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

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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.