Regulation of endothelial CD73 by adenosine: Paracrine pathway for enhanced endothelial barrier functions

During episodes of inflammation, multiple cell types release adenine nucleo tides in the form of ATP, ADP, 5'-AMP, and adenosine, In particular, follow ing activation, polymorphonuclear leukocytes release larger quantities of 5 '-AMP, Extracellular 5'-AMP is metabolized to adenosine by surface-expresse d 5'-ectonucleotidase (CD73), Adenosine liberated by this process activates surface adenosine A,, receptors, results in endothelial junctional reorgan ization, and promotes barrier function. We hypothesized that adenosine sign aling to endothelia provides a paracrine loop for regulated expression of C D73 and enhanced endothelial barrier function. Using an in vitro microvascu lar endothelial model, we investigated the influence of 5'-AMP; adenosine; and adenosine analogues on CD73 transcription, surface expression,and funct ion. Initial experiments revealed that adenosine and adenosine analogues in duce 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. Subsequen t studies revealed that similar exposure conditions increase surface protei n through transcriptional induction of CD73. Analysis of DNA-binding activi ty by EMSA identified a functional role for CD73 cAMP response element and, moreover, indicated that multiple cAMP agonists induce transcriptional act ivation of functional CD73. Induced CD73 functioned to enhance 5'-AMP-media ted promotion of endothelial barrier (measured as a paracellular Bur of 70- kDa FITC-labeled tracer). These results provide an example of transcription al induction of enzyme (CD73) by enzymatic product (adenosine) and define a paracrine pathway for the regulated expression of vascular endothelial CD7 3 and barrier function.