Carvelli Lucia, Morón José A, Kahlig Kristopher M, Ferrer Jasmine V, Sen Namita, Lechleiter James D, Leeb-Lundberg L M Fredrik, Merrill Gerald, Lafer Eileen M, Ballou Lisa M, Shippenberg Toni S, Javitch Jonathan A, Lin Richard Z, Galli Aurelio
Department of Pharmacology, MC 7764, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA.
J Neurochem. 2002 May;81(4):859-69. doi: 10.1046/j.1471-4159.2002.00892.x.
The magnitude and duration of dopamine (DA) signaling is defined by the amount of vesicular release, DA receptor sensitivity, and the efficiency of DA clearance, which is largely determined by the DA transporter (DAT). DAT uptake capacity is determined by the number of functional transporters on the cell surface as well as by their turnover rate. Here we show that inhibition of phosphatidylinositol (PI) 3-kinase with LY294002 induces internalization of the human DAT (hDAT), thereby reducing transport capacity. Acute treatment with LY294002 reduced the maximal rate of [(3) H]DA uptake in rat striatal synaptosomes and in human embryonic kidney (HEK) 293 cells stably expressing the hDAT (hDAT cells). In addition, LY294002 caused a significant redistribution of the hDAT from the plasma membrane to the cytosol. Conversely, insulin, which activates PI 3-kinase, increased [(3)H]DA uptake and blocked the amphetamine-induced hDAT intracellular accumulation, as did transient expression of constitutively active PI 3-kinase. The LY294002-induced reduction in [(3)H]DA uptake and hDAT cell surface expression was inhibited by expression of a dominant negative mutant of dynamin I, indicating that dynamin-dependent trafficking can modulate transport capacity. These data implicate DAT trafficking in the hormonal regulation of dopaminergic signaling, and suggest that a state of chronic hypoinsulinemia, such as in diabetes, may alter synaptic DA signaling by reducing the available cell surface DATs.
多巴胺（DA）信号传导的强度和持续时间由囊泡释放量、DA受体敏感性以及DA清除效率决定，而DA清除效率在很大程度上由DA转运体（DAT）决定。DAT摄取能力由细胞表面功能性转运体的数量及其周转率决定。在此我们表明，用LY294002抑制磷脂酰肌醇（PI）3激酶会诱导人DAT（hDAT）内化，从而降低转运能力。用LY294002急性处理可降低大鼠纹状体突触体和稳定表达hDAT的人胚肾（HEK）293细胞（hDAT细胞）中[³H]DA摄取的最大速率。此外，LY294002导致hDAT从质膜向胞质溶胶显著重新分布。相反，激活PI 3激酶的胰岛素增加了[³H]DA摄取并阻断了苯丙胺诱导的hDAT细胞内积累，组成型活性PI 3激酶的瞬时表达也有同样效果。LY294002诱导的[³H]DA摄取减少和hDAT细胞表面表达受发动蛋白I显性负突变体表达的抑制，表明依赖发动蛋白的转运可调节转运能力。这些数据表明DAT转运参与多巴胺能信号的激素调节，并提示慢性低胰岛素血症状态，如在糖尿病中，可能通过减少可用的细胞表面DAT来改变突触DA信号。
