Regulation of inducible nitric oxide synthase expression by macrophage purinoreceptors and calcium

Plant responses to red and far-red light are mediated by a family of photoreceptors called phytochromes. Arabidopsis thaliana seedlings lacking one of the phytochromes, phyB, have elongated hypocotyls and other tissues, suggesting that they may have an alteration in hormone physiology. We have studied the possibility that phyB mutations affect seedling gibberellin (GA) perception and metabolism by testing the responsiveness of wild-type and phyB seedlings to exogenous GAs. The phyB mutant elongates more than the wild type in response to the same exogenous concentrations of GA3 or GA4, showing that the mutation causes an increase in responsiveness to GAs. Among GAs that we were able to detect, we found no significant difference in endogenous levels between wild-type and phyB mutant seedlings. However, GA4 levels were below our limit of detectability, and the concentration of that active GA could have varied between wild-type and phyB mutant seedlings. These results suggest that, although GAs are required for hypocotyl cell elongation, phyB does not act primarily by changing total seedling GA levels but rather by decreasing seedling responsiveness to GAs.     

Regulation of inducible nitric oxide synthase (iNOS) and GTP cyclohydrolase I (GTP-CH I) gene expression by ox-LDL in rat vascular smooth muscle cells

The generation of nitric oxide is regulated by several factors, including the substrates and cofactors supplementation. Decreased expression and activity of nitric oxide synthase as well as diminished amount of L-arginine or enzyme cofactors results in the inhibition of nitric oxide generation in vascular wall cells. GTP cyclohydrolase 1 is a key enzyme involved in the synthesis of tetrahydrobiopterin, one of the most important cofactors of NO synthases. We have demonstrated that oxidized LDL inhibit not only inducible nitric oxide synthase gene expression but also GTP cyclohydrolase I gene expression in interleukin-1 beta activated rat vascular smooth muscle cells in vitro. It is postulated that diminished availability of tetrahydrobiopterin may additionally impair the generation of nitric oxide in atherosclerosis.     

Regulation by cytokines of the inducible nitric oxide synthase promoter in insulin-producing cells

In this article the complexation of anhydrotetracycline (AHTC), the major toxic decomposition product of the antibiotic tetracycline, with Al(III) has been investigated using the AM1 semiempirical and ab initio Hartree-Fock levels of theory. Different modes of complexation have been considered with the structure of tetra- and pentacoordinated complexes being fully optimized. In the gas phase, processes ii and iii, which lead to the complexes with stoichiometry MHL2+, are favored. Structure II ([AlLH2(OH)(H2O)]2+) has the metal coordinated to the O11 and O12 groups and the O3 group protonated and is the global minimum on the potential energy surface for the interaction. In water solution, the Al(III) is predicted to form predominantly a tetracoordinated complex at the Oam and O3 site (V) of the AHTC with the stoichiometry MH2L3+ (process i). The experimental proposal is the complexed form with the metal ion coordinated to the O11-O12 moiety (site II). The intramolecular proton transfer, which leads to the most stable Al(III)-AHTC MHL2+ complex, has not been considered by the experimentalists. The experimental structure was found to be unfavorable in our calculations in both gas phase and water solution. All the semiempirical results are in perfect agreement with the ab initio calculations. So, we suggest that the experimental assignments should be revised, taking into account the results obtained in the present study.     

The role of inducible nitric oxide synthase in cardiac allograft rejection

Rats were exposed to either a footshock stimulus (FS) or emotional stimulus (ES, forced perception of another rat receiving footshocks) during a daily 10-min session for 5 consecutive days. The consequences of FS and ES on their behavioural responsiveness were assessed at different post-stress intervals using a small open-field. FS induced a decrease in ambulation, rearing and sniffing and an increased immobility in the small open field. These effects were present in rats tested immediately after the last session and remained present for at least 15 days. In contrast, ES induced a transient decrease in ambulation and rearing immediately after the last session, but in the period from half an hour until at least 15 days after the stimulus experience, an increase in ambulation, rearing and sniffing was observed. Exposure to one footshock per session for 5 consecutive days or to 10 footshocks in a single session also resulted in a long-lasting reduction in ambulation and sniffing and an increase in immobility. The former regime did not influence the behavioural response of ES rats, but the latter resulted in an increase in ambulation, rearing and sniffing in ES rats. Naloxone (1 mg/kg s.c.) pretreatment antagonized the increased behavioural activity of the ES rats whereas the activity of control and FS animals was not affected, suggesting an involvement of endogenous opioid systems in the behavioural responses observed in ES rats. It is suggested that the behavioural responses of the ES and FS animals are regulated by different mechanisms.     

Gadolinium chloride inhibits Kupffer cell nitric oxide synthase (iNOS) induction

Kupffer cells (KC) are the phagocytic macrophages of the liver. The rare earth metal, gadolinium (GdCl3), is a lanthanide, which, after phagocytosis by the KC, has been found to alter various aspects of KC physiology. In this study, we describe for the first time that the in vivo administration of GdCl3 to rats decreases the release of NO by isolated rat KC in response to lipopolysaccharide. Western blot analysis shows decreased expression of both inducible nitric oxide synthase as well as total cellular calmodulin after GdCl3 treatment. Possible mechanisms for this phenomenon are suggested.     

Adrenomedullin augments inducible nitric oxide synthase expression in cytokine-stimulated cardiac myocytes

BACKGROUND: Plasma levels of adrenomedullin are increased in patients with congestive heart failure, but there has been no report concerning the effects of adrenomedullin on the heart. We investigated the effects of adrenomedullin on NO synthase activity in cardiac myocytes. METHODS AND RESULTS: We measured the production of nitrite, a stable metabolite of NO, in cultured neonatal rat cardiac myocytes with the Griess reagent. Inducible NO synthase mRNA and protein expression were assayed by Northern and Western blotting, respectively. Incubation of the cultures with interleukin-1 beta (10 ng/mL) for 24 hours caused a significant increase in nitrite accumulation. Adrenomedullin significantly augmented nitrite production by interleukin-1 beta-stimulated but not by unstimulated cardiac myocytes in a dose-dependent manner (10(-10) to 10(-6) mol/L). The adrenomedullin-induced nitrite production by interleukin-1 beta-stimulated cells was accompanied by increased inducible NO synthase mRNA and protein expression. In the presence of dibutyryl cAMP, the interleukin-1 beta-induced nitrite accumulation was increased further, but the stimulatory effect of adrenomedullin on nitrite production was abolished. Adrenomedullin dose-dependently increased intracellular cAMP levels in cardiac myocytes. Addition of the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP[8-37] to the culture dose-dependently inhibited both cAMP and NO generation stimulated by adrenomedullin. CONCLUSIONS: These results indicate that adrenomedullin acts on cardiac myocytes and augments NO synthesis in these cells under cytokine-stimulated conditions, at least partially through a cAMP-dependent pathway.     

Pneumococci stimulate the production of the inducible nitric oxide synthase and nitric oxide by murine macrophages

The role of nitric oxide (NO) in the pathophysiology of gram-positive sepsis is uncertain. In inflammatory conditions, high-output NO production is catalyzed by the enzyme inducible nitric oxide synthase (iNOS). The ability of 2 strains of pneumococci, pneumococcal cell wall preparations, and purified pneumococcal capsule (Pnu-Imune 23) to trigger the production of iNOS protein and NO in RAW 264.7 murine macrophages was tested. Live pneumococci, oxacillin-killed pneumococci, and pneumococcal cell wall preparations stimulated the production of iNOS and NO by RAW 264.7 cells in the presence, but not the absence, of low concentrations of recombinant murine interferon-gamma. In contrast, purified pneumococcal capsule induced little or no iNOS or NO production by these cells. Thus, pneumococci stimulate high-output NO production by murine macrophages. The potential role of NO in the pathogenesis of pneumococcal sepsis deserves further study.     

Streptococcal pyrogenic exotoxins A (SpeA) and C (SpeC) stimulate the production of inducible nitric oxide synthase (iNOS) protein in RAW 264.7 macrophages

Streptococcal pyrogenic exotoxins A (SpeA) and C (SpeC) are members of a family of superantigens produced by group A streptococci that appear to play a key role in the pathogenesis of streptococcal toxic shock syndrome. Since it is known that nitric oxide (NO) and tumor necrosis factor (TNF) are largely responsible for the shock and multiple organ dysfunction of Gram-negative sepsis, we hypothesized that SpeA and/or SpeC could trigger the production of inducible nitric oxide synthase (iNOS) and/or TNF by murine macrophages. We exposed RAW 264.7 macrophages to increasing concentrations of SpeA or SpeC alone and in combination with recombinant murine interferon-gamma (rIFN gamma) for 16-24 h. We found that both SpeA and SpeC triggered iNOS production in the presence of low concentrations of rIFN gamma, while neither provoked iNOS accumulation in the absence of rIFN gamma. Neither SpeA nor SpeC (with or without rIFN gamma) reproducibly induced TNF production by these murine macrophages. These data indicate that two streptococcal exotoxins up-regulate iNOS production by murine macrophages and suggest that nitric oxide production may play an important role in the pathogenesis of streptococcal toxic shock syndrome.     

Therapy of cancer metastasis by activation of the inducible nitric oxide synthase

The process of cancer metastasis consists of multiple sequential and highly selective steps. The vast majority of tumor cells that enter the circulation die rapidly; only a few survive to produce metastases. This survival is not random. Metastases are clonal in origin and are produced by specialized subpopulations of cells that preexist in a heterogeneous primary tumor. Experimental studies concluded that metastatic cells survive in the circulation whereas nonmetastatic cells do not. In part, this difference is due to an inverse correlation between expression of endogenous inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) and metastatic potential. Direct evidence for the role of iNOS in metastasis has been provided by our data on transfection of highly metastatic murine K-1735 clone 4 (C4.P) cells which express low levels of iNOS, with a functional iNOS (C4.L8), inactive mutated iNOS (C4.S2), or neomycin resistance (C4.Neo) genes in medium containing 3 mM of the specific iNOS inhibitor NG-L-methyl arginine (NMA). C4.P, C4.Neo, and C4.S2 cells were highly metastatic, whereas C4.L8 cells were not. Moreover, C4.L8 cells produced slow-growing subcutaneous tumors in nude mice, whereas the other 3 cell lines produced fast-growing tumors. In vitro studies indicated that the expression of iNOS in C4.L8.5 cells was associated with either cytostasis or cytolysis via apoptosis, depending upon NO output. The tumor cells producing high levels of NO underwent autocytolysis and produced cytolysis of bystander cells under both in vitro and in vivo conditions. Multiple i.v. injections of liposomes containing a synthetic lipopeptide upregulated iNOS expression in murine M5076 reticulum sarcoma cells growing as hepatic metastases. The induction of iNOS was associated with the complete regression of the lesions. Transfection of interferon-beta suppressed tumor formation and eradicated metastases, which was apparently linked to iNOS expression and NO production in host cells such as macrophage. Besides mediating cell death, NO produced tumor suppression by regulating expression of genes related to metastasis, e.g., survival, invasion, and angiogenesis. Suppression of metastasis can be achieved through use of immunomodulators that induce iNOS expression in tumor lesions or by the direct delivery of the iNOS gene to tumor cells or host cells through liposome and/or viral vectors.     

Failure of L-NAME to cause inhibition of nitric oxide synthesis: role of inducible nitric oxide synthase

We addressed the hypothesis that administration of nitric oxide synthase inhibitor, NG -nitro-L-arginine methyl ester (L-NAME) does not result in a sustained suppression of nitric oxide (NO) synthesis, because of a compensatory expression of inducible nitric oxide synthase (iNOS). L-NAME was administered in the drinking water (0.1-1.0 mg/ml) for 7 days to guinea pigs and rats. Nitric oxide synthesis was assessed by [1] ex vivo formation of nitrite in blood vessels and intestine [2] tissue levels of cGMP [3] iNOS gene expression by RT-PCR [4] NADPH diaphorase staining [5] direct assessment of NO release in tissue explants using a microelectrode/electrochemical detection system. Chronic L-NAME administration elevated intestinal cGMP and nitrite levels in guinea pigs (p < 0.05). In rats, intestinal nitrite levels were comparable in control and L-NAME treatment groups, whereas direct assessment of NO release defined a marked increase in the L-NAME group. Chronic L-NAME resulted in an induction of iNOS gene expression in rats and guinea pigs and novel sites of NADPH diaphorase staining in the intestine. We conclude that iNOS expression is responsible for a compensatory increase or normalization of NO synthesis during sustained administration of L-NAME.     

Endogenous opioids regulate the expression of inducible nitric oxide synthase by splenocytes

In the present study, we tested the hypothesis that lipopolysaccharide (LPS)-induced expression of nitric oxide synthase (iNOS) by splenocytes is modulated through the activation of endogenous opioids in the central nervous system. The initial studies determined the parameters of LPS-induced expression of iNOS by splenocytes. Rats were injected with LPS at doses of 0, 1, 10, 100, and 1000 microg/kg, and measures of both iNOS mRNA and protein showed a dose-dependent increase in expression. In a time course study, rats received 100 microg/kg LPS and were killed at 0, 2, 4, 8, and 16 h postinjection. Both iNOS mRNA and protein expression was detectable at the 2-h time point, with peak expression occurring at 8 h. To evaluate the involvement of endogenous opioids, the opioid receptor antagonist naltrexone was administered at 0, 0.1, 1, or 10 mg/kg s.c. in combination with LPS (100 microg/kg), with a second injection of naltrexone at the same dose 4 h after the injection of LPS. Naltrexone induced a pronounced dose-dependent reduction in iNOS mRNA and protein expression by splenocytes. The modulation of iNOS expression occurs via central opioid receptors as intracerebroventricular administration but not peripheral administration of N-methylnaltrexone, the quaternary form of naltrexone that does not readily cross the blood-brain barrier, reduced the expression of iNOS. For all of the manipulations, nitrite/nitrate levels in the plasma showed effects similar to those for iNOS mRNA and protein. Collectively, these findings indicate that central opioid receptors are involved in the in vivo regulation of splenic nitric oxide production.     

Impact of surgery on nitric oxide in rats: evidence for activation of inducible nitric oxide synthase

We investigated the effect of euvolemic surgical preparation, on chemical indices of activity of the nitric oxide (NO) system, in anesthetized, acutely prepared rats. The urinary excretion of NO2+NO3 (UNOXV) and cGMP (UcGMPV) increased progressively during the experiment. Pretreatment with aminoguanidine or dexamethasone, inhibitors of inducible NO synthase (iNOS), prevented the increase in UNOXV and UcGMPV but had no impact on mean arterial pressure (BP), renal vascular resistance (RVR) or GFR. Since these variables did not change in the conscious rat, the increased UNOXV results from some aspect of the acute surgical preparation. When acutely prepared rats received L-NAME, a non-specific NOS inhibitor, BP and RVR increased but paradoxical increases in UNOXV and UcGMPV were also seen. Nonselective NOS inhibition (+L-NAME) was fatal in 50% of acutely prepared rats, causing cardiac contracture. The same dose of L-NAME produced no deaths in either conscious chronically catheterized rats or in acutely prepared rats, previously subjected to sterile surgery and acute L-NAME in the conscious state. These data indicate that acute, nonsterile surgery induces expression of iNOS, but that the additional NO generated has no obvious cardiovascular/renal actions. Acute UNOXV and UcGMPV do not predict total NO production, or "hemodynamically active" NO. Generalized NO inhibition in rats acutely stressed by surgery/anesthesia can be fatal.     

Role of nitric oxide in rat nephrotoxic nephritis: comparison between inducible and constitutive nitric oxide synthase

Nitric oxide (NO), generated by inducible NO synthase (iNOS) in migrating macrophages, is increased in glomerulonephritis. This study investigates the effect of NO inhibition on rat nephrotoxic nephritis (NTN) to clarify the role of NO production in glomerular damage. NTN was induced in Sprague Dawley rats by an injection of an anti-glomerular basement membrane (GBM) antibody. Urinary nitrite excretion and nitrite release from kidney slices (5.47 +/- 1.19 versus 2.15 +/- 0.73 nmol/mg protein, NTN versus Control, P < 0.05) were increased in NTN on day 2. Glomerular macrophage infiltration and intercellular adhesion molecule (ICAM)-1 expression increased from day 2. iNOS expression was increased in interstitial macrophages. Glomerular endothelial cell NOS (ecNOS) expression evaluated by counting immunogold particles along GBM was suppressed (0.06 +/- 0.02 versus 0.35 +/- 0.04 gold/micron GBM, P < 0.0001). Glomerular damage developed progressively. NG-nitro-L-arginine methyl ester (L-NAME), which inhibits both iNOS and ecNOS and aminoguanidine (AG), a relatively selective inhibitor for iNOS, equally suppressed nitrite in urine and renal tissue. Glomerular ICAM-1 expression and macrophage infiltration were reduced by L-NAME, but not by AG. Expression of ecNOS was significantly increased by L-NAME (0.91 +/- 0.08, P < 0.0001 versus NTN), but slightly by AG (0.18 +/- 0.04). AG significantly and L-NAME slightly attenuated the glomerular damage at day 4. In conclusion, suppression of iNOS prevents glomerular damage in the early stage of NTN. Treatment by L-NAME reduces macrophage infiltration by suppression of ICAM-1 expression, which may be explained by an increase in ecNOS expression.     

Mechanism of inducible nitric oxide synthase inactivation by aminoguanidine and L-N6-(1-iminoethyl)lysine

The inducible nitric oxide synthase (iNOS) selective inhibitors aminoguanidine (AG) and N6-(1-iminoethyl)-L-lysine (NIL), under conditions that support catalytic turnover, inactivate the enzyme by altering in different ways the functionality of the active site. NIL inactivation of the iNOS primarily targets the heme residue at the active site, as evidenced by a time- and concentration-dependent loss of heme fluorescence that accompanies the loss of NO-forming activity. The NIL-inactivated iNOS dimers that have lost their heme partially disassemble into monomers with no fluorometrically detectable heme. AG inactivation of the iNOS is not accompanied by heme destruction, as evidenced by retention of heme fluorescence and absorbance after complete loss of NO-forming activity. The AG-inactivated iNOS dimers do not disassemble into monomers as extensively as NIL-inactivated dimers. Incubation of the iNOS with 14C-labeled NIL results in no detectable protein-associated radioactivity in the NIL-inactivated iNOS, suggesting that the primary mechanism of the iNOS inactivation by NIL is heme alteration and loss. In contrast, incubations of iNOS with 14C-labeled AG result in the incorporation of radioactivity into both iNOS protein and low molecular weight structures that migrate by SDS-PAGE similarly to free heme. These observations suggest that AG inactivation proceeds through multiple pathways of covalent modification of the iNOS protein and the heme residue at the active site, but which sustain the integrity of the heme porphyrin ring.     

Up-regulation of inducible nitric oxide synthase and production of nitric oxide by the Swarm rat and human chondrosarcoma

Phospholipids are the major constituents of cell membranes, and have numerous structural and functional roles in the nervous system. Although the metabolic pathways responsible for the syntheses of the phosphatides phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), and phosphatidylserine (PtdSer) are well understood, the mechanisms controlling these pathways in neural tissue have not been fully characterized. Recent studies have suggested that the main factors controlling PtdCho and PtdEtn synthesis by the Kennedy cycle tend to be the intracellular levels of key substrates for the biosynthetic enzymes, or changes in the activities of the rate-limiting enzymes. Moreover, different control mechanisms may operate, depending upon the functional state of the tissue.     

Cell-specific effects of pentoxifylline on nitric oxide production and inducible nitric oxide synthase mRNA expression

Cytokine-stimulated astrocytes and macrophages are potent producers of nitric oxide (NO), a free radical proposed to play an important role in organ-specific autoimmunity, including demyelinating diseases of the central nervous system. The aim of this study was to investigate effects of pentoxifylline (PTX), a phosphodiesterase inhibitor with immunomodulatory properties, on NO production and inducible NO synthase (iNOS) mRNA expression in rat astrocytes and macrophages. We have shown that PTX affects cytokine (interferon-gamma, IFN-gamma; interleukin-1, IL-1; tumour-necrosis factor-alpha, TNF-alpha)-induced NO production in both cell types, but in the opposite manner--enhancing in astrocytes and suppressive in macrophages. While PTX did not have any effect on enzymatic activity of iNOS in activated cells, expression of iNOS mRNA was elevated in astrocytes and decreased in macrophages treated with cytokines and PTX. Treatment with PTX alone affected neither NO production nor iNOS mRNA levels in astrocytes or macrophages. This study indicates involvement of different signalling pathways associated with iNOS induction in astrocytes and macrophages, thus emphasizing complexity of regulation of NO synthesis in different cell types.     

Expression of inducible nitric oxide synthase in human burn wounds

A water-soluble synthetic peptide with only nine amino acid residues, comprising the 131-139 sequence region of the cytotoxic protein alpha-sarcin (secreted by the mold Aspergillus giganteus), interacts with large unilamellar vesicles composed of acid phospholipids. It promotes lipid mixing between bilayers and leakage of vesicle aqueous contents, and it also abolishes the phospholipid phase transition. Other larger peptides containing such an amino acid sequence also produce these effects. These peptides acquire alpha-helical conformation in the presence of trifluoroethanol, but display beta-strand conformation in the presence of sodium dodecyl sulfate. The interaction of these peptides with the lipid vesicles also results in beta-structure. The obtained data are discussed in terms of the involvement of the 131-139 stretch of alpha-sarcin in its interaction with lipid membranes.