Equilibrative and concentrative nucleoside transporters mediate influx of extracellular cyclic ADP-ribose into 3T3 murine fibroblasts.
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.
D016475:3T3 Cells; D036541:ADP-ribosyl Cyclase / Q000096:biosynthesis; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis; D001692:Biological Transport; D019556:COS Cells; D002851:Chromatography, High Pressure Liquid; D018920:Coculture Techniques; D036563:Cyclic ADP-Ribose / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007288:Inosine / Q000378:metabolism; D008562:Membrane Glycoproteins; D051379:Mice; D009705:Nucleosides / Q000378:metabolism*; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D012964:Sodium / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014162:Transfection
D015703:Antigens, CD; D008562:Membrane Glycoproteins; D009705:Nucleosides; D012333:RNA, Messenger; D036563:Cyclic ADP-Ribose; D007288:Inosine; D012964:Sodium; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; D051997:ADP-ribosyl Cyclase 1