CYSTINE GLUTAMATE ANTIPORTER EXPRESSION IN RETINAL MULLER GLIAL-CELLS - IMPLICATIONS FOR DL-ALPHA-AMINOADIPATE TOXICITY
CYSTINE GLUTAMATE ANTIPORTER EXPRESSION IN RETINAL MULLER GLIAL-CELLS - IMPLICATIONS FOR DL-ALPHA-AMINOADIPATE TOXICITY
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DOI：
10.1016/0306-4522(93)90080-y
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发表时间：
1993-11-01
期刊：
影响因子：
3.3
通讯作者：
MAWATARI, K
中科院分区：
文献类型：
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作者：
KATO, S;ISHITA, S;MAWATARI, K
最近在N18神经母细胞瘤-大鼠视网膜杂交瘤细胞(N18RE105)和C6胶质瘤细胞中证实了谷氨酸或相关氨基酸(10 MM)对N18神经母细胞瘤-大鼠视网膜杂交细胞(N18RE105)和C6胶质瘤细胞的细胞毒作用。逆向转运蛋白通常将谷氨酸输送到细胞外，将半胱氨酸输送到细胞内，从而维持细胞内谷胱甘肽的浓度。高浓度的谷氨酸抑制了胱氨酸的摄取，并导致细胞内谷胱甘肽水平的耗尽。在相关的氨基酸中，DL-α-氨基己二酸(DL-α-AAA)是众所周知的视网膜中的一种选择性胶质毒素，对这些细胞也有毒性。然而，这并不能解释为什么DL-α-AAA对视网膜起胶质特异性作用。为了回答这个问题，我们首先检测了DL-α-AAA对亲本N18神经母细胞瘤细胞和杂交细胞的大鼠视网膜摄取[S-35]胱氨酸的影响。DL-α-AAA对大鼠视网膜摄取[S-35]半胱氨酸有竞争性抑制作用，但对N18细胞摄取无明显影响。DL-α-AAA对胱氨酸摄取的竞争性抑制也可以在鲤鱼的视网膜中看到。鲤鱼视网膜对半胱氨酸的摄取主要依赖于Na+和Cl-，这已被描述为XcBAR反向转运蛋白的特征离子依赖。接下来，我们研究了外源性半胱氨酸对视网膜谷氨酸释放的影响。胱氨酸(1 MM)实际上诱导了谷氨酸的释放，大约是对照组的两倍。此外，半胱氨酸诱导的谷氨酸释放也是钠非依赖性和氯离子依赖性的，并被DL-α-AAA阻断。鲤鱼视网膜摄取[S-35]胱氨酸的放射自显影显示典型的放射状胶质穆勒细胞。用高压液相色谱方法检测到在1-4-h孵育过程中，[S-35]半胱氨酸大量掺入视网膜谷胱甘肽部分。玻璃体腔注射8-Mumol DL-α-AAA或L-α-AAA后1d，视网膜谷胱甘肽水平明显或大幅下降。谷胱甘肽合成的特异性抑制剂丁硫氨酸亚磺胺(2.5摩尔)在治疗20-30小时后引起视网膜谷胱甘肽水平的大幅下降和视网膜电图b波的丢失。综上所述，我们目前在大鼠和鲤鱼视网膜上的数据有力地表明，胱氨酸/谷氨酸逆向转运体在视网膜中的表达丰富，特别是在具有快速谷胱甘肽周转池的胶质细胞米勒细胞中。胶质毒素DL-α-AAA通过这种逆向转运蛋白抑制神经胶质细胞对半胱氨酸的摄取，并引起细胞内谷胱甘肽水平的降低。胶质毒素DL-α-AAA在视网膜中的定位作用将建立在富含半胱氨酸/谷氨酸逆向转运蛋白表达于胶质细胞的假设基础上。
A cytotoxicity of glutamate or related amino acids (10 mM) mediated by a cystine/glutamate antiporter (system XcBAR) has recently been demonstrated in N18 neuroblastoma-rat retina hybrid (N18RE105) cells and C6 glioma cells. The antiporter usually transports glutamate outside and cystine inside, thereby maintaining cellular concentrations of glutathione. High concentrations of glutamate inhibit cystine uptake and lead to depletion of cellular levels of glutathione. Among related amino acids, DL-alpha-aminoadipic acid (DL-alpha-AAA), which is well known as a selective gliotoxin in the retina, is also toxic to these cells. However, this does not explain why DL-alpha-AAA acts gliospecifically on the retina. To answer this question we first examined the effects Of DL-alpha-AAA on the [S-35]cystine uptake with parental N18 neuroblastoma cells and rat retina of the hybrid cells. DL-alpha-AAA showed a competitive inhibition of [S-35]cystine uptake in the rat retina but not in the N18 cells. Such a competitive inhibition of cystine uptake by DL-alpha-AAA could also be seen in the carp retina. The cystine uptake with carp retina was mainly Na+-independent and Cl--dependent as already described as a characteristic ion dependency of the XcBAR antiporter. We next examined the effects of exogenous cystine on the glutamate release from the retina. Cystine (I mM) actually induced a glutamate release approximately twice that of the control. Furthermore, the glutamate release induced by cystine was also Na+-independent and Cl--dependent, and was blocked by DL-alpha-AAA. An autoradiogram of [S-35]cystine uptake in the carp retina showed typical radial glial Muller cells. A large incorporation of [S-35]cystine into retinal glutathione fraction was detected by a high pressure liquid chromatography method during a 1-4-h incubation. A significant or large decrease of retinal levels of glutathione was observed one day after an intravitreal injection of 8 mumol DL-alpha-AAA or L-alpha-AAA, respectively. Buthionine sulfoximine (2.5 mumol), a specific inhibitor of glutathione synthesis, induced a large decrease of retinal levels of glutathione and a loss of electroretinographic b-wave 20-30 h after treatment.Taken together, our present data with rat and carp retinas strongly indicate that the expression of cystine/glutamate antiporter is enriched in the retina, particularly in the glial Muller cells which have a rapid turnover pool for glutathione. The gliotoxin DL-alpha-AAA inhibits cystine uptake through this antiporter on the glial cells and elicits reduction of cellular levels of glutathione. The site-specific action of gliotoxin DL-alpha-AAA in the retina will be discussed on the basis of the hypothesis that the expression of cystine/glutamate antiporter is enriched in the glial Muller cells.