Visualizing inducible nitric-oxide synthase in living cells with a heme-binding fluorescent inhibitor

Abstract

The study of nitric-oxide synthase (NOS) physiology is constrained by the lack of suitable probes to detect NOS in living cells or animals. Here, we characterized a fluorescent inducible NOS (iNOS) inhibitor called PIF (pyrimidine imidazole FITC) and examined its utility for microscopic imaging of iNOS in living cells. PIF binding to iNOS displayed high affinity, isoform selectivity, and heme specificity, and was essentially irreversible. PIF was used to successfully image iNOS expressed in RAW264.7 cells, HEK293T cells, human A549 epithelial cells, and freshly obtained human lung epithelium. PIF was used to estimate a half-life for iNOS of 1.8 h in HEK293T cells. Our work reveals that fluorescent probes like PIF will be valuable for studying iNOS cell biology and in understanding the pathophysiology of diseases that involve dysfunctional iNOS expression.

GAPDH regulates cellular heme insertion into inducible nitric oxide synthase

“…Cell lysates were processed as described elsewhere (16,25). All of the experiments were repeated at least three times and the average of all is shown in the results.…”

“…RAW264.7 and HEK293 (ATCC) cells were cultured as described elsewhere (25). Cells were pretreated with 2,4-dioxoheptanoic acid (250 μM) for 2-3 d to make heme deficient.…”

Heterodimerization controls localization of Duox-DuoxA NADPH oxidases in airway cells

“…2B). Thus, acquisition of heme by Duox is essential for association with DuoxA, a scenario reminiscent of Nox2-p22 phox heterodimer or iNOS homodimer formation (DeLeo et al, 2000;Panda et al, 2005;Yu et al, 1997). In accord, heme incorporation was essential for H 2 O 2 generation by Duox enzymes (Fig.…”

Mechanism of Inducible Nitric-oxide Synthase Dimerization Inhibition by Novel Pyrimidine Imidazoles

“…Deletion of analogous residues from the N-terminal regions in nNOS and eNOS did not completely abrogate their capability for dimer formation (44,45). Such differences may explain the different affinities and dimerization inhibitory potential of pyrimidine imidazoles for the three NOS isoforms (27,30). In fact, the inhibitory potency of bulky pyrimidine imidazoles was found to be in the order of eNOS/nNOS/iNOS as 1:200:1000 (27).…”

“…Original studies by our group on the effect of N 1 -substituted pyrimidine imidazoles, like clotrimidazole on iNOS-synthesizing RAW cells, revealed that they inhibited iNOS activity by preventing heme insertion (26). Later studies with PID-like compounds showed that they bind to the NOS heme during its expression and produce an irreversible iNOSmonomer-inhibitor complex (28,30). Indeed, among the imidazoles examined for iNOS dimerization inhibition, pyrimidine imidazoles have shown considerable promise to qualify as effective nontoxic iNOS inhibitors (27,28).…”