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 1 - dependent [ 3 H] 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 mani- festations of human alcoholism [8,12]. To date, ®ve Na 1 dependent glutamate transporters, GLT-1, GLAST and EAAC 1, EAAT 4 , and EAAT 5 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 glutamate- mediated 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 C 6 glioma cells [2,6]. Interestingly, PKC activation can also increase glutamate uptake by phos- phorylating non-consensus sites of GLAST [5]. Our labora- tory has also reported increases in glutamate uptake in astrocytes subjected to either phorbol ester or chronic etha- nol. 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 trans- port 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 approxi- mately 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- Neuroscience Letters 269 (1999) 145±148 0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(99)00438-3 * Corresponding author. Tel.: 11-520-792-1450 ext. 6951; fax: 11-520-629-1801.