Jiménez A, Pubill D, Pallàs M, Camins A, Lladó S, Camarasa J, Escubedo E
Unitat de Farmacologia i Farmacognòsia, Facultat de Farmàcia, Nucli Universitat de Pedralbes, 08028 (BCN), Barcelona, Spain.
Eur J Pharmacol. 2000 Jun 9;398(1):31-9. doi: 10.1016/s0014-2999(00)00297-1.
Previous work from our laboratory has demonstrated the presence of high-affinity binding sites for [3H]nitrobenzylthioinosine ([3H]NBTI), a marker of adenosine uptake systems, in the mitochondrial fraction of rat testis. Here, we characterize this system functionally through [3H]adenosine uptake assays. This system (K(m)=2+/-1.3 microM; V(max)=86.2+/-15.5 pmol/mg protein/min) was found to be saturable, non sodium-dependent and sensitive to temperature, pH and osmolarity. [3H]Adenosine incorporation was potently inhibited by hydroxynitrobenzylthioguanosine (HNBTG, IC(50)=3 nM) although NBTI inhibited this uptake weakly (IC(50)=72. 7+/-37.1 microM). Dilazep>dipyridamole>/=hexobendine inhibited [3H]adenosine incorporation at low micromolar concentrations. The nucleosides inosine and uridine were weak inhibitors of this system. The adenosine receptor ligands N(6)-phenylisopropyladenosine (PIA) and 2-chloroadenosine inhibited the uptake only at micromolar concentrations. Neither 5'-(N-ethylcarboxamido)-adenosine (NECA) nor theophylline inhibited adenosine uptake by more than 60% but the mitochodrial benzodiazepine receptor ligands 4'-chloro-diazepam (Ro 5-4864) and 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl) isoquinoline carboxamide (PK 11195) were able to inhibit it. The lack of inhibition by the blockers of the mitochondrial adenine-nucleotide carrier, atractyloside and alpha, beta-methylene-ATP, indicates that [3H]adenosine uptake occurs via a transporter other than this carrier. All these results support the existence of an equilibrative adenosine transport system, which might mediate the passage of adenosine formed in the mitochondria to the cytoplasm.
我们实验室之前的研究表明，大鼠睾丸线粒体部分存在[3H]硝基苄硫基肌苷（[3H]NBTI）的高亲和力结合位点，[3H]NBTI是腺苷摄取系统的标志物。在此，我们通过[3H]腺苷摄取试验对该系统进行功能表征。发现该系统（Km = 2±1.3微摩尔；Vmax = 86.2±15.5皮摩尔/毫克蛋白/分钟）具有饱和性、非钠依赖性，且对温度、pH和渗透压敏感。羟基硝基苄硫基鸟苷（HNBTG，IC50 = 3纳摩尔）能有效抑制[3H]腺苷掺入，而NBTI对这种摄取的抑制作用较弱（IC50 = 72.7±37.1微摩尔）。双嘧达莫>潘生丁≥己氧苯啶在低微摩尔浓度时能抑制[3H]腺苷掺入。肌苷和尿苷这两种核苷是该系统的弱抑制剂。腺苷受体配体N(6)-苯基异丙基腺苷（PIA）和2-氯腺苷仅在微摩尔浓度时抑制摄取。5'-(N-乙基羧酰胺基)-腺苷（NECA）和茶碱对腺苷摄取的抑制均不超过60%，但线粒体苯二氮䓬受体配体4'-氯地西泮（Ro 5-4864）和1-(2-氯苯基)-N-甲基-N-(1-甲基丙基)异喹啉甲酰胺（PK 11195）能够抑制它。线粒体腺嘌呤核苷酸载体的阻断剂苍术苷和α,β-亚甲基-ATP缺乏抑制作用，表明[3H]腺苷摄取是通过该载体以外的转运体进行的。所有这些结果支持存在一种平衡型腺苷转运系统，该系统可能介导线粒体中形成的腺苷向细胞质的转运。
