Yu-Wen Cheng

Mechanism of vasorelaxation of thoracic aorta caused by xanthone

The effect of xanthone on smooth muscle was studied in thoracic aorta isolated from rats. Xanthone relaxed the norepinephrine-induced contraction of rat thoracic aorta. This relaxing effect of xanthone persisted in endothelium-denuded aorta suggesting that the relaxation induced by xanthone is endothelium-independent. The norepinephrine and high-K+-induced vasoconstriction was inhibited dose dependently in aorta pretreated with xanthone with IC50 values of 60.26 +/- 8.43 and 82.9 +/- 13.21 microM, respectively. The inositol 1,4,5-trisphosphate formation induced by norepinephrine (3 microM) in rat aorta was not affected by xanthone (10-100 microM), suggesting that the vasorelaxant effect of xanthone was not exerted on the receptor. Xanthone concentration dependently inhibited the 45Ca2+ influx induced by either norepinephrine or high-K+, suggesting that xanthone might act as a blocker of both receptor-operated and voltage-dependent Ca2+ channels. Furthermore, xanthone caused an increase in the level of intracellular cyclic adenosine 3,5-monophosphate (cAMP), but not cyclic guanosine 3,5-monophosphate (cGMP) content. These data suggested that the mechanism of xanthone-induced vasorelaxation might involve the increase of intracellular cyclic adenosine 3,5-monophosphate (cAMP) content and block of Ca2+ channels.

Physiological and functional interactions between Tcf4 and Daxx in colon cancer cells.

Daxx, a human cell death-associated protein, was isolated as a Tcf4-interacting protein, using a yeast two-hybrid screen. Co-immunoprecipitation in HEK-293T cells and yeast two-hybrid screen in Y190 cells were performed to identify the interaction between Tcf4 with Daxx and to map the binding regions of Tcf4. In the nucleus, Daxx reduced DNA binding activity of Tcf4 and repressed Tcf4 transcriptional activity. Overexpression of Daxx altered the expression of genes downstream of Tcf4, including cyclin D1 and Hath-1, and induced G1 phase arrest in colon cancer cells. A reduction in Daxx protein expression was also observed in colon adenocarcinoma tissue when compared with normal colon tissue. This evidence suggests a possible physiological function of Daxx, via interaction with Tcf4, to regulate proliferation and differentiation of colon cells.

Polycyclic aromatic hydrocarbons-induced vasorelaxation through activation of nitric oxide synthase in endothelium of rat aorta

In the present study, the effect of polycyclic aromatic hydrocarbons (PAHs) on isolated rat aorta was investigated. Acenaphthylene and naphthalene dose-dependently relaxed the phenylephrine-induced contraction of rat aorta with 50% vasorelaxation at 40.8+/-19.83 and 118.75+/-9.83 microM, respectively. The vasorelaxation effect was diminished in the denuded (endothelium removed) aorta suggesting that the relaxation effect of PAHs was endothelium dependent. By comparing PAHs with different ring structures, we have found that acenaphthylene has the highest potency to induce vasorelaxation. Pretreatment with the nitric oxide synthase inhibitor, L-N(G)-nitroarginine methyl ester, and the guanylate cyclase inhibitor, methylene blue, prevents the vasorelaxation induced by PAHs. These results indicate that the vasorelaxation effect of PAHs is mediated by activation of nitric oxide synthase of endothelium.

Emodin‐induced muscle contraction of mouse diaphragm and the involvement of Ca2+ influx and Ca2+ release from sarcoplasmic reticulum

1 The effects on skeletal muscle of emodin, an anthraquinone, were studied in the mouse isolated diaphragm and sarcoplasmic reticulum (SR) membrane vesicles. 2 Emodin dose‐dependently caused muscle contracture, simultaneously depressing twitch amplitude. Neither tubocurarine nor tetrodotoxin blocked the contraction suggesting that it was caused myogenically. 3 The contraction induced by emodin persisted in a Ca2+ free medium with a slight reduction in the maximal force of contraction. The contraction induced by emodin in the Ca2+ free medium was completely blocked when the internal Ca2+ pool of the muscle was depleted by ryanodine. These data suggest that the contraction caused by emodin is due to the release of Ca2+ from the intracellular ryanodine‐sensitive pool. 4 In contrast to the effect seen in the Ca2+ free medium, emodin induced a small but consisted contraction in the ryanodine‐treated muscle in Krebs medium. The contraction was blocked in the presence of dithiothreitol and was partially blocked by nifedipine, suggesting that oxidation of a sulphhydryl group on the external site of dihydropyridine receptor is involved. 5 Emodin dose‐dependently increased Ca2+ release from actively loaded SR vesicles and this effect was blocked by ruthenium red, a specific Ca2+ release channel blocker, and the thiol reducing agent, DTT, suggesting that emodin induced Ca2+ release through oxidation of the critical SH of the ryanodine receptor. 6 [3H]‐ryanodine binding was dose‐dependently potentiated by emodin in a biphasic manner. The degree of potentiation of ryanodine binding by emodin increased dose‐dependently at concentrations up to 10u2003μM but decreased at higher concentrations of 10–100u2003μM. 7 These data suggest that muscle contraction induced by emodin is due to Ca2+ release from the SR of skeletal muscle, as a result of oxidation of the ryanodine receptor and influx of extracellular Ca2+ through voltage‐dependent Ca2+ channels of the plasma membrane.

Induction of skeletal muscle contracture and calcium release from isolated sarcoplasmic reticulum vesicles by sanguinarine.

The benzophenanthrine alkaloid, sanguinarine, was studied for its effects on isolated mouse phrenic‐nerve diaphragm preparations. Sanguinarine induced direct, dose‐dependent effects on muscle contractility. Sanguinarine‐induced contracture was partially inhibited when the extracellular Ca2+ was removed or when the diaphragm was pretreated with nifedipine. Depletion of sarcoplasmic reticulum (SR) internal calcium stores completely blocked the contracture. Sanguinarine induced Ca2+ release from the actively loaded SR vesicles was blocked by ruthenium red and dithiothreitol (DTT), consistent with the ryanodine receptor (RyR) as the site of sanguinarine action. Sanguinarine altered [3H]‐ryanodine binding to the RyR of isolated SR vesicles, potentiating [3H]‐ryanodine binding at lower concentrations and inhibiting binding at higher concentrations. All of these effects were reversed by DTT, suggesting that sanguinarine‐induced Ca2+ release from SR occurs through oxidation of critical SH groups of the RyR SR calcium release channel.

Induction of calcium release from sarcoplasmic reticulum of skeletal muscle by xanthone and norathyriol

1 . Effects of xanthone and its derivative, 1,3,6,7‐tetrahydroxyxanthone (norathyriol), on Ca2+ release and ryanodine binding were studied in isolated sarcoplasmic reticulum (SR) vesicles from rabbit skeletal muscle. 2 . Both xanthone and norathyriol dose‐dependently induced Ca2+ release from the actively loaded SR vesicles which was blocked by ruthenium red, a specific Ca2+ release inhibitor, and Mg2+. 3 . Xanthone and norathyriol also dose‐dependently increased apparent [3H]‐ryanodine binding. Norathyriol, but not xanthone, produced a synergistic effect on binding activation when added concurrently with caffeine. 4 . In the presence of Mg2+, which inhibits ryanodine binding, both caffeine and norathyriol, but not xanthone, could restore the binding to the level observed in the absence of Mg2+. 5 . Xanthone activated the Ca2+‐ATPase activity of isolated SR vesicles dose‐dependently reaching 70% activation at 300 μm. 6 . When tested in mouse diaphragm, norathyriol potentiated the muscle contraction followed by twitch depression and contracture in either a Ca2+‐free bathing solution or one containing 2.5 mM Ca2+. These norathyriol‐induced effects on muscle were inhibited by pretreatment with ruthenium red or ryanodine. 7 . These data suggest that xanthone and norathyriol can induce Ca2+ release from the SR of skeletal muscle through a direct interaction with the Ca2+ release channel, also known as the ryanodine receptor.

Alpha-naphthoflavone induces vasorelaxation through the induction of extracellular calcium influx and NO formation in endothelium

The effect of α-naphthoflavone (α-NF) on vascular function was studied in isolated ring segments of the rat thoracic aorta and in primary cultures of human umbilical vein endothelial cells (HUVECs). α-NF induced concentration-dependent relaxation of the phenylephrine-precontracted aorta endothelium-dependently and -independently at lower and higher concentrations, respectively. The cGMP, but not cAMP, content was increased significantly in α-NF-treated aorta. Pretreatment with Nω-nitro-l-arginine methyl ester (L-NAME) or methylene blue attenuated both α-NF induced vasorelaxation and the increase of cGMP content significantly. The increase of cGMP content induced by α-NF was also inhibited by chelating extracellular Ca2+ with EGTA. These results suggest that the endothelium-dependent vasorelaxation induced by α-NF is mediated most probably through Ca2+-dependent activation of NO synthase and guanylyl cyclase. In HUVECs, α-NF induced concentration-dependent formation of NO and Ca2+ influx. α-NF-induced NO formation was abolished by removal of extracellular Ca2+ and by pretreatment with the Ca2+ channel blockers SKF 96365 and Ni2+, but not by the L-type Ca2+ channel blocker verapamil. The Ca2+ influx, as measured by 45Ca2+ uptake, induced by α-NF was also inhibited by SKF 96365 and Ni2+. Our data imply that α-NF, at lower concentrations, induces endothelium-dependent vasorelaxation by promoting extracellular Ca2+ influx in endothelium and the activation of the NO-cGMP pathway.

Safety assessment of the Cistanche tubulosa health food product Memoregain : Genotoxicity and 28-day repeated dose toxicity test.

The pharmacological effects of Cistanches Herba, known as Ginseng of the desert, have been extensively studied. In this study, we aimed to assess the genotoxic and oral toxic effects of the Cistanche tubulosa health food product Memoregain® using in vitro and in vivo tests. Ames tests using five strains of Salmonella typhimurium showed no signs of increased reverse mutation upon exposure to Memoregain® up to a concentration of 5 mg/plate. Exposure of Chinese hamster ovary (CHO-K1) cells to Memoregain® did not increase the frequency of chromosomal aberrations in vitro. Moreover, Memoregain® treatment did not affect the proportions of immature to total erythrocytes or the number of micronuclei in the immature erythrocytes of ICR mice. Additionally, after 28-day repeated oral dose toxicity tests (0, 0.15, 0.3, and 0.5g/kg body weight) in rats, no observable adverse effects were found. These toxicological assessments supported the safety of Memoregain® for human consumption.