Stimulation of regulatory volume increase (RVI) in avian articular chondrocytes by gadolinium chloride

2010 ◽  
Vol 88 (3) ◽  
pp. 505-512 ◽  
Author(s):  
Sang-Bing Ong ◽  
Dinesh Shah ◽  
Ala Qusous ◽  
Simon M. Jarvis ◽  
Mark J.P. Kerrigan
Keyword(s):  
Actin Cytoskeleton ◽  
Articular Chondrocytes ◽  
Cell Volume Regulation ◽  
Extracellular Calcium ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Gadolinium Chloride ◽  
Volume Increase ◽  
Regulatory Volume Increase ◽  
Volume Recovery

Chondrocytes, the resident cell-type of articular cartilage, are responsible for the regulation of the extracellular matrix (ECM) in response to their physico-chemical environment. Due to the nature of cartilage loading, chondrocytes are exposed to constant changes in extracellular osmolality with a gradual increase throughout the day. As an increase in osmolality attenuates matrix synthesis, we have studied cell volume regulation (regulatory volume increase (RVI)) after hypertonic challenge and the regulation of RVI by the actin cytoskeleton. Using freshly isolated avian articular chondrocytes, changes in actin organisation were studied by confocal laser scanning microscopy following a 43% increase in extracellular osmolality. Using calcein-loading chondrocytes, the capacity for RVI was determined and the rate of volume recovery (t1/2) mathematically extrapolated. Following an increase in extracellular osmolality there was a significant increase (p < 0.05) in cortical actin, inhibited by the removal of extracellular calcium EGTA or by the addition of 100 µmol·L–1 gadolinium chloride. Most cells exhibited slow RVI (t1/2 = 55.5 ± 5.5 min), whereby inhibition of actin polymerisation by gadolinium chloride or the removal of extracellular calcium significantly increased the rate of volume recovery via a bumetanide-sensitive pathway (t1/2 of 29.6 ± 6.5 min and 13.8 ± 3.1 min, respectively). These data suggest the Na+–K+–2Cl– (NKCC) co-transporter regulated by the actin cytoskeleton is involved in avian chondrocyte RVI.

Cytokinesis arrest and redistribution of actin-cytoskeleton regulatory components in cells expressing the Rho GTPase CDC42Hs

1996 ◽  
Vol 109 (2) ◽  
pp. 367-377 ◽  
Author(s):  
H. Dutartre ◽  
J. Davoust ◽  
J.P. Gorvel ◽  
P. Chavrier
Keyword(s):  
Actin Cytoskeleton ◽  
Rho Gtpases ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Rho Gtpase ◽  
Laser Scanning Microscopy ◽  
Regulatory Subunit ◽  
Confocal Laser ◽  
Complex Architectures ◽  
Immunofluorescence Labelling

In mammalian cells, Rho GTPases control the reorganisation of the actin cytoskeleton in response to growth factors. In the cytoplasm, the polymerisation of actin filaments and their organisation into complex architectures is orchestrated by numerous proteins which act either directly, by interacting with actin, or by producing secondary messengers which serve as mediators between signal transduction pathways and the microfilament organisation. We sought to determine whether the intracellular distribution of some of these regulatory components may be controlled by the Rho GTPase CDC42Hs. With this aim, we have established HeLa-derived human cell lines in which expression of a constitutively activated mutant of CDC42Hs is inducible. Morphological analysis by immunofluorescence labelling and confocal laser scanning microscopy revealed a massive reorganisation of F-actin in cortical microspikes as well as podosome-like structures located at the ventral face of the cells. Concomitantly, the cells became giant and multinucleate indicating that cytokinesis was impaired. The actin bundling protein T-plastin, the vasodilatator-stimulated phosphoprotein (VASP), a profilin ligand, as well as the 85 kDa regulatory subunit of the phosphoinosite 3-kinase redistributed with F-actin into the CDC42Hs-induced structures.

scholarly journals Hypertonic stress increases the Na+ conductance of rat hepatocytes in primary culture.

1995 ◽  
Vol 105 (4) ◽  
pp. 507-535 ◽  
Author(s):  
F Wehner ◽  
H Sauer ◽  
R K Kinne
Keyword(s):  
Cell Membrane ◽  
Primary Culture ◽  
Regulatory Volume Decrease ◽  
Rat Hepatocytes ◽  
Laser Scanning Microscopy ◽  
Specific Cell ◽  
Volume Increase ◽  
Regulatory Volume Increase ◽  
Hypertonic Stress ◽  
Cell Volumes

We studied the ionic mechanisms underlying the regulatory volume increase of rat hepatocytes in primary culture by use of confocal laser scanning microscopy, conventional and ion-sensitive microelectrodes, cable analysis, microfluorometry, and measurements of 86Rb+ uptake. Increasing osmolarity from 300 to 400 mosm/liter by addition of sucrose decreased cell volumes to 88.6% within 1 min; thereafter, cell volumes increased to 94.1% of control within 10 min, equivalent to a regulatory volume increase (RVI) by 44.5%. This RVI was paralleled by a decrease in cell input resistance and in specific cell membrane resistance to 88 and 60%, respectively. Ion substitution experiments (high K+, low Na+, low Cl-) revealed that these membrane effects are due to an increase in hepatocyte Na+ conductance. During RVI, ouabain-sensitive 86Rb+ uptake was augmented to 141% of control, and cell Na+ and cell K+ increased to 148 and 180%, respectively. The RVI, the increases in Na+ conductance and cell Na+, as well as the activation of Na+/K(+)-ATPase were completely blocked by 10(-5) mol/liter amiloride. At this concentration, amiloride had no effect on osmotically induced cell alkalinization via Na+/H+ exchange. When osmolarity was increased from 220 to 300 mosm/liter (by readdition of sucrose after a preperiod of 15 min in which the cells underwent a regulatory volume decrease, RVD) cell volumes initially decreased to 81.5%; thereafter cell volumes increased to 90.8% of control. This post-RVD-RVI of 55.0% is also mediated by an increase in Na+ conductance. We conclude that rat hepatocytes in confluent primary culture are capable of RVI as well as of post-RVD-RVI. In this system, hypertonic stress leads to a considerable increase in cell membrane Na+ conductance. In concert with conductive Na+ influx, cell K+ is then increased via activation of Na+/K(+)-ATPase. An additional role of Na+/H+ exchange in the volume regulation of rat hepatocytes remains to be defined.

Articular Cartilage Fragmentation Improves Chondrocyte Migration by Upregulating Membrane Type 1 Matrix Metalloprotease

Cartilage ◽  
2021 ◽  
pp. 194760352110354
Author(s):  
Yunliang Lei ◽  
Jiabin Peng ◽  
Zhu Dai ◽  
Ying Liao ◽  
Quanhui Liu ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Articular Chondrocytes ◽  
Laser Scanning Microscopy ◽  
Gelatin Sponge ◽  
Confocal Laser ◽  
Matrix Metalloprotease ◽  
Membrane Type ◽  
Chondrocyte Migration

Objective This study was undertaken to elucidate the mechanism of improved chondrocyte migration after juvenile articular cartilage fragmentation. Design In vitro organ culture with rabbit cartilage fragments and cell culture with rabbit chondrocytes were performed. In part A, minced juvenile cartilage fragments (~0.5 × 0.5 × 0.5 mm) from rabbits, planted in gelatin sponge and fibrin glue, were cultured for 2, 4, or 6 weeks in vitro and compared with the cartilage chunks (~4 × 4 × 1 mm) and membrane type 1 matrix metalloprotease (MT1-MMP) inhibitor groups. Chondrocyte outgrowth was evaluated on histology and confocal laser scanning microscopy. MT1-MMP expression was compared between the cartilage fragment group and the cartilage chunks group. In part B, articular chondrocytes were harvested from juvenile rabbits, MT1-MMP was transfected into the cells, and cell migration was evaluated using the Transwell and wound healing tests. Results The histology and confocal microscopy results revealed that cell accumulation occurred at the edge of cartilage fragments, and outgrowth was better in the cartilage fragment group than those in the cartilage chunks group. Similar results were observed for MT1-MMP expression. After MT1-MMP inhibition, cells did not accumulate at the edge of the cartilage fragments, and chondrocyte outgrowth did not occur. Furthermore, overexpression of MT1-MMP enhanced the migration of articular chondrocytes. Conclusions Juvenile articular cartilage fragmentation improved chondrocyte migration by upregulating MT1-MMP.

SRC family kinases in cell volume regulation

2005 ◽  
Vol 288 (3) ◽  
pp. C483-C493 ◽  
Author(s):  
David M. Cohen
Keyword(s):  
Tyrosine Kinases ◽  
Volume Regulation ◽  
Cell Volume Regulation ◽  
Src Family Kinases ◽  
Protein Tyrosine Kinases ◽  
Volume Increase ◽  
Regulatory Volume Increase ◽  
Hypotonic Stress ◽  
Cell Functions ◽  
Broad Role

SRC family kinases are a group of nine cytoplasmic protein tyrosine kinases essential for many cell functions. Some appear to be ubiquitously expressed, whereas others are highly tissue specific. The ability of members of the SRC family to influence ion transport has been recognized for several years. Mounting evidence suggests a broad role for SRC family kinases in the cell response to both hypertonic and hypotonic stress, and in the ensuing regulatory volume increase or decrease. In addition, members of this tyrosine kinase family participate in the mechanotransduction that accompanies cell membrane deformation. Finally, at least one SRC family member operates in concert with the p38 MAPK to regulate tonicity-dependent gene transcription.

Regulatory volume increase (RVI) by in situ and isolated bovine articular chondrocytes

2006 ◽  
Vol 209 (2) ◽  
pp. 481-492 ◽  
Author(s):  
Mark J.P. Kerrigan ◽  
Corinne S.V. Hook ◽  
Ala' Qusous ◽  
Andrew C. Hall
Keyword(s):  
Articular Chondrocytes ◽  
Volume Increase ◽  
Regulatory Volume Increase ◽  
Bovine Articular Chondrocytes

Geometrical Micropillars Combined with Chemical Surface Modifications – Independency of Actin Filament Spatial Distribution in Primary Osteoblasts

2014 ◽  
Vol 783-786 ◽  
pp. 1320-1325 ◽  
Author(s):  
J. Barbara Nebe ◽  
Birgit Finke ◽  
Andreas Koertge ◽  
Henrike Rebl ◽  
Susanne Staehlke
Keyword(s):  
Actin Cytoskeleton ◽  
Laser Scanning ◽  
Culture Conditions ◽  
Laser Scanning Microscopy ◽  
Collagen I ◽  
Confocal Laser ◽  
Cell Physiology ◽  
Short Fibers ◽  
Chemical Surface ◽  
Primary Osteoblasts

Cell-biomaterial interactions are strongly affected by topographical and chemical surface characteristics. We found out earlier that geometric titanium (Ti) pillar structures in the micrometer range induce the cells to rearrange their actin cytoskeleton in short fibers solely on the top of the pillars. As a result, cell physiology was hampered concerning collagen I synthesis and spreading capacity. Furthermore, the position-dependent initial cell adhesion strength was declined near the edges. We asked whether these observed cellular effects can be performed only in combination with Ti or occur independently of chemical surface features. In addition, the specific culture conditions, e.g. serum content or influence of gravity, were of interest. Human primary osteoblasts were cultured in Osteoblast Growth Medium with serum containing SupplementMix on pure silicon pillars (5x5x5 μm) or on samples additionally sputtered with Ti (as reference) or gold. To offer the cells ligands for their adhesion receptors, we coated the pillars with collagen I or alternatively with a plasma polymer layer from allylamine. Different from standard culture conditions, the cells were cultured against gravity as well as without serum. The actin cytoskeleton was stained with phalloidin-TRITC after 24 h and analyzed by confocal laser scanning microscopy. Interestingly, on all modifications tested the cell’s actin cytoskeleton was distinctly organized in short fibers on the top of the pillars. Thus, we were able to exclude the influence of (i) the material chemistry (gold, silicon, physical plasma vs. Ti), (ii) the protein deposition on the pillar top and edges, and (iii) the impression caused by gravity.

Electrolyte transport mechanisms involved in regulatory volume increase in C6 glioma cells

1996 ◽  
Vol 271 (4) ◽  
pp. C1041-C1048 ◽  
Author(s):  
I. Mountian ◽  
K. Y. Chou ◽  
W. Van Driessche
Keyword(s):  
Time Course ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
Disulfonic Acid ◽  
Volume Increase ◽  
Regulatory Volume Increase ◽  
Volume Recovery ◽  
Active Substances ◽  
Cell Thickness

Volume regulation of C6 glioma cells was studied with an automatic system for monitoring cell thickness, while increasing bath osmolality from 300 to 440 mosmol/kgH2O. At 37 degrees C, tissues incubated in solutions containing active substances (inositol, D-biotin, hydrocortisone, prostaglandin E1, insulin, transferrin, sodium selenite, and 3,5,3'-triiodothyronine) responded to hyperosmotic challenge with a typical regulatory volume increase (RVI). Lowering temperature or removing the active substances inhibited osmoregulation. Bumetanide, amiloride, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, or ouabain significantly reduced RVI. Ion substitutions of Na+, Cl-, NaCl, or HCO3- also importantly affected the process. Extracellular acidification rate (EAR) was studied by microphysiometry. Hyperosmotic shock induced an increase in EAR with a time course that matched volume recovery. This increase in EAR was prevented by amiloride. The data show that under hyperosmotic conditions C6 cells are able to regulate their volume. Ion substitutions and application of blockers demonstrate that Na+/H+ and Cl-/HCO3- exchangers and Na(+)-K(-)-2Cl- cotransporter are involved in RVI. The rise in EAR is due to the enhanced activity of Na+/H+ antiporter, which seems to be volume dependent but not osmotic dependent.

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

1988 ◽  
Vol 46 ◽  
pp. 96-97
Author(s):  
Thomas M. Jovin ◽  
Michel Robert-Nicoud ◽  
Donna J. Arndt-Jovin ◽  
Thorsten Schormann
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Biochemical Processes ◽  
Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

Sign in / Sign up

Export Citation Format

Share Document