title: OX1 orexin receptors couple to adenylyl cyclase regulation via multiple mechanisms.
abstract: In this study, the mechanism of OX(1) orexin receptors to regulate adenylyl cyclase activity when recombinantly expressed in Chinese hamster ovary cells was investigated. In intact cells, stimulation with orexin-A led to two responses, a weak (21%), high potency (EC(50) approximately 1 nm) inhibition and a strong (4-fold), low potency (EC(50) = approximately 300 nm) stimulation. The inhibition was reversed by pertussis toxin, suggesting the involvement of G(i/o) proteins. Orexin-B was, surprisingly, almost equally as potent as orexin-A in elevating cAMP (pEC(50) = approximately 500 nm). cAMP elevation was not caused by Ca(2+) elevation or by Gbetagamma. In contrast, it relied in part on a novel protein kinase C (PKC) isoform, PKCdelta, as determined using pharmacological inhibitors. Yet, PKC stimulation alone only very weakly stimulated cAMP production (1.1-fold). In the presence of G(s) activity, orexins still elevated cAMP; however, the potencies were greatly increased (EC(50) of orexin-A = approximately 10 nm and EC(50) of orexin-B = approximately 100 nm), and the response was fully dependent on PKCdelta. In permeabilized cells, only a PKC-independent low potency component was seen. This component was sensitive to anti-Galpha(s) antibodies. We conclude that OX(1) receptors stimulate adenylyl cyclase via a low potency G(s) coupling and a high potency phospholipase C --> PKC coupling. The former or some exogenous G activation is essentially required for the PKC to significantly activate adenylyl cyclase. The results also suggest that orexin-B-activated OX(1) receptors couple to G(s) almost as efficiently as the orexin-A-activated receptors, in contrast to Ca(2+) elevation and phospholipase C activation, for which orexin-A is 10-fold more potent.
authors.affiliation: Department of Neuroscience, Unit of Physiology, Uppsala University, BMC, SE-75123 Uppsala, Sweden.
authors.forename: Tomas
authors.initials: T
authors.lastname: Holmqvist
authors.forename: Lisa
authors.initials: L
authors.lastname: Johansson
authors.forename: Marie
authors.initials: M
authors.lastname: Ostman
authors.forename: Sylwia
authors.initials: S
authors.lastname: Ammoun
authors.forename: Karl E O
authors.initials: KE
authors.lastname: Akerman
authors.forename: Jyrki P
authors.initials: JP
authors.lastname: Kukkonen
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002118:Calcium; D000242:Cyclic AMP / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology; D009479:Neuropeptides / Q000502:physiology; D064446:Orexin Receptors; D000068797:Orexins; D011493:Protein Kinase C / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins; D019076:Transgenes; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: C000596229:HCRT protein, human; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides; D064446:Orexin Receptors; D000068797:Orexins; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D011994:Recombinant Proteins; D000242:Cyclic AMP; D011493:Protein Kinase C; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases; D002118:Calcium