Gochenauer G E, Robinson M B
Departments of Pediatrics and Pharmacology, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
J Neurochem. 2001 Jul;78(2):276-86. doi: 10.1046/j.1471-4159.2001.00385.x.
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.
最近的研究表明，N(6),2'-O-二丁酰腺苷3':5'-环一磷酸（dbcAMP）可增加培养星形胶质细胞中钠依赖性谷氨酸转运体特定亚型的表达。我们小组还发现，用dbcAMP处理星形胶质细胞数天会增加L-[3H]谷氨酸的非钠依赖性积累。在本研究中，对这种非钠依赖性积累的特性进行了表征，并研究了dbcAMP上调此过程的机制。在没有Cl(-)的情况下，这种积累明显减少，并且还受到几种阴离子交换抑制剂的抑制，包括4,4'-二异硫氰酸根合芪-2,2'-二磺酸、4,4'-二硝基芪-2,2'-二磺酸和4-乙酰氨基-4'-异硫氰酸根合芪-2,2'-二磺酸，这表明该活性是由Cl(-)依赖性转运体介导的。此外，这种活性受到微摩尔浓度的几种另一种Cl(-)依赖性（非钠依赖性）转运活性抑制剂的抑制，这种转运活性通常称为系统xc(-)（L-胱氨酸、L-α-氨基己二酸、L-高半胱氨酸、喹啉酸、β-N-草酰-L-α,β-二氨基丙酸、鹅膏蕈氨酸）。这种活性受到几种先前被表征为代谢型谷氨酸受体激活抑制剂的苯甘氨酸衍生物的竞争性抑制。比较了dbcAMP处理和未处理的星形胶质细胞对非钠依赖性、Cl(-)依赖性L-[3H]谷氨酸摄取活性的浓度依赖性。用dbcAMP处理使这种Cl(-)依赖性转运活性的V(max)增加了六倍，但对K(m)值没有影响。系统xc(-)需要两个亚基，xCT和4F2hc/CD98，才能重建功能活性。我们发现dbcAMP使xCT mRNA水平增加了两倍，使4F2hc/CD98蛋白水平增加了七倍。本研究表明，dbcAMP上调星形胶质细胞中Cl(-)依赖性L-[3H]谷氨酸转运活性，并表明这种作用与系统xc(-)两个亚基的表达增加有关。因为这种活性被认为对谷胱甘肽的合成和免受氧化损伤很重要，了解系统xc(-)的调节可能会提供限制这种损伤形式的替代方法。
