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.     

Electrical properties of Madin-Darby canine kidney cells

To gain some insight into electrogenic transport processes across the plasma membrane of Madin-Darby canine kidney (MDCK)-cells, continuous measurements of the potential difference across the plasma membrane (PD) were made during step changes of extracellular ion composition as well as application of barium or valinomycin. During control conditions mimicking in vivo extracellular fluid, PD approaches –51.5±0.8 mV (n=62). Step increase of extracellular potassium concentration from 5.4 to 10, to 20 or to 35 mmol/l, depolarizes PD by +5.5±0.8 mV (n=7), by +15.8±0.5 mV (n=64) and by +23.8±1.2 mV (n=12), respectively. 1 mmol/l barium depolarizes PD by +19.8±0.6 mV (n=38) and abolishes the effect of increasing extracellular potassium from 5.4 to 10 mmol/l but not to 35 mmol/l. Ten mol/l valinomycin hyperpolarizes PD to –69.3±2.9 mV (n=7). In the presence of valinomycin, increase of extracellular potassium from 5.4 to 20 mmol/l depolarizes PD by +31.0±1.0 mV (n=7). Ouabain depolarizes PD and reduces the sensitivity of PD to extracellular potassium concentration. Omission of extracellular bicarbonate and carbondioxide as well as increase of extracellular bicarbonate at constant carbondioxide lead to a hyperpolarization and enhanced sensitivity of PD to extracellular potassium. In the presence of barium, the effects of omitted bicarbonate and carbondioxide are only transient. In conclusion, the plasma membrane of MDCK-cells is highly conductive to potassium. At low but not at high extracellular potassium concentrations the potassium conductance can be blocked by barium. The potassium conductance can further be reduced by ouabain as well as acidosis and enhanced by alkalosis as well as omission of extracellular carbondioxide and bicarbonate.     

Influence of barium on the effects of phenylalanine in proximal tubules

The present study was designed to further test for the role of peritubular potassium conductance in the repolarization of peritubular cell membrane during sustained stimulation of sodium coupled transport by phenylalanine. To this end the potential difference across the peritubular cell membrane (PDpt) has been recorded continuously, while 10 mmol/l phenylalanine (Phe) were added to the luminal perfusate, both in the presence or absence of peritubular or luminal barium (1 mmol/l). In the absence of phenylalanine and barium, PDpt amounts to –65.5±2.2 mV. Phe leads to a rapid depolarization of the peritubular cell membrane by +36.2±2.2 mV within 30 s, followed by an almost complete repolarization by –28.9±2.6 mV within 7 min. In the presence of barium in peritubular perfusate, the depolarization following Phe is +24.3±2.6 mV and the repolarization almost abolished (–4.3±0.9 mV). In the presence of barium in luminal perfusate, Phe leads to a depolarization by +35.7±2.4 mV followed by a repolarization of –17.0±3.2 mV within 7 min. It is concluded that the repolarization during sustained stimulation of sodium coupled transport is in large part due to alterations of peritubular potassium conductance.     

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.     

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 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 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.     

Effects of ouabain and temperature on cell membrane potentials in isolated perfused straight proximal tubules of the mouse kidney

In isolated perfused segments of the mouse proximal tubule, the potential difference across the basolateral cell membrane (PDbl) was determined with conventional microelectrodes. Under control conditions with symmetrical solutions it amounted to –62±1 mV (n=118). The potential difference across the epithelium (PDte) was –1.7±0.1 mV (n=45). Transepithelial resistance amounted to 1.82±0.09 k cm (n=28), corresponding to 11.4±0.6 cm². Increasing bath potassium concentration from 5 to 20 mmol/l depolarized PDbl by +24±1 mV (n=103), and PDte by +1.6±0.1 mV (n=19). Thus, the basolateral cell membrane is preferably conductive to potassium. Rapid cooling of the bath perfusate from 38°C to 10°C led to a transient hyperpolarization of PDbl from –60±1 to –65±1 mV (n=21) within 40 s followed by gradual depolarization by +18±1% (n=14) within 5 min. The transepithelial resistance increased significantly from 1.78±0.11 k cm to 2.20±0.21 k cm (n=15). Rapid rewarming of the bath to 38°C caused a depolarization from –61±2 mV (n=17) to –43±2 mV (n=16) within 15 s followed by a repolarization to –59±2 mV (n=10) within 40 s. Ouabain invariably depolarized PDbl. During both, sustained cooling or application of ouabain, the sensitivity of PDbl to bath potassium concentration decreased in parallel to PDbl pointing to a gradual decrease of potassium conductance. Phlorizin hyperpolarized the cell membrane from –59±2 to –66±1 mV (n=13), virtually abolished the transient hyperpolarization under cooling, and significantly reduced the depolarization after rewarming from +17±2 mV (n=16) to +9±3 mV (n=9).The present data indicate that the contribution of peritubular potassium conductance to the cell membrane conductance decreases following inhibition of basolateral (Na⁺+K⁺)-ATPase. Apparently, cooling from 37° to 10°C does not only reduce (Na^–+K⁺)-ATPase activity but in addition luminal sodium uptake mechanisms such as the sodium glucose cotransporter. As a result, cooling leads to an initial hyperpolarization of the cell followed by depolarization only after some delay.Parts of this study have been presented at the 60th and 61th Meeting of the Deutsche Physiologische Gesellschaft, Dortmund 1984 and Berlin 1985     

Electrophysiology of cell volume regulation in proximal tubules of the mouse kidney

The present study has been designed to test for the influence of cell swelling on the potential difference and conductive properties of the basolateral cell membrane in isolated perfused proximal tubules. During control conditions the potential difference across the basolateral cell membrane (PD_bl) is –65±1 mV (n=74). Decrease of peritubular osmolarity by 80 mosmol/l depolarizes the basolateral cell membrane by +7.8±0.5 mV (n=42). An increase of bath potassium concentration from 5 to 20 mmol/l depolarizes the basolateral cell membrane by +25±1 mV (n=11), an increase of bath bicarbonate concentration from 20 to 60 mmol/l hyperpolarizes the basolateral cell membrane by –3.2±0.5 mV (n=13). A decrease of bath chloride concentration from 79.6 to 27 mmol/l hyperpolarizes the basolateral cell membrane by –1.8±0.7 mV (n=6). During reduced bath osmolarity, the influence of altered bath potassium concentration on PD_bl is decreased ( PD_bl=+16±2 mV,n=11), the influence of altered bicarbonate concentration on PDbl is increased ( PD_bl=–6.0±0.8 mV,n=13), and the influence of altered bath chloride concentration on PD_bl is unaffected ( PD_bl=–1.8±0.6 mV,n=6). Barium depolarizes the basolateral cell membrane to –28±2 mV (n=16). In the presence of 1 mmol/l barium, decrease of peritubular osmolarity by 80 mosmol/l leads to a transient hyperpolarization of the basolateral cell membrane by –5.9±0.5 mV (n=16). This transient hyperpolarization is blunted in the absence of extracellular bicarbonate. In conclusion, cell swelling depolarizes straight proximal tubule cells and increases bicarbonate selectivity of the basolateral cell membrane at the expense of potassium selectivity. The data reflect either incrases of bicarbonate conductance or decrease of potassium conductance during exposure of proximal tubule cells to hypotonic media.Parts of this work were presented at the 18th Congress of the Gesellschaft für Nephrologie, Frankfurt/M. 1986 and at the 8th International Symposium on Biochemical Aspects of Kidney Function, Dubrovnik 1986     

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.     

Electrophysiological characterization of rabbit distal convoluted tubule cell

The distal convoluted tubule (DCT) from rabbit kidney were perfused in vitro to study the conductive properties of the cell membranes by using electrophysiological methods. When the lumen and the bath were perfused with a biearbonate free solution buffered with HEPES, the transepithelial voltage (V _T) averaged –2.8±0.6 mV (n=20), lumen negative. The basolateral membrane voltage (V _B) averaged –77.8±1.1 mV (n=33) obtained by intracellular impalement of microelectrodes. Cable analysis performed by injecting a current from perfusion pipette revealed that the transepithelial resistance was 21.8±1.7 ·cm² and the fractional resistance of the luminal membrane was 0.78±0.03 (n=8), indicating the existence of ionic conductances in the luminal membrane. Addition of amiloride (10^–5 mol/l) to the luminal perfusate or Na⁺ removal from the lumen abolished the lumen negativeV _T and hyperpolarized the apical membrane. An increase in luminal K⁺ concentration from 5 to 50 mmol/l reduced the apical membrane potential (V _A) by 37.5±2.6 mV (n=7), whereas a reduction of Cl^– in the luminal perfusate did not changeV _A significantly (0.5±0.5 mV,n=4). Addition of Ba²⁺ to the lumen reducedV _A by 42.6±1.0 mV (n=4). When the bathing fluid was perfused with 50 mmol/l K⁺ solution, the basolateral membrane voltage (V _B) fell from –76.8±1.5 to –31.0±1.3 mV (n=18), and addition of Ba²⁺ to the bath reducedV _B by 18.3±4.8 mV (n=7). Although a reduction of Cl^– in the bathing fluid from 143 to 5 mmol/l did not cause any significant fast initial depolarization (1.8±1.7 mV,n=8), a spike like depolarization (14.0±2.5 mV,n=4) was observed, upon Cl^– reduction in the presence of Ba²⁺ in the bath. From these results, we conclude that the apical membrane of DCT has both K⁺ and Na⁺ conductances and the basolateral membrane has a K⁺ conductance and a small Cl^– conductance.     

A microelectrode for continuous monitoring of redox activity in isolated perfused tubule segments

The design and application of a micro-plantinum electrode for continuous monitoring of reducing activity in the isolated tubule preparation is described. The electrodes response to H₂O₂ up to 0.1 mmol/l, to uric acid up to 0.3 mmol/l, ascorbic acid up to 1.0 mmol/l and cysteine up to 2.0 mmol/l is almost linear. The electrode is insensitive to extracellular ions, to changes of pH (5.5–8.0), CO₂ (1–10%) and O₂ (1–100%). The reading of the electrodes is almost doubled when the temperature is increased from 20–40°C.When reducing substances are omitted from the perfusate for isolated perfused proximal tubules of the mouse, the reading is identical in perfusate and collected fluid, indicating that the tubular epithelium does not produce redox substances in sufficient amount to interfere with the electrode reading at flow rates 10 nl/min. When the tubule is perfused with solutions containing 0.3 mmol/l uric acid, the uric acid concentration in the collected fluid is 0.16±0.01 mmol/l after a contact time of 1.36±0.1 s, revealing net uric acid reabsorption. Adding probenecid to the luminal perfusion fluid, leads to a 37.5±1.0% increase of uric acid concentration in collected fluid, disclosing the inhibitory effect of probenecid on uric acid reabsorption. If 0.3 mmol/l uric acid is added to the bath, 0.017±0.002 mmol/l uric acid is detected in the luminal fluid. The entry of uric acid into the lumen is abolished by 10^–4 mol/l pyrazinamide.Supported by Österr. Forschungsrat, Proj. No. 4366     

Transmitter-induced changes of the membrane voltage of HT29 cells

The colonic carcinoma cell line HT₂₉ was used to examine the influence of agonists increasing cytosolic cAMP and Ca²⁺ activity on the conductances and the cell membrane voltage (V _m). HT₂₉ cells were grown on glass cover-slips. Cells were impaled by microelectrodes 4–10 days after seeding, when they had formed large plaques. In 181 impalements V _m was –51±1 mV. An increase in bath K⁺ concentration from 3.6 mmol/l to 18.6 mmol/l or 0.5 mmol/l Ba²⁺ depolarized the cells by 10±1 mV (n=49) or by 9±2 mV (n=3), respectively. A decrease of bath Cl^– concentration from 145 to 30 mmol/l depolarized the cells by 11±1 mV (n=24). Agents increasing intracellular cAMP such as isobutylmethylxanthine (0.1 mmol/l), forskolin (10 mol/l) or isoprenaline (10 mol/l) depolarized the cells by 6±1 (n=13), 15±3 (n=5) and 6±2 (n=3) mV, respectively. In hypoosmolar solutions (225 mosmol/l) cells depolarized by 9±1 mV (n=6). Purine and pyrimidine nucleotides depolarized the cells dose-dependently with the following potency sequence: UTP > ATP > ITP > GTP > TIP > CTP = 0. The depolarization by ATP was stronger than that by ADP and adenosine. The muscarinic agonist carbachol led to a sustained depolarization by 27±6 mV (n=5) at 0.1 mmol/l, and to a transient depolarization by 12±4 mV (n=5) at 10 mol/l. Neurotensin depolarized with a half-maximal effect at around 5 nmol/l. The depolarization induced by nucleotides and neurotensin was transient and followed by a hyperpolarization. We confirm that HT₂₉ cells possess Cl^–- and K⁺-conductive pathways. The Cl^– conductance is regulated by intracellular cAMP level, cytosolic Ca²⁺ activity, and cell swelling. The K⁺ conductance in HT₂₉ cells is regulated by intracellular Ca²⁺ activity.Supported by DFG Gre 480/10 and GIF Proj. no. I-86-100.10/ 88     

Macula densa cells sense luminal NaCl concentration via furosemide sensitive Na+2Cl−K+ cotransport

The macula densa cells of the juxtaglomerular apparatus probably serve as the sensor cells for the signal which leads to the appropriate tubuloglomerular feedback response. The present study reports basolateral membrane voltage (PD_bl) measurements in macula densa cells. We isolated and perfused in vitro thick ascending limb segments with the glomerulus, and therefore the macula densa cells, and the early distal tubule still attached. Macula densa cells were impaled with microelectrodes under visual control. PD_bl was recorded in order to examine how these cells sense changes in luminal NaCl concentrations. The addition of furosemide, a specific inhibitor of the Na⁺2Cl^–K⁺ cotransporter in the thick ascending limb, to the lumen of the perfused thick ascending limb hyperpolarized PD_bl from –55±5 mV to –79±4 mV (n=7). Reduction of NaCl in the lumen perfusate from 150 mmol/l to 30 mmol/l also hyperpolarized PD_bl from –48±3 mV to –66±5 mV (n=4). A Cl^– concentration step in the bath from 150 mmol/l to 30 mmol/l resulted in a 24±4 mV (n=4) depolarization of PD_bl. This depolarization of PD_bl was absent when furosemide was present during the Cl^– concentration step. These data suggest that the macula densa cells sense changes in luminal NaCl concentration via coupled uptake of Na⁺ and Cl^–. The transport pathways for NaCl transport in macula densa cells are probably identical to those in the thick ascending limb: the (Na⁺+K⁺)-ATPase in the basolateral membrane drives Na⁺ and Cl^– uptake via the luminal Na⁺2Cl^–K⁺ cotransport, Cl^– leaves the cell via basolateral Cl^– channels and K⁺ recycles across the apical membrane via K⁺ channels. Changes in intracellular Cl^– activity as a result of altered luminal NaCl uptake, and thus voltage changes of the basolateral membrane are probably the first signal in the tubuloglomerular feedback regulation.This study was supported by Deutsche Forschungsgemeinschaft Gr. 480/9     

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.     

Isolated perfused rabbit colon crypts: stimulation of Cl− secretion by forskolin

The aim of this study was to characterize ion conductances and carrier mechanisms of isolated in vitro perfused rabbit colonic crypts. Crypts were isolated from rabbit colon mucosa and mounted on a pipette system which allowed controlled perfusion of the lumen. In non-stimulated conditions basolateral membrane voltage (V _b1) was –65±1 mV (n=240). Bath Ba²⁺ (1 mmol/ l) and verapamil (0.1 mmol/l) depolarized V _b1 by 21±2 mV (n=7) and 31±1 (n=4), respectively. Lowering of bath Cl^– concentration hyperpolarized V _b1 from –69±3 to –75±3 mV (n=9). Lowering of luminal Cl^– concentration did not change V _b1. Basolateral application of loop diuretics (furosemide, piretanide, bumetanide) had no influence on V _b1 in non-stimulated crypts. Forskolin (10^–6 mol/l) in the bath depolarized V _b1 by 29±2 mV (n=54) and decreased luminal membrane resistance. In one-third of the experiments a spontaneous partial repolarization of V _b1 was seen in the presence of forskolin. During forskolin-induced depolarization basolateral application of loop diuretics hyperpolarized V _b1 significantly and concentration dependently with a potency sequence of bumetanide > piretanide furosemide. Lowering bath Cl^– concentration hyperpolarized V _b1. Lowering of luminal Cl^– concentration from 120 to 32 mmol/l during forskolin-induced depolarization led to a further depolarization of V_b1 by 7±2 mV (n=10). We conclude that V_b1 of rabbit colonic crypt cells is dominated by a K⁺ conductance. Stimulation of the cells by forskolin opens a luminal Cl^– conductance. Basolateral uptake of Cl^– occurs via a basolateral Na⁺ : 2Cl^– : K⁺ cotransport system.     

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.     

The effect of cyanide on apparent potassium conductance across the peritubular cell membrane of frog proximal tubules

To test for the effect of cyanide on frog proximal renal tubules the potential difference across the peritubular cell membrane (PDpt) has been recorded continuously before and during peritubular application of 1 mmol/l cyanide using conventional microelectrodes.Before application of cyanide PDpt amounts to –61.5 ±2.2 mV in the absence of luminal substrate. Cyanide depolarizes the peritubular cell membrane by +18.8±2.3 mV/10 min in the presence and by +4.5±0.9 mV/10 min in the absence of luminal substrate. The rapid depolarization of the cell membranes to addition of glucose to luminal perfusate is not significantly influenced by exposure to cyanide, whereas the influence of altered peritubular potassium concentration (from 3 to 9 mmol/l) is significantly reduced from +15.2±1.7 mV to +8.7±1.8 mV. Following exposure to cyanide the lumped resistance of the luminal and peritubular cell membranes increases significantly by 36±7%/6 min, and the cellular core resistance significantly by 14±6%/6 min. As a result, cyanide markedly decreases the peritubular potassium conductance, depolarizes the cell membranes and reduces the driving force for sodium coupled transport processes. Thus cyanide fully mimicks the effects of ouabain, although cyanide in contrast to ouabain is expected to deplete the cells from ATP. In conclusion ATP/ADP is not likely to play a major role in the regulation of sodium coupled transport processes and peritubular potassium conductance in amphibian proximal tubules.     

Chloride and potassium conductances of cultured human sweat ducts

The purpose of this study was to characterize the ion conductances, in particular those for Cl^– and K⁺, of human sweat duct cells grown in primary culture. Sweat duct cells from healthy individuals were grown to confluence on a dialysis membrane, which was then mounted in a mini-Ussing chamber and transepithelial and intracellular potentials were measured under open-circuit conditions. Under control conditions the epithelia developed mucosa-negative transepithelial potentials, V _te, of about –10mV. The apical membrane potential, V _a, was –25 mV to –30 mV (n=97) in most cells, but several cells had a higher potential of about –55 mV (n=29). Mucosal amiloride (10 mol/l) hyperpolarized V _a from –31±1 mV to a new sustained level of –46±2 mV (n=36). These changes were accompanied by increase in the fractional resistance of the apical membrane, fR _a, and decreases of V _te and the equivalent short-circuit current, I _sc. In amiloride-treated tissues an increase in mucosal K⁺ concentration (5 mmol/l to 25 mmol/l) depolarized V _a by 5±1 mV (n=8), while the same step on the serosal side depolarized V _a by 20±2 mV (n=8). A Cl^– channel blocker 3,5-dichloro-diphenylamine-2-carboxylate DCl-DPC; 10 mol/l) depolarized V _a by 5±1 mV (n=6), an effect that was lost after amiloride application. The blocker had no effect from the serosal side. Reduction of mucosal Cl^– (from 120 to 30 or 10 mmol/l) depolarized V _a by 9–11 mV (n=35), an effect that was often followed by a secondary hyperpolarization of 10–30 mV (n=27). Isoproterenol (5 mol/l) increased the V _a responses to low Cl^– such that the depolarizing response was increased from 10±1 mV to 19±2 mV (n=8); the hyperpolarizing response seemed to be reduced. With changes in Cl^– concentration on the serosal side, V _a remained relatively constant at –25 mV, while V _te decreased from –8 mV to–3 mV; hence, V _bl depolarized by about 5 mV. Taken together, our results show that the human sweat duct epithelium possesses Na⁺, K⁺ and Cl^– conductances on the luminal membrane and Cl^– and K⁺ conductances on the basolateral membrane. The Cl^– conductances on the luminal membrane is sensitive to DCl-DPC, and can be activated by isoproterenol. The small K⁺ conductance on the luminal membrane could account for some K⁺ secretion in sweat glands.     

A new class of inhibitors of cAMP-mediated Cl− secretion in rabbit colon,acting by the reduction of cAMP-activated K+ conductance

Previously we have shown that arylamino-benzoates like 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which are very potent inhibitors of NaCl absorption in the thick ascending limb of the loop of Henle, are only poor inhibitors of the cAMP-mediated secretion of NaCl in rat colon. This has prompted our search for more potent inhibitors of NaCl secretion in the latter system. The chromanole compound 293 B inhibited the equivalent short-circuit current (I _sc) induced by prostaglandin E₂ (n=7), vasoactive intestinal polypeptide (VIP,n=5), adenosine (n=3), cholera toxin (n=4) and cAMP (n=6), but not by ionomycin (n=5) in distal rabbit colon half maximally (IC₅₀) at 2 mol/l from the mucosal and at 0.7 mol/l from the serosal side. The inhibition was reversible and paralleled by a significant increase in transepithelial membrane resistance [e.g. in the VIP series from 116±16 ·cm² to 136±21 ·cm² (n=5)]. A total of 25 derivatives of 293 B were examined and structure activity relations were obtained. It was shown that the racemate 293 B was the most potent compound with-in this group and that its effect was due to the enantiomer 434 B which acted half maximally at 0.25 mol/l. Further studies in isolated in vitro perfused colonic crypts revealed that 10 mol/l 293 B had no effect on the membrane voltage across the basolateral membrane (V _bl) in non-stimulated crypt cells: –69±3 mV versus –67±3 mV (n=10), whilst in the same cells 1 mmol/l Ba²⁺ depolarised (V _bl) significantly. However, 293 B depolarised (V _bl) significantly in the presence of 1 mol/l forskolin: –45±4mV versus –39±5 mV (n=7). Similar results were obtained with 0.1 mmol/l adenosine. 293 B depolarised (V _bl) from –40±5 mV to –30±4 mV (n=19). This was paralleled by an increase in the fractional resistance of the basolateral membrane. VIP had a comparable effect. The hyperpolarisation induced by 0.1 mmol ATP was not influenced by 10 mol/l 293 B: –75±6 mV versus –75±6 mV (n=6). Also 293 B had no effect on basal K⁺ conductance (n=4). Hence, we conclude that 293 B inhibits the K⁺ conductance induced by cAMP. This conductance is apparently relevant for Cl^– secretion and the basal K⁺ conductance is insufficient to support secretion.