Multiple phospholipase activation by OX₁ orexin/hypocretin receptors

Abstract.

We investigated coupling of OX₁ receptors to phospholipase activation and diacylglycerol generation in Chinese hamster ovary (CHO) cells using both biochemical and fluorescence “real-time” methods. The results indicate that at lowest orexin-A concentrations (highest potency), diacylglycerol generated results from phospholipase D activity. At 10–100-fold higher orexin-A concentrations, phospholipase C is activated, likely hydrolyzing phosphatidylinositol (PI) or phosphatidylinositol monophosphate (PIP) but not phosphatidylinositol bisphosphate (PIP₂). At further 7-fold higher orexin-A concentrations, PIP₂ is hydrolyzed, releasing both diacylglycerol and inositol-1,4,5-trisphosphate. Thus, OX₁ orexin receptors connect to multiple phospholipase activities, apparently composed of at least one phospholipase D and two different phospholipase C activities. At low agonist concentrations, diacylglycerol and phosphatidic acid are the preferred products, and interestingly, it seems that even the primarily activated phospholipase C mainly works to increase diacylglycerol and not inositol-1,4,5-trisphosphate.