Please use this identifier to cite or link to this item: http://hdl.handle.net/20.500.12857/115600
Title: The ecto-enzymes CD73 and adenosine deaminase modulate 5'-AMP-derived adenosine in myofibroblasts of the rat small intestine
Authors: Bin, Anna 
Caputi, Valentina
Bistoletti, Michela
Montopoli, Monica 
Colucci, Rocchina 
Antonioli, Luca
De Martin, Sara 
Castagliuolo, Ignazio 
Orso, Genny 
Giaroni, Cristina
Debetto, Patrizia 
Giron, Maria Cecilia 
Keywords: Adenosine;Adenosine deaminase;Adenosine receptor;Alkaline phosphatase;CD73/ecto-5′nucleotidase;Intestine;Myofibroblasts;Rat
Keywords Plus: NONSPECIFIC ALKALINE-PHOSPHATASE;SMOOTH-MUSCLE-CELLS;MYENTERIC MOTONEURONS;ACETYLCHOLINE-RELEASE;ADENINE-NUCLEOTIDES;ECTO-ATPASE;METABOLISM;RECEPTORS;ECTONUCLEOTIDASES;LOCALIZATION
Mesh headings: 5'-Nucleotidase;Adenosine;Adenosine Deaminase;Intestine, Small;Myofibroblasts
Secondary Mesh headings: Adenosine Monophosphate;Alkaline Phosphatase;Animals;Male;Rats, Wistar
Issue Date: 2018
Publisher: SPRINGER
Journal: Purinergic signalling 
Abstract: 
Adenosine is a versatile signaling molecule recognized to physiologically influence gut motor functions. Both the duration and magnitude of adenosine signaling in enteric neuromuscular function depend on its availability, which is regulated by the ecto-enzymes ecto-5'-nucleotidase (CD73), alkaline phosphatase (AP), and ecto-adenosine deaminase (ADA) and by dipyridamole-sensitive equilibrative transporters (ENTs). Our purpose was to assess the involvement of CD73, APs, ecto-ADA in the formation of AMP-derived adenosine in primary cultures of ileal myofibroblasts (IMFs). IMFs were isolated from rat ileum longitudinal muscle segments by means of primary explant technique and identified by immunofluorescence staining for vimentin and α-smooth muscle actin. IMFs confluent monolayers were exposed to exogenous 5'-AMP in the presence or absence of CD73, APs, ecto-ADA, or ENTs inhibitors. The formation of adenosine and its metabolites in the IMFs medium was monitored by high-performance liquid chromatography. The distribution of CD73 and ADA in IMFs was detected by confocal immunocytochemistry and qRT-PCR. Exogenous 5'-AMP was rapidly cleared being almost undetectable after 60-min incubation, while adenosine levels significantly increased. Treatment of IMFs with CD73 inhibitors markedly reduced 5'-AMP clearance whereas ADA blockade or inhibition of both ADA and ENTs prevented adenosine catabolism. By contrast, inhibition of APs did not affect 5'-AMP metabolism. Immunofluorescence staining and qRT-PCR analysis confirmed the expression of CD73 and ADA in IMFs. Overall, our data show that in IMFs an extracellular AMP-adenosine pathway is functionally active and among the different enzymatic pathways regulating extracellular adenosine levels, CD73 and ecto-ADA represent the critical catabolic pathway.
URI: http://hdl.handle.net/20.500.12857/115600
ISSN: 15739538
DOI: 10.1007/s11302-018-9623-6
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