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.
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.
Wunder
Frank
F
Cardiovascular Research and Screening, Bayer HealthCare AG, D-42096 Wuppertal, Germany. frank.wunder@bayerhealthcare.com <frank.wunder@bayerhealthcare.com>
Buehler
Guido
G
Hüser
Jörg
J
Mundt
Stefan
S
Bechem
Martin
M
Kalthof
Bernd
B
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D001920:Bradykinin / Q000494:pharmacology; D016466:CHO Cells; D002118:Calcium / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D006152:Cyclic GMP / Q000378:metabolism*; D054815:Cyclic Nucleotide-Gated Cation Channels; D006162:Guanylate Cyclase / Q000378:metabolism; D006801:Humans; D007473:Ion Channels / Q000378:metabolism; D008954:Models, Biological; D009569:Nitric Oxide / Q000032:analysis / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011863:Radioimmunoassay; D043782:Receptor, Bradykinin B2 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection
C423582:3-(4-Amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo(3,4-b)pyridine; D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; D011720:Pyrazoles; D011725:Pyridines; D043782:Receptor, Bradykinin B2; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D001920:Bradykinin; D002118:Calcium