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  • Poster presentation
  • Open Access

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

  • 1,
  • 2,
  • 2,
  • 1 and
  • 1Email author
BMC Pharmacology201111 (Suppl 1) :P75

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

  • Published:

Keywords

  • Nucleotide
  • Cell Membrane
  • Membrane Permeability
  • Life Science
  • Enzyme 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 Kw), cell permeability and EIA/RIA specificity of selected cyclic Nucleotide analogs.

Analog

Log Kw

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, Flughafendamm 9a, D-28199 Bremen, Germany

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