Somatostatin receptors in Neuro2A neuroblastoma cells: operational characteristics

1. We have used somatostatin (SRIF) receptor subtype-selective ligands to determine some of the operational characteristics of somatostatin receptors in Neuro2A mouse neuroblastoma cells. The potent SRIF₁-receptor selective ligand, BIM-23027, was able to displace completely the specific binding of radioiodinated somatostatin, [¹²⁵I]-Tyr¹¹-SRIF-14, with a pIC₅₀ of 10.3, suggesting that Neuro2A cells contain predominantly receptors of the SRIF₁ receptor group. The rank order of affinities for several somatostatin analogues tested in competition studies, together with the high affinity of BIM-23027, indicate that the majority of receptors in Neuro2A cells are of the sst₂ subtype.
2. The stable radioligand, [¹²⁵I]-BIM-23027, bound with high affinity (K_d=13 pM, B_max=0.2 pmol mg⁻¹ protein) to Neuro2A cell membranes, but its binding was only partially reversible at room temperature and below. Thus at 4°C, only 36% of the bound ligand dissociated within 2 h. In contrast, 60% of the ligand dissociated at 15°C and 89% of the ligand dissociated at 37°C.
3. Equilibrium binding of [¹²⁵I]-BIM-23027 was partially (25%) inhibited by 10 μM GTP, and by 120 mM NaCl (42% inhibition) but this inhibition was increased to 75% when sodium chloride and GTP were added together. This effect of GTP and sodium chloride was also seen in dissociation experiments. After incubation to equilibrium with [¹²⁵I]-BIM-23027, dissociation was initiated with excess unlabelled ligand in the presence of GTP (10 μM) and sodium chloride (120 mM). Under these conditions 67% of the ligand dissociated at 4°C, 81% at 15°C and 93% at 37°C. Binding was totally inhibited by pretreatment of cells with pertussis toxin.
4. Functionally, BIM-23027 inhibited forskolin-stimulated cyclic AMP accumulation in a concentration-dependent manner with an IC₅₀ of 1.0 nM and a maximal inhibition of 37%. This effect was abolished by pretreatment of the cells with pertussis toxin. However, unlike in studies reported with the recombinant sst₂ receptor, no rise in intracellular calcium concentration was observed with SRIF-14.
5. We conclude that Neuro2A cells provide a stable neuronal cell line for the study of functionally coupled endogenous somatostatin receptors of the sst₂ type. In addition, we have found that activation of the receptor is associated with ligand-receptor internalisation.