PMID- 25275102
OWN - NLM
STAT- MEDLINE
DCOM- 20150602
LR  - 20220129
IS  - 1559-6095 (Electronic)
IS  - 1559-6095 (Linking)
VI  - 2014
IP  - 10
DP  - 2014 Oct 1
TI  - Measurement of luminal pH of acidic stores as a readout for NAADP action.
PG  - pdb.prot076935
LID - 10.1101/pdb.prot076935 [doi]
AB  - In addition to mobilizing Ca(2)(+), 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 Ca(2)(+) 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.
CI  - (c) 2014 Cold Spring Harbor Laboratory Press.
FAU - Galione, Antony
AU  - Galione A
AD  - Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
FAU - Chuang, Kai-Ting
AU  - Chuang KT
AD  - Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
FAU - Funnell, Tim M
AU  - Funnell TM
AD  - Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
FAU - Davis, Lianne C
AU  - Davis LC
AD  - Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
FAU - Morgan, Anthony J
AU  - Morgan AJ
AD  - Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
FAU - Ruas, Margarida
AU  - Ruas M
AD  - Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
FAU - Parrington, John
AU  - Parrington J
AD  - Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
FAU - Churchill, Grant C
AU  - Churchill GC
AD  - Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom.
LA  - eng
GR  - 102828/Wellcome Trust/United Kingdom
GR  - BB/G008523/1/BB_/Biotechnology and Biological Sciences Research Council/United 
      Kingdom
PT  - Journal Article
DEP - 20141001
PL  - United States
TA  - Cold Spring Harb Protoc
JT  - Cold Spring Harbor protocols
JID - 101524530
RN  - 0 (Acids)
RN  - 53-59-8 (NADP)
RN  - 5502-96-5 (NAADP)
SB  - IM
MH  - Acids/*metabolism
MH  - Animals
MH  - *Hydrogen-Ion Concentration
MH  - Male
MH  - NADP/*analogs & derivatives/metabolism
MH  - Organelles/*metabolism
MH  - Sea Urchins
MH  - Signal Transduction
MH  - Spermatozoa/metabolism/ultrastructure
MH  - *Weights and Measures
EDAT- 2014/10/03 06:00
MHDA- 2015/06/03 06:00
CRDT- 2014/10/03 06:00
PHST- 2014/10/03 06:00 [entrez]
PHST- 2014/10/03 06:00 [pubmed]
PHST- 2015/06/03 06:00 [medline]
AID - 2014/10/pdb.prot076935 [pii]
AID - 10.1101/pdb.prot076935 [doi]
PST - epublish
SO  - Cold Spring Harb Protoc. 2014 Oct 1;2014(10):pdb.prot076935. doi: 
      10.1101/pdb.prot076935.