Document Detail


Development and application of an LC-MS/MS method for measuring the effect of (partial) agonists on cAMP accumulation in vitro.
MedLine Citation:
PMID:  20122962     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Cyclic-adenosine monophosphate (cAMP) plays an important role in cell signalling and is widely used as a marker for receptor activation and as a target for treating various diseases. In this paper we present the development and validation of a new method for the determination of cAMP and ATP (adenosine triphosphate) and other nucleotides in a biological system by combining zwitterionic hydrophilic interaction liquid chromatography (HILIC) and tandem mass spectrometry (MS/MS). The HILIC-MS/MS method was developed for the simultaneous quantitative analysis of cAMP and ATP, and was validated by assessment of linearity (over a range from 0.5 to 100nM for cAMP and 50 nM to 50 microM for ATP (r(2)>0.999)), resolution, limit of detection (0.5 and 50 nM for cAMP and ATP, respectively) and reproducibility. Furthermore, the method was validated and applied in vitro to determine cAMP accumulation in biological samples. The effect of several dopamine D(2) (partial) agonists and antagonists on cAMP accumulation was assessed by determination of the cAMP/ATP ratio in cells transfected with the human dopamine D(2L) receptor. Quinpirole, dopamine and ropinirole produced agonist effects on cAMP accumulation, with a potency of quinpirole>ropinirole>dopamine. Lisuride, terguride and bifeprunox were found to be partial agonists with efficacies of lisuride>terguride>bifeprunox. As expected, haloperidol, (-)-sulpiride and LY-741626 were antagonists. These results demonstrate that the present analytical method was robust, fast, sensitive, and selective. Moreover, it showed utility in determining cAMP/ATP in biological systems and the ability to study the effect of (partial) agonists and antagonists which makes it a useful tool for drug discovery.
Authors:
W Goutier; P A Spaans; M A W van der Neut; A C McCreary; J H Reinders
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Validation Studies     Date:  2010-02-01
Journal Detail:
Title:  Journal of neuroscience methods     Volume:  188     ISSN:  1872-678X     ISO Abbreviation:  J. Neurosci. Methods     Publication Date:  2010 Apr 
Date Detail:
Created Date:  2010-03-29     Completed Date:  2010-06-14     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  7905558     Medline TA:  J Neurosci Methods     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  24-31     Citation Subset:  IM    
Copyright Information:
Copyright 2010 Elsevier B.V. All rights reserved.
Affiliation:
Preclinical Candidate Selection Unit, Solvay Pharmaceuticals Research Laboratories, Weesp, The Netherlands.
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MeSH Terms
Descriptor/Qualifier:
Adenosine Triphosphate / analysis*,  metabolism
Animals
CHO Cells
Cells, Cultured
Chromatography, Liquid / methods*
Cricetinae
Cricetulus
Cyclic AMP / analysis*,  metabolism
Dopamine Agonists / pharmacology*
Receptors, Dopamine D2 / physiology
Tandem Mass Spectrometry / methods*
Chemical
Reg. No./Substance:
0/Dopamine Agonists; 0/Receptors, Dopamine D2; 56-65-5/Adenosine Triphosphate; 60-92-4/Cyclic AMP

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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