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

Anal Biochem. 2007 Apr 15;363(2):219-27. doi: 10.1016/j.ab.2007.02.001. Epub 2007 Feb 4.


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, Ca(2+)-dependent and Ca(2+)-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.

MeSH terms

  • Animals
  • Biological Assay / methods*
  • Bradykinin / pharmacology
  • CHO Cells
  • Calcium / pharmacology
  • Cricetinae
  • Cricetulus
  • Cyclic GMP / metabolism*
  • Cyclic Nucleotide-Gated Cation Channels
  • Guanylate Cyclase / metabolism
  • Humans
  • Ion Channels / metabolism
  • Models, Biological
  • Nitric Oxide / analysis
  • Nitric Oxide / metabolism*
  • Nitric Oxide Synthase Type III / genetics
  • Nitric Oxide Synthase Type III / metabolism
  • Phosphorylation / drug effects
  • Pyrazoles / pharmacology
  • Pyridines / pharmacology
  • Radioimmunoassay
  • Receptor, Bradykinin B2 / genetics
  • Receptor, Bradykinin B2 / metabolism
  • Signal Transduction / drug effects
  • Transfection


  • 3-(4-Amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo(3,4-b)pyridine
  • Cyclic Nucleotide-Gated Cation Channels
  • Ion Channels
  • Pyrazoles
  • Pyridines
  • Receptor, Bradykinin B2
  • Nitric Oxide
  • Nitric Oxide Synthase Type III
  • Guanylate Cyclase
  • Cyclic GMP
  • Bradykinin
  • Calcium