calmodulin protein kinase activity in astrocytes chronically exposed to ethanol: influences on glutamate transport

Neuroscience Letters 269 (1999) 145±148

Increased calcium/calmodulin protein kinase activity in astrocytes chronically exposed to ethanol: in¯uences on glutamate transport Thomas L. Smith a, b,*, Edita Navratilova a, b a

Research Service (0±151), Department of Veterans Affairs Medical Center, 3601 S. 6th Avenue, Tucson, AZ 85723, USA b Department of Pharmacology. University of Arizona, Tucson, AZ, USA Received 24 March 1999; received in revised form 14 May 1999; accepted 14 May 1999

Abstract The effects of ethanol exposures on calcium/calmodulin-dependent protein kinase activity as well as its in¯uence on glutamate uptake were determined in astrocytes prepared from neonatal rat cerebral cortex. Acute 15-min exposure to 100 mM ethanol had no effect on Ca 21/CaM-dependent protein kinase activity. However, chronic exposure to 100 mM ethanol for 4 days elicited a signi®cant increase in the activity of this enzyme with no parallel increase in its expression. Ca 21/CaM-independent kinase activity was less than 1% of the Ca 21/CaM-dependent kinase activity and was unaffected by any of the ethanol exposures. Exposure to 100 mM ethanol for four days also resulted in a signi®cant increase in Na 1dependent [ 3H] glutamate uptake which was reversed when ethanol-exposed astrocytes were co-incubated with KN-93, a speci®c inhibitor of Ca 21/CaM kinase. These results suggest that the effects of ethanol on glutamate transport may be mediated in part, by the level of Ca 21/CaM kinase activity. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Astrocytes; Chronic ethanol; Glutamate transport; Ca 21/CaM kinase activity

Glutamate is a major excitatory amino acid found in the mammalian central nervous system. Excessive levels of this transmitter are neurotoxic and have been implicated in neurodegenerative diseases [3] as well as in various manifestations of human alcoholism [8,12]. To date, ®ve Na 1 dependent glutamate transporters, GLT-1, GLAST and EAAC 1, EAAT4, and EAAT5 have been cloned [9]. The clearance of extracellular glutamate is accomplished primarily by the Na 1- dependent glutamate transporters, GLT-1 and GLAST, which are expressed predominantly in glial cells [10,14]. In contrast, EAAC 1 is expressed in neurons and has a minimal in¯uence on chronic glutamatemediated neurotoxcity [13]. Little information has been forthcoming regarding the regulation of the above mentioned transporters. However, each of these transporters has consensus sites for protein kinase C (PKC) and protein kinase A [7,20]. In addition, others have reported that direct activation of PKC with phorbol esters causes a signi®cant increase in glutamate transport activity in C6 glioma cells [2,6]. Interestingly, PKC activation can also increase glutamate uptake by phosphorylating non-consensus sites of GLAST [5]. Our labora* Corresponding author. Tel.: 11-520-792-1450 ext. 6951; fax: 11-520-629-1801.

tory has also reported increases in glutamate uptake in astrocytes subjected to either phorbol ester or chronic ethanol. Moreover, the effects of ethanol on this transport system were reversed by selective PKC inhibitors [18]. These results suggest that PKC may partially mediate the effect of ethanol on glutamate uptake. Although consensus sites for calcium-calmodulin dependent protein kinase have yet to be identi®ed within the glutamate transporter, it is known that multifunctional Ca 21/CaM kinase II is highly concentrated in neurons and glia and recognizes a large array of neuronal substrates, including glutamate receptor/ channels [1,19] and, thus, may also phosphorylate one or more of the glutamate transporters. Therefore, the aims of the present investigation were to determine whether the activity or protein expression of Ca 21/CaM in astrocytes is affected by chronic ethanol exposure and whether the activity of this enzyme can also in¯uence glutamate transport under the same ethanol exposure conditions. Astrocytes were isolated from 0 to 1 day old rat pup cerebral cortices under aseptic conditions as described previously [17]. Cells were plated at a density of approximately 10 5 cells/cm 2 and cultured in T-75 ¯asks with 10 ml Dulbecco's modi®ed Eagles's medium (DMEM) containing 10% fetal calf serum, 50 mg/ml streptomycin and 50 units/ ml penicillin. At con¯uency, Type I astrocytes were subcul-

0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 43 8- 3

146

T.L. Smith, E. Navratilova / Neuroscience Letters 269 (1999) 145±148

Fig. 1. Effects of acute and chronic ethanol exposures on Ca 21/ CaM dependent protein kinase activity of cultured astrocytes. Con¯uent astrocytes were grown in T-25 ¯asks and maintained in DMEM 1 5% FCS in the absence or presence of 100 mM EtOH for 4 days. In parallel experiments, astrocytes grown under the same control conditions were acutely exposed to 100 mM EtOH for 15 min immediately prior to the Ca 21/CaM kinase assays as described in methods. Results represent the mean ^ SD from six to nine individual experiments. One-way ANOVA indicated a signi®cant time effect of ethanol ( ²P , 0:05) with a highly significant effect after 4 days (*P , 0:01, Dunnett's test).

tured into 24-well cluster dishes (Costar) for [ 3H] glutamate uptake or T-25 ¯asks for Ca 21/CaM kinase, electrophoresis and immunoblotting experiments. Con¯uent astrocytes were maintained in DMEM containing 5% FCS in the absence or presence of 100 mM ethanol for four days. Media were replaced each day with fresh ethanol media. To prevent evaporation of ethanol over each 24-h period, wells were placed in a sealed plastic container pre-equilibrated with air-CO2 and maintained at 378C [16]. Multifunctional Ca 21/CaM-dependent protein kinase (CaM kinase II) activity was determined with an assay kit from Gibco BRL according to the manufacturer's instructions. Brie¯y, control and age-matched ethanol exposed astrocytes maintained in T-25 ¯asks were washed three times in 10 ml phosphate-buffered saline (PBS) (no Ca 21, no Mg 21), harvested and homogenized in 1 ml ice-cold buffer containing 20 mM PIPES (pH 7.0), 0.5 mM EGTA, 1 mM EDTA, 10 mg/ml leupetin, 10 mM sodium pyrophosphate, 0.4 mM ammonium molydate and 0.1% Triton X-100. The reaction mixture contained 25 mM PIPES (pH 7.0), 50 mM ATP, 15 mM autocamtide 3, 200 mM BSA, 20 mM MgCl2, 1 mM CaCl2, 20 mg/ml calmodulin and 5±10 mg sample protein in a total volume of 50 ml. Ca 21-calmodulin independent activity was determined by substituting 1 mM EGTA for Ca 21 and calmodulin. Reactions were started with the addition of 20±25 mCi/ml carrier free [ 32P] ATP (3000±6000 Ci/mmol, Amersham) and incubations allowed to proceed for 2 min at 308C. Reactions were stopped with 10 ml ice-cold 15% TCA after which the samples were placed on ice for 30 min. Following centrifugation at 14 000 £ g for 2 min, 30 ml aliquots of the supernatants were spotted on phosphocellulose disks which were then washed twice in 1% H3PO4 solution. Radioactivity remaining on each disk was determined by liquid scintillation spectroscopy. CaM kinase activity was expressed as pmoles P/min/mg protein.

Western blot analysis of Ca 21/CaM kinase II was performed essentially as described previously [6]. Brie¯y, astrocytes cultured in media containing either 0, 25, or 100 mM ethanol for 4 days were harvested and homogenized in 1 ml ice-cold 20 mM TRIS buffer (pH 7.5) containing 4 mM EGTA, 2 mM EDTA, 25 mg/ml each of pepstatin, aprotinin and leupeptin and 1 mM phenylmethylsulfonyl ¯uoride. Aliquots were taken for Bradford protein analysis, after which the remainder of each homogenate was boiled in Laemmli sample buffer for 5 min, aliquoted and stored frozen at 2808C until needed. Total protein samples (10 mg) from each experimental group were separated by SDS-PAGE electrophoresis using 10% acrylamide minigels and transferred to nitrocellulose membranes. Non-speci®c binding sites were blocked with PBS containing 0.05% Tween-20 (PBS-T) and 5% BSA at room temperature for 45 min. Membranes were then incubated with Ca 21/CaM kinase II speci®c antibody (Gibco, BRL) diluted 1:100 000 for 60 min. After several washes with PBS-T, membranes were incubated with a horseradish peroxidase linked secondary rabbit antibody (Amersham) diluted 1:100 000 for 30 min followed by additional washes in PBS-T. Immunoreactivities in the blots were detected using Super Signal Ultra Chemiluminescent Substrate (Pierce) and Hyper®lm (Amersham). Quantitation of immunoblots was performed using a gel scanner (Sharp Model JX-330) and software from Bio Image, Ann Arbor, MI. Data were calculated as percent of control values within a given gel and expressed as mean percent ^ SD. Sodium-dependent glutamate uptake was carried out over a concentration range of 3:5 £ 1027 to 3:5 £ 1024 M [ 3H] glutamate for 4 min at 378C as described previously [16]. Astrocytes cultured in 24-well cluster dishes were maintained in media in the absence or presence of 100 mM ethanol for 4 days as mentioned above. In some experiments, 10 mM KN-93, a speci®c inhibitor of Ca 21/CaM kinase II [20], was added to control and ethanol containing media throughout the above exposure time. All media were washed three times with a HEPES-buffered salt solution immediately prior to the [ 3H] glutamate uptake assays. Km and Vmax values were determined by nonlinear regression analysis of the uptake saturation curves using a curve ®tting program from Graph Pad Prism (San Diego, CA). Vmax values for glutamate uptake are expressed as nmol/mg protein per min. The effects of ethanol exposures on Ca 21/CaM-dependent protein kinase II activities are illustrated in Fig. 1. Acute exposure to 100 mM ethanol for 15 min had no effect on Ca 21/CaM-dependent kinase activity. In contrast, exposure to 100 mM ethanol for 4 days resulted in a modest but signi®cant increase of 25% in Ca 21/CaM-dependent kinase activity (P , 0:01, Dunnett's test). These observations are consonant with earlier work in which Ca 21/CaM-dependent phosphorylation of speci®c synaptic membrane proteins was enhanced in rats that received ethanol by gastric intubation for 4 weeks [15]. Ca 21/CaM-independent activities

T.L. Smith, E. Navratilova / Neuroscience Letters 269 (1999) 145±148

Fig. 2. Effects of chronic ethanol exposures on the protein expression of Ca 21/CaM kinase in astrocytes homogenates. Con¯uent astrocytes were maintained in DMEM 1 5% FCS in the absence or presence of 25 or 100 mM EtOH for 4 days as described in methods. Ten micrograms of denatured sample proteins were applied to each lane. Immunoreactive bands in lanes (A±C) correspond to control, 25 and 100 mM EtOH treated astrocytes, respectively. Under each experimental condition, characteristic a and b subunits of Ca 21/CaM kinase were observed, but their intensities unaffected. The immunoblots shown are representative of results from at least six to eight individual observations.

were 37 ^ 17 and 31 ^ 33 pmol/mg protein per min for control and 4-day ethanol exposed astrocytes, respectively. As these activities were 1% or less of the Ca 21/CaM-dependent activities, the data indicate that under the experimental conditions employed little if any autophosphorylation or autoactivation of Ca 21/CaM kinase occurred in the processing or harvesting of control or ethanol exposed astrocytes. A representative immunoblot of total Ca 21/CaM kinase protein in control and ethanol exposed astrocytes is shown in Fig. 2. The antibody used in the present study detected both a minor a and a major b subunit with approximate molecular weights of 50 and 60 kDa, respectively. Interestingly, the expression of these subunits in astrocytes is quantitatively similar to that observed in 7±8 day old cultured

Fig. 3. The effects of Ca 21/CaM kinase inhibition on Na 1-dependent glutamate uptake in control and EtOH exposed astrocytes. Con¯uent astrocyte were grown in 24-well cluster dishes and maintained in DMEM 1 5% FCS in the absence or presence of 100 mM or EtOH for 4 days as in Fig. 1. In some experiments, 10 mM KN-93 was included in both control and EtOH containing media. Vmax for glutamate uptake was determined using a concentration range of 3:5 £ 1027 to 3:5 £ 1024 M [ 3H] glutamate as described in methods. Results are the mean ^ SD from three to six separate experiments. *P , 0:01 versus EtOH exposure alone (Student's t-test, unpaired).

147

neurons [11]. Visual inspection of Fig. 2 indicates that chronic exposure to 25 or 100 mM ethanol for 4 days did not affect the protein expression of either of the Ca 21/CaM kinase subunits. Quantitative analysis of band intensities as described in Methods con®rmed this observation. Thus, when expressed as percent of paired control values ^ SD, Ca 21/CaM kinase immunoreactivities from astrocytes exposed to 25 and 100 mM ethanol were 103 ^ 10 and 99 ^ 12, respectively (n ˆ 8). It is possible that the 25% increase in Ca 21/CaM kinase activity after ethanol exposure may re¯ect a corresponding increase in its expression, but that such a relatively small increase may not be distinguishable. Therefore, identical protein samples were run side by side at either 5 mg (100%) or 6.25 mg (125%) total sample protein. Ca 21/CaM kinase immunoreactivity from the 6.25 mg protein samples was 115 ^ 5% of paired 5 mg sample values and was statistically signi®cant (P , 0:05, paired ttest, n ˆ 8). These results indicate that our procedures are suf®ciently accurate to detect a possible 25% change in protein expression and support our conclusions that chronic ethanol exposure increases the activity of Ca 21/CaM-dependent protein kinase without altering its expression. The molecular mechanism responsible for such an increase in activity is currently unknown, but may re¯ect an alteration in calmodulin binding or the normal interaction of catalytic and autoinhibitory domains of this enzyme [4]. The effects of the selective Ca 21/CaM kinase inhibitor, KN-93, on maximal [ 3H] glutamate uptake capacity (Vmax) in control and ethanol-exposed astrocytes are illustrated in Fig. 3. Consistent with previous ®ndings from this laboratory [18], Vmax values for [ 3H] glutamate uptake were significantly increased after exposure to 100 mM ethanol for 4 days (P , 0:001, Student's test, unpaired). However, coincubation with 10 mM KN-93 completely reversed the effects of ethanol on [ 3H]glutamate uptake while having no signi®cant effect on corresponding control values. The results of the present investigation suggest that the ethanolinduced increase in Ca 21/CaM kinase activity may mediate, in part, the increase in [ 3H]glutamate uptake observed after ethanol exposure, perhaps by increasing the state of phosphorylation of GLT-1 or GLAST glutamate transporters in a manner similar to that resulting from protein kinase C activation [2]. This hypothesis is supported by the observation that chronic ethanol exposure does not affect the total protein expression of either GLT-1 or GLAST [18]. Further evidence for this hypothesis awaits the development of immunoprecipitation procedures for GLT-1 and GLAST, or availability of phospho-speci®c antibodies directed at these transporters. This project was supported by a medical research grant from the Department of Veterans Affairs, Washington, DC, USA. [1] Bronstein, J., Nishimura, R., Lasher, R., Cole, R., de Vellis, J., Farber, D. and Wasterlain, C., Calmodulin kinase II in

148

[2]

[3] [4] [5] [6]

[7]

[8]

[9] [10]

[11]

T.L. Smith, E. Navratilova / Neuroscience Letters 269 (1999) 145±148 pure cultured astrocytes. J. Neurochem., 50 (1998) 45± 49. Casado, M., Bendahan, A., Zafra, F., Danbolt, N.C., Aragon, C., Gimenez, C. and Kanner, B.I., Phosphorylation and modulation of brain glutamate transporters by protein kinase C. J. Biol. Chem., 268 (1993) 27313±27317. Choi, D.W., Excitotoxic cell death. J. Neurobiol., 23 (1992) 1261±1276. Colbran, R.J., Regulation and role of brain calcium/calmodulin-dependent protein kinase II. Neurochem. Int., 21 (1992) 469±497. Conradt, M. and Stoffel, W., Inhibition of the high af®nity brain glutamate transporter GLAST-1 via direct phosphorylation. J. Neurochem., 68 (1997) 1244±1251. Dowd, L.A. and Robinson, M.B., Rapid stimulation of EAAC 1-mediated Na 1-dependent l-glutamate transport activity in C6 glioma cells by phorbol ester. J. Neurochem., 67 (1996) 508±516. Fairman, W.A., Vandenberg, R.J., Arriza, J.L., Kavanaugh, M.P. and Amara, S.G., An excitatory amino-acid transporter with properties of a ligand-gated chloride channel. Nature, 375 (1995) 599±603. Guochuan, E., Ragan, P., Chang, R., Chen, S., Linnoila, V.M. and Coyle, J.T., Increased glutamatergic neurotransmission and oxidative stress after alcohol withdrawal. Am. J. Psychiat., 155 (1998) 726±732. Gegelashvili, G. and Schousboe, A., High-af®nity glutamate transporters: regulation of expression and activity. Mol. Pharmacol., 52 (1997) 6±15. Lehre, K.P., Levy, L.M., Ottersen, O.P., Storm-Mathisen, J. and Danbolt, N.C., Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. J. Neurosci., 15 (1995) 1835± 1853. Morioka, M., Fukumaga, K., Nagahiro, S., Kurino, M., Ushio, Y. and Miyamoto, E., Glutamate-induced loss of Ca 21/ calmodulin-dependent protein kinase II activity in cultured

[12] [13]

[14]

[15]

[16] [17]

[18] [19]

[20]

rat hippocampal neurons. J. Neurochem., 64 (1995) 2132± 2139. Rossetti, Z.L. and Carboni, S., Ethanol withdrawal is associated with increased extracellular glutamate in the rat brain striatum. Eur. J. Pharmacol., 283 (1998) 177±183. 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. and Welty, D.F., Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron, 16 (1996) 675±686. Rothstein, J.D., Martin, L., Levey, A.I., Dykes-Hoberg, M., Jin, L., Wu, D., Nash, N. and Kuncl, R.W., Localization of neuronal and glial glutamate transporters. Neuron, 13 (1994) 713±725. Shanley, B., Gurd, J. and Kalant, H., Ethanol tolerance and enhanced calcium/calmodulin-dependent phosphorylation of synaptic membrane proteins. Neurosci. Lett., 58 (1985) 55±59. Smith, T.L. and Zsigo, A., Increased Na 1-dependent high af®nity uptake of glutamate in astrocytes chronically exposed to ethanol. Neurosci. Lett., 218 (1996) 142±144. Smith, T.L., Selective effects of ethanol exposure on metabotropic glutamate receptor and guanine nucleotide stimulated phospholipase C activity in primary cultures of astrocytes. Alcohol, 11 (1994) 405±409. Smith, T.L., Regulation of glutamate uptake in astrocytes continuously exposed to ethanol. Life Sci., 61 (1997) 2499±2505. Soderling, T.R., Tan, S.E., McGlade-McCulloh, E., Yamamoto, H. and Fukunaga, K., Excitatory interactions between glutamate receptors and protein kinases. J. Neurobiol., 25 (1994) 304±311. Sumi, M., Kikuchi, K., Ishikawa, T., Ishii, A., Hagirwara, M., Nagatsu, T. and Hidaka, H., The newly synthesized selective Ca 21/calmodulin dependent protein kinase II inhibitor (N-93 reduces dopamine contents in PC12h cells. Biochem. Biophys. Res. Commun., 181 (1991) 968±975.