OXI orexin receptors (OXIR) 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 OXIR 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 OXIR-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 OXIR-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. (c) 2006 Elsevier Inc. All rights reserved.