title: Research Resource: Real-Time Analysis of Somatostatin and Dopamine Receptor Signaling in Pituitary Cells Using a Fluorescence-Based Membrane Potential Assay.
abstract: Stable somatostatin analogues and dopamine receptor agonists are the mainstay for the pharmacological treatment of functional pituitary adenomas; however, only a few cellular assays have been developed to detect receptor activation of novel compounds without disrupting cells to obtain the second messenger content. Here, we adapted a novel fluorescence-based membrane potential assay to characterize receptor signaling in a time-dependent manner. This minimally invasive technique provides a robust and reliable read-out for ligand-induced receptor activation in permanent and primary pituitary cells. The mouse corticotropic cell line AtT-20 endogenously expresses both the somatostatin receptors 2 (sst2) and 5 (sst5). Exposure of wild-type AtT-20 cells to the sst2- and sst5-selective agonists BIM-23120 and BIM-23268, respectively, promoted a pertussis toxin- and tertiapin-Q-sensitive reduction in fluorescent signal intensity, which is indicative of activation of G protein-coupled inwardly rectifying potassium (GIRK) channels. After heterologous expression, sst1, sst3, and sst4 receptors also coupled to GIRK channels in AtT-20 cells. Similar activation of GIRK channels by dopamine required overexpression of dopamine D2 receptors (D2Rs). Interestingly, the presence of D2Rs in AtT-20 cells strongly facilitated GIRK channel activation elicited by the sst2-D2 chimeric ligand BIM-23A760, suggesting a synergistic action of sst2 and D2Rs. Furthermore, stable somatostatin analogues produced strong responses in primary pituitary cultures from wild-type mice; however, in cultures from sst2 receptor-deficient mice, only pasireotide and somatoprim, but not octreotide, induced a reduction in fluorescent signal intensity, suggesting that octreotide mediates its pharmacological action primarily via the sst2 receptor.
authors.affiliation: Department of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena, D-07749 Jena, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Günther
authors.affiliation: Department of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena, D-07749 Jena, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Culler
authors.affiliation: Department of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena, D-07749 Jena, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schulz
mesh_terms: D000818:Animals; D002460:Cell Line; D004298:Dopamine / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology*; D018491:Dopamine Agonists / Q000494:pharmacology; D059290:Dopaminergic Neurons / Q000502:physiology; D004353:Drug Evaluation, Preclinical; D005260:Female; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000378:metabolism; D006801:Humans; D008297:Male; D008564:Membrane Potentials; D008810:Mice, Inbred C57BL; D010902:Pituitary Gland / Q000166:cytology; D061251:Primary Cell Culture; D017448:Receptors, Dopamine D2 / Q000378:metabolism; D017481:Receptors, Somatostatin / Q000378:metabolism; D015398:Signal Transduction; D059010:Single-Cell Analysis; D013004:Somatostatin / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology*; D013050:Spectrometry, Fluorescence
chemical_list: C121506:BIM 23268; D018491:Dopamine Agonists; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D017448:Receptors, Dopamine D2; D017481:Receptors, Somatostatin; C570769:somatostatin receptor 2, mouse; C098681:somatostatin receptor subtype-4; D013004:Somatostatin; C531242:TBR-760; D004298:Dopamine