W. Steigner

Madin-Darby canine kidney cells

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 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.

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.

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

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

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<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.

pHi-dependent membrane conductance of proximal tubule cells in culture (OK) : differential effects on K+ -and Na+-conductive channels

SummaryConfluent monolayers of the established opossum kidney cell line were exposed to NH4Cl pulses (20 mmol/liter) during continuous intracellular measurements of pH, membrane potential (PDm) and membrane resistance (R′m) in bicarbonate-free Ringer. The removal of extracellular NH4Cl leads to an intracellular acidification from a control value of 7.33±0.08 to 6.47±0.03 (n=7). This inhibits the absolute K conductance (gK+), reflected by a decrease of K+ transference number from 71±3% (n=28) to 26±6% (n=5), a 2.6±0.2-fold rise ofR′m, and a depolarization by 24.2±1.5 mV (n=52). In contrast, intracellular acidification during a block ofgK+ by 3 mmol/liter BaCl2 enhances the total membrane conductance, being shown byR′m decrease to 68±7% of control and cell membrane depolarization by 9.8±2.8 mV (n=17). Conversely, intracellular alkalinization under barium elevatesR′m and hyperpolarizes PDm. The replacement of extracellular sodium by choline in the presence of BaCl2 significantly hyperpolarizes PDm and increasesR′m, indicating the presence of a sodium conductance. This conductance is not inhibited by 10−4 mol/liter amiloride (n=7). Patch-clamp studies at the apical membrane (excised inside-out configuration) revealed two Na+-conductive channels with 18.8±1.4 pS (n=10) and 146 pS single-channel conductance. Both channels are inwardly rectifying and highly selective towards Cl−. The low-conductive channel is 4.8 times more permeable for Na+ than for K+. Its open probability rises at depolarizing potentials and is dependent on the pH of the membrane inside (higher at pH 6.5 than at pH 7.8).