Lu Wennan, Reigada David, Sévigny Jean, Mitchell Claire H
Department of Physiology, University of Pennsylvania, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA.
J Pharmacol Exp Ther. 2007 Oct;323(1):157-64. doi: 10.1124/jpet.107.124545. Epub 2007 Jul 12.
Stimulation of receptors for either ATP or adenosine leads to physiologic changes in retinal pigment epithelial (RPE) cells that may influence their relationship with the adjacent photoreceptors. The ectoenzyme nucleoside-triphosphate diphosphohydrolase-1 (NTPDase1) catalyzes the dual dephosphorylation of ATP and ADP to AMP. Although NTPDase1 can consequently control the balance between ATP and adenosine, it is unclear how its expression and activity are regulated. Classic negative feedback theory predicts an increase in enzyme activity in response to enhanced exposure to substrate. This study asked whether exposure to ATP increases NTPDase1 activity in RPE cells. Although levels of NTPDase1 mRNA and protein in cultured human ARPE-19 cells were generally low under control conditions, exposure to slowly hydrolyzable ATPgammaS led to a time-dependent increase in NTPDase1 mRNA that was accompanied by a rise in levels of the functional 78-kDa protein. Neither NTPDase2 nor NTPDase3 mRNA message was elevated by ATPgammaS. The ATPase activity of cells increased in parallel, indicating the up-regulation of NTPDase1 was functionally relevant. The up-regulation of NTPDase1 protein was partially blocked by P2Y1 receptor inhibitors MRS2179 (N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate) and MRS2500 [2-iodo-N6-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bisphosphate] and increased by P2Y1 receptor agonist MRS2365 [(N)-methanocarba-2MeSADP]. In conclusion, prolonged exposure to extracellular ATPgammaS increased NTPDase1 message and protein levels and increased ecto-ATPase activity. This up-regulation reflects a feedback circuit, mediated at least in part by the P2Y1 receptor, to regulate levels of extracellular purines in subretinal space. NTPDase1 levels may thus serve as an index for increased extracellular ATP levels under certain pathologic conditions, although other mechanisms could also contribute.
对ATP或腺苷受体的刺激会导致视网膜色素上皮（RPE）细胞发生生理变化，这可能会影响它们与相邻光感受器的关系。胞外酶核苷三磷酸二磷酸水解酶-1（NTPDase1）催化ATP和ADP双重去磷酸化生成AMP。虽然NTPDase1因此可以控制ATP和腺苷之间的平衡，但其表达和活性是如何调节的尚不清楚。经典的负反馈理论预测，酶活性会随着底物暴露增加而升高。本研究探讨了暴露于ATP是否会增加RPE细胞中NTPDase1的活性。尽管在对照条件下，培养的人ARPE-19细胞中NTPDase1 mRNA和蛋白质水平通常较低，但暴露于缓慢水解的ATPγS会导致NTPDase1 mRNA随时间增加，同时功能性78-kDa蛋白质水平也会升高。ATPγS不会升高NTPDase2和NTPDase3的mRNA水平。细胞的ATP酶活性平行增加，表明NTPDase1的上调在功能上是相关的。NTPDase1蛋白的上调被P2Y1受体抑制剂MRS2179（N6-甲基-2'-脱氧腺苷-3',5'-双磷酸）和MRS2500 [2-碘-N6-甲基-(N)-甲碳环-2'-脱氧腺苷3',5'-双磷酸]部分阻断，并被P2Y1受体激动剂MRS2365 [（N）-甲碳环-2MeSADP]增强。总之，长时间暴露于细胞外ATPγS会增加NTPDase1的信息和蛋白质水平，并增加胞外ATP酶活性。这种上调反映了一种反馈回路，至少部分由P2Y1受体介导，以调节视网膜下间隙中细胞外嘌呤的水平。因此，NTPDase1水平可能作为某些病理条件下细胞外ATP水平升高的指标，尽管其他机制也可能起作用。
