Refinement of a radioreceptor binding assay for nicotinic acid adenine dinucleotide phosphate.
The measurement of changes in nicotinic acid adenine dinucleotide phosphate (NAADP) levels in cells has been, and remains, key to the investigation of the functions of NAADP as a Ca2+ -releasing second messenger. Here we provide details of how to isolate NAADP from cells by extraction with perchloric acid and then measure the NAADP using a radioreceptor assay. We demonstrate that NAADP is neither generated nor broken down during sample processing conditions and that radioreceptor assay is highly selective for the detection of NAADP under cell extract conditions. Furthermore, a number of improvements, such as solid-state detection of the radioactivity, are incorporated to enhance the safety of the procedure. Finally, we have developed a new method to prevent the endogenous metabolism of NAADP by chelating Ca2+ with bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), thereby reducing the difficulty of catching a small transient rise in NAADP levels. In summary, we have refined and improved a method for measuring NAADP levels and presented it in a manner accessible to a wide range of laboratories. It is expected that this will enhance research in the NAADP field.
Lewis
Alexander M
AM
Department of Pharmacology, University of Oxford, Oxford OX1 3QT, UK.
Masgrau
Roser
R
Vasudevan
Sridhar R
SR
Yamasaki
Michiko
M
O'Neill
John S
JS
Garnham
Clive
C
James
Kristin
K
Macdonald
Andrew
A
Ziegler
Mathias
M
Galione
Antony
A
Churchill
Grant C
GC
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002457:Cell Extracts / Q000032:analysis; D002614:Chelating Agents / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004625:Embryo, Nonmammalian; D006863:Hydrogen-Ion Concentration; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008845:Microinjections; D008954:Models, Biological; D009249:NADP / Q000031:analogs & derivatives* / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology; D046790:Pancreas, Exocrine / Q000166:cytology; D010472:Perchlorates / Q000494:pharmacology; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D011485:Protein Binding; D011869:Radioligand Assay; D012617:Sea Urchins / Q000166:cytology / Q000196:embryology; D015290:Second Messenger Systems; D013094:Spermatozoa / Q000378:metabolism; D013997:Time Factors; D017186:Titrimetry
D002457:Cell Extracts; D002614:Chelating Agents; D016877:Oxidants; D010472:Perchlorates; D010761:Phosphorus Radioisotopes; D004533:Egtazic Acid; D009249:NADP; C024376:NAADP; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium