The Effects of Lidocaine on the Activity of Glutamate Transporter EAAT3: The Role of Protein Kinase C and Phosphatidylinositol 3-Kinase

Hwan, Sang; Fang, Hongda; Ham, Byung-Moon; Zuo, Zhiyi

doi:10.1097/00000539-200211000-00030

Cited by 44 publications

(23 citation statements)

References 23 publications

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“…This result was in accordance with our previous studies (Do et al, 2002a;Do et al, 2002b;Kim et al, 2003). When ethanol (50 mM for 96 hr)-treated oocytes were exposed to PMA (100 nM for 10 min), ethanol-induced decrease in EAAT3 activity was abolished (1.05 Ϯ 0.09 C in the PMA plus ethanol group, 0.73 Ϯ 0.08 C in the ethanol group, n ϭ 18, p Ͻ 0.05, Fig.…”

Section: Resultssupporting

confidence: 94%

“…4). Similar to our previous results (Do et al, 2002a), preincubation of the oocytes with a PI3 K inhibitor, wortmannin (1 M for 1 hr), significantly decreased the basal EAAT3 activity. Oocytes exposed to the combination of ethanol and wortmannin had a further decrease in EAAT3 activity compared with the wortmannin or the ethanol alone group (p Ͻ 0.05, Fig.…”

Section: Resultssupporting

confidence: 90%

“…This regulation results in the change of Vmax but not Km of EAATs for glutamate (Davis et al, 1998). We recently demonstrated that an increased Vmax and no change in Km could be common kinetic features of the acute effects of ethanol and various anesthetic agents on EAAT3 for glutamate (Do et al, 2002a;Do et al, 2003;Do et al, 2002b;Kim et al, 2003). In the present study, the kinetic study also showed that ethanol (50 mM) exposure for 96 hr decreased the Vmax but not the Km of glutamate uptake via EAAT3, suggesting that chronic ethanol exposure reduces the available number or the turnover rate of EAAT3 rather than changing the affinity of EAAT3 for glutamate.…”

Section: Discussionmentioning

confidence: 96%

“…Isolation and microinjecion of Xenopus oocytes were performed as described by Do et al (Do et al, 2002a;Do et al, 2002b). Mature female Xenopus laevis frogs were purchased from Purunamu (Seoul, Korea), and fed with regular frog brittle twice weekly.…”

Section: Methodsmentioning

confidence: 99%

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Erratum

2005

Alcoholism Clin Exp Res

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Background: Glutamate transporters (excitatory amino acid transporters, EAAT) regulate extracellular concentrations of glutamate, a major excitatory neurotransmitter. We reported that acute ethanol exposure increases the activity of a major neuronal EAAT, EAAT3. This effect is consistent with the general inhibitory effect of acute alcohol toxicity in the central nervous system (CNS). However, chronic ethanol exposure has CNS presentations different from acute alcohol toxicity. We hypothesize that chronic ethanol exposure will affect the EAAT3 activity differently from acute ethanol exposure.Methods: EAAT3 was expressed in Xenopus oocytes by injection of EAAT3 mRNA. Oocytes were incubated with diluted ethanol for 24 -96 hr. Using two-electrode voltage clamp, membrane currents were recorded after the application of L-glutamate. Responses were quantified by integration of the current trace and reported as microCoulombs (C).Results: Ethanol (10 -100 mM) reduced EAAT3 activity in a time-dependent and reversible manner. After a 96 hr-incubation, the activity was significantly decreased compared to the control values at any concentrations tested in this study. Kinetic study demonstrated that a 96 hr-exposure to 50 mM ethanol significantly decreased Vmax (3.6 Ϯ 0.3 for control versus 2.6 Ϯ 0.3 C for ethanol, n ϭ 20, p Ͻ 0.05) but had no effect on Km (57.6 Ϯ 12.8 for control versus 51.8 Ϯ 16.3 M for ethanol, n ϭ 20, p Ͼ 0.05) of EAAT3 for glutamate. When ethanol (50 mM for 96 hr)-treated oocytes were incubated with phorbol-12-myrisate-13-acetate (50 or 100 nM for 10 min), ethanol-induced decrease in EAAT3 activity was abolished. Preincubation of the oocytes with 100 M chelerythrine significantly decreased EAAT3 activity (1.00 Ϯ 0.08 for control versus 0.51 Ϯ 0.09 C for chelerythrine group, n ϭ 18 -20, p Ͻ 0.05). However, there was no statistical difference among the chelerythrine, ethanol, or chelerythrine plus ethanol groups. Likewise, staurosporine (2 M for 1 hr) significantly decreased EAAT3 activity and there was no statistical difference among the staurosporine, ethanol, or staurosporine plus ethanol groups.Conclusions: Our results show that chronic ethanol exposure decreases EAAT3 activity at clinically relevant concentrations and that this effect may be protein kinase C-dependent. Such an effect could be a neuroadaptive mechanism to overcome the inhibitory effect of ethanol on the excitatory neurotransmission.

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Section: Resultssupporting

confidence: 94%

Section: Resultssupporting

confidence: 90%

Section: Discussionmentioning

confidence: 96%

Section: Methodsmentioning

confidence: 99%

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Erratum

2005

Alcoholism Clin Exp Res

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“…However, other transporters did not change density and/or activity upon PKC stimulation in oocytes, including the endogenous Na ϩ /H ϩ exchanger, the rat GABA transporter GAT1, the glutamate transporter EAAT3, or hSGLT1 (7,10,12,53). In the case of GAT1, even a redistribution of transport proteins from the intracellular pool to the plasma membrane has been reported (10).…”

Section: Regulation Of Heterologously Expressed Transporters By Pkc Imentioning

confidence: 99%

Substrate-induced changes in the density of peptide transporter PEPT1 expressed in Xenopus oocytes

Mertl¹,

Daniel²,

Kottra³

2008

American Journal of Physiology-Cell Physiology

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Mertl M, Daniel H, Kottra G. Substrate-induced changes in the density of peptide transporter PEPT1 expressed in Xenopus oocytes. Am J Physiol Cell Physiol 295: C1332-C1343, 2008. First published September 17, 2008 doi:10.1152/ajpcell.00241.2008.-The adaptation of the capacity of the intestinal peptide transporter PEPT1 to varying substrate concentrations may be important with respect to its role in providing bulk quantities of amino acids for growth, development, and other nutritional needs. In the present study, we describe a novel phenomenon of the regulation of PEPT1 in the Xenopus oocyte system. Using electrophysiological and immunofluorescence methods, we demonstrate that a prolonged substrate exposure of rabbit PEPT1 (rPEPT1) caused a retrieval of transporters from the membrane. Capacitance as a measure of membrane surface area was increased in parallel with the increase in rPEPT1-mediated transport currents with a slope of ϳ5% of basal surface per 100 nA. Exposure of oocytes to the model peptide Gly-L-Gln for 2 h resulted in a decrease in maximal transport currents with no change of membrane capacitance. However, exposure to substrate for 5 h decreased transport currents but also, in parallel, surface area by endocytotic removal of transporter proteins from the surface. The reduction of the surface expression of rPEPT1 was confirmed by presteady-state current measurements and immunofluorescent labeling of rPEPT1. A similar simultaneous decrease of current and surface area was also observed when endocytosis was stimulated by the activation of PKC. Cytochalasin D inhibited all changes evoked by either dipeptide or PKC stimulation, whereas the PKC-selective inhibitor bisindolylmaleimide only affected PKC-stimulated endocytotic processes but not substratedependent retrieval of rPEPT1. Coexpression experiments with human Na ϩ -glucose transporter 1 (hSGLT1) revealed that substrate exposure selectively affected PEPT1 but not the activity of hSGLT1. electrophysiology; dipeptide; presteady-state currents; surface expression; endocytosis THE CAPACITY OF A TRANSPORTER PROTEIN can be regulated either by variation of its membrane abundance or variation of its kinetic properties (e.g., turnover rate, substrate affinity, and ion dependency). Transporter density in the membrane usually involves exocytosis or endocytosis of the proteins delivered to or retrieved from the plasma membrane (26,29,33,53), whereas the kinetic properties can be regulated either by direct phosphorylation and dephosphorylation (8,28) or by the association of cytosolic regulatory proteins (46). A number of observations have suggested that substrates can, via nonkinetic mechanisms, also regulate the activity of their transporters, but the underlying signaling pathways that cause such substrate-dependent protein regulation are mostly unknown. Such transport regulation processes have even been observed in heterologous expression systems. Xenopus laevis oocytes exposed to a mixture of 20 amino acids at concentrations approximating those in Xenopus p...

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