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 (sst1-sst5) which modulate multiple cellular effectors. The aim of this study was to compare two functional effects of the human sst2 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 pEC50 of 8.74 ± 0.03 (n = 52). Five hours after FLIPR II measurements luciferase expression was determined. SRIF-14 concentration-dependently increased luciferase expression (pEC50 = 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 (r2 = 0.83 and 0.90, pEC50 and Emax respectively).

4. Pertussis toxin pre-treatment reduced SRIF-14/octreotide-mediated intracellular calcium increases by 45-47% and luciferase expression by 95-98%.

5. Thapsigargin pre-treatment 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 sst2 receptors in CHO-K1 cells. The increase in luciferase is mediated via Gi/Go while intracellular calcium increase is mediated by both Gi/Go 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.