title: The orexin OX1 receptor activates a novel Ca2+ influx pathway necessary for coupling to phospholipase C.
abstract: Ca(2+) elevations in Chinese hamster ovary cells stably expressing OX(1) receptors were measured using fluorescent Ca(2+) indicators fura-2 and fluo-3. Stimulation with orexin-A led to pronounced Ca(2+) elevations with an EC(50) around 1 nm. When the extracellular [Ca(2+)] was reduced to a submicromolar concentration, the EC(50) was increased 100-fold. Similarly, the inositol 1,4,5-trisphosphate production in the presence of 1 mm external Ca(2+) was about 2 orders of magnitude more sensitive to orexin-A stimulation than in low extracellular Ca(2+). The shift in the potency was not caused by depletion of intracellular Ca(2+) but by a requirement of extracellular Ca(2+) for production of inositol 1,4,5-trisphosphate. Fura-2 experiments with the "Mn(2+)-quench technique" indicated a direct activation of a cation influx pathway by OX(1) receptor independent of Ca(2+) release or pool depletion. Furthermore, depolarization of the cells to +60 mV, which almost nullifies the driving force for Ca(2+) entry, abolished the Ca(2+) response to low concentrations of orexin-A. The results thus suggest that OX(1) receptor activation leads to two responses, (i) a Ca(2+) influx and (ii) a direct stimulation of phospholipase C, and that these two responses converge at the level of phospholipase C where the former markedly enhances the potency of the latter.
authors.affiliation: Department of Physiology, Division of Cell Physiology, Uppsala University, Biomedical Centre (BMC), P.O. Box 572, S-75123 Uppsala, Sweden.
authors.forename: P E
authors.initials: PE
authors.lastname: Lund
authors.forename: R
authors.initials: R
authors.lastname: Shariatmadari
authors.forename: A
authors.initials: A
authors.lastname: Uustare
authors.forename: M
authors.initials: M
authors.lastname: Detheux
authors.forename: M
authors.initials: M
authors.lastname: Parmentier
authors.forename: J P
authors.initials: JP
authors.lastname: Kukkonen
authors.forename: K E
authors.initials: KE
authors.lastname: Akerman
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism* / Q000494:pharmacology; D006224:Cricetinae; D003592:Cytophotometry; D004305:Dose-Response Relationship, Drug; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009479:Neuropeptides / Q000378:metabolism* / Q000494:pharmacology; D064446:Orexin Receptors; D000068797:Orexins; D018408:Patch-Clamp Techniques; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; C000596229:HCRT protein, human; D007295:Inositol Phosphates; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides; D064446:Orexin Receptors; D000068797:Orexins; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D011994:Recombinant Proteins; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D008274:Magnesium; D002118:Calcium