Catecholamines' enhancement of inducible nitric oxide synthase-induced nitric oxide biosynthesis involves CAT-1 and CAT-2A

Catecholamines enhance inducible nitric oxide synthase (iNOS) expression that results in nitric oxide (NO) overproduction in lipopolysaccharide (LPS)-stimulated macrophages. L-arginine transport mediated by cationic amino acid transporters (including CAT-1, CAT-2, CAT-2A, and CAT-2B) is crucial in regulating iNOS activity. We sought to assess the effects of catecholamines on L-arginine transport and CAT isozyme expression in stimulated macrophages. Confluent RAW264.7 cells were cultured with LPS with or without catecholamines (epinephrine or norepinephrine, 5 x 10(-6) M) for 18 h. NO production, L-arginine transport, and enzyme expression were determined. Our data revealed that LPS co-induced iNOS, CAT-2, and CAT-2B expression, whereas CAT-1 and CAT-2A expression remained unaffected. Significant increases in NO production and L-arginine transport (approximately eight-fold and three-fold increases, respectively) were found in activated macrophages. Catecholamines significantly enhanced NO production and L-arginine transport (approximately 30% and 20% increases, respectively) in activated macrophages. Catecholamines also enhanced the expression of iNOS, CAT-1, and CAT-2A but not CAT-2 or CAT-2B in LPS-stimulated macrophages. Furthermore, the enhancement effects of catecholamines were inhibited by either dexamethasone or propranolol. We provide the first evidence to indicate that L-arginine transport in activated macrophages could be enhanced by catecholamines. Furthermore, this catecholamine-enhanced L-arginine transport might involve CAT-1 and CAT-2A but not CAT-2 or CAT-2B.     

NF-kappaB involvement in the induction of high affinity CAT-2 in lipopolysaccharide-stimulated rat lungs

BACKGROUND: Endotoxemia stimulates nitric oxide (NO) biosynthesis through induction of inducible NO synthase (iNOS). Cellular uptake of L-arginine, the sole substrate for iNOS, is an important mechanism regulating NO biosynthesis by iNOS. The isozymes of type-2 cationic amino acid transporters, including CAT-2, CAT-2A, and CAT-2B, constitute the most important pathways responsible for trans-membrane L-arginine transportation. Therefore, regulation of CAT-2 isozymes expression may constitute one of the downstream regulatory pathways that control iNOS activity. We investigated the time course of enzyme induction and the role of nuclear factor-kappaB (NF-kappaB) in CAT-2 isozymes expression in lipopolysaccharide-(LPS) treated rat lungs. METHODS: Adult male Sprague-Dawley rats were randomly given intravenous injections of normal saline (N/S), LPS, LPS plus NF-kappaB inhibitor pre-treatment (PDTC, dexamethasone, or salicylate), or an NF-kappaB inhibitor alone. The rats were sacrificed at different times after injection and enzyme expression and lung injury were examined. Pulmonary and systemic NO production were also measured. RESULTS: LPS co-induced iNOS, CAT-2, and CAT-2B but not CAT-2A expression in the lungs. Furthermore, NF-kappaB actively participated in LPS-induction of iNOS, CAT-2, and CAT-2B. LPS induced pulmonary and systemic NO overproduction and resulted in lung injuries. Attenuation of LPS-induced iNOS, CAT-2, and CAT-2B induction significantly inhibited NO biosynthesis and lessened lung injury. CONCLUSION: NF-kappaB actively participates in the induction of CAT-2 and CAT-2B in intact animals. Our data further support the idea that CAT-2 and CAT-2B are crucial in regulating iNOS activity.     

NF-kappaB inhibitors stabilize the mRNA of high-affinity type-2 cationic amino acid transporter in LPS-stimulated rat liver

BACKGROUND: Induction of inducible nitric oxide synthase (iNOS) results in nitric oxide (NO) overproduction during endotoxemia. Cellular uptake of L-arginine, modulated by the isozymes of type-2 cationic amino acid transporters (CAT), including CAT-2, CAT-2A and CAT-2B, has been reported to be a crucial factor in the regulation of iNOS activity. We sought to elucidate the expression of CAT-2 isozymes and the role of nuclear factor-kappaB (NF-kappaB) in this expression in lipopolysaccharide (LPS)-treated rat liver. METHODS: Adult male Sprague-Dawley rats were randomly given intravenous (i.v.) injections of normal saline (N/S), LPS, LPS preceded by an NF-kappaB inhibitor (PDTC, dexamethasone or salicylate) or an NF-kappaB inhibitor alone. After injection, rats were sacrificed at different times and enzyme expression and liver injury were examined. Hepatic and systemic NO production were also measured. RESULTS: CAT-2, CAT-2A and CAT-2B were constitutively expressed in un-stimulated rat liver. LPS stimulation not only significantly increased iNOS mRNA and NO concentrations but also decreased the mRNA concentrations of CAT-2 and CAT-2B, but not CAT-2A, in a time-dependent manner. LPS-induced hepatic and systemic NO overproduction was associated with hepatocellular injury. Pre-treatment with NF-kappaB inhibitors significantly attenuated LPS-induced iNOS induction as well as CAT-2/CAT-2B mRNA destabilization, which was associated with significant inhibition of NO biosynthesis and less liver injury. CONCLUSION: NF-kappaB inhibitors stabilize CAT-2 and CAT-2B mRNA in LPS-stimulated rat liver. The hepatic CAT-2/CAT-2B pathway may be a constitutive part of cytoprotective mechanisms against sepsis.     

L-Arginine transport is augmented through up-regulation of tubular CAT-2 mRNA in ischemic acute renal failure in rats

BACKGROUND: Ischemic acute renal failure (iARF) is associated with increased nitric oxide (NO) production during the reperfusion period, as endothelial nitric oxide synthase (eNOS) is maximally activated, and renal tubular inducible NOS (iNOS) is stimulated. Increased NO production leads to augmented tubular injury, probably through the formation of peroxynitrite. l-Arginine (l-Arg), the only precursor for NO, is transported into cells by cationic amino acid transporters, CAT-1 and CAT-2. We hypothesized that the increased NO production observed in iARF may result from increased l-Arg uptake, which would be reflected in the augmented expression of l-Arg transporter(s). METHODS: Ischemic acute renal failure was induced in rats by right nephrectomy + left renal artery clamping for 60 minutes. l-Arg uptake was examined in freshly harvested glomeruli and tubuli from control, sham operated, and animals subjected to 15, 30, and 60 minutes, and 24 hours of reperfusion, following 60 minutes of ischemia. Using RT-PCR, renal tissues were examined further for the expression of iNOS, CAT-1, CAT-2, arginase I and arginase II. RESULTS: Tubular expression of iNOS mRNA was initiated by ischemia, continued to increase after 60 minutes of reperfusion, and decreased after 24 hours. l-Arg transport into glomeruli was similar in all experimental groups. l-Arg uptake into tubuli was markedly augmented following the 60-minute reperfusion, while it moderately increased after 24 hours of reperfusion. This was accompanied by a parallel, preferential increase in tubular CAT-2 mRNA expression at 60 minutes of reperfusion. CAT-1 mRNA expression was unchanged, as detected by RT-PCR. In addition, the expression of arginase II and arginase I mRNA was attenuated by 30 minutes and one hour of reperfusion, and returned to baseline values after 24 hours of reperfusion. CONCLUSIONS: Ischemic ARF is associated with augmented tubular CAT-2 mRNA expression, which leads to enhanced l-Arg transport and increased NO production. This may contribute to the renal injury exhibited in iARF.     

Heme oxygenase 1, nuclear factor E2-related factor 2, and nuclear factor kappaB are involved in hemin inhibition of type 2 cationic amino acid transporter expression and L-Arginine transport in stimulated macrophages

BACKGROUND: L-Arginine transport mediated by type 2 cationic amino acid transporter (CAT-2) is one crucial mechanism that regulates nitric oxide production mediated by inducible nitric oxide synthase. Heme oxygenase (HO)-1 induction has been reported to significantly attenuate inducible nitric oxide synthase expression and nitric oxide production. The authors sought to explore the effects of HO-1 induction on CAT-2 expression and L-arginine transport. The effects of HO-1 induction on nuclear factor E2-related factor 2 (Nrf2) and nuclear factor kappaB (NF-kappaB) were also investigated. METHODS: Murine macrophages (RAW264.7 cells) were randomized to receive lipopolysaccharide, lipopolysaccharide plus hemin (an HO-1 inducer; 5, 50, or 500 microm), lipopolysaccharide plus hemin (5, 50, or 500 microm) plus tin protoporphyrin (an HO-1 inhibitor), or lipopolysaccharide plus hemin (5, 50, or 500 microm) plus hemoglobin (a carbon monoxide scavenger). Then, cell cultures were harvested and analyzed. RESULTS: Lipopolysaccharide significantly induced Nrf2 activation and HO-1 expression. Lipopolysaccharide also significantly induced NF-kappaB activation, CAT-2 expression, and L-arginine transport. In a dose-dependent manner, hemin enhanced the lipopolysaccharide-induced Nrf2 activation and HO-1 expression. In contrast, hemin, also in a dose-dependent manner, significantly attenuated the lipopolysaccharide-induced NF-kappaB activation, CAT-2 expression, and L-arginine transport. Furthermore, the effects of hemin were significantly reversed by both tin protoporphyrin and hemoglobin. CONCLUSIONS: HO-1 induction significantly inhibited CAT-2 expression and L-arginine transport in lipopolysaccharide-stimulated macrophages, possibly through mechanisms involved activation of Nrf2 and inhibition of NF-kappaB. In addition, carbon monoxide mediated, at least in part, the effects of HO-1 induction on CAT-2 expression and L-arginine transport.     

Modulation of macrophage nitric oxide production by prostaglandin D2

BACKGROUND: Nitric oxide and prostaglandins readily become activated in response to inflammatory events. The overproduction of nitric oxide is detrimental to the host. The present study was conducted to examine whether prostaglandin D(2) (PGD(2)) modulates nitric oxide production in macrophages in response to an inflammatory stimulus. METHODS: Cultures of RAW 264.7 murine macrophages were exposed to Escherichia coli lipopolysaccharide (LPS, 0.01 and 1.0 microg/ml) before and after exposure to PGD(2) (0.01 to 10 nmol). After 24-h incubation, supernatants were collected and nitrite was quantitated by Greiss reaction as a measure of nitric oxide synthesis. Inducible nitric oxide synthase (iNOS) protein was measured by Western blot analysis. RESULTS: Macrophages exposed to 0.01 and 1.0 microg/ml LPS produced 8.3 +/- 0.2 and 15.0 +/- 1.4 nmol/1.1 x 10(6) cells/24 h of nitrite, respectively. The simultaneous addition of PGD(2) with LPS inhibited nitrite production in a dose-dependent fashion and suppressed iNOS protein expression. A strong time effect was also exhibited when macrophages were incubated with PGD(2) 1 hour before as compared to 7 hours after the addition of LPS (0.01 or 1.0 microg/ml), indicating that the earlier the time PGD(2) was added to the culture media, the greater the inhibition. Prostaglandin D(2) had the capacity to block nitrite synthesis even when added as much as 7 hours after an LPS challenge. Blocking endogenous prostaglandins, using indomethacin (10 microM), suppressed nitrite production. CONCLUSION: Exogenous PGD(2) caused dose- and time-dependent decreases in LPS-stimulated nitrite production by RAW 264.7 macrophages by hindering iNOS protein expression. Conversely, the endogenous prostaglandins released by these same cells in response to an LPS challenge stimulated nitrite production, which may consequently dampen the inhibitory actions of exogenous PGD(2).     

NF-κB信号通路在异丙酚抑制脂多糖诱导RAW264.7细胞诱导型一氧化氮合酶基因表达上调中的作用

目的 探讨NF-κB信号通路在异丙酚抑制脂多糖(LPS)诱导RAW264.7细胞诱导型一氧化氮合酶( iNOS)基因表达上调中的作用.方法 体外培养RAW264.7细胞,以5×105/ml密度接种于6 cm培养皿(3 ml/皿)或6孔板(2 ml/孔),采用随机数字表法,将其随机分为3组(n=18):正常对照组(C组)、LPS组(L组)和LPS+异丙酚(LP组).C组不做任何处理;L组和LP组均加入1μg/mlLPS,LP组于加入LPS前2h加入50 μmol/L异丙酚.于LPS孵育30 min时,每组取6皿和6孔,收集细胞,分别采用免疫印迹法测定磷酸化IκB激酶(p-IKK)和NF-κB活性;于LPS孵育6h时,每组取6皿,收集细胞,测定iNOS mRNA表达.结果 与C组比较,L组p-IKK和iNOS mRNA表达上调,NF-κB活性升高(P＜0.05);与L组比较,LP组p-IKK和iNOS mRNA表达下调,NF-κB活性降低(P＜0.05).结论 NF-κB信号通路参与了异丙酚抑制脂多糖诱导的RAW264.7细胞iNOS基因表达上调.     

Nitric oxide boosts TLR‐4 mediated lipocalin 2 expression in chondrocytes

Lipocalin 2 (LCN2) has recently emerged as a novel adipokine involved in different processes including arthritis and chondrocyte inflammatory response. However, little is known about its activity on chondrocyte homeostasis and its regulation by nitric oxide (NO) Hence, we performed a set of experiments aimed to achieve a better understanding of this relationship. Cell vitality was tested in the ATDC5 cell line by the MTT colorimetric assay. Protein expression and gene expression was evaluated by Western blot and real time RT‐PCR, respectively. NO production (determined as nitrite accumulation) was assayed by the Griess reaction. First, we demonstrated that LCN2 decreased murine chondrocytes vitality. Next, LCN2 co‐stimulation with LPS enhanced NOS2 protein expression by murine chondrocytes. In addition, inhibition of LPS‐induced nitric oxide production by aminoguanidine, a selective NOS2 inhibitor, significantly reduced LPS‐mediated LCN2 expression. In contrast, treatment of murine chondrocytes with sodium nitroprussiate (SNP), a classic NO donor, scarcely induced LCN2 expression. Intriguingly, SNP addition to LPS‐challenged chondrocytes, treated with aminoguanidine, provoked a strong induction of LCN2 expression. Finally, murine ATDC5 cells, co‐cultured with LPS pre‐challenged macrophages, had higher LCN2 expression in comparison with murine chondrocytes co‐cultured with non pre‐challenged macrophages. In this work we have described for the first time that NO is able to exert a control on LCN2 expression, suggesting the existence of a feedback loop regulating its expression. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1046–1052, 2013     

氯化镧对内毒素/脂多糖诱导巨噬细胞株一氧化氮合酶表达的抑制作用

目的了解氯化镧（LaCl3）对内毒素／脂多糖（LPS）刺激的巨噬细胞诱导型一氧化氮合酶（iNOS）表达的影响，并探讨其机制。方法将小鼠巨噬细胞株RAW264．7分为空白对照组、LaCl、组、LPS组和LaCl3＋LPS组。前3组细胞分别用常规培养液、含2．50μmol／L LaCl3的培养液、含1mg／L LPS的培养液培养24h，LaCl3＋LPS组用含2．5μmol／LLaCl，的培养液培养24h后，换为含1mg／L LPS的培养液培养24h。采用免疫细胞化学染色法检测iNOS在各组细胞中的表达强度；蛋白质印迹法检测iNOS的蛋白表达水平；反转录一PCR测定iNOS的mRNA表达水平；硝酸还原酶法测定各组细胞培养上清液中一氧化氮（NO）含量。结果免疫细胞化学染色结果显示，iNOS主要分布于各组细胞的胞质中，空白对照组和LaCl3组荧光强度极弱；LPS组荧光强度最强，阳性细胞百分率为44．4％，明显高于LaCl3＋LPS组（11．8％，P〈0．05）。LPS组iNOS蛋白及其mRNA表达量和细胞培养上清液中NO含量均高于其余各组（P〈0．05）。结论LaCl3可在mRNA水平和蛋白水平抑制LPS诱导的iNOS过度表达，减少NO生成，提示LaCl3能拮抗LPS诱导的iNOS过度活化。     

Vaccinia virus-induced inhibition of nitric oxide production

BACKGROUND: The role of nitric oxide (NO) in the host defense against viruses has not been well defined. Several studies have implicated NO as responsible for the destruction of a variety of viruses. However, others have reported that certain viruses can impair the ability of macrophages to produce NO. This study was initiated to determine the ability of macrophages to produce NO in response to vaccinia virus infection. METHODS: RAW 264.7 murine macrophages in minimum essential medium were exposed to virus-containing supernatants for 1 h before stimulation with Escherichia coli lipopolysaccharide (LPS, 0.001 and 1.0 microg/ml). After further 24-h incubations, nitrite concentration, cell viability, and inducible nitric oxide synthase (iNOS) were quantitated. RESULTS: The viral preparation alone did not stimulate nitric oxide synthesis (measured as nitrite) by macrophages. However, macrophages exposed to 0.001 and 1.0 microg/ml LPS produced 7.7 +/- 0.6 and 16.6 +/- 0.8 nmole/1.1 x 10(6) cells/24-h nitrite, respectively. Production of nitrite caused cell death. Macrophages incubated with vaccinia virus prior to exposure to LPS resulted in a dose-dependent decrease in nitrite production. An 80% inhibition of nitrite was noted when macrophages were exposed to vaccinia virus (m.o.i. 10(-4)) plus LPS (1.0 microg/ml) (P < 0.05). Further study showed that this inhibition was not associated with changes in cell viability or substrate availability, but was associated with a marked reduction in iNOS protein. When the virus was inactivated with UV-irradiation, the same incubation caused a 46% inhibition of nitrite production (P < 0.05 vs active virus). However, this effect occurred without altering the quantity of iNOS protein. CONCLUSION: These results indicate that active vaccinia virus inhibits the ability of stimulated macrophages to produce NO by hindering iNOS protein expression. Because live viral particles were not entirely required for this inhibition, it is possible that by products of viral infection, such as soluble viral proteins, may also be responsible for this effect.     

Acupuncture stimulation of ST-36 (Zusanli) significantly mitigates acute lung injury in lipopolysaccharide-stimulated rats

BACKGROUND: We sought to investigate the potential therapeutic effects of acupuncture stimulation of ST-36 (Zusanli) on endotoxemia-induced acute lung injury in lipopolysaccharide (LPS)-stimulated rats. METHODS: Sixty rats were randomized into six groups (n = 10): (i) lipopolysaccharide (LPS) control group, (ii) normal saline (N/S) control group, (iii) LPS plus ST-36 group, (iv) N/S plus ST-36 group, (v) LPS plus sham point (Sham) group, and (vi) N/S plus Sham group. Manual acupuncture stimulation of ST-36 (designated as 'ST-36') or a 'non-acupoint' (designated as 'Sham') was performed in lightly immobilized rats for 30 min. Then, LPS injection was employed to induce sepsis. Rats were killed at 6 h after LPS injection and lung injury, nitric oxide (NO) biosynthesis and inducible NO synthase (iNOS) expression were assayed. RESULTS: Significant lung injury, pulmonary iNOS expression and systemic and pulmonary NO biosynthesis were noted in the LPS groups. Rats in the LPS plus Sham group had lung injury, pulmonary iNOS expression, systemic and pulmonary NO biosynthesis similar to those observed in the LPS group. However, the degree of lung injury, pulmonary iNOS expression and pulmonary NO biosynthesis, but not systemic NO biosynthesis, were significantly attenuated in the LPS plus ST-36 group as compared with those in both the LPS group and the LPS plus Sham group. CONCLUSION: Acupuncture stimulation of ST-36 may be effective as a prophylaxis measure against sepsis. However, results from this study do not support the use of acupuncture for the treatment of sepsis.     

Heme Oxygenase 1, Nuclear Factor E2-related Factor 2, and Nuclear Factor κB Are Involved in Hemin Inhibition of Type 2 Cationic Amino Acid Transporter Expression and l-Arginine Transport in Stimulated Macrophages

Background: l-Arginine transport mediated by type 2 cationic amino acid transporter (CAT-2) is one crucial mechanism that regulates nitric oxide production mediated by inducible nitric oxide synthase. Heme oxygenase (HO)-1 induction has been reported to significantly attenuate inducible nitric oxide synthase expression and nitric oxide production. The authors sought to explore the effects of HO-1 induction on CAT-2 expression and l-arginine transport. The effects of HO-1 induction on nuclear factor E2-related factor 2 (Nrf2) and nuclear factor [kappa]B (NF-[kappa]B) were also investigated.

Methods: Murine macrophages (RAW264.7 cells) were randomized to receive lipopolysaccharide, lipopolysaccharide plus hemin (an HO-1 inducer; 5, 50, or 500 [mu]m), lipopolysaccharide plus hemin (5, 50, or 500 [mu]m) plus tin protoporphyrin (an HO-1 inhibitor), or lipopolysaccharide plus hemin (5, 50, or 500 [mu]m) plus hemoglobin (a carbon monoxide scavenger). Then, cell cultures were harvested and analyzed.

Results: Lipopolysaccharide significantly induced Nrf2 activation and HO-1 expression. Lipopolysaccharide also significantly induced NF-[kappa]B activation, CAT-2 expression, and l-arginine transport. In a dose-dependent manner, hemin enhanced the lipopolysaccharide-induced Nrf2 activation and HO-1 expression. In contrast, hemin, also in a dose-dependent manner, significantly attenuated the lipopolysaccharide-induced NF-[kappa]B activation, CAT-2 expression, and l-arginine transport. Furthermore, the effects of hemin were significantly reversed by both tin protoporphyrin and hemoglobin.     

Nitric oxide promotes infectious bone resorption by enhancing cytokine-stimulated interstitial collagenase synthesis in osteoblasts.

This experiment was undertaken to determine the role of macrophage-derived nitric oxide (NO) in mediating lipopolysaccharide (LPS)-induced bone resorption by using an in vitro co-culture system and an in vivo model of infectious bone resorption. Our results demonstrated that LPS stimulated the expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-a mRNAs and nitrite synthesis in the J774 mouse macrophage cell line but not in the UMR-106 (rat) and MC3T3-E1 (mouse) osteoblast cell lines. Conditioned media (CM) from LPS-stimulated J774 triggered only low to moderate levels of iNOS mRNAs in MC3T3-E1 and a trivial effect in UMR-106. On the other hand, CM induced matrix metalloproteinase-1 (MMP-1) gene expression in both osteoblast cell lines. The NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) did not alter this effect in MC3T3-E1 and UMR-106, whereas TNF-a antibody diminished the CM-induced MMP-1 gene expression in both cell lines. Interestingly, SNAP, a NO donor, although by itself is not a MMP-1 stimulator for UMR-106, augmented the TNF-alpha-stimulated MMP-1 mRNA production in UMR-106. In a J774/UMR-106 co-culture system, LPS stimulated significant MMP-1 gene expression in UMR-106, and this upregulation was abolished by L-NMMA and TNF-alpha antibodies. Immunohistochemical analysis in a rat model of infectious bone resorption (periapical lesion) showed co-distributions of iNOS+ macrophages and MMP-1+ osteoblasts around the osteolytic areas. Administration of L-NMMA markedly reduced the extent of bone loss and the percentage of MMP-1-synthesizing osteoblasts. These data suggest that NO derived from macrophages after LPS stimulation may enhance bone loss by augmenting the cytokine-induced MMP-1 production in osteoblasts.