The role of endoplasmic reticular Ca2+ stores in cell viability and tumor necrosis factor-α production of the murine macrophage RAW 264.7 cell line

Thapsigargin (TG), an endoplasmic reticular (ER) Ca(2+)-ATPase inhibitor, can increase the intracellular calcium concentration and then deplete the TG-sensitive intracellular Ca(2+) pool. In this study, we investigated the effects of TG on cell viability and tumor necrosis factor-alpha (TNF-alpha) production in the murine macrophage RAW 264.7 cell line. We found that treatment with TG (10-800 nM) induced apoptosis in RAW 264.7 cells in a dose-dependent manner (IC(50), 200 nM). Lipopolysaccharide (LPS, 1 microg/ml) markedly potentiated low concentrations of TG (10-75 nM) in inducing apoptosis (IC(50), 20 nM) as revealed by the DNA ladder. Polymycin B (an LPS receptor antagonist) inhibited the cytotoxic effect induced by LPS plus TG. Although TG, A23187 and ionomycin all definitely increased intracellular Ca(2+) concentrations, neither A23187 nor ionomycin mimicked TG in inducing apoptotic events in LPS-activated RAW 264.7 cells. Moreover, the production of TNF-alpha induced by LPS was profoundly potentiated by TG but not by A23187 or by ionomycin. We conclude from these combined results that TG-sensitive ER Ca(2+) stores play a pivotal role in modulating cell viability and TNF-alpha production. The mutual potentiation between the LPS receptor signaling pathway and the depletion of ER Ca(2+) stores implies the existence of cross-talk between these multiregulatory mechanisms in this murine macrophage RAW 264.7 cell line.     

Inhibitory effects of phloroglucinol derivatives from Mallotus japonicus on nitric oxide production by a murine macrophage-like cell line,RAW 264.7, activated by lipopolysaccharide and interferon-γ

An aqueous acetone extract of the pericarps of Mallotus japonicus (MJE) inhibited nitric oxide (NO) production by a murine macrophage-like cell line, RAW 264.7, which was activated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Seven phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against NO production. Among these phloroglucinol derivatives, isomallotochromanol exhibited strong inhibitory activity toward NO production, exhibiting an IC₅₀ of 10.7 μM. MJE and the phloroglucinol derivatives significantly reduced both the induction of inducible nitric oxide synthase (iNOS) protein and iNOS mRNA expression. NO production by macrophages preactivated with LPS and IFN-γ for 16 h was also inhibited by MJE and the phloroglucinol derivatives. Furthermore, MJE and the derivatives directly affected the conversion of L-[¹⁴C]arginine to L-[¹⁴C]citrulline by the cell extract. These results suggest that MJE and the phloroglucinol derivatives have the pharmacological ability to suppress NO production by activated macrophages. They inhibited NO production by two mechanisms: reduction of iNOS protein induction and inhibition of enzyme activity.     

Effect of diabetes on aortic nitric oxide synthesis in spontaneously hypertensive rats; does captopril modulate this effect?

Nitric oxide (NO) is a potent regulator in the cardiovascular system; it is generated by the nitric oxide synthase (NOS) family of proteins. NO produced in endothelial cells plays a crucial role in vascular functions. The aim of this study was to clarify the effect of diabetes on aortic NO synthesis in a model of genetic hypertension and determine whether captopril modulates this effect. Diabetes was induced in ten weeks old spontaneously hypertensive rats (SHR) by streptozotocin injection. The rats were allocated into 3 groups: control group 1, non-diabetic SHR; group 2, diabetic SHR; group 3, diabetic SHR group receiving captopril at 80 mg/kg in drinking water for 4 weeks. Mean blood pressure (MBP) was measured once a week by tail-cuff method. Aortic NO metabolities (nitrite/nitrate) and endothelial NOS (NOS-3) were assayed by Griess reaction and by immunoblotting and immunohistochemistry, respectively. There was a significant decrease in nitrite/nitrate (NOx) in aortas of diabetic SHR compared with controls. The decrease of aortic NOx in diabetic SHR was accompanied by a decrease in NOS-3 expression. Captopril treatment reduced MBP without affecting either NOx level or NOS-3 expression in aortas of diabetic SHR. We conclude that STZ-induced diabetes decreased NO in aortas of SHR that may reflect endothelial cell dysfunction; captopril administration decreased MBP without affecting NO level in aortas of diabetic SHR which suggest that the blood pressure-lowering effects of captopril were independent of NO.     

Effect of 17β-estradiol on the human osteosarcoma cell line MG-63

We present evidence that 17β-estradiol (17β-E₂) regulates 1,25(OH)₂D₃-induced alkaline phosphatase synthesis and osteocalcin secretion by the human osteosarcoma cell line MG-63. When cells were pre-treated with 17β-E₂ for 48 h prior to treatment with 1,25(OH)₂D₃ (50 nM) for another 48 h, alkaline phosphatase activity increased by 40% (P < 0.025) with 2 nM 17β-E₂ and plateaued at levels of 20 and 200 nM 17β-E₂. Under the same experimental conditions, osteocalcin secretion was enhanced by 37% (P < 0.005) with 2 nM E₂. However, 17β-E₂ had no effect on basal alkaline phosphatase or on osteocalcin secretion. Moreover, simultaneous addition of 17β-E₂ and 1,25(OH)₂D₃ to cells did not result in any additional effect over l,25(OH)₂D₃ treatment alone. Tamoxifen (10 nM) inhibited 17β-E₂-induced activities in l,25(OH)₂D₃-treated cells while not affecting control cells. Dexamethasone pretreat-ment (100 nM, 48 h) also stimulated alkaline phosphatase activity in MG-63 cells. Moreover, dexamethasone pretreatment followed by treatment with 17β-E₂ and l,25(OH)₂D₃ gave an additive effect for alkaline phosphatase activity. 17α-Estradiol (17α-E₂), a less active form of estrogen, failed to modify, at low concentrations, control or l,25(OH)₂D₃-induced alkaline phosphatase synthesis and osteocalcin secretion. In fact, a 100–1000-fold higher concentration of 17α-E₂ was necessary to reproduce the effects of 17β-E₂ on osteocalcin secretion. The addition of insulin-like growth factor I (IGF-I) for 24 h (1–50 ng/ml) to MG-63 cells did not modify 1,25(OH)₂D₃-induced osteocalcin release from these cells. However, longer incubations with 50 ng/ml IGF-I did reproduce some of the effects observed with 17β-E₂. Thus, the effects of 17β-E₂ are probably not related to IGF-I production in MG-63 cells since under these conditions the addition of IGF-I alone should have produced a response at shorter incubation times and in the presence of lower concentrations of IGF-I. Since 17β-E₂ pretreatment was necessary to observe any effects on l,25(OH)₂D₃-induced activities, we hypothesized that 17β-E₂ regulated 1,25(OH)₂D₃ receptors in MG-63 cells. When cells were treated with 100 nM 17β-E₂ for 48 h, the binding affinity was unchanged: 37.3 ± 1.9 versus 35.1 ± 0.4 pM for cells whether treated or not with l7β-E₂, respectively. In contrast, a significant increase in binding capacity (B_max) was noted (15 ± 3.5%; P < 0.025). These results suggest that the estrogen analogue 17β-E₂ induces the differentiation of MG-63 cells into a more osteoblastic-like phenotype while 17α-E₂ is without physiological effect. They also suggest that estrogens may regulate bone remodeling by modulating hormonal-induction of proteins involved in bone mineralization. This effect is indirect since it does not modify basal activities, but involves a regulation of 1,25(OH)₂D₃ receptor levels in these MG-63 cells.     

Effect of corticosteroids on nitric oxide production in inflammatory bowel disease: are leukocytes the site of action?

Nitric oxide (NO) production is increased in the human colonic mucosa in intestinal inflammation. We examined the effect of corticosteroids and the role of mononuclear cells in this production. Colonic biopsies from patients with ulcerative colitis and normal controls were cultured with either budesonide or prednisolone in the presence of proinflammatory cytokines. Human mixed mononuclear cells (MMCs) were cocultured with HT-29 cells stimulated with IFN-gamma and LPS in the presence or absence of corticosteroids. Nitrite production was measured in supernatants by a modification of the Griess reaction, and inducible NO synthase (iNOS) mRNA expression was studied in colonic tissue by RT-PCR. Both steroids significantly suppressed the nitrite production and iNOS mRNA expression in inflamed colonic biopsies from ulcerative colitis patients and in cytokine-stimulated normal colonic biopsies but not in cytokine-stimulated HT-29 cells. Nitrite production by HT-29 cells was significantly increased (P < 0.01) in cocultures with MMCs stimulated with IFN-gamma and LPS. The presence of either prednisolone or budesonide significantly (P < 0.01) suppressed nitrite production from cocultures of HT-29 cells and MMCs but not from cultures of HT-29 cells stimulated with conditioned media from activated MMCs. Interestingly, stimulation of HT-29 with conditioned media from MMCs pretreated with steroids before stimulation with LPS and IFN-gamma induced a significantly (P < 0.01) lower nitrite production. These results suggest that the inhibitory effect of corticosteroids on the NO production in the intestinal inflammation might be via the inhibition of MMC-produced mediators responsible for NO production by colonic epithelial cells.     

Effect of 5′-methylthioadenosine on induction of murine erythroleukemia cell differentiation

The polyamines putrescine, spermidine and spermine have been implicated in the regulation of proliferation and differentiation. The present study has monitored the effects of 5′-methylthioadenosine, the metabolic product of spermidine and spermine synthesis, on the appearance of a differentiated murine erythroleukemia cell phenotype. The results demonstrate that increasing concentrations of 5′-methylthioadenosine (1 × 10^?6 to 5 × 10^?4M) progressively inhibit murine erythroleukemia cell heme synthesis and hemoglobin production. The results also demonstrate that this inhibition of differentiation is not related to depletion of intracellular spermidine or cytostasis. Since 5′-methylthioadenosine is also a known inhibitor of DNA methylation, this naturally occurring nucleoside may be an intermediate involved in both murine erythroleukemia cell proliferation and differentiation.     

A role for PPARα in the regulation of arginine metabolism and nitric oxide synthesis

The pleiotropic effects of PPARα may include the regulation of amino acid metabolism. Nitric oxide (NO) is a key player in vascular homeostasis. NO synthesis may be jeopardized by a differential channeling of arginine toward urea (via arginase) versus NO (via NO synthase, NOS). This was studied in wild-type (WT) and PPARα-null (KO) mice fed diets containing either saturated fatty acids (COCO diet) or 18:3 n-3 (LIN diet). Metabolic markers of arginine metabolism were assayed in urine and plasma. mRNA levels of arginases and NOS were determined in liver. Whole-body NO synthesis and the conversion of systemic arginine into urea were assessed by using ¹⁵N₂-guanido-arginine and measuring urinary ¹⁵NO₃ and [¹⁵N]-urea. PPARα deficiency resulted in a markedly lower whole-body NO synthesis, whereas the conversion of systemic arginine into urea remained unaffected. PPARα deficiency also increased plasma arginine and decreased citrulline concentration in plasma. These changes could not be ascribed to a direct effect on hepatic target genes, since NOS mRNA levels were unaffected, and arginase mRNA levels decreased in KO mice. Despite the low level in the diet, the nature of the fatty acids modulated some effects of PPARα deficiency, including plasma arginine and urea, which increased more in KO mice fed the LIN diet than in those fed the COCO diet. In conclusion, PPARα is largely involved in normal whole-body NO synthesis. This warrants further study on the potential of PPARα activation to maintain NO synthesis in the initiation of the metabolic syndrome.     

Effects of inhibition of nitric oxide synthesis on TNFα serum levels in e. coli endotoxemic rats

We investigated the effects of nitric oxide (NO) synthesis inhibition on mortality rate and TNFa serum levels in rats inoculated with E. Coli endotoxin (30 mg/kg i.V.). Pre-treatment of endotoxemic rats with NG-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthesis by both the constitutive and the inducible isoforms of the NO synthase, did not change the mortality rate but significantly reduced TNFa serum levels. By contrast, administration of aminoguanidine, a more specific inhibitor of the inducible NO synthase, did not modifiy serum TNFα. These results suggest that, in E. Coli endotoxemic rats, NO synthetized by the constitutive isoform of the NO synthase positively modulates TNFa synthesis.     

Picrasma quassioides inhibits LPS- and IFN-γ-stimulated nitric oxide production and inflammatory response in RAW264.7 macrophage cells

The present study was designed to investigate the anti-inflammatory effects of Picrasma quassioides (P. quassioides) in lipopolysaccharide (LPS)- and interferon (IFN)-γ-stimulated RAW 264.7 cells. P. quassioides has been used as a traditional medicine for the treatment of gastro-enteritis, eczema, and snakebite. P. quassioides significantly decreased LPS- and IFN-γ-stimulated nitric oxide (NO) production in a concentration-dependent manner. Real-time PCR or Western blotting confirmed that the expression of the extra-cellular signal-regulated kinase (ERK), p42 and, p38 mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinase, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) mediated MAPK signaling pathways in LPS- and IFN-γ-stimulated RAW264.7 cells. Mechanistic studies revealed the activities of nuclear factor kappa B (NF-κB). As P. quassioides regulated the gene expression of iNOS and COX-2 in RAW264.7 cells, it might be a promising agent for the prevention and/or treatment of various inflammatory diseases.     

NO signal at the crossroads: polyamine-induced nitric oxide synthesis in plants?

Polyamines, such as spermine, spermidine and putrescine, are ubiquitous polycationic compounds that are produced by almost all living organisms, including plants, animals, fungi and bacteria. Polyamines are multifunctional and interact with polyanionic biomolecules such as DNA or protein. However, despite their potential significance, the polyamine-dependent signal transduction system has not been revealed yet. Ni Ni Tun and colleagues have recently reported a possible linkage between polyamine and nitric oxide (NO), another ubiquitous signalling molecule.     

Kinetics of retroviral production from the amphotropic ΨCRIP murine producer cell line

Rapidly expanding development and practice of gene therapy requires the availability of large quantities of high titer retroviral supernatants. One way to achieve high retroviral titers is through improved understanding of the kinetics of retroviral production and decay, and the subsequent development of improved cell culture methods. In the present study we investigated the effects of different operational modes on the retroviral production of the NIH 3T3 fibroblast derived amphotropic murine retroviral producing cell line pMFG/CRIP. Semi-continuous culture (exchange of 50% of medium volume daily) was found to promote cell growth and enhance retroviral production. The rapid medium exchange resulted in significantly larger amounts of high titer supernatants and an extended production phase as compared to the batch control cultures. The specific viral productivity of the pMFG/CRIP cells was in the range of 10 to 40 infectious viruses produced per thousand producer cells per day. The CV-1 African Green Monkey kidney cell line was used as the infection target. Lowering the serum level form 20% to 10% improved retroviral production slightly. However, at lower serum levels (1%, 5% and 10% (v/v)) growth of the producer cell line, and thus retroviral production, was directly proportional to the serum level. The half-life of the virus at 37°C was found to be 5.5 hours. Promoting the growth of producer cell lines can improve retroviral vectors titers and viral production. High cell density systems that allow for rapid cell growth and waste product removal are likely to be used to generate high-titer retroviral supernatants.     

Monitoring the polysaccharide quality of Agaricus blazei in submerged culture by examining molecular weight distribution and TNF-α release capability of macrophage cell line RAW 264.7

A method for monitoring the biological activity of broth polysaccharides of Agaricus blazei (AB-BP) in a submerged culture is described. The TNF-alpha releasing capability of AB-BP on the macrophage cell line, RAW 264.7, correlated with the molecular weight of AB-BP. The quality of polysaccharide in a submerged culture of Agaricus blazei was indirectly monitored by analyzing the distribution of its molecular weight within 1 h. The harvest time of the maximum polysaccharide production did not coincide with that of the maximum biological characteristics in the batch culture.     

Effect of dehydroepiandrosterone derivatives on the activity of 5α-reductase isoenzymes and on cancer cell line PC-3

It is well known that testosterone (T) under the influence of 5α-reductase enzyme is converted to dihydrotestosterone (DHT), which causes androgen-dependent diseases. The aim of this study was to synthesize new dehydroepiandrosterone derivatives (3a–e, 4a–i, 6 and 7) having potential inhibitory activity against the 5α-reductase enzyme. This paper also reports the in vivo pharmacological effect of these steroidal molecules. The results from this study showed that all compounds exhibited low inhibitory activity for 5α-reductase type 1 and 2 enzymes and they failed to bind to the androgen receptor. Furthermore, in the in vivo experiment, steroids 3b, 4f, and 4g showed comparable antiandrogenic activity to that of finasteride; only derivatives 4d and 7 produced a considerable decrease in the weight of the prostate gland of gonadectomized hamsters treated with (T). On the other hand, compounds 4a, f and h showed 100% inhibition of the growth of prostate cancer cell line PC-3, with compound 4g having a 98.2% antiproliferative effect at 50 μM. The overall data indicated that these steroidal molecules, having an aromatic ester moiety at C-3 (4f–h), could have anticancer properties.     

Anti-inflammatory effect of soyasaponins through suppressing nitric oxide production in LPS-stimulated RAW 264.7 cells by attenuation of NF-κB-mediated nitric oxide synthase expression

The anti-inflammatory properties of soyasaponins (especially soyasaponins with different chemical structures) have scarcely been investigated. We investigated the inhibitory effects of five structural types of soyasaponins (soyasaponin A¹, A², I and soyasapogenol A, B) on the induction of nitric oxide (NO) and inducible NO synthase (iNOS) in murine RAW 264.7 cells activated with lipopolysaccharide (LPS). Soyasaponin A¹, A² and I (25-200 μg/mL) dose-dependently inhibited the production of NO and tumor necrosis factor α (TNF-α) in LPS-activated macrophages, whereas soyasapogenol A and B did not. Furthermore, soyasaponin A¹, A² and I suppressed the iNOS enzyme activity and down-regulated the iNOS mRNA expression both in a dose-dependent manner. The reporter gene assay revealed that soyasaponin A¹, A² and I decreased LPS-induced nuclear factor kappa B (NF-κB) activity. Soyasaponin A¹, A² and I exhibit anti-inflammatory properties by suppressing NO production in LPS-stimulated RAW 264.7 cells through attenuation of NF-κB-mediated iNOS expression. It is proposed that the sugar chains present in the structures of soyasaponins are important for their anti-inflammatory activities. These results have important implication for using selected soyasaponins towards the development of effective chemopreventive and anti-inflammatory agents.     

A role for tyrosine kinase activation in interleukin-1β induced nitric oxide production in the insulin producing cell line RINm-5F

The aim of this investigation was to study the putative role of protein phosphorylation in interleukin-1 (IL-1) induced signal transduction in insulin producing cells. For this purpose, insulin producing RINm-5F cells were exposed to IL-1 for 7 hours with or without different agonists and antagonists to protein kinases and phosphatases and the production of nitrite was subsequently determined. It has been shown earlier that IL-1 will stimulate the production of nitrite in such cells. It was found that EDTA, TPA and staurosporine did not affect IL-1 induced nitrite production. However, the tyrosine kinase antagonist tyrphostin inhibited, whereas sodium orthovanadate, okadaic acid and cyclosporin A, all inhibitors of protein phosphatases, potentiated IL-1 induced nitrite release to the medium. The tyrosine kinase antagonist genistein potentiated at a low concentration and inhibited at a high concentration the IL-1 effect. It is concluded that protein phosphorylation events, mediated either by protein kinases or phosphatases on both tyrosine and serine/threonine residues, may mediate or antagonize IL-1 induced signal transduction in insulin producing cells.     

Effect of nitric oxide – releasing compounds on phytochrome – controlled germination of Empress tree seeds

Using different nitric oxide releasing compounds and appropriate controls we have obtained data strongly suggesting the involvement of nitric oxide in the phytochrome controlled germination of Paulownia tomentosa seeds. Direct detection of nitric oxide, under various experimental conditions, was performed by a spin-trapping technique combined with electron paramagnetic resonance (EPR) spectroscopy. The addition of methylene blue prevented light-induced and NO donors-potentiated germination of P. tomentosa seeds. This inhibition could be completely overcome by addition of gibberellin. The promotive effect of nitrite was pH dependent, maximally pronounced at the pH range where nitrite undergoes dismutation and liberates nitric oxide. Under these conditions, nitrite exerted its efficacy at the same concentrations at which nitric oxide releasing compounds such as sodium nitroprusside (SNP), S-nitroso acetylpenicillamine (SNAP), and 3-morpholinosydnonimine (SIN-1), were the most effective. Likewise, the potentiation of P. tomentosa seed germination could be achieved by chemical reduction of nitrite with Na2S2O4 during which liberation of nitric oxide could be detected.     

Are nitric oxide donors a valuable tool to study the functional role of nitric oxide in plant metabolism?

In the present work, we tested known nitric oxide (NO) modulators generating the NO+ (sodium nitroprusside, SNP) and NO˙ forms (S-nitroso-N-acetyl-D-penicillamine, SNAP and nitrosoglutathione, GSNO). This allowed us to compare downstream NO-related physiological effects on proteins found in leaves of pelargonium (Pelargonium peltatum L.). Protein modification via NO donors generally affects plant metabolism in a distinct manner, manifested by a lower thiobarbituric acid reactive substance (TBARS) content and lipoxygenase (LOX) activity in response to SNAP and GSNO. This is in contrast to the response observed for SNP treatment. Most changes in enzyme activity (GR, glutathione reductase; GST, glutathione-S-transferase; GPX, glutathione peroxidase) are most spectacular and repeatable during the first 8 h of incubation, which is explained by the half-life of the applied donors. In particular, a close dependence was found between the time-course of NO emission from the applied donors and the temporary inhibition of antioxidant enzymes, such as catalase (CAT) and ascorbate peroxidase (APX). The observed changes were accompanied by time-dependent alterations in protein accumulation as analysed by two-dimensional gel electrophoresis (2-DE) in pelargonium leaves treated with NO donors (SNP, SNAP and GSNO). Using proteomics, different proteins were found to be down- and up-regulated. However, no new protein spots characteristic of all three donors were found. These results indicate that the form of NO emitted from the donor structure plays a key role in switching on appropriate metabolic modifications. It has been noted that several NO-affected metabolomic changes induced by the used donors were not comparable, which confirms the need to maintain caution when interpreting results obtained using the pharmacological approach with different NO modulator compounds.     

Predominance of IL-10 and TGF-β production from the mouse macrophage cell line, RAW264.7, in response to crude antigens from Clonorchis sinensis

Parasitic helminths are well-known to have the ability to modulate host immune responses. In this study, we investigated the fundamental immunoregulatory mechanism of the liver fluke Clonorchis sinensis (C. sinensis) using a murine macrophage RAW 264.7 (RAW) cell line and mouse bone marrow derived macrophages (BMDMs). We found that C. sinensis crude antigen (CA) is able to differentiate macrophage RAW cells into dendritic-like cells that can be detected by morphological observations. In addition, CA induces prominent secretion of anti-inflammatory cytokines such as IL-10 and TGF-β; however, we did not observe changes in cell surface markers that are involved in antigen recognition, antigen presentation, and T cell activation. Additionally, CA treatment induced ERK and JNK phosphorylation, and unexpectedly, elevated levels of IL-10 and TGF-β were inhibited by the addition of an ERK-specific inhibitor. Taken together, these data demonstrate that CA from C. sinensis may modulate host immune responses by upregulating anti-inflammatory cytokines via the regulation of ERK.