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

doi:10.1016/j.ab.2004.12.025

Analytical Biochemistry

Volume 339, Issue 1, 1 April 2005, Pages 104-112

https://doi.org/10.1016/j.ab.2004.12.025 Get rights and content

Abstract

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.

Section snippets

Generation of the recombinant sGC-overexpressing CHO cell line

A recombinant cytosolic apoaequorin-expressing Chinese hamster ovary (CHO) cell line was cotransfected with the plasmids pcDNAI-olf (bovine CNGA2 [10], accession no. X55010, in pcDNAI) and pZeoSV (zeocin resistance). After selection with zeocin, several CNGA2 channel-expressing clones were identified by stimulation with 1 mM 8-Br-cGMP, a membrane-permeable analog of cGMP. Positive clones were purified by the limited dilution technique and were cotransfected with the rat sGC α1-subunit cDNA in

Generation of the recombinant cGMP readout and sGC-overexpressing cell line

Two different CNG channels, CNGA2 and CNGA3, were stably expressed in a CHO cell line already expressing cytosolic apoaequorin. Primary clones from both transfection experiments were then stimulated with 8-Br-cGMP, a membrane-permeable cGMP analog, and positive clones were purified by a limited dilution step. Because CNGA2-expressing clones regularly showed much stronger aequorin responses than did CNGA3-expressing clones (data not shown), only CNGA2 positive clones were further characterized.

Discussion

Cyclic nucleotide-gated channels have properties that led us to investigate their utility as cGMP sensors in an HTS assay. In this study, we have demonstrated that olfactory cyclic nucleotide-gated channels can be used in an ultra-high-throughput format to monitor intracellular cGMP levels via Ca²⁺ influx. Stimulation of the recombinant sGC screening cell line with different classes of stimulators and activators of the soluble guanylate cyclase yields dose-dependent luminescence signals with a

Acknowledgments

We thank Annegret Rebmann, Dorota Thomas, Martina Barg, Yvonne Keim, and Georg Schmidt for their outstanding technical assistance, and we thank Dave Wood for critical comments on the manuscript. We also thank Ulrich Benjamin Kaupp and Rupert Gerzer, who kindly provided the cDNAs encoding CNGA2 and sGC, respectively.

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

Abstract

Section snippets

Generation of the recombinant sGC-overexpressing CHO cell line

Generation of the recombinant cGMP readout and sGC-overexpressing cell line

Discussion

Acknowledgments

Neuron

Trends Biotechnol.

FEBS Lett.

Nitric Oxide

Bioorg. Med. Chem. Lett.

J. Biol. Chem.

Eur. J. Pharmacol.

Endothelium-derived relaxing factor: Discovery, early studies, and identification as nitric oxide [Nobel lecture]

Biosci. Rep.

Discovery of some of the biological effects of nitric oxide and its role in cell signaling [Nobel lecture]

Biosci. Rep.