Dibutyryl-cAMP (dbcAMP) up-regulates astrocytic chloride-dependent L-[3H]glutamate transport and expression of both system xc(-) subunits.
Recent studies have shown that N(6),2'-O-dibutyryladenosine 3':5' cyclic monophosphate (dbcAMP) increases the expression of specific subtypes of Na(+)-dependent glutamate transporters in cultured astrocytes. Our group also found that treatment of astrocytes with dbcAMP for several days increases the Na(+)-independent accumulation of L-[3H]glutamate. In this study, the properties of this Na(+)-independent accumulation were characterized, and the mechanism by which dbcAMP up-regulates this process was investigated. This accumulation was markedly reduced in the absence of Cl(-) and was also inhibited by several anion-exchange inhibitors, including 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, 4,4'-dinitrostilbene-2,2'-disulfonic acid and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, suggesting that this activity is mediated by a Cl(-)-dependent transporter. In addition, this activity was inhibited by micromolar concentrations of several inhibitors of another Cl(-)-dependent (Na(+)-independent) transport activity frequently referred to as system xc(-) (L-cystine, L-alpha-aminoadipate, L-homocysteate, quisqualate, beta-N-oxalyl-l-alpha,beta-diaminopropionate, ibotenate). This activity was competitively inhibited by several phenylglycine derivatives previously characterized as inhibitors of metabotropic glutamate receptor activation. The concentration-dependence for Na(+)-independent, Cl(-)-dependent L-[3H]glutamate uptake activity was compared for dbcAMP-treated and untreated astrocytes. Treatment with dbcAMP increased the V(max) of this Cl(-)-dependent transport activity by sixfold but had no effect on the K(m) value. System xc(-) requires two subunits, xCT and 4F2hc/CD98, to reconstitute functional activity. We found that dbcAMP caused a twofold increase in the levels of xCT mRNA and a sevenfold increase in the levels of 4F2hc/CD98 protein. This study indicates that dbcAMP up-regulates Cl(-)-dependent L-[3H]glutamate transport activity in astrocytes and suggests that this effect is related to increased expression of both subunits of system xc(-). Because this activity is thought to be important for the synthesis of glutathione and protection from oxidant injury, understanding the regulation of system xc(-) may provide alternate approaches to limit this form of injury.
Gochenauer
G E
GE
Departments of Pediatrics and Pharmacology, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Robinson
M B
MB
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D027322:Amino Acid Transport System X-AG; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001692:Biological Transport; D003994:Bucladesine / Q000494:pharmacology*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018846:Excitatory Amino Acids / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D018698:Glutamic Acid / Q000378:metabolism*; D007700:Kinetics; D021122:Protein Subunits; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects; D014316:Tritium
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D002712:Chlorides; D018690:Excitatory Amino Acid Agonists; D018846:Excitatory Amino Acids; D021122:Protein Subunits; D012333:RNA, Messenger; D014316:Tritium; D018698:Glutamic Acid; D003994:Bucladesine