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
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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