Inducible nitric oxide synthase (iNOS): More than an inducible enzyme? Rethinking the classification of NOS isoforms

Abstract

Nitric oxide (NO) is a critical signaling molecule synthesized from L-arginine by nitric oxide synthase (NOS). The three NOS isoforms-neuronal NOS (nNOS; NOS1), inducible NOS (iNOS; NOS2), and endothelial NOS (eNOS; NOS3)-have traditionally been classified as either constitutive (nNOS and eNOS) or inducible (iNOS). However, this binary classification oversimplifies their functions, particularly by neglecting the physiological roles of iNOS and misrepresenting its involvement in pathological processes. Increasing evidence demonstrates that all three isoforms can exhibit both constitutive and inducible expression. Notably, iNOS is constitutively expressed at low levels in several tissues, including blood, heart, bone marrow, lung, brain, spinal cord, retina, colonic mucosa, liver, ileum, skeletal muscle, epidermis, adipose tissue, endometrium, ovary, and kidney under normal physiological conditions, a form we refer to as constitutive iNOS (ciNOS). This basal expression contributes to essential functions such as heart rate regulation, respiratory exchange, and microbiome balance in the gut. Moreover, in certain pathological contexts, iNOS may exert protective rather than harmful effects, challenging the prevailing view that it is solely a pro-inflammatory mediator. Current drug development strategies targeting NOS are largely based on the outdated dichotomy of constitutive "physiologic" versus inducible "pathologic" isoforms, focusing primarily on iNOS inhibition. The failure of iNOS inhibitors in most clinical trials highlights the limitations of this approach. To address these gaps, we propose a revised nomenclature that incorporates both gene expression mode (constitutive vs. inducible) and discovery order, offering a more nuanced framework for understanding NOS isoforms in both health and disease.

Keywords: Constitutive inducible nitric oxide synthase; Constitutivity; Inducibility; Nitric oxide.

Fig. 1.

Regulation of inducible nitric oxide synthase (iNOS) at the transcriptional (a), post-transcriptional (b), translational (c), and post-translational (d) levels. (a) Transcriptional regulation: iNOS gene expression is modulated by various transcription factors. Additionally, iNOS-derived NO can S-nitrosylate and inhibit NF-κB, providing a negative feedback mechanism. (b) Post-transcriptional regulation: Alternative splicing of iNOS mRNA gives rise to different isoforms, including variants lacking exon 5 (5-), which acts as a negative regulator of iNOS expression, and exons 8 and 9 (8- and 9-), which disrupt dimerization and thus enzyme function. (c) Translational regulation: Certain microRNAs (miRNAs), such as miR-939 and miR-26a, bind to the untranslated regions (UTRs) of iNOS mRNA to suppress translation and protein synthesis. (d) Post-translational regulation: iNOS activity is further modulated by mechanisms involving protein degradation, dimerization, and cofactor (e.g., BH4) binding capacity. Abbreviations: eIF-2α, eukaryotic translation initiation factor 2α; IFN-γ, interferon-gamma; IL-1β, interleukin-1 beta; iNOS, inducible nitric oxide synthase; IRF-1, interferon regulatory factor 1; LPS, lipopolysaccharide; miR, microRNA; NAP110, NOS-associated protein 110 kDa; NF-κB, nuclear factor κB; NO, nitric oxide; P-eIF-2α, phosphorylated eIF-2α; STAT-1, signal transducer and activator of transcription 1; TGF-β, transforming growth factor-beta; TNF-α, tumor necrosis factor-alpha; UTR, untranslated region. Created with BioRender.com.

Fig. 2.

Cross-talk between iNOS-derived NO and other cell signaling pathways, including nuclear factor κB (NF-κB) (a), hypoxia-inducible factor-1 (HIF-1) (b), AMP-activated protein kinase (AMPK) (c), and Janus kinase/signal transducer and activator of transcription (JAK/STAT) (d). AMP, Adenosine monophosphate; ATP, Adenosine triphosphate; IFN-γ, interferon-gamma; IκB, inhibitor of kappa B; IKK, IκB kinase; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NO, nitric oxide; PHD, prolyl hydroxylase; P-IκB, phosphorylated IκB; P-JAK, phosphorylated Janus kinase; P-STAT, phosphorylated signal transducer and activator of transcription; TNF-α, tumor necrosis factor-alpha. Created with BioRender.com.

Fig. 3.

Role of iNOS- and eNOS-derived NO in forming DNICs in activated macrophages and endothelial cells and their function in macrophage-induced cytotoxicity and NO storage and transport. Apo-Tf, apo-transferrin; DMT1, divalent metal transporter 1; DNICs, dinitrosyl-dithiol-iron complexes; Fpn1, ferroportin; Fe²⁺, ferrous iron; Fe³⁺, ferric iron; NO, nitric oxide; NOS, NO synthase; iNOS, inducible NOS; eNOS, endothelial NOS; Tf, transferrin; TfR1, transferrin receptor 1. Created with BioRender.com.

Fig. 4.

Human NOS gene, promoter and regulatory sequences related to constitutive expression. AABS, A-activator binding site; C/EBPβ, CCAAT-enhancer box binding protein β; Elf-1, E74-like factor 1; Ets-1, E26 transformation-specific-1; NRE, negative regulatory element; NRF, NF-κB-repressing factor; PRD, positive regulatory domain; Sp1, specificity protein 1; Sp3, specificity protein 3; TBP, TATA-binding protein; TFIID, transcription factor IID; YY1, Yin Yang 1. Created with BioRender.com.

Fig. 5.

Relative gene expression of nitric oxide (NO) synthase (NOS) enzyme. Figure shows both constitutive and inducible expression of all three isoforms. c, constitutive; i, inducible; NOS, nitric oxide synthase. Created with BioRender.com.