Effects of epinephrine on electrical properties of Madin-Darby canine kidney cells

The present study has been performed, to test for the influence of epinephrine on the potential difference across the cell membrane (PD) of Madin-Darby canine kidney (MDCK) cells. Under control conditions, mimicking the in vivo situation, PD averages –53.3±0.9 mV (n=37). Increasing extracellular potassium concentration from 5.4 to 10 and 20 mmol/l depolarizes the cell membrane by +4.3±0.4 mV (n=5) and +15.8±1.2 mV (n=5), respectively. The application of 1 mol/l epinephrine leads to sustained hyperpolarization of the cell membrane to –71.5±0.7 mV (n=37). In the presence of epinephrine, increasing extracellular potassium concentration from 5.4 to 20 mmol/l depolarizes the cell membrane by +30.6 ±0.2 mV (n=5); 1 mmol/l barium depolarizes the cell membrane by +14.8±0.7 mV (n=20) and abolishes the effect of step increases of extracellular potassium concentration from 5.4 to 10 mmol/l. In the presence of barium, epinephrine leads to a transient hyperpolarization by –31.2 ±1.2 mV (n=18). During this transient hyperpolarization, the cell membrane is sensitive to extracellular potassium concentration despite the continued presence of barium; 10 mol/l verapamil depolarizes the cell membrane to –41.0±2.6 mV (n=11). In the presence of verapamil, the hyperpolarizing effect of epinephrine is only transient; 10 mol/l phentolamine depolarizes the cell membrane by +3.0±0.6 mV (n=8). In the presence of phentolamine, the effect of epinephrine is virtually abolished (+0.4±0.6 mV,n=8); 1 mol/l isoproterenol depolarizes the cell membrane by +2.8±0.8 mV (n=8). In the norminal absence of extracellular calcium, epinephrine leads to a transient hyperpolarization, which can only be elicited once. In conclusion, cpinephrine hyperpolarizes MDCK cells by increasing the apparent potassium conductance. This effect is transmitted by -receptors and may be mediated by increases of intracellular calcium activity.     

Effects of bradykinin on electrical properties of Madin-Darby canine kidney epithelioid cells

In the present study we have investigated the influence of bradykinin on the potential difference across the cell membrane (PD) of Madin Darby Canine Kidney (MDCK)-cells. In the absence of bradykinin PD averages –52.6±0.9 mV (n=52). Increasing extracellular potassium concentration from 5.4 to 10 and 20 mmol/l depolarizes the cell membrane by +5.2±0.3 mV (n=8) and +14.9±1.0 mV (n=9), respectively. The application of 0.1 mol/l bradykinin leads to a transient hyperpolarization of the cell membrane to –70.3±0.6 mV (n=30). During this transient hyperpolarization increasing extracellular potassium concentration from 5.4 to 10 and 20 mmol/l depolarizes the cell membrane by +10.4±0.7 mV (n=10) and +29.2±0.8 mV (n=8) respectively. Application of fragments of bradykinin (0.1 mol/l) are without significant effect on the potential difference across the cell membrane. 1 mmol/l barium depolarizes the cell membrane by +15.8±1.2 mV (n=9) and abolishes the effect of step increase of extracellular potassium concentration from 5.4 to 10 mmol/l. In the presence of barium, bradykinin leads to a transient hyperpolarization by –24.7±1.3 mV (n=7). During this transient hyperpolarization, the cell membrane is sensitive to extracellular potassium concentration despite the continued presence of barium. In the nominal absence of extracellular calcium, bradykinin leads to a transient hyperpolarization, which can be elicited only once. The transient hyperpolarization is not affected by the presence of verapamil or indomethacin. In conclusion, bradykinin hyperpolarizes MDCK-cells by increasing the apparent potassium conductance. This effect is probably mediated by increase of intracellular calcium activity.     

Effects of serotonin on electrical properties of Madin-Darby canine kidney cells

The present study has been performed to test for the influence of serotonin on the potential difference across the cell membrane (PD) of Madin-Darby canine kidney (MDCK)-cells. Under control conditions PD averages –48.6±0.6 mV (n=98). Increasing extracellular potassium concentration from 5.4 to 10 and 20 mmol/l depolarizes the cell membrane by +6.3±0.6 mV (n=6) and +14.1±1.0 mV (n=12), respectively. The cell membrane is transiently hyperpolarized to –67.8±0.8 mV (n=63) by 1 mol/l serotonin. In the presence of serotonin, increasing extracellular potassium concentration from 5.4 to 20 mmol/l depolarizes the cell membrane by +26.4±1.0 mV (n=11). 1 mmol/l barium depolarizes the cell membrane by +15.7±1.3 mV (n=17) and abolishes the effect of step increases of extracellular potassium concentration from 5.4 to 10 mmol/l. In the presence of barium, serotonin leads to a transient hyperpolarization by –26.3±1.0 mV (n=16). During this transient hyperpolarization, the cell membrane is sensitive to extracellular potassium concentration despite the continued presence of barium. 10 mol/l methysergide hyperpolarize the cell membrane by –7.2±2.0 mV (n=6). In the presence of 10mol/l methysergide, the effect of serotonin is virtually abolished (+0.4±0.9 mV,n=6). 1 mol/l ketanserin, a 5-HT₂ receptor blocking agent, ICS 205-930, a 5-HT₃ receptor blocking agent, and phentolamine, an unspecific -receptor blocking agent, do not significantly modify the effect of serotonin. In the nominal absence of extracellular calcium, the effect of serotonin is markedly reduced. In conclusion, serotonin hyperpolarizes MDCK-cells by increasing apparent potassium conductance. This effect is transmitted by 5-HT₁ receptors and depends on extracellular calcium.     

Electrical properties of Madin-Darby-canine-kidney cells

In incompletely confluent madin Darby canine kidney cells continuous measurements of the potential difference across the cell membrane (PD) were made with conventional microelectrodes during rapid changes of extracellular sodium and/or calcium concentration. During control conditions PD averages –50.6±0.7 mV. Reduction of extracellular sodium concentration from 131.8 to 17.8 mmol/l leads to a reversible hyperpolarization of the cell membrane to –65.3±1.1 mV. This hyperpolarization is not significantly reduced by omission of glucose or presence of amiloride (1 mmol/l) in the perfusates. Instead, 1 mmol/l amiloride depolarizes the cell membrane by +5.2±0.4 mV. 1 mmol/l barium depolarizes the cell membrane to –31.3±1.1 mV. Step increases of extracellular potassium concentration from 5.4 to 10 and 20 mmol/l depolarize the cell membrane by +5.5±0.5 mV and +16.5±1.8 mV respectively. In the presence of barium, the depolarizing effect of increasing extracellular potassium concentration and of amiloride is almost abolished. Reduction of extracellular sodium concentration in the presence of barium, however, leads to a transient hyperpolarization of the cell membrane. During this transient hyperpolarization, increasing extracellular potassium concentration depolarizes the cell membrane despite the continued presence of barium. Omission of extracellular calcium (EDTA) depolarizes the cell membrane by +36.7±3.2 mV. In the absence of extracellular calcium, the hyperpolarizing effect of reduced extracellular sodium concentration is markedly reduced (–4.5±1.2 mV). 2 mol/l A23187 in the presence of extracellular calcium hyperpolarizes the cell membrane to –72.5±0.6 mV. In conclusion, reduction of extracellular sodium concentration increases the potassium conductance of the cell membrane, possibly by increasing intracellular calcium activity via an influence on the sodium/calcium-exchange.     

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.     

Electrical properties of Ehrlich ascites tumor cells

The cell membrane potential (PD) of Ehrlich ascites tumor cells was measured continuously at 37°C with conventional microelectrodes during rapid alterations of extracellular fluid composition. At extracellular electrolyte composition mimicking the in vivo situation PD is –56.7±0.7 mV and the apparent membrane resistance is 62.2±2.2 M. Increasing extracellular potassium concentration from 5.4 to 20.0 mmol/l depolarizes the cell membrane by +18.4±0.5 mV. Thus, the transference number for potassium (tk, apparent slope potassium conductance over slope membrane conductance) is 0.53±0.01. A significant correlation is observed between tk and PD: tk=–(0.014±0.001) [1/mV]·PD [mV] –(0.243±0.051). 0.7 mmol/l barium depolarizes the cell membrane by +28.2±0.7 mV, increases the apparent membrane resistance by a factor of 2.6±0.1 and abolishes the apparent potassium conductance. Reduction of extracellular sodium concentration from 141 to 21 mmol/l depolarizes the cell membrane by +3.1±1.3 mV. Similarly, 0.1 mmol/l amiloride depolarizes the cell membrane by +3.3±0.7 mV. Reduction of extracellular chloride concentration from 128 to 67 mmol/l hyperpolarizes the cell membrane by –2.5±0.2 mV. 1 mmol/l anthracene-9-COOH does not significantly alter PD. Temporary omission of glucose from the extracellular fluid has no appreciable effect on PD. In conclusion, PD of Ehrlich ascites tumor cells is in the range of other mammalian epithelial cells and is generated mainly by potassium diffusion, while the conductances to sodium and chloride appear to be small.     

Epinephrine activates outward rectifying K channel in Madin-Darby canine kidney cells

Patch-clamp recordings were used to study the epinephrine dependent activation of ion channels in the cell membrane of cultured subconfluent renal epithelial (MDCK) cells. The patch-current was dominated by two populations of K channels. The spontaneously active population of K channels shows an inward rectifying behavior. Addition of epinephrine to the cell exterior, after the patchpipette had been sealed to the cell membrane, increased the open probability of the inward rectifying K channel and shifted the membrane potential in the hyperpolarizing direction. The epinephrine induced hyperpolarization occurs in the range of seconds and is caused by activation of outward-rectifying K channels. The outward-rectifying K channel could not be observed under control conditions. Epinephrine activated channels always appeared in clusters of four to nine channels. Both populations of K channels are modulated in their open probability by cytoplasmic free calcium and voltage.     

The effect of hypoosmolarity on the electrical properties of Madin Darby canine kidney cells

The present study has been performed to test for the effect of hypotonic extracellular fluid on the electrical properties of Madin Darby canine kidney (MDCK)-cells. The volume of suspended MDCK-cell is 1,892±16 fl (n=8) in isotonic (298.7 mosmol/l) extracellular fluid. Exposure of the cells to hypotonic (230.7 mosmol/l) extracellular fluid is followed by cellular swelling to 2,269±18 fl (n=4) and subsequent volume regulatory decrease to 2,052±22 fl (n=4) within 512 s. Volume regulatory decrease is abolished by quinidine (1 mmol/l) and by lipoxygenase inhibitor nordihydroguaiaretic acid (50 mol/l). The potential difference across the cell membrane averages –53.6±0.9 mV (n=49) in isotonic extracellular perfusates. Reduction of extracellular osmolarity depolarizes the cell membrane by +25.7±0.8 mV (n=67), reduces the apparent potassium selectivity of the cell membrane, from 0.55±0.07 (n=9) to 0.09±0.01 (n=26), and increases the apparent chloride selectivity from close to zero to 0.34±0.02 (n=21). Potassium channel blocker barium (1 mmol/l) depolarizes the cell membrane by +15.2±1.1 mV (n=13). In the presence of barium, reduction of extracellular osmolarity leads to a further depolarization by +14.0±1.4 mV (n=12). Addition of chloride channel blocker anthracene-9-COOH (1 mmol/l) leads to a hyperpolarization of the cell membrane by –6.7±2.2 mV (n=11). In the presence of anthracene-9-COOH, reduction of the extracellular osmolarity leads to a depolarization by +22.4±1.7 mV (n=11). Application of 1 mmol/l quinidine depolarizes the cell membrane to –6.6±0.5 mV (n=8) and virtually abolishes the effect of reduced extracellular osmolarity on cell membrane potential. Nordihydroguaiaretic acid (50 mol/l), a substance known to inhibit lipoxygenase, increases steady state cell membrane potential in isotonic extracellular fluid to –58.8±1.8 mV (n=10) and blunts the depolarizing effect of hypotonic extracellular fluid (+5.4±1.5 mV,n=10). In conclusion, exposure of MDCK-cells to hypotonic media depolarizes the cell membrane by activation of a conductive pathway, which is insensitive to both barium and anthracene-9-COOH. The conductive pathway is possibly activated by leucotrienes.Parts of this work were presented at the 8th International Symposium on Biochemical Aspects of Kidney Function, Dubrovnik, 1986.     

Extracellular detection of K+ release during migration of transformed Madin-Darby canine kidney cells

 Madin Darby canine kidney cells transformed by alkaline stress (MDCK-F cells) constitutively migrate at a rate of about 1 μm·min^–1. Migration depends on the intermittent activity of a Ca²⁺-stimulated, 53-pS K⁺ channel (K_Ca channel) that is inhibitable by charybdotoxin. In the present study we examined whether this intermittent K_Ca channel activity results in a significant K⁺ loss across the plasma membrane. K⁺ efflux from MDCK-F cells should result in a transient increase of extracellular K⁺ ([K⁺]_e) in the close vicinity of a migrating cell. However, due to the rapid diffusion of K⁺ ions into the virtually infinite extracellular space, such a transient increase in [K⁺]_e was too small to be detected by conventional K⁺-selective electrodes. Therefore, we developed a ”shielded ion-sensitive microelectrode” (SIM) that limited diffusion to a small compartment, formed by a shielding pipette which surrounded the tip of the K⁺-sensitive microelectrode. The SIM improved the signal to noise ratio by a factor of at least three, thus transient increases of [K⁺]_e in the vicinity of MDCK-F cells became detectable. They occurred at a rate of 1.3 min^–1. The cell releases 40 fmol K⁺ during each burst of intermittent K_Ca channel activity, which corresponds to about 15% of the total cellular K⁺ content. Since transmembrane K⁺ loss must be accompanied by anion loss and therefore leads to a decrease of cell volume, these findings support the hypothesis that intermittent volume changes are a prerequisite for the migration of MDCK-F cells. Received: 15 April 1996 / Received after revision: 18 June 1996 / Accepted: 23 July 1996     

Polarized ion transport during migration of transformed Madin-Darby canine kidney cells

Epithelial cells lose their usual polarization during carcinogenesis. Although most malignant tumours are of epithelial origin little is known about ion channels in carcinoma cells. Previously, we observed that migration of transformed Madin-Darby canine kidney (MDCK-F) cells depended on oscillating K⁺ channel activity. In the present study we examined whether periodic K⁺ channel activity may cause changes of cell volume, and whether K⁺ channel activity is distributed in a uniform way in MDCK-F cells. After determining the average volume of MDCK-F cells (2013±270 m³; n=8) by means of atomic force microscopy we deduced volume changes by calculating the K⁺ efflux during bursts of K⁺ channel activity. Therefore, we measured the membrane conductance of MDCK-F cells which periodically rose by 22.3±2.5 nS from a resting level of 6.5±1.4 nS (n=12), and we measured the membrane potential which hyperpolarized in parallel from –35.4±1.2 mV to –71.6±1.8 mV (n=11). The distribution of K⁺ channel activity was assessed by locally superfusing the front or rear end of migrating MDCK-F cells with the K⁺ channel blocker charybdotoxin (CTX). Only exposure of the rear end to CTX inhibited migration providing evidence for horizontal polarization of K⁺ channel activity in transformed MDCK-F cells. This is in contrast to the vertical polarization in parent MDCK cells. We propose that the asymmetrical distribution of K⁺ channel activity is a prerequisite for migration of MDCK-F cells.     

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 mol/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.     

Ouabain decreases apparent potassium-conductance in proximal tubules of the amphibian kidney

According to a previous study from this laboratory, the electrochemical gradient for potassium across the peritubular cell membrane of proximal tubules in the isolated perfused frog kidney increases following the application of ouabain. In order to test, if this phenomenon were due to a decrease of potassium conductance, the effects of ouabain on cell membrane resistances and the sensitivity of the peritubular cell membrane potential difference (PD_pt) to step changes of peritubular potassium and bicarbonate concentration were studied. In the absence of ouabain, PD_pt averaged –60±3 mV (n=25). A step increase of peritubular potassium concentration from 3 to 18 mmol/l (pH 8.07) depolarises PD_pt (PD_k) by +24±2 mV (n=8). An increase of bicarbonate from 20 to 40 mmol/l (pH 8.07) hyperpolarises PD_pt (PD_b) by –2.8±0.4 mV (n=9). The resistance of the luminal and peritubular cell membranes in parallel (R _m) amounts to 45±9 k cm (tubule length) (n=4) and the voltage divider ratio (VDR) to 1.4±0.2 (n=7). The resistance of the cellular cable (cellular core,R _c) approaches 131±37 M/cm (n=4). Peritubular application of 0.1 mmol/l ouabain leads to a gradual decline of PD_pt (t _1/2 approx. 30 min), to an increase ofR _m, a decrease of PD_k and an increase of PD_b. VDR andR _c are not changed significantly. The data point to a functional link between the sodium/potassium ATPase and the potassium conductance of the peritubular cell membrane.     

Cell transformation induces a cytoplasmic Ca2+ oscillator in Madin-Darby canine kidney cells

Alkaline stress transforms Madin-Darby canine kidney (MDCK) cells as indicated by loss of epithelial structure, multilayer cell growth and formation of foci. In the present study we report that transformed MDCK cells (MDCK-F cells) exhibit spontaneous and lasting oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i), which are absent in non-transformed cells. Oscillations, as revealed by Fura-2 video imaging, were due to the activity of an inositol 1,4,5-trisphosphate-(InsP ₃)-sensitive Ca²⁺ store since their frequency was dependent on bradykinin concentration and they were abolished by the phosphoinositidase C inhibitor U73122. Moreover, blockers of the cytoplasmic Ca²⁺-ATPase, thapsigargin and 2,5-di-(tetr-butyl)-1,4-benzohydroquinone inhibited oscillatory activity. In contrast, neither injection of ruthenium red, ryanodine nor caffeine had any effect on oscillations. Analysis of the spatial distribution of [Ca²⁺]_i showed that Ca²⁺ transients originated from an initiation site constant for a given cell and spread through the cell as an advancing Ca²⁺ wave. Oscillations started in a random manner from single cells and spread over neighbouring cells, suggesting a kind of intercellular communication. We conclude that MDCK-F cells have acquired the ability for endogenous Ca²⁺ release through transformation. Oscillations are primarily due to the activity of an InsP ₃-sensitive cytosolic Ca²⁺ oscillator.     

The effect of phenylalanine on the electrical properties of proximal tubule cells in the frog kidney

The present study was designed to elucidate the effects of sodium-coupled transport on the electrical properties of proximal tubule cells in the isolated perfused frog kidney. Cable analysis techniques have been employed to determine the resistance of the luminal and peritubular cell membranes in parallel (R _m) and the apparent ratio of the luminal over the peritubular cell membrane resistance (VDR). Furthermore, the sensitivity of the potential difference across the peritubular cell membrane (PD_pt) to 6-fold increases of peritubular potassium concentration (PD_k) was taken as a measure of the relative potassium conductance of this membrane. In the absence of luminal phenylalanine, PD_pt amounts to –60±1 mV (n=90),R _m to 36±3 k cm (n=22), VDR to 1.81±0.14 (n=20), and PD_k to 15.0±0.9 mV (n=25). The application of 10 mmol/l phenylalanine replacing 10 mmol/l raffinose leads to a rapid (within 30 s) depolarisation of PD_pt to 50±5% of its control value and to a delayed (within 12 min) recovery to 95±5% of control. The rapid depolarisation is associated with a decline ofR _m and VDR, indicating a decrease mainly of the luminal cell membrane resistance. During recovery of PD_pt there is a parallel increase of VDR and a further decline ofR _m pointing to a decline of the basolateral cell membrane resistance. PD_k is decreased during rapid depolarisation but increases again during the recovery phase. Thus, phenylalanine initially decreases but then increases above control the apparent potassium conductance. Removal of phenylalanine leads to a transient hyperpolarisation and increased apparent potassium conductance. If a cell is depolarised by current injection into a neighbouring cell, a similar decrease of PD_k is observed which shows also a similar recovery (partial repolarisation) despite continued injection of constant current. The data point to a potential-dependent peritubular K⁺-conductance (of the inwardly rectifying type) and to a regulatory increase within some ten minutes, when the cell is depolarised either by sodium entry across the luminal cell membrane or by current injection into a neighbouring cell.     

Liquid flow through monolayers of cultured Madin-Darby canine kidney cells.

The rate of liquid flow per unit area (Jv/A) through Madin-Darby canine kidney cell monolayers has been studied at temperatures between 0 and 38 degrees C and at transmonolayer hydrostatic pressures between 14 and 44 cmH2O. Jv/A decreased exponentially with time during application of a constant pressure to the free surface of each monolayer. This behaviour resembles the sealing of cultured vascular endothelium. For monolayers sealed between 33-38 degrees C and 30-33 cmH2O, the mean (+/- S.E.M.) half-time (t1/2) of sealing was 228 (+/- 88) s (n = 6). The decrease in Jv/A during sealing can be expressed as a fraction of the initial Jv/A. Between 33-38 degrees C and 30-33 cmH2O, the mean (+/- S.E.M.) sealing fraction was 0.58 (+/- 0.06; n = 6). Mean sealing t1/2 was longer at lower temperatures, and longer for glutaraldehyde-fixed than for unfixed monolayers, but did not vary with transmonolayer pressure. Sealing fraction was not affected by variations in temperature or transmonolayer pressure, or by glutaraldehyde fixation of monolayers. It is argued that sealing is a physical, rather than a biological, phenomenon and that monolayers have non-linear mechanical properties.     

Influence of potassium depletion on potassium conductance in proximal tubules of frog kidney

In order to test for the contribution of intracellular potassium activity to the link of sodium/potassium-ATPase activity and potassium conductance, studies with conventional and potassium selective microelectrodes were performed on proximal tubules of the isolated perfused frog kidney. The peritubular transference number for potassium (t _k), i.e., the contribution of peritubular slope potassium conductance to the slope conductance of the cell membranes (luminal and peritubular), was estimated from the influence of peritubular potassium concentration on the potential difference across the peritubular cell membrane (PD _pt). During control conditions,PD _pt is –65±1 mV, intracellular potassium activity (K _i) 57±2 mmol/l andt _k 0.41±0.05. The resistance in parallel of the luminal and peritubular cell membranes (R _m) is 44±4 kcm, the resistance of the cellular cable (R _c) 137±13 M/cm. When the cells are exposed 10 min to potassium free perfusates (series I),PD _pt increases by –28±3 mV within 2 min and then decreases gradually to approach the control value within 10 min.K _i decreases by 22±3 mmol/l andR _c increases by 35±10%. After a transient decrease,R _m increases by 36±9%. Readdition of peritubular potassium leads to a transient increase ofPD _pt, a gradual decrease ofR _m andR _c as well as a gradual increase ofK _i t _k recovers only slowly to approach 65±8% of control value within 3 and 79±10% within 6 min. When the cells are exposed 10 min to potassium free perfusates containing 1 mmol/l barium (series II),PD _pt depolarizes by +28±4 mV andK _i decreases by 7±1 mmol/l within 10 min. Within 2 min of reexposure to control perfusatesPD _pt approaches the control value.t _k recovers significantly faster than in series I and approaches 92±8% of control value within 3 min and 107±8% within 6 min reexposure to control perfusates. In conclusion, the effect of potassium free perfusates on peritubular potassium conductance depends on the degree of potassium depletion of the cell.     

Determination of cell membrane resistance in cultured renal epithelioid (MDCK) cells: effects of cadmium and mercury ions

Previous studies have indicated that the cell membrane of Madin Darby Canine Kidney (MDCK) cells is hyperpolarized by a number of hormones and trace elements, in parallel with an enhancement of potassium selectivity. Without knowledge of the cell membrane resistance (R _m), however, any translation of potassium selectivity into potassium conductance remains equivocal. The present study was performed to determine the R _m of MDCK cells by cellular cable analysis. To this end, three microelectrodes were impaled into three different cells of a cell cluster; current was injected via one microelectrode and the corresponding voltage deflections measured by the other two microelectrodes. In order to extract the required specific resistances, the experimental data were analysed mathematically in terms of an electrodynamical model derived from Maxwell's equations. As a result, a mean R _m of 2.0±0.2 kcm² and an intercellular coupling resistance (R _c) of 6.1±0.8 M were obtained at a mean potential difference across the cell membrane of -47.0±0.6 mV. An increase of the extracellular K⁺ concentration from 5.4 to 20 mmol/l depolarized the cell membrane by 16.2±0.5 mV and decreased R _m by 30.6±3.0%; 1 mmol/l barium depolarized the cell membrane by 20.1±1.1 mV and increased R _m by 75.9±14.3%. Omission of extracellular bicarbonate and carbon dioxide at constant extracellular pH caused a transient hyperpolarization (up to –60.4±1.4 mV), a decrease of R _m (by 75±4.5%) and a decrease of R _c (by 23.1±8.4%). The changes in R _m and R _c were probably the result of intracellular alkalosis. Cadmium ions (1 mol/l) led to a sustained, reversible hyperpolarization (to –64.8±1.3 mV) and to a decrease of R _m (by 77.0±2.7%); mercury ions (1 mol/l) cause a sustained hyperpolarization (to –60.1±1.2 mV) and a decrease of R _m (by 76.3±3.9%). Neither manoeuvre significantly altered R _c. We have previously shown that both cadmium and mercury hyperpolarize the cell membrane potential and increase its potassium selectivity; the decrease of the R _m observed in the present study indicates that these effects are due to an increase of the potassium-selective conductance of the cell membrane.     

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 (V _m) was measured by means of microelectrodes. In contrast to non-transformed MDCK cells, V _M 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.     

JAM4 enhances hepatocyte growth factor-mediated branching and scattering of Madin-Darby canine kidney cells

Junctional adhesion molecule (JAM) 4 is a member of immunoglobulin superfamily that interacts with MAGI-1, a membrane-associated guanylate kinase protein at tight junctions in epithelial cells. We prepared Madin-Darby canine kidney II (MDCK) cells expressing JAM4 (MDCK-JAM4) and compared them with wild MDCK cells. The treatment of hepatocyte growth factor (HGF) induced more prominent branching and scattering in MDCK-JAM4 cells. Subsequently we attempted to identify signalling pathways modified by JAM4. The over-expression of JAM4 induced the formation of protrusions in COS-7 cells. Although those protrusions were different from typical lamellipodia, the dominant negative mutant of Rac suppressed them. The pull-down assay using CDC42 and Rac interactive binding domain of PAK also supports that Rac is activated in COS-7 cells expressing JAM4. Taken together, JAM4 itself activates Rac and may augment Rac activation by HGF, resulting in the enhancement of branching and scattering.     

Aldosterone actions on basolateral Na+/H+ exchange in Madin-Darby canine kidney cells

In recent studies, there has been a re-evaluation of the polarity of Na⁺/H⁺ exchange in Madin-Darby canine kidney (MDCK) cells. This study was designed to examine aldosterone actions on basolaterally located Na⁺/H⁺ exchange of MDCK cell monolayers grown on permeant filter supports; pH_i was analysed in the absence of bicarbonate by using the pH-sensitive fluorescent probe 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein. Pre-exposure of MDCK cells to aldosterone led within 10–20 min to an alkalization of pH_i ( 0.3 pH unit); this effect is prevented by an addition of dimethylamiloride to the basolateral superfusate. Addition of aldosterone led to stimulation of the basolaterally located Na⁺/H⁺ exchange activity (Na⁺-dependent recovery from an acid load); this effect required preincubation (more then 3 min) and was observed at 0.1 nM aldosterone. Preexposure (15 min) of MDCK monolayers to phorbol 12-myristate 13-acetate also led to an activation of Na⁺/H⁺ exchange; pre-exposure to 8-bromo-cAMP led to inhibition of Na⁺/H⁺ exchange activity. An inhibitory effect of aldosterone was observed if Na⁺/H⁺ exchange activity was analysed in the presence of aldosterone; the highest inhibitory effects (20%–30%) occurred at concentrations of 5 nM and higher. Aldosterone-dependent inhibition does not require preincubation and is fully reversible; it was only observed at low (20 mM) but not at high Na⁺ concentrations (130 mM). The data suggest that aldosterone has an instantaneous inhibitory effect on basolaterally located Na⁺/H⁺ exchange activity under conditions of low Na⁺, but stimulates the rate of transport activity upon preincubation under conditions of physiological Na⁺ concentrations.