The ecto-enzymes CD73 and adenosine deaminase modulate 5′-AMP-derived adenosine in myofibroblasts of the rat small intestine

Anna Bin, Valentina Caputi, Michela Bistoletti, Monica Montopoli, Rocchina Colucci, Luca Antonioli, Sara De Martin, Ignazio Castagliuolo, Genny Orso, Cristina Giaroni, Patrizia Debetto, Maria Cecilia Giron

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)409-421
Number of pages13
JournalPurinergic Signalling
Volume14
Issue number4
DOIs
Publication statusPublished - 2018

Keywords

  • Adenosine
  • Adenosine deaminase
  • Adenosine receptor
  • Alkaline phosphatase
  • CD73/ecto-5′nucleotidase
  • Intestine
  • Myofibroblasts
  • Rat

ASJC Scopus subject areas

  • Molecular Biology
  • Cellular and Molecular Neuroscience
  • Cell Biology

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