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.
Keywords. Orexin, hypocretin, phospholipase C, phospholipase D, phosphoinositides, phosphatidylcholine, diacylglycerol, live cell imaging
Footnotes
Received 17 April 2008; received after revision 5 May 2008; accepted 6 May 2008