The effect of DA-9701 on 5-hydroxytryptamine-induced contraction of feline esophageal smooth muscle cells.

Serotonin, or 5-hydroxytryptamine (5-HT), is a monoamine neurotransmitter found in blood platelets, the gastrointestinal (GI) tract, and the central nervous system (CNS) of animals and humans. The signaling pathways of 5-hydroxytryptamine (5-HT)-induced contractions in cat esophageal smooth muscle cell (ESMC)s have been identified, but the downstream components of the 5-HT signaling pathway remain unclear. DA-9701 is the standardized extract of the Pharbitis nil Choisy seed (Pharbitidis Semen, Convolvulaceae) and the root of Corydalis yahusuo W.T. Wang (Corydalis Tuber, Papaveraceae). DA-9701 is known to have strong gastroprokinetic effects and a good safety profile. In this study, we investigated the 5-HT signaling pathway at the G-protein level, and we explored the mechanisms by which DA-9701 induces smooth muscle contraction. Freshly isolated smooth muscle cells were harvested from the feline esophagus, and cells were permeabilized to measure their length. 5-HT produced esophageal smooth muscle contractions in a dose-dependent manner. Furthermore, 5-HT produced a relatively long-acting contraction. 5-HT binds to the 5-HT2, 5-HT3 and 5-HT4 receptors to induce smooth muscle contraction in feline ESMCs. These receptors, which are located in esophageal smooth muscle, are coupled to Gαq, Gαo and Gαs. These G proteins activate PLC, which leads to Ca2+/calmodulin-dependent MLCK activation, resulting in MLC20 phosphorylation and cell contraction. Conversely, DA-9701 inhibits 5-HT-induced contraction by inhibiting MLC20 phosphorylation.

Honokiol attenuates vascular contraction through the inhibition of the RhoA/Rho-kinase signalling pathway in rat aortic rings.

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 20kDa myosin light chains (MLC(20) ), myosin phosphatase targeting subunit 1 (MYPT1) and protein kinase C (PKC)-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase (MLCP) of 17kDa (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 A(2) agonist U46619. Honokiol decreased the phosphorylation levels of MLC(20) , MYPT1(Thr855) and CPI17(Thr38) as well as the activation of RhoA induced by 8.0mm NaF or 30nm U46619. CONCLUSIONS: These results demonstrated that honokiol attenuated vascular contraction through the inhibition of the RhoA/Rho-kinase signalling pathway.

HMC05 attenuates vascular contraction through inhibition of RhoA/Rho-kinase signaling pathway.

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.

Effects of gomisin A on vascular contraction in rat aortic rings.

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 A(2) agonist U46619, but not to phorbol 12, 13-dibutyrate (PDBu). GA decreased the activation of RhoA as well as the phosphorylation level of MLC20, MYPT1(Thr855), 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.

Anti-allergic inflammatory activity of the fruit of Prunus persica: role of calcium and NF-kappaB.

Mast cell-mediated allergic symptoms are involved in many diseases, such as asthma and sinusitis. In this study, we investigated the effect of ethanol extract of fruits of Prunus persica (L) Batsch (FPP) on the mast cell-mediated allergic inflammation and studied the possible mechanism of action. FPP dose-dependently inhibited compound 48/80-induced systemic anaphylaxis and immunoglobulin E-mediated local allergic reactions. Histamine releasing from mast cells was reduced by FPP, which was mediated by modulation of intracellular calcium. In addition, FPP attenuated the phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-stimulated expression and secretion of pro-inflammatory cytokines in human mast cells. The inhibitory effect of FPP on pro-inflammatory cytokines was nuclear factor (NF)-kappaB dependent. Our findings provide evidence that FPP inhibits mast cell-derived allergic inflammation and involvement of calcium and NF-kappaB in these effects.

Suppression of mast cell-mediated allergic reaction by Amomum xanthiodes.

The mast cell-mediated immediate-type allergic reaction is involved in many allergic diseases such as asthma, allergic rhinitis and sinusitis. Stimulation of mast cells starts the process of degranulation resulting in release of mediators such as histamine and an array of inflammatory cytokines. In this report, we investigated the effect of aqueous extract of Amomum xanthiodes (Zingiberaceae) (AXE) on the mast cell-mediated allergy model and studied its possible mechanisms of action. AXE inhibited compound 48/80-induced systemic reactions and serum histamine release in mice. AXE decreased immunoglobulin E (IgE)-mediated passive cutaneous anaphylaxis reaction. AXE reduced histamine release and intracellular calcium from rat peritoneal mast cells activated by compound 48/80. Furthermore, AXE decreased the activation of p38 mitogen-activated protein kinase (MAPK) but not extracellular signal-regulated kinase and c-jun N-terminal kinase, and downstream tumor necrosis factor (TNF)-alpha production in phorbol 12-myristate 13-acetate and calcium ionophore A23187-stimulated human mast cells. Our findings provide evidence that AXE inhibits mast cell-derived allergic reactions, and that intracellular calcium, TNF-alpha, and p38 MAPK are involved in these effects.

Upregulation of PBR mRNA expression in human neuroblastoma cells by flavonoids.

To investigate the putative mediation of peripheral benzodiazepine receptor (PBR) in the cytotoxicity of flavonoids, in this study, modulatory effects of several flavonoids on the lipid peroxide (LPO) production and PBR mRNA expression of human neuroblastoma cells were observed. Elevated levels of peroxidated products in cancer cells may activate pro-apoptotic and anti-proliferative signaling pathways. Treatment of 10(-6) M 4'-chlorodiazepam and PK 11195 ligands of the PBR for 6 days enhanced the generation of LPO of the human neuroblastoma cells. Several flavonoids, well-known cytotoxic substances, potentiated the enhancement of LPO production by PBR ligands. Treatment of 10(-6) M flavonoids for 6 days elevated the expression of PBR mRNA in cells. These findings indicate that the potential of flavonoids to induce apoptosis in cancer cells is strongly associated with their PBR-inducing properties, thereby providing a new mechanism by which polyphenolic compounds may exert their cancer-preventive and anti-neoplastic effects.

Vasorelaxation by Samhwangsasim-tang, an herb medicine, is associated with decreased phosphorylation of the myosin phosphatase target subunit.

Samhwangsasim-tang (SST) is a widely used herbal medicine with vasodilatory actions in oriental countries. We hypothesized that SST modulates vascular contractility by decreasing phosphorylation of the myosin phosphatase target subunit. Rat aortic ring preparations were mounted in organ baths and subjected to contractions or relaxations. Phosphorylation of 20kDa myosin light chains (MLC(20)) and MYPT1, a target subunit of myosin phosphate 1, were examined with immunoblots. SST relaxed aortic ring preparations precontracted with phenylephrine whether endothelium was intact or denuded. Treatment of aortic rings with N(ω)-nitro-l-arginine methyl ester (l-NAME), an inhibitor of endothelial nitric oxide synthase or methylene blue, an inhibitor of guanylyl cyclase, did not affect the relaxing action of SST. Furthermore, SST inhibited vascular contractions induced by NaF or phenylephrine, but not by phorbol dibutyrate. SST also decreased vascular tension precontracted by 8.0mmol/L NaF or 1.0µmol/L phenylephrine, but not by 1.0µmol/L phorbol dibutyrate. In vascular strips, SST decreased the phosphorylation level of both MLC(20) and MYPT1 induced by 8.0mmol/L NaF. In conclusion, SST inhibited vascular contraction by decreasing phosphorylation of the myosin phosphatase target subunit.

Sequential induction of heme oxygenase-1 and manganese superoxide dismutase protects cultured astrocytes against nitric oxide.

Nitric oxide (NO) is a widely recognized mediator of physiological and pathophysiological signal transmission. In an attempt to better understand the molecular actions of NO in astrocytes, stress protein expression in response to NO donor sodium nitroprusside was investigated. Heme oxygenase-1 (HO-1) has been identified as an inducer of manganese superoxide dismutase (MnSOD), playing a cytoprotective role under the condition of nitrosative stress. We present evidence that the sequential induction of HO-1 and MnSOD protects astrocytes from NO toxicity: (1) both HO-1 and MnSOD expression were induced by NO; (2) NO-mediated increase in MnSOD activity was partly abolished by HO-1 inhibitor Zn(II) protoporphyrin IX (ZnPP); (3) pretreatment of astrocytes with a nontoxic dose of NO protected the cells against the later treatment with a toxic dose of NO; (4) inhibition of HO-1 by ZnPP sensitized astrocytes to the nontoxic dose of NO resulting in a marked cytotoxicity; and (5) adenovirus-mediated overexpression of MnSOD protected astrocytes from the NO toxicity. The molecular action of NO in astrocytes appears to be dose-dependent. While a high dose of NO exerts cytotoxicity leading to the tissue damage in the central nervous system, a low dose of NO may act as an important signaling molecule in astrocytes with concurrent induction of cytoprotective proteins such as HO-1 and MnSOD.