Comparison of functional profiles at human recombinant somatostatin sst 2 receptor: Simultaneous determination of intracellular Ca 2+ 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.