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Journal of Biomolecular Screening
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A Homogeneous Fluorescent Cell-Based Assay for Detection of Heterologously Expressed Nitric Oxide Synthase Activity

Michael W. Wood

Richard C. Hastings

Linda A. Sygowski

Arhodamine-derived, membrane-permeable fluorophore (DAR-4MAM) 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 cellswas 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 µ 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.

Key Words: neuronal nitric oxide synthase • cell-based assay • small-molecule inhibitors

This version was published on December 1, 2005

Journal of Biomolecular Screening, Vol. 10, No. 8, 849-855 (2005)
DOI: 10.1177/1087057105280640
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