| Reference | [1]. J Cell Sci. 2001 Mar;114(Pt 5):1025-36.<br />
Effects of cytochalasin D and latrunculin B on mechanical properties of cells.<br />
Wakatsuki T(1), Schwab B, Thompson NC, Elson EL.<br />
Author information: (1)Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.<br />
Actin microfilaments transmit traction and contraction forces generated within a cell to the extracellular matrix during embryonic development, wound healing and cell motility, and to maintain tissue structure and tone. Therefore, the state of the actin cytoskeleton strongly influences the mechanical properties of cells and tissues. Cytochalasin D and Latrunculin are commonly used reagents that, by different mechanisms, alter the state of actin polymerization or the organization of actin filaments. We have investigated the effect of a wide range of Cytochalasin D and Latrunculin B concentrations (from 40 pM to 10 microM) on the mechanical properties of the cells within fibroblast populated collagen matrices. Contractile force and dynamic stiffness were measured by uniaxial stress-strain testing. The range of effective concentrations of Cytochalasin D (200 pM-2 microM) was broader than that of Latrunculin B (20 nM-200 nM). Activating the cells by serum did not change the effective range of Cytochalasin D concentrations but shifted that of Latrunculin B upward by tenfold. Simple mathematical binding models based on the presumed mechanisms of action of Cytochalasin D and Latrunculin B simulated the concentration-dependent mechanical changes reasonably well. This study shows a strong dependence of the mechanical properties of cells and tissues on the organization and degree of polymerization of actin filaments.<br />
PMID: 11181185<br />
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[2]. Exp Eye Res. 2018 May;170:101-107. doi: 10.1016/j.exer.2018.02.003. Epub 2018 Feb 6.<br />
Latrunculin B and substratum stiffness regulate corneal fibroblast to myofibroblast transformation.<br />
Thomasy SM(1), Raghunathan VK(2), Miyagi H(3), Evashenk AT(4), Sermeno JC(4), Tripp GK(4), Morgan JT(4), Murphy CJ(5).<br />
Author information: (1)Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA; Department of Ophthalmology & Vision Science, School of Medicine, University of California, Davis, Davis, CA, USA. Electronic address: [email protected]. (2)Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA; Department of Basic Sciences, The Ocular Surface Institute, College of Optometry, University of Houston, Houston, TX, USA. (3)Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA; Department of Ophthalmology and Visual Sciences, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Kasumi 1-2-3, Hiroshima, 7348551, Japan. (4)Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA. (5)Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, Davis, CA, USA; Department of Ophthalmology & Vision Science, School of Medicine, University of California, Davis, Davis, CA, USA.<br />
The transformation of keratocytes and fibroblasts to myofibroblasts is important to corneal wound healing as well as formation of stromal haze. The purpose of this study was to determine the effect of latrunculin B, an actin cytoskeleton disruptor in conjunction with a fundamental biophysical cue, substrate stiffness, on myofibroblast transformation in vitro and in vivo. Rabbit corneal fibroblasts were cultured on substrates of differing compliance (1.5, 22, and 71 kPa) and tissue culture plastic (TCP; > 1 GPa) in media containing 0 or 10 ng/ml TGFβ1 for 72 h. Cells were treated with 0.4 μM Lat-B or DMSO for 30 min every 24 h for 72 h. RNA was collected from cells and expression of alpha-smooth muscle actin (α-SMA), keratocan, and ALDH1A1 determined using qPCR; immunocytochemistry was used to assess α-SMA protein expression. A rabbit phototherapeutic keratectomy (PTK) model was used to assess the impact of 0.1% Lat-B (n = 3) or 25% DMSO (vehicle control, n = 3) on corneal wound healing by assessment of epithelial wound size with fluorescein stain and semi-quantitative stromal haze scoring by an observer masked to treatment group as well as Fourier-domain optical coherence tomography (FD-OCT) at set time points. Statistical analysis was completed using one-way or two-way analysis of variance. Treatment with Lat-B versus DMSO resulted in significantly less αSMA mRNA (P ≤ 0.007) for RCF cells grown on 22 and 71 kPa substrates as well as TCP without or with TGFβ1, and significantly decreased α-SMA protein expression in RCFs cultured on the intermediate (22 kPa) stiffness in the absence (P = 0.028) or presence (P = 0.018) of TGFβ1. Treatment with Lat-B versus DMSO but did not significantly alter expression of keratocan or ALDH1A1 mRNA in RCFs (P > 0.05) in the absence or presence of TGFβ1, but RCFs grown on stiff hydrogels (71 kPa) had significantly more keratocan mRNA expression versus the 22 kPa hydrogel or TCP (P < 0.001) without TGFβ1. Administration of topical Lat-B BID was well tolerated by rabbits post-PTK but did not significantly alter epithelial wound closure, stromal haze score, stromal haze thickness as measured by FD-OCT in comparison to DMSO-treated rabbits. When corneal stromal cells are cultured on substrates possessing biologically relevant substratum stiffnesses, Lat-B modulates mRNA and protein expression of α-SMA and thus modulates myofibroblast transformation. At a dose and dose-frequency that reduced IOP in human glaucoma patients, Lat-B treatment did not substantially impact corneal epithelial or stromal wound healing in a rabbit PTK model. While a significant impact on wound healing was observed at the concentration and dose frequency reported here was not found, encouraging in vitro data support further investigations of topically applied Lat-B to determine if this compound can reduce stromal fibrosis.<br />
DOI: 10.1016/j.exer.2018.02.003 PMCID: PMC5924616 PMID: 29421383<br />
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[3]. Clin Sci (Lond). 2016 May;130(9):721-32. doi: 10.1042/CS20150593. Epub 2016 Feb 2.<br />
Latrunculin B modulates electrophysiological characteristics and arrhythmogenesis in pulmonary vein cardiomyocytes.<br />
Lu YY(1), Lin YK(2), Wen ZH(3), Chen YC(4), Chen SA(5), Chen YJ(6).<br />
Author information: (1)Division of Cardiology, Department of Internal Medicine, Sijhih Cathay General Hospital, New Taipei City, Taiwan School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan. (2)Division of Cardiovascular Medicine, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan. (3)Department of Marine Biotechnology and Resources, Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung, Taiwan. (4)Department of Biomedical Engineering, National Defense Medical Center, Taipei, Taiwan [email protected]. (5)National Yang-Ming University, School of Medicine; Division of Cardiology and Cardiovascular Research Center, Veterans General Hospital-Taipei, Taipei, Taiwan. (6)Division of Cardiovascular Medicine, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.<br />
AF (atrial fibrillation) is the most common sustained arrhythmia, and the PVs (pulmonary veins) play a critical role in triggering AF. Stretch causes structural remodelling, including cytoskeleton rearrangement, which may play a role in the genesis of AF. Lat-B (latrunculin B), an inhibitor of actin polymerization, is involved in Ca(2+) regulation. However, it is unclear whether Lat-B directly modulates the electrophysiological characteristics and Ca(2+) homoeostasis of the PVs. Conventional microelectrodes, whole-cell patch-clamp, and the fluo-3 fluorimetric ratio technique were used to record ionic currents and intracellular Ca(2+) within isolated rabbit PV preparations, or within isolated single PV cardiomyocytes, before and after administration of Lat-B (100 nM). Langendorff-perfused rabbit hearts were exposed to acute and continuous atrial stretch, and we studied PV electrical activity. Lat-B (100 nM) decreased the spontaneous electrical activity by 16±4% in PV preparations. Lat-B (100 nM) decreased the late Na(+) current, L-type Ca(2+) current, Na(+)/Ca(2+) exchanger current, and stretch-activated BKCa current, but did not affect the Na(+) current in PV cardiomyocytes. Lat-B reduced the transient outward K(+) current and ultra-rapid delayed rectifier K(+) current, but increased the delayed rectifier K(+) current in isolated PV cardiomyocytes. In addition, Lat-B (100 nM) decreased intracellular Ca(2+) transient and sarcoplasmic reticulum Ca(2+) content in PV cardiomyocytes. Moreover, Lat-B attenuated stretch-induced increased spontaneous electrical activity and trigger activity. The effects of Lat-B on the PV spontaneous electrical activity were attenuated in the presence of Y-27632 [10 μM, a ROCK (Rho-associated kinase) inhibitor] and cytochalasin D (10 μM, an actin polymerization inhibitor). In conclusion, Lat-B regulates PV electrophysiological characteristics and attenuates stretch-induced arrhythmogenesis.<br />
DOI: 10.1042/CS20150593 PMID: 26839418<br />
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[4]. J Appl Physiol (1985). 2005 Feb;98(2):489-97. doi: 10.1152/japplphysiol.01378.2003. Epub 2004 Oct 1.<br />
Latrunculin B increases force fluctuation-induced relengthening of ACh-contracted, isotonically shortened canine tracheal smooth muscle.<br />
Dowell ML(1), Lakser OJ, Gerthoffer WT, Fredberg JJ, Stelmack GL, Halayko AJ, Solway J, Mitchell RW.<br />
Author information: (1)Section of Pulmonary and Critical Care Medicine, Univ. of Chicago, MC6026, 5841 S. Maryland Ave., Chicago, IL 60637, USA.<br />
We hypothesized that differences in actin filament length could influence force fluctuation-induced relengthening (FFIR) of contracted airway smooth muscle and tested this hypothesis as follows. One-hundred micromolar ACh-stimulated canine tracheal smooth muscle (TSM) strips set at optimal reference length (Lref) were allowed to shorten against 32% maximal isometric force (Fmax) steady preload, after which force oscillations of +/-16% Fmax were superimposed. Strips relengthened during force oscillations. We measured hysteresivity and calculated FFIR as the difference between muscle length before and after 20-min imposed force oscillations. Strips were relaxed by ACh removal and treated for 1 h with 30 nM latrunculin B (sequesters G-actin and promotes depolymerization) or 500 nM jasplakinolide (stabilizes actin filaments and opposes depolymerization). A second isotonic contraction protocol was then performed; FFIR and hysteresivity were again measured. Latrunculin B increased FFIR by 92.2 +/- 27.6% Lref and hysteresivity by 31.8 +/- 13.5% vs. pretreatment values. In contrast, jasplakinolide had little influence on relengthening by itself; neither FFIR nor hysteresivity was significantly affected. However, when jasplakinolide-treated tissues were then incubated with latrunculin B in the continued presence of jasplakinolide for 1 more h and a third contraction protocol performed, latrunculin B no longer substantially enhanced TSM relengthening. In TSM treated with latrunculin B + jasplakinolide, FFIR increased by only 3.03 +/- 5.2% Lref and hysteresivity by 4.14 +/- 4.9% compared with its first (pre-jasplakinolide or latrunculin B) value. These results suggest that actin filament length, in part, determines the relengthening of contracted airway smooth muscle.<br />
DOI: 10.1152/japplphysiol.01378.2003 PMID: 15465883<br />
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[5]. Exp Eye Res. 2006 Feb;82(2):236-46. doi: 10.1016/j.exer.2005.06.017. Epub 2005 Jul 27.<br />
Latrunculin B effects on trabecular meshwork and corneal endothelial morphology in monkeys.<br />
Sabanay I(1), Tian B, Gabelt BT, Geiger B, Kaufman PL.<br />
Author information: (1)Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.<br />
To determine the mechanism of latrunculin B (LAT-B)-induced decrease in outflow resistance and the effect of LAT-B on the cornea, structural changes of the trabecular meshwork (TM) and the corneal endothelium following LAT-B were studied in the live monkey eye. LAT-B (0.5 microM) and vehicle were administered by anterior chamber exchange and infusion with cationized and non-cationized gold solution in opposite eyes. The eyes were fixed by infusing Ito's solution and enucleated. Anterior segments were quadrisected and embedded in Epon-Embed 812. Morphology of the TM and the corneal endothelium was studied by light and electron microscopy. LAT-B-induced morphological changes in the TM included: (1) loss of microfilament integrity in cells, especially in TM cells on the collagen beams; (2) development of numerous cytoplasmic projections of the sub-canalicular cells (SUB); (3) reorganization of intermediate filaments in Schlemm's canal inner wall (IW) cells; (4) massive 'ballooning' of the juxtacanalicular (JXT) region, leading to a substantial expansion of the space between the IW of Schlemm's canal and the trabecular collagen beams; and (5) retention of extracellular matrix (ECM), trapped between the SUB cell layer and IW cells. No detrimental effects on tight junctions, giant vacuoles, and cell-cell and cell-ECM adhesions were observed. Endocytosis of gold particles was not affected. Morphology of the corneal endothelium of the LAT-B-treated eye was unchanged. In conclusion, TM changes in the LAT-B-treated eye suggest that the expansion of the JXT space may account for the decrease in outflow resistance induced by latrunculins. The outflow-effective concentration of LAT-B administered intracamerally does not significantly affect the corneal endothelium.<br />
DOI: 10.1016/j.exer.2005.06.017 PMID: 16054137
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