Endogenous and exogenous Na-K-Cl cotransporter expression in a low K-resistant mutant MDCK cell line

A low K-resistant mutant Madin-Darby canine kidney (MDCK) cell line, LK-C1, has been shown previously to lack functional Na-K-Cl cotransporter (NKCC) activity, indicating that it may be a useful NKCC “knockout” cell line for structure-function studies. Using immunological probes, we first characterized the defect in the endogenous NKCC protein of the LK-C1 cells and then fully restored NKCC activity in these cells by stably expressing the human secretory NKCC1 protein (hNKCC1). The endogenous NKCC protein of the LK-C1 cells was expressed at significantly lower levels than in wild-type MDCK cells and was not properly glycosylated. This latter finding indicated that the lack of functional NKCC activity in the LK-C1 cells may be due to the inability to process the protein to the plasma membrane. In contrast, exogenously expressed hNKCC1 protein was properly processed and fully functional at the plasma membrane. Significantly, the exogenous hNKCC1 protein was regulated in a manner similar to the protein in native secretory cells as it was robustly activated by cell shrinkage, calyculin A, and low-Cl incubation. Furthermore, when the LK-C1 cells formed an epithelium on permeable supports, the exogenous hNKCC1 protein was properly polarized and functional at the basolateral membrane. The low levels of endogenous NKCC protein expression, the absence of any endogenous NKCC transport activity, and the ability to form a polarized epithelium indicate that the LK-C1 cells offer an excellent expression system with which to study the molecular physiology of the cation Cl cotransporters.

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Assessment of aspects of the toxicity of Clostridium perfringens E-toxin using the MDCK cell line

Human & Experimental Toxicology ◽

10.1177/096032719601501107 ◽

1996 ◽

Vol 15 (11) ◽

pp. 904-908 ◽

Cited By ~ 20

Author(s):

Christopher D Lindsay

Keyword(s):

Cell Line ◽

Clostridium Perfringens ◽

Mechanism Of Action ◽

Sodium Azide ◽

Mdck Cell ◽

Mdck Cells ◽

Neutral Red ◽

Temperature Sensitive ◽

Mdck Cell Line ◽

Neutral Red Assay

1 The epithelial Madin Darby Canine Kidney (MDCK) cell line was used to study the toxicity of ∈-toxin from Clostridium perfringens. The epithelial MDCK cell line is known to be sensitive to ∈-toxin of Clostridium perfringens and to investigate its mechanism of action, the neutral red assay has been used to determine the viability of cultures of this cell line. 2 Comparison of the LC 50s obtained at 34°C and 0°C showed that the lethality of ∈-toxin was reduced by 18- fold at the lower temperature. The effect of tempera ture on ∈-toxin lethality is unlikely to be due to reductions in membrane fluidity for the addition of Ca2+ or Mg2+ (2 mM) to buffer containing toxin was without effect. Varying the pH of the toxin-containing buffer from 6.9 to 8.7 did not increase the lethality of the toxin, though the most acidic pH used (5.8) was found to potentiate its action on MDCK cells. 3 The effect of inhibiting endocytosis on the lethality ofe toxin was also investigated by incubating cultures of MDCK cells with and without sodium azide over a range of concentrations of toxin. The co-administra tion of sodium azide did not reduce the toxicity of ∈ toxin, suggesting that energy-dependent uptake pro cesses such as endocytosis were unlikely to be involved in its mechanism of action. The results are, however, consistent with known receptor-based me chanisms of uptake and with other mechanisms of internalisation across the plasma membrane. ∈-toxin thus interacts with cell surfaces by a temperature sensitive mechanism potentiated by low pH.

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C-Met signalling in an HGF/SF-insensitive variant MDCK cell line with constitutive motile/invasive behaviour

Journal of Cell Science ◽

10.1242/jcs.109.9.2371 ◽

1996 ◽

Vol 109 (9) ◽

pp. 2371-2381

Author(s):

C.P. Webb ◽

K. Lane ◽

A.P. Dawson ◽

G.F. Vande Woude ◽

R.M. Warn

Keyword(s):

Growth Factor ◽

Tyrosine Kinase ◽

Cell Line ◽

Tyrosine Phosphorylation ◽

Mdck Cell ◽

Mdck Cells ◽

Cell Surface Expression ◽

Cell Phenotype ◽

Mdck Cell Line

The Met protein is a receptor tyrosine kinase for hepatocyte growth factor/scatter factor (HGF/SF), a multifunctional growth factor with mitogenic, motogenic and morphogenic properties. A morphologically altered variant of the MDCK cell line, MDCK-1, spontaneously exhibits a number of features associated with a partial HGF/SF-Met induced phenotype (less adhesive colonies in culture, enhanced invasion and motility, nascent tubule formation), but paradoxically does not respond to HGF/SF treatment. Although the overall cell surface expression and distribution of Met were found to be similar in parental MDCK cells and the MDCK-1 cell line, p145met autophosphorylation (+/ HGF/SF) was significantly reduced in MDCK-1 cells in vitro and in vivo when compared with parental MDCK cells. In contrast, EGF induced cell proliferation and EGF receptor autophosphorylation to similar levels in both cell lines. The basal levels of protein tyrosine phosphorylation were higher in MDCK-1 cells when compared with parental MDCK cells, including that of two prominent proteins with molecular masses of approximately 185 kDa and 220 kDa. Moreover, both p185 and p220 are present and tyrosine phosphorylated in Met immunoprecipitates from MDCK-1 cells (+/-HGF/SF), but not parental MDCK cells. In addition, Met immunocomplexes from MDCK-1 cells exhibited an approximately 3-fold increased tyrosine kinase activity in vitro when compared with MDCK cells, correlating with the higher basal levels of total phosphotyrosine. Treatment of MDCK-1 cells with the tyrosine kinase inhibitor herbimycin A reverted the cell phenotype to a more MDCK-like morphology in culture, with a concomitant reduction in the tyrosine phosphorylation predominantly of p220. Taken together these data suggest that aberrations in Met activity and associated signalling render MDCK-1 cells insensitive to HGF/SF, and may also mediate alterations in MDCK-1 cell behaviour.

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Antioxidant potential and gap junction-mediated intercellular communication as early biological markers of mercuric chloride toxicity in the MDCK cell line

Toxicology in Vitro ◽

10.1016/s0887-2333(02)00030-9 ◽

2002 ◽

Vol 16 (4) ◽

pp. 457-465 ◽

Cited By ~ 23

Author(s):

M.F Aleo ◽

F Morandini ◽

F Bettoni ◽

S Tanganelli ◽

A Vezzola ◽

...

Keyword(s):

Gap Junction ◽

Cell Line ◽

Mercuric Chloride ◽

Biological Markers ◽

Intercellular Communication ◽

Mdck Cell ◽

Antioxidant Potential ◽

Mdck Cell Line ◽

Chloride Toxicity

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Establishment and characterization of an MDCK cell line stably-transfected with chicken Abcb1 encoding P-glycoprotein

Research in Veterinary Science ◽

10.1016/j.rvsc.2016.03.004 ◽

2016 ◽

Vol 106 ◽

pp. 37-44 ◽

Cited By ~ 3

Author(s):

Yong Sun ◽

Tingting Guo ◽

Dawei Guo ◽

Li Guo ◽

Li Chen ◽

...

Keyword(s):

Cell Line ◽

Mdck Cell ◽

Mdck Cell Line ◽

P Glycoprotein

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GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells

The Journal of Cell Biology ◽

10.1083/jcb.200507116 ◽

2006 ◽

Vol 172 (7) ◽

pp. 1023-1034 ◽

Cited By ~ 75

Author(s):

Simona Paladino ◽

Thomas Pocard ◽

Maria Agata Catino ◽

Chiara Zurzolo

Keyword(s):

Plasma Membrane ◽

Mdck Cells ◽

Basolateral Membrane ◽

Apical Surface ◽

Madin Darby Canine Kidney ◽

Biochemical Assays ◽

Intracellular Sorting ◽

Apical Sorting ◽

Golgi Network ◽

Darby Canine Kidney

The polarity of epithelial cells is dependent on their ability to target proteins and lipids in a directional fashion. The trans-Golgi network, the endosomal compartment, and the plasma membrane act as sorting stations for proteins and lipids. The site of intracellular sorting and pathways used for the apical delivery of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are largely unclear. Using biochemical assays and confocal and video microscopy in living cells, we show that newly synthesized GPI-APs are directly delivered to the apical surface of fully polarized Madin–Darby canine kidney cells. Impairment of basolateral membrane fusion by treatment with tannic acid does not affect the direct apical delivery of GPI-APs, but it does affect the organization of tight junctions and the integrity of the monolayer. Our data clearly demonstrate that GPI-APs are directly sorted to the apical surface without passing through the basolateral membrane. They also reinforce the hypothesis that apical sorting of GPI-APs occurs intracellularly before arrival at the plasma membrane.

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Immunocytochemical studies of the Na-K-Cl cotransporter of shark kidney

AJP Renal Physiology ◽

10.1152/ajprenal.1996.270.6.f927 ◽

1996 ◽

Vol 270 (6) ◽

pp. F927-F936 ◽

Cited By ~ 4

Author(s):

D. Biemesderfer ◽

J. A. Payne ◽

C. Y. Lytle ◽

B. Forbush

Keyword(s):

Plasma Membrane ◽

Immunoelectron Microscopy ◽

Basolateral Membrane ◽

Distal Tubule ◽

Rectal Gland ◽

Apical Plasma Membrane ◽

Secretory Cells ◽

Weak Staining ◽

Basolateral Plasma Membrane ◽

Kidney Functions

The Na-K-Cl cotransporter (NKCC or BSC) has been described in numerous secretory and reabsorptive epithelia and is an important part of the mechanism of NaCl reabsorption in both the mammalian and elasmobranch kidneys. We have recently developed a panel of four monoclonal antibodies (MAbs) raised to the 195-kDa Na-K-Cl cotransport protein of the shark rectal gland (sNKCC1), which is expressed along the basolateral plasma membrane of secretory cells in this tissue (29). Here, we report immunologic studies of the Na-K-Cl cotransporter in the kidney of the dogfish shark Squalus acanthias. Western blot analysis of shark renal microsomes using MAbs J3, J7, and J25 identified proteins of approximately 195 and 150 kDa, whereas MAb J4 was not reactive. To define the cellular and subcellular distribution of the cotransport protein, immunofluorescence and immunoelectron microscopy studies were performed on fixed kidneys. Immunofluorescence microscopy on semithin (0.5-micron) cryosections demonstrated that MAbs J3, J7, and J25 intensely stained the apical plasma membrane of all distal tubule segments. Weak staining was also seen along the basolateral membrane of most distal nephrons. Immunoelectron microscopy confirmed this observation and showed that some of these segments were morphologically similar to diluting segments from other species. MAbs also reacted with the brush border and, to a lesser extent, the basolateral membrane of proximal tubules. This study supports the hypothesis that the lateral bundle zone of the elasmobranch kidney functions as a countercurrent exchanger and is consistent with the presence of multiple isoforms of the Na-K-Cl cotransporter in the shark kidney.

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