Soltoff S P, McMillian M K, Talamo B R, Cantley L C
Department of Medicine, Beth Israel Hospital, Boston, MA 02115.
Biochem Pharmacol. 1993 May 5;45(9):1936-40. doi: 10.1016/0006-2952(93)90455-6.
Extracellular ATP activates a P2Z-type purinergic receptor (purinoceptor) in rat parotid acinar cells that increases the intracellular free Ca2+ concentration via the entry of extracellular Ca2+ through an ATP-sensitive cation channel (Soltoff et al., Am J Physiol 262: C934-C940, 1992). To learn more about the ATP binding site of the purinoceptor, we examined the effects of several stilbene isothiocyanate analogs of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), which block the binding of [32P]ATP to intact parotid cells (McMillian et al., Biochem J 255:291-300, 1988) and blocked the activation of the P2Z purinoceptor. The ATP-stimulated 45Ca2+ uptake was blocked by DIDS, H2DIDS (dihydro-DIDS; 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid), and SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid), but not by DNDS (4,4'-dinitrostilbene-2,2'-disulfonic acid), a stilbene disulfonate compound lacking isothiocyanate (SCN-) groups, or by KSCN. The potency of the stilbene disulfonates was related to the number of isothiocyanate groups on each compound. Under the experimental conditions, the IC50 value of DIDS (approximately 35 microM), which has two SCN-groups, was much lower than that of SITS (approximately 125 microM), which has only one SCN-group. The inhibitory effects of DIDS appeared to be much more potent than those of SITS due to the kinetics of their binding to the purinoceptors. Eosin-5-isothiocyanate (EITC) and fluorescein-5-isothiocyanate (FITC), non-stilbene isothiocyanate compounds with single SCN-groups, also blocked the response to ATP and were less potent than DIDS. Trinitrophenyl-ATP (TNP-ATP), an ATP derivative that is not an effective agonist of the parotid P2Z receptor, blocked the covalent binding of DIDS to the plasma membrane, suggesting that ATP and DIDS bind to the same site. Reactive Blue 2 (Cibacron Blue 3GA), an anthraquinone-sulfonic acid derivative that is a noncovalent purinergic antagonist, also blocked the covalent binding of DIDS to the plasma membrane. These results suggest that isothiocyanate compounds interact with the ATP binding site of this P2 purinoceptor, and that isothiocyanate groups make an important contribution in determining the effectiveness of the stilbene disulfonate compounds in blocking the binding of nucleotide agonists to this purinoceptor.
细胞外ATP可激活大鼠腮腺腺泡细胞中的P2Z型嘌呤能受体（嘌呤受体），该受体通过细胞外Ca²⁺经ATP敏感阳离子通道内流来增加细胞内游离Ca²⁺浓度（索尔托夫等人，《美国生理学杂志》262:C934 - C940，1992年）。为了更多地了解嘌呤受体的ATP结合位点，我们研究了几种二硫代二苯乙烯异硫氰酸酯类似物（DIDS，4,4'-二异硫氰酸根合芪-2,2'-二磺酸）的作用，这些类似物可阻断[³²P]ATP与完整腮腺细胞的结合（麦克米利安等人，《生物化学杂志》255:291 - 300，1988年），并阻断P2Z嘌呤受体的激活。ATP刺激的⁴⁵Ca²⁺摄取被DIDS、H2DIDS（二氢-DIDS；4,4'-二异硫氰酸根合二氢芪-2,2'-二磺酸）和SITS（4-乙酰氨基-4'-异硫氰酸根合芪-2,2'-二磺酸）阻断，但未被DNDS（4,4'-二硝基芪-2,2'-二磺酸）阻断，DNDS是一种不含异硫氰酸根（SCN⁻）基团的二硫代二苯乙烯化合物，也未被KSCN阻断。二硫代二苯乙烯磺酸盐的效力与每种化合物上异硫氰酸根的数量有关。在实验条件下，具有两个SCN基团的DIDS（约35微摩尔）的IC50值远低于仅具有一个SCN基团的SITS（约125微摩尔）。由于DIDS与嘌呤受体结合的动力学，其抑制作用似乎比SITS更强。曙红-5-异硫氰酸酯（EITC）和荧光素-5-异硫氰酸酯（FITC），具有单个SCN基团的非二硫代二苯乙烯异硫氰酸酯化合物，也阻断了对ATP的反应，且效力低于DIDS。三硝基苯基-ATP（TNP-ATP），一种不是腮腺P2Z受体有效激动剂的ATP衍生物，阻断了DIDS与质膜的共价结合，表明ATP和DIDS结合到同一位点。活性蓝2（汽巴克隆蓝3GA），一种蒽醌磺酸衍生物，是非共价嘌呤能拮抗剂，也阻断了DIDS与质膜的共价结合。这些结果表明异硫氰酸酯化合物与该P2嘌呤受体的ATP结合位点相互作用，并且异硫氰酸根在确定二硫代二苯乙烯磺酸盐化合物阻断核苷酸激动剂与该嘌呤受体结合的有效性方面起重要作用。
