The ecto-enzymes CD73 and adenosine deaminase modulate 5′-AMP-derived adenosine in myofibroblasts of the rat small intestine
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.
KeywordsIntestine Adenosine CD73/ecto-5′nucleotidase Adenosine deaminase Alkaline phosphatase Adenosine receptor Rat Myofibroblasts
α,β-methyleneadenosine 5′-diphosphate sodium salt
High-pressure liquid chromatography
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.
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.
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.
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.
- 4.Antonioli L, Colucci R, Pellegrini C, Giustarini G, Tuccori M, Blandizzi C, Fornai M (2013) The role of purinergic pathways in the pathophysiology of gut diseases: pharmacological modulation and potential therapeutic applications. Pharmacol Ther 139(2):157–188. https://doi.org/10.1016/j.pharmthera.2013.04.002 CrossRefPubMedGoogle Scholar
- 5.Zoppellaro C, Bin A, Brun P, Banzato S, Macchi V, Castagliuolo I, Giron MC (2013) Adenosine-mediated enteric neuromuscular function is affected during herpes simplex virus type 1 infection of rat enteric nervous system. PLoS One 8(8):e72648. https://doi.org/10.1371/journal.pone.0072648 CrossRefPubMedPubMedCentralGoogle Scholar
- 8.Duarte-Araujo M, Nascimento C, Alexandrina Timoteo M, Magalhaes-Cardoso T, Correia-de-Sa P (2004) Dual effects of adenosine on acetylcholine release from myenteric motoneurons are mediated by junctional facilitatory A(2A) and extrajunctional inhibitory A(1) receptors. Br J Pharmacol 141(6):925–934. https://doi.org/10.1038/sj.bjp.0705697 CrossRefPubMedPubMedCentralGoogle Scholar
- 9.Correia-de-Sa P, Adaes S, Timoteo MA, Vieira C, Magalhaes-Cardoso T, Nascimento C, Duarte-Araujo M (2006) Fine-tuning modulation of myenteric motoneurons by endogenous adenosine: on the role of secreted adenosine deaminase. Autonomic neuroscience: basic & clinical 126–127:211–224. https://doi.org/10.1016/j.autneu.2006.02.004 CrossRefGoogle Scholar
- 11.Fukuuchi T, Kobayashi M, Yamaoka N, Kaneko K (2016) Evaluation of cellular purine transport and metabolism in the Caco-2 cell using comprehensive high-performance liquid chromatography method for analysis of purines. Nucleosides Nucleotides Nucleic Acids 35(10–12):663–669. https://doi.org/10.1080/15257770.2016.1205195 CrossRefPubMedGoogle Scholar
- 12.Antonioli L, Pellegrini C, Fornai M, Tirotta E, Gentile D, Benvenuti L, Giron MC, Caputi V, Marsilio I, Orso G, Bernardini N, Segnani C, Ippolito C, Csóka B, Németh ZH, Haskó G, Scarpignato C, Blandizzi C, Colucci R (2017) Colonic motor dysfunctions in a mouse model of high-fat diet-induced obesity: an involvement of A(2B) adenosine receptors. Purinergic Signal 13(4):497–510CrossRefGoogle Scholar
- 13.Duarte-Araujo M, Nascimento C, Timoteo MA, Magalhaes-Cardoso MT, Correia-de-Sa P (2009) Relative contribution of ecto-ATPase and ecto-ATPDase pathways to the biphasic effect of ATP on acetylcholine release from myenteric motoneurons. Br J Pharmacol 156(3):519–533. https://doi.org/10.1111/j.1476-5381.2008.00058.x CrossRefPubMedPubMedCentralGoogle Scholar
- 17.Pacheco R, Martinez-Navio JM, Lejeune M, Climent N, Oliva H, Gatell JM, Gallart T, Mallol J, Lluis C, Franco R (2005) CD26, adenosine deaminase, and adenosine receptors mediate costimulatory signals in the immunological synapse. Proc Natl Acad Sci U S A 102(27):9583–9588. https://doi.org/10.1073/pnas.0501050102 CrossRefPubMedPubMedCentralGoogle Scholar
- 19.Vieira C, Magalhaes-Cardoso MT, Ferreirinha F, Silva I, Dias AS, Pelletier J, Sevigny J, Correia-de-Sa P (2014) Feed-forward inhibition of CD73 and upregulation of adenosine deaminase contribute to the loss of adenosine neuromodulation in postinflammatory ileitis. Mediat Inflamm 2014:254640. https://doi.org/10.1155/2014/254640 CrossRefGoogle Scholar
- 20.Synnestvedt K, Furuta GT, Comerford KM, Louis N, Karhausen J, Eltzschig HK, Hansen KR, Thompson LF, Colgan SP (2002) Ecto-5′-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia. J Clin Investig 110(7):993–1002. https://doi.org/10.1172/jci15337 CrossRefPubMedGoogle Scholar
- 23.Caputi V, Marsilio I, Filpa V, Cerantola S, Orso G, Bistoletti M, Paccagnella N, De Martin S, Montopoli M, Dall'Acqua S, Crema F, Di Gangi IM, Galuppini F, Lante I, Bogialli S, Rugge M, Debetto P, Giaroni C, Giron MC (2017) Antibiotic-induced dysbiosis of the microbiota impairs gut neuromuscular function in juvenile mice. Br J Pharmacol 174(20):3623–3639CrossRefGoogle Scholar
- 25.Achenbach JE, Topliff CL, Vassilev VB, Donis RO, Eskridge KM, Kelling CL (2004) Detection and quantitation of bovine respiratory syncytial virus using real-time quantitative RT-PCR and quantitative competitive RT-PCR assays. J Virol Methods 121(1):1–6. https://doi.org/10.1016/j.jviromet.2004.05.004 CrossRefPubMedGoogle Scholar
- 26.Qin X, Liu S, Lu Q, Zhang M, Jiang X, Hu S, Li J, Zhang C, Gao J, Zhu MS, Feil R, Li H, Chen M, Weinstein LS, Zhang Y, Zhang W (2017) Heterotrimeric G stimulatory protein alpha subunit is required for intestinal smooth muscle contraction in mice. Gastroenterology 152(5):1114–1125.e1115. https://doi.org/10.1053/j.gastro.2016.12.017 CrossRefPubMedGoogle Scholar
- 27.Brun P, Giron MC, Zoppellaro C, Bin A, Porzionato A, De Caro R, Barbara G, Stanghellini V, Corinaldesi R, Zaninotto G, Palù G, Gaion RM, Tonini M, De Giorgio R, Castagliuolo I (2010) Herpes simplex virus type 1 infection of the rat enteric nervous system evokes small-bowel neuromuscular abnormalities. Gastroenterology 138(5):1790–1801. https://doi.org/10.1053/j.gastro.2010.01.036 CrossRefPubMedGoogle Scholar
- 28.Chen W, Lu C, Hirota C, Iacucci M, Ghosh S, Gui X (2017) Smooth muscle hyperplasia/hypertrophy is the most prominent histological change in Crohn’s fibrostenosing bowel strictures: a semiquantitative analysis by using a novel histological grading scheme. J Crohn’s Colitis 11(1):92–104. https://doi.org/10.1093/ecco-jcc/jjw126 CrossRefGoogle Scholar
- 30.Severi C, Sferra R, Scirocco A, Vetuschi A, Pallotta N, Pronio A, Caronna R, Di Rocco G, Gaudio E, Corazziari E, Onori P (2014) Contribution of intestinal smooth muscle to Crohn’s disease fibrogenesis. Eur J Histochem 58(4):2457. https://doi.org/10.4081/ejh.2014.2457 CrossRefPubMedPubMedCentralGoogle Scholar
- 33.Antonioli L, Colucci R, Pellegrini C, Giustarini G, Sacco D, Tirotta E, Caputi V, Marsilio I, Giron MC, Németh ZH, Blandizzi C, Fornai M (2016) The AMPK enzyme-complex: from the regulation of cellular energy homeostasis to a possible new molecular target in the management of chronic inflammatory disorders. Expert Opin Ther Targets 20(2):179–191. https://doi.org/10.1517/14728222.2016.1086752 CrossRefPubMedGoogle Scholar
- 37.Strohmeier GR, Lencer WI, Patapoff TW, Thompson LF, Carlson SL, Moe SJ, Carnes DK, Mrsny RJ, Madara JL (1997) Surface expression, polarization, and functional significance of CD73 in human intestinal epithelia. J Clin Invest 99(11):2588–2601. https://doi.org/10.1172/jci119447 CrossRefPubMedPubMedCentralGoogle Scholar
- 43.Bilski J, Mazur-Bialy A, Wojcik D, Zahradnik-Bilska J, Brzozowski B, Magierowski M, Mach T, Magierowska K, Brzozowski T (2017) The role of intestinal alkaline phosphatase in inflammatory disorders of gastrointestinal tract. Mediat Inflamm 2017:9074601. https://doi.org/10.1155/2017/9074601 CrossRefGoogle Scholar
- 44.Bhattarai S, Freundlieb M, Pippel J, Meyer A, Abdelrahman A, Fiene A, Lee SY, Zimmermann H, Yegutkin GG, Strater N, El-Tayeb A, Muller CE (2015) alpha, beta-Methylene-ADP (AOPCP) derivatives and analogues: development of potent and selective ecto-5′-nucleotidase (CD73) inhibitors. J Med Chem 58(15):6248–6263. https://doi.org/10.1021/acs.jmedchem.5b00802 CrossRefPubMedGoogle Scholar
- 46.Antonioli L, Fornai M, Awwad O, Giustarini G, Pellegrini C, Tuccori M, Caputi V, Qesari M, Castagliuolo I, Brun P, Giron MC, Scarpignato C, Blandizzi C, Colucci R (2014) Role of the A(2B) receptor-adenosine deaminase complex in colonic dysmotility associated with bowel inflammation in rats. Br J Pharmacol 171(5):1314–1329. https://doi.org/10.1111/bph.12539 CrossRefPubMedPubMedCentralGoogle Scholar
- 48.Street SE, Kramer NJ, Walsh PL, Taylor-Blake B, Yadav MC, King IF, Vihko P, Wightman RM, Millan JL, Zylka MJ (2013) Tissue-nonspecific alkaline phosphatase acts redundantly with PAP and NT5E to generate adenosine in the dorsal spinal cord. J Neurosci 33(27):11314–11322. https://doi.org/10.1523/jneurosci.0133-13.2013 CrossRefPubMedPubMedCentralGoogle Scholar
- 49.Araujo CL, Quintero IB, Kipar A, Herrala AM, Pulkka AE, Saarinen L, Hautaniemi S, Vihko P (2014) Prostatic acid phosphatase is the main acid phosphatase with 5′-ectonucleotidase activity in the male mouse saliva and regulates salivation. Am J Physiol Cell Physiol 306(11):C1017–C1027. https://doi.org/10.1152/ajpcell.00062.2014 CrossRefPubMedGoogle Scholar
- 51.Caputi V, Marsilio I, Cerantola S, Roozfarakh M, Lante I, Galuppini F, Rugge M, Napoli E, Giulivi C, Orso G, Giron MC (2017) Toll-like receptor 4 modulates small intestine neuromuscular function through nitrergic and purinergic pathways. Front Pharmacol 8:350. https://doi.org/10.3389/fphar.2017.00350 CrossRefPubMedPubMedCentralGoogle Scholar
- 52.Brun P, Gobbo S, Caputi V, Spagnol L, Schirato G, Pasqualin M, Levorato E, Palu G, Giron MC, Castagliuolo I (2015) Toll like receptor-2 regulates production of glial-derived neurotrophic factors in murine intestinal smooth muscle cells. Mol Cell Neurosci 68:24–35. https://doi.org/10.1016/j.mcn.2015.03.018 CrossRefPubMedGoogle Scholar
- 53.Vieira C, Ferreirinha F, Magalhaes-Cardoso MT, Silva I, Marques P, Correia-de-Sa P (2017) Post-inflammatory ileitis induces non-neuronal purinergic signaling adjustments of cholinergic neurotransmission in the myenteric plexus. Front Pharmacol 8:811. https://doi.org/10.3389/fphar.2017.00811 CrossRefPubMedPubMedCentralGoogle Scholar