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  • 1
    Digitale Medien
    Digitale Medien
    Springer
    Journal of molecular medicine 68 (1990), S. 1087-1090 
    ISSN: 1432-1440
    Schlagwort(e): Aldosterone ; MDCK cells ; Na+/H+ exchange ; K+ channels
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Medizin
    Notizen: Summary Madin-Darby canine kidney (MDCK) cells resemble intercalated cells of the renal collecting duct. In these cultured epithelial cells aldosterone activates apical Na+/H+ exchange, initiating a cascade of intracellular events such as cell growth, epithelial cell polarity, and stimulation of transepithelial ion transport. Transepithelial K+ secretion is triggered by the insertion of new ion channels and the activation of previously quiescent channels with increasing cytoplasmic pH. Aldosterone supplies the cell with ion transporters necessary for adequate function of the renal collecting duct when the organism is metabolically challenged.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 2
    ISSN: 1432-2013
    Schlagwort(e): Key words Atomic force microscopy ; Patch clamp ; MDCK cells ; Plasma membrane ; Membrane protein
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Medizin
    Notizen: Abstract  We combined the patch-clamp technique with atomic force microscopy (AFM) to visualize plasma membrane proteins protruding from the extracellular surface of cultured kidney cells (MDCK cells). To achieve molecular resolution, patches were mechanically isolated from whole MDCK cells by applying the patch-clamp technique. The excised inside-out patches were transferred on freshly cleaved mica and imaged with the AFM in air and under physiological conditions (i.e. in fluid). Thus, the resolution could be increased considerably (lateral and vertical resolutions 5 and 0.1 nm, respectively) as compared to experiments on intact cells, where plasma membrane proteins were hardly detectable. The apical plasma membrane surface of the MDCK cells showed multiple protrusions which could be identified as membrane proteins through the use of pronase. These proteins had a density of about 90 per μm², with heights between 1 and 9 nm, and lateral dimensions of 20–60 nm. Their frequency distribution showed a peak value of 3 nm for the protein height. A simplified assumption – modelling plasma membrane proteins as spherical structures protruding from the lipid bilayer – allowed an estimation of the possible molecular weights of these proteins. They range from 50 kDa to 710 kDa with a peak value of 125 kDa. We conclude that AFM can be used to study the molecular structures of membranes which were isolated with the patch-clamp technique. Individual membrane proteins and protein clusters, and their arrangement and distribution in a native plasma membrane can be visualized under physiological conditions, which is a first step for their identification.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 3
    Digitale Medien
    Digitale Medien
    Springer
    Pflügers Archiv 419 (1991), S. 43-50 
    ISSN: 1432-2013
    Schlagwort(e): Hyperosmolarity ; Volume regulation ; MDCK cells ; Na+/H+ antiporter ; Na+/K+-ATPase ; Renal medulla
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Medizin
    Notizen: Abstract We investigated mechanisms of regulatory volume increase in fused Madin-Darby canine kidney (MDCK) cells, a cell line originally derived from renal collecting duct. The intracellular ion concentrations as well as the concentration of the volume marker tetramethylammonium+ were measured by means of ion-selective microelectrodes. Application of hypertonic Ringer bicarbonate solution (+150 mmol/l mannitol) resulted in cell shrinkage to 84±2% of the initial cell volume (shrinkage expected for an ideal osmometer = 66%), indicating a significant regulatory volume increase. During the first 90 s of the hypertonic stress, a transient increase in intracellular Na+ and HCO 3 − concentrations was observed. It was followed by a sustained increase in intracellular K+ and Cl− concentrations. Ouabain (0.1 mmol/l) as well as amiloride (1 mmol/l) reduced K+ accumulation significantly, whereas the H+ /K+-ATPase inhibitor SCH 28080 had no effect. Hypertonic stress hyperpolarized the cell membrane potential by 19±2 mV, owing to the decrease of the ratio of Cl− conductance to K+ conductance of the cell membrane. We conclude: (a) acute hypertonic stress activates Na+/H+ exchange in MDCK cells; (b) transient alteration of intracellular Na+ and pH stimulates Na+/K+-ATPase and Cl−/HCO 3 − exchange, both leading to the sustained intracellular accumulation of KCl; (c) a high intracellular KCl concentration is maintained by the partial reversion of the Cl−/K+ conductance ratio of the plasma membrane.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 4
    Digitale Medien
    Digitale Medien
    Springer
    Pflügers Archiv 416 (1990), S. 526-532 
    ISSN: 1432-2013
    Schlagwort(e): Aldosterone ; MDCK cells ; Dome formation ; Differentiation
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Medizin
    Notizen: Abstract Vectorial transport of salt and water in the Madin-Darby canine kidney (MDCK) cell line is indicated by the formation of domes when a monolayer is grown on an impermeable support. We investigated aldosterone-induced dome formation and evaluated the dome as an experimental model. Transepithelial dome resistance was about 80 Ωcm2 and constant when dome size exceeded 2 · 10−4 cm2. The relative ion conductances (expressed as transference numbers) across the dome epithelium were t Na∶t Cl∶t K= 0.64∶0.24∶0.06. They reflect the permeability properties of the paracellular shunt pathway tested at physiological concentrations of the individual ions. Aldosterone accelerated dome formation in serum-deprived MDCK monolayers. Prostaglandin E1 and transferrin were supportive but not essential for aldosterone-induced dome formation. After 72 h dome density was equal in monolayers cultured in serum-supplemented medium either in the presence or absence of mineralocorticoids. We conclude that aldosterone induces cell polarization in MDCK monolayers, leading to the formation of domes. The dome epithelium appears to be electrically isolated from the adjacent monolayer and can be studied by microelectrode techniques.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 5
    Digitale Medien
    Digitale Medien
    Springer
    Pflügers Archiv 416 (1990), S. 540-547 
    ISSN: 1432-2013
    Schlagwort(e): MDCK cells ; Omeprazole ; H+-K+ pump ; Carbonic anhydrase ; Intercalated cells
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Medizin
    Notizen: Abstract Functionally and morphologically, Madin-Darby canine kidney (MDCK) cells resemble intercalated cells of urinary epithelia. Experiments were performed on domes of confluent MDCK monolayers to test for apical H+ secretion. Apical application of 10−3 mol/l amiloride or of Na+-free solution significantly reduced the limiting pH gradient across the dome epithelium (Δ pHd) consistent with inhibition of apical Na+/H+ exchange. Short-circuit current (SCC) measurements disclosed an acetazolamide-sensitive, (basolateral to apical) positive transepithelial current stimulated by 10−7 mol/l aldosterone and inhibited by acidification of apical medium to pH=4.5. Histochemical evaluation of carbonic anhydrase (CA) activity revealed cytoplasmic and apicalmembrane-bound CA particularly in dome-forming cells. Apical substitution of Na+ by K+ increased Δ pHd, whereas a reduction of K+ concentration to 0.5 mmol/l or addition of barium or omeprazole (10−5 mol/l) to the apical superfusate reduced Δ pHd by at least 75%. Aldosterone-stimulated SCC was completely abolished by the apical application of barium. We conclude that besides Na+/H+ exchange MDCK cells can express an apically located H+-K+ pump stimulated by aldosterone and inhibited directly by the anti-ulcer agent omeprazole or indirectly, either by blocking apical K+ recycling or by interfering with the CA-dependent intracellular formation of H+ ions.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 6
    ISSN: 1432-2013
    Schlagwort(e): Endothelin ; MDCK cells ; Cell differentiation ; Peanut lectin ; Na+/H+ antiporter ; Renal medulla
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Medizin
    Notizen: Abstract We investigated the effects of endothelin-1 (ET-1) on Madin-Darby canine kidney (MDCK) cells, a cell line originating from the renal collecting duct. The activity of transepithelial transport was assessed as the rate of dome formation in monolayers grown on solid support. The pH value of the dome fluid (dome pH) was measured by means of pH-selective microelectrodes. Differentiation of monolayer cells was estimated as the peanut-lectin(PNA)-binding capacity of the apical membrane. Confluent monolayers were incubated for 12–72 h in serum-free medium at various concentrations of ET-1. Exposure to 1 nmol/l ET-1 reduced dome formation by a maximum of 41±8% (n=4; P〈0.02) after 24 h. ET-1 (10 nmol/l; 24 h) decreased dome pH from 7.52±0.02 (n=53) to 7.36±0.03 (n=51; P〈0.02). Apical application of amiloride (1 mmol/l) reduced dome pH in both ET-1-treated and non-treated domes to essentially the same level, 7.25±0.03 (n=19) and 7.23±0.03 (n=17) respectively. ET-1 (10 nmol/l; 24 h) reduced PNA-binding capacity by 19±3% (n=5; P 〈 0.02). Moreover, ET-1 prevented the increase in PNA binding (+53±7%; n=5) induced by 0.1 μmol/l aldosterone. We conclude that ET-1 inhibits transepithelial transport and PNA binding via inhibition of apical Na+/H+ exchange, thus antagonizing aldosterone action in MDCK cells.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 7
    Digitale Medien
    Digitale Medien
    Springer
    The journal of membrane biology 139 (1994), S. 183-190 
    ISSN: 1432-1424
    Schlagwort(e): Ochratoxin A ; MDCK cells ; Intracellular chloride ; Intracellular pH ; DNDS
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie , Chemie und Pharmazie
    Notizen: Abstract Ochratoxin A (OTA) is a nephrotoxin which blocks plasma membrane anion conductance in Madin-Darby canine kidney (MDCK) cells. Added to the culture medium, OTA transforms MDCK cells in a manner similar to exposure to alkaline stress. By means of video-imaging and microelectrode techniques, we investigated whether OTA (1 μmol/liter) affects intracellular pH (pH.), Cl− (Cl i − ) or cell volume of MDCK cells acutely exposed to normal (pHnorm=7.4) and alkaline (pHalk=7.7) conditions. At pHnorm, OTA increased Cl i − by 2.6±0.4 mmol/liter (n=14, P〈0.05) but had no effect on pH i . At pHalk, application of OTA increased Cl i − by 8.6±2.6 mmol/liter (n=10, P〈 0.05) and raised pH i by 0.11±0.03 (n= 8, P〈0.05). The Cl−HCO 3 − exchange inhibitor DNDS (4,4′-dinitro-stilbene-2, 2′-disulfonate; 10 μmol/liter) eliminated the OTA-induced changes of pH i and Cl i − . OTA did not affect cell volume under both pHnorm and pHalk conditions. We conclude that the OTA-induced blockade of plasma membrane anion conductance increases Cl i − without changing cell volume. The driving force of plasma membrane Cl−/HCO 3 − exchange dissipates, leading to a rise of pH i when cells are exposed to an acute alkaline load. Thus, OTA interferes with pH i and Cl i − homeostasis leading to morphological and functional alterations in MDCK cells. The work was supported by the Deutsche Forschungsgemeinschaft (DFG, Si 170/7-1). We thank the Zeiss Company (Oberkochen, Germany) for providing the Attofluor™ video-imaging system for the intracellular Ca2+ measurements.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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