Modulation of monocarboxylic acid transporter-1 kinetic function by the cAMP signaling pathway in rat brain endothelial cells
Modulation of monocarboxylic acid transporter-1 kinetic function by the cAMP signaling pathway in rat brain endothelial cells
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DOI：
10.1074/jbc.m511577200
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发表时间：
2006-01-27
影响因子：
4.8
通讯作者：
Drewes, LR
中科院分区：
文献类型：
--
作者：
Smith, JP;Drewes, LR
MCT1（单羧酸转运蛋白1）促进单羧酸跨膜双向转运。MCT1的功能和调节在脑内皮细胞中尚未被描述，但在正常的脑能代谢和脑疾病（如中风）期间可能很重要。本研究利用胞质pH指示剂2′，7′-双-（2-羧基乙基）-5-（和6）-羧基荧光素-乙酰氧基甲酯，利用大鼠脑内皮细胞（RBE4）血脑运输模型，测量了单羧酸依赖的胞质酸化的初始速率，作为MCT1体外动力学功能的指标。细胞内cAMP依赖性细胞信号通路的激动剂（二丁基cAMP、福斯克林和异丙肾上腺素）孵育5-10分钟后，l -乳酸依赖性酸化的初始速率被显著抑制。异丙肾上腺素降低了V-max，但不影响K-m值。蛋白激酶A抑制剂H89完全逆转了福斯克林的作用，而单独H89则增加了转运率。放射免疫法测定，福斯克林或异丙肾上腺素均能提高细胞内cAMP水平，而心得安可抑制异丙肾上腺素的作用。与MCT1无关的细胞内pH控制机制与福斯克林或H89对MCT1动力学功能的影响无关，这是通过amiloride、单羧酸无关的酸负载或运输抑制剂α -氰基-4-羟基肉桂酸测定的。这些数据表明，已知影响β -肾上腺素能受体/腺苷酸环化酶/cAMP/蛋白激酶A细胞内信号通路的药物直接调节脑内皮细胞的MCT1动力学功能。
MCT1 ( monocarboxylic acid transporter 1) facilitates bidirectional monocarboxylic acid transport across membranes. MCT1 function and regulation have not been characterized previously in cerebral endothelial cells but may be important during normal cerebral energy metabolism and during brain diseases such as stroke. Here, by using the cytoplasmic pH indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-acetoxymethyl ester, the initial rates of monocarboxylate-dependent cytoplasmic acidification were measured as an indication of MCT1 kinetic function in vitro using the rat brain endothelial cell (RBE4) model of blood-brain transport. The initial rate of L-lactate-dependent acidification was significantly inhibited by 5-10-min incubations with agonists of intracellular cAMP-dependent cell signaling pathways as follows: dibutyryl cAMP, forskolin, and isoproterenol. Isoproterenol reduced V-max but did not affect K-m values. The effects of forskolin were completely reversed by the protein kinase A inhibitor H89, whereas H89 alone increased transport rates. Cytoplasmic cAMP levels, measured by radioimmunoassay, were increased by forskolin or isoproterenol, and the effect of isoproterenol was inhibited by propranolol. MCT1-independent intracellular pH control mechanisms did not contribute to the forskolin or H89 effects on MCT1 kinetic function as determined with amiloride, monocarboxylate-independent acid loading, or the transport inhibitor alpha-cyano-4-hydroxycinnamate. The data demonstrate the direct modulation of MCT1 kinetic function in cerebral endothelial cells by agents known to affect the beta-adrenergic receptor/adenylyl cyclase/cAMP/protein kinase A intracellular signaling pathway.