Höpfner M, Lemmer K, Jansen A, Hanski C, Riecken E O, Gavish M, Mann B, Buhr H, Glassmeier G, Scherübl H
Abteilung Innere Medizin/Gastroenterologie, Abteilung Allgemein-, Gefäss-, und Thoraxchirugie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, Berlin, 12200, Germany.
Biochem Biophys Res Commun. 1998 Oct 29;251(3):811-7. doi: 10.1006/bbrc.1998.9555.
Primary cell cultures of human colorectal carcinomas were established and characterized immunocytochemically. In the isolated cancer cells intracellular Ca2+ concentrations ([Ca2+]i) were measured by the fura-2 method. Stimulation with either extracellular ATP or UTP caused a biphasic rise of [Ca2+]i in a dose-dependent manner and cross-desensitization between both nucleotides was observed. The rank order of potency was ATP >== UTP > ATP-gamma-S > ADP > adenosine which is characteristic for a P2U-receptor subtype. Selective agonists of P1-, or P2X- purinoceptors had no effect on [Ca2+]i. The initial rise in [Ca2+]i was independent of extracellular calcium [Ca2+]e, whereas the second phase was not observed under [Ca2+]e-free conditions suggesting a capacitative Ca2+-entry-mechanism. Intracellular Ca2+ mobilization was proven by use of the Ca2+-ATPase inhibitor thapsigargin. P2U-specific mRNA could be detected by RT-PCR in both colorectal tumor tissues and in the human colorectal cancer cell line HT 29. In HT 29 cells, the hydrolysis-resistant ATP analog ATP-gamma-S inhibited cell proliferation and, also, induced apoptosis in a dose-dependent manner. Thus, human colorectal cancer cells express functional P2U-receptors which may play a role in the regulation of cell proliferation and apoptosis.
建立了人结肠直肠癌的原代细胞培养物，并通过免疫细胞化学进行了表征。在分离的癌细胞中，采用fura-2方法测量细胞内Ca2+浓度（[Ca2+]i）。细胞外ATP或UTP刺激均以剂量依赖性方式引起[Ca2+]i双相升高，且观察到两种核苷酸之间存在交叉脱敏现象。效力顺序为ATP＞UTP＞ATP-γ-S＞ADP＞腺苷，这是P2U受体亚型的特征。P1或P2X嘌呤受体的选择性激动剂对[Ca2+]i无影响。[Ca2+]i的初始升高与细胞外钙[Ca2+]e无关，而在无[Ca2+]e条件下未观察到第二阶段，提示存在钙池调控的Ca2+内流机制。通过使用Ca2+-ATP酶抑制剂毒胡萝卜素证实了细胞内Ca2+的动员。通过RT-PCR在结肠肿瘤组织和人结肠癌细胞系HT 29中均可检测到P2U特异性mRNA。在HT 29细胞中，抗水解的ATP类似物ATP-γ-S抑制细胞增殖，并以剂量依赖性方式诱导细胞凋亡。因此，人结肠癌细胞表达功能性P2U受体，其可能在细胞增殖和凋亡的调控中发挥作用。
