title: Measurement of luminal pH of acidic stores as a readout for NAADP action.
abstract: In addition to mobilizing Ca²⁺, NAADP plays a role in modulating the luminal pH (pHL) of acidic stores of the endolysosomal system. The effects of NAADP on pHL have been most extensively studied in the sea urchin egg, both in the intact egg and in egg homogenates. Related observations have also been made in mammalian systems (e.g., guinea pig atrial myocytes and pancreatic acinar cells). Although the connection between Ca²⁺ mobilization and increase in pHL is not understood, pHL can be a useful parameter to measure when studying NAADP-mediated signaling. This protocol describes the fluorescent measurement of pHL of acidic stores. It relies on the use of acridine orange (AO), a standard dye for pHL. AO selectively accumulates to high concentrations in the lumen of organelles as a function of acidity; at these high concentrations it self-quenches. When pHL increases, some AO is lost from the vesicle. As a result, the lower luminal AO concentration relieves the quenching and fluorescence increases in the lumen.
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
authors.forename: Antony
authors.initials: A
authors.lastname: Galione
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
authors.forename: Kai-Ting
authors.initials: KT
authors.lastname: Chuang
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
authors.forename: Tim M
authors.initials: TM
authors.lastname: Funnell
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
authors.forename: Lianne C
authors.initials: LC
authors.lastname: Davis
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Morgan
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
authors.forename: Margarida
authors.initials: M
authors.lastname: Ruas
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
authors.forename: John
authors.initials: J
authors.lastname: Parrington
authors.affiliation: Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
authors.forename: Grant C
authors.initials: GC
authors.lastname: Churchill
mesh_terms: D000143:Acids / Q000378:metabolism*; D000818:Animals; D006863:Hydrogen-Ion Concentration; D008297:Male; D009249:NADP / Q000031:analogs & derivatives* / Q000378:metabolism; D015388:Organelles / Q000378:metabolism*; D012617:Sea Urchins; D015398:Signal Transduction; D013094:Spermatozoa / Q000378:metabolism / Q000648:ultrastructure; D014894:Weights and Measures
chemical_list: D000143:Acids; D009249:NADP; C024376:NAADP