title: Somatostatin receptors in Neuro2A neuroblastoma cells: ligand internalization.
abstract: 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.
authors.affiliation: Glaxo Institute of Applied Pharmacology.
authors.forename: J A
authors.initials: JA
authors.lastname: Koenig
authors.forename: J M
authors.initials: JM
authors.lastname: Edwardson
authors.forename: P P
authors.initials: PP
authors.lastname: Humphrey
mesh_terms: D000818:Animals; D001932:Brain Neoplasms / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D008024:Ligands; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism*; D010456:Peptides, Cyclic; D017481:Receptors, Somatostatin / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: C091738:BIM 23027; D007457:Iodine Radioisotopes; D008024:Ligands; D010456:Peptides, Cyclic; D017481:Receptors, Somatostatin; D019204:GTP-Binding Proteins