Regulatory mechanism of phospholipase D activity and its physiological role in rat parotid glands

在这项研究中，为了阐明控制胞吐部位PLD活性的机制，我们研究了各种因素对大鼠腮腺顶端质膜PLD活性的影响。我们还研究了PLD激活和唾液分泌之间的生理关系。结果如下：1.PLD的激活机制：游离脂肪酸与磷脂酰肌醇-4，5-二磷酸(PIP_2)协同激活PLD，而磷脂酰肌醇(PI)抑制PLD的激活。PLD也被PLD本身产生的磷脂酸(PA)激活。这些结果表明，PLD活性的调节是复杂的，至少有三个因素协同作用：(1)磷脂酶A_2产生脂肪酸，(2)平衡从PI转移到PIP_2，(3)产物介导的正反馈回路。磷脂酰肌醇和磷脂酰肌醇激酶之间的信号串扰：有趣的是，磷脂酰肌醇5-激酶(PIP5K)合成PIP_2也被PLD来源的PA激活。相反，PA抑制为PIP5K提供底物的PI激酶。另一方面，有人认为在PA存在或不存在的情况下，PIP倾向于在分泌颗粒膜中积累。这些结果表明，PIP5K来源的PIP_2刺激PLD，PLD来源的PA刺激PIP5K，从而实现正前馈调节。在胞吐部位，分泌颗粒膜可提供PIP5K底物。PLD信号与胞吐作用的相关性：在体外融合系统中用PLD处理细胞膜时，促进融合的作用与膜上PA含量成正比。此外，当PIP2直接导入腮腺细胞时，异丙肾上腺素刺激的淀粉酶分泌增加。这些结果表明，PLD和PIP5K共同作用，诱导某些生理功能，如胞吐。

In this study, to clarify mechanisms controlling PLD activity at the exocytotic site, we investigated the effects of various factors on the PLD activity associated with apical plasma membranes from rat parotid glands. We also investigated the physiological relationship between PLD activation and saliva secretion. An outline of the results is as follows :1. The mechanism of PLD activation :PLD was activated synergistically by free fatty acids and phosphatidylinositol-4, 5-diphosphate (PIP_2), whereas it was inhibited by phosphatidylinositol(PI). PLD was also activated by phosphatidic acid(PA), which is produced by PLD itself. These results suggest that the regulation of PLD activity is complex, and at least three factors act in a synergistic manner as follows: (1) the production of fatty acids by phospho-lipase A_2, (2) a shift in the balance from PI to PIP_2 and (3) a product-mediated positive feedback loop.2. Signal cross-talk between PLD and phosphoinositide kinases :Interestingly, the synthesis of PIP_2 by phosphoinositide 5-kinase (PIP5K) was also activated by PLD-derived PA. On the contrary, PA inhibited PI kinase, which supplies the substrate to PIP5K. On the other hand, it was thought that PIP tended to accumulate in secretory granular membranes in the presence or absence of PA. These results suggest that positive feed-forward regulation is achieved through the stimulation of PLD by PIP5K-derived PIP_2, and of PIP5K by PLD-derived PA. At the site of exocytosis, secretory granular membranes may supply the PIP5K substrate.3. The correlation of PLD signaling with exocytosis :When the membranes were treated with PLD in the in vitro fusion system, fusion was facilitated in proportion to the PA content of the membrane. Moreover, when PIP2 was introduced directly into the parotid cells, the amylase secretion stimulated by isoproterenol was increased.These results suggest that PLD and PIP5K act together to induce some physiological function, such as exocytosis.