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A Homogeneous Fluorescent Cell-Based Assay for Detection of Heterologously Expressed Nitric Oxide Synthase Activity
AstraZeneca R&D Wilmington, Wilmington, DE.
* To whom correspondence should be addressed. E-mail: michael.wood{at}astrazeneca.com.
A rhodamine-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 IC50 = 3.0 mM). 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
First published on October 18, 2005, doi:10.1177/1087057105280640 |
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