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.     

Influence of mepacrine,indomethacin, and nordihydroguaiaretic acid on the electrical properties of frog renal proximal tubules

In proximal renal tubules of the frog kidney, stimulation of sodium-coupled transport leads to a depolarization of the peritubular cell membrane, followed by partial repolarization. These alterations of the potential difference across the peritubular cell membrane (PD_pt,) are in part the result of altered peritubular potassium conductance. The repolarization has been blunted by the phospholipase A₂ inhibitor mepacrine, but not by the cyclooxygenase inhibitor indomethacin. In the present study the effect of mepacrine, indomethacin and the lipoxygenase inhibitor nordihydroguaiaretic acid on the electrical properties of proximal renal tubules has been tested in the presence and absence of stimulated sodium-coupled transport. In the absence of inhibitors, addition of 10 mmol/l phenylalanine to the luminal perfusate leads to a rapid depolarization and partial repolarization of the peritubular cell membrane, a decrease of the luminal cell membrane resistance (R _a) and a small increase of the cellular core resistance (R _c). Removal of phenylalanine leads to rapid hyperpolarization, increase of R _a and decline R _c. Mepacrine (100 ol/l) depolarizes the cell membrane and increases the peritubular cell membrane resistance (R _b), R _c and the intracellular pH. In the presence of mepacrine, phenylalanine leads to a sustained depolarization and a transient decrease of R _a. Indomethacin (10 mol/l) does not significantly modify PD_pt, the lumped resistance of both cell membranes (R _m) or R _c in the presence or absence of phenylalanine. Nordihydroguaiaretic acid (50 mol/l) does not alter significantly PD_pt, R _a, R _b or R _c prior to phenylalanine. However, in the presence of nordihydroguaiaretic acid, the repolarization upon phenylalanine is significantly more rapid, and the removal of phenylalanine in the presence of nordihydroguaiaretic acid is followed by a significant decrease of both, R _a and R _b. The observations point to an involvement of eicosanoids in the regulation of ion conductances during stimulation of sodium-coupled transport.     

The effect of phenylalanine on intracellular pH and sodium activity in proximal convoluted tubule cells of the frog kidney

The present study was performed to test the influence of sodium coupled transport of neutral substrates on intracellular pH and sodium activity in proximal tubules of the amphibian kidney. To this end, kidneys of rana esculenta have been isolated and perfused both through the portal vein (peritubular capillaries) and the aorta (luminal perfusate). The potential difference across the peritubular membrane of proximal tubule cells has been redorced with conventional (PD_pt) as well as with sodium (PD_na) and hydrogen ion (PD_h) selective microelectrodes continuously before during and after the luminal application of 10 mmol/l phenylalanine, replacing 10 mmol/l raffinose. PD_b and PD_na allowed the calculation of intracellular pH (pH_i) and sodium activity (Na_i), respectively. In the absence of phenylalanine in the tubule lumen, PD_pt approximates –57.5±2.3 mV (n=27), pH_i 7.73±0.04 (n=14, extracellular pH 7.77), and Na_i 13.3±0.9 mmol/l (n=13, extracellular sodium activity 74 mmol/l). Within 1 min the luminal application of phenylalanine leads to a depolarisation of PD_pt by +32±2 mV, as well as an increase of pH_i by 0.24±0.04 and of Na_i by 5.2±1.0 mmol/l. At 8 min from luminal application of phenylalanine, Na_i plateaus 5±1 mmol/l above control value, PD_pt increases again to a value of +12±2 mV below and pH_i decreases to a value 0.04±0.07 above their respective control values. All changes are fully reversed after removal of phenylalanine from the tubule lumen. The steady state of intracellular sodium activity might be explained by an extrusion of sodium via the sodium/potassium-ATPase, which approaches the entry across the luminal membrane, the intracellular alkalinisation is probably due to the reduced exit of bicarbonate across the peritubular cell membrane following the depolarisation of PD_pt.     

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.     

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.     

On the nature of delayed repolarization during sustained sodium coupled transport in frog proximal tubules

In proximal tubules of the frog kidney, stimulation of coupled transport of sodium with phenylalanine leads to depolarization of the cell membrane, followed by repolarization within a few minutes. The repolarization is due to a delayed increase of potassium conductance at the peritubular cell membrane. The present study was designed to test for the role of depolarization, of calmodulin and of arachidonic acid metabolites for the delayed increase of potassium conductance. To this end, the potential difference across the peritubular cell membrane of proximal convoluted tubules (PD_pt) has been recorded continuously during exposure of the lumen to phenylalanine or during galvanic current injection into a neighbouring cell. During control conditions, PD_pt averages –68.6±1.0 mV (n=45). Phenylalanine leads to a depolarization of the peritubular cell membrane by +31.5±1.3 mV (n=20), followed by a repolarization by –12.9±1.1 mV (n=20) within 3 min. Injection of currents from 10 to 80 nAmps leads to a depolarization by + 0.83±0.01 mV/nAmps which is again followed by repolarization. A linear correlation is observed between the magnitude of depolarization (dep) and repolarization (rep)within 3 min: rep (mV)= –(0.24±0.01) dep (mV) + (2.45±0.12) mV (r=0.09). Thus, depolarization is capable to trigger delayed repolarization. The extent of repolarization is a function of the magnitude of depolarization. The possible involvement of calmodulin or arachidonic acid metabolites has been tested for by inducing sodium coupled transport in the presence of 100 mol/l mepacrine, 10 mol/l indomethacin or 10 mol/l trifluoperazine. As a result, indomethacin and trifluoperazine do not significantly interfere with either depolarization or repolarization of the cell membrane during stimulation of sodium coupled transport while mepacrine significantly reduces repolarization.     

The effect of shear stress on the basolateral membrane potential of proximal convoluted tubule of the rat kidney

As consequence of glomerular filtration the viscosity of blood flowing through the efferent arteriole increases. Recently, we found that shear stress modulates proximal bicarbonate reabsorption and nitric oxide (NO·) was the chemical mediator of this effect. In the present work, we found that agonists of NO· production affected basolateral membrane potential (V _blm) of the proximal convoluted tubule (PCT) epithelium. Using paired micropuncture experiments, we perfused peritubular capillaries with solutions with different viscosity while registering the V _blm. Our results showed that a 50% increment in the viscosity, or the addition of bradykinin (10⁻⁵ M) to the peritubular perfusion solution, induced a significant and similar hyperpolarization of the V _blm at the PCT epithelium of 6 ± 0.7 mV (p < 0.05). Both hyperpolarizations were reverted by l-NAME (10⁻⁴ M). Addition of 2,2′-(hydroxynitrosohydrazino) bis-ethanamine (NOC-18) 3 × 10⁻⁴ M to the peritubular perfusion solution induced a hyperpolarization of the same magnitude of that high viscosity or bradykinin. These results strongly suggest the involvement of NO· in the effect of high viscosity solutions. This effect seems to be mediated by activation of channels as glybenclamide (5 × 10⁻⁵ M) added to peritubular solutions induced a larger depolarization of the V _blm with high viscosity solutions. Acetazolamide (5 × 10⁻⁵ M) added to high viscosity solutions induced a larger hyperpolarization (8 ± 1 mV; p < 0.05), suggesting that depolarizing current due to exit across the basolateral membrane damps the hyperpolarizing effect of high viscosity. Considering that Na⁺ and consequently water reabsorption is highly dependent on electrical gradient, the present data suggest that the endothelium of kidney vascular bed interacts in paracrine fashion with the epithelia, affecting V _blm and thus modulating PCT reabsorption.     

Potassium-selective channels in the basolateral membrane of single proximal tubule cells of frog kidney

The membrane potential of proximal tubule cells is dominated by the potassium conductance of the basolateral membrane. In the present paper the nature of this conductance is investigated by the patch-clamp technique. Only one type of K channel was found in the basolateral membranes of freshly isolated proximal cells. In cell-attached patches, the current/voltage relationship is markedly non-linear with much larger inward (30 pS) than outward ( 6 pS) conductances, even in the presence of roughly symmetrical K concentrations. Thus the channels show inward rectification. The determination of the conductance for outward current flow is complicated since the current/voltage curves show an area of negative conductance. Nevertheless, taking the conductance for outward current flow and the density of the channels it is possible to account for all of the previously reported potassium conductance of amphibian proximal tubule cells. The open probability of the channels was found not to depend upon the membrane potential. However, the non-linearity of the current/voltage relationships will confer upon the channel the same voltage dependence as that seen in intact proximal tubules, i.e. the conductance decreases with depolarisation. Incubation of cells in Ringer with no substrates or in the presence of alanine and/or glucose showed no change in the activity of the channels. These findings suggest that, although these channels may represent the basolateral conductance of frog proximal tubule cells, they are not involved in the well-established coupling between transport rate and potassium conductance.This work was supported by the Wellcome Trust     

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.     

Intracellular potassium in cells of the proximal tubule of necturus maculosus

Summary Using double-barreled K⁺ selective liquid ion-exchange microelectrodes intracellular K⁺ activity and the peritubular potential difference (PD) were measured simultaneously in single cells of Necturus proximal tubules. Proximal tubular fluid K⁺ activity and the transepithelial PD were also measured simultaneously. Kidney slices analyzed by flame photometry yielded a mean K⁺ concentration of 103.0±1.8 mM per Kg cell water. This electrometric study yielded a mean K⁺ activity of 58.7±2.3 mM, thus giving a low value of 0.57 for the mean ionic activity coefficient. The electrometric mean proximal tubule fluid K⁺ activity of 5.4±0.1 mM and plasma K⁺ activity of 2.8±0.3 mM yield a fluid/plasma activity ratio of 1.9±0.2. The calculated K⁺ equilibrium potentials (as calculated from the activity ratios) across the whole proximal tubular epithelium, its luminal cell boundary and its peritubular cell boundary are not significantly different from their respective measured membrane PDs. This signifies that K⁺ is in electro-chemical equilibrium distribution across the boundaries that separate the different compartments of the proximal tubular system.     

Effects of membrane potential changes on electrical cell-to-cell coupling in proximal tubule

Single proximal convoluted tubules (PCT) of Necturus kidney were impaled with three microelectrodes in the sequence M1, M2, M3. M1 was used for injecting short DC current pulses, M2 for recording peritubular membrane potential, V, and M3 for injecting longer DC current steps and thereby shifting V to a new baseline potential, V. We define the p.d. changes at M2 due to M1-induced pulses as V and V (for baselines V and V, respectively). Our objective was to test whether V was equal to V. The main finding is that when V depolarized by 10 to 80 mV V/V remained close to 1.00. Care was taken to ensure that this apparent stability of the pulse ratio was not due (i) to opposite changes of apical and basolateral membrane conductances (g(A) and g(B) respectively), (ii) to changes of the sum g(A)+g(B) compensated for by changes of the cell-to-cell junctional conductance, g(j), or (iii) to a distortion of the V/V ratio as a function of interelectrode distance, masking voltage-dependent changes of cell membrane conductances. Hyperpolarization of V produced gradual electrical uncoupling between cells as V became increasingly negative, by a mechanism yet to be determined.Supported by GRECO 24 (CNRS).     

Transepithelial potential difference in the proximal tubule of Necturus kidney

Summary Transepithelial potential difference (p.d.) was measured in the proximal tubule of Necturus kidney in vivo, by means of microelectrodes filled either with a 3M KCl solution or with a Ringer's solution for amphibians. The average transepithelial p.d., measured with KCl-tips, was: –1.4±2.4 mV (early convolutions), –0.1±2.0 mV (middle convolutions) and +0.1±2.4 mV (straight segment). The corresponding values obtained with Ringer's-filled microelectrodes were –2.3±1.8 mV, –1.3±1.1 mV and +0.1±1.2 mV, respectively. Tip localization into the lumen was ascertained by luminal injection of either oil (KCl electrode measurements) or artificial solutions which produced a measurable shift of transepithelial p.d. (determinations obtained with Ringer's-tips). Transepithelial p.d. in split-drops (mean reabsorptive half time 27.1±2.5 min) was –1.8±1.1 mV. The magnitude of transepithelial p.d. is discussed with respect to an equivalent electrical circuit; it is shown that high transepithelial p.d.'s are inconsistent with the known values of relative conductances of cell membranes in series and shunt pathway, respectively.     

The electrical profile of the distal tubule in triturus kidney

The transepithelial potential difference (V _TE) and transepithelial resistance (R _TE) were determined along the length of the distal tubule of the amphibianTriturus alpestris. The site of impalements was determined at the end of each experiment by latex injection and microdissection. Two segments, differing by their distribution at the surface of the kidney, by their respective diameters and by their electrical properties could be identified: the early distal tubule (EDT) and the late distal tubule (LDT).V _TE was invariably positive in the EDT; it increased from +5 mV to +22 mV within the first 30% of this segment and remained roughly constant distally to this site.R _TE was estimated at 57 ·cm² in the EDT. The LDT exhibited essentially negativeV _TE figures with the exception of its very initial portion; the peak negativity at the end of the LDT was –35 mV.R _TE was assessed from 8 measurements performed in the first 30% of the LDT: the tentative transepithelial resistance was 530 ·cm². Provisonal evidence suggests that the transepithelial resistance of the terminal portion of the LDT may be substantially larger than the resistance measured in other parts of this segment.C.N.R.S. GRECO 24     

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     

Stretch-activated channels in the basolateral membrane of single proximal cells of frog kidney

Epithelial cells are capable of regulating their volume in response to osmotic swelling or shrinkage. In the present paper a channel is described which may be involved in such a volume-regulatory response. Channels were studied in cell-attached patches of the basolateral membrane of cells isolated from frog kidneys using the patch-clamp technique. The open probability of the channels is increased by the application of negative pressure to the rear of the patch pipette or by bathing the cells in hypotonic fluid. In addition, the channels are voltagesensitive, such that depolarisation increases the open probability. The channels have a conductance of 25 pS with amphibian Ringer as the pipette solution and appear not to discriminate between potassium and sodium. Replacement of chloride by gluconate as the dominant anion in the pipette solution did not affect the current/voltage relationship, suggesting that the channels are cation-nonselective. Inward currents are observed at the resting membrane potential with either potassium or sodium as the dominant cation in the pipette solution: this obviates the channels serving a role as the route for solute exit from the cell during a volume-regulatory decrease response and suggests that they may act as the transduction mechanism sensing changes in cell volume.     

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.     

Intracellular bicarbonate in single cells of Necturus kidney proximal tubule

Summary The intracellular bicarbonate concentration in the cytoplasmic water of kidney cells of Necturus was determined by means of double-barreled HCO₃ ^–-selective liquid ion-exchange microelectrodes. These microelectrodes permit the simultaneous determination of intracellular HCO₃ ^– and membrane PD of single cells. The fact that these double-barreled microelectrodes yielded a normal peritubular cell membrane electrical PD (70 m V) may be taken as evidence against any significant cellular damage by the electrodes. This electrometric study yielded a mean intracellular [HCO₃ ^–] in single proximal tubule cells of Necturus of 11.1±0.6 mM, a value which is more than an order of magnitude higher than that predicted for a Donnan-type electrochemical equilibrium distribution of HCO₃ ^– ions. Thus there is a net electrochemical gradient favoring the passive efflux of HCO₃ ^– ions across both individual cell membranes. The movement of HCO₃ ^– ions from cell-to-interstitium would contribute to the renal acidification function. Across the luminal cell membrane the two possible mechanisms are either active reabsorption (lumen-to-cell) of HCO₃ ^– ions as such or active H⁺ secretion (cell-to-lumen). Our directly measured relatively high intracellular [HCO₃ ^–] and the associated calculated relatively alkaline kidney cell pH of 7.44 are both more consistent with the H⁺ secretion hypothesis.     

Electrochemical potentials of potassium in proximal renal tubule of rat

Summary By means of double-barreled K⁺ selective liquid ion-exchange microelectrodes, the electrical potential differences across individual cell membranes were determined simultaneously with the K⁺ concentration in single cellular elements of the proximal tubular epithelium of the rat. Proximal tubular fluid [K⁺] and plasma [K⁺] were also determined electrometrically. Thin cortical slices of the rat kidney analyzed by flame photometry yielded a mean [K⁺] of 136.3±4.2 mM per kg cell water. This electrometric study yielded a mean intracellular [K⁺] of 54.4±2.5 mM, a value which is about 1/3 of the total K⁺ content of proximal tubule cells. The electrometric mean proximal tubule fluid (second half) [K⁺] was 3.7±0.1 mM while plasma [K⁺] was 4.3±0.1 mM, yielding a fluid/plasma concentration ratio of 0.85±0.02. The calculated K⁺ equilibrium potentials (E _K )across the two individual from their respective measured membrane electrical PDs. This signifies that K⁺ exhibits an electrochemical equilibrium distribution across the luminal and peritubular cell boundaries of the proximal tubular epithelium. Thus it is no longer necessary to postulate the presence of an active K⁺ pump in either the luminal or peritubular cell membranes.     

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     

Effects of mucosal lanthanum on electrical parameters of isolated frog skin

The effect of mucosal La³⁺ on electrical parameters of isolated frog skins was studied on isolated frog skins with normally polarized or depolarized apical membrane. La³⁺ increases R_s, the paracellular or shunt resistance and diminishes R_Na, the resistance of the active sodium path, in both polarized and depolarized skins.The stimulatory effect of La³⁺ on short-circuit current (I_s.c.) is correlated with this decrease in R_Na. The characteristics of the stimulatory effect are: very rapid onset, ionic strength dependency, the possibility of being elicited by many other ions besides La³⁺. These features allow us to postulate that La³⁺ might affect the external interfacial potential which in turn affects the resistance of the sodium path.