Expression of 5-HT1C receptors in transfected MDCK cells is functionally and anatomically asymmetric

Madin-Darby canine kidney (MDCK) cells were transfected with the cDNA for the rat 5-HT1C receptor (pMV7-SR1c) using electroporation. Cells that survived G418 selection medium were loaded with indo-1 and run through a fluorescence-activated cell sorter (FACS); 10% responded to serotonin (5-HT) with an increase in intracellular Ca2+ concentration ([Ca2+]i). Responding cells were separated with the FACS, grown to confluence, and resorted two more times until a clone of 100% respondents was obtained (SR-MDCK). In SR-MDCK cells grown on porous filters, [Ca2+]i increased only when 5-HT was applied to the basolateral membrane (change in [Ca2+]i = 190 +/- 43 nM); there was no response of [Ca2+]i to apical application of 5-HT. The asymmetric response to 5-HT was likely due to targeting of 5-HT1C receptors exclusively to the basolateral membrane of SR-MDCK cells; 125I-labeled lysergic acid diethylamide binding sites, a marker of high-affinity 5-HT receptors, were located only in the basolateral membrane. These experiments demonstrate that epithelial cells can be stably transfected to express G protein-linked, calcium-mobilizing receptors and that the receptors may be targeted asymmetrically to specific domains of the plasma membrane.

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The MAL Proteolipid Is Necessary for Normal Apical Transport and Accurate Sorting of the Influenza Virus Hemagglutinin in Madin-Darby Canine Kidney Cells

The Journal of Cell Biology ◽

10.1083/jcb.145.1.141 ◽

1999 ◽

Vol 145 (1) ◽

pp. 141-151 ◽

Cited By ~ 128

Author(s):

Rosa Puertollano ◽

Fernando Martín-Belmonte ◽

Jaime Millán ◽

María del Carmen de Marco ◽

Juan P. Albar ◽

...

Keyword(s):

Influenza Virus ◽

Mdck Cells ◽

Basolateral Membrane ◽

Ectopic Expression ◽

Apical Surface ◽

Madin Darby Canine Kidney ◽

Transport Pathway ◽

Influenza Virus Hemagglutinin ◽

Darby Canine Kidney ◽

Canine Kidney

The MAL (MAL/VIP17) proteolipid is a nonglycosylated integral membrane protein expressed in a restricted pattern of cell types, including T lymphocytes, myelin-forming cells, and polarized epithelial cells. Transport of the influenza virus hemagglutinin (HA) to the apical surface of epithelial Madin-Darby canine kidney (MDCK) cells appears to be mediated by a pathway involving glycolipid- and cholesterol- enriched membranes (GEMs). In MDCK cells, MAL has been proposed previously as being an element of the protein machinery for the GEM-dependent apical transport pathway. Using an antisense oligonucleotide-based strategy and a newly generated monoclonal antibody to canine MAL, herein we have approached the effect of MAL depletion on HA transport in MDCK cells. We have found that MAL depletion diminishes the presence of HA in GEMs, reduces the rate of HA transport to the cell surface, inhibits the delivery of HA to the apical surface, and produces partial missorting of HA to the basolateral membrane. These effects were corrected by ectopic expression of MAL in MDCK cells whose endogenous MAL protein was depleted. Our results indicate that MAL is necessary for both normal apical transport and accurate sorting of HA.

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A basolateral lactate/H+ co-transporter in Madin-Darby Canine Kidney (MDCK) cells

Biochemical Journal ◽

10.1042/bj2890263 ◽

1993 ◽

Vol 289 (1) ◽

pp. 263-268 ◽

Cited By ~ 15

Author(s):

S O Rosenberg ◽

T Fadil ◽

V L Schuster

Keyword(s):

Apical Membrane ◽

Mdck Cells ◽

Basolateral Membrane ◽

Ionic Diffusion ◽

Madin Darby Canine Kidney ◽

Apical Compartment ◽

Disulphonic Acid ◽

Component Carrier ◽

Darby Canine Kidney ◽

Canine Kidney

Monolayers of Madin-Darby Canine Kidney (MDCK) cells grown on permeable filters generated lactate aerobically and accumulated it preferentially in the basolateral compartment, suggesting the presence of a lactate carrier. The mechanism of lactate transport across apical and basolateral membranes was examined by determining intracellular pH (pHi) microspectrofluorimetrically after addition of lactate to the extracellular solutions and by measuring uptake of [14C]lactate. Addition of 20 mM lactate to the apical compartment produced no change in pHi, whereas lactate added to the basolateral compartment rapidly and reversibly lowered pHi. Pyruvate produced similar results. Inhibitors of lactate/H+ co-transporters, alpha-cyano-4-hydroxycinnamate (CnCN) and quercetin, partially inhibited the fall in pHi produced by basolateral lactate. In contrast, the disulphonic stilbene. DIDS (4,4′-di-isothiocyanostilbene-2,2′-disulphonic acid) produced no inhibition at 0.5 mM. Kinetic analysis was performed by applying basolateral lactate at various concentrations and measuring the rate of entry (delta pHi/min) in the presence and absence of CnCN. Lactate flux was shown to occur by both non-ionic diffusion and a alpha-cyano-4-hydroxycinnamate-sensitive component (carrier). The latter has a Km of approximately 7 mM for the lactate anion. Propionate, but not formate, lowered pHi to the same degree as did equimolar lactate, but the propionate effect was not inhibited by CnCN. Influx of [14C]lactate was substantially greater across the basolateral membrane than across the apical membrane and occurred in the absence of Na+. We conclude that MDCK cells grown on permeable filters generate lactate aerobically and transport it across the basolateral membrane by way of a lactate/H+ cotransporter.

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Association of Rab25 and Rab11a with the Apical Recycling System of Polarized Madin–Darby Canine Kidney Cells

Molecular Biology of the Cell ◽

10.1091/mbc.10.1.47 ◽

1999 ◽

Vol 10 (1) ◽

pp. 47-61 ◽

Cited By ~ 301

Author(s):

James E. Casanova ◽

Xiaoye Wang ◽

Ravindra Kumar ◽

Sheela G. Bhartur ◽

Jennifer Navarre ◽

...

Keyword(s):

Mdck Cells ◽

Basolateral Membrane ◽

Dominant Negative ◽

Epithelial Cell Line ◽

Madin Darby Canine Kidney ◽

Recycling Endosome ◽

Recycling System ◽

Endosomal Compartment ◽

Darby Canine Kidney ◽

Canine Kidney

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin–Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.

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Polarized endocytosis by Madin-Darby canine kidney cells transfected with functional chicken liver glycoprotein receptor.

The Journal of Cell Biology ◽

10.1083/jcb.109.6.2809 ◽

1989 ◽

Vol 109 (6) ◽

pp. 2809-2816 ◽

Cited By ~ 37

Author(s):

L Graeve ◽

K Drickamer ◽

E Rodriguez-Boulan

Keyword(s):

Mdck Cells ◽

Basolateral Membrane ◽

Cell Surface Receptor ◽

Madin Darby Canine Kidney ◽

Surface Receptors ◽

Surface Receptor ◽

The Stability ◽

Darby Canine Kidney ◽

Canine Kidney

We have studied the expression of the chicken hepatic glycoprotein receptor (chicken hepatic lectin [CHL]) in Madin-Darby canine kidney (MDCK) cells, by transfection of its cDNA under the control of a retroviral promotor. Transfected cell lines stably express 87,000 surface receptors/cell with a kd = 13 nM. In confluent monolayers, approximately 40% of CHL is localized at the plasma membrane. 98% of the surface CHL is expressed at the basolateral surface where it performs polarized endocytosis and degradation of glycoproteins carrying terminal N-acetylglucosamine at a rate of 50,000 ligand molecules/h. Studies of the half-life of metabolically labeled receptor and of the stability of biotinylated cell surface receptor after internalization indicate that transfected CHL performs several rounds of uptake and recycling before it gets degraded. The successful expression of a functional basolateral receptor in MDCK cells opens the way for the characterization of the mechanisms that control targeting and recycling of proteins to the basolateral membrane of epithelial cells.

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Selective Alterations in Biosynthetic and Endocytic Protein Traffic in Madin-Darby Canine Kidney Epithelial Cells Expressing Mutants of the Small GTPase Rac1

Molecular Biology of the Cell ◽

10.1091/mbc.11.1.287 ◽

2000 ◽

Vol 11 (1) ◽

pp. 287-304 ◽

Cited By ~ 51

Author(s):

Tzuu-Shuh Jou ◽

Som-Ming Leung ◽

Linette M. Fung ◽

Wily G. Ruiz ◽

W. James Nelson ◽

...

Keyword(s):

Apical Membrane ◽

Mdck Cells ◽

Basolateral Membrane ◽

Membrane Traffic ◽

Small Gtpase ◽

Apical Plasma Membrane ◽

Membrane Domains ◽

Madin Darby Canine Kidney ◽

Darby Canine Kidney ◽

Canine Kidney

Madin-Darby canine kidney (MDCK) cells expressing constitutively active Rac1 (Rac1V12) accumulate a large central aggregate of membranes beneath the apical membrane that contains filamentous actin, Rac1V12, rab11, and the resident apical membrane protein GP-135. To examine the roles of Rac1 in membrane traffic and the formation of this aggregate, we analyzed endocytic and biosynthetic trafficking pathways in MDCK cells expressing Rac1V12 and dominant inactive Rac1 (Rac1N17). Rac1V12 expression decreased the rates of apical and basolateral endocytosis, whereas Rac1N17 expression increased those rates from both membrane domains. Basolateral-to-apical transcytosis of immunoglobulin A (IgA) (a ligand for the polymeric immunoglobulin receptor [pIgR]), apical recycling of pIgR-IgA, and accumulation of newly synthesized GP-135 at the apical plasma membrane were all decreased in cells expressing Rac1V12. These effects of Rac1V12 on trafficking pathways to the apical membrane were the result of the delivery and trapping of these proteins in the central aggregate. In contrast to abnormalities in apical trafficking events, basolateral recycling of transferrin, degradation of EGF internalized from the basolateral membrane, and delivery of newly synthesized pIgR from the Golgi to the basolateral membrane were all relatively unaffected by Rac1V12 expression. Rac1N17 expression had little or no effect on these postendocytic or biosynthetic trafficking pathways. These results show that in polarized MDCK cells activated Rac1 may regulate the rate of endocytosis from both membrane domains and that expression of dominant active Rac1V12 specifically alters postendocytic and biosynthetic membrane traffic directed to the apical, but not the basolateral, membrane.

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The cytoplasmic domain of the interleukin-6 receptor gp80 mediates its basolateral sorting in polarized madin-darby canine kidney cells

Journal of Cell Science ◽

10.1242/jcs.113.20.3593 ◽

2000 ◽

Vol 113 (20) ◽

pp. 3593-3602 ◽

Cited By ~ 3

Author(s):

A.S. Martens ◽

J.G. Bode ◽

P.C. Heinrich ◽

L. Graeve

Keyword(s):

Mdck Cells ◽

Basolateral Membrane ◽

Signal Transducer ◽

Madin Darby Canine Kidney ◽

Interleukin 6 Receptor ◽

Basolateral Plasma Membrane ◽

First Time ◽

Transcription 3 ◽

Darby Canine Kidney ◽

Canine Kidney

The IL-6 receptor complex is expressed in different polarized epithelial cells such as liver hepatocytes and intestinal cells. It consists of two subunits: gp80, which binds the ligand, and gp130, which is responsible for signal transduction. In stably transfected Madin-Darby canine kidney (MDCK) cells we have studied the localization of the human IL-6 receptor subunits and found that gp80 and gp130 are predominantly expressed at the basolateral membrane. Analysis of MDCK cells expressing truncated forms of gp80 or gp130 showed that loss of the cytoplasmic domains results in apical delivery. Expression of deletion mutants of gp80 in MDCK cells led to the identification of two discontinous motifs responsible for basolateral sorting: a membrane-proximal tyrosine-based motif (YSLG) and a more membrane-distal dileucine-type motif (LI). Activation of signal transducer and activator of transcription-3 (STAT-3) only occurred via basolaterally located gp80, suggesting that endogenous gp130 is also constrained to the basolateral plasma membrane. Our identification of a basolateral sorting signal within the cytoplasmic region of gp80 for the first time attributes a function to this domain.

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Mammalian Homolog of Drosophila Tumor Suppressor Lethal (2) Giant Larvae Interacts with Basolateral Exocytic Machinery in Madin-Darby Canine Kidney Cells

Molecular Biology of the Cell ◽

10.1091/mbc.01-10-0496 ◽

2002 ◽

Vol 13 (1) ◽

pp. 158-168 ◽

Cited By ~ 130

Author(s):

Anne Mu∸sch ◽

David Cohen ◽

Charles Yeaman ◽

W. James Nelson ◽

Enrique Rodriguez-Boulan ◽

...

Keyword(s):

Epithelial Cell ◽

Tumor Suppressor ◽

Cell Polarity ◽

Mdck Cells ◽

Basolateral Membrane ◽

Epithelial Cell Line ◽

Madin Darby Canine Kidney ◽

Late Step ◽

Darby Canine Kidney ◽

Canine Kidney

The Drosophila tumor suppressor protein lethal (2) giant larvae [l(2)gl] is involved in the establishment of epithelial cell polarity during development. Recently, a yeast homolog of the protein has been shown to interact with components of the post-Golgi exocytic machinery and to regulate a late step in protein secretion. Herein, we characterize a mammalian homolog of l(2)gl, called Mlgl, in the epithelial cell line Madin-Darby canine kidney (MDCK). Consistent with a role in cell polarity, Mlgl redistributes from a cytoplasmic localization to the lateral membrane after contact-naive MDCK cells make cell-cell contacts and establish a polarized phenotype. Phosphorylation within a highly conserved region of Mlgl is required to restrict the protein to the lateral domain, because a recombinant phospho-mutant is distributed in a nonpolar manner. Membrane-bound Mlgl from MDCK cell lysates was coimmunoprecipitated with syntaxin 4, a component of the exocytic machinery at the basolateral membrane, but not with other plasma membrane solubleN-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins that are either absent from or not restricted to the basolateral membrane domain. These data suggest that Mlgl contributes to apico-basolateral polarity by regulating basolateral exocytosis.

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

The journal of nutrition health & aging ◽

10.1007/s12603-015-0604-y ◽

2015 ◽

Vol 20 (5) ◽

pp. 486-493 ◽

Cited By ~ 4

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

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

Biochemistry and Cell Biology ◽

10.1139/o96-030 ◽

1996 ◽

Vol 74 (2) ◽

pp. 283-287 ◽

Cited By ~ 12

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.

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