High Performance liquid Chromatography Analysis of Nucleosides and Nucleobases in Mouse Plasma and Xenopus oocytes

• Author / Creator

  Altaweraqi, Reema A

• In humans, there are three members of the cation-coupled concentrative nucleoside transporter CNT (SLC28) family, hCNT1-3: hCNT1 is selective for pyrimidine nucleosides but also transports adenosine, hCNT2 transports purine nucleosides and uridine, and hCNT3 transports both pyrimidine and purine nucleosides. hCNT1/2 transport nucleosides using the transmembrane Na+ electrochemical gradient, while hCNT3 is both Na+- and H+-coupled. By producing recombinant hCNT3 in Xenopus laevis oocytes, I used radiochemical high performance liquid chromatography (HPLC) analysis to investigate the metabolic fate of transported radiolabelled [3H] and [14C] pyrimidine and purine nucleosides inside cells. My findings suggest that transported nucleosides in oocytes after 30 minute of incubation are generally subject to minimal intracellular metabolism (uridine 7%, cytidine 0%, thymidine 0%, inosine 16% and guanosine 0%). The exception was adenosine, for which only 49% remained unmetabolized. I also used radiochemical HPLC analysis to study mechanisms by which adenosine functions as an atypical low Km, low Vmax permeant of pyrimidine nucleoside-selective hCNT1. Oocytes producing recombinant hCNT1 were pre-loaded with [3H]uridine, after which efflux of accumulated radioactivity was measured in transport medium alone, or in the presence of extracellular non-radiolabelled adenosine or uridine. The results found that hCNT1-mediated [3H]-efflux was stimulated by extracellular uridine, but inhibited by extracellular adenosine, with > 95% of the radioactivity exiting cells being unmetabolized radiolabelled uridine. This suggests that the low Vmax for adenosine transport by hCNT1 is a consequence of low transmembrane mobility of the hCNT1/adenosine complex. Humans also possess four members of the equilibrative nucleoside transporter ENT (SLC29) family, hENT1-4. hENT1 and hENT2 function to transport both nucleosides and nucleobases into and out of cells, but their relative contributions to nucleoside and nucleobase homeostasis and, in particular, to adenosine signaling via purinoreceptors, are not known. My second project addressed this question by using HPLC to compare plasma nucleoside and nucleobase concentrations in wild-type, mENT1-, mENT2- and mENT1/mENT2-knockout (KO) mice. Results from these studies demonstrated the importance of ENT1 relative to ENT2 in determining plasma adenosine concentrations, indicating a modest role of both transporters in inosine homeostasis, and suggested that neither is a major participant in handling of nucleobases.

• Subjects / Keywords

  • Mouse Plasma
  • Nucleosides
  • Nucleobases
  • Xenopus Oocytes
  • HPLC

• Graduation date

  Fall 2017

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3S17T651

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.