1.Effect of nitric oxide on HaCaT cell migration.
Shi-wei YANG ; Jun WU ; Gao-xing LUO ; Xiao-rong ZHANG ; Xiao-hong HU ; Yan-meng PENG ; Jun-jie YANG ; Xiao-li LUO ; Ying WANG
Chinese Journal of Burns 2010;26(2):146-149
OBJECTIVETo investigate the effect of exogenous nitric oxide (NO) on the migration of HaCaT cell and its possible mechanism.
METHODSSodium nitroprusside (SNP) was used as the donor of NO. Different concentrations of SNP (0.1, 1.0, 10.0, 100.0, 1000.0 micromol/L) were added into nutrient culture medium of HaCaT cells. Cell migration rate was observed and calculated at post scratching hour (PSH) 0 (immediately after scratching), 6, 12, 24, 48. The most suitable concentration of SNP and culture duration were selected as stimulation condition. Cytoskeletons of HaCaT cells were observed under confocal laser scanning microscope. The expressions of integrin beta 1, RhoA, Rac1 and Cdc42 of cells in experiment group (cultured with 10.0 micromol/L SNP for 24 hours) and negative control group were determined at mRNA and protein levels with RT-PCR and Western blot respectively. Data were processed with one-way analysis of variance (ANOVA) and repeated measure ANOVA.
RESULTSMigration rate of HaCaT cells in each group increased gradually as time after scratching went on. There were significant differences between PSH 6-48 and PSH 0 in cells cultured with 10.0 micromol/L SNP (F = 31.002, P values all below 0.05). Pili were rarely observed in negative control group with slender stress fibers in cells. In comparison, the amount of pili amount increased obviously in experiment group with thickened stress fibers. Compared with those of cells in control group (RhoA protein expression = 0.64 +/- 0.04), integrin beta 1 expression decreased obviously (F = 8.25, P = 0.015), RhoA (0.92 +/- 0.04), Cdc42 and Rac1 were up-regulated at both protein (with F value respectively 7.25, 14.10, 6.50, P values all below 0.05) and mRNA levels (with F value respectively 23.67, 10.39, 9.52, P values all below 0.05).
CONCLUSIONSExogenous NO in suitable concentration can promote the proliferation and migration of HaCaT cell, suggesting it exerts significant effect in wound repair. The changed cytoskeletons and the down-regulated integrin beta 1 expression may be involved in this process.
Cell Line ; Cell Movement ; drug effects ; Cytoskeleton ; drug effects ; metabolism ; Humans ; Nitric Oxide ; pharmacology ; RNA, Messenger ; genetics ; rhoA GTP-Binding Protein ; genetics ; metabolism
2.Staurosporine and cytochalasin D induce chondrogenesis by regulation of actin dynamics in different way.
Minjung KIM ; Kyung SONG ; Eun Jung JIN ; Jongkyung SONN
Experimental & Molecular Medicine 2012;44(9):521-528
Actin cytoskeleton has been known to control and/or be associated with chondrogenesis. Staurosporine and cytochalasin D modulate actin cytoskeleton and affect chondrogenesis. However, the underlying mechanisms for actin dynamics regulation by these agents are not known well. In the present study, we investigate the effect of staurosporine and cytochalasin D on the actin dynamics as well as possible regulatory mechanisms of actin cytoskeleton modulation. Staurosporine and cytochalasin D have different effects on actin stress fibers in that staurosporine dissolved actin stress fibers while cytochalasin D disrupted them in both stress forming cells and stress fiber-formed cells. Increase in the G-/F-actin ratio either by dissolution or disruption of actin stress fiber is critical for the chondrogenic differentiation. Cytochalasin D reduced the phosphorylation of cofilin, whereas staurosporine showed little effect on cofilin phosphorylation. Either staurosporine or cytochalasin D had little effect on the phosphorylation of myosin light chain. These results suggest that staurosporine and cytochalasin D employ different mechanisms for the regulation of actin dynamics and provide evidence that removal of actin stress fibers is crucial for the chondrogenic differentiation.
Actin Cytoskeleton/*drug effects
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Actins/metabolism
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Animals
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Cell Differentiation/*drug effects
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Cells, Cultured
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Chickens
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Chondrogenesis/*drug effects
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Cytochalasin D/*pharmacology
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Mesoderm/cytology/drug effects
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Myosin Light Chains/metabolism
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Nucleic Acid Synthesis Inhibitors/*pharmacology
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Phosphorylation
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Staurosporine/*pharmacology
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Stress Fibers/drug effects
3.Effects of the tyrosine protein kinase inhibitor, genistein on the filamentous-actin and cytosolic calcium in rat hepatic stellate cells.
Li YANG ; Xiao-jing LIU ; Qiong WANG ; Ming-hui HUANG ; Yi-ping WANG ; Hong-bin WU
Chinese Journal of Hepatology 2003;11(10):634-635
Actins
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metabolism
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Animals
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Calcium
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metabolism
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Cells, Cultured
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Cytoskeleton
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metabolism
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Enzyme Inhibitors
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pharmacology
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Genistein
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pharmacology
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Hepatocytes
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drug effects
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metabolism
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Male
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Protein-Tyrosine Kinases
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antagonists & inhibitors
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Rats
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Rats, Wistar
4.Curcumin inhibits cellular condensation and alters microfilament organization during chondrogenic differentiation of limb bud mesenchymal cells.
Dongkyun KIM ; Song Ja KIM ; Shin Sung KANG ; Eun Jung JIN
Experimental & Molecular Medicine 2009;41(9):656-664
Curcumin is a well known natural polyphenol product isolated from the rhizome of the plant Curcuma longa, anti-inflammatory agent for arthritis by inhibiting synthesis of inflammatory prostaglandins. However, the mechanisms by which curcumin regulates the functions of chondroprogenitor, such as proliferation, precartilage condensation, cytoskeletal organization or overall chondrogenic behavior, are largely unknown. In the present report, we investigated the effects and signaling mechanism of curcumin on the regulation of chondrogenesis. Treating chick limb bud mesenchymal cells with curcumin suppressed chondrogenesis by stimulating apoptotic cell death. It also inhibited reorganization of the actin cytoskeleton into a cortical pattern concomitant with rounding of chondrogenic competent cells and down-regulation of integrin beta1 and focal adhesion kinase (FAK) phosphorylation. Curcumin suppressed the phosphorylation of Akt leading to Akt inactivation. Activation of Akt by introducing a myristoylated, constitutively active form of Akt reversed the inhibitory actions of curcumin during chondrogenesis. In summary, for the first time, we describe biological properties of curcumin during chondrogenic differentiation of chick limb bud mesenchymal cells. Curcumin suppressed chondrogenesis by stimulating apoptotic cell death and down-regulating integrin-mediated reorganization of actin cytoskeleton via modulation of Akt signaling.
Animals
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Anti-Inflammatory Agents, Non-Steroidal/*pharmacology
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Apoptosis/drug effects
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Cells, Cultured
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Chick Embryo
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Chondrogenesis/*drug effects
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Curcumin/*pharmacology
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Cytoskeleton/*drug effects/metabolism
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Limb Buds/*cytology
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Mesenchymal Stem Cells/cytology/*drug effects
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Proto-Oncogene Proteins c-akt/metabolism
5.Functions of thrombin receptors in the reversible distribution of platelet surface glycoprotein I balpha in activated platelets.
Yue HAN ; J M PASQUET ; A NURDEN ; Zhao-yue WANG ; Chang-geng RUAN
Chinese Journal of Hematology 2005;26(3):152-156
OBJECTIVETo detect the redistribution of platelet surface glycoprotein (GP)Ib alpha and cytoskeleton reorganization in the course of thrombin receptor activation, and investigate the mechanism of GPIb alpha re-translocation and the role of thrombin receptors in platelet signal transduction.
METHODSThe thrombin receptor activating peptide (PAR1-AP, TRAP) was used for stimulating platelet at different time points (0 - 60 min), then the platelet surface GPIb alpha and P-selectin were examined with flow cytometry, and the alterations of GPIb alpha, actin and myosin were analyzed in cytoskeleton by Western blot and GPIb alpha immunoprecipitation. Cytochalasin D and/or Apyrase VII were used for investigating their inhibitory effect on platelet activation.
RESULTSAn increase of P-selectin and reversible internalization of GPIb alpha were observed within platelets upon TRAP activation, and transient changes of actin, myosin and GPIb alpha/myosin, GPIb alpha/actin association were also found in this course. These changes were apparently blocked by cytochalasin D, which inhibited the incorporation of GPIb alpha, actin and myosin into cytoskeleton. Apyrase VII had a weak effect on GPIb alpha internalization, although it accelerated the return of GPIb alpha to platelet surface. In addition, Apyrase VII also quickened the GPIb alpha disappearance in cytoskeleton and the dissociation of GPIb/myosin or GPIb/actin during activation.
CONCLUSIONThrombin receptor activation takes part in platelet signal transduction, inducing a reversible redistribution of GPIb alpha. This process is related to cytoskeleton reorganisation and ADP.
Actins ; metabolism ; Blood Platelets ; cytology ; drug effects ; metabolism ; Blotting, Western ; Cells, Cultured ; Cytoskeleton ; metabolism ; Humans ; Myosins ; metabolism ; P-Selectin ; metabolism ; Peptide Fragments ; pharmacology ; Platelet Activation ; drug effects ; physiology ; Platelet Glycoprotein GPIb-IX Complex ; metabolism ; Receptors, Thrombin ; metabolism ; physiology
6.Suilysin remodels the cytoskeletons of human brain microvascular endothelial cells by activating RhoA and Rac1 GTPase.
Qingyu LV ; Huaijie HAO ; Lili BI ; Yuling ZHENG ; Xuyu ZHOU ; Yongqiang JIANG
Protein & Cell 2014;5(4):261-264
Brain
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Cholesterol
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chemistry
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Cytoskeleton
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drug effects
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Endothelial Cells
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cytology
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metabolism
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Hemolysin Proteins
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chemistry
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pharmacology
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Humans
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Phalloidine
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pharmacology
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Pseudopodia
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drug effects
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Stress Fibers
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drug effects
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rac1 GTP-Binding Protein
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metabolism
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rhoA GTP-Binding Protein
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metabolism
7.Protective effect of catalpolon destruction of tight junctions of high glucose induced BMECs.
Li ZOU ; Ke LIU ; Hui-Feng ZHU ; Shan FENG
China Journal of Chinese Materia Medica 2018;43(20):4118-4124
This paper aimed to observe the protective effect of catalpol on the high glucose induced destruction of tight junctions of rat primary brain microvascular endothelial cells (BMECs). Catalpol co-administrated with high glucose increased BMECs survival, decreased its ET-1 secretion, and improved transmembrane electrical resistance in a time-dependent manner. Furthermore, transmission electron microscopy was used to observe catalpol's protective effect on tight junction. Fluorescence staining displayed that catalpol reversed the rearrangement of the cytoskeleton protein F-actin and up-regulated the tight junction proteins claudin-5 and ZO-1, which were further demonstrated by the mRNA expression levels of claudin-5, occludin, ZO-1, ZO-2, ZO-3, -actintin, vinculin and cateinins. This study indicated that catalpol reverses the disaggregation of cytoskeleton actin in BMECs and up-regulates the expression of tight junction proteins, such as claudin-5, occludin, and ZO-1, and finally alleviates the increase in high glucose-induced BMECs injury.
Actin Cytoskeleton
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drug effects
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Actins
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metabolism
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Animals
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Brain
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cytology
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Cells, Cultured
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Claudin-5
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metabolism
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Endothelial Cells
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drug effects
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Glucose
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Iridoid Glucosides
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pharmacology
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Phosphoproteins
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Rats
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Tight Junctions
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drug effects
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Zonula Occludens-1 Protein
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metabolism
8.Experimental studies on male reproductive toxicity of bisphenol A in vitro and vivo.
Mao-xian DENG ; De-sheng WU ; Xiang-gui CHEN ; Li-shi ZHANG ; Pei-yu XU
Chinese Journal of Preventive Medicine 2004;38(6):383-387
OBJECTIVETo explore the effects of Bisphenol A in adult rats and its possible mechanisms.
METHODSBPA (in corn oil) was administered orally to 9-week-old male Sprague-Dawley rats for 14 days (0, 1 and 5 g/kg bw), and incubated primary Sertoli cells from pubertal SD rats with 0, 10(-7), 10(-6), 10(-5), 10(-4) mol/L BPA.
RESULTSAfter oral administration, a significant decrease in right testis weight was observed in 5 g/kg dose group, but not in the 1 g/kg bw dose group. Germ cells were detached from basement membrane of seminiferous tubules and Sertoli cells in BPA-treated groups. Administration of BPA at 1 g/kg bw and 5 g/kg bw produced both nucleus pycnosis and vacuolized nucleus in germ cells and Sertoli cells. A marked loss in vimentin staining in Sertoli cells from testis of BPA-treated rats was detected. No change in levels of serum estradiol and testosterone was observed after two-week exposure to BPA. In Sertoli cell primary culture, BPA destroyed the cytoskeleton and cell-cell junctions, and elongated Sertoli cells.
CONCLUSIONThese results suggest that BPA may injure reproductive function of male rats by destroying the cytoskeleton and changing the form of Sertoli cells.
Animals ; Benzhydryl Compounds ; Cells, Cultured ; Cytoskeleton ; drug effects ; Male ; Organ Size ; drug effects ; Phenols ; toxicity ; Rats ; Rats, Sprague-Dawley ; Sertoli Cells ; cytology ; drug effects ; Testis ; anatomy & histology ; cytology ; drug effects ; Vimentin ; metabolism
9.Bombesin increases cytokeratin expression and free calcium concentration in PC-3 cell line.
Bin WU ; He WANG ; Wei-jun QING
National Journal of Andrology 2007;13(4):332-334
OBJECTIVETo observe the effect of bombesin noncytoskeleton form and intracellular free calcium ([Ca2+]i) concentration in PC-3 prostate cancer cell line.
METHODSImmunofluorescent histochemistry (IH) combined with laser scanning confocal microscopy (LSCM) was used to examine the expression of cytokeratin (CK) in PC-3 cells treated with definite concentrations of BBS and observe its effect on cytoskeleton form. Fluo-3/AM fluorescence technique and LSCM were adopted to measure the [Ca2+]i concentration after different concentrations (10(-9), 10(-7) and 10(-5) mol/L) of BBS were added in PC-3 cells.
RESULTSBBS (10(-5) mol/L) stimulated the expression of CK in PC-3 cells and the formation of lamellipodium, and increased the [Ca2+]i concentration, with concentration dependence.
CONCLUSIONDefinite concentrations of BBS could obviously enhance the [Ca2+] i concentration, CK expression and cytoskeleton morphology of PC-3 cells. The results provide a basis for further studies on the role of BBS in tumour researches as well as in intracellular signal transmission.
Bombesin ; pharmacology ; Calcium ; analysis ; Cytoskeleton ; drug effects ; metabolism ; Fluoroimmunoassay ; Humans ; Keratins ; biosynthesis ; Male ; Microscopy, Confocal ; Prostatic Neoplasms ; metabolism ; Serum Albumin, Bovine ; Tumor Cells, Cultured
10.Rho-kinase signaling pathway participates in endothelin-1-induced human airway smooth muscle cell migration and cytoskeletal reorganization.
Zhen-Xing LI ; Ya-Ling LUO ; Jian XU ; Dun-Qiang REN ; Yan-Xia ZHAO
Journal of Southern Medical University 2008;28(6):1031-1034
OBJECTIVETo investigate the role of Rho-kinase signaling pathway in human airway smooth muscle cell (ASMCs) migration and cytoskeletal reorganization induced by endothelin-1 (ET-1).
METHODSPrimary cultured human ASMCs obtained by tracheal explant culture method were examined for cell migration in response to ET-1 treatment using modified Boyden chambers. The changes in actin cytoskeletal reorganization were observed under confocal laser scanning microscope, and the phosphorylation of myosin-phosphatase target 1 (p-MYPT1) was examined using Western blot analysis.
RESULTSAt the concentration of 0.1, 1, 10, and 100 nmol/L, ET-1 induced migration of the ASMCs, and 10 nmol/L ET-1 produced the most obvious effect (P<0.01). Rho-kinase inhibitor Y-27632 showed a dose-dependent inhibitory effect on ET-1-induced ASMC migration, and in cells exposed to 10 nmol/L ET-1, Y-27632 at 10 micromol/L significantly blocked ASMC migration (P<0.01). ET-1 (10 nmol/L) exposure resulted in reorganization of actin cytoskeleton and formation of stress fibers in the ASMCs, which were obviously inhibited by Y-27632. Compared with the control group, the AMSCs showed significant enhancement of p-MYPT1 protein expression after ET-1 exposure for 15 and 30 min (P<0.01), but prolonged exposure failed to result in the expression enhancement (P>0.05).
CONCLUSIONRho-kinase signaling pathway may play an important role in ET-1-induced ASMC migration and reorganization of actin cytoskeleton.
Amides ; pharmacology ; Bronchi ; cytology ; Cell Movement ; drug effects ; Cells, Cultured ; Cytoskeleton ; drug effects ; metabolism ; Endothelin-1 ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Humans ; Microscopy, Confocal ; Muscle, Smooth ; cytology ; Pyridines ; pharmacology ; Signal Transduction ; drug effects ; rho-Associated Kinases ; antagonists & inhibitors ; metabolism