PubMed Health⌕ Search

Biomedical subjects

H Oberleithner

Publications and source records attributed to H Oberleithner.

At least 91 records · Page 5Linked to original sources
PubMed1993

Spontaneously oscillating K+ channel activity in transformed Madin-Darby canine kidney cells.

Intracellular alkalinization is known to be associated with tumorigenic transformation. Besides phenotypical alterations alkali-transformed Madin-Darby canine kidney (MDCK) cells exhibit a spontaneously oscillating cell membrane potential (PD). Using single-channel patch clamp techniques, it was the aim of this study to identify the ion channel underlying the rhythmic hyperpolarizations of the PD. In the cell-attached patch configuration, we found that channel activity was oscillating. The frequency of channel oscillations is 1.1 +/- 0.1 min-1. At the peak of oscillatory channel activity, single-channel current was -2.7 +/- 0.05 pA, and in the resting state it was -1.95 +/- 0.05 pA. Given the single-channel conductance of 53 +/- 3 pS for inward (and of 27 +/- 5 pS for outward) current the difference of single-channel current amplitude corresponded to a hyperpolarization of approximately 14 mV. The channel is selective for K+ over Na+. Channel kinetics are characterized by one open and by three closed time constants. The channel is Ca2+ sensitive. Half maximal activation in the inside-out patch mode is achieved at a Ca2+ concentration of 10 mumol/liter. In addition, we also found a 13-pS K+ channel that shows no oscillatory activity in the cell-attached patch configuration and that was not Ca2+ sensitive. We conclude that the Ca(2+)-sensitive 53-pS K+ channel is underlying spontaneous oscillations of the PD. It has virtually identical biophysical properties as a Ca(2+)-sensitive K+ channel in nontransformed parent MDCK cells. Hence, alkali-induced transformation of MDCK cells did not affect the channel protein itself but its regulators thereby causing spontaneous fluctuations of the PD.

Animals↗
PubMed1992

Patchy accumulation of apical Na+ transporters allows cross talk between extracellular space and cell nucleus.

Intracellular Na+ activities and local current densities were measured in fused Madin-Darby canine kidney cells using Na+ and voltage-sensing microelectrodes. Na+ that enters the cell across the apical plasma membrane accumulates initially in the nucleoplasm, several seconds ahead of its appearance in the cell cytoplasm. The spatial distribution of Na+ currents, produced by a local superfusion of the cell surface, indicates a nonuniform, patchy accumulation of apical Na+ transporters in the vicinity of the nucleus. Such pathways for direct Na+ flux between extracellular space and cell nucleus could be potentially important for gene activation.

Animals↗
PubMed1992

Renal potassium bicarbonate release in humans exposed to an acute volume load.

Cells of the renal medulla regulate their volume by transmembrane ion movements when exposed to large changes in osmolality. Since renal cells in culture release KHCO3 in response to hypotonic stress [11], we investigated the effect of an acute water load on urinary KHCO3 excretion in 5 healthy individuals. Water diuresis was induced by the ingestion of 1.5 l hypoosmolal fluid (22 mosm/kg H2O) over 15 min. The rate of urinary volume excretion increased from an initial value of 1.4 ml/min to 9.3 ml/min after 75 min. Urinary osmolality dropped from an initial value of 940 +/- 32 mosm/kg H2O to 74 +/- 4 mosm/kg H2O (n = 5). The decrease of osmolality was accompanied by the transient release of potassium and bicarbonate. Peak values of KHCO3 excretion were observed between 30 and 45 min after the onset of the experiment corresponding to the drop of urinary osmolality. The magnitude of renal potassium release correlated significantly (r = 0.93; P less than 0.05) with endogenous plasma aldosterone concentrations measured prior to the experiment in the 5 volunteers. We conclude that medullary epithelial cells release KHCO3 when exposed to hypotonic stress. The volume regulatory response is upregulated by aldosterone.

Adult↗
PubMed1992

Oscillations: a key event in transformed renal epithelial cells.

Intracellular pH (pHi) plays a critical role in the entry of cells into the DNA-synthesis phase of the cell cycle. Alterations in pHi may contribute to abnormal proliferative responses such as those seen in tumorigenic cells. We observed that alkaline stress leads to genomic transformation of Madin-Darby canine kidney (MDCK) cells. Transformed cells (F cells) form "foci" in culture, lack contact inhibition, and are able to migrate, typical characteristics of dedifferentiated tumorigenic cells. F cells exhibit spontaneous biorhythmicity. Rhythmic transmembrane Ca2+ flux activates plasma membrane K+ channels and Na+/H+ exchange. This leads to periodic changes of membrane voltage and pHi at about one cycle per minute. We conclude that endogenous oscillatory activity could be a trigger mechanism for DNA synthesis, proliferation, and abnormal growth of renal epithelial cells in culture.

Alkalosis↗
PubMed1992

Endothelin-1 blunts transepithelial transport and differentiation of Madin-Darby canine kidney cells.

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 less than 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 less than 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 less than 0.02). Moreover, ET-1 prevented the increase in PNA binding (+ 53 +/- 7%; n = 5) induced by 0.1 mumol/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.

Animals↗
PubMed1992

Spontaneous membrane potential oscillations in Madin-Darby canine kidney cells transformed by alkaline stress.

High pH is known to be associated with normal cell growth and neoplastic transformation. We observed that Madin-Darby canine kidney (MDCK) cells grown under sustained alkaline stress (pH 7.7) develop "foci" composed of spindle-shaped cells lacking contact inhibition and exhibiting only poor adhesion to the culture support. Foci-developing (F) cells were cloned and grown in control medium (pH 7.4), where they maintained their neoplastic features indicating a stable pH-induced genetic transformation. After F cells had been fused to giant cells with polyethylene glycol, the cell membrane potential (Vm) was measured by means of microelectrodes. In contrast to non-transformed MDCK cells, Vm of F cells showed spontaneous biorhythmicity caused by periodic opening of Ca2(+)-activated K+ channels. Spiking activity was blunted by the Ca2+ channel blocker nifedipine, by the K+ channel blocker Ba2+, by the Na+/H+ exchange blocker amiloride and its analogue ethylisopropylamiloride, and by an extracellular pH of 7.6 and 6.8. We conclude that MDCK cells transformed by sustained alkaline stress have lost their stable plasma membrane potential but, instead, exhibit endogenous Ca2(+)- and pH-sensitive oscillations.

Animals↗
PubMed1991

Alkaline stress transforms Madin-Darby canine kidney cells.

Similar to growth factors aldosterone stimulates Na+/H+ exchange in renal target cells leading to cytoplasmic alkalinization. An alkaline intracellular pH reduces the H+ bonds between repressor proteins and DNA leading to the destabilization of the nuclear chromatin. We observed that sustained alkaline stress "per se" can lead to malignant transformation of Madin-Darby canine kidney (MDCK) cells. Cells grown for two weeks in alkaline culture medium (pH 7.8) developed multiple "foci" composed of spindle-shaped pleomorphic cells lacking contact inhibition and exhibiting poor adhesion to the culture support, typical characteristics of dedifferentiated tumor cells. "Focus" cells were cloned and grown in standard medium (pH 7.4). Cells maintained their abnormal growth pattern, indicating stable pH-induced genetic transformation. Cells were fused with polyethylene glycol to giant cells and impaled with microelectrodes. In contrast to non-transformed giant MDCK cells the plasma membrane potential showed spontaneous oscillations that could be virtually abolished by the omission of extracellular Ca2+ or by the addition of the K+ channel blocker Ba2+. We conclude that sustained alkaline stress can induce malignant transformation in MDCK cells indicated by an abnormal growth pattern and by membrane potential oscillations most likely due to Ca2+ activated K+ channels in the plasma membrane.

Alkalies↗
PubMed1991

Hypertonicity in fused Madin-Darby canine kidney cells: transient rise in NaHCO3 followed by sustained KCl accumulation.

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 HCO3- 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-/HCO3- exchange, 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.

Adenosine Triphosphatases↗
PubMed1991

Hypotonic stress-induced release of KHCO3 in fused renal epitheloid (MDCK) cells.

Mechanisms of cell volume regulation induced by the reduction of the osmolality of the Ringer solution by one-third were studied in fused Madin-Darby canine kidney (MDCK) cells. Intracellular HCO3-, K+ and Cl- concentrations [ion]i in parallel with cell membrane potential (PD), cell membrane conductance (Gm) and conductances of individual ions (Gmion) were evaluated with microelectrode techniques. Fused cells regulate their cell volume by about 50%. Gm increased from 0.43 +/- 0.03 mS/cm2 in isotonic Ringer solution to 4.3 +/-0.3 mS/cm2 in the steady state phase of cell swelling. GmCl was 0.31 +/- 0.03 mS/cm2 in isotonic Ringer solution and thus was the dominant individual ion conductance. In the initial phase of cell swelling GmK increased transiently 64-fold to 0.32 +/- 0.03 mS/cm2, and consequently PD hyperpolarized. At peak hyperpolarization GmCl transiently decreased by 15%. Cell swelling increased GmCl 11-fold and GmHCO3 28-fold to 0.95 +/- 0.1 mS/cm2 in the steady state phase of cell swelling. In this phase GmCl and GmHCO3 were dominating, whereas GmK was only slightly increased compared to isotonic conditions. The hyperpolarization of PD was paralleled by cytoplasmic acidification. At peak acidification [HCO3-]i decreased by 6.4 mmol/kg H2O. Cl- extrusion was not detectable in the initial phase of cell swelling. In isotonic Ringer solution [K+]i was 125 +/- 5 mmol/kg H2O. During the initial phase of cell swelling 23 +/- 5 mmol/kg H2O K+ was extruded, indicating that yet unknown anions participated in cell volume regulation in this phase of cell swelling. In the steady state phase of cell swelling [pH]i was normalized by replenishing [HCO3-]i, whereas Cl- was extruded. We conclude that fused renal epitheloid cells acutely release KHCO3 in response to hypotonicity, but then regain pH homeostasis in the steady state phase of cell swelling.

Animals↗
PubMed1990

Aldosterone-regulated ion transporters in the kidney.

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.

Aldosterone↗
PubMed1990

Madin-Darby canine kidney cells. I. Aldosterone-induced domes and their evaluation as a model system.

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 omega cm2 and constant when dome size exceeded 2.10(-4) cm2. The relative ion conductances (expressed as transference numbers) across the dome epithelium were tNa:tCl:tk = 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.

Aldosterone↗
PubMed1990

Madin-Darby canine kidney cells. II. Aldosterone stimulates Na+/H+ and Cl-/HCO3- exchange.

Experiments in dome epithelium of Madin-Darby canine kidney (MDCK) cells were performed to elucidate aldosterone action on acid-base transport. By means of pH-sensitive microelectrodes the pH of the dome fluid was measured while the apical plasma membrane was superfused. In the absence of HCO3- the dome fluid (facing the basolateral cell membrane) alkalinized in response to 10(-7) mol/l aldosterone. Amiloride (10(-3) mol/l) inhibited dome formation and pH recovery of the dome fluid from an extracellular acid load. In the presence of HCO3- dome fluid acidified in response to aldosterone. The stilbene derivative diisothiocyanate-stilbene-2,2'-disulphonic acid (DIDS) or removal of Cl- from the apical perfusate inhibited this dome acidification. In aldosterone-depleted MDCK monolayers HCO3- was actively accumulated in the dome fluid in contrast to aldosterone-supplemented cells. The results indicate that aldosterone stimulates both amiloride-sensitive Na+/H+ exchange and DIDS-sensitive Cl-/HCO3- exchange in the apical cell membrane of MDCK cells. In the absence of aldosterone the HCO3- extrusion process is localized in the basolateral membrane in series with apical Na+/H+ exchange, while in the presence of aldosterone Cl-/HCO3- is mainly localized in the apical membrane in parallel with Na+/H+ exchange. Cl- exits the cell through apical Cl- channels and is absorbed via the paracellular route.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1990

Madin-Darby canine kidney cells. III. Aldosterone stimulates an apical H+/K+ pump.

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 (delta 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 apical-membrane-bound CA particularly in dome-forming cells. Apical substitution of Na+ by K+ increased delta 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 delta 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.

Aldosterone↗
PubMed1989

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

The 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.

Animals↗
PubMed1989

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

We have chosen the MDCK cell line to investigate aldosterone action on H+ transport and its role in regulating cell membrane K+ conductance (GKm). 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) and GKm were measured simultaneously while the cell interior was acidified gradually by an extracellular acid load. We found a steep sigmoidal relationship between pHi and GKm (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/HCO-3 buffer system, respectively. We conclude that the hormone-induced cytoplasmic alkalinization in the presence of CO2/HCO-3 is limited by the increased activity of a pHi-regulating HCO-3 extrusion system. Since GKm 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.

Aldosterone↗