N-monomethyl-L-arginine Research Articles

Effect of nitric oxide synthase induction on the expression of mitochondrial respiratory chain enzyme subunits in mixed cortical and astroglial cell cultures

In the present study we evaluated the effects of NO synthase (NOS) induction on the regulation of cytochrome c oxidase (CO) and F 0F 1-ATPase subunit expression in astroglial and mixed cortical cell cultures. In mixed cortical cell cultures, 18 h of treatment with lipopolysaccharide (LPS, 0.1 μg/mL) plus interferon-γ (INFγ, 10 U/mL) caused an increase of mRNAs for CO-I, F 0F 1-ATPase 6 and also for iNOS at 20 DIV. The induction of both CO-I and F 0F 1-ATPase 6 was abolished by the NOS inhibitor N-monomethyl-L-arginine (NMMA) or by the enzymatic scavenger superoxide dismutase/catalase (SOD/CAT). In primary astroglial cell cultures, treatment for 18 h with increasing concentrations of LPS and INFγ, produced an increase in the amount of mitochondrial encoded CO-I and -II subunits, with no significant modifications of nuclear encoded subunits IV. An increase was also observed at level of transcription for CO-I and -II, and F 0F 1-ATPase 6 mRNAs. These effects were abolished by addition of NMMA or SOD/CAT. mRNA induction of CO-I was higher in mixed cortical than in astroglial cell cultures while that of F 0F 1-ATPase 6 was similar in both cell types. These results suggest that the expression of mitochondrial encoded subunits (CO-I, CO-II and F 0F 1-ATPase 6) is up-regulated in response to oxygen and NO reactive species. The activity of cytochrome c oxidase decreased after LPS/INFγ treatment in both astroglial and mixed cortical cultures. The activity of ATP synthase was unmodified, while ATP content drastically decreased after LPS/INFγ treatment, in both astroglial and mixed cortical cultures. The enzymatic activities of catalase and Mc-SOD (mitochondrial) showed a significant increase after LPS/INFγ treatment, which was abolished by NMMA.

Nitric oxide-mediated modulation of interleukin-8 production by a human glioblastoma cell line, T98G, cocultured with myeloid and monocytic cell lines.

Coculture of T98G glioblastoma cells with the myeloid and monocytic cell lines, HL-60, and THP-1 produced minimal amounts of interleukin-8 (IL-8). Pretreatment of HL-60 or THP-1 cells with phorbol myristate acetate (PMA) enhanced their capacity to induce IL-8 production by T98G cells. In contrast, the murine macrophage cell lines J774 A.1 and RAW 264.7 induced high levels of IL-8 production by T98G cells without PMA activation. To determine the molecules responsible for the induction of IL-8 by T98G cells, we carried out coculture experiments with a membrane fraction prepared from RAW cells and indicated that membrane-associated and free forms of murine IL-1alpha acted on human T98G cells to produce IL-8. RAW cells were unique in that increasing the number of RAW cells relative to the number of T98G cells (RAW/T98G ratio > 4:1) significantly suppressed IL-8 production by T98G cells. Because RAW cells produce large amounts of nitric oxide (NO), we assumed that the suppression of IL-8 production was ascribable to the NO produced by the RAW cells. This was supported by the inverse relationship between increasing concentrations of NO and IL-8 production seen in this coculture system. The involvement of NO in the suppression of IL-8 production was confirmed by the finding that N-monomethyl-L-arginine (NMMA), which inhibits NO production, reversed this suppression, whereas S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a strong NO generator, suppressed IL-8 production. Our results indicate that high levels of NO suppress IL-8 production by T98G cells, and murine IL-1alpha plays a major role in the induction of IL-8 production by T98G cells. It is, therefore, possible that excessive production of NO during the interaction of glioma cells with macrophages may play a regulatory role in chemokine production, thus mitigating inflammatory responses.

L-Arginine uptake in rat cerebral mitochondria

The kinetics of L-[14C] arginine ( L-[14C] Arg) uptake and the effects of potential competitors on the uptake were analysed in nonsynaptic mitochondria isolated from rat cerebral hemispheres. Analysis of uptake kinetics revealed a high affinity component with a mean K m=0.08 mM, and V max=1.89 nmoles⧹min⧹mg, and a very low affinity component probably manifesting diffusion. The uptake of 25 mM L-Arg was strongly inhibited by a 20-fold excess of L-lysine ( L-Lys) and L-ornithine ( L-Orn), but not by D-Arg nor any neutral amino acid, which resembles the characteristics of the y+ transport system operating in the nerve- and glia cell-, and synaptic plasma membranes. Also in consistence with the other y + systems, L-Arg uptake to mitochondria was inhibited by a nitric oxide synthase (NOS) inhibitor L-N-monomethyl arginine ( L-NMMA), but not by another NOS inhibitor N G-nitro- L-arginine ( L-NNA). However, the uptake was very little affected by 20-fold excess of L-histidine ( L-His), L-glutamate ( L-Glu) or L-glutamine ( L-Gln), which is in contrast to the nonmitochondrial systems. The uptake was only marginally influenced by cytoplasmic L-Arg metabolites: ammonia, creatine, putrescine, or the mitochondrial L-Arg decarboxylation product, agmatine.

Evidence that S-adenosyl-L-methionine diastereoisomers may reduce ischaemia-reperfusion injury by interacting with purinoceptors in isolated rat liver.

1. Mechanisms underlying the haemodynamic activity of diastereoisomers of S-adenosyl-L-methionine (SAM) were investigated using inhibitors of purinoceptors and nitric oxide (NO) synthase in perfused rat livers damaged by sequential 24 h cold and 20 min rewarming ischaemia + reperfusion. 2. Stored livers were flushed with 10 ml saline alone (control) or with added (R,S) or (S,S) SAM (100 microM) and reperfused in the absence (control) or presence of 10 microM 8-phenyltheophylline (8-PT) or 100 microM L-N-monomethylarginine (L-NMMA). 3. Both SAM diastereoisomers rapidly increased blood flow and bile production versus controls (P<0.001) but the (R,S) isomer induced greater increases in blood flow and the (S,S) isomer greater increases in bile production: 625 versus 596 versus 518 ml blood flow and 100 versus 119 versus 56 mg bile production per g liver over 3 h in (R,S), (S,S) and control, respectively. 4. 8-PT prevented the enhancement of blood flow by (S,S) SAM (529 versus 596 ml g(-1) liver over 3 h for (S,S) SAM alone, P<0.001), but was without effect in control livers. 8-PT also reduced SAM-enhanced bile production: 51 versus 119 mg g(-1) liver over 3 h, P<0.001. L-NMMA reduced blood flow and bile production similarly in the absence or presence of (S,S) SAM. 5. Thus, SAM may improve liver perfusion after ischaemia-reperfusion injury via stimulation of P, (A2) purinoceptors at which SAM shows activity. The choleretic activity of (S,S) SAM is disproportionately greater than enhanced blood flow and may occur independently of a NO-dependent component of bile production.

The inhibitory effect of interleukin 1beta on rat hepatocyte DNA synthesis is mediated by nitric oxide.

Interleukin 1beta (IL-1beta) and nitric oxide (NO) have potent growth-regulatory effects on different cell types. We found that epidermal growth factor-induced DNA synthesis in primary cultures of adult rat hepatocytes was inhibited by NO when it was provided by addition to the cultures of S-nitroso-N-acetyl-penicillamine (SNAP), an NO donor, as well as by addition of IL-1beta in a dose-dependent manner. IL-1beta also induced NO production and inducible NO synthase (iNOS) gene expression. The inhibition of DNA synthesis by IL-1beta was completely abrogated when NO production was inhibited by N-monomethyl-L-arginine (NMA), a competitive inhibitor of iNOS. IL-1beta-receptor antagonist (IL-1ra), which interferes with the interaction of IL-1beta with target cells, also abolished the inhibitory effects of IL-1beta on hepatocyte DNA synthesis as well as IL-1beta-induced iNOS gene expression. We also found that hepatocyte DNA synthesis inhibition by IL-1beta was completely antagonized by providing deoxynucleosides to bypass the block in ribonucleotide reductase, a rate-limiting step in DNA synthesis, thus implicating this enzyme in the mechanism of growth inhibition by IL-1beta. These experiments extended prior observations on the growth-inhibitory actions of IL-1beta on hepatocyte DNA synthesis, involving the IL-1beta receptor, NO production, and ribonucleotide reductase.

Antigen-specific lymphocytes enhance nitric oxide production in Mycobacterium bovis BCG-infected bovine macrophages.

Nitric oxide (NO) production was evaluated in macrophages isolated from Mycobacterium bovis bacille Calmette-Guérin (BCG)-immunized, and control non-immunized, cattle. Incubation of M. bovis BCG-infected macrophages with recombinant bovine IFN-gamma led to increased nitrite levels in culture supernatants. It was also demonstrated that NO production by autologous M. bovis BCG-infected macrophages increased in a linear relationship with the number of antigen-specific lymphocytes added to cultures. The elevated NO levels were also associated with increased IFN-gamma secretion. Treatment of cultures with the NO inhibitor, N-monomethyl L-arginine (L-NMMA), reduced the levels of NO without affecting the metabolic activity of internalized M. bovis BCG. Our results suggest that synthesis of NO may constitute an integral part of the cell-mediated antigen-specific response against M. bovis BCG. However, although the presence of lymphocytes does partially inhibit multiplication of M. bovis BCG in macrophages, it appears that the activity of NO, or the levels produced in monocyte-derived macrophages, may be insufficient to influence the growth of the intracellular mycobacteria.

There is no Regulatory Role for Induced Nitric Oxide in the Regulation of the in vitro Proliferative Response of Bovine Mononuclear Cells to Mitogens, Alloantigens or Superantigens

Nitric oxide (NO) is a potent cellular mediator which has been shown to modulate several immune mechanisms. Between species, however, there are considerable differences regarding the signals required for induction of NO as well as the kind of cells capable of producing NO. The object of this study was to determine the kinetics of NO production of bovine blood mononuclear cells (boMNC) stimulated in vitro and to investigate whether it modulates their proliferative response following allogeneic (mixed leukocyte cultures, aMLC), mitogenic (PWM, Con A) or superantigenic (SEA, SEB) stimulation. NO production was indirectly determined with the Griess reagent measuring nitrite (NO − 2). Significant but low amounts of NO could be detected as early as day 3 after in vitro stimulation and did only slightly increase during the 6–8 day culture period. Superantigens (SEA, SEB) and aMLCs (4.3–5.2 µM NO − 2) induced a significantly higher nitrite accumulation compared to Con A (2.6 µM N − 2. Generation of nitrite, most likely produced by monocytes/macrophages, could be inhibited by 1 mM N-monomethyl-L-arginine (NMLA). Flow cytometric characterization of various cellular responses revealed no differences between cultures with or without NMLA. This included the determination of blastogenesis, absolute numbers of viable cells, expression density of activation markers (MHC class II, IL-2Rα) and cellular subpopulations (CD4 +, CD8 +, sIg +) among blasts. In addition, exogenously provided NO via SNOG in non-toxic concentrations (10 −5–10 −4 M) did not alter the proliferative reaction of boMNC in vitro. The results suggest that NO is induced after in vitro stimulation of boMNC, however, at a low level, and without having any positive or suppressive effects on the so far tested cellular parameters of activation and proliferation.

Does Lipoprotein(a) Impair Endothelial Function?

Objectives. This study was undertaken to test the hypothesis that lipoprotein(a) [Lp(a)] impairs endothelial function.Background. Elevated Lp(a) plasma levels have been demonstrated to be associated with an increased risk of coronary heart disease. In atherosclerosis, endothelial dysfunction is known to be an early indicator of vascular changes. However, the effect of Lp(a) on nitric oxide (NO)-dependent vasodilator response has not yet been determined. We therefore examined the influence of Lp(a) on basal and stimulated NO-mediated vasodilator response in the forearm vascular bed.Methods. Strain gauge plethysmography was used to measure changes in forearm blood flow produced by intraarterial infusion of increasing doses of acetylcholine (3, 12, 24 and 48 μg/min), sodium nitroprusside (200, 800 and 3,200 ng/min) and N-monomethyl l-arginine (l-NMMA) (1, 2 and 4 μmol/min) in 57 white subjects (mean age ± SD 37 ± 14 years). Lp(a) plasma concentrations were determined by rocket immunoelectrophoresis.Results. Endothelium-dependent vasodilation tested by intraarterial acetylcholine and endothelium-independent vascular relaxation tested by intraarterial sodium nitroprusside were not correlated with Lp(a). Similarly, no significant differences in forearm blood flow changes were observed when patients were classified into tertiles according to their individual Lp(a) concentration. In contrast, changes in forearm blood flow after intraarterial l-NMMA indicating basal production and release of NO differed significantly among tertiles. Patients in the highest Lp(a) tertile (49.2 ± 20.3 mg/dl) had a much greater vasoconstrictive response to l-NMMA than patients in the lowest Lp(a) tertile (4.8 ± 2.5 mg/dl): 2 μmol/min of l-NMMA, −23.6 ± 22.5% vs. −10.4 ± 9.1% (p < 0.02); 4 μmol/min of l-NMMA, −27.8 ± 10.3% vs. −17.6 ± 9.9% (p < 0.03). Lp(a) plasma level consistently correlated negatively with the forearm blood flow responses to 4 μmol/min of intraarterial l-NMMA (r = −0.38, p < 0.01). Multiple stepwise regression analysis of variables, including total and high and low density lipoprotein cholesterol, further confirmed that plasma Lp(a) remained a significant independent determinant of forearm blood flow changes in response to l-NMMA (p < 0.02).Conclusions. The endothelium-dependent vasoconstrictive response to l-NMMA was enhanced in subjects with relatively high Lp(a) plasma levels, suggesting an increased basal production and release of NO. This response seemed to reflect a compensatory mechanism of the endothelium to yet unknown Lp(a)-induced atherosclerotic effects.

Suppression of lymphoproliferation by alveolar macrophages in the guinea pig

Setting : The relationship between alveolar macrophages (AM) and lymphocytes may be important in the early establishment of infection with Mycobacterium tuberculosis . AM in several species have been shown to suppress lymphoproliferation by producing inhibitors that include nitric oxide (NO).Objective : To study this phenomenon in the guinea pig, the mitogen-induced proliferation of splenic lymphocytes was quantified under various conditions of co-culture with resident AM.Results : Guinea pig AM consistently and profoundly suppressed proliferation in the co-cultures at AM:lymphocyte ratios of 1:4 or greater. The inclusion of a NO synthesis inhibitor, N-monomethyl-L arginine (NMMA), in the co-culture medium did not influence the suppression of Con A-induced lymphoproliferation by resident guinea pig AM. No nitrite could be detected in supernatant fluids of co-cultured AM and splenocytes. Attempts to stimulate guinea pig AM with LPS in combination with recombinant murine and human IFN--γ, infection with live Listeria monocytogenes , or incubation with the supernatants from ConA-activated guinea pig lymphocytes failed to generate NO metabolites. The addition of catalase or indomethacin to the Con A-induced AM-splenocyte co-cultures, to inhibit hydrogen peroxide (H2O2) or prostaglandin E2(PGE2), respectively, did not counteract the suppression mediated by AM. Cell contact was necessary for the co-cultures to generate their inhibitory effects on lymphoproliferation, however, the suppression was actually mediated, at least in part, by soluble factors produced in the co-cultures.Conclusion : These results suggest that resident alveolar macrophages suppress lymphocyte proliferation in the guinea pig, but that the effect is not mediated by NO, PGE2or H2O2. The failure to demonstrate NO synthesis under a variety of stimulatory conditions, which resulted in macrophage activation, suggests that the guinea pig is similar to the human in that regard.

Nitric oxide mediates plateletactivating factor stimulatory action on uterine prostaglandin production

Accumulated evidence suggests that platelet-activating factor (PAF) may have a role in implantation by stimulating prostaglandin (PG) production. Since we had demonstrated that nitric oxide (NO) can increase uterine PGs, the aim of this study was to explore whether or not NO could mediate rat uterus responses to PAF on day 5 of gestation, when implantation takes place. Uterine motility was enhanced by PAF as compared to controls. This action was abolished by either the arginine analogue, N-monomethyl L-arginine (L-NMMA) or the cyclooxygenase inhibitor, indomethacin. On the other hand, NOS activity was detected in uterine strips and could be stimulated by PAF. The cyclooxygenase product PGE 2 was also significantly stimulated by PAF. Inhibition of endogenous NO formation abolished the PAF effect on PG synthesis. Our results suggest that NO is an important intermediate in the interaction between PAF and PGs.

Nitric oxide-independent suppression of P450 2C11 expression by interleukin-1β and endotoxin in primary rat hepatocytes

Hepatic expression of multiple cytochrome P450 genes is suppressed in the livers of rats undergoing an inflammatory response. Nitric oxide (NO) released during inflammation has been implicated in the decreased activities and expression of several cytochrome P450 isozymes. We examined the role of cytokine-mediated NO release on cytochrome P450 2C11 expression in rat hepatocytes cultured on Matrigel. Lipopolysaccharide (LPS), interleukin-1β (IL-lβ), IL-6, and tumor necrosis factor-α (TNF-α), but not interferon-γ (IFN-γ), suppressed the expression of P450 2C11 mRNA. Neither IL-6 nor IFN-γ caused NO release into the medium or induction of inducible nitric oxide synthase (iNOS) mRNA. IL-1β and LPS were the most effective in causing NO release and iNOS induction, and in down-regulating P450 2C11 mRNA expression. Combinations of the cytokines, IFN-γ, and LPS produced an additive release of NO but did not synergize to further suppress P450 2C11 mRNA. To investigate the role of NO in the IL-1β- or LPS-mediated suppression of P450 2C11, N-monomethyl- l-arginine (NMA) was administered at concentrations ranging from 30 to 300 μM. Three hundred micromolar NMA returned NO release back to control levels, but did not affect the IL-1β- or LPS-mediated down-regulation of P450 2C11 mRNA or protein expression. Our results suggest that NO is not required for IL-1β- or LPS-mediated down-regulation of P450 2C11 expression in cultured hepatocytes.

Role of nitric oxide on oxytocin-evoked contractions and prostaglandin synthesis in isolated pregnant rat uterus

Uterine contractions elicited by oxytocin (OT), possibly linked with uterus prostaglandin (PG) release, are involved in the final pathway of labor. It is known that nitric oxide (NO) may contribute to the maintenance of uterine contractile quiescence during gestation. Therefore in this study the effect of the inhibition of NO synthase (NOS), with N-monomethyl L-arginine (L-NMMA), on the ability of OT to stimulate uterine contractions and PG synthesis was investigated in isolated rat uterus at days 13 and 21 of pregnancy. L-NMMA did not modify the frequency and the force of contractions elicited by OT at day 13. On day 21 the frequency of contractions evoked by OT were better sustained in the presence of L-NMMA. PGs were not affected by OT on day 13. OT stimulated PGF 2α on day 21 when NOS had been inhibited with L-NMMA, but not in the absence of L-NMMA. NOS activity was stimulated by OT at day 21 of gestation. In summary these findings indicate that near term NO can regulate OT PGF 2α induced contractions and PG synthesis in isolated pregnant rat uterus.

Arginine metabolism in keratinocytes and macrophages during nitric oxide biosynthesis: Multiple modes of action of nitric oxide synthase inhibitors

Nitric oxide is an important cellular mediator produced in keratinocytes and macrophages from arginine by the enzyme nitric oxide synthase during inflammatory reactions in the skin. We found that γ-interferon stimulated nitric oxide production and the expression of inducible nitric oxide synthase in both cell types. However, macrophages produced more nitric oxide and nitric oxide synthase protein, and at earlier times than keratinocytes. Keratinocytes treated with γ-interferon took up more arginine than macrophages; however, they were less efficient in metabolizing this amino acid and exhibited reduced nitric oxide synthase enzyme activity. In both cell types, the nitric oxide synthase inhibitors, N°-monomethyl- L-arginine (NMMA), L-N 5-(iminoethyl)ornithine, L-canavanine, and Nω-nitro- L-arginine, as well as lysine, ornithine, and homoarginine markedly reduced arginine uptake. In contrast, Nω-nitro- L-arginine methyl ester and Nω-nitro- L-arginine benzyl ester were poor inhibitors of arginine uptake, while aminoguanidine had no effect on uptake of arginine by the cells. Moreover, NMMA was found to inhibit simultaneously arginine uptake and nitric oxide synthase enzyme activity in both cell types, whereas aminoguanidine only affected nitric oxide synthase activity. No major differences were observed between keratinocytes and macrophages. Taken together, these data demonstrate that, although keratinocytes and macrophages both synthesize nitric oxide, its production is regulated distinctly in these two cell types. Furthermore, in these cells, nitric oxide synthase inhibitors such as NMMA exhibit at least two sites of action: inhibition of nitric oxide synthase and cellular uptake of arginine.

Induction of Adhesion/Differentiation of Human Neuroblastoma Cells by Tumour Necrosis Factor‐α Requires the Expression of an Inducible Nitric Oxide Synthase

Neuroblastoma cell lines (SK-N-SH and SK-N-MC) were induced to differentiate, as detected by the expression of neurofilament proteins of 68 and 200 kDa, and to express adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule) after stimulation with tumour necrosis factor-alpha (TNF-alpha). This induction was accompanied by the arrest of cell growth. The induction of neuroblastoma adhesion by TNF-alpha could be inhibited by the nitric oxide synthase inhibitors, L-N-monomethyl arginine (L-NMMA) and L-N6-(1-imidoethyl)-lysine (highly specific for the inducible enzyme), but not by the inactive enantiomer D-NMMA. These results indicate that TNF-alpha induces the adhesion of neuroblastoma cells via nitric oxide. This was confirmed by the finding that the adhesion/differentiation of SK-N-SH and SK-N-MC cells can be directly induced by the addition of nitric oxide donors, sodium nitroprusside and S-nitroso-N-acetyl-penicillamine, into the culture medium. The isoform of the nitric oxide synthase induced in human neuroblastoma cells by TNF-alpha treatment was identified enzymatically as isoform II by Western blotting and by the polymerase chain reaction. Thus TNF-alpha induces the in vitro adhesion/differentiation of human neuroblastoma cells through nitric oxide synthesized by a calcium-independent inducible form of nitric oxide synthase, clearly indicating that isoform II of nitric oxide synthase can be expressed in human neuronal cell types.

CYTOKINE-MEDIATED NEURONAL APOPTOSIS

Cytokines have been reported to induce neuronal injury via the free radical nitric oxide (NO); however, the precise mechanism underlying cytokine-mediated neurotoxicity is unclear. We investigated the hypothesis that cytokine-mediated neurotoxicity in primary cultures of human fetal neurons occurs via an apoptotic mechanism triggered by NO. Treatment of mixed neuronal/glial cell cultures with interferon (IFN)-γ plus interleukin (IL)-1β for 13 days induced a high output of NO accompanied by marked neuronal loss. The NO synthase inhibitor N-monomethyl- l-arginine (NMMA) significantly attenuated cytokine-induced neuronal loss, confirming the involvement of NO. Cytokine-mediated neuronal injury was accompanied by morphologic changes and a DNA fragmentation pattern consistent with apoptosis. Treatment of neuronal cell cultures with NMMA protected against cytokine-mediated apoptotic death. These findings, using primary human neuronal cell cultures, support the hypothesis that cytokine-mediated neurotoxicity involving NO proceeds via an apoptotic mechanism. These findings could lead to the development of new therapies for neurodegenerative diseases involving glia, cytokines, and NO. © 1997 Elsevier Science Ltd. All rights reserved

ROLE OF ENDOGENOUS NITRIC OXIDE SYNTHASE INHIBITION IN PERSISTENT PULMONARY HYPERTENSION OF THE NEWBORN (PPHN). 464

Nitric oxide is believed to be responsible for the normal 8 to 10 fold decrease in the pulmonary vascular resistance that occurs during the transition at birth from fetal to postnatal circulation. NO is produced in the endothelial cells from L-arginine by nitric oxide synthase (NOS). Chronic inhibition of NOS in utero by L-arginine analogs results in PPHN(Fineman, JCI, 1994). The existence of endogenous arginine analogs such as N-monomethyl L-arginine (L-NMMA) has been demonstrated by Vallance et al.(1991) who suggested that its accumulation in chronic renal failure may be responsible for systemic hypertension. To determine the role by endogenous L-NMMA inhibition of NOS in PPHN, we collected aortic blood samples at 0, 15, 60, 240 and 420 minutes after birth from control lambs and lambs in whom PPHN was induced by prenatal ductal ligation. The assay for L-NMMA was carried out by high performance liquid chromatography (HPLC) after derivatization with dabsyl chloride. ˙V NO was measured from expired gas by chemiluminescence.Figure

The role of nitric oxide in proteoglycan turnover by bovine articular cartilage organ cultures.

Monolayer cultures of articular chondrocytes synthesize large amounts of nitric oxide (NO) following exposure to IL-1. The latter has antianabolic and procatabolic activities on these cells, but little is known about the role, if any, of NO in the integrated metabolic pathways of the chondrocyte. In the present study, the role of endogenously produced NO in both the synthesis and degradation of proteoglycans was investigated for the first time. Bovine articular cartilage slices exposed to 20 U/ml human rIL-1 beta (hrIL-1 beta) synthesized large amounts of NO for 1 to 2 days, after which production fell to a steady state level approximately 20% of the peak value for the remainder of the 14-day incubation. The NO synthase inhibitor, N-monomethyl L-arginine (L-NMA, 1 mM), blocked NO production and enhanced the acute catabolic effects of hrIL-1 beta in cartilage derived from both cartilage derived from both calves and adult animals. However, in late cultures, release of proteoglycans was reduced in the presence of L-NMA. The proteolytic activity in conditioned medium of these cultures (measured as caseinolytic activity) was enhanced by L-NMA; however, this inhibitor did not affect the rates of synthesis of proteoglycans. Although NO is widely assumed to be a mediator of cartilage catabolism, our data suggest that it may instead have an acute protective effect. Whether this effect is maintained chronically is less clear.

Increase in brain nitric oxide synthase activity in daunorubicin-treated rats.

Anthracyclines such as daunorubicin are very effective anticancer agents. These drugs are known to cause side effects including cardiotoxicity. Anthracyclines are neurotoxic to laboratory animals. Nitric oxide is a novel and very important chemical messenger in the brain. However, at higher levels, nitric oxide causes well defined neurotoxicity. Therefore, we determined nitric oxide synthase activity in rat brain after daunorubicin treatment in an effort to explain the neurotoxicity produced by anthracyclines. Male Sprague-Dawley rats were treated with different subcutaneous doses of daunorubicin (0.1-4.0 mg/kg/week for five weeks) while control animals were injected with phosphate buffered saline. There was a significant increase (80%) of nitric oxide synthase activity in daunorubicin-treated animals as compared to controls. This activity was inhibited by N-monomethyl-L-arginine (NMMA), nitroarginine, N-6-aminohexyl-5-chloro-1-napthalene sulfonamide (W-7), a calmodulin antagonist, suggesting that the nitric oxide synthase activity is calmodulin dependent. Further, our in vitro studies demonstrated that daunorubicin interacted with calmodulin as measured by N-phenyl-1-napthylamine (NPN) fluorescence. These results indicate that daunorubicin increases nitric oxide synthase activity in rat brain which may increase the levels of nitric oxide. The increased levels of nitric oxide may cause neurotoxicity. Our results further indicate that daunorubicin interacts with calmodulin and enhances nitric oxide synthase activity which is dependent on calmodulin.

Induction of nitric oxide and nitric oxide synthase mRNA by silica and lipopolysaccharide in PMA-primed THP-1 cells.

Nitric oxide (NO), a nitrogen-free radical, plays an important role in mediating inflammatory reaction and cytotoxicity of tissue. To determine whether NO was involved in silica-induced pulmonary tissue damage, we studied the effects of silica on nitric oxide (NO) production and inducible NO synthase (iNOS) mRNA expression by THP-1 cells, a monocyte-like cell line with properties of the pulmonary alveolar macrophage. Experimental results showed that silica elicited a marked stimulation of nitric oxide production in a time-dependent manner by THP-1 cells in vitro following the priming of these cells with the phorbol ester PMA. Both nitric oxide synthase inhibitor N-monomethyl-L-arginine (NMMA) and xanthine oxidase inhibitor allopurinol can partially suppress silica-induced NO production in PMA-primed THP-1 cells. Northern blot analysis indicated that, after 2 h of silica exposure, PMA-primed THP-1 cells began to express iNOS mRNA, which reached peak expression at 8 h. Endotoxin treatment of these cells produced a similar effect. These results indicated that silica is a potent inducer of NO production in macrophages and its ability to induce tissue damage may partially be attributed to its ability to initiate excessive production of nitric oxide from macrophages.

In vivo regulation of replicative Legionella pneumophila lung infection by endogenous tumor necrosis factor alpha and nitric oxide

The in vivo role of endogenous tumor necrosis factor alpha (TNF-alpha) and reactive nitrogen intermediates (RNIs) in modulation of growth of Legionella pneumophila in the lung was assessed using a murine model of replicative L. pneumophila lung infection. Intratracheal inoculation of mice with L. pneumophila resulted in induction of endogenous TNF-alpha, which preceded clearance of L. pneumophila from the lung. Inhibition of endogenous TNF-alpha activity, via in vivo administration of TNF-alpha neutralizing antibody, or inhibition of endogenous RNIs, via administration of the nitric oxide (NO) synthetase inhibitor N-monomethyl-L-arginine (NMMA), resulted in enhanced growth of L. pneumophila in the lung at > or = 3 days postinfection (when compared with untreated L. pneumophila-infected mice). Because of the similar kinetics of enhanced pulmonary growth of L. pneumophila in mice treated in vivo with either anti-TNF-alpha antibody or NMMA, the immunomodulatory effect of NO on endogenous TNF-alpha activity in the lung was assessed. Administration of NMMA to L. pneumophila-infected mice resulted in a significant decrease in endogenous TNF-alpha activity in the lung during replicative L. pneumophila infections in vivo. However, administration of exogenous TNF-alpha to NMMA-treated mice failed to significantly enhance clearance of L. pneumophila from the lung. Results of these studies indicate that both endogenous NO and TNF-alpha facilitate resolution of replicative L. pneumophila lung infections and that regulation of L. pneumophila replication by TNF-alpha is mediated, at least in part, by NO.