A homogeneous fluorescent cell-based assay for detection of heterologously expressed nitric oxide synthase activity

Abstract

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-lold 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 μM). 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. © 2005 The Society for Biomolecular Screening.