title: Absence of equilibrative nucleoside transporter 1 in ENT1 knockout mice leads to altered nucleoside levels following hypoxic challenge.
abstract: OBJECTIVE
  Equilibrative nucleoside transporters (ENT) modulate the flux of adenosine. The ENT1-null (KO) mouse heart is endogenously cardioprotected but the cellular basis of this phenotype is unknown. Therefore, we investigated the cellular mechanisms underlying ENT1-mediated cardioprotection.
  
  
  METHODS
  Circulating adenosine levels were measured in WT and KO mice. Cellular levels of nucleosides and nucleotides were investigated in isolated adult cardiomyocytes from WT and KO mice using HPLC following hypoxic challenge (30 min, 2% O(2)). Changes in hypoxic gene expression were analyzed by PCR arrays and cAMP levels were measured to investigate contributions from adenosine receptors.
  
  
  RESULTS
  Circulating adenosine levels were significantly higher in KO (416±42nmol/l, n=12) compared to WT animals (208±21, n=13, p<0.001). Absence of ENT1 led to an elevated expression of genes involved in cardioprotective pathways compared to WT cardiomyocytes. Following hypoxic challenge, extracellular adenosine levels were significantly elevated in KO (4360±1840 pmol/mg protein) versus WT cardiomyocytes (3035±730 pmol/mg protein, n≥12, p<0.05). This effect was enhanced in the presence of dipyridamole (30 μM), which inhibits ENT1 and ENT2. Enhanced extracellular adenosine levels in ENT1-null cardiomyocytes appeared to come from a pool of extracellular nucleotides including IMP, AMP and ADP. Adenosine receptor (AR) activation mimicked increases in cAMP levels due to hypoxic challenge suggesting that ENT1 modulates AR-dependent signaling.
  
  
  CONCLUSIONS
  ENT1 contributes to modulation of extracellular adenosine levels and subsequent purinergic signaling via ARs. ENT1-null mice possess elevated circulating adenosine levels and reduced cellular uptake resulting in a perpetually cardioprotected phenotype.
authors.affiliation: Department of Biology, Muscle Health Research Centre, York University, Toronto, ON, Canada.
authors.forename: Jennifer B
authors.initials: JB
authors.lastname: Rose
authors.forename: Zlatina
authors.initials: Z
authors.lastname: Naydenova
authors.forename: Andrew
authors.initials: A
authors.lastname: Bang
authors.forename: Azza
authors.initials: A
authors.lastname: Ramadan
authors.forename: Jost
authors.initials: J
authors.lastname: Klawitter
authors.forename: Kristin
authors.initials: K
authors.lastname: Schram
authors.forename: Gary
authors.initials: G
authors.lastname: Sweeney
authors.forename: Almut
authors.initials: A
authors.lastname: Grenz
authors.forename: Holger
authors.initials: H
authors.lastname: Eltzschig
authors.forename: James
authors.initials: J
authors.lastname: Hammond
authors.forename: Doo-Sup
authors.initials: DS
authors.lastname: Choi
authors.forename: Imogen R
authors.initials: IR
authors.lastname: Coe
mesh_terms: D000241:Adenosine / Q000378:metabolism; D000818:Animals; D015687:Cell Hypoxia; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D033721:Equilibrative Nucleoside Transporter 1 / Q000235:genetics* / Q000378:metabolism; D005260:Female; D017353:Gene Deletion; D005786:Gene Expression Regulation; D007288:Inosine / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D009705:Nucleosides / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D011983:Receptors, Purinergic / Q000378:metabolism
chemical_list: D033721:Equilibrative Nucleoside Transporter 1; D009705:Nucleosides; D011983:Receptors, Purinergic; D007288:Inosine; D000242:Cyclic AMP; D011494:Protein Kinases; D000241:Adenosine