A cell-based cGMP assay useful for ultra-high-throughput screening and identification of modulators of the nitric oxide/cGMP pathway.

We have established a rapid, homogeneous, cell-based, and highly sensitive assay for guanosine 3'-5'-cyclic monophosphate (cGMP) that is suitable for fully automated ultra-high-throughput screening. In this assay system, cGMP production is monitored in living cells via Ca2+ influx through the olfactory cyclic nucleotide-gated cation channel CNGA2, acting as the intracellular cGMP sensor. A stably transfected Chinese hamster ovary (CHO) cell line was generated recombinantly expressing soluble guanylate cyclase, CNGA2, and aequorin as a luminescence indicator for the intracellular calcium concentration. This cell line was used to screen more than 900,000 compounds in an automated ultra-high-throughput screening assay using 1536-well microtiter plates. In this way, we have been able to identify BAY 58-2667, a member of a new class of amino dicarboxylic acids that directly activate soluble guanylate cyclase. The assay system allows the real-time cGMP detection within living cells and makes it possible to screen for activators and inhibitors of enzymes involved in the nitric oxide/cGMP pathway.

[1]  R. Gerzer,et al.  Generation and characterization of a stable soluble guanylate cyclase-overexpressing CHO cell line. , 1999, Nitric oxide : biology and chemistry.

[2]  O. Civelli,et al.  High-throughput real-time monitoring of Gs-coupled receptor activation in intact cells using cyclic nucleotide-gated channels. , 2003, European journal of pharmacology.

[3]  Stephan Frings,et al.  Profoundly different calcium permeation and blockage determine the specific function of distinct cyclic nucleotide-gated channels , 1995, Neuron.

[4]  J. Schaack,et al.  In Vivo Assessment of Local Phosphodiesterase Activity Using Tailored Cyclic Nucleotide–Gated Channels as Camp Sensors , 2001, The Journal of general physiology.

[5]  R. Furchgott,et al.  Endothelium-Derived Relaxing Factor: Discovery, Early Studies, and Identification as Nitric Oxide , 1999, Bioscience reports.

[6]  F Murad.  Discovery of some of the biological effects of nitric oxide and its role in cell signaling. , 1999, Bioscience reports.

[7]  L. Ignarro.  Nitric oxide: a unique endogenous signaling molecule in vascular biology. , 1999, Bioscience reports.

[8]  U. Kaupp,et al.  Cyclic nucleotide-gated ion channels. , 2002, Physiological reviews.

[9]  K. Yau,et al.  Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons , 1990, Nature.

[10]  J. Stasch,et al.  NO-independent stimulators of soluble guanylate cyclase. , 2001, Bioorganic & medicinal chemistry letters.

[11]  K Jones,et al.  Glowing jellyfish, luminescence and a molecule called coelenterazine. , 1999, Trends in biotechnology.

[12]  L. Ignarro.  Nitric Oxide: A Unique Endogenous Signaling Molecule in Vascular Biology , 1999 .

[13]  J. Stasch,et al.  NO‐ and haem‐independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle , 2002, British journal of pharmacology.

[14]  K. Yau,et al.  Calcium-Calmodulin Modulation of the Olfactory Cyclic Nucleotide-Gated Cation Channel , 1994, Science.

[15]  S. Moncada,et al.  Nitric oxide: physiology, pathophysiology, and pharmacology. , 1991, Pharmacological reviews.

[16]  D. Lancet,et al.  Primary structure of cAMP‐gated channel from bovine olfactory epithelium , 1990, FEBS letters.

[17]  J. Stasch,et al.  Identification of Residues Crucially Involved in the Binding of the Heme Moiety of Soluble Guanylate Cyclase* , 2004, Journal of Biological Chemistry.

[18]  R. Gerzer,et al.  NO-independent regulatory site on soluble guanylate cyclase , 2001, Nature.