Quantification of cAMP and cGMP analogs in intact cells: pitfals in enzyme immunoassays for cyclic nucleotides

Katharina Werner; Frank Schwede; Hans-Gottfried Genieser; Jörg Geiger; Elke Butt

doi:10.1186/1471-2210-11-S1-P75

Volume 11 Supplement 1

5th International Conference on cGMP: Generators, Effectors and Therapeutic Implications

Poster presentation

Open Access

Quantification of cAMP and cGMP analogs in intact cells: pitfals in enzyme immunoassays for cyclic nucleotides

Katharina Werner¹,
Frank Schwede²,
Hans-Gottfried Genieser²,
Jörg Geiger¹ and
Elke Butt¹Email author

BMC Pharmacology201111(Suppl 1):P75

https://doi.org/10.1186/1471-2210-11-S1-P75

Published: 1 August 2011

Keywords

NucleotideCell MembraneMembrane PermeabilityLife ScienceEnzyme Immunoassay

Background

The present work evaluates the cross-reactivity of commercially available cyclic nucleotide analogs with cAMP- and cGMP-immunoassays from Cayman, IBL (both IBL International, Hamburg, Germany) and ENZO Life Sciences (Loerrach, Germany).

Results and conclusion

Most of the tested cyclic nucleotide analogs showed low degree competition with the antibodies; however, with Rp-cAMPS, 8-Br-cGMP and 8-pCPT-cGMP a strong cross-reactivity with the ENZO cAMP- respectively cGMP-EIA and the IBL cGMP-RIA was observed (Table 1). As a consequence we tested these derivatives with the Cayman cGMP-EIA. This assay is less sensitive to cGMP (1.0 pmol/ml) than the ENZO cGMP-EIA (0.01 pmol/ml), however the specificity concerning cGMP-analogs is superior and therefore advantageous when measuring cGMP in the presence of 8-Br-cGMP or 8-pCPT-cGMP.

The determined EIA binding constants enabled the measurement of the intracellular cyclic nucleotide concentrations and revealed a time- and lipophilicity-dependent cell membrane permeability of the compounds in the range of 10-30 % of the extracellular applied concentration after 20 min (Table 1).

Table 1

Lipophilicity (log K_w), cell permeability and EIA/RIA specificity of selected cyclic Nucleotide analogs.

Analog	Log K_w	Permeability	Specificity ENZO cAMP-EIA	Specificity ENZO cAMP-EIA	Specificity IBL cGMP-RIA	Specificity Cayman cGMP-EIA
2’-dcGMP	0.65	0%		5.21%
cGMP	0.77			100%	100%	100%
Rp-cGMPS	0.89			0.27%	10.6%
2’-dcAMP		0%	2.4%
cAMP	1.09		100%
8-Br-cGMP	1.17	12.1%		490%	20%	0.5%
Rp-cAMPS	1.21	12.2%	68%
8-Br-cAMP	1.35	8.0%	0.4%
Rp-8-Br-cAMPS	1.47		0.3%
6-MB-cAMP	1.64		0.4%
6-Bnz-cAMP	1.9		0.6%
8-pCPT-cGMP	2.52	19.6%		240%	30%	0.008%
8-pCPT-cAMP	2.65	22.0%	0.05%
8-Br-PET-cGMP	2.83	30.9%		10%	0.15%	1.6%
Rp-8-Br-PET-cGMPS	2.83			0.2%
8-pCPT-2’-OMe-cAMP (Epac Activator)	2.94		0.03%	0.02%
Sp-5,6-DCI-cBIMPS	2.99		<0.001%

Authors’ Affiliations

(1)

Institute for Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Germany

(2)

Biolog Life Science Institute, Bremen, Germany

Copyright

This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.