title: Comparison of functional profiles at human recombinant somatostatin sst2 receptor: simultaneous determination of intracellular Ca2+ and luciferase expression in CHO-K1 cells.
abstract: 1. Somatostatin (somatotropin release inhibiting factor; SRIF) acts via five G protein-coupled receptors (sst(1)-sst(5)) that modulate multiple cellular effectors. The aim of this study was to compare two functional effects of the human sst(2) receptor stably expressed in CHO-K1 cells in a single experiment using a duplex assay for intracellular calcium and serum response element (SRE)-driven luciferase expression. 2. Intracellular calcium was measured using a fluorometric imaging plate reader II (FLIPR II). SRIF-14 rapidly and transiently increased intracellular calcium with a pEC(50) of 8.74+/-0.03 (n=52). At 5 h after FLIPR II measurements, luciferase expression was determined. SRIF-14 concentration-dependently increased luciferase expression (pEC(50)=9.06+/-0.03, n=52). 3. Natural and synthetic agonist/antagonist ligands for SRIF receptors were tested in the duplex assay. Correlation of agonist potencies and efficacies between the two responses were significant (r(2)=0.83 and 0.90, pEC(50) and E(max), respectively). 4. Pertussis toxin pretreatment reduced SRIF-14/octreotide-mediated intracellular calcium increases by 45-47% and luciferase expression by 95-98%. 5. Thapsigargin pretreatment abolished the SRIF-14/octreotide-mediated intracellular calcium increase but had no effect on luciferase expression. 6. In conclusion, SRIF stimulates an increase in intracellular calcium and SRE-luciferase expression via human sst(2) receptors in CHO-K1 cells. The increase in luciferase is mediated via G(i)/G(o) while intracellular calcium increase is mediated by both G(i)/G(o) proteins and pertussis toxin-insensitive G proteins, and is mainly via release of calcium from intracellular stores. SRIF ligands display a similar recognition profile suggesting that the ligand/receptor/G protein/effector interaction is similar for the two parameters.
authors.affiliation: Neuroscience Research, Novartis Institutes for Biomedical Research, Novartis Pharma AG, CH-4002, Basel, Switzerland.
authors.forename: Caroline
authors.initials: C
authors.lastname: Nunn
authors.forename: Davide
authors.initials: D
authors.lastname: Cervia
authors.forename: Daniel
authors.initials: D
authors.lastname: Langenegger
authors.forename: Laurent
authors.initials: L
authors.lastname: Tenaillon
authors.forename: Rochdi
authors.initials: R
authors.lastname: Bouhelal
authors.forename: Daniel
authors.initials: D
authors.lastname: Hoyer
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism*; D008156:Luciferases / Q000096:biosynthesis* / Q000235:genetics; D020033:Protein Isoforms / Q000819:agonists / Q000502:physiology; D017481:Receptors, Somatostatin / Q000819:agonists / Q000502:physiology*; D013004:Somatostatin / Q000378:metabolism / Q000494:pharmacology
chemical_list: D020033:Protein Isoforms; D017481:Receptors, Somatostatin; D013004:Somatostatin; C093733:somatostatin receptor 2; D008156:Luciferases; D002118:Calcium