Potassium Channels Lost During Harvesting of Epithelial Cells are Restored with a Kinetics that Depends on Channel Species

García-Villegas, Refugio; Escamilla, Juan C.; Fiorentino, Rosana; Cereijido, Marcelino

doi:10.1159/000107525

Cited by 6 publications

(5 citation statements)

References 35 publications

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“…It was widely believed that the membrane potential of MDCK cells is determined by K + and Cl − conductances and their transmembrane concentration gradients [36, 41]. Our extended observations show both agreements and discrepancies with previous reports.…”

Section: Discussionsupporting

confidence: 63%

“…Second, previous reports showed that MDCK cells exhibit transcription signals and single channel activity of the TEA-sensitive big conductance Ca 2+ -activated K + channels (BK) [41, 45, 46]. However, we observed only a small (2–4 mV) depolarization by 10 mM TEA in a small portion of cells tested, indicating only a small contribution of this channel to the membrane potential.…”

Section: Discussionmentioning

confidence: 49%

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Bumetanide Hyperpolarizes Madin–Darby Canine Kidney Cells and Enhances Cellular Gentamicin Uptake by Elevating Cytosolic Ca2+ Thus Facilitating Intermediate Conductance Ca2+-Activated Potassium Channels

Yang

Karasawa

Wang

et al. 2012

Cell Biochem Biophys

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Loop diuretics such as bumetanide and furosemide enhance aminoglycoside ototoxicity when co-administered to patients and animal models. The underlying mechanism(s) is poorly understood. We investigated the effect of these diuretics on cellular uptake of aminoglycosides, using Texas Red-tagged gentamicin (GTTR), and intracellular/whole-cell recordings of Madin-Darby Canine kidney (MDCK) cells. We found that bumetanide and furosemide concentration-dependently enhanced cytoplasmic GTTR fluorescence by ~60%. This enhancement was suppressed by La3+, a non-selective cation channel (NSCC) blocker, and by K+ channel blockers Ba2+ and clotrimazole, but not by tetraethylammonium (TEA), 4-aminopyridine (4-AP) or glipizide, nor by Cl− channel blockers diphenylamine-2-carboxylic acid (DPC), niflumic acid (NFA), and CFTRinh-172. Bumetanide and furosemide hyperpolarized MDCK cells by ~14 mV, increased whole-cell I/V slope conductance; the bumetanide-induced net current I/V showed a reversal potential (Vr) ~−80 mV. Bumetanide-induced hyperpolarization and I/V change was suppressed by Ba2+ or clotrimazole, and absent in elevated [Ca2+]i, but not affected by apamin, 4-AP, TEA, glipizide, DPC, NFA or CFTRinh-172. Bumetanide and furosemide stimulated a surge of Fluo-4-indicated cytosolic Ca2+. Ba2+ and clotrimazole alone depolarized cells by ~18 mV and reduced I/V slope with a net current Vr near −85 mV, and reduced GTTR uptake by ~20%. La3+ alone hyperpolarized the cells by ~−14 mV, reduced the I/V slope with a net current Vr near −10 mV, and inhibited GTTR uptake by ~50%. In the presence of La3+, bumetanide caused negligible potential or I/V change. We conclude that NSCCs constitute a major cell entry pathway for cationic aminoglycosides; bumetanide enhances aminoglycoside uptake by hyperpolarizing cells that increases cation influx driving force; and bumetanide-induced hyperpolarization is caused by elevating the intracellular Ca2+ and thus a facilitation of the intermediate conductance Ca2+-activated K+ channels.

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Section: Discussionsupporting

confidence: 63%

Section: Discussionmentioning

confidence: 49%

Bumetanide Hyperpolarizes Madin–Darby Canine Kidney Cells and Enhances Cellular Gentamicin Uptake by Elevating Cytosolic Ca2+ Thus Facilitating Intermediate Conductance Ca2+-Activated Potassium Channels

Yang

Karasawa

Wang

et al. 2012

Cell Biochem Biophys

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“…For qPCR of LDH‐H mRNA, amplification primers were LDH‐1 and LDH‐4 as previously described (Hernández‐Quintero et al, 2002). We used as internal control, the primers PRP0‐F and PRP0‐R (García‐Villegas et al, 2007) specific for the acidic ribosomal phosphoprotein P0.…”

Section: Methodsmentioning

confidence: 99%

Pax‐6 is expressed early in the differentiation of a corneal epithelial model system

García-Villegas

Escamilla

Sánchez-Guzmán

et al. 2009

Journal Cellular Physiology

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Pax-6 is a regulatory gene with a major role during visual system development, but its association with corneal epithelial differentiation is not clearly established. Using the RCE1-(5T5) cell line, which mimics corneal epithelial differentiation, we analyzed Pax-6 biological role. Immunostaining of proliferating colonies and confluent sheets showed that Pax-6-positive cells were also K3 keratin-positive, suggesting that Pax-6 is expressed in differentiating cells. Pax-6 mRNA was barely expressed in early cell cultures; but after confluence, its levels raised up to fivefold as demonstrated by Northern blot and RT-qPCR. The raise in Pax-6 expression preceded for 9 h the increase in LDH-H and LDH-M mRNAs, previously shown as early markers of corneal epithelial cell differentiation. The full-length mRNAs encoding for the two major Pax-6 isoforms were found at very low levels in proliferating cells, and abundantly expressed in the confluent stratified epithelia; Pax-6 mRNA was 2- to 2.5-fold more abundant than Pax-6(5a) mRNA. The ectopic expression of Pax-6 or Pax-6(5a) decreased proliferative ability leading to the formation of abortive, non-proliferative colonies. In contrast, culture conditions that delay or block corneal epithelial cell differentiation reduced or inhibited the expression of Pax-6. Collectively, results show that Pax-6 is the earlier differentiation marker expressed by corneal epithelial cells, and open the possibility for a major role of Pax-6 as the main driver of the differentiation of corneal epithelial cells.

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“…We used the acidic ribosomal phosphoprotein P0 (PR‐P0) as an internal control to normalize for sample‐to‐sample variations in total RNA amounts and for reaction efficiency (Marone et al, 2001). PR‐P0 was chosen as internal control since its mRNA levels do not change during growth and differentiation of corneal epithelial cells (García‐Villegas et al, 2009); primers specific for PR‐P0 were previously reported (García‐Villegas et al, 2007) (Table 1).…”

Section: Methodsmentioning

confidence: 99%

Asymmetrical cell division and differentiation are not dependent upon stratification in a corneal epithelial cell line

Gómez-Flores

Sánchez-Guzmán

Castro‐Muñozledo

2010

Journal Cellular Physiology

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To determine whether asymmetrical cell division takes place during growth and differentiation of corneal epithelial cells, we analyzed the expression of some proteins required for the correct execution of the asymmetric division in cultured RCE1-(5T5) cells, which mimic the differentiation of corneal epithelial cells. RT-PCR and immunostaining showed that Par-3, LGN (GPSM2), NuMA, and the mammalian homolog of inscuteable (Insc) are expressed by the cultured cells. Semi-quantitative RT-PCR demonstrated that Insc mRNA levels were stable throughout the experiment. Conversely, LGN and NuMA mRNAs increased slightly and steadily in proliferative cells, reaching a peak of about 20% above basal levels when cells were confluent. At later times, LGN and NuMA mRNAs decreased to become barely detectable when cells organized into a four-layered epithelium and expressed terminal phenotype as indicated by the highest expression of LDH-H mRNA. Cultivation under low Ca2+ conditions (0.09 mM) reduced about 50% Insc mRNA expression both in proliferating and confluent cultures, but did not affect the levels of LGN and NuMA mRNAs. Hence, asymmetric cell division seems to take place with a lower frequency in cells grown with low Ca2+ concentrations, in spite of the absence of stratification. Immunostaining experiments raise the possibility of an interaction between k3/K12 keratin cytoskeleton and Par-3. The results show for the first time the coordination between the expression of corneal epithelial cell differentiation and the expression of cell polarity machinery. They also suggest that asymmetric division does not depend on stratification; instead, it seems to be part of the differentiation program.

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Potassium Channels Lost During Harvesting of Epithelial Cells are Restored with a Kinetics that Depends on Channel Species

Cited by 6 publications

References 35 publications

Bumetanide Hyperpolarizes Madin–Darby Canine Kidney Cells and Enhances Cellular Gentamicin Uptake by Elevating Cytosolic Ca2+ Thus Facilitating Intermediate Conductance Ca2+-Activated Potassium Channels

Bumetanide Hyperpolarizes Madin–Darby Canine Kidney Cells and Enhances Cellular Gentamicin Uptake by Elevating Cytosolic Ca2+ Thus Facilitating Intermediate Conductance Ca2+-Activated Potassium Channels

Pax‐6 is expressed early in the differentiation of a corneal epithelial model system

Asymmetrical cell division and differentiation are not dependent upon stratification in a corneal epithelial cell line

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