Dibutyryl‐cAMP (dbcAMP) up‐regulates astrocytic chloride‐dependent l‐[³H]glutamate transport and expression of both system x_c⁻ subunits

Abstract

Recent studies have shown that N 6 ,2 H -O-dibutyryladenosine 3 H :5 H cyclic monophosphate (dbcAMP) increases the expression of speci®c subtypes of Na 1 -dependent glutamate transporters in cultured astrocytes. Our group also found that treatment of astrocytes with dbcAMP for several days increases the Na 1 -independent accumulation of L-[ 3 H]glutamate. In this study, the properties of this Na 1 -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 2 and was also inhibited by several anion-exchange inhibitors, including 4,4 H -diisothiocyanatostilbene-2,2 H -disulfonic acid, 4,4 H -dinitrostilbene-2,2 H -disulfonic acid and 4-acetamido-4 Hisothiocyanatostilbene-2,2 H -disulfonic acid, suggesting that this activity is mediated by a Cl 2 -dependent transporter. In addition, this activity was inhibited by micromolar concentrations of several inhibitors of another Cl 2 -dependent (Na 1 -independent) transport activity frequently referred to as system x c 2 (L-cystine, L-a-aminoadipate, L-homocysteate, quisqualate, b-N-oxalyl-l-a,b-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 1 -independent, Cl 2 -dependent L-[ 3 H]glutamate uptake activity was compared for dbcAMP-treated and untreated astrocytes. Treatment with dbcAMP increased the V max of this Cl 2 -dependent transport activity by sixfold but had no effect on the K m value. System x c 2 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 2 -dependent L-[ 3 H]glutamate transport activity in astrocytes and suggests that this effect is related to increased expression of both subunits of system x c 2 . 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 2 may provide alternate approaches to limit this form of injury. Keywords: carboxyphenylglycine, CD98, cystine, 4F2hc, glutamate transport, system x c 2 .Effective clearance of glutamate, a major excitatory amino acid in the mammalian CNS, from the synaptic cleft is necessary to maintain ef®cient synaptic transmission. It is thought that the main mechanism of the removal of glutamate is a family of ®ve recently cloned high-af®nity Na 1 -dependent transporters (for review, see Danbolt 1994;Sims and Robinson 1999). The transporters GLT-1 and GLAST are primarily found in astrocytes, EAAC1 and EAAT4 are predominantly found in neurons and EAAT5 is found only in retina. These transporters are responsible for preventing the accumulation of excitotoxic levels of glutamate as well as limitin...

Cystine/Glutamate Exchange Regulates Metabotropic Glutamate Receptor Presynaptic Inhibition of Excitatory Transmission and Vulnerability to Cocaine Seeking

“…Although in the physiological range and consistent with concentrations of cystine used in vivo to elevate extracellular glutamate in cocaine-withdrawn rats (Baker et al, 2003), the concentration of cystine used in the present study was lower than the reported K m values (ϳ2-100 M) for [35]Scystine or Na ϩ -independent [3]H-glutamate uptake via xc-in experiments using cultured cells (Patel et al, 2004). The reason for this difference between in vivo and in vitro physiological studies and uptake measurements made in vitro is unclear but may reflect differences in the phosphorylation or trafficking of the exchanger between preparations (Gochenauer and Robinson, 2001;Baker et al, 2003).…”

Distribution of the Cystine/Glutamate Antiporter System x−c in the Brain, Kidney, and Duodenum

“…System x c -in the Brain, Duodenum, and Kidney 2001), in mouse kidney and intestine by RT-PCR (Bassi et al 2001), in primary neuronal cultures (Shih et al 2003), immortalized (Tetsuka et al 2001) and primary (Gochenauer and Robinson 2001) astrocyte cultures, and in the HT22 neuronal cell line (Lewerenz et al 2003). One would expect that 4F2hc would be more widely distributed than xCT, because it is paired with numerous other amino acid light chain transporters and, in general, that is what we found.…”

Astrocyte-Derived MCP-1 Mediates Neuroprotective Effects of Noradrenaline

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