GABA transport ity [9], whereas 3 caused by an increase in Vma x value [10]. PKC is known to comprise a multigene family that encodes several distinct isoforms [21].…”

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confidence: 99%

“…Stimulation of PKC has been shown to downregulate transporter-mediated neurotransmitter uptake into different subcellular fractions and cellular systems, including (i) glycine uptake in human placental choriocarcinoma, C6 glioblastoma cells and mammalian cells expressing a recombinant glycine transporter [7,8,24], (ii) high-affinity GABA uptake in rat cortical synaptosomes, primary neurons and glial cells from rat brain cortex [9,25], (iii) serotonin uptake into human platelets [6,26], and (iv) dopamine uptake into COS cells expressing a recombinant dopamine transporter [5]. In case of serotonin and dopamine uptake, it could be shown that the observed reduction in Vmax values did not result from a decreased number of transporter molecules on the cell surface, since PMA treatment had no effect on the Bmax values of radiolabelled antagonists [5,6,26].…”

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confidence: 99%

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The recombinant GABA transporter GAT1 is downregulated upon activation of protein kinase C

1995

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Treatment of human embryonic kidney 293 cells expressing the rat ~/-aminobutyric acid (GABA) transporter 1 (GATI) with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) was found to decrease the velocity of specific [3H]GABA uptake. This downregulation varied with extracellular GABA concentration and was blocked by the PKC inbibitors 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine 017) and staurosporine. An about 50% reduction of uptake velocity by PMA treatment was observed at GABA concentrations > 1 pM, whereas only a minor effect was seen at low substrate concentrations. These data indicate that GATI activity is downregulated by PKC activation.Key words: GABA uptake; Neurotransmitter transporter; Protein kinase C; Rat system, Corey et al. [10] found an upregulation of GABA uptake caused by an increase in the maximal transport velocity (Vmax) upon phorbol ester treatment. This discrepancy may reflect differences in drug application, since the former investigators added the ester to the incubation medium, whereas Corey et al. injected it directly into the oocytes. Here, we used a mammalian cell expression system to re-analyse PKC modulation of this transporter protein. The human embryonic kidney cell line 293 (HEK-293) has been widely employed to express brain membrane proteins [11,12], and we therefore transfected GAT1 cDNA into these cells for investigating the effect of phorbol ester. Our data show that the velocity of GABA uptake mediated by GAT1 is decreased upon activation of PKC. Materials and methods

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confidence: 76%

Section: Introductionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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The recombinant GABA transporter GAT1 is downregulated upon activation of protein kinase C

1995

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“…In addition to the translocation of GAT1 by PMA observed in our studies, independent investigations have shown that, under certain conditions and in different cell types, phorbol esters can exert other effects on GABA transporter function, including: (1) increasing the K m for GABA in oocytes twofold (Osawa et al, 1994); and (2) decreasing V max 50% (at GABA concentrations Ͼ1 M) in 293 cells (Sato et al, 1995). To further understand the mechanisms underlying these multiple effects of phorbol ester treatment and to minimize their impact on the present studies, we examined changes in [ 3 H]GABA uptake and resting membrane potential after different PMA applications.…”

Section: Coinjection Of Rat Brain Mrna Permits Pma-induced Modulationmentioning

confidence: 93%

Second Messengers, Trafficking-Related Proteins, and Amino Acid Residues that Contribute to the Functional Regulation of the Rat Brain GABA Transporter GAT1

et al. 1997

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Recent evidence indicates that several members of the Na ϩ -coupled transporter family are regulated, and this regulation in part occurs by redistribution of transporters between intracellular locations and the plasma membrane. We elucidate components of this process for both wild-type and mutant GABA transporters (GAT1) expressed in Xenopus oocytes using a combination of uptake assays, immunoblots, and electrophysiological measurements of membrane capacitance, transportassociated currents, and GAT1-specific charge movements. At low GAT1 expression levels, activators of protein kinase C (PKC) induce redistribution of GAT1 from intracellular vesicles to the plasma membrane; at higher GAT1 expression levels, activators of PKC fail to induce this redistribution. However, coinjection of total rat brain mRNA with GAT1 permits PKCmediated modulation at high transporter expression levels. This effect of brain mRNA on modulation is mimicked by coinjection of syntaxin 1a mRNA and is eliminated by injecting synaptophysin or syntaxin antisense oligonucleotides. Additionally, botulinum toxins, which inactivate proteins involved in vesicle release and recycling, reduce basal GAT1 expression and prevent PKC-induced translocation. Mutant GAT1 proteins, in which most or all of a leucine heptad repeat sequence was removed, display altered basal distribution and lack susceptibility to modulation by PKC, delineating one region of GAT1 necessary for its targeting. Thus, functional regulation of GAT1 in oocytes occurs via components common to transporters and to trafficking in both neural and non-neural cells, and suggests a relationship between factors that control neurotransmitter secretion and the components necessary for neurotransmitter uptake.

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Neuronal and glial localization of GAT-1, a high-affinity ?-aminobutyric acid plasma membrane transporter, in human cerebral cortex: With a note on its distribution in monkey cortex

et al. 1998

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High-affinity gamma-aminobutyric (GABA) plasma membrane transporters (GATs) influence the action of GABA, the main inhibitory neurotransmitter in the human cerebral cortex. In this study, the cellular expression of GAT-1, the main cortical GABA transporter, was investigated in the human cerebral cortex by using immunocytochemistry with affinity-purified polyclonal antibodies directed to the C-terminus of rat GAT-1. In temporal and prefrontal association cortex (Brodmann's areas 21 and 46) and in cingulofrontal transition cortex (area 32), specific GAT-1 immunoreactivity (ir) was localized to numerous puncta and fibers in all cortical layers. GAT-1+ puncta were distributed homogeneously in all cortical layers, although they were slightly more numerous in layers II-IV, and appeared to have a preferential relationship to the somata and proximal dendrites of unlabeled pyramidal cells, even though, in many cases, they were also observed around nonpyramidal cells. Electron microscopic observations showed that GAT-1+ puncta were axon terminals that formed exclusively symmetric synapses. In addition, some distal astrocytic processes also contained immunoreaction product. Analysis of the patterns of GAT-1 labeling in temporal and prefrontal association areas (21 and 46), in cingulofrontal transition areas (32), and in somatic sensory and motor areas (1 and 4) of the monkey cortex revealed that its distribution varies according to the type of cortex examined and indicated that the distribution of GAT-1 is similar in anatomically corresponding areas of different species. The present study demonstrates that, in the human homotypical cortex, GAT-1 is expressed by both inhibitory axon terminals and astrocytic processes. This localization of GAT-1 is compatible with a major role for this transporter in GABA uptake at GABAergic synapses and suggests that GAT-1 may contribute to determining GABA levels in the extracellular space.

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Phorbol ester-induced inhibition of GABA uptake by synaptosomes and by Xenopus oocytes expressing GABA transporter (GAT1)

Cited by 42 publications

References 25 publications

The recombinant GABA transporter GAT1 is downregulated upon activation of protein kinase C

The recombinant GABA transporter GAT1 is downregulated upon activation of protein kinase C

Second Messengers, Trafficking-Related Proteins, and Amino Acid Residues that Contribute to the Functional Regulation of the Rat Brain GABA Transporter GAT1

Neuronal and glial localization of GAT-1, a high-affinity ?-aminobutyric acid plasma membrane transporter, in human cerebral cortex: With a note on its distribution in monkey cortex

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