title: Pharmacological characterisation of the goldfish somatostatin sst5 receptor.
abstract: Somatostatin (somatotropin release inhibiting factor, SRIF), exerts its effects via specific G protein coupled receptors of which five subtypes have been cloned (sst1-5). Recently, SRIF receptors have also been cloned from fish tissues. In this study, goldfish sst5 receptors (gfsst5) were expressed and characterised in the Chinese hamster lung fibroblast cell line, that harbours the luciferase reporter gene driven by the serum responsive element (CCL39-SRE-Luci). The agonist radioligands [125I]-LTT-SRIF-28 ([Leu8, DTrp22, 125I-Tyr25]SRIF-28) and [125I][Tyr10]cortistatin-14 labelled similar receptor densities with high affinity and in a saturable manner (pKd: 9.99-9.71; Bmax: 300-350 fmol mg-1). 5'-Guanylyl-imidodiphosphate inhibited radioligand binding to some degree (38.5-57.9%). In competition binding studies, the pharmacological profile of SRIF binding sites defined with [125I]LTT-SRIF-28 and [125I][Tyr10]cortistatin-14 correlated significantly (r2=0.97, n=20). Pharmacological profiles of human and mouse sst5 receptors expressed in CCL39 cells correlated markedly less with those of the gfsst5 profile (r2=0.52-0.78, n > or = b16). Functional expression of the gfsst5 receptor was examined by measurement of agonist-induced luciferase expression and stimulation of [35S]GTPgammaS ([35S]guanosine 5'-O-(3-thiotriphosphate) binding. Profiles were similar to those achieved in radioligand binding studies (r2=0.81-0.93, n=20), although relative potency (pEC50) was reduced compared to pKd values. Relative efficacy profiles of luciferase expression and [35S]GTPgammaS binding, were rather divergent (r2=0.48, n=20) with peptides showing full agonism at one pathway and absence of agonism at the other. BIM 23056 (D-Phe-Phe-Tyr-D-Trp-Lys-Val-Phe-D-Nal-NH2) acted as an antagonist on the effects of SRIF-14 (pKB=6.74 +/- 0.23) on stimulation of [35S]GTPgammaS binding. Pertussis toxin abolished the effect of SRIF-14 on luciferase expression and [35S]GTPgammaS binding suggesting coupling of the receptor to G(i)/G(o) proteins. In summary, the present studies demonstrate that the gfsst5 receptor has a similar pharmacological profile and transductional properties to mammalian sst5 receptors. The difference in efficacy profiles defined using different functional assays suggests numerous, agonist specific, conformational receptor states, and/or ligand-dependent receptor trafficking.
authors.affiliation: Nervous System Research, Novartis Pharma AG, CH-4002, Basel, Switzerland.
authors.forename: Caroline
authors.initials: C
authors.lastname: Nunn
authors.forename: Dominik
authors.initials: D
authors.lastname: Feuerbach
authors.forename: Xinwei
authors.initials: X
authors.lastname: Lin
authors.forename: Richard
authors.initials: R
authors.lastname: Peter
authors.forename: Daniel
authors.initials: D
authors.lastname: Hoyer
mesh_terms: D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006054:Goldfish; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D008024:Ligands; D008156:Luciferases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D009479:Neuropeptides / Q000378:metabolism / Q000494:pharmacology; D011869:Radioligand Assay; D017481:Receptors, Somatostatin / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000378:metabolism; D013004:Somatostatin / Q000378:metabolism / Q000494:pharmacology
chemical_list: D008024:Ligands; D009479:Neuropeptides; D017481:Receptors, Somatostatin; D011993:Recombinant Fusion Proteins; C114430:cortistatin(14), Tyr(10)-; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D013004:Somatostatin; C094573:somatostatin receptor 5; D008156:Luciferases