High-output Nitric Oxide Research Articles

Arginase 1 Overexpression in Psoriasis: Limitation of Inducible Nitric Oxide Synthase Activity as a Molecular Mechanism for Keratinocyte Hyperproliferation

Abnormal proliferation of keratinocytes in the skin appears crucial to the pathogenesis of psoriasis, but the underlying mechanisms remain unknown. Nitric oxide (NO), released from keratinocytes at high concentrations, is considered a key inhibitor of cellular proliferation and inducer of differentiation in vitro. Although high-output NO synthesis is suggested by the expression of inducible NO synthase (iNOS) mRNA and protein in psoriasis lesions, the pronounced hyperproliferation of psoriatic keratinocytes may indicate that iNOS activity is too low to effectively deliver anti-proliferative NO concentrations. Here we show that arginase 1 (ARG1), which substantially participates in the regulation of iNOS activity by competing for the common substrate L-arginine, is highly overexpressed in the hyperproliferative psoriatic epidermis and is co-expressed with iNOS. Expression of L-arginine transporter molecules is found to be normal. Treatment of primary cultured keratinocytes with Th1-cytokines, as present in a psoriatic environment, leads to de novo expression of iNOS but concomitantly a significant down-regulation of ARG1. Persistent ARG1 overexpression in psoriasis lesions, therefore, may represent a disease-associated deviation from normal expression patterns. Furthermore, the culturing of activated keratinocytes in the presence of an ARG inhibitor results in a twofold increase in nitrite accumulation providing evidence for an L-arginine substrate competition in human keratinocytes. High-output NO synthesis is indeed associated with a significant decrease in cellular proliferation as shown by down-regulation of Ki67 expression in cultured keratinocytes but also in short-term organ cultures of normal human skin. In summary, our data demonstrate for the first time a link between a human inflammatory skin disease, limited iNOS activity, and ARG1 overexpression. This link may have substantial implications for the pathophysiology of psoriasis and the development of new treatment strategies.

Overexpression of the human inducible nitric oxide synthase gene enhances radiation-induced apoptosis in colorectal cancer cells via a caspase-dependent mechanism

Nitric oxide (NO) has been reported to sensitize cancer cells to radiation. Since delivery of NO to tumors is limited in vivo by systemic toxicity of NO, we examined the potential of gene delivery of the human inducible nitric oxide synthase (iNOS) gene as a means of achieving high output NO production. We successfully transduced two colorectal cancer cell lines as evidenced by increased iNOS protein accumulation and nitrite production. We found that overexpression of iNOS enhanced the effects of radiation on apoptosis in both cell lines in a caspase-dependent fashion. Gene transfer of iNOS holds much promise as a potential radiosensitizer of cancer cells since it increases apoptosis in an additive manner with radiation.

Cytosolic and mitochondrial glutathione in microglial cells are differentially affected by oxidative/nitrosative stress

Glutathione (GSH), the major cellular protectant against reactive oxygen and nitrogen species, is compartmentalized in a cytosolic (c) and a mitochondrial (mt) pool. We investigated how c-GSH and mt-GSH are differentially affected by endogenously produced nitric oxide (NO). Microglial cell line (N9) cultures were immunostimulated with lipopolysaccharide/interferon-γ to elicit the inducible isoform of NO synthase (iNOS). Despite a significant reduction in total GSH, the mt-GSH remained nearly unaffected by iNOS-mediated NO production. To investigate possible consequences of GSH depletion on the mitochondrial membrane potential, we used buthionine sulfoximine (BSO) to reduce separately the c-GSH, whereas ethacrynic acid (EA) was applied to deplete both mt-GSH and c-GSH. The mitochondrial membrane potential was more vulnerable to NO exposure in EA-pretreated cultures than in BSO-pretreated cultures, indicated by a potentiated release of tetramethylrhodamine from mitochondria into the cytosol. To relate the EA-mediated decrease in mitochondrial membrane potential to the oxidant buildup after GSH depletion, we loaded the cells with the oxidant-sensitive fluorochrome 2 ′,7 ′-dihydrodichlorofluorescein (DCF) diacetate. EA treatment caused an increase in DCF fluorescence over time that was potentiated when the iNOS expression was stimulated. Inhibition of NO production abolished this effect. We conclude that endogenous NO production in microglial cells does not compromise the mt-GSH pool which, in turn, might explain the ability of these cells to combat high-output NO production.

Cat2l-arginine transporter-deficient fibroblasts can sustain nitric oxide production

High-output nitric oxide (NO) production by nitric oxide synthase 2 (NOS2) contributes to normal cellular processes and pathophysiological conditions. The transport of l-arginine, the substrate for NOS2, is required for sustained NO production by NOS2. l-Arginine can be transported by several kinetically defined transport systems, although the majority of arginine uptake is mediated by transport system y +, encoded by the Cat1–3 gene family. Using macrophages from Cat2-deficient mice, we previously determined that arginine uptake via CAT2 is absolutely required for sustained NO production. Because NO production by fibroblasts is important in wound healing, we sought to determine whether CAT2 is required for NO production in cytokine-stimulated Cat2-deficient and wild-type embryonic fibroblasts. Although macrophages and fibroblasts both required extracellular l-arginine for NO production, NO synthesis by activated Cat2 −/− fibroblasts was reduced only 19%, whereas Cat2 −/− macrophages were virtually unable to produce NO. As expected, activated Cat2 −/− fibroblasts had reduced system y +-mediated arginine uptake. However, their reduced NO output was not the result of a significant difference in intracellular l-arginine levels following cytokine stimulation. Uptake experiments revealed that the l-arginine transport system y +L was the major cationic amino acid carrier in fibroblasts of both genotypes. We conclude that NO production in embryonic fibroblasts is only partially dependent on CAT2 and that other compensating transporters provide arginine for NOS2-mediated NO synthesis. The data demonstrate that fibroblasts and macrophages have differential dependence on CAT2-mediated l-arginine transport for NO synthesis. The important physiological implication of this finding is discussed.

Revisiting an old antimicrobial drug: amphotericin B induces interleukin-1-converting enzyme as the main factor for inducible nitric-oxide synthase expression in activated endothelia.

We have investigated the impact of the widely used antifungal agent Amphotericin B (AmB) on cytokine activated aortic endothelial cells (AEC) and their inflammatory response as monitored by cytokine and inducible nitric-oxide synthase (iNOS) expression as well as high-output nitric oxide synthesis. Because both blood-borne infections and systemically administered drugs will first encounter vessel lining endothelial cells, this cell type represents an important participant in innate immune reactions against xenobiotics. Culturing cytokine-activated AEC in the presence of 1.25 microg/ml AmB, a concentration equivalent to serum levels during patient treatment, we find increases in iNOS promoter activity up to 120%, in iNOS mRNA or protein expressions by factors of up to 3.5 +/- 1.1, and in iNOS activity of up to 180% compared with cells with cytokines only. In parallel, a strong increase in endothelial interleukin (IL)-1beta-converting enzyme (ICE) and IL-1beta expression and activity was observed. Specific inhibition of ICE activity or IL-1beta functionality significantly reduces expression and activity of the iNOS to control values. Because ICE activity is essential for the endogenous synthesis of active IL-1beta, ICE overexpression represents the key signal in the AmB-induced and IL-1beta-mediated effects on iNOS activity. In summary, in endothelial cells, AmB strongly augments cytokine-induced iNOS expression and activity by increasing the expression and activity of the ICE. This adjuvant activity for augmented endogenous cytokine processing adds to the efficacy of the antimycotic activity of AmB. Furthermore, our data underline the relevance of the endothelial iNOS as a potent effector of the innate immune system.

Interactions between inducible nitric oxide synthase and heme oxygenase-1 in glomerulonephritis

In anti-glomerular basement membrane (GBM) nephritis, inducible nitric oxide synthase (iNOS) and heme oxygenase (HO-1) are co-induced. Moreover, in glomerular mesangial cells iNOS-derived nitric oxide (NO) production stimulates HO-1 while HO-1 activation reduces iNOS expression/activity. These observations prompted us to explore regulatory interactions between iNOS and HO-1 in anti-GBM nephritis. Rats with anti-GBM nephritis were pretreated with the iNOS inhibitor l-N6-(1-iminoethyl) lysine (L-NIL) or with the HO-1 inducer hemin. Glomerular HO-1 levels were assessed by Western blot analysis. iNOS activity was assessed by calculating conversion of l-arginine to l-citrulline. iNOS inhibition reduced glomerular HO-1 levels without altering the inflammatory response to anti-GBM antibody induced injury. Induction of HO-1 reduced glomerular iNOS activity. In anti-GBM nephritis iNOS up-regulates HO-1 presumably via high output NO production. Suprainduction of HO-1 attenuates iNOS activity. This negative feedback interaction points to HO-1 as a target for pharmacologic manipulation to reduce activity of prooxidant heme containing enzymes such as iNOS.

Induced nitric oxide (NO) synthesis in heterologous nephrotoxic nephritis; effects of selective inhibition in neutrophil-dependent glomerulonephritis.

Increased NO synthesis, due to inducible NO synthase (iNOS) activity, is found in macrophage-associated glomerulonephritis. Little is known about NO in neutrophil-dependent immune complex inflammation, and its role remains controversial. We therefore studied early phase heterologous nephrotoxic nephritis (HNTN) induced in rats by nephrotoxic globulin and the effects of selective iNOS inhibition of this model. At 2 h of the model iNOS mRNA was induced and nitrite (NO-2) was generated in glomeruli incubated ex vivo (5.2 +/- 1.0 nmol/2000 glomeruli per 24 h). There were 14.7 +/- 2.2 polymorphonuclear neutrophils (PMN)/glomerulus (normal controls 0.1 +/- 0.1). At 8 h urinary protein was 69 +/- 15.3 (normal controls 0. 6 +/- 0.2 mg/24 h). Peritoneal PMN expressed iNOS and produced significant NO-2 (basal 11.2 +/- 0.3 nmol/106 cells per 24 h). Selective iNOS inhibition with L-N6-(1-iminoethyl)-lysine (L-NIL) in vitro inhibited nephritic glomerular and PMN NO-2 synthesis. In HNTN L-NIL in vivo significantly suppressed elevated plasma NO-2/NO-3 levels (representative experiment: 17 +/- 2 microM, untreated 40 +/- 4 microM, P = < 0.01, normal control 18 +/- 2 microM). This inhibition did not affect leucocyte infiltration into glomeruli or induce thrombosis. There was no consistent effect on proteinuria. This is the first demonstration of glomerular iNOS induction and high output NO production in the acute phase of PMN-dependent acute immune complex glomerulonephritis. Selective iNOS inhibition does not affect the primary mechanism of injury (leucocyte infiltration) in this model.

Helicobacter pylori arginase inhibits nitric oxide production by eukaryotic cells: a strategy for bacterial survival.

The antimicrobial effect of nitric oxide (NO) is an essential part of innate immunity. The vigorous host response to the human gastric pathogen Helicobacter pylori fails to eradicate the organism, despite up-regulation of inducible NO synthase (iNOS) in the gastric mucosa. Here we report that wild-type strains of H. pylori inhibit NO production by activated macrophages at physiologic concentrations of l-arginine, the common substrate for iNOS and arginase. Inactivation of the gene rocF, encoding constitutively expressed arginase in H. pylori, restored high-output NO production by macrophages. By using HPLC analysis, we show that l-arginine is effectively consumed in the culture medium by wild-type but not arginase-deficient H. pylori. The substantially higher levels of NO generated by macrophages cocultured with rocF-deficient H. pylori resulted in efficient killing of the bacteria, whereas wild-type H. pylori exhibited no loss of survival under these conditions. Killing of the arginase-deficient H. pylori was NO-dependent, because peritoneal macrophages from iNOS(-/-) mice failed to affect the survival of the rocF mutant. Thus, bacterial arginase allows H. pylori to evade the immune response by down-regulating eukaryotic NO production.

Nitric Oxide Inhibits Endothelial IL-1β-induced ICAM-1 Gene Expression at the Transcriptional Level Decreasing Sp1 and AP-1 Activity

Nitric oxide (NO) has frequently been shown to inhibit leukocyte adherence to activated endothelium thus displaying anti-adhesive and immunosuppressive activities. A molecular mechanism contributing to this effect is described. Primary murine aortic endothelial cells were activated with interleukin (IL)-1beta to express intercellular adhesion molecule-1 (ICAM-1) mRNA in the presence or absence of the physiological spontaneous NO-donor S-nitrosocysteine. Subsequently, semiquantitative RT-PCR and gel shift assays with nuclear extracts were performed to analyse the effects of NO on ICAM-1 mRNA expression and on the activity of transcription factors involved in ICAM-1 transcription. In addition, luciferase reporter gene activity of cytokine-activated cells transiently transfected with an ICAM-1 promoter-luciferase construct and cultured in the presence of the slow-releasing NO-donor DETA/NO was determined. NO at subtoxic concentrations decreases IL-1beta-induced endothelial ICAM-1 mRNA expression. This inhibition occurs at the transcriptional level, as NO affects IL-1b-induced ICAM-1 promoter activity in transiently transfected cells. Using gel-shift assays and double-stranded oligonucleotide consensus sequences of the known transcription factor binding sites of the ICAM-1 promoter, Sp1 and AP-1 were identified as transcriptional activators of IL-1beta-driven ICAM-1 expression. The DNA binding of both of these transcription factors to specific binding sites of the ICAM-1 promoter was decreased in MAEC exposed to NO. Our studies indicate that the anti-adhesive effect of NO concentrations equivalent to high-output NO synthesis is mediated, at least in part, by inhibition of ICAM-1 expression via a concerted action of NO on the redox-sensitive transcriptional activators Sp1 and AP-1. This molecular mechanism may contribute to the anti-inflammatory actions of NO synthesized by the inducible NO synthase.

Nitric oxide: not just a negative inotrope.

Nitric oxide (NO) appears to play a role in modulating cardiac function in both health and disease. Early studies in isolated rodent cardiac myocytes demonstrated a depressant effect of NO supplied by NO donors (exogenous) as well as NO generated within myocytes (endogenous). There is increasing evidence for a functional NO generating system within the human myocardium, which appears upregulated in certain disease states. Induction of the high output nitric oxide synthase isoform (iNOS) has been demonstrated in the failing myocardium, though its functional significance remains unproven. More recently published data have contradicted the notion that NO acts solely as a negative inotrope demonstrating positive inotropy in both isolated rodent and human ventricular myocytes in response to a range of NO donors. Different NO donors have different NO release kinetics and generate a range of NO species (NO., NO+ and NO-) which may interact at a number of subcellular targets. The observed response of any cardiac preparation to an NO donor represents the net effect of activation of different effector targets and may explain the contradictory reported effects of NO. To realise the therapeutic potential of NO will require specific targeting at a subcellular level.

Induction of iNOS in human monocytes infected with different Legionella species

The contribution of nitric oxide (NO) radicals to the suppression of intracellular replication of Legionella has been well established in rodents but remained questionable in humans. Considering the fact that human monocytes do not exhibit a high-output NO production, we used sensitive methods such as detection of inducible NO synthase (iNOS) mRNA by reverse transcription-PCR and demonstration of iNOS protein expression by means of flow cytometry and Western blot to compare the levels of iNOS induced by Legionella species which, in accordance to their human prevalence, show different multiplication rates within human monocytic cells. The expression of iNOS in Mono Mac 6 (MM6) cells showed an only moderate inverse correlation to the intracellular replication rate of a given Legionella species in the protein expression assays. However, stimulation of host cells with 1,25-dihydroxyvitamin D 3 to enhance NO production and inhibition of NO production by treatment of host cells with N G-methyl- L-arginine were not able to modify the intracellular multiplication of legionellae within MM6 cells. Therefore, NO production does not seem to play a crucial role for the restriction of intracellular replication of Legionella bacteria within human monocytic cells. Rodent models in investigations which are supposed to clarify the involvement of NO radicals in defense mechanisms against Legionella infections in humans are of doubtful significance.

Specific association of nitric oxide synthase-2 with Rac isoforms in activated murine macrophages.

Nitric oxide synthase-2 (NOS2) is responsible for high-output nitric oxide production important in renal inflammation and injury. Using a yeast two-hybrid assay, we identified Rac2, a Rho GTPase member, as a NOS2-interacting protein. NOS2 and Rac2 proteins coimmunoprecipitated from activated RAW 264.7 macrophages. The two proteins colocalized in an intracellular compartment of these cells. Glutathione-S-transferase (GST) pull-down assays revealed that both Rac1 and Rac2 associated with GST-NOS2 and that the NOS2 oxygenase domain was necessary and sufficient for the interaction. [(35)S]methionine-labeled NOS2 interacted directly with GST-Rac2 in the absence of GTP, calmodulin, or NOS2 substrates or cofactors. Stable overexpression of Rac2 in RAW 264.7 cells augmented LPS-induced nitrite generation (~60%) and NOS2 activity (~45%) without measurably affecting NOS2 protein abundance and led to a redistribution of NOS2 to a high-speed Triton X-100-insoluble fraction. We conclude that Rac1 and Rac2 physically interact with NOS2 in activated macrophages and that the interaction with Rac2 correlates with a posttranslational stimulation of NOS2 activity and likely its spatial redistribution within the cell.

Protection of Primary Glial Cells by Grape Seed Proanthocyanidin Extract against Nitrosative/ Oxidative Stress

Previous studies showed that proanthocyanidins provide potent protection against oxidative stress. Here we investigate the effects of grape seed proanthocyanidin extract (GSPE) as a novel natural antioxidant on the generation and fate of nitric oxide (NO) in rat primary glial cell cultures. GSPE treatment (50 mg/L) increased NO production (measured by NO2− assay) by stimulation of the inducible isoform of NOS. However, GSPE failed to affect the LPS/IFN-γ-induced NO production or iNOS expression. Similar responses were found in the murine macrophage cell line RAW264.7. GSPE did not show any effect on dihydrodichlorofluorescein fluorescence (ROS marker with high sensitivity toward peroxynitrite) either in control or in LPS/IFN-γ-induced glial cultures even in the presence of a superoxide generator (PMA). GSPE treatment alone had no effect on the basal glutathione (GSH) status in glial cultures. Whereas the microglial GSH level declined sharply after LPS/IFN-γ treatment, the endogenous GSH pool was protected when such cultures were treated additionally with GSPE, although NO levels did not change. Glial cultures pretreated with GSPE showed higher tolerance toward application of hydrogen peroxide (H2O2) and tert-butylhydroperoxide. Furthermore, GSPE-pretreated glial cultures showed improved viability after H2O2-induced oxidative stress demonstrated by reduction in lactate dehydrogenase release or propidium iodide staining. We showed that, in addition to its antioxidative property, GSPE enhances low-level production of intracellular NO in primary rat astroglial cultures. Furthermore, GSPE pretreatment protects the microglial GSH pool during high output NO production and results in an elevation of the H2O2 tolerance in astroglial cells.

Nitric Oxide is Involved in the Immunomodulating Activities of Acidic Polysaccharide from Panax ginseng

The effects of an acidic polysaccharide isolated from the ethanol-insoluble and water-soluble fraction of Panax ginseng C. A. Meyer on immunomodulating activities were investigated. A high output nitric oxide synthase (iNOS) was shown in female BALB/c mice administered intraperitoneally with the acidic polysaccharide from ginseng. Newly synthesized iNOS protein was also observed in peritoneal macrophages cultured with interferon-gamma and the acidic polysaccharide. Spleen cells from acidic polysaccharide-treated mice did not proliferate in response to concanavalin A, but restored the responsiveness by the cotreatment of NG-monomethyl-L-arginine (NMMA) with concanavalin A. The treatment of mice with aminoguanidine, a specific iNOS inhibitor, alleviated the acidic polysaccharide-induced suppression of antibody response to sheep red blood cells. Present results suggest that the immunomodulating activities of the acidic polysaccharide were mediated by the production of nitric oxide.

Cooperation between reactive oxygen and nitrogen intermediates in killing of Rhodococcus equi by activated macrophages.

Rhodococcus equi is a facultative intracellular bacterium of macrophages which can infect immunocompromised humans and young horses. In the present study, we examine the mechanism of host defense against R. equi by using a murine model. We show that bacterial killing is dependent upon the presence of gamma interferon (IFN-gamma), which activates macrophages to produce reactive nitrogen and oxygen intermediates. These two radicals combine to form peroxynitrite (ONOO(-)), which kills R. equi. Mice deficient in the production of either the high-output nitric oxide pathway (iNOS(-/-)) or the oxidative burst (gp91(phox-/-)) are more susceptible to lethal R. equi infection and display higher bacterial burdens in their livers, spleens, and lungs than wild-type mice. These in vivo observations, which implicate both nitric oxide (NO) and superoxide (O(2)(-)) in bacterial killing, were reexamined in cell-free radical-generating assays. In these assays, R. equi remains fully viable following prolonged exposure to high concentrations of either nitric oxide or superoxide, indicating that neither compound is sufficient to mediate bacterial killing. In contrast, brief exposure of bacteria to ONOO(-) efficiently kills virulent R. equi. The intracellular killing of bacteria in vitro by activated macrophages correlated with the production of ONOO(-) in situ. Inhibition of nitric oxide production by activated macrophages by using N(G)-monomethyl-L-arginine blocks their production of ONOO(-) and weakens their ability to control rhodococcal replication. These studies indicate that peroxynitrite mediates the intracellular killing of R. equi by IFN-gamma-activated macrophages.

The preferred source of arginine for high-output nitric oxide synthesis in blood vessels

L-arginine is the substrate for nitric oxide (NO) production by each of the 3 NO synthase (NOS) isoforms encoded by the mammalian genome. Despite the pivotal roles of NO in mammalian physiology and pathophysiology, the source of arginine for NO synthesis is not clearly defined. In this context, it is notable that cell types that do not have a complete urea cycle often possess the urea cycle enzymes argininosuccinate synthase and argininosuccinate lyase; together, these enzymes confer the ability to regenerate arginine from the NOS product, L-citrulline. Herein, the authors summarize evidence to support the view that argininosuccinate synthase and argininosuccinate lyase function in an arginine-citrulline cycle, providing a ready source of arginine for high-output NO synthesis. The arginine-citrulline cycle is induced in vascular cells by the same cytokines that trigger iNOS expression and provides the preferred source of substrate for NO production. Evidence suggests that argininosuccinate synthase activity is rate-limiting to high-output NO synthesis and, hence, represents a novel target for the treatment of pathophysiological conditions arising from NO overproduction.

Gene regulation of high-output nitric oxide synthesis

Gene regulation of high-output nitric oxide synthesis

Induction of Intracellular Arginase Activity Does not Diminish the Capacity of Macrophages to Produce Nitric Oxide in vitro

The hypothesis was tested that induction of arginase expression in macrophages (Mφ) diminishes nitric oxide (NO) synthesis due to intracellular competition between arginase and inducible nitric oxide synthase (iNOS) for L-arginine (L-arg). Murine Mφ cell lines and bone marrow-derived Mφ (BMM) were stimulated to express either 1NOS or arginase or to coexpress these two enzymes. The response pattern obtained was complex but allowed the following conclusions: (1) 1NOS and arginase are differentially regulated. (ii) High intracellular arginase levels do not limit the capacity of Mφ to synthesize NO even when the L-arg concentration in the culture medium is lowered to physiological levels. (iii) Arginase levels in BMM pre-exposed to either Mφ colony-stimulating factor (M-CSF) or granulocyte-Mφ colony-stimulating factor (GM-CSF) differ markedly, but 1NOS expression and NO synthesis by the two BMM types is similar. (iv) Regulation of 1NOS and arginase differs between primary murine bone marrow Mφ and murine Mφ cell lines. (v) Arginase activity appears to be inhibited during high-output NO synthesis. Taken together, our results show that NO production by Mφ is not compromised by conditions that increase intracellular arginase activity.

The inducible nitric oxide synthase in vascular and cardiac tissue

Expression of the inducible form of nitric oxide synthase (iNOS) has been reported in a variety of cardiovascular diseases. The resulting high output nitric oxide (NO) formation, besides the level of iNOS expression, depends also on the expression of the metabolic pathways providing the enzyme with substrate and cofactor. NO may trigger short and long term effects which are either beneficial or deleterious, depending on the molecular targets with which it interacts. These interactions are governed by local factors (like the redox state). In the cardiovascular system, the major targets involve not only guanylyl cyclase, but also other haem proteins, protein thiols, iron–non-haem complexes, and superoxide anion (forming peroxynitrite). The latter has several intracellular targets and may be cytotoxic, despite the existence of endogenous defence mechanisms. These interactions may either trigger NO effects or represent releasable NO stores, able to buffer NO and prolong its effects in blood vessels and in the heart. Besides selectively inhibiting iNOS, a number of other therapeutic strategies are conceivable to alleviate deleterious effects of excessive NO formation, including peroxynitrite (ONOO −) scavenging and inhibition of metabolic pathways triggered by ONOO −. When available, these approaches might have the advantage to preserve beneficial effects of iNOS induction. Counteracting vascular hyper-responsiveness to endogenous vasoconstrictor agonists in septic shock, or inducing cardiac protection against ischaemia–reperfusion injury are examples of such beneficial effects of iNOS induction.

Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Prevent Inducible Nitric Oxide Synthase Transcription in Macrophages by Inhibiting NF-κB and IFN Regulatory Factor 1 Activation

AbstractHigh-output nitric oxide (NO) production from activated macrophages, resulting from the induction of inducible NO synthase (iNOS) expression, represents a major mechanism for macrophage cytotoxicity against pathogens. However, despite its beneficial role in host defense, sustained high-output NO production was also implicated in a variety of acute inflammatory diseases and autoimmune diseases. Therefore, the down-regulation of iNOS expression during an inflammatory process plays a significant physiological role. This study examines the role of two immunomodulatory neuropeptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), on NO production by LPS-, IFN-γ-, and LPS/IFN-γ-stimulated peritoneal macrophages and the Raw 264.7 cell line. Both VIP and PACAP inhibit NO production in a dose- and time-dependent manner by reducing iNOS expression at protein and mRNA level. VPAC1, the type 1 VIP receptor, which is constitutively expressed in macrophages, and to a lesser degree VPAC2, the type 2 VIP receptor, which is induced upon macrophage activation, mediate the effect of VIP/PACAP. VIP/PACAP inhibit iNOS expression and activity both in vivo and in vitro. Two transduction pathways appear to be involved, a cAMP-dependent pathway that preferentially inhibits IFN regulatory factor-1 transactivation and a cAMP-independent pathway that blocks NF-κB binding to the iNOS promoter. The down-regulation of iNOS expression, together with previously reported inhibitory effects on the production of the proinflammatory cytokines IL-6, TNF-α, and IL-12, and the stimulation of the anti-inflammatory IL-10, define VIP and PACAP as “macrophage deactivating factors” with significant physiological relevance.