Abstract
Cardiovascular diseases are the number one death worldwide. Nitric oxide (NO)—NO-sensitive (soluble) guanylyl cyclase (sGC)—cyclic guanosine monophosphate (cGMP) pathway regulates diverse set of important physiological functions, including maintenance of cardiovascular homeostasis. Resting and activated sGC enzyme converts guanosine triphosphate to an important second messenger cGMP. In addition to traditional NO generators, a number of sGC activators and stimulators are currently in clinical trials aiming to support or increase sGC activity in various pathological conditions. cGMP-specific phosphodiesterases (PDEs), which degrade cGMP to guanosine monophosphate, play key role in controlling the cGMP level and the strength or length of the cGMP-dependent cellular signaling. Thus, PDE inhibitors also have clear clinical applications. Here, we introduce a homogeneous quenching resonance energy transfer (QRET) for cGMP to monitor both sGC and PDE activities using high throughput screening adoptable method. We demonstrate that using cGMP-specific antibody, sGC or PDE activity and the effect of small molecules modulating their function can be studied with sub-picomole cGMP sensitivity. The results further indicate that the method is suitable for monitoring enzyme reactions also in complex biological cellular homogenates and mixture.
Highlights
Cyclic 3 ́,5 ́-guanosine monophosphate is an important intracellular second messenger involved in various aspects of cell physiology and tissue homeostasis[1,2,3]
The paramembrane pool of natriuretic peptide activated guanylyl cyclases (GCs) generate cyclic guanosine monophosphate (cGMP), which is controlled by PDE2, whereas the pool of cytosolic cGMP generated in response to Nitric oxide (NO) is targeted mainly by PDE57–10
High cGMP concentration results in low time-resolved fluorescence (TRF)-signal since cGMP efficiently blocks Eu(III)-cGMP from binding to the cGMP-specific antibody, leaving the Eu(III)-cGMP in solution where it is quenched by the presence of modulator, and vice versa
Summary
Cyclic 3 ́,5 ́-guanosine monophosphate (cGMP) is an important intracellular second messenger involved in various aspects of cell physiology and tissue homeostasis[1,2,3]. Most of the current methods for cGMP detection and quantification are based on direct cGMP concentration monitoring using heterogeneous enzyme-linked immunosorbent assays (ELISA) utilizing cGMP antibodies, radioimmunoassay (RIA) or HPLC-based cGMP detection[20,21,22,23] Sensitive, these methods contain various time- and labor-consuming separation steps. The strategy utilizes cyclic nucleotide-gated channels and Ca2+-sensitive photoprotein as an indirect way to monitor small molecule induced cGMP production in real-time using designed recombinant cell line. It does not allow for quantification of cGMP levels or measurement of PDE activity. Because of the indirect strategy of cGMP detection, the method has limited potential in quantifying cGMP levels
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