title: Identification and characterisation of heterogeneous somatostatin binding sites in rat distal colonic mucosa.
abstract: We have previously shown that the somatostatin (SRIF) sst2 receptor-selective peptide, BIM-23027, is a potent antisecretory agent in rat isolated distal colonic mucosa (RDCM) and in radioligand binding studies in RDCM membranes, it only maximally inhibited approximately 40% of [125I]-Tyr11-SRIF-14 binding (McKeen ES, Feniuk W, Humphrey PPA (1995) Naunyn-Schmiedeberg's Arch Pharmacol 352:402-411). The aim of this study was to characterise the BIM-23027-sensitive and -insensitive SRIF binding sites in more detail and to compare their properties with those of the recombinant sst2 receptor stably expressed in mouse fibroblast (Ltk-) cells. SRIF-14, SRIF-28, CGP-23996 and D Trp8-SRIF-14 abolished [125I]-Tyr11-SRIF-14 binding (pIC50 values, 8.7-9.7) but the competition curves had Hill slopes which were less than unity. Octreotide and L-362,855 inhibited binding over a wide concentration range (0.1 nM-1 microM) and inhibition of binding was incomplete at the highest concentration studied. BIM-23056 (pIC50 < 6.5) was a weak inhibitor of [125]-Tyr11-SRIF-14 binding. GTP gamma S decreased [125I]-Tyr11-SRIF-14 binding by 40%. Further binding experiments with [125I]-Tyr11-SRIF-14 were carried out in RDCM in the continuous presence of BIM-23027 (1 microM). Under these conditions, seglitide had no effect on [125I]-Tyr11-SRIF-14 binding at concentrations up to 10 microM, whilst SRIF-14 and SRIF-28 abolished specific [125I]-Tyr11-SRIF-14 binding in a manner which was consistent with the ligand binding to two sites. SRIF-14 and SRIF-28 displayed high affinity (pIC50 values of 7.8 and 7.3) for the remaining sites. Octreotide, L-362,855 and BIM-23056 were weak inhibitors of [125I]-Tyr11-SRIF-14 binding (pIC50 < 6.5). [125I]-BIM-23027 labelled a single population of SRIF binding sites in RDCM membranes and mouse fibroblast (Ltk-) cells stably expressing the human recombinant sst2 receptor. There was a significant correlation between the affinity estimates of a range of SRIF analogues at inhibiting [125I]-BIM-23027 binding in RDCM membranes and binding to the recombinant sst2 receptor in Ltk- cells, suggesting that the sites labelled by [125I]-BIM-23027 in RDCM are similar to the sst2 receptor. GTP gamma S (100 microM) decreased [125I]-BIM-23027 binding in RDCM by 60%. The results from these studies demonstrate that [125I]-Tyr11-SRIF-14 labels a heterogeneous population of high affinity SRIF binding sites in RDCM membranes. The majority of these sites are insensitive to GTP gamma S and display negligible affinity for the cyclic hexapeptides, BIM-23027 and seglitide. The remaining high affinity binding sites can be selectively labelled with [125I]-BIM-23027, are sensitive to GTP gamma S and show similar characteristics to the recombinant sst2 receptor which appears to mediate the antisecretory effects of SRIF in the mucosa (McKeen ES, Feniuk W, Humphrey PPA (1995) Naunyn-Schmiedeberg's Arch Pharmacol 352:402-411).
authors.affiliation: Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, UK.
authors.forename: E S
authors.initials: ES
authors.lastname: McKeen
authors.forename: W
authors.initials: W
authors.lastname: Feniuk
authors.forename: A D
authors.initials: AD
authors.lastname: Michel
authors.forename: E J
authors.initials: EJ
authors.lastname: Kidd
authors.forename: P P
authors.initials: PP
authors.lastname: Humphrey
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D003106:Colon / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005765:Gastrointestinal Agents / Q000378:metabolism; D006727:Hormone Antagonists / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D051379:Mice; D015282:Octreotide / Q000378:metabolism; D010456:Peptides, Cyclic / Q000037:antagonists & inhibitors / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D017481:Receptors, Somatostatin / Q000378:metabolism* / Q000502:physiology; D013004:Somatostatin / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: C091738:BIM 23027; D005765:Gastrointestinal Agents; D006727:Hormone Antagonists; C091739:L 362855; D010456:Peptides, Cyclic; D017481:Receptors, Somatostatin; D013004:Somatostatin; D015282:Octreotide