Pharmacological characterisation of native somatostatin receptors in AtT-20 mouse tumour corticotrophs

The mouse corticotroph tumour cell line AtT-20 is a useful model to investigate the physiological role of native somatostatin (SRIF, Somatotropin release inhibitory factor) receptor subtypes (sst1 – sst5). The objective of this study was to characterise the pharmacological features and the functional effects of SRIF receptors expressed by AtT-20 cells using radioligand binding and cAMP accumulation. [125I]LTT-SRIF-28, [125I]CGP 23996, [125I]Tyr10-cortistatin-14 and [125I]Tyr3-octreotide labelled SRIF receptor binding sites with high affinity and in a saturable manner (Bmax=315, 274, 239 and 206 fmol mg−1, respectively). [125I]LTT-SRIF-28 labels significantly more sites than [125I]Tyr10 -cortistatin-14 and [125I]Tyr3 -octreotide as seen previously in cells expressing pure populations of sst2 or sst5 receptors. SRIF analogues displaced the binding of the four radioligands. sst2/5 receptor-selective ligands showed much higher affinity than sst1/3/4 receptor-selective ligands. The binding profile of [125I]Tyr3-octreotide was different from that of [125I]LTT-SRIF-28, [125I]CGP 23996 and [125I]Tyr10-cortistatin-14. The sst5/1 receptor-selective ligand L-817,818 identified two binding sites, one with subnanomolar affinity (sst5 receptors) and one with micromolar affinity (sst2 receptors); however, the proportions were different: 70 – 80% high affinity with [125I]LTT-SRIF-28, [125I]CGP 23996, [125I]Tyr10-cortistatin-14, but only 20% with [125I]Tyr3-octreotide. SRIF analogues inhibited the forskolin-stimulated cAMP levels depending on concentration. sst2/5 receptor-selective ligands were highly potent, whereas sst1/3/4 receptor-selective ligands had no significant effects. The sst2 receptor antagonist D-Tyr8-CYN 154806 competitively antagonised the effects of SRIF-14 and sst2 receptor-preferring agonists, but not those of L-817,818. The complex binding properties of SRIF receptor analogues indicate that sst2 and sst5 receptors are the predominant SRIF receptors expressed on AtT-20 cell membranes with no or only negligible presence of sst1, sst3 and sst4 receptors. In the functional studies using cAMP accumulation, only sst2 and sst5 receptors appear to play a role. However, the ‘predominant’ receptor appears to be the sst2 receptor, although sst5 receptors can also mediate the effect, when the ligand is not able to activate sst2 receptors. This clearly adds flexibility to SRIF-mediated functional effects and suggests that the physiological role of SRIF and its analogues may be mediated preferentially via one subtype over another. British Journal of Pharmacology (2003) 139, 109–121. doi:10.1038/sj.bjp.0705235