A homogeneous fluorescent cell-based assay for detection of heterologously expressed nitric oxide synthase activity.
Arhodamine-derived, membrane-permeable fluorophore (DAR-4M AM) sensitive to nitric oxide production has been developed recently. The authors evaluated this reagent in both 96 and 384-well formats using heterologously expressed neuronal nitric oxide synthase (nNOS). nNOS transfected into HEK-293T cells was stimulated by the addition of ionomycin. The calcium mobilization resulting from ionomycin treatment of nNOS-expressing 293T cells induced a robust increase in emission intensity, as measured using a standard rhodamine filter set. The effect was time dependent, and a 3 to 4-fold stimulation could be achieved in a 2-h time period. Ionomycin-dependent nitric oxide (NO) production was completely inhibited by several arginine analogs at micromolar concentrations (e.g., L-NAME IC 50=3.0 micro M). Several arginine analog inhibitors of nNOS were revealed to be differentially reversible over increasing substrate concentrations. The assay is a facile method for characterizing inhibitors of nNOS in a relatively unperturbed cell environment.
Wood
Michael W
MW
AstraZeneca R&D Wilmington, DE 19850, USA. michael.wood@astrazeneca.com
Hastings
Richard C
RC
Sygowski
Linda A
LA
D002460:Cell Line; D003001:Cloning, Molecular; D004353:Drug Evaluation, Preclinical; D005453:Fluorescence; D006801:Humans; D020128:Inhibitory Concentration 50; D015759:Ionomycin / Q000494:pharmacology; D007668:Kidney / Q000166:cytology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis*; D052248:Nitric Oxide Synthase Type I / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism*; D014162:Transfection
D012235:Rhodamines; C508638:diaminorhodamine-4M; D009569:Nitric Oxide; D015759:Ionomycin; D052248:Nitric Oxide Synthase Type I; D019331:NG-Nitroarginine Methyl Ester