Convergence of nitric oxide and lipid signaling: anti-inflammatory nitro-fatty acids

Abstract

The signaling mediators nitric oxide ( NO) and oxidized lipids, once viewed to transduce metabolic and inflammatory information via discrete and independent pathways, are now appreciated as interdependent regulators of immune response and metabolic homeostasis. The interactions between these two classes of mediators result in reciprocal control of mediator synthesis that is strongly influenced by the local chemical environment. The relationship between the two pathways extends beyond coregulation of NO and eicosanoid formation to converge via the nitration of unsaturated fatty acids to yield nitro derivatives (NO(2)-FA). These pluripotent signaling molecules are generated in vivo as an adaptive response to oxidative inflammatory conditions and manifest predominantly anti-inflammatory signaling reactions. These actions of NO(2)-FA are diverse, with these species serving as a potential chemical reserve of NO, reacting with cellular nucleophiles to posttranslationally modify protein structure, function, and localization. In this regard these species act as potent endogenous ligands for peroxisome proliferator-activated receptor gamma. Functional consequences of these signaling mechanisms have been shown in multiple model systems, including the inhibition of platelet and neutrophil functions, induction of heme oxygenase-1, inhibition of LPS-induced cytokine release in monocytes, increased insulin sensitivity and glucose uptake in adipocytes, and relaxation of preconstricted rat aortic segments. These observations have propelled further in vitro and in vivo studies of mechanisms of NO(2)-FA signaling and metabolism, highlighting the therapeutic potential of this class of molecules as anti-inflammatory drug candidates.

Fig. 1. Sources of ^·NO₂

During host inflammatory responses, the activities of NADPH oxidase and nitric oxide synthase-2 are increased elevating steady state levels of superoxide (O₂^·−) and (^·NO). Reaction between these two free radicals generates peroxynitrite (ONOO⁻), which when protonated at neutral pH, homolyzes to form the hydroxyl radical (^·OH) and nitrogen dioxide(^·NO₂). Also, reaction of ONOO⁻ with CO₂ yields nitrosoperoxocarbonate (ONOOCO₂⁻) that undergoes homolytic scission to carbonate radical (HCO₃^·) and ^·NO₂. Dietary-derived nitrite and nitrates can be converted to ^·NO₂ in the presence of bacterial flora and acidic conditions found in the gastric compartment.

Fig. 2. Fatty acid oxidation pathways

The PLA₂-mediated arachidonic acid release from phospholipids provides the precursor to a diverse array of oxidized lipids including the prostaglandins, leukotrienes and isoprostanes. The eicosanoids and pathways illustrated herein are not comprehensive but are most directly relevant to this review. Virtually any fatty acid containing a 1,4-pentadienyl bond configuration (e.g., linoleic, linolenic, eicosapentaenoic and docosahexaenoic acids) can be oxidized via these oxidation pathways to generate a vast spectrum of lipid oxidation products. The ribbon structure of PGHS is courtesy of Dr. R.M. Garavito, Michigan State University.

Fig. 3. The effect of ^·NO on PGHS1/2 activity

A) Oxidation of arachidonic acid occurs at two separate but interdependent regions of PGHS that catalyze cyclooxygenation to PGG₂ and peroxidation to PGH₂. B) The activity of PGHS1/2 is modulated by ^·NO and secondary oxides of nitrogen (i.e., ONOO⁻) by acting as a substrate to the peroxidase activity, which ‘primes’ the cyclooxygenase activity of the enzyme. The level of ONOO⁻ depends on the balance of O₂^·− and ^·NO concentrations. At high and possibly non-biological concentrations of ONOO⁻, however, PGHS becomes nitrated, resulting in inhibition of the enzyme.

Fig. 4. Fatty acid nitration

Unsaturated fatty acids can be nitrated by a variety of free radical-mediated mechanisms, the majority of which involve ^·NO₂. The diversity of molecular species generated is driven by the complex redox environment that lipids can be exposed to, double bond rearrangement, secondary metabolism (i.e., β-oxidation, saturation, desaturation) and reaction with nucleophiles.

Fig. 5. Nitric oxide release by nitroalkenes

Two mechanisms have been proposed to explain the release of ^·NO from nitroalkenes in an aqueous environment. In mechanism I, a nitroso intermediate can be formed during aqueous decay. The C—N bond of the nitroso compound is expected to be very weak and can homolyze to form ^·NO and a carbon radical stabilized by the conjugated double bond and the hydroxyl group (107). Mechanism II proposes a nitroalkene rearrangement to form a nitrite ester. This species can also homolyze to form ^·NO and a stabilized radical (96).

Fig. 6. Induction of HO-1 in rat aortic endothelial and smooth muscle cells

Nitrated linoleic acid induces expression of HO-1, which catalyzes the breakdown of heme to CO, a molecule that also mediates anti-inflammatory signaling actions (118). Aortic segments were treated with vehicle or LNO₂ for 16 h, and prepared for imaging. Immunofluorescence detection of HO-1 protein (green) and DAPI nuclear staining was performed on sections with nonimmune rabbit IgG as a negative control (Center). (Upper Panels) A higher magnification of the endothelial layer is shown.

Fig. 7. Cell signaling activity of nitrated lipids

The diverse signaling actions of nitroalkenes is driven primarily by electrophilic reactions with cellular proteins that either inhibit (e.g., blocking NF-κB translocation to the nucleus) or activate (e.g., the Keap1/Nrf2 pathways) their signaling actions. Nitroalkenes also bind with high affinity to cellular lipid receptors (e.g., PPARγ) which may be independent of its electrophilic nature. Finally, nitroalkene levels in the cell are regulated by metabolism to short chain nitroalkenes and reaction with low molecular weight thiols such as GSH.

Fig. 8. LNO₂ bound the ligand binding domain of PPARγ

A) The ribbon structure of the ligand binding domain of PPARγ is shown with bound LNO₂. The nitro-fatty acid ligand is stabilized by different amino acids than the synthetic ligand Rosiglitizone, which may explain the differential signaling actions and potencies of the two compounds. B) A closer view of bound LNO₂, which is presented in green.