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Unpredictable climate change might cause serious lack of food in the world. Therefore, in the present world, it is urgent to prepare countermeasures to solve problems in terms of human survival. In this research, quantitative trait loci (QTLs) were analyzed when rice attacked by white backed planthopper (WBPH) were analyzed using 120 Cheongcheong/Nagdong double haploid lines. Moreover, from the detected QTLs, WBPH resistance-related genes were screened in large candidate genes. Among them, OsCM, a major gene in the synthesis of Cochlioquinone-9 (cq-9), was screened. OsCM has high homology with the sequence of chorismate mutase, and exists in various functional and structural forms in plants that produce aromatic amino acids. It also induces resistance to biotic stress through the synthesis of secondary metabolites in plants. The WBPH resistance was improved in rice overexpressed through map-based cloning of the WBPH resistance-related gene OsCM, which was finally detected by QTL mapping. In addition, cq-9 increased the survival rate of caecal ligation puncture (CLP)-surgery mice by 60%. Moreover, the aorta of rat treated with cq-9 was effective in vasodilation response and significantly reduced the aggregation of rat platelets induced by collagen treatment. A cq-9, which is strongly associated with resistance to WBPH in rice, is also associated with positive effect of CLP surgery mice survival rate, vasodilation, and significantly reduced rat platelet aggregation induced by collagen treatment. Therefore, cq-9 presents research possibilities as a substance in a new paradigm that can act on both Plant-Insect in response to the present unpredictable future.

Here, we report that LMK235, a class I and histone deacetylase (HDAC6)‐preferential HDAC inhibitor, reduces hypertension via inhibition of vascular contraction and vessel hypertrophy. Angiotensin II‐infusion mice and spontaneously hypertensive rats (SHRs) were used to test the anti‐hypertensive effect of LMK235. Daily injection of LMK235 lowered angiotensin II‐induced systolic blood pressure (BP). A reduction in systolic BP in SHRs was observed on the second day when SHRs were treated with 3 mg/kg LMK235 every 3 days. However, LMK235 treatment did not affect angiotensin‐converting enzyme 1 and angiotensin II receptor mRNA expression in either hypertensive model. LMK235, acting via the nitric oxide pathway, facilitated the relaxing of vascular contractions induced by a thromboxane A2 agonist in the rat aortic and mesenteric artery ring test. In addition, LMK235 increased nitric oxide production in HUVECs and inhibited the increasing of aortic wall thickness in both animal hypertensive models. LMK235 decreased the enhanced cell cycle‐related genes cyclin D1 and E2F3 in angiotensin II‐infusion mice and restored the decreased p21 expression. In addition, LMK235 suppressed calcium calmodulin‐dependent protein kinase II (CaMKII) α, which is related to vascular smooth muscle cell proliferation. Inhibition or knockdown of HDAC5 blocked the CaMKIIα‐induced cell cycle gene expression. Immunoprecipitation demonstrated that class I HDACs were involved in the inhibition of CaMKII α‐induced HDAC4/5 by LMK235. We suggest that LMK235 should be further investigated for its use in the development of new therapeutic options to treat hypertension via reducing vascular hyperplasia or vasoconstriction.

Chungkookjang is a beneficial fermented soybean food with anticancer, anti-inflammatory, and antioxidant activities. High oleic acid (HO)-containing diets are known to help in preventing cardiovascular disease (CVD). In the present study, we assessed functional quality characteristics of Chungkookjang made from soybean seeds differing in oleic acid concentration. Chungkookjang made from HO and normal oleic acid (NO) soybeans did not differ significantly in moisture content, pH, viable cell number, protease activity, amino type nitrogen, and polyphenol activity. However, the diphenylpicrylhydrazyl (DPPH)-scavenging activity and flavonoid contents of Chungkookjang made from HO soybeans were lower than those from NO soybeans. The oleic acid concentrations were not significantly altered by fermentation. Additionally, Chungkookjang from the HO soybean cultivar ‘Hosim’ affected the vascular relaxation of endothelium-intact rat aortic rings at concentrations as low as 0.03 mg/mL. The results showed that Chungkookjang from HO soybeans possesses characteristics that might be potentially helpful in reducing the risk of CVD.

Mountain ginseng has been used generally as a pharmacopuncture for cancer therapy in clinical practice in Northeast Asia. Nonetheless, there have been few scientific reports for the anticancer action of mountain ginseng. In this study, we investigated whether mountain ginseng extract (MGE) could inhibit the growth of breast cancer in in vitro and in vivo models. MGE showed stronger cytotoxicity than farm-cultivated ginseng extract (FGE) through promoting ROS generation. Also MGE dose-dependently brought about mitochondrial dysfunction in MCF-7 cells. In addition, MGE induced apoptosis through enhancing the activities of caspase-3/7 by regulation of expression of Bcl-2, Bax, cytochrome c, and cleaved caspase-3 in the MCF-7 cells. Consistent with the in vitro results, MGE significantly reduced tumor weights compared with FGE in mice transplanted with MCF-7 cells, and it regulated the expression of apoptosis-related proteins, such as Bcl-2, Bax, cytochrome c, cleaved caspase-3, and cleaved PARP, in the tumor tissues. Additionally, MGE included higher total ginsenoside contents than FGE. In conclusion, MGE, which is richer in ginsenosides, exerts a stronger anticancer action than FGE in breast cancer. The anticancer action of MGE may be closely correlated with caspase-mediated apoptosis through upregulating ROS generation. Therefore, these findings may be helpful for a clinical understanding of the anticancer mechanism of MGE for breast cancer patients.

Background The dysregulation of histone deacetylase (HDAC) protein expression or its enzyme activity is implicated in a variety of diseases. Cardiac HDAC6 and HDAC8 enzyme activity induced by deoxycorticosterone acetate (DOCA) hypertension was attenuated by sodium valproate, a pan-HDAC inhibitor. However, the HDAC6-selective inhibitor, tubastatin A, did not attenuate angiotensin II-induced hypertension. The purpose of this study was to investigate whether PCI34051, an HDAC8-selective inhibitor, can modulate angiotensin II-induced hypertension and its regulatory mechanism. Methods An angiotensin II-regulated mouse model was used in this study. Animals received vehicle or PCI34051 (3 mg·kg − 1·day− 1) via intraperitoneal injection. Systolic blood pressure was measured by the tail-cuff method. Blood vessel thickness was measured following hematoxylin and eosin staining, VCAM-1 immunohistochemistry was performed in the aortas, and mRNA expression of renin-angiotensin system components, inflammation markers, and NADPH oxidase (Nox) was determined by RT-PCR. The effect of PCI34051 on vasorelaxation was studied in rat aortic rings, and its effect on nitric oxide (NO) production was determined using DAF-FM DA, a fluorescent dye, in human umbilical vascular endothelial cells (HUVECs). Results PCI34051 administration reduced systolic blood pressure via downregulation of angiotensin II receptor type 1 (AT1) mRNA expression. PCI34051 treatment attenuated vascular hypertrophy by decreasing E2F3 and GATA6 mRNA expression. Vascular relaxation after PCI34051 treatment was more dependent on vascular endothelial cells and it was blocked by an NO synthase (NOS) inhibitor. In addition, NO production increased in HUVECs after PCI34051 treatment; this was decreased by the NOS inhibitor. The expression of inflammatory molecules and adhesion molecules VCAM-1 and ICAM-1 decreased in the aortas of angiotensin II-infused mice after PCI34051 administration. However, PCI34051 did not affect Nox or its regulatory subunits. Conclusions PCI34051 lowered high blood pressure through modulation of arterial remodeling, vasoconstriction, and inflammation in an angiotensin II-induced hypertension model. We suggest that HDAC8 could be a potential therapeutic target for hypertension.

Ethnopharmacological relevance Mountain ginseng (Panax ginseng C.A. Meyer) is a medicinal herb with immune effects, muscle damage protection and energy metabolism effects. However, the pharmacological role of mountain ginseng in dexamethasone (DEXA)-induced muscle atrophy through the forkhead box O (FOXO) family is not understood. Therefore, we hypothesized that mountain ginseng inhibits skeletal muscle atrophy by decreasing muscle RING finger protein-1 (MuRF1) and atrogin1 through FOXO3 in L6 myotubes. Methods Rat myoblast (L6) cells or Sprague-Dawley (SD) rats were exposed to DEXA and mountain ginseng. The expressions of muscle atrophy targets such as MuRF1, atrogin1, MyHC (myosin heavy chain), HSP90, p-Akt, Akt, p-ERK1/2, ERK, FOXO3a, FOXO1, myostatin, and follistatin were analyzed by using Western blot analysis or real-time PCR. The diameter of myotubes was measured. Recruitment of glucocorticoid receptor (GR) or FOXO3a was analyzed by performing a chromatin immunoprecipitation (ChIP) assay. Results Mountain ginseng treatment reduced muscle weight loss and collagen deposition in DEXA-induced rats. Mountain ginseng treatment led to decreases in MuRF1, atrogin1, p-ERK1/2, FOXO3a, FOXO1, and myostatin. Also, mountain ginseng treatment led to increases in the diameter of myotubes, MyHC, HSP90, p-Akt, and follistatin. Treatment with mountain ginseng reduced enrichment of GR, FOXO3a, and RNA polymerase II on the promoters. Conclusions These results suggest that mountain ginseng inhibits skeletal muscle atrophy by decreasing MuRF1 and atrogin1 through FOXO3a in L6 myotubes.

Combined oral contraceptive (COC) use is associated with increased risk of developing hypertension. Activation of the intrarenal renin-angiotensin system (RAS) and endothelial dysfunction play an important role in the development of hypertension. We tested the hypothesis that COC causes hypertension that is associated with endothelial dysfunction and upregulation of intrarenal angiotensin-converting enzyme 1 (Ace1) and angiotensin II type 1 receptor (At1r). Female Sprague-Dawley rats aged 12 weeks received (p.o.) olive oil (control) and a combination of 0.1 μg ethinylestradiol and 1.0 μg norgestrel (low COC) or 1.0 μg ethinylestradiol and 10.0 μg norgestrel (high COC) daily for 6 weeks. Blood pressure was recorded by tail cuff plethysmography. Expression of genes in kidney cortex was determined by quantitative real-time polymerase chain reaction. COC treatment led to increased blood pressure, circulating uric acid, C-reactive protein and plasminogen activator inhibitor-1, renal uric acid, and expression of renal Ace1 and At1r. COC treatment resulted in increased contractile responses to phenylephrine in endothelium-denuded aortic rings. Endothelium-dependent relaxation responses to acetylcholine, but not endothelium-independent relaxation responses to nitric oxide (NO) donation by sodium nitroprusside, were attenuated in COC-exposed rings. Impaired relaxation responses to acetylcholine were masked by the presence of NO synthase inhibitor (L-NAME) in the COC-exposed rings, whereas the responses to acetylcholine in the presence of selective cyclooxygenase-2 inhibitor (NS-398) were enhanced. These findings indicate that COC induces hypertension that is accompanied by endothelial dysfunction, upregulated intrarenal Ace1 and At1r expression, and elevated proinflammatory biomarkers.

Inhibition of lysine deacetylase (KDAC) attenuated development of hypertension in spontaneously hypertensive rats (SHRs). We hypothesized that KDAC inhibition attenuates hypertension and is accompanied by acetylation of mineralocorticoid receptors (MR) instead of histone acetylation in SHRs. Valproate (VPA, 0.71 % wt/vol), an inhibitor of class I KDACs, was administered in drinking water to 7-week-old SHRs and Wistar Kyoto rats for 11 weeks. MR acetylation was determined by immunoprecipitation with anti-MR antibody followed by western blot with anti-acetyl-lysine antibody. Expression levels of acetylated histone H3, KDACs, MR target genes, or MR corepressors in the kidney cortex were measured by using western blot analysis or real-time PCR. Recruitment of MR and RNA polymerase II (Pol II) and histone modifications on promoters of target genes were analyzed by performing a chromatin immunoprecipitation (ChIP) assay. Treatment of SHR with VPA increased MR acetylation without affecting MR expression, which attenuated development of hypertension in SHR VPA decreased expression of KDAC class I but globally increased acetylated histone H3. Although VPA treatment increased histone 3 acetylation (H3Ac) and trimethylation of the fourth lysine (H3K4me3) in the promoter regions of MR target genes, it decreased the expression of target genes as well as recruitment of MR and Pol II. These results suggest that KDAC inhibition attenuates the development of hypertension in SHRs and is accompanied by acetylation of MR that is independent of histone acetylation.

Type 2 diabetes mellitus (T2DM) is a chronic disease manifested by hyperglycemia. It is essential to effectively control hyperglycemia to prevent complications of T2DM. Here, we hypothesize that repression of transcriptional activity of forkhead box O1 (FoxO1) via histone deacetylase inhibitors (HDACi) ameliorates hyperglycemia in T2DM rats. Methods: Male Long-Evans Tokushima Otsuka (LETO) and Otsuka Long-Evans Tokushima Fatty (OLETF) rats aged 14 weeks were administered sodium valproate (VPA, 0.71% w/v) dissolved in water for 20 weeks. Electrophoretic mobility shift assay (EMSA) and luciferase assay were performed for elucidation of transcriptional regulation through acetylation of FoxO1 by HDACi. Results: VPA attenuated blood glucose levels in accordance with a decrease in the expression of gluconeogenic genes in hyperglycemic OLETF rats. It has been shown that HDAC class I-specific and HDAC class IIa-specific inhibitors, as well as pan-HDAC inhibitors decrease FoxO1 enrichment at the cis-element of target gene promoters. Mutations in FoxO1 prevent its acetylation, thereby increasing its transcriptional activity. HDAC3 and HDAC4 interact with FoxO1, and knockdown of HDAC3, HDAC4, or their combination increases FoxO1 acetylation, thereby decreasing the expression of gluconeogenic genes. Conclusions: These results indicate that HDACi attenuates the transcriptional activity of FoxO1 by impeding deacetylation, thereby ameliorating hyperglycemia in T2DM rats.

Gallic acid (GA) has been reported to have beneficial effects on cancer, vascular calcification, and diabetes-induced myocardial dysfunction. We hypothesized that GA controls hypertension via oxidative stress response regulation in an animal model for essential hypertension. Spontaneously hypertensive rats (SHRs) were administered GA for 16 weeks. GA treatment lowered elevated systolic blood pressure in SHRs through the inhibition of vascular contractility and components of the renin-angiotensin II system. In addition, GA administration reduced aortic wall thickness and body weight in SHRs. In SHRs, GA attenuated left ventricular hypertrophy and reduced the expression of cardiac-specific transcription factors. NADPH oxidase 2 (Nox2) and GATA4 mRNA expression was induced in SHR hearts and angiotensin II-treated H9c2 cells; this expression was downregulated by GA treatment. Nox2 promoter activity was increased by the synergistic action of GATA4 and Nkx2-5. GA seems to regulate oxidative stress by inhibiting the DNA binding activity of GATA4 in the rat Nox2 promoter. GA reduced the GATA4-induced Nox activity in SHRs and angiotensin II-treated H9c2 cells. GA administration reduced the elevation of malondialdehyde levels in heart tissue obtained from SHRs. These findings suggest that GA is a potential therapeutic agent for treating cardiac hypertrophy and oxidative stress in SHRs.

Objectives The G protein-coupled oestrogen receptor-1 (GPER-1) agonist G1 induces endothelium-dependent relaxation. Activation of the epidermal growth factor (EGF) receptor leads to transduction of signals from the plasma membrane for the release of nitric oxide. We tested the hypothesis that G1 induces endothelium-dependent vasorelaxation through activation of the EGF receptor. Methods Rat aortic rings were mounted in organ baths. After pretreatment with various inhibitors, aortic rings contracted with 11,9-epoxymethano-prostaglandin F2α or KCl were subjected to relaxation by G1. Key findings G1 induced endothelium-dependent vasorelaxation, which was attenuated by pretreatment with either L-Nω-nitroarginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, or (3aS,4R,9bR)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline HB-EGF, heparin-binding EGF-like growth factor, a GPER-1 antagonist. Neither a general oestrogen receptor antagonist, ICI 182 780, nor a selective oestrogen receptor-α antagonist, methyl-piperidino-pyrazole dihydrochloride (MPP), had an effect on G1-induced vasorelaxation. However, pretreatment with EGF receptor blockers, AG1478 or DAPH, resulted in attenuated G1-induced vasorelaxation. In addition, pretreatment with Src inhibitor 4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine or Akt inhibitor VIII also resulted in attenuated vascular relaxation induced by the cumulative addition of G1. However, neither phosphatidylinositol-3 kinase inhibitors LY294002 and wortmannin nor an extracellular signal-regulated kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene monoethanolate had effect on vascular relaxation induced by the cumulative addition of G1. Conclusions G1 induces endothelium-dependent vasorelaxation through Src-mediated activation of the EGF receptor and the Akt pathway in rat aorta.

It is of interest to investigate whether synthetic thioflavonoids have vasorelaxant actions as natural flavonoids. We tested the hypothesis that 3',4'-dimethoxythioflavone induces endothelium-dependent vasorelaxation through activation of epidermal growth factor (EGF) receptor. Rat aortic rings were mounted in organ baths and subjected to relaxation upon contraction. 3',4'-Dimethoxythioflavone induced endothelium-dependent vasorelaxation, which was attenuated by pretreatment with either L-N (ω)-nitroarginine methyl ester, an inhibitor of nitric oxide synthase, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase. 3',4'-Dimethoxythioflavone-induced vasorelaxation was not affected by pretreatment with a general estrogen receptor antagonist ICI 182,780, a selective estrogen receptor-α antagonist methyl-piperidino-pyrazole dihydrochloride, or a G protein-coupled receptor 30 antagonist G15. However, pretreatment with EGF receptor blockers AG1478 or DAPH, but not with a phosphatidylinositol-3 kinase inhibitor LY294002 or an Akt1/2 kinase inhibitor Akt inhibitor VIII, attenuated 3',4'-dimethoxythioflavone-induced vasorelaxation. In addition, pretreatment with a Src inhibitor PP2 or an ERK inhibitor U0126 also attenuated vascular relaxation induced by the cumulative addition of 3',4'-dimethoxythioflavone. However, neither a mitochondrial electron transport inhibitor rotenone, an NADPH oxidase inhibitor apocynin, nor a superoxide dismutase mimetic MnTMPyP affected the vascular relaxation induced by the cumulative addition of 3',4'-dimethoxythioflavone. In conclusion, 3',4'-dimethoxythioflavone induces endothelium-dependent vasorelaxation through activation of EGF receptor and Src/ERK pathway in rat aorta.

Clinical studies have shown that combined oral contraceptive (COC) use is associated with cardiometabolic disturbances. Elevated renin-angiotensin system (RAS) and plasminogen activator inhibitor-1 (PAI-1) have also been implicated in the development of cardiometabolic events.To determine the effect of COC treatment on cardiac RAS and PAI-1 gene expressions, and whether the effect is circulating aldosterone or corticosterone dependent.Female rats were treated (p.o.) with olive oil (vehicle) or COC (1.0 µg ethinylestradiol and 10.0 µg norgestrel) daily for six weeks.COC treatment led to increases in blood pressure, HOMA-IR, Ace1 mRNA, Atr1 mRNA, Pai1 mRNA, cardiac PAI-1, plasma PAI-1, C-reactive protein, uric acid, insulin and corticosterone. COC treatment also led to dyslipidemia, decreased glucose tolerance and plasma 17β-estradiol.These results demonstrates that hypertension and insulin resistance induced by COC is associated with increased cardiac RAS and PAI-1 gene expression, which is likely to be through corticosterone-dependent but not aldosterone-dependent mechanism.

The Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) is upregulated in diverse models of hypertension. We hypothesized that NKCC1 is upregulated via histone modification in the aortas of angiotensin II (Ang II)-induced hypertensive rats. An osmotic mini-pump containing Ang II was implanted in the subcutaneous tissues of the backs of Sprague-Dawley (SD) rats for 7 days. The systolic blood pressure was recorded every day by the tail-cuff method. On days 3 and 7, the mesenteric arteries were excised, cut into rings, mounted in organ baths and subjected to vascular contraction. The levels of Nkcc1 mRNA and protein in the aortas were measured using real-time PCR and Western blotting, respectively. The histone modifications and recruited proteins at the Nkcc1 promoter were determined by chromatin immunoprecipitation. The inhibition of concentration-response curves to phenylephrine by bumetanide, an inhibitor of NKCCs, was greater in Ang II-infused rats than in sham-operated (sham) rats . The levels of Nkcc1 mRNA and protein in the aortas increased gradually as Ang II was infused into the rats. Acetylated histone H3 (H3Ac), an activating histone code, was increased but trimethylated histone H3 at lysine 27 (H3K27me3), a repressive histone code, was greatly decreased in Ang II-infused rats compared with sham. RNA polymerase II was recruited to the Nkcc1 promoter with increased KDM6b. We conclude that the NKCC1 is upregulated via histone modification in the aortas of Ang II-induced hypertensive rats. Thus, we suggest that this ion transporter is epigenetically upregulated by histone modification or DNA demethylation upon the development of hypertension.

1. Magnolol (5,5'-diallyl-2,2'-dihydroxybiphenyl) is a major phenolic compound purified from Magnolia officinalis. The aim of the present study was to elucidate the effects of magnolol on vascular contractions. 2. Rat aortic rings were mounted in organ baths. Magnolol was added cumulatively (0.3-30 μmol/L) to elicit relaxation in endothelium-intact and -denuded rat aortic rings precontracted with U46619 (30 nmol/L, 20 min), NaF (8.0 mmol/L, 40 min), phenylephrine (1.0 or 0.1 μmol/L, 15 min) or phorbol-12,13-dibutyrate (PDBu, 0.3 or 0.1 μmol/L, 40 min). In separate experiments, cumulative concentrationresponse curves were obtained for NaF (2.0-12 mmol/L), U46619 (1.0 nmol/L1.0 μmol/L) or PDBu (1.0 nmol/L1.0 μmol/L) after pretreatment with either magnolol or vehicle for 30 min in endothelium-denuded aortic rings. After completion of the functional study, we measured the amount of guanosine triphosphate (GTP) RhoA by using a G-LISA RhoA Activation Assay, as well as the phosphorylation of 20 kDa myosin light chain (MLC₂₀), myosin phosphatase-targeting subunit 1 (MYPT1) and protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light-chain phosphatase of 17 kDa (CPI-17) by immunobloting. 3. Magnolol (0.3-30 μmol/L) reduced vascular tension induced by the thromboxane A₂ agonist U46619 (30 nmol/L), sodium fluoride (NaF) (8.0 mmol/L) and the α₁ -adrenoceptor agonist phenylephrine (1.0 or 0.1 μmol/L) in both endothelium-intact and -denuded rings. The magnitude of the relaxation effects of magnolol on the contraction induced by each of the drugs were similar. The magnitude of the effect of magnolol in endothelium-intact and -denuded rings were similar. 4. Pretreatment of rat aortic rings with 1.0, 3.0 or 10 μmol/L magnolol for 30 min dose-dependently inhibited the maximum response on the concentration-response curves to NaF and U46619, but not to phorbol-12,13-dibutyrate (PDBu). 5. Magnolol (3.0 or 10 μmol/L) decreased RhoA activation, as well as the phosphorylation of MLC₂₀ , MYPT1(Thr855) and CPI-17(Thr38) induced by either 8.0 mmol/L NaF or 30 nmol/L U46619. In contrast, magnolol did not affect PDBu (0.1 μmol/L)-induced phosphorylation of CPI-17(Thr38) . 6. In conclusion, magnolol reduces vascular contraction by inhibiting the RhoA/Rho kinase pathway in endothelium-denuded rat aorta.

3',4'-Dihydroxyflavonol (DiOHF) exerts endothelium-independent relaxation in rat aortic rings. In this study, we hypothesized that DiOHF reduces vascular contraction through Ca²⁺ desensitization in permeabilized third-order branches of rat mesenteric arteries. The third-order branches of rat mesenteric arteries were permeabilized with β-escin and subjected to tension measurement. Cumulative addition of phenylephrine (0.3-30 μM) produced concentration-dependent vascular contraction of endothelium-intact and endothelium-denuded arterial rings, which were inhibited by pretreatment with DiOHF (10, 30, or 100 μM). In addition, DiOHF dose-dependently decreased vascular contractions induced by 3.0 μM phenylephrine. β-Escin-permeabilized third-order branches of mesenteric arteries were contracted with Ca²⁺, NaF, or guanosine-5'-(γ-thio)triphosphate (GTPγS) 30 min after pretreatment with DiOHF or vehicle. Pretreatment with DiOHF for 30 min inhibited vascular contraction induced by cumulative additions of Ca²⁺ (pCa 9.0-6.0) or NaF (4.0-16.0 mM) in permeabilized arterial rings. Cumulative addition of DiOHF also reduced vascular contraction induced by Ca²⁺-controlled solution of pCa 6.0, 16.0 mM NaF, or 100 μM GTPγS in permeabilized arterial rings. DiOHF inhibited the increase in vascular tension provoked by calyculin A, even though it did not affect vascular tension already produced by calyculin A. DiOHF accelerated the relaxation induced by rapidly lowering Ca²⁺. DiOHF reduced vascular contraction through Ca²⁺ desensitization in permeabilized third-order branches of rat mesenteric arteries. These results suggest that DiOHF may have a therapeutic potential in the treatment of cardiovascular diseases.

Objectives Honokiol is a small-molecule polyphenol isolated from the species Magnolia obovata. We hypothesized that honokiol attenuated vascular contractions through the inhibition of the RhoA/Rho-kinase signalling pathway. Methods Rat aortic rings were denuded of endothelium, mounted in organ baths, and subjected to contraction or relaxation. Phosphorylation of 20 kDa myosin light chains (MLC20), myosin phosphatase targeting subunit 1 (MYPT1) and protein kinase C (PKC)-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase (MLCP) of 17 kDa (CPI17) were examined by immunoblot. We also measured the amount of guanosine triphosphate RhoA as a marker for RhoA activation. Key findings Pretreatment with honokiol dose-dependently inhibited the concentration–response curves in response to sodium fluoride (NaF) or thromboxane A2 agonist U46619. Honokiol decreased the phosphorylation levels of MLC20, MYPT1Thr855 and CPI17Thr38 as well as the activation of RhoA induced by 8.0 mm NaF or 30 nm U46619. Conclusions These results demonstrated that honokiol attenuated vascular contraction through the inhibition of the RhoA/Rho-kinase signalling pathway.

金沢大学医薬保健研究域医学系, Gomisin A (GA) is an active ingredient of the fruits of Schisandra chinensis which has been widely used as a tonic in traditional Korean medicine. GA induces not only endothelium-dependent but also endothelium-independent relaxation in an isolated rat's thoracic aorta. This study was aimed to investigate the molecular mechanism by which GA induces endothelium-independent vasorelaxation. Rat aortic rings were denuded of endothelium, mounted in organ baths, and subjected to contraction or relaxation. We measured the amount of GTP RhoA as well as the phosphorylation level of 20 kDa myosin light chains (MLC20), myosin phosphatase-targeting subunit 1 (MYPT1) and protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light-chain phosphatase of 17 kDa (CPI17). Pretreatment with GA dose-dependently inhibited the concentration-response curves in response to sodium fluoride (NaF) or thromboxane A2 agonist U46619, but not to phorbol 12, 13-dibutyrate (PDBu). GA decreased the activation of RhoA as well as the phosphorylation level of MLC20, MYPT1Thr855, and CPI17 induced by 8.0 mM NaF or 30 nM U46619. However, K+ channel blockers such as glibenclamide, apamin, or charybdotoxin did not affect the vascular relaxation induced by GA. Furthermore, GA did not affect the level of phosphorylation of CPI17 induced by PDBu. GA reduces vascular contraction through inhibition of RhoA/Rho-kinase pathway in endothelium-denuded rat aorta. © 2010 Springer-Verlag.

The Na+–K+–2Cl− cotransporter 1 (NKCC1) is one of several transporters that have been implicated for development of hypertension since NKCC1 activity is elevated in hypertensive aorta and vascular contractions are inhibited by bumetanide, an inhibitor of NKCC1. We hypothesized that promoter hypomethylation upregulates the NKCC1 in spontaneously hypertensive rats (SHR). Thoracic aortae and mesenteric arteries were excised, cut into rings, mounted in organ baths and subjected to vascular contraction. The expression levels of nkcc1 mRNA and protein in aortae and heart tissues were measured by real-time PCR and Western blot, respectively. The methylation status of nkcc1 promoter region was analyzed by combined bisulfite restriction assay (COBRA) and bisulfite sequencing. Phenylephrine-induced vascular contraction in a dose-dependent manner, which was inhibited by bumetanide. The inhibition of dose–response curves by bumetanide was much greater in SHR than in Wistar Kyoto (WKY) normotensive rats. The expression levels of nkcc1 mRNA and of NKCC1 protein in aortae and heart tissues were higher in SHR than in WKY. Nkcc1 gene promoter was hypomethylated in aortae and heart than those of WKY. These results suggest that promoter hypomethylation upregulates the NKCC1 expression in aortae and heart of SHR.

The purpose of this paper was to describe the diversity of learners within the Korean community colleges. Students were mostly from the Seoul metropolitan city and the Gyeonggi Province areas, and foreign students were mostly from China. The results show that college students’ learning styles were mainly of diverger and accommodator types. Seventy percent of the students planned to gain employments after graduation. The diversity of students was not a big issue in the college teaching and learning situations, but the college-level support for teaching and learning existed. The results point to some suggestions for the education of various learners.

1. Phorbol-12,13-dibutyrate (PDBu) is an activator of protein kinase C (PKC) that causes contractions in both physiological salt solutions and Ca(2+)-depleted solutions. In the present study, we tested the hypothesis that Rho-kinase plays a role in Ca(2+)-independent contractions induced by PDBu in vascular smooth muscles. 2. In Ca(2+)-free solution, 0.1 and 1 micromol/L PDBu induced contraction and myosin light chain (MLC(20)) phosphorylation, both of which were approximately 40% of responses obtained in normal Krebs' solution. Hydroxyfasudil (H1152; 1 micromol/L), an inhibitor of Rho-kinase, but not ML7 (10 micromol/L), an inhibitor of myosin light chain kinase, inhibited Ca(2+)-independent contractions induced by PDBu. 3. In Ca(2+)-free solution, PDBu increased phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and CPI-17 (PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa). This action was inhibited by H1152, with the phosphorylation of CPI-17 almost completely abolished by 1 micromol/L Ro31-8220, an inhibitor of PKC. 4. In Ca(2+)-free solution, PDBu increased the amount of GTP-RhoA (an activated form of RhoA). This increase was blocked by the PKC inhibitor Ro31-8220, but not by the Rho kinase inhibitor H1152. 5. In conclusion, RhoA/Rho-kinase plays an important role in Ca(2+)-independent contractions induced by PDBu in vascular smooth muscles. The results of the present study suggest that PDBu induces Ca(2+)-independent contractions by inhibiting myosin light chain phospatase (MLCP) through activation of GTP-RhoA and subsequent phosphorylation of MYPT1 and CPI-17.

Isoflavones decrease blood pressure, improve lipid profiles, and restore vascular function. We hypothesized that isoflavone attenuates vascular contraction by inhibiting RhoA/Rho-kinase signaling pathway. Rat aortic rings were denuded of endothelium, mounted in organ baths, and contracted with 11,9 epoxymethano-prostaglandin F(2alpha) (U46619), a thromboxane A2 analog, or KCl 30 min after the pretreatment with genistein (4',5,7-trihydroxyisoflavone), daidzein (4',7-dihydroxyisoflavone), or vehicle. We determined the phosphorylation level of the myosin light chain (MLC(20)), myosin phosphatase-targeting subunit 1 (MYPT1), and protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light-chain phosphatase of 17 kDa (CPI17) by means of the Western blot. We also measured the amount of GTP RhoA as a marker regarding RhoA activation. The cumulative additions of U46619 or KCl increased vascular tension in a concentration-dependent manner, which were inhibited by pretreatment with genistein or daidzein. Both U46619 (30 nM) and KCl (50 mM) increased MLC(20) phosphorylation levels, which were inhibited by genistein and daidzein. Furthermore, both genistein and daidzein decreased the amount of GTP RhoA activated by either U46619 or KCl. U46619 (30 nM) increased phosphorylation of the MYPT1(Thr855) and CPI17(Thr38), which were also inhibited by genistein or daidzein. However, neither genistein nor daidzein inhibited phorbol 12,13-dibutyrate-induced vascular contraction and CPI17 phosphorylation. In conclusion, isoflavone attenuates vascular contraction, at least in part, through inhibition of the RhoA/Rho-kinase signaling pathway.

Renal fibrosis is highly implicated as a cause of chronic renal failure, for which suitable therapeutics have not yet been developed. Recently, it was reported that Wen-pi-tang-Hab-Wu-ling-san (WHW) extract attenuates epithelial cells undergoing mesenchymal transition in cultured Madin-Darby canine kidney cells. This study investigated whether WHW extract prevents renal fibrosis induced by ischemia/reperfusion (I/R) in mice. Ischemia/reperfusion resulted in kidney fibrosis at 14 days after the procedure. When WHW was administered orally to mice beginning from 2 days after the onset of ischemia until killing, the fibrosis was significantly reduced. WHW administration significantly prevented a post-ischemic decrease of copper-zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD) activities, leading to decreased lipid peroxidation and hydrogen peroxide production. In addition, WHW administration attenuated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase 1/2 (JNK1/2) and attenuated the activation of nuclear factor-kappa B (NF-κB) in the kidneys subjected to ischemia. In conclusion, WHW extract attenuated the renal fibrosis and the attenuation was associated with a reduction of oxidative stress and an inhibition of ERK1/2, JNK1/2, p38 and NF-κB activation. WHW extract may be an attractive agent to attenuate the progression of fibrosis. Copyright © 2008 John Wiley & Sons, Ltd.

The purpose of this study was to investigate the effects of Wen-pi-tang-Hab-Wu-ling-san (WHW) extract, which has been used for treatment of renal diseases, on kidney ischemia/reperfusion (I/R) injury. Thirty minutes of bilateral renal ischemia resulted in disruption of kidney tubular epithelial cells and increased plasma creatinine levels in mice, however these effects were significantly attenuated when WHW was administered prior to I/R. WHW-administration also inhibited post-ischemic decreases of catalase, copper–zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD) activity in kidney tissue, leading to decreased tissue hydrogen peroxide levels and lipid peroxidation. Post-ischemic increases of heat-shock protein (HSP)-27 and -72 expressions were greater in mouse kidneys that received WHW. In conclusion, WHW-administration reduced kidney susceptibility to I/R injury, and this reduced susceptibility was associated with greater post-ischemic activation of catalase, CuZnSOD and MnSOD, resulting in reduced hydrogen peroxide levels and lipid peroxidation, as well as higher post-ischemic expression of HSP-27 and -72.

Our previous study demonstrated that heat shock augmented vascular contraction. In the present study, we hypothesized that heat shock augments myosin phosphatase target-subunit (MYPT1) phosphorylation resulting in augmented vascular contraction. Endothelium-denuded rat aortic rings were mounted in organ baths, exposed to heat shock (42 degrees C for 45 min), and subjected to contraction 4 h after the heat shock followed by Western blot analysis for MLC(20) (the 20 kDa light chains of myosin II) or MYPT1. The contractile responses in both control and heat shock-treated aorta were inhibited by Y27632, an inhibitor of Rho-kinase. The level of the MLC(20) and MYPT1(Thr855) phosphorylation in response to KCl was higher in heat shock-treated aorta than that in timed-control. The increased MYPT1(Thr855) phosphorylation was inhibited by Y27632 (1.0 microM) in parallel with inhibition of MLC(20) phosphorylation and vascular contraction. These results indicate that heat shock augments MYPT1 phosphorylation resulting in augmented vascular contraction.

Clinical studies have shown that the use of combined oral contraceptive in pre-menopausal women is associated with fluid retention. However, the molecular mechanism is still elusive. We hypothesized that combined oral contraceptive (COC) ethinyl estradiol (EE) and norgestrel (N) synergistically activates mineralocorticoid receptor (MR) through histone code modifications. Twelve-week-old female Sprague-Dawley rats were treated with olive oil (control), a combination of 0.1µg EE and 1.0µg N (low COC) or 1.0µg EE and 10.0µg N (high COC) as well as 0.1 or 1.0µg EE and 1.0 or 10.0µg N daily for 6 weeks. Expression of MR target genes in kidney cortex was determined by quantitative real-time polymerase chain reaction. MR was quantified by western blot. Recruitment of MR and RNA polymerase II (Pol II) on promoters of target genes as well as histone code modifications was analyzed by chromatin immunoprecipitation assay. Treatment with COC increased renal cortical expression of MR target genes such as serum and glucocorticoid-regulated kinase 1 (Sgk-1), glucocorticoid-induced leucine zipper (Gilz), epithelial Na(+)channel (Enac) and Na(+)-K(+)-ATPase subunit α1 (Atp1a1). Although COC increased neither serum aldosterone nor MR expression in kidney cortex, it increased recruitment of MR and Pol II in parallel with increased H3Ac and H3K4me3 on the promoter regions of MR target genes. However, treatment with EE or N alone did not affect renal cortical expression of Sgk-1, Gilz, Enac or Atp1a1. These results indicate that COC synergistically activates MR through histone code modifications.

Inhibition of histone deacetylases (HDACs) by valproic acid (VPA) attenuates inflammatory, hypertrophic, and fibrotic responses in the hearts of spontaneously hypertensive rats (SHRs); however, the molecular mechanism is still unclear. We hypothesized that HDAC inhibition (HDACi) attenuates cardiac hypertrophy and fibrosis through acetylation of mineralocorticoid receptor (MR) in SHRs. Seven-week-old SHRs and Wistar-Kyoto rats were treated with an HDAC class I inhibitor (0.71% w/v in drinking water; VPA) for 11 weeks. Sections of heart were visualized after trichrome stain as well as H&E stain. Histone modifications, such as acetylation (H3Ac [acetylated histone 3]) and fourth lysine trimethylation (H3K4me3) of histone 3, and recruitment of MR and RNA polymerase II (Pol II) into promoters of target genes were measured by quantitative real-time polymerase chain reaction after chromatin immunoprecipitation assay. MR acetylation was determined by Western blot with anti-acetyl-lysine antibody after immunoprecipitation with anti-MR antibody. Treatment with VPA attenuated cardiac hypertrophy and fibrosis. Although treatment with VPA increased H3Ac and H3K4me3 on promoter regions of MR target genes, expression of MR target genes as well as recruitment of MR and Pol II on promoters of target genes were decreased. Although HDACi did not affect MR expression, it increased MR acetylation. These results indicate that HDACi attenuates cardiac hypertrophy and fibrosis through acetylation of MR in spontaneously hypertensive rats.

Males are much more susceptible to ischemia/reperfusion (I/R)-induced kidney injury when compared with females. Recently we reported that the presence of testosterone, rather than the absence of estrogen, plays a critical role in gender differences in kidney susceptibility to I/R injury in mice. Although reactive oxygen species and antioxidant defenses have been implicated in I/R injury, their roles remain to be defined. Here we report that the orchiectomized animal had significantly less lipid peroxidation and lower hydrogen peroxide levels in the kidney 4 and 24 h after 30 min of bilateral renal ischemia when compared with intact or dihydrotestosterone-treated orchiectomized males. The post-ischemic kidney expression and activity of manganese superoxide dismutase (MnSOD) in orchiectomized mice was much greater than in intact or dihydrotestosterone-administered orchiectomized mice. Four hours after 30 min of bilateral ischemia, superoxide formation was significantly lower in orchiectomized mice than in intact mice. In Madin-Darby canine kidney cells, a kidney epithelial cell line, 1 mm H(2)O(2) decreased MnSOD activity, an effect that was potentiated by pretreatment with dihydrotestosterone. Orchiectomy prevented the post-ischemic decrease of catalase activity. Treatment of male mice with manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a SOD mimetic, reduced the post-ischemic increase of plasma creatinine, lipid peroxidation, and tissue hydrogen peroxide. These results suggest that orchiectomy accelerates the post-ischemic activation of MnSOD and reduces reactive oxygen species and lipid peroxidation, resulting in reduced kidney susceptibility to I/R injury.

SUMMARY 1 In a previous study, we demonstrated that heat shock augments the contractility of vascular smooth muscle through the stress response. 2 In the present study, we investigated whether Rho-kinases play a role in heat shock-induced augmentation of vascular contractility in rat isolated aorta. 3 Rat aortic strips were mounted in organ baths, exposed to 42C for 45 min and subjected to contractile or relaxant agents 5 h later. 4 The level of expression of Rho-kinases in heat shock-exposed tissues was no different to that of control tissues, whereas heat shock induced heat shock protein (Hsp) 72 at 3 and 5 h. Heat shock resulted in an increase in vascular contractility in response to phenylephrine 5 h later. 5 The Rho-kinase inhibitors Y27632 (30 nmol/L-10 mmol/L) or HA 1077 (10 nmol/L-10 mmol/L) relaxed 1.0 mmol/L phenylephrine-precontracted vascular strips in a concentration-dependent manner; these effects were attenuated in heat shock-exposed strips. Pretreatment with Y27632 resulted in greater inhibition of the maximum contraction in control strips compared with those in heat shock-exposed strips. 6 The results of the present study suggest that Rho-kinases are unlikely to be involved in heat shock-induced augmentation of vascular contractility.

Summary Acetylation of the mineralocorticoid receptor (MR) by inhibition of lysine deacetylases attenuates MR's transcriptional activity. However, the specific lysine acetyltransferases that are responsible for acetylation of the MR remain unknown. We hypothesized that the acetyltransferases cyclic adenosine monophosphate response element-binding binding protein (CBP) and acetyltransferase p300 (p300) attenuate transcriptional activity of the MR through its acetylation. Expression of MR target genes was measured by quantitative real-time polymerase chain reaction. Recruitment of MR and RNA polymerase II (Pol II) on promoters of target genes was analysed by chromatin immunoprecipitation. Acetylation of the MR was determined by western blot with an anti-acetyl-lysine antibody after immunoprecipitation with an anti-MR antibody. In human embryonic kidney (HEK) 293 cells, overexpression of CBP or p300, but not p300/CBP-associated factor, increased MR acetylation and decreased expression of MR target genes. The downregulation of target genes coincided with a decrease in the recruitment of MR and Pol II to specific hormone response elements. These results demonstrate that overexpression of CBP or p300 attenuates the transcriptional activity of the MR through its acetylation in HEK 293 cells. Our data provide strong evidence identifying CBP and p300 as lysine acetyltransferases responsible for the regulation of MR that may provide new therapeutic targets for the treatment of hypertension.

Brief exposure to cobalt chloride augmented vascular contractility. We hypothesized that endothelial dysfunction plays a role in the augmentation of aortic contractility, after brief exposure to cobalt chloride. Rat aortic ring preparations were mounted in organ baths, exposed to cobalt chloride (0.3–300 μmol/L) for 30 min, and then subjected to contractile agents or relaxants 1 and 5 h after the end of exposure. Presence of cobalt chloride did not affect the contractile response to phenylephrine. Brief exposure to cobalt chloride, however, even at 5 h after the end of exposure, not only augmented contractile responses to KCl or phenylephrine but also attenuated the relaxant response to acetylcholine. The mechanical denudation of endothelium or inhibition of endothelial nitric oxide synthase with 100 μmol/L Nω-nitro- l -arginine methyl ester abolished the augmentation of contractile responses. Pre-treatment with 150 units/mL of superoxide dismutase also abrogated the augmented contractile responses. Brief exposure to cobalt chloride did not affect the contractile response to phorbol dibutyrate in the presence or absence of calcium, or the expression of HSP70. In conclusion, endothelial dysfunction plays an important role in the augmentation of aortic contractility, after brief exposure to cobalt chloride.

Isoflavone consumption correlates with reduced rates of cardiovascular disease. Epidemiological studies and clinical data provide evidence that isoflavone metabolites, such as the isoflavan equol, contribute to these beneficial effects. In this study we developed a new route to isoflavans and isoflavenes via 2-morpholinoisoflavenes derived from a condensation reaction of phenylacetaldehydes, salicylaldehydes and morpholine. We report the synthesis of the isoflavans equol and deoxygenated analogues, and the isoflavenes 7,4'-dihydroxyisoflav-3-ene (phenoxodiol, haganin E) and 7,4'-dihydroxyisoflav-2-ene (isophenoxodiol). Vascular pharmacology studies reveal that all oxygenated isoflavans and isoflavenes can attenuate phenylephrine-induced vasoconstriction, which was unaffected by the estrogen receptor antagonist ICI 182,780. Furthermore, the compounds inhibited U46619 (a thromboxane A(2) analogue) induced vasoconstriction in endothelium-denuded rat aortae, and reduced the formation of GTP RhoA, with the effects being greatest for equol and phenoxodiol. Ligand displacement studies of rat uterine cytosol estrogen receptor revealed the compounds to be generally weak binders. These data are consistent with the vasorelaxation activity of equol and phenoxodiol deriving at least in part by inhibition of the RhoA/Rho-kinase pathway, and along with the limited estrogen receptor affinity supports a role for equol and phenoxodiol as useful agents for maintaining cardiovascular function with limited estrogenic effects.

17β-Estradiol (E2) exerts rapid non-genomic vascular effects through activation of its plasma membrane receptors. We tested the hypothesis that E2 induces vasorelaxation through activation of the G-protein-coupled receptor 30 (GPR30) in rat aorta. Rat aortic rings were mounted in organ baths and subjected to contraction followed by relaxation. Whether endothelium was intact or denuded, both E2 and G1, a GPR30 agonist, induced vasorelaxation in concentration-dependent manners. Although G15, a specific GPR30 antagonist, blocked G1-induced vasorelaxation, it did not block E2-induced vasorelaxation. In conclusion, 17β-estradiol induces vasorelaxation in a GPR30-independent manner in rat aorta.

1. Magnolol (5,5'-diallyl-2,2'-dihydroxybiphenyl) is a major phenolic compound purified from Magnolia officinalis. The aim of the present study was to elucidate the effects of magnolol on vascular contractions. 2. Rat aortic rings were mounted in organ baths. Magnolol was added cumulatively (0.3-30 μmol/L) to elicit relaxation in endothelium-intact and -denuded rat aortic rings precontracted with U46619 (30 nmol/L, 20 min), NaF (8.0 mmol/L, 40 min), phenylephrine (1.0 or 0.1 μmol/L, 15 min) or phorbol-12,13-dibutyrate (PDBu, 0.3 or 0.1 μmol/L, 40 min). In separate experiments, cumulative concentrationresponse curves were obtained for NaF (2.0-12 mmol/L), U46619 (1.0 nmol/L1.0 μmol/L) or PDBu (1.0 nmol/L1.0 μmol/L) after pretreatment with either magnolol or vehicle for 30 min in endothelium-denuded aortic rings. After completion of the functional study, we measured the amount of guanosine triphosphate (GTP) RhoA by using a G-LISA RhoA Activation Assay, as well as the phosphorylation of 20 kDa myosin light chain (MLC₂₀), myosin phosphatase-targeting subunit 1 (MYPT1) and protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light-chain phosphatase of 17 kDa (CPI-17) by immunobloting. 3. Magnolol (0.3-30 μmol/L) reduced vascular tension induced by the thromboxane A₂ agonist U46619 (30 nmol/L), sodium fluoride (NaF) (8.0 mmol/L) and the α₁ -adrenoceptor agonist phenylephrine (1.0 or 0.1 μmol/L) in both endothelium-intact and -denuded rings. The magnitude of the relaxation effects of magnolol on the contraction induced by each of the drugs were similar. The magnitude of the effect of magnolol in endothelium-intact and -denuded rings were similar. 4. Pretreatment of rat aortic rings with 1.0, 3.0 or 10 μmol/L magnolol for 30 min dose-dependently inhibited the maximum response on the concentration-response curves to NaF and U46619, but not to phorbol-12,13-dibutyrate (PDBu). 5. Magnolol (3.0 or 10 μmol/L) decreased RhoA activation, as well as the phosphorylation of MLC₂₀ , MYPT1(Thr855) and CPI-17(Thr38) induced by either 8.0 mmol/L NaF or 30 nmol/L U46619. In contrast, magnolol did not affect PDBu (0.1 μmol/L)-induced phosphorylation of CPI-17(Thr38) . 6. In conclusion, magnolol reduces vascular contraction by inhibiting the RhoA/Rho kinase pathway in endothelium-denuded rat aorta.

3',4'-Dihydroxyflavonol (DiOHF) exerts endothelium-independent relaxation in rat aortic rings. In this study, we hypothesized that DiOHF reduces vascular contraction through Ca²⁺ desensitization in permeabilized third-order branches of rat mesenteric arteries. The third-order branches of rat mesenteric arteries were permeabilized with β-escin and subjected to tension measurement. Cumulative addition of phenylephrine (0.3-30 μM) produced concentration-dependent vascular contraction of endothelium-intact and endothelium-denuded arterial rings, which were inhibited by pretreatment with DiOHF (10, 30, or 100 μM). In addition, DiOHF dose-dependently decreased vascular contractions induced by 3.0 μM phenylephrine. β-Escin-permeabilized third-order branches of mesenteric arteries were contracted with Ca²⁺, NaF, or guanosine-5'-(γ-thio)triphosphate (GTPγS) 30 min after pretreatment with DiOHF or vehicle. Pretreatment with DiOHF for 30 min inhibited vascular contraction induced by cumulative additions of Ca²⁺ (pCa 9.0-6.0) or NaF (4.0-16.0 mM) in permeabilized arterial rings. Cumulative addition of DiOHF also reduced vascular contraction induced by Ca²⁺-controlled solution of pCa 6.0, 16.0 mM NaF, or 100 μM GTPγS in permeabilized arterial rings. DiOHF inhibited the increase in vascular tension provoked by calyculin A, even though it did not affect vascular tension already produced by calyculin A. DiOHF accelerated the relaxation induced by rapidly lowering Ca²⁺. DiOHF reduced vascular contraction through Ca²⁺ desensitization in permeabilized third-order branches of rat mesenteric arteries. These results suggest that DiOHF may have a therapeutic potential in the treatment of cardiovascular diseases.

Diabetes is a well-known independent risk factor for vascular disease. However, its underlying mechanism remains unclear. It has been reported that increased influx of the hexosamine biosynthesis pathway (HBP) induces O-GlcNAcylation of proteins, leading to insulin resistance. In this study, we determined whether or not O-GlcNAc modification of proteins could increase vessel contraction. Using an endothelium-denuded aortic ring, we observed that glucosamine induced OGlcNAcylation of proteins and augmented vessel contraction stimulated by U46619, a thromboxane A(2) agonist, via augmentation of the phosphorylation of MLC(20), MYPT1(Thr855), and CPI17, but not phenylephrine. Pretreatment with OGT inhibitor significantly ameliorated glucosamine-induced vessel constriction. Glucosamine treatment also increased RhoA activity, which was also attenuated by OGT inhibitor. In conclusion, glucosamine, a product of glucose influx via the HBP in a diabetic state, increases vascular contraction, at least in part, through activation of the RhoA/Rho kinase pathway, which may be due to O-GlcNAcylation.

金沢大学医薬保健研究域医学系, Rho-mediated inhibition of myosin light chain (MLC) phosphatase (MLCP), together with Ca-dependent MLC kinase activation, constitutes the major signaling mechanisms for vascular smooth muscle contraction. We recently unveiled the involvement of Ca-induced, phosphoinositide 3-kinase (PI3K) class IIα isoform (PI3K-C2α)-dependent Rho activation and resultant Rho kinase-dependent MLCP suppression in membrane depolarization- and receptor agonist-induced contraction. It is unknown whether Ca- and PI3K-C2α- dependent regulation of MLCP is altered in vascular smooth muscle of hypertensive animals and is involved in hypertension. Therefore, we studied the role of the Ca-PI3K-C2α-Rho-MLCP pathway in spontaneously hypertensive rats (SHRs). PI3K-C2α was readily detected in various vascular beds of Wistar-Kyoto rats and activated by high KCl. High KCl also stimulated vascular Rho activity and phosphorylation of the MLCP regulatory subunit MYPT1 at Thr in a PI3K inhibitor wortmannin-sensitive manner. In mesenteric and other vessels of SHRs at the hypertensive but not the prehypertensive stage, the activity of PI3K-C2α but not class I PI3K p110α was elevated with concomitant rises of Rho activity and Thr-phosphorylation of MYPT1, as compared with normotensive controls. Infusion of the Ca channel antagonist nicardipine reduced blood pressure with suppression of vascular activity of PI3K-C2α-Rho and phosphorylation of MYPT1 in hypertensive SHRs. Infusion of wortmannin lowered blood pressure with inhibition of PI3K-C2α-Rho activities and MYPT1 phosphorylation in hypertensive SHRs. These observations suggest that an increased activity of the Ca-PI3K-C2α-Rho signaling pathway with resultant augmented MLCP suppression contributes to hypertension in SHRs. The Ca- and PI3K-C2α-dependent Rho stimulation in vascular smooth muscle may be a novel, promising target for treating hypertension. © 2010 American Heart Association, Inc.

Aim of the study HMC05, an extract from eight different herbal mixtures, has been developed to treat cardiovascular disease. This extract has a vasorelaxant and anti-atherosclerotic action. We hypothesized that HMC05 attenuates vascular contraction through inhibition of the RhoA/Rho-kinase signaling pathway. Materials and methods Rat aortic ring preparations were mounted in organ baths and subjected to contraction and relaxation. Phosphorylation of 20 kDa myosin light chains (MLC 20 ) and myosin phosphatase targeting subunit 1 (MYPT1) were examined by immunoblot. We also measured the amount of GTP RhoA as a marker for RhoA activation. Results In endothelium-denuded aortic ring preparations, HMC05 relaxed vascular contraction induced by 6.0 mM NaF, 100 nM phenylephrine, 30 nM thromboxane A 2 agonist U46619 or 1.0 μM protein kinase C (PKC) activator phorbol-12,13-dibutyrate (PDBu) in a decreasing order. HMC05 relaxed aortic ring preparations precontracted with sodium fluoride (NaF) whether endothelium was intact or denuded. Pre-incubation with HMC05 for 30 min dose-dependently inhibited the NaF-induced contractile response. In vascular strips, HMC05 decreased the phosphorylation level of both MLC 20 and MYPT1 Thr855 induced by 6.0 mM NaF. Furthermore, HMC05 decreased the amount of GTP RhoA activated by NaF. Conclusions HMC05 attenuates vascular contraction through inhibition of the RhoA/Rho-kinase signaling pathway. HMC05 may be useful for the treatment and/or prevention of cardiovascular diseases associated with activation of RhoA/Rho-kinase signaling pathway.

Recently, kidney fibrosis following transplantation has become recognized as a main contributor of chronic allograft nephropathy. In transplantation, transient ischemia is an inescapable event. Reactive oxygen species (ROS) play a critical role in ischemia and reperfusion (I/R)-induced acute kidney injury, as well as progression of fibrosis in various diseases such as hypertension, diabetes, and ureteral obstruction. However, a role of ROS/oxidative stress in chronic kidney fibrosis following I/R injury remains to be defined. In this study, we investigated the involvement of ROS/oxidative stress in kidney fibrosis following kidney I/R in mice. Mice were subjected to 30 min of bilateral kidney ischemia followed by reperfusion on day 0 and then administered with either manganese (III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP, 5 mg/kg body wt ip), a cell permeable superoxide dismutase (SOD) mimetic, or 0.9% saline (vehicle) beginning at 48 h after I/R for 14 days. I/R significantly increased interstitial extension, collagen deposition, apoptosis of tubular epithelial cells, nitrotyrosine expression, hydrogen peroxide production, and lipid peroxidation and decreased copper-zinc SOD, manganese SOD, and glucose 6-phosphate dehydrogenase activities in the kidneys 16 days after the procedure. MnTMPyP administration minimized these postischemic changes. In addition, MnTMPyP administration significantly attenuated the increases of α-smooth muscle actin, PCNA, S100A4, CD68, and heat shock protein 47 expression following I/R. We concluded that kidney fibrosis develops chronically following I/R injury, and this process is associated with the increase of ROS/oxidative stress.

1. Phorbol-12,13-dibutyrate (PDBu) is an activator of protein kinase C (PKC) that causes contractions in both physiological salt solutions and Ca(2+)-depleted solutions. In the present study, we tested the hypothesis that Rho-kinase plays a role in Ca(2+)-independent contractions induced by PDBu in vascular smooth muscles. 2. In Ca(2+)-free solution, 0.1 and 1 micromol/L PDBu induced contraction and myosin light chain (MLC(20)) phosphorylation, both of which were approximately 40% of responses obtained in normal Krebs' solution. Hydroxyfasudil (H1152; 1 micromol/L), an inhibitor of Rho-kinase, but not ML7 (10 micromol/L), an inhibitor of myosin light chain kinase, inhibited Ca(2+)-independent contractions induced by PDBu. 3. In Ca(2+)-free solution, PDBu increased phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and CPI-17 (PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa). This action was inhibited by H1152, with the phosphorylation of CPI-17 almost completely abolished by 1 micromol/L Ro31-8220, an inhibitor of PKC. 4. In Ca(2+)-free solution, PDBu increased the amount of GTP-RhoA (an activated form of RhoA). This increase was blocked by the PKC inhibitor Ro31-8220, but not by the Rho kinase inhibitor H1152. 5. In conclusion, RhoA/Rho-kinase plays an important role in Ca(2+)-independent contractions induced by PDBu in vascular smooth muscles. The results of the present study suggest that PDBu induces Ca(2+)-independent contractions by inhibiting myosin light chain phospatase (MLCP) through activation of GTP-RhoA and subsequent phosphorylation of MYPT1 and CPI-17.

1. Flavonoids modulate vascular tone through an endothelium-dependent or -independent mechanism. Although a few mechanisms for endothelium-independent relaxation have been suggested, such as interference with protein kinase C or cAMP or cGMP phosphodiesterase, the inhibition of Ca(2+) release from intracellular stores or Ca(2+) influx from extracellular fluids, the mode of action of flavonoids remains elusive. 2. We hypothesized that treatment with flavone inhibits vascular smooth muscle contraction by decreasing the phosphorylation of the myosin phosphatase target subunit (MYPT1). 3. Rat aortic rings were denuded of endothelium, mounted in organ baths and contracted with U46619, a thromboxane A(2) analogue. 4. Flavone dose-dependently inhibited the U46619-induced contractile response and myosin light chain (MLC(20)) phosphorylation. At 10(-7) mol/L, U46619 induced vascular contraction with the concomitant phosphorylation of MYPT1 at Thr855, but not at Thr697. Incubation with flavone (100 or 300 micromol/L) for 30 min attenuated the phosphorylation of MYPT1(Thr855), but not MYPT1(Thr697). 5. It is concluded that treatment with flavone inhibits vascular smooth muscle contraction by decreasing the phosphorylation of the MYPT1. These results suggest that flavone causes endothelium-independent relaxation through, at least in part, the inhibition of p160 Rho-associated coiled-coil-containing protein kinase (ROCK) signalling.