Isoflurane Induces a Protein Kinase C -Dependent Increase in Cell-Surface Protein Level and Activity of Glutamate Transporter Type 3

Glutamate transporters regulate extracellular concentrations of glutamate, an excitatory neurotransmitter in the central nervous system. We have shown that the commonly used anesthetic isoflurane increased the activity of glutamate transporter type 3 (excitatory amino acid transporter 3, EAAT3) possibly via a protein kinase C (PKC)-dependent pathway. In this study, we showed that isoflurane induced a timeand concentrationdependent redistribution of EAAT3 to the cell membrane in C6 glioma cells. This redistribution was inhibited by staurosporine, a pan PKC inhibitor, or by 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö6976) at a concentration that selectively inhibits conventional PKC isozymes (PKC , , and ). This isoflurane-induced EAAT3 redistribution was also blocked when the expression of PKC but not PKC proteins was down-regulated by the respective antisense oligonucleotides. The isoflurane-induced increase of glutamate uptake by EAAT3 was abolished by the down-regulation of PKC expression. Immunoprecipitation with an anti-EAAT3 antibody pulled down more PKC in cells exposed to isoflurane than in control cells. Isoflurane also increased the phosphorylated EAAT3 and the redistribution of PKC to the particulate fraction of cells. Consistent with the results in C6 cells, isoflurane also increased EAAT3 cell-surface expression and enhanced the association of PKC with EAAT3 in rat hippocampal synaptosomes. Our results suggest that the isoflurane-induced increase in EAAT3 activity requires an increased amount of EAAT3 protein in the plasma membrane. These effects are PKC -dependent and may rely on the formation of an EAAT3-PKC complex. Together, these results suggest an important mechanism for the regulation of glutamate transporter functions and expand our understanding of isoflurane pharmacology at cellular and molecular levels. Glutamate transporters (also called excitatory amino acid transporters, EAAT) are important in regulating extracellular concentrations of glutamate (Danbolt, 2001), a major excitatory neurotransmitter in the mammalian central nervous system (CNS). By transporting glutamate from extracellular to intracellular space under physiological conditions, EAATs prevent extracellular glutamate accumulation and regulate glutamate neurotransmission. Five EAATs have been identified: EAAT1 to EAAT5 (Danbolt, 2001). In rats, EAAT1 and EAAT2 are found in glial cells, and EAAT3 and EAAT4 are mainly expressed in neurons, whereas EAAT5 is located in neurons and glial cells of retina (Rothstein et al., 1994; Lehre et al., 1995; Arriza et al., 1997). The transporting functions of all five EAATs are sodium-dependent. They use the transmembrane gradient of Na , K , and H as a driving force to uptake glutamate (Billups et al., 1998; Danbolt, 2001). Studies on regional distribution of EAATs have shown that EAAT3 is widely distributed in forebrain, hippocampus, and cerebellum and that EAAT4 is largely restricted to the molecular cell layer of cerebellum (Rothstein et al., 1994; Danbolt, 2001). Thus, EAAT3 is the major neuronal EAAT in the CNS. We have shown that isoflurane, a commonly used volatile anesthetic in clinical practice, increases EAAT3 activity and that these effects are possibly protein kinase C (PKC)dependent (Do et al., 2002). It has been shown that PKC activation-induced increase of EAAT3 activity is associated with an increase of EAAT3 proteins in the plasma membrane (Davis et al., 1998) and that PKC is involved in regulating EAAT3 expression in the plasma membrane (Gonzalez et al., 2002). In this study, we determined whether isoflurane inThis study was supported by National Institutes of Health grants R01GM065211 and R01-NS045983 (to Z.Z.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.104.007443. ABBREVIATIONS: EAAT, excitatory amino acid transporter; CNS, central nervous system; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; IPSC, inhibitory postsynaptic current; AMPA, -amino3-hydroxy-5-methyl-4-isoxazolepropionic acid; Gö6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)carbazole; PBS-Ca/Mg, phosphate-buffered saline containing calcium and magnesium. 0026-895X/05/6705-1522–1533$20.00 MOLECULAR PHARMACOLOGY Vol. 67, No. 5 Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics 7443/1198168 Mol Pharmacol 67:1522–1533, 2005 Printed in U.S.A. 1522 at A PE T Jornals on O cber 9, 2017 m oharm .aspeurnals.org D ow nladed from creases cell-surface expression of EAAT3 proteins and whether the isoflurane-induced increase of EAAT3 activity and cell-surface expression is PKC isozyme-specific. We used C6 glioma cells as one of our study models. C6 cells have been shown to express EAAT3 only and have been frequently used in studies of EAAT3 activity and cell-surface expression (Davis et al., 1998; Gonzalez et al., 2002). We then used rat hippocampal synaptosomes to determine whether the results observed in C6 cells are relevant to the CNS. Our results indicate that isoflurane induced a PKC -dependent increase of EAAT3 activity and cell-surface expression and that isoflurane also increased the coimmunoprecipitation of PKC with EAAT3, which may suggest an increased interaction between these two proteins in cells. Materials and Methods Materials. Ham’s F-10 nutrient mixture was from Invitrogen (Carlsbad, CA). Six-well tissue culture plates and 25and 75-cm tissue culture flasks were manufactured by Corning Glassworks (Corning, NY). L-[H]Glutamate (specific activity, 56 Ci/mM) was purchased from Amersham Biosciences Inc. (Piscataway, NJ). Wizard Plus Minipreps were from Promega (Madison, WI). Anti-PKC , PKC I, PKC , and PKC antibodies and Protein A/G Plus-Agarose were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Affinity-purified polyclonal rabbit anti-EAAT1 and anti-EAAT3 antibodies were from Alpha Diagnostics International (San Antonio, TX). Sulfo-N-hydroxysulfosuccinimidobiotin and immunopureimmobilized monomeric avidin were from Pierce (Rockford, IL). Fluorescent antibodies were purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). Vectashield mounting medium was from Vector Laboratories (Burlingame, CA). Oligonucleotides were prepared by the University of Virginia Biomolecular Research Facility (Charlottesville, VA). Nitrocellulose membranes for Western blot were from Bio-Rad (Hercules, CA). Sytox green nucleic acid stain, ProLong Antifade Kit, and Pro-Q Diamond dye were from Molecular Probes (Eugene, OR). Rat C6 glioma cells were from American Type Culture Collection (Manassas, VA). Sprague-Dawley rats were from Hilltop Laboratory Animals, Inc. (Scottsdale, PA). Complete protease inhibitors were from Roche Applied Science (Indianapolis, IN). Gö6976 was from BIOMOL Research Laboratories (Plymouth Meeting, PA). Other reagents were purchased from Sigma-Aldrich (St. Louis, MO). Cell Culture and Isoflurane Incubation. Rat C6 glioma cells were cultured in flasks (for biotinylation and immunoprecipitation experiments) or on coverslips (for immunocytochemistry experiments, coverslips were placed in six-well plates) in Ham’s F-10 nutrient mixture containing 15% horse serum and 2.5% fetal bovine serum at 37°C in a 95% air/5% CO2 incubator. When cells were 80% confluent, the culture medium was replaced with a serum-free medium (Ham’s F-10 mixture only) for 24 h before isoflurane incuba-