MDCK II cells

Luo, F., P. Paranjpe, A. Guo, E. Rubin, and P. Sinko. Intestinal transport of irinotecan in Caco-2 cells and MDCK II cells overexpressing efflux transporters Pgp, cMOAT, and MRP1., Drug Metab. Dispos. 2002, 30, 763-770... 

It is also important to predict the in vivo biliary excretion clearance in humans, and for this purpose MDCK II cell lines expressing both uptake and efflux transporters may be used (Fig. 12.3) [92, 93]. It has been shown that MRP2 is expressed on the apical membrane, whereas OATP2 and 8 are expressed on the basolateral membrane after cDNA transfection (Fig. 12.3) [92, 93]. The transcellular transport across such double-transfected cells may correspond to the excretion of ligands from blood into bile across hepatocytes. Indeed, the vectorial transport from the basal to apical side was observed for pravastatin only in OATP2- and MRP2-expressing... 

MDCK II cells (Fig. 12.3) [93], Kinetic analysis revealed that the Km value for transcellular transport (24 pM) was similar to the Km for OATP2 (34 pM) [93], Moreover, the efflux across the bile canalicular membrane was not saturated under these experimental conditions. These in vitro observations are consistent with in vivo experimental results in rats which showed that the rate-determining process for the biliary excretion of pravastatin is uptake across the sinusoidal membrane. By normalizing the expression level between the double transfectant and human hepatocytes, it might be possible to predict in vivo hepatobiliary excretion. 

Fig. 12.3. Transcellular transport of pravastatin across MDCK II cells. Upper panel schematic diagram illustrating the expression of transporters in MDCK II cells after transfection of OATP2 and MRP2 cDNAs. Lower...

Figure 6 Directional transport of pravastatin in Oatplb2/Mrp2 double transfectants in the apical direction (A), and comparison of in vivo biliary excretion clearance and in vitro transcellular transport clearance across the double transfectant (B). (A) Transcellular transport across the monolayers of MDCK II cells was determined in the basal-to-apical and the opposite direction. (B) The x axis represents CLint determined in vitro multiplied by /B and the scaling factor, and the y axis represents the in vivo biliary clearance defined for the blood ligand concentrations. The symbol ( ) represents data whose x axis values were corrected for the scaling factor (a = 17.9). The solid line represents the theoretical curve, and the symbol (o), the observed data. Source From Ref. 59.

Gebhard, D., de Morais, S. and Duignan, D.B. (2006) A 96-well efflux assay to identify ABCG2 substrates using a stably transfected MDCK II cell line. Molecular Pharmacology, 3, 45-54. 

Fig. 3 Time course of attachment and spreading when equal amounts of MDCK II cells were seeded into the quartz dish in presence of the soluble peptides Arg-Gly-Asp (RGD), Arg-Gly-Asp-Ser (RGBS), Gly-Arg-Gly-Asp-Ser (GRGDS), and Ser-Asp-Gly-Arg-Gly (SDGRG). The concentration of each peptide was 1 mM, the cell densitiy was adjusted...

Fig. 4 RICM micrographs of MDCK II cells 200 min after seeding. The cells in a were suspended in serum-containing medium with no inhibitory peptides included, while the cells in b were exposed to 1 mM RODS in the bathing fluid. The black arrow indicates the presence of a cell body that does not make close contact with the surface. Scale bar represents 10 xm...

Fig. 5 Slope of the attachment curve of MDCK II cells as a function of RGBS concentra-...

Fig. 12 Fluorescence micrographs of confluent MDCK II cell monolayers after the actin cytoskeleton has been stained by fluorescence-labeled phalloidin. a Control cells were not exposed to Cytochalasin D. b Cells were exposed to 5 xM Cytochalasin D for 100 min. The staining confirms that actin filaments have been degraded to small actin aggregates. The scale bar represents 25 xm. c Magnitude of the load impedance A 2l as a function of time when confluent MDCK II cell monolayers were exposed to 5 xM Cytochalasin D at the time indicated by the arrow. The value of Zi at the beginning of the experiment was set to zero...

We consider these studies to be strong evidence that the protein content of the cell, and in particular the cytoskeleton, is a prominent if not the predominant contributor to the acoustic load that is created on shear wave resonators by adherent cells. The reduction of A Zl after disassembly of the actin cytoskeleton supports this hypothesis. Please note that all the experiments described in this paragraph have been performed with MDCK II cells. In order to exclude that the observed increase in acoustic load after fixation is a cell-type-specific phenomenon of MDCK II cells, analog experiments were performed with other cell types and returned the same answer. Only the numerical value of the increase in A Zl varied to some extend. 

Fig. 15 Attachment, spreading, and differentiation of MDCK II cells followed by the combined QCM-ECIS approach. The change in load impedance A Zl (open circles) reports on attachment, spreading, and reorganization of the actin cytoskeleton. The electrical impedance at 400 Hz (filled symbols) mirrors the establishment of barrier-forming cellcell contacts and, thus, differentiation. The fluorescence micrographs in the right panel visualize the status of the actin cytoskeleton 3 h and 10 h after cell seeding. These time points are indicated in the graph by the dashed lines marked as 1 and 2...

Bre MH, Pepperkok R, Hill AM, Levilliers N, Ansorge W, Stelzer EHK, Karsenti E. Regulation of microtubule dynamics and nucleation during polarization in MDCK II cells. J Cell Biol 1990 111 3013-3021. 

Table 1 compares the QCM-based cell density on the surface for MDCK cells strain I (MDCK I), MDCK cells strain II (MDCK II) and 3T3 fibroblasts with the outcome of cell density estimates derived from microscopic images [12]. The final shiffs in resonance frequency that we observed when the resonator was completely covered by a continuous cell monolayer of either kind (A/confi) are also summarized in Table 1. Interestingly, different cell...

Figure 8.4 Palmostatin B specifically inhibits depalmitoylation. (a) Schematic illustration of the Cy3-labeled semisynthetic lipoproteins (Cy3 is a fluorescent label). The two differentially lipid-modified carboxyterminal N-Ras heptapeptides were coupled via a maleimido-caproyl linker to the carboxyterminal cysteine of recombinant expressed N-Ras (1 -181). GalT-CFP is a marker for the Golgi system. Citrine-N-Ras is a fluorescently labeled N-Ras construct, (b) Confocal time-lapse images of MDCK cells expressing the Golgi marker GalT-CFP and Citrine-N-Ras before and after microinjection of CysFar (i) or PalFar (ii). Cells were incubated for 80 min with 1 [iM palmostatin B before the experiment, (c) Quantitative ratiometric...

MDCK II Madin—Darby canine kidney II cell... 

The evaluation of the apparent ionization constants (i) can indicate in partition experiments the extent to which a charged form of the drug partitions into the octanol or liposome bilayer domains, (ii) can indicate in solubility measurements, the presence of aggregates in saturated solutions and whether the aggregates are ionized or neutral and the extent to which salts of dmgs form, and (iii) can indicate in permeability measurements, whether the aqueous boundary layer adjacent to the membrane barrier, Umits the transport of drugs across artificial phospholipid membranes [parallel artificial membrane permeation assay (PAMPA)] or across monolayers of cultured cells [Caco-2, Madin-Darby canine kidney (MDCK), etc.]. 

Soldner A, Benet L, Mutschler E, Christians U (2000) Active transport of the angiotensine II antagonist losartan and its main metabolite EXP 3174 across MDCK-MDR1 and CACO-2 cell monolayers. Br J Pharmacol 129 1235-1243... 

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