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

Abstract

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.

Keywords

neuronal nitric oxide synthase cell-based assay small-molecule inhibitors

References

Hobbs AJ , Higgs A , MoncadaS : Inhibition of nitric oxide synthase as a potential therapeutic target. Annu Rev Pharmacol Toxicol 1999;39:191-220.

Alderton WK , Cooper CE , Knowles RG : Nitric oxide synthases: structure, function and inhibition. Biochem J 2001;357:3-615.

Prast H , Philippu A : Nitric oxide as modulator of neuronal function. Prog Neurobiol 2001;64:51-68.

Moore PK , Handy RL : Selective inhibitors of neuronal nitric oxide synthase—is no NOS really good NOS for the nervous system? TiPS 1997;18:204-211.

Salerno L , Sorrenti V , DiGiacomo C , Romeo G , Siracusa MA : Progress in the development of selective nitric oxide synthase (NOS) inhibitors. Curr Pharm Design 2002;8:177-200.

Hassid A : Nitric Oxide Protocols. Totowa, NJ: Humana, 2004.

Kojima H , Nakatsubo N , Kikuchi K , Kawahara S , Kirino Y , Nagoshi H , et al: Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal Chem 1998;70:2446-2453.

Kojima H , Hirotani M , Nakatsubo N , Kikuchi K , Urano Y , Higuchi T , et al: Bioimaging of nitric oxide with fluorescent indicators based on the rhodamine chromophore. Anal Chem 2001;73:1967-1973.

Liu C , Hermann TE : Characterization of ionomycin as a calcium ionophore. J Biol Chem 1978;253:4-4.

10.

Boer R , Ulrich WR , Klein T , Mirau B , Haas S , Baur I : The inhibitory potency and selectivity of arginine substrate site nitric-oxide synthase inhibitors is solely determined by their affinity toward the different isoenzymes. Mol Pharm 2000;58:1026-1034.

11.

Bryk R , Wolff DJ : Pharmacological modulation of nitric oxide synthesis by mechanism-based inactivators and related inhibitors. Pharmacol Ther 1999;84:157-178.

12.

Cooper GR , Barr A , Wolff DJ : Neuronal nitric oxide synthase is refractory to mechanism-based inactivation in GH3 pituitary cells. Arch Biochem Biophys 1998;357:195-206.

13.

Klatt P , Schmidt K , Brunner F , Mayer B : Inhibitors of brain nitric oxide synthase: binding kinetics, metabolism, and enzyme inactivation. J Biol Chem 1994;269:1674-1680.

14.

Wiesinger H : Argininemetabolismand the synthesis of nitric oxide in the nervous system. Prog Neurobiol 2001;64:365-391.

15.

Schmidt K , List BM , Klatt P , Mayer B : Characterization of neuronal amino acid transporters: uptake of nitric oxide synthase inhibitors and implication for their biological effects. J Neurochem 1995;64:1469-1475.