Equilibrative and concentrative nucleoside transporters mediate influx of extracellular cyclic ADP-ribose into 3T3 murine fibroblasts

J Biol Chem. 2002 Dec 6;277(49):47097-105. doi: 10.1074/jbc.M207793200. Epub 2002 Oct 3.

Abstract

In mammals cyclic ADP-ribose (cADPR), a universal calcium mobilizer from intracellular stores, is generated from NAD(+) at the outer cell surface by the multifunctional ectoenzyme CD38 and by related ADP-ribosyl cyclases. Recently, influx of extracellular cADPR has been observed in 3T3 murine fibroblasts, where it elicits Ca(2+)-mediated enhancement of proliferation. Here we addressed the nature and the properties of cADPR influx into CD38(-) 3T3 cells, which showed pleiotropic mechanisms of both equilibrative and concentrative transport. Based on selective inhibitors or experimental conditions (e.g. abrogation of Na(+)-dependent active symport processes and transient transfection experiments) and on reverse transcriptase-polymerase chain reaction analysis of transcripts in 3T3 fibroblasts and comparatively in HeLa cells, we identified cADPR-transporting activities with specific nucleoside transporters (NT), both equilibrative (ENT2) and concentrative (CNT2 and a nitrobenzylthioinosine (NBMPR)-inhibitable NT). A reciprocal inhibition relationship was observed between inosine and cADPR fluxes across these NT species. Concentrative (but not equilibrative) transport of nanomolar extracellular cADPR took place in CD38(-) 3T3 cells co-cultured for 48 h in transwells on feeders of CD38-transfected, cADPR-generating 3T3 fibroblasts. These results suggest possible, hitherto unrecognized, correlations between ectocellular metabolism of nucleotides/nucleosides and cADPR-mediated regulation of intracellular calcium homeostasis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • ADP-ribosyl Cyclase / biosynthesis
  • ADP-ribosyl Cyclase 1
  • Animals
  • Antigens, CD / biosynthesis
  • Biological Transport
  • COS Cells
  • Chromatography, High Pressure Liquid
  • Coculture Techniques
  • Cyclic ADP-Ribose / metabolism*
  • Dose-Response Relationship, Drug
  • Erythrocytes / metabolism
  • Fibroblasts / metabolism
  • HeLa Cells
  • Humans
  • Inosine / metabolism
  • Membrane Glycoproteins
  • Mice
  • Nucleosides / metabolism*
  • Protein Transport
  • RNA, Messenger / metabolism
  • Sodium / chemistry
  • Temperature
  • Time Factors
  • Transfection

Substances

  • Antigens, CD
  • Membrane Glycoproteins
  • Nucleosides
  • RNA, Messenger
  • Cyclic ADP-Ribose
  • Inosine
  • Sodium
  • ADP-ribosyl Cyclase
  • CD38 protein, human
  • Cd38 protein, mouse
  • ADP-ribosyl Cyclase 1