Cunha-Melo J R, Dean N M, Moyer J D, Maeyama K, Beaven M A
J Biol Chem. 1987 Aug 25;262(24):11455-63.
We have re-examined, by high pressure liquid chromatographic (HPLC) procedures, the hydrolysis of 3H-labeled inositol phospholipids in rat basophilic leukemia (RBL-2H3) cells. Previous studies showed no clear correlation between the release of any particular inositol metabolite and the calcium signal in these cells. Paradoxically no responses were observed when the cells were stimulated with the antigen, aggregated ovalbumin, in the absence of external Ca2+. We report here that in the absence of external Ca2+ aggregation of the IgE receptor by agents other than aggregated ovalbumin causes the release of small amounts of [3H]inositol phosphates and a small increase in levels of cytosol Ca2+ (approximately 25 nM). The response, however, varied with the type of stimulant used. Within seconds after addition of 24 mol of dinitrophenol conjugated with 1 mol of bovine serum albumin to cells primed with dinitrophenol-specific IgE there was a small burst in release of [3H]inositol 1,4,5-triphosphate, [3H]inositol 1,3,4,5-tetrakisphosphate, and [3H]inositol 1,3,4-trisphosphate which was followed by a gradual rise in inositol 1,3,4-trisphosphate, inositol bisphosphate, and inositol monophosphate. Eventually, all inositol phosphates reached different steady state levels which were maintained for at least 40 min. In contrast, the initial response to oligomeric IgE, which aggregates receptors at a relatively slow rate, was muted although the subsequent development of the response was the same. The levels of inositol pentakisphosphate and hexakisphosphate remained unchanged. These and other studies with cell extracts support the conclusion that inositol 1,4,5-trisphosphate, a putative messenger for release of intracellular Ca2+, was converted to inositol 1,3,4,5-tetrakisphosphate and thence to inositol 1,3,4-trisphosphate. Both trisphosphate metabolites were dephosphorylated in sequential fashion by phosphatase enzymes in the cytosolic and membrane fractions. However, the appearance of several isomers of inositol monophosphates and bisphosphates suggested that degradation proceeded through multiple pathways in the intact cell.
我们运用高压液相色谱（HPLC）方法，重新检测了大鼠嗜碱性白血病（RBL - 2H3）细胞中3H标记的肌醇磷脂的水解情况。先前的研究表明，在这些细胞中，任何特定肌醇代谢产物的释放与钙信号之间均无明显关联。矛盾的是，当细胞在无细胞外Ca2+的情况下用抗原（聚合卵清蛋白）刺激时，未观察到任何反应。我们在此报告，在无细胞外Ca2+的情况下，除聚合卵清蛋白外的其他试剂使IgE受体聚集会导致少量[3H]肌醇磷酸的释放以及胞质溶胶Ca2+水平的小幅升高（约25 nM）。然而，该反应会因所用刺激剂的类型而异。在用二硝基苯酚特异性IgE致敏的细胞中加入24摩尔与1摩尔牛血清白蛋白偶联的二硝基苯酚后数秒内，[3H]肌醇1,4,5 - 三磷酸、[3H]肌醇1,3,4,5 - 四磷酸和[3H]肌醇1,3,4 - 三磷酸的释放出现一个小高峰，随后肌醇1,3,4 - 三磷酸、肌醇二磷酸和肌醇单磷酸逐渐升高。最终，所有肌醇磷酸达到不同的稳态水平，并维持至少40分钟。相比之下，对以相对较慢速率使受体聚集的寡聚IgE的初始反应较为微弱，尽管随后反应的发展情况相同。肌醇五磷酸和六磷酸的水平保持不变。这些以及对细胞提取物的其他研究支持以下结论：肌醇1,4,5 - 三磷酸（一种推测的细胞内Ca2+释放信使）先转化为肌醇1,3,4,5 - 四磷酸，进而转化为肌醇1,3,4 - 三磷酸。这两种三磷酸代谢产物在胞质溶胶和膜部分被磷酸酶依次去磷酸化。然而，肌醇单磷酸和二磷酸的几种异构体的出现表明，在完整细胞中降解通过多种途径进行。
