Seyfred M A, Wells W W
J Biol Chem. 1984 Jun 25;259(12):7666-72.
The intracellular site of vasopressin-induced phosphoinositide breakdown in rat hepatocytes was investigated. After 45 s of vasopressin treatment of hepatocytes prelabeled with 32Pi, the levels of 32P-labeled phosphatidylinositol 4-phosphate (PI-P) and phosphatidylinositol 4,5-bisphosphate (PI-P2) in the plasma membrane decreased by approximately 40%, then gradually returned to near control levels after 10 min of treatment. Only small changes in the levels of [32P] PI-P and [32P]PI-P2 were observed in the other subcellular fractions, and were attributed to contamination of these fractions by plasma membranes. The level of 32P-labeled phosphatidylinositol in the plasma membrane decreased by 15% after 45 s of vasopressin treatment and then increased above control levels at later times while 32P-labeled phosphatidic acid levels in the plasma membrane gradually increased to 2-fold greater than control after 5 min of treatment. Using 32P-labeled plasma membranes obtained from prelabeled hepatocytes, it was found that PI-P and PI-P2 were rapidly degraded by a calcium-dependent polyphosphoinositide-specific phosphodiesterase. The enzyme was activated by physiological concentrations (200 nM) of free calcium when assayed at low ionic strength, but the calcium requirement shifted to micromolar concentrations under isosmotic, intracellular-like, ionic conditions. Addition of vasopressin (200 nM) to the 32P-labeled plasma membranes stimulated the breakdown of 20% of the [32P]PI-P2 present in the plasma membranes in 1 min when assayed under isosmotic conditions in the presence of 2 nM MgCl2 and approximately 200 nM free calcium. This suggests that the phosphoinositide-specific phosphodiesterase is not active under normal cellular conditions, but is activated upon the addition of vasopressin to the intact cell.
研究了血管加压素诱导大鼠肝细胞中磷酸肌醇分解的细胞内位点。用³²P标记预先标记的肝细胞，经血管加压素处理45秒后，质膜中³²P标记的磷脂酰肌醇4-磷酸（PI-P）和磷脂酰肌醇4,5-二磷酸（PI-P2）水平下降约40%，处理10分钟后逐渐恢复到接近对照水平。在其他亚细胞组分中，仅观察到[³²P]PI-P和[³²P]PI-P2水平有微小变化，这归因于这些组分被质膜污染。血管加压素处理45秒后，质膜中³²P标记的磷脂酰肌醇水平下降15%，随后在稍后时间升至对照水平以上，而质膜中³²P标记的磷脂酸水平在处理5分钟后逐渐增加至对照水平的2倍。使用从预先标记的肝细胞获得的³²P标记质膜，发现PI-P和PI-P2被钙依赖性多磷酸肌醇特异性磷酸二酯酶快速降解。在低离子强度下测定时，该酶被生理浓度（200 nM）的游离钙激活，但在等渗、类似细胞内的离子条件下，对钙的需求转变为微摩尔浓度。在含有2 nM MgCl₂和约200 nM游离钙的等渗条件下测定时，向³²P标记的质膜中添加血管加压素（200 nM）可在1分钟内刺激质膜中20%的[³²P]PI-P2分解。这表明磷酸肌醇特异性磷酸二酯酶在正常细胞条件下不活跃，但在向完整细胞中添加血管加压素后被激活。
