Elsevier

Analytical Biochemistry

Volume 363, Issue 2, 15 April 2007, Pages 219-227
Analytical Biochemistry

A cell-based nitric oxide reporter assay useful for the identification and characterization of modulators of the nitric oxide/guanosine 3′,5′-cyclic monophosphate pathway

https://doi.org/10.1016/j.ab.2007.02.001Get rights and content

Abstract

Nitric oxide (NO) plays an important role in protection against the onset and progression of various cardiovascular disorders. Therefore, the NO/guanosine 3′,5′-cyclic monophosphate (cGMP) pathway has gained considerable attention and has become a target for new drug development. We have established a rapid, homogeneous, cell-based, and highly sensitive reporter assay for NO generated by endothelial nitric oxide synthase (eNOS). In a coculture system, NO production is indirectly monitored in living cells via soluble guanylyl cyclase (sGC) activation and calcium influx mediated by the olfactory cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the intracellular cGMP sensor. Using this NO reporter assay, we performed a fully automated high-throughput screening campaign for stimulators of NO synthesis. The coculture system reflects most aspects of the natural NO/cGMP pathway, namely, Ca2+-dependent and Ca2+-independent regulation of eNOS activity by G protein-coupled receptor agonists, oxidative stress, phosphorylation, and cofactor availability as well as NO-mediated stimulation of cGMP synthesis by sGC activation. The NO reporter assay allows the real-time detection of NO synthesis within living cells and makes it possible to identify and characterize activators and inhibitors of enzymes involved in the NO/cGMP signaling pathway.

Section snippets

Generation of the recombinant eNOS cell line

A recombinant Chinese hamster ovary (CHO) cell line expressing the human bradykinin B2 receptor (acc. no. NM_000623) was cotransfected with a pcDNA1.1/Amp plasmid construct containing the human eNOS cDNA (acc. no. NM_000603) and pZeoSV (zeocin resistance). Positive clones were identified by bradykinin stimulation in coculture experiments (data not shown) using a recombinant sGC reporter cell line that was generated as described previously [21] (referred to here as the sGC cell line). Positive

Generation of the recombinant eNOS cell line

A CHO cell line expressing the human bradykinin B2 receptor was cotransfected with an expression construct containing the human eNOS cDNA and a plasmid providing zeocin resistance. Positive clones with high eNOS expression were identified by bradykinin stimulation in coculture experiments using a recombinant sGC reporter cell line [21] (referred to here as the sGC cell line [data not shown]). Positive clones were further purified by the limited dilution technique, and one clonal cell line

Discussion

The challenge for the identification and characterization of NO synthesis modulators is the development of a sensitive NO reporter assay. Due to its very short biological half-life, the real-time detection of NO in living cells is extremely difficult. In addition, assay development for endothelial NOS is particularly challenging because the cellular NO production by eNOS is fairly low as compared with that by iNOS [2].

Several methods for direct or indirect NO detection, including the Griess

Acknowledgments

The authors thank Damaris Hucke and Annegret Rebmann for their excellent technical assistance and thank Dave Wood for critical comments on the manuscript. In addition, we thank Hartmut Kleinert, who kindly provided the cDNA encoding human eNOS.

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