Duncan R A, Davis J S
Department of Veterans Affairs Medical Center, Wichita, Kansas.
Endocrinology. 1991 Mar;128(3):1519-26. doi: 10.1210/endo-128-3-1519.
The present studies were conducted to further evaluate inositol phosphate formation and metabolism in prostaglandin F2 alpha (PGF2 alpha)-stimulated bovine luteal cells. Corpora lutea were dispersed with collagenase, and luteal cells were prelabeled for 3 h with [3H]inositol. Inositol phosphates produced in response to PGF2 alpha were analyzed by ion exchange column chromatography and HPLC. Time-course experiments revealed that significant increases in inositol trisphosphate (InsP3) were apparent within 5 sec of incubation with PGF2 alpha. Increases in inositol bisphosphate (InsP2) were also apparent within 5 sec. InsP1 and InsP4 were observed after a short (5-sec) lag period. HPLC revealed that PGF2 alpha provoked rapid (5 sec) increases in inositol 1,4,5-trisphosphate (Ins 1,4,5-P3), which was rapidly converted to inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5-P4) and inositol 1,3,4-trisphosphate (Ins 1,3,4-P3). The primary inositol bisphosphate isomer present in PGF2 alpha-stimulated bovine luteal cells was inositol 1,4-bisphosphate (Ins 1,4-P2), with lesser amounts of Ins 1,3-P2. Inositol monophosphates were also increased. These findings were confirmed in studies in which the metabolism of purified [3H]Ins 1,4,5-P3 was followed temporally in saponin-permeabilized bovine luteal cells. Additional studies demonstrated the presence of an enzyme, InsP3-3-kinase, in the cytosolic fraction of bovine corpora lutea. InsP3-3-kinase phosphorylated Ins 1,4,5-P3 to form Ins 1,3,4,5-P4. The activity of InsP3-3-kinase was calcium dependent and was enhanced by calmodulin at low calcium concentrations. Calmidazolium, a calmodulin inhibitor, reduced InsP3-3-kinase activity in a concentration-dependent manner. These results demonstrate the presence of multiple polyphosphorylated inositol phosphates in PGF2 alpha-stimulated bovine luteal cells. The isomers were formed via the action of a specific calcium/calmodulin-regulated kinase (InsP3-3-kinase), which phosphorylated Ins 1,4,5-P3 during agonist-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. These data suggest that the inositol tris/tetrakisphosphate pathway is an important sequelae to PGF2 alpha-stimulated inositol phospholipid hydrolysis, and that the pathway may be activated during agonist-mediated calcium mobilization.
本研究旨在进一步评估前列腺素F2α（PGF2α）刺激的牛黄体细胞中肌醇磷酸的形成和代谢。黄体用胶原酶分散，黄体细胞用[3H]肌醇预标记3小时。通过离子交换柱色谱和高效液相色谱分析PGF2α刺激产生的肌醇磷酸。时间进程实验表明，与PGF2α孵育5秒内，肌醇三磷酸（InsP3）显著增加。肌醇二磷酸（InsP2）在5秒内也明显增加。InsP1和InsP4在短暂（5秒）延迟期后被观察到。高效液相色谱显示，PGF2α促使肌醇1,4,5-三磷酸（Ins 1,4,5-P3）迅速（5秒）增加，其迅速转化为肌醇1,3,4,5-四磷酸（Ins 1,3,4,5-P4）和肌醇1,3,4-三磷酸（Ins 1,3,4-P3）。PGF2α刺激的牛黄体细胞中存在的主要肌醇二磷酸异构体是肌醇1,4-二磷酸（Ins 1,4-P2），Ins 1,3-P2含量较少。肌醇单磷酸也增加。在皂角苷通透的牛黄体细胞中对纯化的[3H]Ins 1,4,5-P3的代谢进行时间跟踪的研究中证实了这些发现。进一步的研究表明，牛黄体的胞质部分存在一种酶，即InsP3-3-激酶。InsP3-3-激酶将Ins 1,4,5-P3磷酸化形成Ins 1,3,4,5-P4。InsP3-3-激酶的活性依赖于钙，在低钙浓度下被钙调蛋白增强。钙调蛋白抑制剂氯米达唑以浓度依赖的方式降低InsP3-3-激酶的活性。这些结果表明，在PGF2α刺激的牛黄体细胞中存在多种多磷酸化的肌醇磷酸。这些异构体是通过一种特定的钙/钙调蛋白调节激酶（InsP3-3-激酶）的作用形成的，该激酶在激动剂介导的磷脂酰肌醇4,5-二磷酸水解过程中将Ins 1,4,5-P3磷酸化。这些数据表明，肌醇三/四磷酸途径是PGF2α刺激的肌醇磷脂水解的重要后续反应，并且该途径可能在激动剂介导的钙动员过程中被激活。
