title: Somatostatin receptors in Neuro2A neuroblastoma cells: operational characteristics.
abstract: 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.
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: D000255:Adenosine Triphosphate / Q000494:pharmacology / Q000502:physiology; D000818:Animals; D001932:Brain Neoplasms / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology / Q000502:physiology; D007700:Kinetics; D008024:Ligands; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism*; D010456:Peptides, Cyclic; D017481:Receptors, Somatostatin / Q000819:agonists / Q000378:metabolism*; D015290:Second Messenger Systems / Q000187:drug effects; D013004:Somatostatin / Q000031:analogs & derivatives / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: C091738:BIM 23027; D008024:Ligands; D010456:Peptides, Cyclic; D017481:Receptors, Somatostatin; D013004:Somatostatin; C032367:somatostatin, Tyr(11)-; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D002118:Calcium