Bi-directional flux of 2-chloroadenosine by equilibrative nucleoside transporter 4 (ENT4)

• Author / Creator

  Tandio, David

• Introduction: Cardiac ischaemia-reperfusion injury (IRI) remains a life-threatening injury with no effective treatments. Though the mechanisms remain unknown, we know that ischaemic tissues are acidic. This is where equilibrative nucleoside transporter 4 (ENT4), which mediates an increased adenosine influx under acidic conditions (Barnes et al., 2006), can potentially be a therapeutic target for cardiac IRI. It has been found to be expressed in cardiomyocytes, and vascular smooth muscle cells. Although ENT4 is part of the nucleoside transporter family, it was initially only found to transport monoamines, and therefore it is also identified as the plasma membrane monoamine transporter (Engel et al., 2004). We hypothesise that ENT4 activity increases following ischaemia and that ENT4 may mediate the loss of intracellular adenosine during reperfusion which is detrimental for the recovery of contractile function in cardiomyocytes.

  Methods: To better understand ENT4 as a nucleoside transporter and to study ENT4 in isolation, the pig kidney epithelial nucleoside transporter deficient cells (PK15-NTD) was stably transfected with ENT4 to create the PK15-hENT4 cells. To investigate if ENT4 plays a significant role in ischaemia-reperfusion injury, HL-1 cardiomyocytes were used to explore how ENT4 activity changes during ischaemia-reperfusion injury. In collaborative studies, hearts and mesenteric vascular beds isolated from ENT4-knockout (KO) and control mice were subjected to global no-flow ischaemia (20 min) and reperfusion (30 min), and assessed for their functional recovery. Therefore, we investigated any compensatory messenger ribonucleic acid (mRNA) changes in the ENT4 KO animals using quantitative reverse transcription polymerase chain reaction (RT-qPCR).
  Results: Our data shows that ENT4 is capable of mediating not just adenosine uptake, but also a more stable adenosine analogue, 2-chloroadenosine (2CADO). 2CADO was then used as a substitute for the more labile adenosine to show that ENT4 mediates nucleoside efflux in an acidic pH-sensitive manner and that a proton gradient is not needed for its pH sensitivity. This suggests that ENT4 is capable of mediating bi-directional adenosine flux in ischaemic tissues, and during early reperfusion, before the recovery from acidic pH. In HL-1 cells, an acidic pH-sensitive purine influx was observed. This acidic pH-sensitive influx was reduced during ischaemia, but recovered following reperfusion; this was supported by reduced ENT4 transcript levels in ischaemic conditions. In ENT4 KO animals, compensatory changes in adenosine related genes (and not those related to monoamines) was mostly observed.

  Conclusion: Our data showed that ENT4 can mediate adenosine efflux under acidic conditions and, therefore, blocking ENT4 during early reperfusion may prevent the rapid loss of intracellular adenosine pools that limits the recovery of contractile function. The suppression of ENT4 activity and expression following simulated ischaemia suggests that this might be a protective mechanism that the heart adopts to prevent further loss of intracellular adenosine pools. As an acidic pH-sensitive adenosine uptake was still observed following simulated ischaemia, this also suggests that inhibition of ENT4 will still be more selective for ischaemic tissues (compared to ENT1, for e.g.). This was further supported by data from ENT4 KO animals which suggests that the adenosine transport function of ENT4 might play an important role in vivo. The findings in this thesis add to our current understanding of ENT4, with data from a cellular to an organ level to determine if ENT4 can be used as a novel therapeutic target for a clinically unmet condition.

• Subjects / Keywords

  • Nucleoside Transporter
  • Membrane Protein

• Graduation date

  Fall 2019

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-df00-2h51

• License

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