Heung-Mook Shin

Differential Association and Localization of Myosin Phosphatase Subunits During Agonist-Induced Signal Transduction in Smooth Muscle

It has been known for some time that agonist-induced contractions of vascular smooth muscle are often associated with a sensitization of the contractile apparatus to intracellular Ca2+. One mechanism that has been suggested to explain Ca2+ sensitization is inhibition of myosin phosphatase activity. In the present study, we tested the hypothesis that differential localization of the phosphatase might be associated with its inhibition. Quantitative confocal microscopy of freshly dissociated, fully contractile smooth muscle cells was used in parallel with measurements of myosin light chain and myosin phosphatase phosphorylation. The results indicate that, in the smooth muscle cells, the catalytic and targeting subunits of the phosphatase are dissociated from each other in an agonist-specific manner and that the dissociation is accompanied by a slower rate of myosin phosphorylation. Targeting of myosin phosphatase to the cell membrane precedes the dissociation of subunits and is associated with phosphorylation of the targeting subunit at a Rho-associated kinase (ROK) phosphorylation site. The phosphorylation and membrane translocation of the targeting subunit are inhibited by a ROK inhibitor. This dissociation of subunits may provide a mechanism for the decreased phosphatase activity of phosphorylated myosin phosphatase.

FLAVONE INHIBITS VASCULAR CONTRACTION BY DECREASING PHOSPHORYLATION OF THE MYOSIN PHOSPHATASE TARGET SUBUNIT

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 Ca2+ release from intracellular stores or Ca2+ 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 A2 analogue. 4 Flavone dose‐dependently inhibited the U46619‐induced contractile response and myosin light chain (MLC20) 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 µmol/L) for 30 min attenuated the phosphorylation of MYPT1Thr855, but not MYPT1Thr697. 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.

Heat-shock response is associated with decreased production of interleukin-6 in murine aortic vascular smooth muscle cells

Heat shock has been known to change cellular responses to noxious stimuli by inducing heat-shock proteins (Hsps). We hypothesized that a heat-shock response modulates cytokine production in murine aortic vascular smooth muscle cells (VSMCs). VSMCs were exposed to 44°C for 15–60xa0min, and subjected to interleukin-1β (IL-1β) or tumor necrosis factor α (TNFα), which induced interleukin-6 (IL-6) production. Expression of Hsps was examined with immunoblots, immunocytochemistry, or enzyme-linked immunosorbent assay (ELISA), and that of IL-6 with reverse transcription-polymerase chain reaction (RT-PCR) or ELISA. Heat shock (44°C for 45xa0min) induced Hsp72 in VSMCs at 4xa0h and elicited its maximal expression at 8xa0h after the end of heat shock. Treatment with IL-1β increased IL-6 transcription in VSMCs up to 24xa0h in an incubation time-dependent manner. Treatment with IL-1β or TNFα caused a concentration-dependent increase in IL-6 production in culture medium, which was attenuated by heat shock. Although treatment with Hsp72 or Hsp60 alone did not significantly affect basal IL-6 release into culture medium statistically, cotreatment with IL-1β and Hsp72, but not Hsp60 or boiled Hsp72, decreased IL-1β-induced IL-6 production in culture medium. Introduction of Hsp72, but not Hsp60, into VSMCs decreased IL-1β-induced IL-6 production in culture medium. These results indicate that the heat-shock response transcriptionally attenuated production of IL-6 in murine aortic VSMCs.

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

AIM OF THE STUDYnHMC05, 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.nnnMATERIALS AND METHODSnRat 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.nnnRESULTSnIn 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.nnnCONCLUSIONSnHMC05 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.

Simultaneous Determination of Alkaloids and Flavonoids in HMC05 Preparation by HPLC-DAD

Abstract High performance liquid chromatography with diode array detector (HPLC-DAD) was developed for simultaneous determination of hesperidin, coptisine, palmatine, and berberine in HMC05, a standardized extract of eight different herbs. This method was validated in terms of specificity, linearity (r2 > 0.9995), precision (<5.0% RSD), and recoveries (96.6–110.5%). The LOD of these compounds were ranged from 101.6 to 171.7 ng. In addition, these four compounds in HMC05 were identified or tentatively characterized using HPLC-DAD-electrospray mass spectrometry (HPLC-DAD-ESI-MS).

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.

Astragali Radix and its compound formononetin ameliorate diesel particulate matter-induced skin barrier disruption by regulation of keratinocyte proliferation and apoptosis

ETHNOPHARMACOLOGICAL RELEVANCEnAstragali Radix (AR), the root of Astragalus mongholicus Bunge, is widely applied in traditional medicine to promote skin health and tissue regeneration.nnnAIM OF THE STUDYnThis study investigated the effects of AR and its active compound, formononetin (FMT), on skin barrier defects in keratinocytes exposed to diesel particulate matter (PM).nnnMATERIALS AND METHODSnHaCaT cells and three-dimensional (3D) human skin reconstructed model were pre-treated with AR (50, 100u202fμg/ml) and FMT (30, 50u202fμM), then treated with PM (200u202fμg/ml).nnnRESULTSnAR and FMT significantly enhanced the expression of Keratin (KRT) 16 in PM stimulated HaCaT cells. PM increased p53 and Bax expression as well as the subsequent cleavage of caspase 3 and PARP in HaCaT cells, while this was inhibited by AR and FMT treatment. In vitro studies using the PM stimulated 3D human skin reconstructed model revealed that AR and FMT increased the expression of KRT 16 and KRT 17. Histological examination of the 3D human skin reconstructed model showed that AR and FMT up-regulated the expression of Ki67, but down-regulated the expression of cleaved caspase 3. Both AR and FMT significantly inhibited phosphorylation of ERK, but not JNK and p38 MAPK in PM stimulated HaCaT cells.nnnCONCLUSIONSnThese results suggest that AR and FMT act as anti-pollution agents and alleviate PM induced skin barrier defects through regulation of apoptosis and proliferation in keratinocytes.

Endothelium-dependent vasodilatory effect of Smilax china Linn. water extract via PI3K/Akt signaling

Abstract Objective: The objective of this study was to investigate the pharmacological effect of Smilax china Linn. water extract (SCLWE) on vascular relaxation and its underlying biochemical mechanisms. Methodology: Isolated rat aortic rings were pre-constricted with phenylephrine (PE). This was followed by the cumulative addition of SCLWE. The effect of endothelial nitric oxide and PI3K/Akt on the SCLWE-induced vasodilation was investigated by the pretreatment of endothelium-intact aortic strips with or without NG-nitro-L-arginine methyl ester (L-NAME) or wortmanin before constriction with PE. Results: Treatment of PE (1u2009μM)-pre-contracted aortic strips with SCLWE induced endothelium-dependent relaxation, which was attenuated by L-NAME and wortmanin. Further studies using HUVECs indicated that nitrite production, eNOS and PI3K/PKB (Akt) phosphorylations were increased after exposure to SCLWE but was attenuated by pretreatment with wortmanin. Conclusion: These results suggest that SCLWE induces vasodilation by augmenting NO production in endothelial cells via PI3K/Akt-dependent eNOS phosphorylation.

In vitro Antitubercular Activity of 3‐Deoxysappanchalcone Isolated From the Heartwood of Caesalpinia sappan Linn.

Responsible for nearly 1.5 million deaths every year, the infectious disease tuberculosis remains one of the most serious challenges to global health. The emergence of multidrug‐resistant tuberculosis and, more recently, extensively drug‐resistant tuberculosis poses a significant threat in our effort to control this epidemic. New drugs are urgently needed to combat the growing threat of antimicrobial resistance. To achieve this goal, we screened approximately 500 species of medicinal plant methanol extracts and their solvent partitioned fractions for potential inhibitors of Mycobacterium tuberculosis growth. Using microdilution screening, the ethyl acetate solvent partitioned fraction from the heartwood of Caesalpinia sappan exhibited strong antitubercular activity. We isolated the active compound and identified it as 3‐deoxysappanchalcone. The extracted 3‐deoxysappanchalcone possessed activity against both drug‐susceptible and drug‐resistant strains of M. tuberculosis at MIC50s of 3.125–12.5 μg/mL in culture broth and MIC50s of 6.25–12.5 μg/mL inside macrophages and pneumocytes. 3‐Deoxysappanchalcone was also found to act in partial synergy with streptomycin/ethambutol against M. tuberculosis H37Rv. 3‐Deoxysappanchalcone had no cytotoxicity against the A549 cell line up to a concentration of 100 μg/mL (selectivity index > 8–32). Further studies are warranted to establish the in vivo effect and therapeutic potential of 3‐deoxysappanchalcone. Copyright

Effects of Constituent Compounds of Smilax china on Nicotine-Induced Endothelial Dysfunction in Human Umbilical Vein Endothelial Cells

This study investigated the effects of compounds isolated from 70% ethanol (EtOH) extraction of Smilax china L. (SCE), a plant belonging to the family Smilacaceae on nicotine-induced endothelial dysfunction (ED) in human umbilical vein endothelial cells. We isolated 10 compounds from ethyl acetate (EtOAc) fraction of 70% EtOH extract of SCE and investigated their inhibitory effect on nicotine-induced ED in endothelial cells. Kaempferol, kaempferol 7-O-α-L-rhamnopyranoside, puerarin and ferulic acid showed strong inhibition of nicotine-induced vascular cell adhesion molecule (VCAM-1) expression while kaempferol, kaempferin, and caffeic acid attenuated intercellular adhesion molecule (ICAM-1) expression. Lepidoside, caffeic acid and methylsuccinic acid caused the highest up-regulated expression of endothelial nitric oxide synthase at the protein level with caffeic acid and ferulic acid showing strong inhibitory effects on inducible nitric oxide synthase (iNOS) expression. In addition, ferulic acid and kaempferol showed inhibition against interleukin-8 (IL-8) and interleukin-1β (IL-1β) expression while ferulic acid and caffeic acid showed comparatively higher inhibition of ED associated tumor necrosis factor-α (TNF-α) expression. These results show the potential of the aforementioned compounds to reverse the toxic effects of nicotine on the endothelium.