Narravula S, Lennon P F, Mueller B U, Colgan S P
Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital, and Division of Hematology and Oncology, Beth Israel-Deaconess Medical Center and Harvard Medical School, Boston, MA 02115, USA.
J Immunol. 2000 Nov 1;165(9):5262-8. doi: 10.4049/jimmunol.165.9.5262.
During episodes of inflammation, multiple cell types release adenine nucleotides in the form of ATP, ADP, 5'-AMP, and adenosine. In particular, following activation, polymorphonuclear leukocytes release larger quantities of 5'-AMP. Extracellular 5'-AMP is metabolized to adenosine by surface-expressed 5'-ectonucleotidase (CD73). Adenosine liberated by this process activates surface adenosine A(2B) receptors, results in endothelial junctional reorganization, and promotes barrier function. We hypothesized that adenosine signaling to endothelia provides a paracrine loop for regulated expression of CD73 and enhanced endothelial barrier function. Using an in vitro microvascular endothelial model, we investigated the influence of 5'-AMP; adenosine; and adenosine analogues on CD73 transcription, surface expression, and function. Initial experiments revealed that adenosine and adenosine analogues induce CD73 mRNA (RT-PCR), surface expression (immunoprecipitation of surface biotinylated CD73), and function (HPLC analysis of etheno-AMP conversion to ethenoadenosine) in a time- and concentration-dependent fashion. Subsequent studies revealed that similar exposure conditions increase surface protein through transcriptional induction of CD73. Analysis of DNA-binding activity by EMSA identified a functional role for CD73 cAMP response element and, moreover, indicated that multiple cAMP agonists induce transcriptional activation of functional CD73. Induced CD73 functioned to enhance 5'-AMP-mediated promotion of endothelial barrier (measured as a paracellular flux of 70-kDa FITC-labeled tracer). These results provide an example of transcriptional induction of enzyme (CD73) by enzymatic product (adenosine) and define a paracrine pathway for the regulated expression of vascular endothelial CD73 and barrier function.
在炎症发作期间，多种细胞类型会以三磷酸腺苷（ATP）、二磷酸腺苷（ADP）、5'-单磷酸腺苷（5'-AMP）和腺苷的形式释放腺嘌呤核苷酸。特别是，多形核白细胞激活后会释放大量的5'-AMP。细胞外的5'-AMP通过表面表达的5'-核苷酸酶（CD73）代谢为腺苷。这一过程释放的腺苷激活表面腺苷A(2B)受体，导致内皮细胞连接重组，并促进屏障功能。我们推测，腺苷向内皮细胞发出的信号提供了一个旁分泌环，用于调节CD73的表达并增强内皮屏障功能。我们使用体外微血管内皮模型，研究了5'-AMP、腺苷和腺苷类似物对CD73转录、表面表达和功能的影响。初步实验表明，腺苷和腺苷类似物以时间和浓度依赖性方式诱导CD73信使核糖核酸（RT-PCR）、表面表达（表面生物素化CD73的免疫沉淀）和功能（高效液相色谱分析乙烯基-AMP转化为乙烯基腺苷）。后续研究表明，类似的暴露条件通过CD73的转录诱导增加表面蛋白。通过电泳迁移率变动分析（EMSA）对DNA结合活性的分析确定了CD73环磷酸腺苷（cAMP）反应元件的功能作用，此外，还表明多种cAMP激动剂诱导功能性CD73的转录激活。诱导的CD73发挥作用，增强5'-AMP介导的内皮屏障促进作用（以70 kDa异硫氰酸荧光素（FITC）标记示踪剂的细胞旁通量来衡量）。这些结果提供了酶促产物（腺苷）对酶（CD73）进行转录诱导的实例，并定义了一种旁分泌途径，用于调节血管内皮CD73的表达和屏障功能。
