• The Korean Journal of Physiology and Pharmacology
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• v.17 no.4
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• pp.315-320
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• 2013

Here, we show that radicicol, a fungal antibiotic, resulted in marked inhibition of inducible nitric oxide synthase (iNOS) transcription by the pancreatic beta cell line MIN6N8a in response to cytokine mixture (CM: TNF-${\alpha}$, IFN-${\gamma}$, and IL-$1{\beta}$). Treatment of MIN6N8a cells with radicicol inhibited CM-stimulated activation of NF-${\kappa}B$/Rel, which plays a critical role in iNOS transcription, in a dose-related manner. Nitrite production in the presence of PD98059, a specific inhibitor of the extracellular signal-regulated protein kinase-1 and 2 (ERK1/2) pathway, was dramatically diminished, suggesting that the ERK1/2 pathway is involved in CM-induced iNOS expression. In contrast, SB203580, a specific inhibitor of p38, had no effect on nitrite generation. Collectively, this series of experiments indicates that radicicol inhibits iNOS gene expression by blocking ERK1/2 signaling. Due to the critical role that NO release plays in mediating destruction of pancreatic beta cells, the inhibitory effects of radicicol on iNOS expression suggest that radicicol may represent a useful anti-diabetic activity.

• Preventive Nutrition and Food Science
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• v.12 no.2
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• pp.74-78
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• 2007

Fucoidan, that is high-molecular-weight sulfated polysaccharides extracted from brown seaweeds has been shown to elicit various biological activities. Here, we investigated the effects of fucoidan on cell proliferation and nitric oxide (NO) production in neuronal blastoma cell (SH-SY5Y). In the present study, we demonstrated that fucoidan treatment resulted in increase of cell proliferation and NO production. When cells were treated with amyloid-${\beta}$ (A${\beta}$) in the absence or presence of fucoidan, fucoidan recovered the cell viability decreased by A${\beta}$ peptides. To further determine whether nitric oxide synthase (NOS) is involved in proliferative effect of fucoidan, cells were treated with NOS inhibitors in the absence or presence of fucoidan. Selective constitutive nitric oxide synthase (cNOS) inhibitor, diphenylene iodonium chloride (DPI), caused a decrease of cell viability, whereas cell viability was increased by specific inducible nitric oxide synthase (iNOS) inhibitor, S-methylisothiourea (SMT), in the fucoidan-untreated cells. Treatment with fucoidan inhibited the cell viability decreased in DPI-exposed cells. In contrast, fucoidan had no effect on cell growth in SMT-treated cells, indicating that cNOS may not play a role in the proliferation of fucoidan-treated cells. The present data suggest that fucoidan has proliferative and neuroprotective effects and these effects may be associated with iNOS.

• The Korean Journal of Physiology and Pharmacology
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• v.8 no.3
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• pp.161-165
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• 2004

Exogenous carbon monoxide (0.2%) increases L-type calcium $(Ca^{2+})$ current in human jejunal circular smooth muscle cells. The stimulatory effect of carbon monoxide (CO) on L-type $Ca^{2+}$ current is inhibited by pre-application of L-NNA, a classical competitive inhibitor of nitric oxide synthase (NOS) with no significant isoform selectivity (Lim, 2003). In the present study, we investigated which isoform of NOS affected CO induced stimulation of L-type $Ca^{2+}$ current in human jejunal circular smooth muscle cells. Cells were voltage clamped by whole-cell mode patch clamp technique, and membrane currents were recorded with 10 mM barium as the charge carrier. Before the addition of CO, cells were pretreated with each inhibitor of three NOS isoforms for 15 minutes. CO-stimulating effect on L-type $Ca^{2+}$ current was partially blocked by N-(3-(Amino-methyl) benzyl) acetamidine 2HCl (1400W, an iNOS inhibitor). On the other hand, 3-bromo-7-nitroindazole (BNI, a nNOS inhibitor) or $N^5-(1-Iminoethyl)-L-ornithine$ dihydrochloride (L-NIO, an eNOS inhibitor) completely blocked the CO effect. These data suggest that low dose of exogenous CO may stimulate all NOS isoforms to increase L-type $Ca^{2+}$ channel through nitric oxide (NO) pathway in human jejunal circular smooth muscle cells.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.4
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• pp.343-351
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• 2001

This study was undertaken to investigate the mechanism of lipopolysaccharide (LPS) and nitric oxide (NO) as a regulator of vascular smooth muscle cell (VSMC) proliferation. VSMC was primarily cultured from rat aorta and confirmed by the immunocytochemistry with anti-smooth muscle myosin antibody. The number of viable VSMCs were counted, and lactate dehydrogenase (LDH) activity was measured to assess the degree of cell death. Concentrations of nitrite in the culture medium were measured as an indicator of NO production. LPS was introduced into the medium to induce the inducible nitric oxide synthase (iNOS) in VSMC, and Western blot for iNOS protein and RT-PCR for iNOS mRNA were performed to confirm the presence of iNOS. Inhibitors of iNOS and soluble guanylate cyclase (sGC), sodium nitroprusside (SNP) and L-arginine were employed to observe the action of LPS on the iNOS-NO-cGMP signalling pathway. LPS and SNP decreased number of VSMCs and increased the nitrite concentration in the culture medium, but there was no significant change in LDH activity. A cell permeable cGMP derivative, 8-Bromo-cGMP, decreased the number of VSMCs with no significant change in LDH activity. L-arginine, an NO substrate, alone tended to reduce cell count without affecting nitrite concentration or LDH level. Aminoguanidine, an iNOS specific inhibitor, inhibited LPS-induced reduction of cell numbers and reduced the nitrite concentration in the culture medium. LY 83583, a guanylate cyclase inhibitor, suppressed the inhibitory actions of LPS and SNP on VSMC proliferation. LPS increased amounts of iNOS protein and iNOS mRNA in a concentration-dependent manner. These results suggest that LPS inhibits the VSMC proliferation via production of NO by inducing iNOS gene expression. The cGMP which is produced by subsequent activation of guanylate cyclase would be a major mediator in the inhibitory action of iNOS-NO signalling on VSMC proliferation.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.6
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• pp.353-358
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• 2010

This study demonstrates the ability of magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, to inhibit LPS-induced expression of iNOS gene and activation of NF-${\kappa}B$/Rel in RAW 264.7 cells. Immunohisto-chemical staining of iNOS and Western blot analysis showed magnolol to inhibit iNOS gene expression. Reporter gene assay and electrophoretic mobility shift assay showed that magnolol inhibited NF-${\kappa}B$/Rel transcriptional activation and DNA binding, respectively. Since p38 is important in the regulation of iNOS gene expression, we investigated the possibility that magnolol to target p38 for its anti-inflammatory effects. A molecular modeling study proposed a binding position for magnolol that targets the ATP binding site of p38 kinase (3GC7). Direct interaction of magnolol and p38 was further confirmed by pull down assay using magnolol conjugated to Sepharose 4B beads. The specific p38 inhibitor SB203580 abrogated the LPS-induced NF-${\kappa}B$/Rel activation, whereas the selective MEK-1 inhibitor PD98059 did not affect the NF-${\kappa}B$/Rel. Collectively, the results of the series of experiments indicate that magnolol inhibits iNOS gene expression by blocking NF-${\kappa}B$/Rel and p38 kinase signaling.

• IMMUNE NETWORK
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• v.4 no.2
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• pp.108-115
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• 2004

Background: Production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathology of autoimmune disease. It is unknown whether iNOS expression is increased within testes and whether iNOS and NO have essential roles in the pathogenesis of EAO. Methods: EAO was induced in guinea pig testes at 17 days after secondary immunization by administration of crude extract (CE) and purified glycoprotein 1 (GP1) from normal guinea pig testes. iNOS gene expression was assessed by RT-PCR and Northern blot analysis in testes. Localization of iNOS and Mac-1 and the indicator of NO-mediated tissue injury, nitrotyrosine, were detected in the testicular lesion by immunohistochemistry. Results: In control testes, inflammation and iNOS gene expression were not detected, whereas, in CE- and GP1-injected testes, inflammation and marked iNOS gene expression were evident at day 17 after secondary immunization. Immunohistochemistry of Mac-1 showed the colocalization with iNOS protein and nitrotyrosyl proteins in intertubules, suggesting that NO produced by infiltrated macrophages may be involved in inflammatory lesions of intertubules. Intraperitoneal administration of aminoguanidine significantly prevented EAO with reduction of inflammation, iNOS expression and nitrotyrosine formation. Conclusion: These results suggest that NO production by macrophages may be important in the pathogenesis of CE- and GP1-induced EAO. Furthermore, this study demonstrated the therapeutic potential of iNOS inhibitor in the treatment of inflammatory and autoimmune mediated-diseases.

• Archives of Pharmacal Research
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• v.26 no.4
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• pp.301-305
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• 2003

Nitric oxide (NO) is an important bioactive agent that mediates a wide variety of physiological and pathophysiological events. NO overproduction by inducible nitric oxide synthase (iNOS) results in severe hypotension and inflammation. This investigation is part of a study to discover new iNOS inhibitors from medicinal plants using a macrophage cell culture system. Two sesquiterpenes (1 and 2) were isolated from Artemisia iwayomogi (Compositae) and were found to inhibit NO synthesis ($IC_{50} 3.64 \mu g/mL and 2.81 \mu$g/mL, respectively) in lipopolysaccharide (LPS)-activated RAW 264.7 cells. Their structures were identified as 3-Ο-methyl-iso-secotanapartholide (1) and iso-secotanapartholide (2). Compounds 1 and 2 inhibited the LPS-induced expression of the iNOS enzyme in the RAW 264.7 cells. The inhibition of NO production via the down regulation of iNOS expression may substantially modulate the inflammatory responses.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.1
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• pp.55-63
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• 2001

In macrophages, lipopolysaccharide (LPS) alone or in combination with $interferon-{\gamma}\;(IFN-{\gamma})$ has been shown to release a nitric oxide (NO) through the increase of the transcription of the inducible nitric oxide synthase (iNOS) gene. To investigate the exact intracellular signaling pathway of the regulation of iNOS gene transcription by LPS plus $IFN-{\gamma},$ the effects of protein tyrosine kinase (PTK) inhibitor and protein kinase C (PKC) inhibitors on NO production, iNOS mRNA expression, nuclear $factor-_{\kappa}B\;(NF-_{\kappa}B)$ binding activity and the promoter activity of iNOS gene containing two $NF-_{\kappa}B$ sites have been examined in a mouse macrophage RAW 264.7 cells. LPS or $IFN-{\gamma}$ stimulated NO production, and their effect was enhanced synergistically by mixture of LPS and $IFN-{\gamma}.$ The PTK inhibitor such as tyrphostin reduced LPS plus $IFN-{\gamma}-induced$ NO production, iNOS mRNA expression and $NF-_{\kappa}B$ binding activity. In contrast, PKC inhibitors such as H-7, Ro-318220 and staurosporine did not show any effect on them. In addition, transfection of RAW 264.7 cells with iNOS promoter linked to a CAT reporter gene revealed that tyrphostin inhibited the iNOS promoter activity through the $NF-_{\kappa}B$ binding site, whereas PKC inhibitors did not. Taken together, these suggest that PTK, but not PKC pathway, is involved in the regulation of the iNOS gene transcription through the $NF-_{\kappa}B$ sites of iNOS promoter in RAW 264.7 macrophages by LPS plus $IFN-{\gamma}$.

• The Korean Journal of Physiology and Pharmacology
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• v.11 no.6
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• pp.253-257
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• 2007

Among the arthritis symptoms, chronic pain is the most serious, and it can profoundly affect the quality of human life. Unfortunately, the mechanism of development in arthritis and arthritic pain has not yet been precisely elucidated. Accumulating evidence indicates that nitric oxide (NO) plays a pivotal role in nociceptive processing in the spinal cord. However, the modulation mechanism of NO in the peripheral site of arthritis and arthritic pain has not been clarified. Therefore, I determined in the present study which nitric oxide synthase (NOS) was involved in the induction of arthritis and arthritic pain. Monoarthritis was induced by intra-articular injection of carrageenan (2%, $50{\mu}l$) into rats, and resulted in the reduction of weight load on the injected leg, increase of knee joint diameter and inflammatory response. Pre-treatment of rats with L-N6-(1-iminoethyl)-lysine (L-NIL, $500{\mu}g$, in $50{\mu}l$), an inhibitor of inducible NOS (iNOS), partially prevented the induction of pain-related behavior and partially reduced inflammatory response in the synovial membrane in the knee joint. These results suggest that iNOS in the knee joint may play an important role in the induction of pain-related behavior and inflammation, and that NO produced by iNOS may be associated with nociceptive signaling in the peripheral site.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.1
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• pp.93-98
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• 2001

The precise mechanism of set-point regulation in hypothalamus was not elucidated. Nitric oxide synthases(NOS) were detected in hypothalamus, however, the roles of NO in hypothalamus was not fully studied. So, we tested the effects of NO on body temperature because preoptic-anterior hypothalamus was known as the presumptive primary fever-producing site. NO donor sodium nitroprusside (SNP, 4 nmol, i.c.v.) elicited marked febrile response, and this febrile response was completely blocked by indomethacin (a cyclooxygenase inhibitor). But, ODQ (selective guanylate cyclase inhibitor, $50\;{\mu}g,$ i.c.v.) did not inhibit fever induced by SNP. The cyclic GMP analogue dibutyryl-cGMP $(100\;{\mu}g,\;i.c.v.)$ induced significant pyreses, which is blocked by indomethacin. $N^G-nitro-L-arginine$ methyl ester (L-NAME, non selective NOS inhibitor) inhibited fever induced by $interleukin-1{\beta}\;(IL-1{\bata},\;10\;ng,\;i.c.v.),$ one of endogenous pyrogens. These results indicate that NO may have an important role, not related to stimulation of soluble guanylate cyclase, in the signal pathway of thermoregulation in hypothalamus.