Equilibrative and concentrative nucleoside transporters mediate influx of extracellular cyclic ADP-ribose into 3T3 murine fibroblasts
Equilibrative and concentrative nucleoside transporters mediate influx of extracellular cyclic ADP-ribose into 3T3 murine fibroblasts
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DOI：
10.1074/jbc.m207793200
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发表时间：
2002-12-06
影响因子：
4.8
通讯作者：
De Flora, A
中科院分区：
文献类型：
--
作者：
Guida, L;Bruzzone, S;De Flora, A
在哺乳动物中，环ADP-核糖（cADPR）是一种来自细胞内储存的通用钙动员剂，通过多功能胞外酶CD 38和相关的ADP-核糖基环化酶由细胞外表面的NAD（+）产生。最近，已在3 T3鼠成纤维细胞中观察到细胞外cADPR的流入，其中其促进Ca 2+介导的增殖增强。在这里，我们解决的性质和性质的cADPR流入CD 38（-）3 T3细胞，这表明多效性机制的平衡和集中运输。基于选择性抑制剂或实验条件（例如Na+依赖性主动共转运过程的消除和瞬时转染实验）和对3 T3成纤维细胞和HeLa细胞中的转录物的逆转录酶-聚合酶链反应分析，我们鉴定了具有特异性核苷转运蛋白（NT）的cADPR转运活性，平衡型（ENT 2）和浓缩型（CNT 2和硝基苄基硫代肌苷（NBMPR）-可降解的NT）。在这些NT物种中观察到肌苷和cADPR通量之间的相互抑制关系。在transwell中与CD 38转染的、产生cADPR的3 T3成纤维细胞饲养层共培养48 h的CD 38（-）3 T3细胞中，发生了纳摩尔细胞外cADPR的集中（但非平衡）转运。这些结果表明，可能的，迄今未被认识到的，细胞外代谢的核苷酸/核苷和cADPR介导的调节细胞内钙稳态之间的相关性。
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.