Institute of Pharmacology, Medical School of Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
Naunyn Schmiedebergs Arch Pharmacol. 2012 Jul;385(7):671-83. doi: 10.1007/s00210-012-0759-6. Epub 2012 May 3.
Hypoxanthine phosphoribosyl transferase (HPRT) deficiency results in Lesch-Nyhan disease (LND). The link between the HPRT defect and the self-injurious behavior in LND is still unknown. HPRT-deficient rat B103 neuroblastoma cells serve as a model system for LND. In B103 cell membranes, HPRT deficiency is associated with a decrease of basal and guanosine triphosphate-stimulated adenylyl cyclase (AC) activity (Pinto and Seifert, J Neurochem 96:454-459, 2006). Since recombinant AC2 possesses a high basal activity, we tested the hypothesis that AC2 function and expression is impaired in HPRT deficiency. We examined AC regulation in B103 cell membranes, cAMP accumulation in intact B103 cells, AC isoform expression, and performed morphological studies. As most important pharmacological tool, we used 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene forskolin (BODIPY-FS) that inhibits recombinant AC2 but activates ACs 1 and 5 (Erdorf et al., Biochem Pharmacol 82:1673-1681, 2011). In B103 control membranes, BODIPY-FS reduced catalysis, but in HPRT(-) membranes, BODIPY-FS was rather stimulatory. 2'(3')-O-(N-methylanthraniloyl) (MANT)-nucleoside 5'-[γ-thio]triphosphates inhibit recombinant ACs 1 and 5 more potently than AC2. In B103 control membranes, MANT-guanosine 5'-[γ-thio]triphosphate inhibited catalysis in control membranes less potently than in HPRT(-) membranes. Quantitative real-time PCR revealed that in HPRT deficiency, AC2 was virtually absent. In contrast, AC5 was up-regulated. Forskolin (FS) and BODIPY-FS induced cell clustering and rounding and neurite extension in B103 cells. The effects of FS and BODIPY-FS were much more prominent in control than in HPRT(-) cells, indicative for a differentiation defect in HPRT deficiency. Neither FS nor BODIPY-FS significantly changed cAMP concentrations in intact B103 cells. Collectively, our data show that HPRT deficiency in B103 cells is associated with impaired AC2 function and expression and reduced sensitivity to differentiation induced by FS and BODIPY-FS. We discuss the pathophysiological implications of our data for LND.
次黄嘌呤磷酸核糖转移酶 (HPRT) 缺陷导致 Lesch-Nyhan 病 (LND)。HPRT 缺陷与 LND 中的自伤行为之间的联系尚不清楚。HPRT 缺陷的大鼠 B103 神经母细胞瘤细胞可作为 LND 的模型系统。在 B103 细胞膜中，HPRT 缺陷与基础和鸟苷三磷酸刺激的腺苷酸环化酶 (AC) 活性降低有关 (Pinto 和 Seifert，J Neurochem 96:454-459, 2006)。由于重组 AC2 具有很高的基础活性，我们检验了 AC2 功能和表达在 HPRT 缺陷中受损的假设。我们研究了 B103 细胞膜中的 AC 调节、完整 B103 细胞中的 cAMP 积累、AC 同工型表达，并进行了形态学研究。作为最重要的药理学工具，我们使用了 4,4-二氟-4-硼-3a,4a-二氮杂-s-茚并菲醇 forskolin (BODIPY-FS)，它抑制重组 AC2 但激活 ACs 1 和 5 (Erdorf 等人，Biochem Pharmacol 82:1673-1681, 2011)。在 B103 对照膜中，BODIPY-FS 降低了催化作用，但在 HPRT(-) 膜中，BODIPY-FS 则具有刺激性。2'(3')-O-(N-甲基邻氨基苯甲酰基) (MANT)-核苷 5'-[γ-硫]三磷酸对重组 ACs 1 和 5 的抑制作用比对 AC2 更强。在 B103 对照膜中，MANT-鸟苷 5'-[γ-硫]三磷酸对对照膜的抑制作用比对 HPRT(-) 膜的抑制作用弱。实时定量 PCR 显示，在 HPRT 缺陷中，AC2 几乎不存在。相比之下，AC5 上调。福司可林 (FS) 和 BODIPY-FS 诱导 B103 细胞聚类和圆化以及神经突延伸。FS 和 BODIPY-FS 的作用在对照细胞中比在 HPRT(-) 细胞中更为明显，表明 HPRT 缺陷中存在分化缺陷。FS 和 BODIPY-FS 均未显著改变完整 B103 细胞中的 cAMP 浓度。总的来说，我们的数据表明，B103 细胞中的 HPRT 缺陷与 AC2 功能和表达受损以及对 FS 和 BODIPY-FS 诱导的分化的敏感性降低有关。我们讨论了我们的数据对 LND 的病理生理意义。
