TY  - JOUR
A1  - Werner, Katharina
A1  - Schwede, Frank
A1  - Genieser, Hans-Gottfried
A1  - Geiger, Jörg
A1  - Butt, Elke
T1  - Quantification of cAMP and cGMP analogs in intact cells: pitfalls in enzyme immunoassays for cyclic nucleotides
JF  - Naunyn-Schmiedeberg's Archives of Pharmacology
N2  - Immunoassays are routinely used as research tools to measure intracellular cAMP and cGMP concentrations. Ideally, this application requires antibodies with high sensitivity and specificity. The present work evaluates the cross-reactivity of commercially available cyclic nucleotide analogs with two non-radioactive and one radioactive cAMP and cGMP immunoassay. 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 corresponding cAMP and cGMP, respectively, immunoassays was observed. 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, thus allowing a more accurate prediction of the intracellular analog levels in a given experiment.
KW  - Cyclic nucleotides
KW  - Enzyme immunoassay
KW  - Lipophilicity
KW  - Cell permeability
Y1  - 2011
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-141828
VL  - 384
IS  - 2
ER  - 
TY  - JOUR
A1  - Wangorsch, Gaby
A1  - Butt, Elke
A1  - Mark, Regina
A1  - Hubertus, Katharina
A1  - Geiger, Jörg
A1  - Dandekar, Thomas
A1  - Dittrich, Marcus
T1  - Time-resolved in silico modeling of fine-tuned cAMP signaling in platelets: feedback loops, titrated phosphorylations and pharmacological modulation
N2  - Background: Hemostasis is a critical and active function of the blood mediated by platelets. Therefore, the prevention of pathological platelet aggregation is of great importance as well as of pharmaceutical and medical interest. Endogenous platelet inhibition is predominantly based on cyclic nucleotides (cAMP, cGMP) elevation and subsequent cyclic nucleotide-dependent protein kinase (PKA, PKG) activation. In turn, platelet phosphodiesterases (PDEs) and protein phosphatases counterbalance their activity. This main inhibitory pathway in human platelets is crucial for countervailing unwanted platelet activation. Consequently, the regulators of cyclic nucleotide signaling are of particular interest to pharmacology and therapeutics of atherothrombosis. Modeling of pharmacodynamics allows understanding this intricate signaling and supports the precise description of these pivotal targets for pharmacological modulation. Results: We modeled dynamically concentration-dependent responses of pathway effectors (inhibitors, activators, drug combinations) to cyclic nucleotide signaling as well as to downstream signaling events and verified resulting model predictions by experimental data. Experiments with various cAMP affecting compounds including antiplatelet drugs and their combinations revealed a high fidelity, fine-tuned cAMP signaling in platelets without crosstalk to the cGMP pathway. The model and the data provide evidence for two independent feedback loops: PKA, which is activated by elevated cAMP levels in the platelet, subsequently inhibits adenylyl cyclase (AC) but as well activates PDE3. By multi-experiment fitting, we established a comprehensive dynamic model with one predictive, optimized and validated set of parameters. Different pharmacological conditions (inhibition, activation, drug combinations, permanent and transient perturbations) are successfully tested and simulated, including statistical validation and sensitivity analysis. Downstream cyclic nucleotide signaling events target different phosphorylation sites for cAMP- and cGMP-dependent protein kinases (PKA, PKG) in the vasodilator-stimulated phosphoprotein (VASP). VASP phosphorylation as well as cAMP levels resulting from different drug strengths and combined stimulants were quantitatively modeled. These predictions were again experimentally validated. High sensitivity of the signaling pathway at low concentrations is involved in a fine-tuned balance as well as stable activation of this inhibitory cyclic nucleotide pathway. Conclusions: On the basis of experimental data, literature mining and database screening we established a dynamic in silico model of cyclic nucleotide signaling and probed its signaling sensitivity. Thoroughly validated, it successfully predicts drug combination effects on platelet function, including synergism, antagonism and regulatory loops.
KW  - Vasodilatator-stimuliertes Phosphoprotein
KW  - VASP
KW  - cyclic nucleotide signaling
KW  - silico model
Y1  - 2011
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-69145
ER  -