Multiple phospholipase activation by OX₁ orexin/hypocretin receptors

• Published in: Cellular and molecular life sciences : CMLS Vol. 65; no. 12; pp. 1948 - 1956
• Main Authors: Johansson, L, Ekholm, M. E, Kukkonen, J. P
• Format: Journal Article
• Language: English
• Published: Basel Basel : SP Birkhäuser Verlag Basel 01.06.2008; SP Birkhäuser Verlag Basel; Springer Nature B.V
• Subjects: agonists; animal ovaries; Biochemistry; Biomedical and Life Sciences; Biomedicine; Cell Biology; Cellular biology; Chinese hamsters; diacylglycerol; Diglycerides; Enzymes; Fluorescence; hypocretin; Inositol 1,4,5-trisphosphate receptors; Inositols; Life Sciences; live cell imaging; Orexin; Orexin receptors; Orexins; Phosphatidic acid; phosphatidylcholine; Phosphatidylinositol; Phosphatidylinositol 4,5-diphosphate; phosphoinositides; Phospholipase; Phospholipase C; Phospholipase D; Receptors; Research Article; phospholipase C; phospholipase D; phosphatidylcholine; hypocretin; live cell imaging; Orexin; phosphoinositides; diacylglycerol
• ISSN: 1420-682X; 1420-9071
• DOI: 10.1007/s00018-008-8206-z

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.