title: Real-time detection of acetylcholine release from the human endocrine pancreas.
abstract: Neurons, sensory cells and endocrine cells secrete neurotransmitters and hormones to communicate with other cells and to coordinate organ and system function. Validation that a substance is used as an extracellular signaling molecule by a given cell requires a direct demonstration of its secretion. In this protocol we describe the use of biosensor cells to detect neurotransmitter release from endocrine cells in real-time. Chinese hamster ovary cells expressing the muscarinic acetylcholine (ACh) receptor M3 were used as ACh biosensors to record ACh release from human pancreatic islets. We show how ACh biosensors loaded with the Ca(2+) indicator Fura-2 and pressed against isolated human pancreatic islets allow the detection of ACh release. The biosensor approach is simple; the Ca(2+) signal generated in the biosensor cell reflects the presence (release) of a neurotransmitter. The technique is versatile because biosensor cells expressing a variety of receptors can be used in many applications. The protocol takes ∼3 h.
authors.affiliation: Diabetes Research Institute, Miller School of Medicine, University of Miami, Miami, Florida, USA.
authors.forename: Rayner
authors.initials: R
authors.lastname: Rodriguez-Diaz
authors.forename: Robin
authors.initials: R
authors.lastname: Dando
authors.forename: Y Anthony
authors.initials: YA
authors.lastname: Huang
authors.forename: Per-Olof
authors.initials: PO
authors.lastname: Berggren
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Roper
authors.forename: Alejandro
authors.initials: A
authors.lastname: Caicedo
mesh_terms: D000109:Acetylcholine / Q000032:analysis*; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D016466:CHO Cells; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000032:analysis; D016257:Fura-2 / Q000032:analysis; D006801:Humans; D007515:Islets of Langerhans / Q000378:metabolism*; D043587:Receptor, Muscarinic M3 / Q000235:genetics / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D043587:Receptor, Muscarinic M3; D000109:Acetylcholine; D002118:Calcium; D016257:Fura-2