Purinergic Signalling

, Volume 14, Issue 4, pp 409–421 | Cite as

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 GironEmail author
Original Article


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.


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



Adenosine deaminase


α,β-methyleneadenosine 5′-diphosphate sodium salt




Erythro-9-(2-hydroxy-3-nonyl) adenine


Equilibrative transporters


High-pressure liquid chromatography


Ileal myofibroblasts


Alkaline phosphatase


Prostatic acid phosphatase



We thank Dr. Francesca Patrese and Dr. Ludovico Scenna for veterinary assistance; Mauro Berto, Massimo Rizza, and Andrea Pagetta for technical assistance in animal handling and experimental procedures.

Author contributions

Conceived and designed the experiments: MCG, AB, VC. Performed the experiments: AB, VC, IC, GO, MB. Analyzed the data: MCG, AB, VC, IC, GO, SDM, MM, LA, CG, RC, PD. Contributed reagents/materials/analysis tools: MCG, GO, IC, PD, CG. Wrote the manuscript: MCG, AB, VC. All the authors reviewed the manuscript.

Funding information

This work was supported by grants from University of Padova (UNIPD-CPDR155591/15 Assegno di Ricerca 2016, UNIPD-DSF-DOR-2016 and 2017 funds, and UNIPD-DSF-PRID-2017) and from San Camillo Hospital, Treviso (Italy) to MCG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Ethical approval

All experimental protocols were approved by the Animal Care and Use Committee of the University of Padova and by the Italian Ministry of Health and were in compliance with the national and EU guidelines for handling and use of experimental animals.


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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Anna Bin
    • 1
  • Valentina Caputi
    • 1
    • 2
  • Michela Bistoletti
    • 3
  • Monica Montopoli
    • 1
  • Rocchina Colucci
    • 1
  • Luca Antonioli
    • 4
  • Sara De Martin
    • 1
  • Ignazio Castagliuolo
    • 5
  • Genny Orso
    • 1
    • 6
  • Cristina Giaroni
    • 3
  • Patrizia Debetto
    • 1
  • Maria Cecilia Giron
    • 1
    Email author
  1. 1.Department of Pharmaceutical and Pharmacological SciencesUniversity of PadovaPadovaItaly
  2. 2.APC Microbiome IrelandUniversity College CorkCorkIreland
  3. 3.Department of Medicine and SurgeryUniversity of InsubriaVareseItaly
  4. 4.Department of Clinical and Experimental MedicineUniversity of PisaPisaItaly
  5. 5.Department of Molecular MedicineUniversity of PadovaPadovaItaly
  6. 6.IRCCS “E. Medea” Bosisio PariniLeccoItaly

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