title: Calcium affects OX1 orexin (hypocretin) receptor responses by modifying both orexin binding and the signal transduction machinery.
abstract: OBJECTIVE
  One of the major responses upon orexin receptor activation is Ca(2+) influx, and this influx seems to amplify the other responses mediated by orexin receptors. However, the reduction in Ca(2+) , often used to assess the importance of Ca(2+) influx, might affect other properties, like ligand-receptor interactions, as suggested for some GPCR systems. Hence, we investigated the role of the ligand-receptor interaction and Ca(2+) signal cascades in the apparent Ca(2+) requirement of orexin-A signalling.
  
  
  METHODS
  Receptor binding was assessed in CHO cells expressing human OX1 receptors with [(125) I]-orexin-A by conventional ligand binding as well as scintillation proximity assays. PLC activity was determined by chromatography.
  
  
  RESULTS
  Both orexin receptor binding and PLC activation were strongly dependent on the extracellular Ca(2+) concentration. The relationship between Ca(2+) concentration and receptor binding was the same as that for PLC activation. However, when Ca(2+) entry was reduced by depolarizing the cells or by inhibiting the receptor-operated Ca(2+) channels, orexin-A-stimulated PLC activity was much more strongly inhibited than orexin-A binding.
  
  
  CONCLUSIONS
  Ca(2+) plays a dual role in orexin signalling by being a prerequisite for both ligand-receptor interaction and amplifying orexin signals via Ca(2+) influx. Some previous results obtained utilizing Ca(2+) chelators have to be re-evaluated based on the results of the current study. From a drug discovery perspective, further experiments need to identify the target for Ca(2+) in orexin-A-OX1 receptor interaction and its mechanism of action.
authors.affiliation: Biochemistry and Cell Biology, Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland.
authors.forename: Jaana
authors.initials: J
authors.lastname: Putula
authors.forename: Tero
authors.initials: T
authors.lastname: Pihlajamaa
authors.forename: Jyrki P
authors.initials: JP
authors.lastname: Kukkonen
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D003412:Cricetulus; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D009479:Neuropeptides / Q000378:metabolism*; D064446:Orexin Receptors / Q000378:metabolism*; D000068797:Orexins; D011485:Protein Binding; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: C000596229:HCRT protein, human; C578630:HCRTR1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides; D064446:Orexin Receptors; D000068797:Orexins; D010738:Type C Phospholipases; D002118:Calcium