Somatostatin receptors in Neuro2A neuroblastoma cells: ligand internalization

• Published in: British journal of pharmacology Vol. 120; no. 1; pp. 52 - 59
• Main Authors: Koenig, J A, Edwardson, J M, Humphrey, P P A
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.01.1997; Nature Publishing
• Subjects: [125I]‐BIM‐23027; Animals; Biological and medical sciences; Brain Neoplasms - metabolism; Cell Membrane - drug effects; Cell Membrane - metabolism; endocytosis; GTP-Binding Proteins - metabolism; internalization; Iodine Radioisotopes; Kinetics; Ligands; Medical sciences; Mice; Neuro2A cells; neuroblastoma; Neuroblastoma - metabolism; Neuropharmacology; Neurotransmitters. Neurotransmission. Receptors; Peptidergic system (neuropeptide, opioid peptide, opiates...). Adenosinergic and purinergic systems; Peptides, Cyclic; Pharmacology. Drug treatments; Receptors, Somatostatin - agonists; Receptors, Somatostatin - antagonists & inhibitors; Receptors, Somatostatin - metabolism; somatostatin receptors; Tumor Cells, Cultured; Cell culture; Ligand binding; Agonist; Neuron; Internalization; Peptide hormone; Peptidergic receptor; Binding site; Somatostatin; In vitro
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1038/sj.bjp.0700859

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. Incubation of Neuro2A cells with [125I]‐BIM‐23027 at 37°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°C. internalization of [125]‐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. 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. After internalization of [125I]‐BIM‐23027, approximately half of the ligand was recycled back to the extracellular medium within 20 min at 37°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. 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. British Journal of Pharmacology (1997) 120, 52–59; doi:10.1038/sj.bjp.0700859