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...

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Published inAnalytical biochemistry Vol. 371; no. 1; pp. 26 - 36
Main Authors Lewis, Alexander M., Masgrau, Roser, Vasudevan, Sridhar R., Yamasaki, Michiko, O’Neill, John S., Garnham, Clive, James, Kristin, Macdonald, Andrew, Ziegler, Mathias, Galione, Antony, Churchill, Grant C.
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 01.12.2007
Academic Press
Subjects
Animals
Calcium - metabolism
Calcium Signaling - physiology
Cell Extracts - analysis
Chelating Agents - pharmacology
Chromatography, High Pressure Liquid
Egtazic Acid - analogs & derivatives
Egtazic Acid - pharmacology
Embryo, Nonmammalian
Hydrogen-Ion Concentration
Male
Mammalian
Mice
Mice, Inbred Strains
Microinjections
Models, Biological
NAADP
NADP - analogs & derivatives
NADP - analysis
NADP - isolation & purification
NADP - metabolism
Oocytes - metabolism
Oxidants - pharmacology
Pancreas, Exocrine - cytology
Perchlorates - pharmacology
Phosphorus Radioisotopes - metabolism
Protein Binding
Radioligand Assay
Radioreceptor
Sea Urchins - cytology
Sea Urchins - embryology
Second messenger
Second Messenger Systems
Spermatozoa - metabolism
Time Factors
Titrimetry
NAADP
Second messenger
Mammalian
Radioreceptor
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Abstract 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.
AbstractList 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.
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 Ca 2+ -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 Ca 2+ 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.
Author Garnham, Clive
Yamasaki, Michiko
Macdonald, Andrew
Ziegler, Mathias
Vasudevan, Sridhar R.
Churchill, Grant C.
O’Neill, John S.
Masgrau, Roser
Lewis, Alexander M.
James, Kristin
Galione, Antony
AuthorAffiliation b Department of Molecular Biology, University of Bergen, N-5008 Bergen, Norway
a Department of Pharmacology, University of Oxford, Oxford OX1 3QT, UK
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Issue 1
Keywords NAADP
Second messenger
Mammalian
Radioreceptor
Language English
License http://creativecommons.org/licenses/by/3.0
Open Access under CC BY 3.0 license
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Notes These authors contributed equally to this work.
Current address: ResVerLogix Corporation, Calgary, Alberta T2L 2K7, Canada.
Current address: Institut de Neurosciences, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès (Barcelona), Catalonia, Spain.
Current address: MRC Laboratory of Molecular Biology, Neurobiology Division, Cambridge CB2 2QH, UK.
Current address: Department of Neurobiology and Behavior, University of California, Irvine, Irvine, CA 92697, USA.
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Snippet The measurement of changes in nicotinic acid adenine dinucleotide phosphate (NAADP) levels in cells has been, and remains, key to the investigation of the...
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StartPage 26
SubjectTerms Animals
Calcium - metabolism
Calcium Signaling - physiology
Cell Extracts - analysis
Chelating Agents - pharmacology
Chromatography, High Pressure Liquid
Egtazic Acid - analogs & derivatives
Egtazic Acid - pharmacology
Embryo, Nonmammalian
Hydrogen-Ion Concentration
Male
Mammalian
Mice
Mice, Inbred Strains
Microinjections
Models, Biological
NAADP
NADP - analogs & derivatives
NADP - analysis
NADP - isolation & purification
NADP - metabolism
Oocytes - metabolism
Oxidants - pharmacology
Pancreas, Exocrine - cytology
Perchlorates - pharmacology
Phosphorus Radioisotopes - metabolism
Protein Binding
Radioligand Assay
Radioreceptor
Sea Urchins - cytology
Sea Urchins - embryology
Second messenger
Second Messenger Systems
Spermatozoa - metabolism
Time Factors
Titrimetry
Title Refinement of a radioreceptor binding assay for nicotinic acid adenine dinucleotide phosphate
URI https://dx.doi.org/10.1016/j.ab.2007.08.030
https://www.ncbi.nlm.nih.gov/pubmed/17919448
https://pubmed.ncbi.nlm.nih.gov/PMC2518627
Volume 371
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