Simultaneous measurement of ligand-induced intracellular calcium increase and serum response element driven luciferase expression at human sst2 receptors C. Nunn, D. Cervia, R. Bouhelal & D. Hoyer. Novartis Pharma AG, CH 4002 Basel, Switzerland.	Print Abstract Search PubMed for: Nunn C Cervia D Bouhelal R Hoyer D

Somatostatin (SRIF, somatotropin release inhibiting factor) acts via 5 G-protein coupled receptors (sst_1-5) to modulate various effectors including adenylate cyclase, phospholipase C, protein phosphatases and potassium and calcium channels (Patel, 1999). The aim of this study was to measure ligand induced changes in intracellular calcium and serum-response element (SRE)-driven luciferase expression simultaneously in CHO cells expressing human sst₂ (hsst₂) receptors in order to reduce differences due to assay conditions.

CHO-hsst₂ cells were cultured as previously described (Nunn et al., 2003). Cells seeded (9,000 cells
well^-1) in black walled clear base 384 well plates were incubated for 1 h (5% CO₂/95% O₂/37°C) with Hanks buffered salt solution (pH 7.4) containing Hepes (20mM), Fluo-4-AM (2mM) and probenecid (0.1mM). The cells were washed once and refilled with buffer before being placed into a FLIPR^TM to monitor fluorescence (Laser excitation 488nm at 1W, CCD camera opening of 0.4s) before and after addition of ligand. The plates were then incubated for 5 h before lysis and measurement of luciferase expression as previously described (Nunn et al., 2003). Data was expressed as % increase over basal and analysed using Graph Pad Prism.

SRIF-14 concentration-dependently increased both intracellular calcium and luciferase expression (pEC₅₀: 8.74±0.03 & 9.06±0.03, n=52 respectively). Intracellular calcium increase was rapid with maximal peak after 10 s followed by a sustained plateau. The natural ligand SRIF-28, and SRIF analogues for sst₂ receptors (BIM 23027, L 363,301). and sst₂/sst₅ receptors (octreotide, RC 160) had high potency and efficacy similar to that of SRIF-14 in both measurements. Although rank orders of ligands were different in calcium stimulation compared to luciferase expression, correlations between the two measurements were high (r²: 0.83 & 0.9, n=15, pEC₅₀ and E_max respectively).

Pertussis toxin pre-incubation (100 ng ml^-1, 18 h) almost abolished the effect of SRIF-14 on luciferase expression (E_max=11.9±1.3% relative to SRIF-14 alone, n=15) but only partially inhibited the increase in intracellular calcium (E_max=44.9±8.4% relative to SRIF-14 alone, n=15). Thapsigargin pre-treatment (1 µM, 1 h) abolished, while removal of extracellular calcium slightly reduced (E_max=86.0±5.0 relative to SRIF-14 alone, n=14), the increase of intracellular calcium by SRIF. Thapsigargin did not affect luciferase expression (E_max=107.9±3.6 relative to SRIF-14 alone, P=0.82, n=12).

In summary, when SRIF-induced increase of intracellular calcium and luciferase expression via the human sst2 receptor are measured in the same cell population the profiles of SRIF ligands are similar. Luciferase expression is mediated solely via G_i/G_o proteins, while intracellular calcium is mediated via both G_i/G_o proteins and pertussis toxin insensitive G proteins. The calcium increase is mainly from intracellular stores.

Supported by EC Contract QLG3-CT-1999-00908 and Swiss grant BBW 00-0427.

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Nunn, C., et al., (2003). Naunyn-Schmiedeberg's Arch. Pharmacol., 367, 1-9.