Koichiro Tsuchiya

Nitrate and nitrite in biology, nutrition and therapeutics

Inorganic nitrate and nitrite from endogenous or dietary sources are metabolized in vivo to nitric oxide (NO) and other bioactive nitrogen oxides. The nitrate-nitrite-NO pathway is emerging as an important mediator of blood flow regulation, cell signaling, energetics and tissue responses to hypoxia. The latest advances in our understanding of the biochemistry, physiology and therapeutics of nitrate, nitrite and NO were discussed during a recent 2-day meeting at the Nobel Forum, Karolinska Institutet in Stockholm.

Roles of nitric oxide in compression injury of rat spinal cord

Nitric oxide (NO) was measured directly after spinal cord injury (SCI) in rats by an ESR spin-trapping technique using Fe2+ and diethyldithiocarbamate (DETC). The levels of NO and lipid peroxides expressed as thiobarbituric acid reactive substances (TBARS) were increased by SCI in the injured region and the adjacent central region. Pretreatment with 30 mg/kg of NG-nitro-L-arginine methylester (L-NAME), an inhibitor of NO synthase, accelerated increases of the TBARS level and myeloperoxidase (MPO) activity in the injured tissue and caused deterioration of hind limb motor function after SCI, suggesting that NO formation by constitutive NO synthase (c-NOS) has a protective effect against cellular damage resulting from ischemia-reperfusion after SCI. Though c-NOS mRNA expression was not altered after SCI, inducible NO synthase (i-NOS) mRNA expression increased to a maximum of 24 h after SCI with progress of motor dysfunction. Intravenous injection of L-NAME (0.1 mg/kg) 6, 24, 48, and 72 h after SCI reduced the motor disturbance. These results indicate that NO induced by i-NOS may be neurotoxic in the subacute phase after SCI.

Quercetin glucuronide prevents VSMC hypertrophy by angiotensin II via the inhibition of JNK and AP-1 signaling pathway

We previously reported that quercetin, a bioflavonoid belonging to polyphenols, inhibited Angiotensin II (Ang II)-induced vascular smooth muscle cell (VSMC) hypertrophy through the inhibition of c-Jun N-terminal kinase (JNK) activation. However, we recently found that orally administered quercetin appeared in plasma as glucuronide-conjugated forms in rats and humans. Therefore we examined the effect of chemically synthesized quercetin glucuronide on Ang II-induced mitogen-activated protein (MAP) kinase activation and hypertrophy of cultured rat aortic smooth muscle cells (RASMC). Ang II activated extracellular signal-regulated kinase (ERK)1/2, JNK, and p38 in RASMC. Ang II-induced JNK activation was inhibited by quercetin 3-O-beta-d-glucuronide (Q3GA) whereas ERK1/2 and p38 activations were not affected. Q3GA scavenged 1,1-diphenyl-2-picrylhydrazyl radical measured by a method of electron paramagnetic resonance. Q3GA also inhibited Ang II-induced increases in activator protein-1 (AP-1) DNA binding, a downstream transcription factor of JNK. Finally, Ang II-induced [3H]leucine incorporation into RASMC was abolished by Q3GA. These findings suggest that the preventing effect of Q3GA on Ang II-induced VSMC hypertrophy is attributable in part to its inhibitory effect on JNK and the AP-1 signaling pathway. Q3GA would be an active metabolite of quercetin in plasma and may possess a preventing effect for cardiovascular diseases relevant to VSMC growth.

Ebselen attenuates oxidative stress-induced apoptosis via the inhibition of the c-Jun N-terminal kinase and activator protein-1 signalling pathway in PC12 cells.

Ebselen (2‐phenyl‐1,2‐benzisoselenazol‐3[2H]‐one) is a selenoorganic compound exhibiting both glutathione peroxidase activity and antioxidant activity. Although it has been reported that ebselen is effective for oxidative stress‐induced neuronal damage both in vivo and clinically, the precise mechanisms of the efficacy have not yet been elucidated. Thus, we hypothesized that ebselen may affect reactive oxygen species‐induced mitogen‐activated protein (MAP) kinase activation in cultured PC12 cells. Our findings showed that hydrogen peroxide (H2O2) stimulated rapid and significant activation of extracellular signal‐regulated kinase (ERK)1/2, c‐Jun N‐terminal kinase (JNK) and p38 in PC12 cells, which is a model of catecholamine‐containing neurons. H2O2‐induced JNK activation was inhibited by ebselen, whereas ERK1/2 and p38 activation by H2O2 were not affected by ebselen. Inhibition by ebselen of H2O2‐induced hydroxyl radical generation in PC12 cells was observed using electron paramagnetic resonance measurements. Ebselen also inhibited H2O2‐induced increases in DNA binding activity of activator protein‐1 (AP‐1), a downstream transcription factor of JNK, composed of the c‐Jun homo/heterodimer. Finally, pretreatment of cells with ebselen resulted in a significant recovery from cell death including apoptosis by H2O2 in PC12 cells. These findings suggest that ebselen attenuates oxidative stress‐induced neuronal cell death through the inhibition of the JNK and AP‐1 signalling pathway. Thus, inhibition of JNK by ebselen may imply its usefulness for treatment of ischaemic cerebral diseases relevant to neuronal cell death.

Aldosterone Stimulates Vascular Smooth Muscle Cell Proliferation Via Big Mitogen-Activated Protein Kinase 1 Activation

The nongenomic effects of aldosterone have been implicated in the pathogenesis of various cardiovascular diseases. Aldosterone-induced nongenomic effects are attributable in part to the activation of extracellular signal-regulated kinase 1/2 (ERK1/2), a classical mitogen-activated protein (MAP) kinase. Big MAP kinase 1 (BMK1), a newly identified MAP kinase, has been shown to be involved in cell proliferation, differentiation, and survival. We examined whether aldosterone stimulates BMK1-mediated proliferation of cultured rat aortic smooth muscle cells (RASMCs). Mineralocorticoid receptor (MR) expression and localization were evaluated by Western blotting analysis and fluorolabeling methods. ERK1/2 and BMK1 activities were measured by Western blotting analysis with the respective phosphospecific antibodies. Cell proliferation was determined by Alamar Blue colorimetric assay. Aldosterone (0.1 to 100 nmol/L) dose-dependently activated BMK1 in RASMCs, with a peak at 30 minutes. To clarify whether aldosterone-induced BMK1 activation is an MR-mediated phenomenon, we examined the effect of eplerenone, a selective MR antagonist, on aldosterone-induced BMK1 activation. Eplerenone (0.1 to 10 &mgr;mol/L) dose-dependently inhibited aldosterone-induced BMK1 activation in RASMCs. Aldosterone also stimulated RASMC proliferation, which was inhibited by eplerenone. Aldosterone-mediated phenomena were concluded to be attributable to a nongenomic effect because cycloheximide failed to inhibit aldosterone-induced BMK1 activation. Transfection of dominant-negative MAP kinase/ERK kinase 5 (MEK5), which is an upstream regulator of BMK1, partially inhibited aldosterone-induced RASMC proliferation, which was almost completely inhibited by MEK inhibitor PD98059. In addition to the classical steroid activity, rapid nongenomic effects induced by aldosterone may represent an alternative etiology for vascular diseases such as hypertension.

Effect of endothelin‐1 (1‐31) on extracellular signal‐regulated kinase and proliferation of human coronary artery smooth muscle cells

1 We have previously found that human chymase cleaves big endothelins (ETs) at the Tyr31‐Gly32 bond and produces 31‐amino acid ETs (1‐31), without any further degradation products. In this study, we investigated the effect of synthetic ET‐1 (1‐31) on the proliferation of cultured human coronary artery smooth muscle cells (HCASMCs). 2 ET‐1 (1‐31) increased [3H]‐thymidine incorporation and cell numbers to a similar extent as ET‐1 at 100 nM. This ET‐1 (1‐31)‐induced [3H]‐thymidine uptake was not affected by phosphoramidon, an inhibitor of ET‐converting enzyme. It was, however, inhibited by BQ123, an endothelin ETA receptor antagonist, but not by BQ788, an endothelin ETB receptor antagonist. 3 By using an in‐gel kinase assay, we demonstrated that ET‐1 (1‐31) activated extracellular signal‐regulated kinase 1/2 (ERK1/2) in a concentration‐dependent manner (100 pM to 1 μM) in HCASMCs. ET‐1 (1‐31)‐induced ERK1/2 activation was inhibited by BQ123, but not by BQ788 and phosphoramidon. Inhibition of protein kinase C (PKC) and ERK kinase also caused a reduction of ET‐1 (1‐31)‐induced ERK1/2 activation, whereas tyrosine kinase inhibition had little effect. 4 Gel‐mobility shift analysis revealed that the ERK1/2 activation was followed by an increase in transcription factor activator protein‐1 DNA binding activity in HCASMCs. 5 Our results strongly suggest that ET‐1 (1‐31) itself stimulates HCASMC proliferation probably through endothelin ETA or ETA‐like receptors. The underlining mechanism of cell growth by ET‐1 (1‐31) may be explained in part by PKC‐dependent ERK1/2 activation. Since human chymase has been proposed to play a role in atherosclerosis, ET‐1 (1‐31) may be one of the mediators.

Estrogen Regulates Hepcidin Expression via GPR30-BMP6-Dependent Signaling in Hepatocytes

Hepcidin, a liver-derived iron regulatory protein, plays a crucial role in iron metabolism. It is known that gender differences exist with respect to iron storage in the body; however, the effects of sex steroid hormones on iron metabolism are not completely understood. We focused on the effects of the female sex hormone estrogen on hepcidin expression. First, ovariectomized (OVX) and sham-operated mice were employed to investigate the effects of estrogen on hepcidin expression in an in vivo study. Hepcidin expression was decreased in the livers of OVX mice compared to the sham-operated mice. In OVX mice, bone morphologic protein-6 (BMP6), a regulator of hepcidin, was also found to be downregulated in the liver, whereas ferroportin (FPN), an iron export protein, was upregulated in the duodenum. Both serum and liver iron concentrations were elevated in OVX mice relative to their concentrations in sham-operated mice. In in vitro studies, 17β-estradiol (E2) increased the mRNA expression of hepcidin in HepG2 cells in a concentration-dependent manner. E2-induced hepatic hepcidin upregulation was not inhibited by ICI 182720, an inhibitor of the estrogen receptor; instead, hepcidin expression was increased by ICI 182720. E2 and ICI 182720 exhibit agonist actions with G-protein coupled receptor 30 (GPR30), the 7-transmembrane estrogen receptor. G1, a GPR30 agonist, upregulated hepcidin expression, and GPR30 siRNA treatment abolished E2-induced hepcidin expression. BMP6 expression induced by E2 was abolished by GPR30 silencing. Finally, both E2 and G1 supplementation restored reduced hepatic hepcidin and BMP6 expression and reversed the augmentation of duodenal FPN expression in the OVX mice. In contrast, serum hepcidin was elevated in OVX mice, which was reversed in these mice with E2 and G1. Thus, estrogen is involved in hepcidin expression via a GPR30-BMP6-dependent mechanism, providing new insight into the role of estrogen in iron metabolism.

Antioxidants inhibit endothelin-1 (1-31)-induced proliferation of vascular smooth muscle cells via the inhibition of mitogen-activated protein (MAP) kinase and activator protein-1 (AP-1).

We previously found that human chymase cleaves big endothelins (ETs) at the Tyr(31)-Gly(32) bond and produces 31-amino acid ETs (1-31), without any further degradation products. In the present study, we investigated the effects of various antioxidants on the ET-1 (1-31)-induced change in intracellular signaling and proliferation of cultured rat aortic smooth muscle cells (RASMC). ET-1 (1-31) stimulated rapid and significant activation of the mitogen-activated protein (MAP) kinase family, i.e. extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH(2)-terminal kinase (JNK), and p38 MAPK, in RASMC to an extent similar to that of ET-1. All of the antioxidants examined, i.e. N-acetyl-L-cysteine (NAC), diphenyleneiodonium chloride (DPI), and L-(+)-ascorbic acid (ascorbic acid), inhibited both ET-1 (1-31)- and ET-1-induced JNK and p38 MAPK activation but not ERK1/2 activation. Electron paramagnetic resonance (EPR) spectroscopy measurements revealed that NAC, DPI, and ascorbic acid inhibited xanthine oxidase-induced superoxide (O(2)(.-)) generation in a cell-free system. ET-1 (1-31) in addition to ET-1 increased the generation of cellular reactive oxygen species (ROS) in RASMC. ET-1 (1-31)- and ET-1-induced cellular ROS generation was inhibited similarly by NAC, DPI, and ascorbic acid in RASMC. Gel-mobility shift analysis showed that ET-1 (1-31) and ET-1 caused an increase in activator protein-1 (AP-1)-DNA binding activity in RASMC that was inhibited by the above three antioxidants. ET-1 (1-31) increased [3H]thymidine incorporation into cells to an extent similar to that of ET-1. This ET-1 (1-31)-induced increase in [3H]thymidine incorporation was also inhibited by NAC and DPI, but not by ascorbic acid. These results suggest that antioxidants inhibit ET-1 (1-31)-induced RASMC proliferation by inhibiting ROS generation within the cells. The underlying mechanisms of the inhibition of cellular proliferation by antioxidants may be explained, in part, by the inhibition of JNK activation and the resultant inhibition of AP-1-DNA binding.

Sensitive quantitation of nitric oxide by EPR spectroscopy

A recent method for NO detection is electron paramagnetic resonance (EPR) with ferrous and mononitrosyl dithiocarbamate (Fe2+ (DETC)2) for spin trapping [Menon, N.K., et al., J.Mol. Cell Cardiol., 23:389; 1991]. However, by this technique, we failed to detect the spectrum of the NOFe2+ (DETC)2 complex in biological systems because of the low solubility of Fe2+ (DETC)2 and rapid oxidation of NOFe2+ (DETC)2 complex. To overcome these problems, we modified this method by adding albumin to solubilize Fe2+ (DETC)2 and Na2S2O4 as a strong reductant to increase the sensitivity and stability of the EPR spectrum of the NOFe2+ (DETC)2 complex. The optimal concentrations of these reagents were 3.3 mM of Fe2+ and DETC, 33 mg/ml albumin and 2 M Na2S2O4. The detection limit was less than 10 pmol/ml under these conditions. By this modified method, we succeeded in quantifying NO production from porcine aorta induced by forskolin.

Endothelium-dependent relaxation by cilostazol, a phosphodiesteras III inhibitor, on rat thoracic aorta.

The relaxation effect of cilostazol, a phosphodiesterase III inhibitor, on the thoracic aorta was investigated. Cilostazol induced the relaxation of the thoracic aorta precontracted by phenylephrine in a concentration-dependent manner. The concentration-dependent relaxation was shifted to the right in the endothelium denuded aorta compared with that of intact endothelium, suggesting that this relaxation was partly dependent on endothelium. Cilostazol-induced relaxation of thoracic aorta tone was reversed by treatment with N(G)-nitro L-arginine (L-NNA), a competitive inhibitor of nitric oxide (NO) synthase. Cilostazol also significantly increased the NO level in the porcine thoracic aorta. In rats treated with cilostazol, the urinary excretion of nitrites, a stable metabolite of NO, and basal production of NO of the aortic ring were significantly greater than in those without treatment. These findings indicate that cilostazol-induced vasodilation of the rat thoracic aorta was dependent on the endothelium, which released NO from aortic endothelial cells.