IP3-independent signalling of OX1 orexin/hypocretin receptors to Ca2+ influx and ERK.
OX1 orexin receptors (OX1R) have been shown to activate receptor-operated Ca2+ influx pathways as their primary signalling pathway; however, investigations are hampered by the fact that orexin receptors also couple to phospholipase C, and therewith inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ release. We have here devised a method to block the latter signalling in order to focus on the mechanism of Ca2+ influx activation by OX1R in recombinant systems. Transient expression of the IP3-metabolising enzymes IP3-3-kinase-A (inositol-1,4,5-trisphosphate-->inositol-1,3,4,5-tetrakisphosphate) and type I IP3-5-phosphatase (inositol-1,4,5-trisphosphate-->inositol-1,4-bisphosphate) almost completely attenuated the OX1R-stimulated IP3 elevation and Ca2+ release from intracellular stores. Upon attenuation of the IP3-dependent signalling, the receptor-operated Ca2+ influx pathway became the only source for Ca2+ elevation, enabling mechanistic studies on the receptor-channel coupling. Attenuation of the IP3 elevation did not affect the OX1R-mediated ERK (extracellular signal-regulated kinase) activation in CHO cells, which supports our previous finding of the major importance of receptor-operated Ca2+ influx for this response.
Ekholm
Marie E
ME
Department of Neuroscience, Division of Physiology, Uppsala University, BMC, PO Box 572, SE-75123 Uppsala, Sweden.
Johansson
Lisa
L
Kukkonen
Jyrki P
JP
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000493:pharmacokinetics*; D006224:Cricetinae; D003412:Cricetulus; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D064446:Orexin Receptors; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D018013:Receptors, Neuropeptide / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
D053496:Inositol 1,4,5-Trisphosphate Receptors; D064446:Orexin Receptors; D019269:Phosphatidylinositol 4,5-Diphosphate; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium