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

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.

Table 1.

Lipophilicity (log K_w), 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%

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).