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

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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 Ca2+-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.

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Guida, L., Bruzzone, S., Sturla, L., Franco, L., Zocchi, E., & De Flora, A. (2002). Equilibrative and concentrative nucleoside transporters mediate influx of extracellular cyclic ADP-ribose into 3T3 murine fibroblasts. Journal of Biological Chemistry, 277(49), 47097–47105. https://doi.org/10.1074/jbc.M207793200

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