Dibutyryl-cAMP (dbcAMP) up-regulates astrocytic chloride-dependent L-[H-3]glutamate transport and expression of both system x(c)(-) subunits

Recent studies have shown that N-6,2 ' -O-dibutyryladenosine 3 ' :5 ' cycli c monophosphate (dbcAMP) increases the expression of specific subtypes of N a+-dependent glutamate transporters in cultured astrocytes. Our group also found that treatment of astrocytes with dbcAMP for several days increases t he Na+-independent accumulation of L-[H-3]glutamate. In this study, the pro perties of this Na+-independent accumulation were characterized, and the me chanism by which dbcAMP up-regulates this process was investigated. This ac cumulation was markedly reduced in the absence of Cl- and was also inhibite d by several anion-exchange inhibitors, including 4,4 ' -diisothiocyanatost ilbene-2,2 ' -disulfonic acid, 4,4 ' -dinitrostilbene-2,2 ' -disulfonic aci d and 4-acetamido-4 ' -isothiocyanatostilbene-2,2 ' -disulfonic acid, sugge sting that this activity is mediated by a Cl--dependent transporter. In add ition, this activity was inhibited by micromolar concentrations of several inhibitors of another Cl--dependent (Na+-independent) transport activity fr equently 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 phenylgly cine derivatives previously characterized as inhibitors of metabotropic glu tamate receptor activation. The concentration-dependence for Na+-independen t, Cl--dependent L-[H-3]glutamate uptake activity was compared for dbcAMP-t reated and untreated astrocytes. Treatment with dbcAMP increased the V-max of this Cl--dependent transport activity by sixfold but had no effect on th e K-m value. System x(c)(-) requires two subunits, xCT and 4F2hc/CD98, to r econstitute functional activity. We found that dbcAMP caused a twofold incr ease in the levels of xCT mRNA and a sevenfold increase in the levels of 4F 2hc/CD98 protein. This study indicates that dbcAMP up-regulates Cl--depende nt 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 g lutathione and protection from oxidant injury, understanding the regulation of system x(c)(-) may provide alternate approaches to limit this form of i njury.