Effect of brefeldin A on heparan sulphate biosynthesis in Madin–Darby canine kidney cells

2002 ◽  
Vol 362 (2) ◽  
pp. 359-366 ◽  
Author(s):  
Svein Olav KOLSET ◽  
Kristian PRYDZ ◽  
Katja FJELDSTAD ◽  
Fariba SAFAIYAN ◽  
Tram Thu VUONG ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Heparan Sulphate ◽  
Brefeldin A ◽  
Sodium Chlorate ◽  
Cell Types ◽  
Madin Darby Canine Kidney ◽  
Golgi Stack ◽  
Golgi Network ◽  
Darby Canine Kidney ◽  
Canine Kidney

Brefeldin A (BFA) perturbs the organization of the Golgi apparatus, such that Golgi stack components are fused with the endoplasmic reticulum (ER) and separated from the trans-Golgi network. In many cell types, BFA blocks the secretion of macromolecules but still allows the action of Golgi enzymes in the ER. Treatment of cells with BFA has been reported to inhibit the secretion of heparan sulphate (HS) proteoglycans and alter the structure of their HS components, but the nature of such structural alterations has not been characterized in detail. We analysed the effect of BFA on HS biosynthesis in Madin—Darby canine kidney (MDCK) cells, in which the Golgi complex is more resistant towards BFA than in most other cell types. We found that MDCK cells were able to secrete HS proteoglycans in spite of BFA treatment. However, the secretion of HS was reduced and the secreted HS differed from that produced by untreated cells. In BFA-treated cells, two structurally distinct pools of HS were generated. One pool was similar to HS from control cells, with the exception that the 6-O-sulphation of glucosamine (GlcN) residues was reduced. In contrast, the other pool consisted of largely unmodified N-acetylheparosan polymers with a low (<20%) proportion of N-sulphated GlcN residues but a substantial proportion of N-unsubstituted GlcN units, indicating that it had been acted upon by N-deacetylases and partly by the N-sulphotransferases, but not by O-sulphotransferases. Together, these findings represent a previously unrecognized alteration in HS biosynthesis caused by BFA, and differ dramatically from our previous findings in MDCK cells pertaining to the undersulphation of HS caused by sodium chlorate treatment.

scholarly journals Effect of brefeldin A on heparan sulphate biosynthesis in Madin‒Darby canine kidney cells

2002 ◽  
Vol 362 (2) ◽  
pp. 359 ◽  
Author(s):  
Svein Olav KOLSET ◽  
Kristian PRYDZ ◽  
Katja FJELDSTAD ◽  
Fariba SAFAIYAN ◽  
Tram Thu VUONG ◽  
...  
Keyword(s):  
Heparan Sulphate ◽  
Brefeldin A ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

scholarly journals Selective Perturbation of Apical Membrane Traffic by Expression of Influenza M2, an Acid-activated Ion Channel, in Polarized Madin–Darby Canine Kidney Cells

1998 ◽  
Vol 9 (9) ◽  
pp. 2477-2490 ◽  
Author(s):  
Jennifer R. Henkel ◽  
Gerard Apodaca ◽  
Yoram Altschuler ◽  
Stephen Hardy ◽  
Ora A. Weisz
Keyword(s):  
Ion Channel ◽  
Mdck Cells ◽  
Endocytic Pathway ◽  
Apical Plasma Membrane ◽  
Madin Darby Canine Kidney ◽  
Recycling Endosome ◽  
Trans Golgi Network ◽  
Golgi Network ◽  
Darby Canine Kidney ◽  
Canine Kidney

The function of acidification along the endocytic pathway is not well understood, in part because the perturbants used to modify compartmental pH have global effects and in some cases alter cytoplasmic pH. We have used a new approach to study the effect of pH perturbation on postendocytic traffic in polarized Madin–Darby canine kidney (MDCK) cells. Influenza M2 is a small membrane protein that functions as an acid-activated ion channel and can elevate the pH of the trans-Golgi network and endosomes. We used recombinant adenoviruses to express the M2 protein of influenza virus in polarized MDCK cells stably transfected with the polymeric immunoglobulin (Ig) receptor. Using indirect immunofluorescence and immunoelectron microscopy, M2 was found to be concentrated at the apical plasma membrane and in subapical vesicles; intracellular M2 colocalized partly with internalized IgA in apical recycling endosomes as well as with the trans-Golgi network marker TGN-38. Expression of M2 slowed the rate of IgA transcytosis across polarized MDCK monolayers. The delay in transport occurred after IgA reached the apical recycling endosome, consistent with the localization of intracellular M2. Apical recycling of IgA was also slowed in the presence of M2, whereas basolateral recycling of transferrin and degradation of IgA were unaffected. By contrast, ammonium chloride affected both apical IgA and basolateral transferrin release. Together, our data suggest that M2 expression selectively perturbs acidification in compartments involved in apical delivery without disrupting other postendocytic transport steps.

scholarly journals Influenza M2 Proton Channel Activity Selectively Inhibits Trans-Golgi Network Release of Apical Membrane and Secreted Proteins in Polarized Madin-Darby Canine Kidney Cells

2000 ◽  
Vol 148 (3) ◽  
pp. 495-504 ◽  
Author(s):  
Jennifer R. Henkel ◽  
Gregory A. Gibson ◽  
Paul A. Poland ◽  
Mark A. Ellis ◽  
Rebecca P. Hughey ◽  
...  
Keyword(s):  
Ion Channel ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Protein Sorting ◽  
Madin Darby Canine Kidney ◽  
Trans Golgi Network ◽  
Golgi Network ◽  
Kinetics Of ◽  
Darby Canine Kidney ◽  
Canine Kidney

The function of acidification in protein sorting along the biosynthetic pathway has been difficult to elucidate, in part because reagents used to alter organelle pH affect all acidified compartments and are poorly reversible. We have used a novel approach to examine the role of acidification in protein sorting in polarized Madin-Darby canine kidney (MDCK) cells. We expressed the influenza virus M2 protein, an acid-activated ion channel that equilibrates lumenal and cytosolic pH, in polarized MDCK cells and examined the consequences on the targeting and delivery of apical and basolateral proteins. M2 activity affects the pH of only a subset of acidified organelles, and its activity can be rapidly reversed using ion channel blockers (Henkel, J.R., G. Apodaca, Y. Altschuler, S. Hardy, and O.A. Weisz. 1998. Mol. Biol. Cell. 8:2477–2490; Henkel, J.R., J.L. Popovich, G.A. Gibson, S.C. Watkins, and O.A. Weisz. 1999. J. Biol. Chem. 274:9854–9860). M2 expression significantly decreased the kinetics of cell surface delivery of the apical membrane protein influenza hemagglutinin, but not of the basolaterally delivered polymeric immunoglobulin receptor. Similarly, the kinetics of apical secretion of a soluble form of γ-glutamyltranspeptidase were reduced with no effect on the basolaterally secreted fraction. Interestingly, M2 activity had no effect on the rate of secretion of a nonglycosylated protein (human growth hormone [hGH]) that was secreted equally from both surfaces. However, M2 slowed apical secretion of a glycosylated mutant of hGH that was secreted predominantly apically. Our results suggest a role for acidic trans-Golgi network pH in signal-mediated loading of apical cargo into forming vesicles.

Presence of a novel influx pathway for Mg2+ in MDCK cells

1990 ◽  
Vol 259 (3) ◽  
pp. C521-C525 ◽  
Author(s):  
G. A. Quamme ◽  
L. J. Dai
Keyword(s):  
Cell Line ◽  
Mdck Cell ◽  
Mdck Cells ◽  
Cell Types ◽  
Madin Darby Canine Kidney ◽  
Entry Pathway ◽  
Electrical Gradient ◽  
Free Media ◽  
Darby Canine Kidney ◽  
Canine Kidney

Basal free Mg2+ concentration was 0.49 +/- 0.03 mM in normal single Madin-Darby canine kidney (MDCK) cells as measured by fluorescence with the aid of mag-fura-2. Accordingly, Mg2+ may enter the cell down a transmembrane electrical gradient. The present study describes some aspects of Mg2+ entry into the established MDCK cell line. MDCK cells were Mg2(+)-depleted (0.26 +/- 0.01 mM) by culturing in Mg2(+)-free media for 16-20 h. Cells were subsequently exposed to 5 mM MgCl2, and intracellular Mg2+ concentration ([Mg2+]i) was monitored with fluorescence. [Mg2+]i returned to normal basal levels, 0.56 +/- 0.05 mM, with a refill rate of 272 +/- 39 nM/s, n = 4. Mg2+ entry was not changed by 5.0 mM external Ca2+ but was completely inhibited with 5.0 mM La3+. Intracellular Ca2+ concentration was not altered by Mg2+ depletion or during Mg2+ repletion. Mg2+ uptake was inhibited by verapamil (0 +/- 27 nM/s, n = 3), was inhibited less so by diltiazem (141 +/- 34 nM/s, n = 3), and was not affected by nifedipine (300 +/- 53 nM/s, n = 6). These inhibitors were fully reversible on removal, and [Mg2+]i returned to normal levels. These data indicate the presence of a unique Mg2+ entry pathway in MDCK cells that may be important in Mg2+ homeostasis. The model of Mg2+ refill into Mg2(+)-depleted cells may be useful in other cell types.

scholarly journals Structure and assembly of desmosome junctions: biosynthesis and turnover of the major desmosome components of Madin-Darby canine kidney cells in low calcium medium.

1987 ◽  
Vol 105 (5) ◽  
pp. 2327-2334 ◽  
Author(s):  
E J Penn ◽  
I D Burdett ◽  
C Hobson ◽  
A I Magee ◽  
D A Rees
Keyword(s):  
Mdck Cells ◽  
Cell Types ◽  
Proteolytic Processing ◽  
Turnover Rates ◽  
Madin Darby Canine Kidney ◽  
Cell Monolayers ◽  
Low Calcium ◽  
High Calcium ◽  
Darby Canine Kidney ◽  
Canine Kidney

Neither stratifying (primary keratinocytes) nor simple (Madin-Darby canine kidney [MDCK] and Madin-Darby bovine kidney [MDBK]) epithelial cell types from desmosomes in low calcium medium (LCM; less than 0.1 mM), but they can be induced to do so by raising the calcium level to physiological concentrations (standard calcium medium [SCM], 2 mM). We have used polyclonal antisera to the major bovine epidermal desmosome components (greater than 100 kD) in a sensitive assay involving immunoprecipitation of the components from metabolically labeled MDCK cell monolayers to investigate the mechanism of calcium-induced desmosome formation. MDCK cells, whether cultured in LCM or SCM, were found to synthesize the desmosome protein, DPI and desmosome glycoproteins DGI and DGII/III with identical electrophoretic mobility, and also, where relevant, with similar carbohydrate addition/processing and proteolytic processing. The timings of these events and of transport of DGI to the cell surface were similar in low and high calcium. Although the rates of synthesis of the various desmosome components were also similar under both conditions, the glycoprotein turnover rates increased dramatically in cells cultured in LCM. The half-lives decreased by a factor of about 7 for DGI and 12 for DGII/III and, consistent with this, MDCK cells labeled for 48 h in SCM had three and six times the amount of DGI and DGII/III, respectively, as cells labeled for 48 h in LCM. The rate of turnover and the levels of DPI were changed in the same direction, but to much lesser extents. Possible mechanisms for the Ca2+-dependent control of desmosome formation are discussed in the light of this new evidence.

scholarly journals Inhibition by brefeldin A of protein secretion from the apical cell surface of Madin-Darby canine kidney cells.

1991 ◽  
Vol 266 (27) ◽  
pp. 17729-17732 ◽  
Author(s):  
S.H. Low ◽  
S.H. Wong ◽  
B.L. Tang ◽  
P. Tan ◽  
V.N. Subramaniam ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Secretion ◽  
Apical Cell ◽  
Brefeldin A ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

Differential sensitivity of Madin-Darby canine kidney (MDCK) cells to epinephrine

2015 ◽  
Vol 20 (5) ◽  
pp. 486-493 ◽  
Author(s):  
P. Muthuraman ◽  
P. C. Nagajyothi ◽  
M. Chandrasekaran ◽  
G. Enkhtaivan ◽  
B. Venkitasamy ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Differential Sensitivity ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

Accumulation of natural and synthetic betaines by a mammalian renal cell line

1996 ◽  
Vol 74 (2) ◽  
pp. 283-287 ◽  
Author(s):  
K. Randall ◽  
M. Lever ◽  
B. A. Peddie ◽  
S. T. Chambers
Keyword(s):  
Osmotic Stress ◽  
Glycine Betaine ◽  
Mdck Cells ◽  
Intracellular Accumulation ◽  
Madin Darby Canine Kidney ◽  
Renal Cell Line ◽  
High Concentrations ◽  
Inhibition Studies ◽  
Darby Canine Kidney ◽  
Canine Kidney

Intracellular accumulation of different betaines was compared in osmotically stressed Madin Darby canine kidney (MDCK) cells to model the betaine accumulation specificity of the mammalian inner medulla and to show how this accumulation differed from that of bacteria. All betaines accumulated less than glycine betaine. Arsenobetaine (the arsenic analogue of glycine betaine) accumulated to 12% of the glycine betaine levels and the sulphur analogue dimethylthetin accumulated to >80%. Most substituted glycine betaine analogues accumulated to 2–5% of intracellular glycine betaine concentrations, however, serine betaine accumulated to <0.5% of glycine betaine levels. Inhibition studies to distinguish the betaine ports were performed by the addition of proline. Butyrobetaine and carnitine accumulation was not proline sensitive, whereas that of omer betaines was. As with glycine betaine, the accumulation of propionobetaine and dimethylthetin was proline sensitive and osmoregulated. Pyridinium betaine was accumulated by both proline-sensitive and -insensitive systems, with a small increase under osmotic stress. High concentrations (10 times that of glycine betaine) of the dietary betaines proline betaine and trigonelline inhibited total betaine accumulation. Because α-substituted betaines are accumulated by bacteria and not by MDCK cells, these betaines may be the basis for design of antimicrobial agents.Key words: MDCK cells, betaine accumulation, osmolytes, betaine analogues.

Damaging effects of high energy shock waves on cultured Madin Darby Canine Kidney (MDCK) cells

1990 ◽  
Vol 18 (4) ◽  
pp. 255-258 ◽  
Author(s):  
W. L. Strohmaier ◽  
K. -H. Bichler ◽  
P. Deetjen ◽  
S. Kleinknecht ◽  
M. Pedro ◽  
...  
Keyword(s):  
Shock Waves ◽  
Mdck Cells ◽  
High Energy ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
Damaging Effects
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