Inazu Masato, Takeda Hiroshi, Maehara Katsuyuki, Miyashita Kyoji, Tomoda Akio, Matsumiya Teruhiko
Department of Pharmacology, Tokyo Medical University, Japan.
J Neurochem. 2006 Apr;97(2):424-34. doi: 10.1111/j.1471-4159.2006.03757.x. Epub 2006 Mar 15.
In this study, we sought to identify the transporters that mediate the uptake of L-carnitine and acetyl-L-carnitine in cultured rat cortical astrocytes. L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake were both saturable, and mediated by a single Na(+)-dependent transport system. Uptake of both was inhibited by L-carnitine, D-carnitine, acetyl-L-carnitine and various organic cations. Acylcarnitines (acetyl-, butyryl-, hexanoyl-, octanoyl- and palmitoyl-L-carnitine) also interacted with L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine transport. 2-Amino-2-norbornane carboxylic acid, a known inhibitor of amino acid transporter B(0,+) (ATB(0,+)), did not cause any significant inhibition. A highly significant correlation was found between the potencies of acylcarnitines in the inhibition of L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake and the acyl chain length of acylcarnitines. The expression of mRNA for organic cation/carnitine transporters (OCTNs), carnitine transporter 2 (CT2) and ATB(0,+) in astrocytes was investigated by reverse transcription (RT)-PCR. OCTN2 mRNA was expressed in astrocytes, whereas the expression of OCTN1, OCTN3 and CT2 mRNA could not be detected. ATB(0,+) mRNA was expressed at very low levels in astrocytes. Western blotting analysis indicated that anti-OCTN2 polyclonal antibody recognized a band of 70 kDa in both kidney and astrocyte preparations. OCTN2 immunoreactivity was detected in rat astrocytes by immunocytochemical staining. Inhibition of OCTN2 expression by RNA interference significantly inhibited L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake into astrocytes. These results suggest that OCTN2 is functionally expressed in rat astrocytes, and is responsible for L-carnitine and acetyl-L-carnitine uptake in these cells.
在本研究中，我们试图鉴定介导培养的大鼠皮质星形胶质细胞摄取左旋肉碱和乙酰左旋肉碱的转运体。L-[(3)H]肉碱和乙酰-L-[(3)H]肉碱的摄取均具有饱和性，且由单一的Na(+)依赖性转运系统介导。二者的摄取均受到左旋肉碱、右旋肉碱、乙酰左旋肉碱和各种有机阳离子的抑制。酰基肉碱（乙酰基-、丁酰基-、己酰基-、辛酰基-和棕榈酰基-L-肉碱）也与L-[(3)H]肉碱和乙酰-L-[(3)H]肉碱的转运相互作用。2-氨基-2-降冰片烷羧酸是一种已知的氨基酸转运体B(0,+)（ATB(0,+)）抑制剂，未引起任何显著抑制作用。在酰基肉碱对L-[(3)H]肉碱和乙酰-L-[(3)H]肉碱摄取的抑制效力与酰基肉碱的酰基链长度之间发现了高度显著的相关性。通过逆转录（RT）-PCR研究了星形胶质细胞中有机阳离子/肉碱转运体（OCTNs）、肉碱转运体2（CT2）和ATB(0,+)的mRNA表达。OCTN2 mRNA在星形胶质细胞中表达，而未检测到OCTN1、OCTN3和CT2 mRNA的表达。ATB(0,+) mRNA在星形胶质细胞中的表达水平非常低。蛋白质印迹分析表明，抗OCTN2多克隆抗体在肾脏和星形胶质细胞制剂中均识别出一条70 kDa 的条带。通过免疫细胞化学染色在大鼠星形胶质细胞中检测到OCTN2免疫反应性。RNA干扰对OCTN2表达的抑制显著抑制了星形胶质细胞对L-[(3)H]肉碱和乙酰-L-[(3)H]肉碱的摄取。这些结果表明，OCTN2在大鼠星形胶质细胞中功能性表达，并负责这些细胞中左旋肉碱和乙酰左旋肉碱的摄取。
