Nitrite is an alternative source of NO in vivo

In this study, we investigated whether orally administered nitrite is changed to NO and whether nitrite attenuates hypertension in a dose-dependent manner. We utilized a stable isotope of [15N]nitrite (15NO2-) as a source of nitrite to distinguish between endogenous nitrite and that exogenously administered and measured hemoglobin (Hb)-NO as an index of circulating NO in whole blood using electron paramagnetic resonance (EPR) spectroscopy. When 1 mg/kg Na15NO2 was orally administered to rats, an apparent EPR signal derived from Hb15NO (A(Z) = 23.4 gauss) appeared in the blood. The peak blood HbNO concentration occurred at the first measurement after intake (5 min) for treatment with 1 and 3 mg/kg (HbNO: 4.93 +/- 0.52 and 10.58 +/- 0.40 microM, respectively) and at 15 min with 10 mg/kg (HbNO: 38.27 +/- 9.23 microM). In addition, coadministration of nitrite (100 mg/l drinking water) with N(omega)-nitro-L-arginine methyl ester (L-NAME; 1 g/l) for 3 wk significantly attenuated the L-NAME-induced hypertension (149 +/- 10 mmHg) compared with L-NAME alone (170 +/- 13 mmHg). Furthermore, this phenomenon was associated with an increase in circulating HbNO. Our findings clearly indicate that orally ingested nitrite can be an alternative to L-arginine as a source of NO in vivo and may explain, at least in part, the mechanism of the nitrite/nitrate-rich Dietary Approaches to Stop Hypertension diet-induced hypotensive effects.

American Journal of Physiology, Heart and Circulatory Physiology


Nitrite-derived nitric oxide formation following ischemia-reperfusion injyury in kidney.

Nitric oxide (NO) is synthesized from l-arginine by nitric oxide synthase (NOS), and nitrite and nitrate are believed to be waste forms of NO. We previously reported an enzyme-independent pathway of NO generation from nitrite in acidic conditions. In this study, we show nitrite-derived NO formation in renal ischemia-reperfusion injury using electron paramagnetic resonance (EPR) spectroscopy. In this experiment, we utilized a stable isotope of [(15)N]nitrite as a source of nitrite to distinguish l-arginine-derived NO from [(15)N]nitrite-derived (15)NO. Intravenous infusion of a stable isotope of [(15)N]nitrite ((15)NO(2)(-)) facilitated the formation of Hb(15)NO during renal ischemia, which demonstrated that the origin of NO was nitrite. The EPR signal of Hb(15)NO in kidney appeared after 40 min of renal ischemia, and renal reperfusion decreased the Hb(15)NO level in the kidney and increased it in blood by contrast. In addition, the amount of HbNO was nitrite concentration dependent, and this formation was NOS independent. Our findings suggest that nitrite can be an alternative source of NO in ischemic kidney and that it binds with hemoglobin and then is spread by the circulation after reperfusion.

American Journal of Physiology, Renal Physiology


Malfunction of vascular control in lifestyle-related diseases : formation of systemic hemoglobin-nitric oxide Complex(HbNO) From Dietary Nitrite

Nitric oxide (NO) has many physiological functions. It is believed to be produced from L-arginine by nitric oxide synthase (NOS), and nitrite and nitrate are waste forms of it. By the way, nitrate and nitrite are abundant in vegetables and fruits, especially leafy vegetables and pickled vegetables. Orally-ingested nitrate is changed to nitrite by micro-organelles living in the hypopharynx area, and nitrite is expected to change to NO in the stomach due to its low pH. Indeed, some researchers reported that NO is produced in the gastric cavity, although few reports mentioned the physiological meanings of this NO formation. Therefore, we investigated whether the nitrite-derived NO can shift to the circulation and acts like NOS-derived NO does in tissues. We adopted a stable isotope of nitrite (15NO2-) in order to distinguish between the endogenous nitrite and the exogenously administered one and measured nitrosyl hemoglobin (HbNO) as an index of circulating NO using electron paramagnetic resonance spectroscopy. It appeared that the oral administration of 15N-nitrite formed the Hb15NO in rat blood and decreased the blood pressure of chronic L-NAME treated rats. Our findings suggest that the intake of nitrite (or nitrate)-rich foods such as vegetables and fruits would alter the systemic HbNO dynamism, resulting in the improvement of cardiovascular diseases.


Antioxidant effects of stereoisomers of N-acetylcysteine (NAC), L-NAC and D-NAC, on angiotensin II-stimulated MAP kinase activation and vascular smooth muscle cell proliferation

We examined the effects of the stereoisomers of N-acetylcysteine (NAC), L-NAC and D-NAC, on cellular glutathione (GSH) concentration and whether NAC-regulated cellular GSH levels are directly associated with angiotensin II (Ang II)-induced intracellular signaling events in vascular smooth muscle cells (VSMC). Both L-NAC and D-NAC similarly increased intracellular GSH concentration. We found that L-NAC and D-NAC both inhibited Ang II-induced c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activation and [(3)H]-thymidine incorporation in VSMC. Our present study indicates the comparable effects of NAC stereoisomers in regulating intracellular GSH and the redox-dependent intracellular signaling mechanisms in VSMC.


Atheroprotective effects of antioxidants through inhibition of mitogen-activated protein kinases

Reactive oxygen species (ROS) have been known to play an important role in the pathogenesis of atherosclerosis and several other cardiovascular diseases. It is now apparent that ROS induce endothelial cell damage and vascular smooth muscle cell (VSMC) growth and cardiac remodeling, which are associated with hypertension, atherosclerosis, heart failure, and restenosis. Several lines of evidence have indicated that ROS and mitogen-activated protein (MAP) kinases were involved in vascular remodeling under various pathological conditions. Recently, it was also reported that MAP kinases were sensitive to oxidative stress. MAP kinases play an important role in cell differentiation, growth, apoptosis, and the regulation of a variety of transcription factors and gene expressions. Bioflavonoids and polyphenolic compounds are believed to be beneficial for the prevention and treatment of atherosclerosis and cardiovascular diseases. One of the most widely distributed bioflavonoids, 3,3',4',5,7-penta-hydroxyflavone (quercetin) and its metabolite quercetin 3-O-beta-D-glucuronide (Q3GA) inhibited Angiotensin II-stimulated JNK activation and resultant hypertrophy of VSMC. Several studies have suggested that various antioxidants including probucol, N-acetyl-L-cysteine, diphenylene iodonium, Trolox C (vitamin E analogue), and vitamin C inhibit VSMC growth, which is associated with pathogenesis of cardiovascular diseases. Therefore, inhibition of MAP kinases by antioxidant treatment may prove to be a therapeutic strategy for cardiovascular diseases. In contrast, some clinical studies have reported that antioxidant vitamins did not show beneficial effects in coronary artery disease or in a number of high-risk people. Thus, further studies are needed to clarify why antioxidants showed beneficial effects in vitro, whereas less satisfactory results were obtained in some clinical conditions.


Dual effects of endothelin-1 (1-31): induction of mesangial cell migration and facilitation of monocyte recruitment through monocyte chemoattractant protein-1 production by mesangial cells

We previously found that human chymase selectively cleaves big endothelin-1 (ET-1) at the Tyr31-Gly32 bond and produces 31-amino acid endothelins, ET-1 (1-31), without any further degradation products. In this study, we investigated the effect of ET-1 (1-31) on the migration of cultured rat mesangial cells (RMCs) and on cells of the human monocytic cell line, THP-1. In addition, we examined the interaction between RMCs and THP-1 cells using conditioned media from ET-1 (1-31)-stimulated RMCs. ET-1 (1-31) caused an increase in RMC migration in a concentration-dependent manner, and the degree of increase was similar to those by ET-1 and angiotensin II (All). The ET-1 (1-31)-induced increase in RMC migration was inhibited by BQ123, an endothelin ETA receptor antagonist, but not by BQ788, an endothelin ETB receptor antagonist. ET-1 (1-31) alone did not cause significant migration of THP-1 cells. However, significant recruitment of THP-1 cells was observed with conditioned media taken from ET-1 (1-31)-stimulated RMCs. The conditioned media-induced migration of THP-1 cells was inhibited by BQ123, but not by BQ788. Western blotting analysis of the lysate of RMCs revealed that the expression of monocyte chemoattractant protein-1 (MCP-1) protein in RMCs was increased by treatment with ET-1 (1-31). The addition of neutralizing antibody for MCP-1 to the medium inhibited the migration of THP-1 cells induced by conditioned media from ET-1 (1-31)-stimulated RMCs. These findings suggest that ET-1 (1-31) play a role in glomerulonephritis (GN) via dual effects that directly cause the migration of mesangial cells (MCs) and may be responsible for the recruitment of mononuclear cells mediated through the activation of MCs. Since human chymase has been reported to be involved in glomerular disease, ET-1 (1-31) may be among the mediators.


BMK1 is activated in glomeruli of diabetic rats and in mesangial cells by high glucose conditions

High glucose causes renal cell injury through various signal transduction pathways, including mitogen-activated protein (MAP) kinases cascades. Big MAP kinase 1 (BMK1), also known as extracellular signal-regulated kinase 5 (ERK5), is a recently identified MAP kinase family member and was reported to be sensitive to osmotic and oxidative stress. However, the role of BMK1 in diabetic nephropathy has not been elucidated yet. We investigated whether BMK1 is activated in the glomeruli of Otsuka Long Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes mellitus in comparison with the control Long Evans Tokushima Otsuka (LETO) rats. We also examined the effect of high glucose on BMK1 activity in cultured rat mesangial cells. BMK1 and ERK1/2 but not p38 were activated in the glomeruli of OLETF rats, which showed diabetic nephropathy at 52 weeks of age. High glucose, in addition to a high concentration of raffinose, caused rapid and significant activation of BMK1 in rat mesangial cells. MAP kinase/ERK kinase (MEK) inhibitors, U0126 and PD98059, both inhibited BMK1 activation by high glucose in a concentration-dependent manner. Protein kinase C (PKC) inhibition by GF109203X and PKC down-regulation with long-time phorbol myristate acetate (PMA) treatment both inhibited BMK1 and Src kinase activation. Src kinase inhibitors, herbimycin A and PP2, also inhibited high glucose-induced BMK1 activation. PKC inhibitors, Src inhibitors and MEK inhibitors, all inhibited cell proliferation by high glucose. Finally, transfection of dominant-negative MEK5, which is an upstream regulator of BMK1, abolished the BMK1-mediated rat mesangial cell proliferation stimulated by high glucose. In the present study, we demonstrated that high glucose activates BMK1 both in vivo and in vitro. It was suggested that high glucose induces PKC- and c-Src-dependent BMK1 activation. It could not be denied that BMK1 activation is induced through an osmotic stress-sensitive mechanism. BMK1-mediated mesangial cell growth may be involved in the pathogenesis of diabetic nephropathy.


Src and Cas are essentially but differentially involved in angiotensin II-stimulated migration of vascular smooth muscle cell via extracellur signal-regulated kinase 1/2 and c-Jun NH2-terminal kinase activation

Angiotensin II (Ang II) plays an important role in several cardiovascular diseases associated with vascular smooth muscle cell (VSMC) growth and migration. Src activity is known to be required for the migration of a number of cell types. p130Cas was reported to be essential for cell migration and actin filament reorganization. Mitogen-activated protein (MAP) kinases were also reported to be critical regulatory factors for growth and migration of VSMC. However, precise intracellular mechanisms involving c-Src, p130Cas, and MAP kinases in Ang II-stimulated migration of VSMC have not been well elucidated. Here we demonstrated that Ang II rapidly and significantly stimulated tyrosine phosphorylation of Src and Cas and their association in rat aortic smooth muscle cells (RASMC). Ang II-stimulated tyrosine phosphorylation of Src and Cas and activation of ERK1/2 and JNK, but not p38, were potently inhibited by Src family tyrosine kinase inhibitors, herbimycin A (HA) and PP2. Ang II-stimulated Src and Cas association, tyrosine phosphorylation of Cas, and activation of ERK1/2 and JNK were suppressed in kinase-inactive Src (KI Src)-overexpressed RASMC. Ang II-stimulated JNK activation but not ERK1/2 activation was blocked in substrate domain-deleted Cas (DeltaSD Cas)-overexpressed RASMC. In addition, HA, PP2, ERK1/2 inhibitor, 2'-amino-3'-methoxyflavone (PD98059) and JNK inhibitor, and anthra[1,9-cd]pyrazol-6(2H)-one (SP600125) significantly inhibited Ang II-stimulated migration of RASMC. Ang II-induced colocalization of Src and Cas and migration were inhibited in both KI Src- and DeltaSD Cas-overexpressed RASMC. These findings suggest that Src and Cas are essentially but differentially involved in Ang II-stimulated migration of VSMC through the activation of ERK1/2 and JNK.


Ebsalen inhibits p38MAP kinase-mediated endothelial cell death by hydrogen peroxide.

Ebselen (2-phenyl-1, 2-benzisoselenazol-3[2H]-one) is a seleno-organic compound exhibiting both glutathione peroxidase and antioxidant activity. Although it has been reported that ebselen is effective against hydrogen peroxide (H(2)O(2))-induced cell death in several cell types, its effect on endothelial cell damage has not yet been elucidated. In the present study, we examined the effect of ebselen on H(2)O(2)-induced human umbilical vein endothelial cells (HUVECs) death, and its intracellular mechanism. Our findings showed that pretreatment of HUVECs with ebselen resulted in a significant recovery from H(2)O(2)-induced cell death in a concentration-dependent manner. In addition to the inhibition of lactate dehydrogenase (LDH) leakage, ebselen inhibited H(2)O(2)-induced cytochrome c release and caspase-3 activation and the resultant apoptosis in HUVECs. Moreover, it was observed that H(2)O(2) significantly stimulated activation of mitogen-activated protein (MAP) kinases, i.e., p38 MAP kinase, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK1/2). Ebselen inhibited H(2)O(2)-induced p38 MAP kinase, but not JNK or ERK1/2 activation. Furthermore, SB203580 (4-[4-fluorophenyl]-2-[4-methylsulfinylphenyl]-5-[4-pyridyl]-1H-imidazole), a specific p38 MAP kinase inhibitor, inhibited H(2)O(2)-induced p38 MAP kinase phosphorylation, cytochrome c release, caspase-3 activation, as well as cell death in HUVECs. These findings suggest that ebselen attenuates H(2)O(2)-induced endothelial cell death through the inhibition of signaling pathways mediated by p38 MAP kinase, caspase-3, and cytochrome c release. Thus, inhibition of p38 MAP kinase by ebselen may imply its usefulness for prevention and/or treatment of endothelial cell dysfunction, which was suggested to be the first step in the development of atherosclerosis.


Evaluation of Serial Intra-Arterial Indocyanine Green Videoangiography in the Surgical Treatment of Cranial and Craniocervical Junction Arteriovenous Fistulae: A Case Series

Intravenous indocyanine green (IV-ICG) videoangiography is commonly performed to detect blood flow in the microscopic view. However, intra-arterial ICG (IA-ICG) videoangiography provides high-contrast imaging, repeatability within a short period of time, and clear-cut separation of the arterial and venous phases compared with IV-ICG. These features are useful for detecting retrograde venous drainage (RVD) and shunt occlusion in arteriovenous fistulae (AVF) surgery. This study aimed to investigate whether IA-ICG videoangiography can be repeatable within a short period of time and be useful for detecting RVD and shunt occlusion in cranial- and craniocervical junction (CCJ)-AVF surgery. Between January 2012 and December 2022, 50 patients were treated with endovascular or surgical intervention for cranial- and CCJ-AVF at Tokushima University Hospital. Of these, 5 patients (6 lesions) underwent open surgery with IA-ICG videoangiography in a hybrid operating room. We analyzed the data of these 5 patients (6 lesions). There were 4/patient (median, range 2-12) and 3.5/lesion (median, range 2-10) intraoperative IA-ICG runs. IA-ICG videoangiography detected RVD in all patients. Clearance of IA-ICG-induced fluorescence was achieved within 30 seconds in all patients at each region of interest. After the disconnection of the fistulae, IA-ICG videoangiography and intraoperative digital subtraction angiography (DSA) confirmed the disappearance of RVD in all patients. There were no complications associated with IA-ICG videoangiography. This study showed that IA-ICG videoangiography is repeatable within a short period of time before and after obliteration and can be useful for detecting RVD and shunt occlusion in cranial- and CCJ-AVF surgery. IA-ICG videoangiography also allows intraoperative DSA studies in a hybrid operating room. Considering the recent advancements in hybrid operating rooms, combining IA-ICG videoangiography with intraoperative DSA is a useful strategy for cranial- and CCJ-AVF surgery.


Evaluation of Blood Flow and Plaque Vulnerability in Carotid Artery Stenosis Focusing on Morphological and Component Characteristics

Proceedings of the 2022 ISMRM & SMRT ANNUAL MEETING & EXHIBITION, No.3783, 2022


Lysophosphatidylcholine(LPC)transactivates vascular endothelial growth factor(VEGF) receptor via c-Src in HUVEC

ヒト臍帯静脈内皮細胞(HUVEC)における Lysophosphatidylcholine(LPC)刺激によるVEGFレセプターのトランスアクチベーション

One of the major lipid components of oxidized low density lipoprotein, lysophosphatidylcholine(LPC)is involved in numerous biological processes as a bioactive lipid molecule and has beenshown to be involved in the progression of atherosclerosis. As counter-ligands, G2A and GPR4wereidentified with high binding affinity for LPC that are belonging to orphan G-protein-coupledreceptors(GPCRs)at plasma membranes. Although several GPCR ligands transactivate receptortyrosine kinases (RTKs), such as epidermal growth factor receptor, transactivation of RTK byLPC has not yet been reported. Here we observed for the first time that LPC treatment inducestyrosyl phosphorylation of vascular endothelial growth factor(VEGF)receptor2(fetal liverkinase-1/kinase-insert domain-containing receptor, Flk-1/KDR)in human umbilical vein endothelialcells(HUVEC). VEGF receptor tyrosine kinase inhibitors, SU1498and VTKi inhibited Flk-1/KDRtransactivation by LPC. Furthermore, we examined the effect of the Src family kinases inhibitors,Herbimycin A and PP2 on LPC-induced Flk-1/KDR transactivation. Herbimycin A and PP2inhibited Flk-1/KDR transactivation in HUVEC, suggesting that c-Src is involved in LPC-inducedFlk-1/KDR transactivation. Kinase-inactive(KI)Src transfection also inhibited LPC-induced Flk-1/KDR transactivation. In addition, LPC activated extracellular signal-regulated kinase1/2and Akt,which are downstream effectors of Flk-1/KDR, and these were inhibited by SU1498，VTKi,Herbimycin A, PP2and KI Src transfection in HUVEC. LPC-mediated HUVEC proliferation wasshown to be secondary to transactivation because it was suppressed by SU1498，VTKi, HerbimycinA, PP2and KI Src transfection. It is concluded that c-Src-mediated Flk-1/KDR transactivation byLPC may have important implications for the progression of atherosclerosis.


生体内における亜硝酸由来のNO産生と，その循環動態への作用検討

生体内における亜硝酸由来のNO産生と，その循環動態への作用検討

Japanese Journal of Circulation Research


Ⅴ．エンドセリン 薬理作用と生理作用 心筋障害作用

Nihon Rinsho. Japanese Journal of Clinical Medicine