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To elucidate the effects of acetylcholine on the electrical properties of incompletely confluent Madin Darby canine kidney (MDCK) cells continuous measurements of the potential difference across the cell membrane (PD) were made with conventional microelectrodes during rapid changes of extracellular fluid composition. During control conditions PD averages -48.9 +/- 1.0 mV (n = 51). 1 mumol/l acetylcholine leads to a sustained but reversible hyperpolarization of the cell membrane by -17.9 +/- 0.7 mV (n = 51). Half-maximal effect is observed at some 100 nmol/l. 1 mumol/l atropine does not significantly alter the potential difference across the cell membrane, but abolishes reversibly the hyperpolarizing effect of acetylcholine. Increase of extracellular potassium concentration from 5.4 mmol/l to 20 mmol/l depolarizes the cell membrane by +12.1 +/- 1.1 mV (n = 12) in the absence and by +25.7 +/- 0.9 mV (n = 12) in the presence of acetylcholine. Within 80 s removal of extracellular calcium leads to a depolarization of the cell membrane by +16.2 +/- 3.2 mV (n = 9). In the nominal absence of extracellular calcium acetylcholine leads to a transient hyperpolarization by -13.8 +/- 1.8 mV (n = 9), which can be elicited only once. In conclusion, acetylcholine hyperpolarizes the plasma membrane of MDCK cells by calcium-dependent enhancement of potassium conductance.

The cell membrane potential (PD) of Ehrlich ascites tumor cells was measured continuously at 37 degrees 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 omega. 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] X 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.

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 mumol/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 mumol/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.(ABSTRACT TRUNCATED AT 250 WORDS)

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 mumol/l A23187 in the presence of extracellular calcium hyperpolarizes the cell membrane to -72.5 +/- 0.6 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of basolateral Na+/K+ ATPase by ouabain eventually abolishes transport of glucose. The present study was performed to test, if this effect is due to a dissipation of the electrochemical gradient for sodium or due to a regulatory inhibition of sodium-coupled glucose entry across the luminal membrane at increasing intracellular sodium activity. To this end, proximal convoluted tubules of the doubly perfused isolated frog kidney were perfused alternatively with solutions containing either 5 mmol/l glucose or raffinose. The potential difference across the peritubular cell membrane (PDpt) and across the epithelium (PDte) has been recorded with conventional and across the peritubular cell membrane with ion selective microelectrodes (PDpt). In the absence of luminal glucose PDpt is (+/- SEM) -54.0 +/- 2.4 mV, PDte = -1.2 +/- 2.0 mV and PDNapt = -96 +/- 5 mV. The electrochemical gradient for sodium (mu Na+) amounts to 95 mV and intracellular sodium activity to 14 mmol/l (extracellular sodium activity is 74 mmol/l). Luminal application of glucose leads to a rapid depolarisation of PDpt (delta PDpt = 8.6 +/- 0.9 mV and PDNapt (delta PDNapt = 11.1 +/- 3.0 mV) and to hyperpolarisation of PDte (delta PDte = -0.8 +/- 0.2 mV). The peritubular application of ouabain leads to a gradual, reversible and proportional decline of PDpt, PDNapt and mu Na+. Glucose induced delta PDpt and delta PDNapt decrease in parallel to PDpt and PDNapt, resp. In a separate series, the lumped conductance (Gm) of the luminal and basolateral cell membrane has been determined, which amounts to 2.4 +/- 0.3 microS/mm (tubule length). Gm decreases 23 +/- 4%, when PDpt is decreased to half.(ABSTRACT TRUNCATED AT 250 WORDS)

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 (PDpt) to step changes of peritubular potassium and bicarbonate concentration were studied. In the absence of ouabain, PDpt averaged -60 +/- 3 mV (n = 25). A step increase of peritubular potassium concentration from 3 to 18 mmol/l (pH 8.07) depolarizes PDpt (delta PDk) by +24 +/- mV (n = 8). An increase of bicarbonate from 20 to 40 mmol/l (pH 8.07) hyperpolarizes PDpt (delta PDb) by -2.8 +/- 0.4 mV (n = 9). The resistance of the luminal and peritubular cell membranes in parallel (Rm) amounts to 45 +/- 9 k omega 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, Rc) approaches 131 +/- 37 M omega/cm (n = 4). Peritubular application of 0.1 mmol/l ouabain leads to a gradual decline of PDpt (t1/2 approx. 30 min), to an increase of Rm, a decrease of delta PDk and an increase of delta PDb. VDR and Rc 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.

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 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 mumol/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 carbon dioxide as well as increase of extracellular bicarbonate at constant carbon dioxide lead to a hyperpolarization and enhanced sensitivity of PD to extracellular potassium. In the presence of barium, the effects of omitted bicarbonate and carbon dioxide of MDCK-cells is highly conductive to potassium.(ABSTRACT TRUNCATED AT 250 WORDS)

Both proximal renal tubule cells and cultured Madin-Darby canine kidney (MDCK) cells are capable of regulating their volume in hypotonic media. Regulatory cell volume decrease in proximal straight tubules is impaired by barium, amiloride and acetazolamide and depends on the presence of bicarbonate and of sodium, whereas it is unaffected by complete removal of extracellular chloride. The observations may point to parallel loss of potassium through potassium channels as well as of bicarbonate and sodium via a bicarbonate-sodium cotransport. Alternatively, potassium/hydrogen ion exchange or potassium bicarbonate cotransport could be involved. In MDCK cells, exposure to hypotonic media apparently leads to the activation of an anion channel, while potassium conductance is rather decreased. In both proximal tubules and MDCK cells, volume regulatory decrease is possibly triggered by leucotrienes, which may be released during cell swelling. Cell volume is altered in a variety of conditions even at isotonic extracellular fluid and cell volume-regulatory mechanisms are likely to participate in regulation of renal transepithelial transport.

1. The present study has been performed to test for an influence of extracellular ATP on the potential differences across the cell membrane (PD) in subconfluent MDCK cells utilizing conventional microelectrodes. 2. In the absence of ATP, the mean measured PD was -47.5 +/- 0.3 mV (+/- S.E.M., n = 320). Application of 10 mumol/l ATP leads to rapid (less than 2 s) hyperpolarization of the cell membrane by -18.5 +/- 0.4 mV (n = 221), reduction of input resistance by 14 +/- 1 M omega (n = 106) and increase of the sensitivity of PD to alterations of extracellular potassium. 3. The concentration needed for half-maximal effect (K1/2) of ATP is approximately 0.5 mumol/l. ATP-gamma-S (K1/2 approximately 0.4 mumol/l) aand ADP (K1/2 approximately 0.9 similarly effective, whereas up to 1 mmol/l AMP or adenosine does not significantly alter PD. Application of 10 mumol/l theophylline, 1 mumol/l phentolamine and 10 mumol/l indomethacin does not blunt the hyperpolarizing effect of ATP. 4. The ATP-induced hyperpolarization is completely abolished in the presence of 1 mmol/l quinidine but only incompletely by 0.1 mmol/l quinidine or 1 mmol/l barium. In calcium-free extracellular fluid (1 mmol/l EDTA added) PD is 18.5 +/- 1.7 mV (n = 18). With reduced extracellular calcium, the hyperpolarizing effect of ATP is blunted (-12.3 +/- 1.6 mV, n = 18) and only transient. 5. In conclusion, ATP hyperpolarizes MDCK cells by increasing the potassium conductance. The activation of potassium channels requires calcium.

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 patch-pipette 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 present study has been performed to test for the involvement of pertussis toxin-sensitive GTP-binding proteins (G-proteins) in the cellular transduction of hormone-induced activation of potassium channels. In Madin Darby canine kidney (MDCK) cells, a permanent cell line from dog kidney, epinephrine, acetylcholine, bradykinin, serotonin and ATP hyperpolarize the cell membrane by activation of potassium channels. In cells pretreated with pertussis toxin the hyperpolarizations elicited by either acetylcholine or serotonin are completely abolished; that following epinephrine is blunted and only transient. The hyperpolarizing effects of ATP or bradykinin are not affected by pertussis toxin. Thus, in MDCK cells both pertussis toxin-dependent and -independent mechanisms operate in parallel to enhance the potassium conductance of the cell membrane.

In incompletely confluent Madin Darby canine kidney (MDCK)-cells continuous measurements of the potential difference across the cell membrane (PD) were made with conventional microelectrodes during rapid changes of extracellular chloride concentration. During control conditions mimicking in vivo situation, PD averages -50.3 +/- 0.7 mV. Reduction of extracellular chloride concentration from 122 mmol/l to 64.5 mmol/l depolarizes the cell membrane by +1.8 +/- 0.2 mV while reduction to 16 mmol/l leads to a transient, variable depolarization followed by a hyperpolarization of the cell membrane by -11.8 +/- 1.4 mV. 1 mmol/l anthracene-9-COOH hyperpolarizes the cell membrane by -10.7 +/- 1.0 mV, and abolishes the effect of altered extracellular chloride concentration (-0.6 +/- 0.5 mV), 1 mumol/l diphenylamine-2-carboxylate hyperpolarizes the cell membrane by -11.7 +/- 1.4 mV. 10 mumol/l furosemide hyperpolarize the cell membrane by -11.4 +/- 1.4 mV. Step increases of extracellular potassium concentration from 5.4 to 20 mmol/l depolarize the cell membrane by +14.9 +/- 1.0 mV in the absence of inhibitors, by +24.2 +/- 1.3 mV in the presence of anthracene-9-COOH and by +28.8 +/- 0.7 mV in the presence of furosemide. 10 mumol/l isoproterenol depolarize the cell membrane by +2.4 +/- 0.3 mV and increase the depolarizing effect of reducing extracellular chloride concentration to 64.5 mmol/l (+2.9 +/- 0.4 mV). 1 mumol/l forskolin depolarizes the cell membrane by +5.8 +/- 1.0 mV. In conclusion, chloride conductance of subconfluent MDCK-cells may be small during control conditions, is apparently decreased by anthracene-9-COOH and reduction of extracellular chloride concentration but is enhanced by isoproterenol.

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-cells 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 mumol/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).(ABSTRACT TRUNCATED AT 250 WORDS)

The present study has been performed to test for the effect of intracellular calcium and of serotonin on the channel activity in patches from subconfluent MDCK-cells. In inside-out patches, inwardly rectifying potassium-selective channels are observed with open probabilities of 0.01 +/- 0.01, 0.24 +/- 0.03 and 0.39 +/- 0.07, at 100 nmol/liter, 1 mumol/liter or 10 mumol/liter calcium activity, respectively. The single-channel slope conductance is 34 +/- 2 pS, if the potential difference across the patch (Vp) is zero, and approaches 59 +/- 1 pS, if Vp is -50 mV, cell negative. In the cell-attached mode, little channel activity is observed prior to application of serotonin (open probability = 0.03 +/- 0.03). If 1 mumol/liter serotonin is added to the bath perfusate, the open probability increases rapidly to a peak value of 0.34 +/- 0.04 within 8 sec. In continued presence of the hormone, the open probability declines to approach 0.06 +/- 0.02 within 30 sec. At zero potential difference between pipette and reference in the bath (i.e., the potential difference across the patch is equal to the potential difference across the cell membrane), the single-channel conductance is 59 +/- 4 pS. In conclusion, inwardly rectifying potassium channels have been identified in the cell membrane of subconfluent MDCK-cells, which are activated to a similar extent by increase of intracellular calcium activity to 1 mumol/liter and by extracellular application of 1 mumol/liter serotonin.

Fura2 - fluorescence was utilized to test for the effect of extracellular nucleotides on intracellular calcium concentration of subconfluent Madin-Darby Canine Kidney (MDCK)-cells. Extracellular ATP (10 mumol/l) and UTP (10 mumol/l) lead to rapid (within seconds), sustained, and fully reversible enhancement of intracellular calcium concentration from 138 +/- 9 nmol/l (n = 27), to 1561 +/- 260 nmol/l (n = 10) and 3435 +/- 949 nmol/l (n = 5), respectively. Half maximal effects are observed at some 1 mumol/l. In the absence of extracellular calcium the effect of ATP is transient, pointing to release of intracellular calcium. The sustained effect in the presence of extracellular calcium indicates that the nucleotides in addition recruit calcium from extracellular space.

Extracellular ATP has been shown to stimulate transepithelial chloride transport in confluent Madin-Darby canine kidney (MDCK) cell layers and to enhance potassium conductance in subconfluent MDCK cells. The present study has been performed to test for the effect of extracellular ATP on channel activity in patches from subconfluent MDCK cells. Within 8 s, addition of extracellular ATP (10 mumol/l) leads to a sustained, but fully reversible, appearance of potassium-selective channels in cell-attached patches [increase of open probability from 0.03 +/- 0.02 (n = 10) to 0.50 +/- 0.07 (n = 6)]. With the use of pipettes filled with 145 mmol/l KCl, inwardly rectifying property of the channels is disclosed with a single-channel conductance of 65.7 +/- 3.1 pS (n = 9) at zero potential difference between pipette and bath and with a reversal potential of 75.4 +/- 2.0 mV (n = 5; pipette negative vs. reference in the bath). The open probability of the channels is not significantly modified by altering pipette potential from -50 mV, pipette positive, to 50 mV, pipette negative. At extracellular calcium activities of less than 10 nmol/l, ATP leads to a transient activation of channels. In conclusion, extracellular ATP activates inwardly rectifying potassium channels in the cell membrane of subconfluent MDCK cells. A sustained activation of the channels requires the presence of extracellular calcium and is probably mediated by increases in intracellular calcium.