title: Na(+)-dependent and -independent uridine uptake in an established renal epithelial cell line, OK, from the opossum kidney.
abstract: The characteristics of Na(+)-dependent and Na(+)-independent uridine uptake at 22 degrees C were determined for monolayers of OK renal epithelial cells. The majority of uridine influx in subconfluent to early confluent (day 1 postconfluency) OK monolayers was mediated via a facilitated-diffusion pathway (apparent Km 160 +/- 41 microM, Vmax 610 +/- 100 pmol/mg protein per min). This system was inhibited with high affinity by nitrobenzylthioinosine (NBMPR) (IC50 value 1.5 nM) and by purine and pyrimidine nucleosides. Specific [3H]NBMPR binding sites were detected in OK monolayers (apparent Kd 0.67 +/- 0.25 nM, Bmax 90 +/- 19 fmol/mg protein) yielding a turnover number for the carrier of 112 uridine molecules/site per s at 22 degrees C. Na(+)-dependent uridine uptake was minor in subconfluent OK monolayers, but increased 8-fold with time after confluency reaching a stable plateau at 8 days postconfluency. Inhibition of Na(+)-dependent 1 microM uridine uptake by inosine, guanosine, adenosine and uridine was biphasic with approx. 40% of the total uptake inhibited with high affinity (IC50 value 2 to 14 microM). Concentrations of thymidine and cytidine up to 1 mM had no effect on Na(+)-dependent uridine uptake and no Na(+)-dependent thymidine influx by confluent OK monolayers was detected. Using cell monolayers grown on a permeable filter support, Na(+)-dependent uridine uptake occurred preferentially from the apical surface. This high affinity component of Na(+)-dependent uridine uptake is suggested to represent the Na(+)-dependent purine preferring N1 nucleoside transporter. The Na+/uridine stoichiometry for this system was consistent with 1:1. The remaining component of Na(+)-dependent uridine uptake was inhibited by some nucleosides, such as guanosine and inosine, with low affinity (IC50 values of 0.6 to 5 mM). Other nucleosides showed little specific inhibition. We propose that this component of uridine uptake represents a mutated carrier that binds nucleosides but is defective in the translocation of permeant.
authors: Biological Laboratory, University of Kent, Canterbury, UK.
authors: A J
authors: AJ
authors: Doherty
authors: S M
authors: SM
authors: Jarvis
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D016922:Cellular Senescence; D004848:Epithelium / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D033703:Nucleoside Transport Proteins; D009893:Opossums; D011684:Purine Nucleosides / Q000378:metabolism; D012964:Sodium / Q000737:chemistry / Q000494:pharmacology*; D013868:Thioinosine / Q000031:analogs & derivatives / Q000494:pharmacology; D014529:Uridine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D011684:Purine Nucleosides; D013868:Thioinosine; D012964:Sodium; C001789:4-nitrobenzylthioinosine; D014529:Uridine