RESUMEN
Cyclic nucleotide regulation is an important target for drug development, particularly for treatment and prophylaxis of cardiovascular diseases. Determination of cyclic nucleotide levels for screening and monitoring of cyclic nucleotide modulating drug action is necessary, yet the techniques available are cumbersome and not sufficiently accurate. Here we present an approach based on the detection of cyclic nucleotide-dependent protein phosphorylation. By use of a common substrate of cyclic nucleotide-dependent protein kinases, the protein vasodilator-stimulated phosphoprotein (VASP) featuring two phosphorylation sites specifically phosphorylated by these kinases, an assay was developed for the monitoring of intracellular cyclic nucleotide levels. The assay was tested with human platelets ex vivo treated with stimulants of nucleotide cyclases, kinases, and phosphodiesterase inhibitors. Phosphorylation of the protein VASP correlates with intracellular cyclic nucleotide concentration (R(2)>0.90 for cGMP and cAMP); however, VASP phosphorylation is more sensitive to elevated cyclic nucleotide levels and significantly more stable over time. Quantification of VASP phosphorylation offers a reliable and robust tool for fast and easy monitoring of cyclic nucleotide levels and is also applicable to unprocessed biological matrices. Owing to these properties, VASP is a promising biomarker for screening and monitoring of cyclic nucleotide modulating drugs.