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-[H-3]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 x(c)(-) (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-[H-3]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 x(c)(-) 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-[H-3]glutamate transport activity in astrocytes and suggests that this effect is related to increased expression of both subunits of system x(c)(-). Because this activity is thought to be important for the synthesis of glutathione and protection from oxidant injury, understanding the regulation of system x(c)(-) may provide alternate approaches to limit this form of injury.