Ulrich Kersting

Intracellular Ca2+ distribution in migrating transformed renal epithelial cells

Abstract Migration of transformed Madin-Darby canine kidney (MDCK-F) cells depends on the polarized activity of a Ca2+-sensitive K+ channel. We tested whether a gradient of intracellular Ca2+ concentration ([Ca2+]i) underlies the horizontal polarization of K+ channel activity. [Ca2+]i was measured with the fluorescent dye fura-2/AM. Spatial analysis of [Ca2+]i indicated that a horizontal gradient exists, with [Ca2+]i being higher in the cell body than in the lamellipodium. Resting and maximal levels during oscillations of [Ca2+]i in the cell body were found to be 135 ± 34 and 405 ± 59 nmol/l, respectively, whereas they were 79 ± 18 and 307 ± 102 nmol/l in the lamellipodium. This gradient can partially explain the preferential activation of K+ channels in the plasma membrane of the cell body. We applied a local superfusion technique during migration experiments and measurements of [Ca2+]i to test whether its maintenance is due to an uneven distribution of Ca2+ influx into migrating MDCK-F cells. Locally superfusing the cell body of migrating MDCK-F cells with La3+ alone or together with charybdotoxin, a specific blocker of Ca2+-sensitive K+ channels, slowed migration to 47 ± 10% and 9 ± 5% of control, respectively. Local blockade of Ca2+ influx into the cell body and the lamellipodium with La3+ was followed by a decrease of [Ca2+]i at both cell poles. This points to Ca2+ influx occurring over the entire cell surface. This conclusion was confirmed by locally superfusing Mn2+ over the cell body and the lamellipodium. Fura-2 fluorescence was quenched in both areas, the decrease of fluorescence being two to three times faster in the cell body than in the lamellipodium. However, this difference is insufficient to account for the observed gradient of [Ca2+]i. We hypothesize that the polarized distribution of intracellular Ca2+ stores contributes significantly to the generation of a gradient of [Ca2+]i.

Fusion of cultured dog kidney (MDCK) cells: II. Relationship between cell pH and K+ conductance in response to aldosterone.

SummaryWe have chosen the MDCK cell line to investigate aldosterone action on H+ transport and its role in regulating cell membrane K+ conductance (GmK). Cells grown in a monolayer respond to aldosterone indicated by the dose-dependent formation of domes and by the alkalinization of the dome fluid. The pH sensitivity of the plasma membrane K+ channels was tested in “giant cells” fused from individual MDCK cells. Cytoplasmic pH (pHi) andGmK were measured simultaneously while the cell interior was acidified gradually by an extracellular acid load. We found a steep signoidal relationship between pHi andGmK (Hill coefficient 4.4±0.4), indicating multiple H+ binding sites at a single K+ channel. Application of aldosterone increased pHi within 120 min from 7.22±0.04 to 7.45±0.02 and from 7.15±0.03 to 7.28±0.02 in the absence and presence of the CO2/HCO3− buffer system, respectively. We conclude that the hormone-induced cytoplasmic alkalinization in the presence of CO2/ HCO3− is limited by the increased activity of a pHi-regulating HCO3− extrusion system. SinceGmK is stimulated half-maximally at the pHi of 7.18±0.04, internal H+ ions could serve as an effective intracellular signal for the regulation of transepithelial K+ flux.

Fusion of cultured dog kidney (MDCK) cells. I: Technique, fate of plasma membranes and of cell nuclei

SummaryThe evaluation of the intracellular signal train and its regulatory function in controlling transepithelial transport with electrophysiological methods often requires intracellular measurements with microelectrodes. However, multiple impalements in epithelial cells are hampered by the small size of the cells. In an attempt to avoid these problems we fused cells of an established cell line, Madin Darby canine kidney cells, originally derived from dog kidney, to “giant” cells by applying a modified polyethylene glycol method. During trypsin-induced detachment from the ground of the petri dish, individual cells grown in a monolayer incorporate volume and mainly lose basolateral plasma membrane by extrusion. By isovolumetric cell-to-cell fusion, spherical “giant” cells are formed within 2 hr. During this process a major part of the individual cell plasma membranes is internalized. Over three weeks following cell plasma membrane fusion degradation of single cell nuclei and cell nuclear fusion occurs. We conclude that this experimental approach opens the possibility to investigate ion transport of epithelia in culture by somatic cell genetic techniques.

Evidence for an acid pH in rat renal inner medulla: paired measurements with liquid ion-exchange microelectrodes on collecting ducts and vasa recta.

We reevaluated the pH in the renal medulla in rats. pH of the vasa recta blood was about 1 pH unit acidic in comparison to the pH of renal artery blood. During furosemideinduced diuresis pH of vasa recta blood increased whereas pH of collecting duct urine further decreased. The acidic pH in the rat renal inner medulla during antidiuresis raises important questions about the source of H+ in inner medulla.

Carbonic Anhydrase Activity in Madin Darby Canine Kidney Cells

Madin Darby canine kidney (MDCK) renal epithelial cell cultures have been investigated with respect to their potency to express carbonic anhydrase activity using histochemical methods. Acetazolamide inhibitable carbonic anhydrase activity could be detected in the cytoplasmic compartment as well as in the apical membrane of cells when grown on solid culture supports. Cells forming domes in MDCK monolayers exhibit the highest histochemically detectable enzyme activity. The attempt to subculture clonal cell lines from MDCK monolayer cultures resulted in the establishment of 5 clones, slightly different with respect to size and shape of cells and their potency to form domes. Scanning electron microscopy ensured the identification of one clone (1A4), which distinctly differed from the others with respect to the apical membrane architecture. Co-localization of peanut agglutinin and carbonic anhydrase activity at the plasma membrane always revealed a combined occurrence of enzyme reactivity and lectin binding in the apical membrane domain. Both, lectin binding and carbonic anhydrase activity were distinctly more intense in plasma membrane regions equipped with microvilli. From the results it is concluded that MDCK cells in tissue culture retained properties of intercalated cells of the nephron collecting duct segment.

Differentiation of Madin-Darby Canine Kidney Cells Depends on Cell Culture Conditions

We used Madin-Darby canine kidney (MDCK) cells as a model to study changes in renal epithelial cell structure and function induced by different environments. We identined two cell types, MDCK-1 and MD

Changes in the countercurrent system in the renal papilla: diuresis increases pH and HCO3− gradients between collecting duct and vasa recta

This study was designed to elucidate the acidbase balance local to the collecting duct urine (CD) and vasa recta blood (VR) in the rat renal papilla in diuresis. The pH changes were measured in both a furosemide-induced and a volume-load-induced diuresis, whereas the PCO2 (i.e., CO2 tension) and HCO3− concentration were measured only in a furosemideinduced diuresis. In an antidiuresis, the pH of the VR was more acidic than that of the systemic arterial blood (ΔpH = 0.44–0.73). Additionally, the pH of the ascending VR was significantly lower than that of the descending VR (ΔpH = 0.14–0.16). In diuresis, the pH of the CD decreased (ΔpH = 0.81–0.97), while the pH of the descending and the ascending VR increased; however, the increase was only significant in the ascending VR (ΔpH = 0.23–0.30). Consequently, the significant difference in the pH gradient between the descending and the ascending VR was eliminated. The PCO2 values in the CD and the ascending VR were not different from those in antidiuresis, while the HCO3− concentration in the CD and the ascending VR, respectively, decreased and increased significantly. Thus, in diuresis, the decrease in the pH of the CD and the increase in the pH of the ascending VR result, respectively, from the decrease and the increase in the HCO3− concentration, with no changes in the PCO2 values.

Low pH and Hyperosmolality Determine the Differentiation of MDCK Cells Typical for the Outer Medullary Collecting Duct

The Madin-Darby canine kidney (MDCK) cell line resembles cortical collecting duct epithelium, forming cells with properties corresponding to principal (P) and intercalated (IC) cells. Cells express ca