Intracellular Assembly of Inducible NO Synthase Is Limited by Nitric Oxide-mediated Changes in Heme Insertion and Availability

Abstract

Cytokines induce the mouse macrophage cell line RAW 264.7 to express cytokine-inducible nitric oxide synthase (iNOS), which is active only in dimeric form. Because dimerization of purified iNOS subunits requires tetrahydrobiopterin, heme, and L-arginine, we investigated if availability of these factors also influences intracellular assembly of dimeric iNOS. Following exposure to cytokines, iNOS protein was found to accumulate in a near linear manner over 16 h of further culture. In contrast, dimeric iNOS accumulated at a slower rate that continuously decreased during culture, resulting in only 25% of the accumulated iNOS protein being in dimeric form by 16 h. Further experiments argued against dimer instability or L-arginine and tetrahydrobiopterin availability as factors limiting iNOS dimer accumulation. Blocking cellular NO synthesis with N -nitro-L-arginine methyl ester (L-NAME) greatly increased iNOS dimer assembly, indicating NO synthesis limited iNOS dimerization. NO synthesis was found to prevent an increase in soluble heme level that was associated with iNOS induction in N -nitro-L-arginine methyl ester-treated cells and also diminished heme insertion into iNOS. These NO-related defects were not reversed by adding heme precursors or hemin to the activated cell cultures. Measurement of iron release from activated cells demonstrated that endogenous NO synthesis substantially increased the release of 59 Fe to the medium. These observations suggest that iNOS dimerization is limited to a large extent by iNOS NO synthesis. NO appears to limit intracellular assembly of dimeric iNOS by preventing heme insertion and decreasing heme availability.

Nitric oxide suppresses inducible nitric oxide synthase expression by inhibiting post-translational modification of IκB

“…Their data demonstrated an NO-mediated 40-60% suppression of iNOS enzyme activity. Another possible negative feedback inhibitory mechanism of NO can be associated with limitation of intracellular assembly of active dimeric iNOS by preventing heme insertion and decreasing heme availability (Kim et al, 1995;Albakri et al, 1996). We here examined if NO inhibited iNOS activity in CM-stimulated hepatocytes.…”

Tumor microenvironment-based feed-forward regulation of NOS2 in breast cancer progression

“…Nitric oxide participates in positive and negative feedback regulation of NOS2 through diverse mechanisms involving translation (17,18), posttranslational modification, and enzymatic activity (19). The affect of AG on NOS2 induction was examined by confocal microscopy of IFN-γ/SWtreated MDA-MB-231 cells.…”

Autoinhibition of neuronal nitric oxide synthase: distinct effects of reactive nitrogen and oxygen species on enzyme activity

“…However, it is also important to elucidate at the biochemical level the precise mechanism(s) by which NOS is autoinhibited, in addition to previously described nitrosyl-haem formation [26]. Other mechanisms might include changes in post-translational haem insertion [45], autonitrosylation of essential thiols [8,46,47] or auto-nitration of essential protein tyrosine moieties [31,48]. Finally, a complete understanding of NOS autoinhibition is not only of mechanistic importance but is also relevant to cell biology and, in particular, pathophysiology, i.e.…”