bim-23027 and Neuroblastoma

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 (Kd = 13 pM, Bmax = 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 degrees C, only 36% of the bound ligand dissociated within 2 h. In contrast, 60% of the ligand dissociated at 15 degrees C and 89% of the ligand dissociated at 37 degrees C. 3. Equilibrium binding of [125I]-BIM-23027 was partially (25%) inhibited by 10 microM 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 microM) and sodium chloride (120 mM). Under these conditions 67% of the ligand dissociated at 4 degrees C, 81% at 15 degrees C and 93% at 37 degrees 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.

Topics: Adenosine Triphosphate; Animals; Brain Neoplasms; Calcium; Cell Membrane; Cyclic AMP; Guanosine Triphosphate; Kinetics; Ligands; Mice; Neuroblastoma; Peptides, Cyclic; Receptors, Somatostatin; Second Messenger Systems; Somatostatin; Tumor Cells, Cultured

1. Receptor-dependent internalization of somatostatin (SRIF) agonists has been a matter of controversy probably because [125I]Tyr11-SRIF-14 is rapidly degraded. We have studied the internalization of a stable somatostatin analogue, [125I]-BIM-23027, in a neuronal cell line, Neuro2A, which natively expresses somatostatin sst2 receptors. 2. Incubation of Neuro2A cells with [125I]-BIM-23027 at 37 degrees C resulted in a time-dependent internalization of the ligand, which reached a maximum at 30 min. Acid-washing showed that cell-surface binding of the ligand accounted for only 34% of total binding at this time. Internalization was dramatically reduced at 15 degrees C. 3. Internalization of [125I]-BIM-23027 was prevented by inclusion of unlabelled somatostatin receptor agonists in a concentration-dependent manner. The IC50 values for inhibition of [125I]-BIM-23027 internalization were approximately 100 fold lower than for inhibition of [125I]-BIM-23027 binding to membrane homogenates but followed the same rank order of potencies. 4. Disruption of G-protein coupling by treatment with pertussis toxin caused a 60% reduction in internalization of ligand. A combination of antimycin (50 nM) and deoxyglucose (50 mM) pretreatment, which leads to a depletion of cellular ATP, decreased internalization of [125I]-BIM-23027 by 66% of control and increased the proportion of surface-bound ligand. Hypertonic sucrose, which prevents clathrin-mediated endocytosis, reversibly abolished the internalization of ligand without increasing the proportion bound at the cell surface. 5. After internalization of [125I]-BIM-23027, approximately half of the ligand was recycled back to the extracellular medium within 20 min at 37 degrees C. This finding suggests that the intracellular content of [125I]-BIM-23027 reaches a steady state which is determined by the rates of both internalization and recycling of the ligand. In contrast to studies in which the internalization of [125I]-Tyr11-SRIF-14 was examined, neither internalized nor recycled [125I]-BIM-23027 was degraded to its component amino acids. 6. These findings indicate that the somatostatin agonist, [125I]-BIM-23027, is internalized in a receptor-dependent manner which involves clathrin-coated pits in Neuro2A cells. Furthermore, much of the internalized ligand is rapidly recycled back to the extracellular medium without undergoing significant degradation.

Topics: Animals; Brain Neoplasms; Cell Membrane; GTP-Binding Proteins; Iodine Radioisotopes; Kinetics; Ligands; Mice; Neuroblastoma; Peptides, Cyclic; Receptors, Somatostatin; Tumor Cells, Cultured