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H Oberleithner

Publications and source records attributed to H Oberleithner.

At least 109 records · Page 6Linked to original sources
PubMed1988

Aldosterone-controlled linkage between Na+/H+ exchange and K+ channels in fused renal epithelial cells.

Aldosterone maintains acid-base balance and K+ homeostasis by controlling H+ and K+ secretion in renal epithelial cells. We have shown recently in the amphibian distal nephron that aldosterone activates a Na+/H+ exchange system in the luminal cell membrane, leading to transepithelial H+ secretion and cytoplasmic alkalinization. Since H+ secretory fluxes are paralleled by K+ secretion, it was postulated that the hormone-induced increase of intracellular pH activates the luminally located K+ channels. In 'giant' cells fused from individual cells of the distal nephron, we measured simultaneously cytoplasmic pH and cell membrane K+ conductance during acidification of the cell cytoplasm. The experiments demonstrate that cell membrane K+ conductance is half-maximal at an intracellular pH of 7.42, and that a positive cooperative interaction exists between K+ channel proteins and H+ ions (Hill coefficient = 6.5). Moreover, the cellular K+ conductance is most sensitive to cytoplasmic pH in the range modified by aldosterone. This supports the hypothesis that intracellular H+ activity, regulated by the Na+/H+ exchanger, serves as the signal to couple aldosterone-induced K+ secretory flux to H+ secretion in renal tubules.

Aldosterone↗
PubMed1988

Trans- and paracellular K+ transport in diluting segment of frog kidney.

In frog diluting segment transepithelial K+ net flux (JKte) occurs via trans- and paracellular transport routes. Inhibition of transcellular K+ transport discloses JKte across the shunt-pathway. By means of K+-sensitive microelectrodes we have measured secretory JKte induced by an acute K+ load, in the diluting segment of the isolated and doubly-perfused frog kidney. Transcellular K+ transport was inhibited by blocking the luminal K+ permeability either directly by barium or indirectly by the diuretic drug amiloride (via intracellular acidification induced by inhibition of Na+/H+ exchange), by the Na+/K+ pump inhibitor ouabain or by inducing an acute acid load. All experimental maneouvers led to a reduction of secretory JKte to about 50% of the control JKte. The apparent permeability coefficient for K+ of this nephron portion after inhibition of transcellular secretory JKte was reduced to a similar extent. We conclude: In frog diluting segment the ratio of trans- over paracellular JKte is close to unity. This ratio represents a minimum estimate because inhibition of the transcellular K+ pathway by barium, amiloride or an acute acid load may have been incomplete. Acidosis and/or amiloride exert large antikaliuretic effects due to the inhibition of the luminal K+ permeability.

Amiloride↗
PubMed1988

Fused cells of frog proximal tubule: II. Voltage-dependent intracellular pH.

Experiments were performed in intact proximal tubules of the doubly perfused kidney and in fused proximal tubule cells of Rana esculenta to evaluate the dependence of intracellular pH (pHi) on cell membrane potential applying pH-sensitive and conventional microelectrodes. In proximal tubules an increase of the K+ concentration in the peritubular perfusate from 3 to 15 mmol/liter decreased the peritubular cell membrane potential from -55 +/- 2 to -38 +/- 1 mV paralleled by an increase of pHi from 7.54 +/- 0.02 to 7.66 +/- 0.02. The stilbene derivative DIDS hyperpolarized the cell membrane potential from -57 +/- 2 to -71 +/- 4 mV and led to a significant increase of the K+-induced cell membrane depolarization, but prevented the K+-induced intracellular alkalinization. Fused proximal tubule cells were impaled by three microelectrodes simultaneously and cell voltage was clamped stepwise while pHi changes were monitored. Cell membrane hyperpolarization acidified the cell cytoplasm in a linear relationship. This voltage-induced intracellular acidification was reduced to about one-third when HCO-3 ions were omitted from the extracellular medium. We conclude that in proximal tubule cells pHi depends on cell voltage due to the rheogenicity of the HCO-3 transport system.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

Cytoplasmic pH determines K+ conductance in fused renal epithelial cells.

The mineralocorticoid hormone aldosterone maintains acid-base balance and K+ homeostasis by regulating H+ and K+ secretory mechanisms in kidney epithelial cells. We have shown recently in the amphibian distal nephron that aldosterone activates a Na+/H+ exchange system in the luminal cell membrane, thus leading to transepithelial H+ secretion and cytoplasmic alkalinization. Since H+ secretory fluxes were paralleled by K+ secretion, it was postulated that the hormone-induced increase of intracellular pH activates the luminally located K+ channels. In "giant" cells fused from individual cells of the distal nephron, we measured simultaneously cytoplasmic pH and cell membrane K+ conductance during acidification of the cell cytoplasm. The experiments show that cell membrane K+ conductance is half-maximal at an intracellular pH of 7.42 and that a positive cooperative interaction exists between K+-channel proteins and H+ (Hill coefficient = 6.5). Moreover, the cellular K+ conductance is most sensitive to cytoplasmic pH in the range modified by aldosterone. This supports the hypothesis that intracellular H+ activity, regulated by the Na+/H+ exchanger, serves as the signal to couple aldosterone-induced K+ secretory flux to H+ secretion in renal tubules.

Animals↗
PubMed1988

Electrode sandwich technique: a method for studying K+ transport in renal cells.

Distal tubules were harvested from frog kidney and placed on the membrane of a K+-selective macroelectrode. Then the renal tissue was covered with a dialysis membrane to produce a closed extracellular compartment with a constant volume (40 microliter). K+ fluxes in and out of the cells could be determined, since the steady-state K+ activity during constant perfusion changed to a new steady state when perfusion was stopped. Inhibition of passive K+ permeability by the addition of Ba2+ resulted in K+ uptake by the cells because of the function of the Na+-K+ pump. Inhibition of the pump by the addition of ouabain led to K+ efflux from cells reflecting the passive K+ permeability. Because K+ net movement under control conditions (no Ba2+ or ouabain) results from both uptake and efflux, subtraction of K+ uptake (in the presence of Ba2+) from control K+ net flux reveals the passive K+ efflux. This value agrees well with that obtained with ouabain. Furosemide led to a significant K+ shift from the extracellular compartment into the intracellular compartment. Reduction of extracellular pH from 7.8 to 6.0 decreased the rate of K+ uptake by 39 +/- 7% and the K+ leak by 51 +/- 11%. We conclude that K+ uptake and K+ release can be functionally separated. This so-called "electrode sandwich technique" permits evaluation of pump and leak independently in the same cell population.

Animals↗
PubMed1988

The amphibian diluting segment.

Diluting segments function to reabsorb NaCl and to reduce the osmolality of tubule fluid. These segments in amphibians are important in the conservation of NaCl. The diluting segment of mammals, the thick ascending limb, besides being an important site for the reabsorption of NaCl, supplies the energy that enables the kidney to excrete a concentrated urine. We focus in this review on the mechanisms involved in cellular and paracellular transport of Na+, K+, Cl-, and H+ in the amphibian diluting segment and how the transport of these ions is regulated. Also discussed is the action of loop diuretics and hormones on both transepithelial and cellular function. The large size of amphibian cells and their viability in various artificial conditions have allowed experiments to be performed that are not possible in the mammalian kidney, providing important information on the mechanisms of ion transport common to both mammalian thick ascending limb and amphibian diluting segment.

Aldosterone↗
PubMed1988

Whole-cell potassium currents in single early distal tubule cells.

The apical membrane potassium conductance of amphibian early distal tubules is sensitive to changes in the intracellular pH, with cellular acidification causing a decreased conductance. With the whole-cell patch-clamp technique, we have measured the total potassium conductance of single isolated early distal tubule cells of the frog. With symmetrical potassium gluconate solutions, the whole-cell current was found to be strongly rectifying, with an inward conductance of 12.9 nS (at intracellular pH between 7.6 and 8.0) and an outward conductance of 1.0 nS. The inward current was almost totally inhibited by the addition of 10 mM Ba2+ to the bath solution. The use of pipette solutions with pH between 7.0 and 8.0 showed a positive correlation between intracellular pH and conductance. In contrast, acidification of the extracellular solution caused no significant change in conductance.

Animals↗
PubMed1987

Activation of luminal Na+/H+ exchange in distal nephron of frog kidney. An early response to aldosterone.

Increased chronic intake of K+ induces H+ and K+ secretion in amphibian distal tubule, paralleled by an elevation of plasma aldosterone. The present experiments test whether the mineralocorticoid hormone is responsible for the alteration of ion transport. The blood capillaries of the isolated kidneys of NaCl-adapted (i.e. aldosterone-suppressed) Rana pipiens were perfused with HEPES-buffered amphibian Ringer solution (pH 7.8). Limiting intraluminal pH (pHlu) was measured continuously with pH-sensitive microelectrodes while aldosterone (3 X 10(-7) to 3 X 10(-6) mol/l) was applied in the peritubular perfusate. Concomitant with a decrease of the lumen-positive transepithelial potential (Vte) from 8.5 +/- 1.1 mV to 4.0 +/- 0.6 mV pHlu dropped from 7.73 +/- 0.02 to a new steady-state value of 7.17 +/- 0.05 within 60 to 180 min of aldosterone administration. Significant luminal acidification occurred already 20 min after application of aldosterone. Luminal addition of 10(-3) mol/l amiloride reversed luminal acidification to a pHlu of 7.68 +/- 0.04; at the same time Vte recovered partially. Pretreatment of the distal tubules with spironolactone prevented the aldosterone-induced acidification of the tubule fluid. We conclude that in early distal tubule of the amphibian kidney aldosterone--after interaction with cytoplasmic receptors--activates the luminal, amiloride-inhibitable Na+/H+ exchanger. This mechanism could explain enhanced H+ secretion found in the K+ adapted animal.

Aldosterone↗
PubMed1987

Ca2+ transport in diluting segment of frog kidney.

In the isolated-perfused frog (Rana pipiens) kidney the question of whether transepithelial transport of Ca2+ is a passive voltage driven process or involves active mechanisms was investigated. With conventional and ion-sensitive microelectrodes transepithelial electrical and electrochemical potential differences were measured. Luminal activities and transepithelial net fluxes of Ca2+ and Cl- were evaluated. Different transepithelial electrical voltages in a wide range (+20 to -4 mV) were generated by "chemical voltage clamping" and the dependence of Ca2+ net fluxes on these voltages investigated. The hormonal control of both Cl- and Ca2+ transport was studied by evaluating the effect of the cell-permeable cAMP analogue, db-cAMP and of the adenylate cyclase stimulator, forskolin. The experiments reveal that: (a) Ca2+ is reabsorbed along the diluting segment of frog kidney. (b) Ca2+ reabsorption is inhibited by furosemide because of the elimination of the transepithelial voltage. (c) There is a direct relationship between transepithelial voltage and Ca2+ reabsorption. (d) Neither Cl- nor Ca2+ reabsorption are affected by db-cAMP or forskolin. We conclude that Ca2+ reabsorption is passive, driven by the lumen-positive transepithelial voltage. It most likely occurs via the paracellular shunt pathway.

Absorption↗
PubMed1987

Cell membrane potential: a signal to control intracellular pH and transepithelial hydrogen ion secretion in frog kidney.

The dependence of intracellular pH (pHi) and transepithelial H+ secretion on the cell membrane potential (Vm) was tested applying pH-sensitive and conventional microelectrodes in giant cells fused from single epithelial cells of the diluting segment and in intact tubules of the frog kidney. An increase of extracellular K+ concentration from 3 to 15 mmol/l decreased Vm from -49 +/- 4 to -29 +/- 1 mV while pHi increased from 7.44 +/- 0.04 to 7.61 +/- 0.06. Addition of 1 mmol/l Ba2+ depolarized Vm from -45 +/- 3 to -32 +/- 2 mV, paralleled by an increase of pHi from 7.46 +/- 0.04 to 7.58 +/- 0.03. Application of 0.05 mmol/l furosemide hyperpolarized Vm from -48 +/- 3 to -53 +/- 3 mV and decreased pHi from 7.47 +/- 0.05 to 7.42 +/- 0.05. In the intact diluting segment of the isolated-perfused frog kidney an increase of peritubular K+ concentration from 3 to 15 mmol/l increased the luminal pH from 7.23 +/- 0.08 to 7.41 +/- 0.08. Addition of Ba2+ to the peritubular perfusate also increased luminal pH from 7.35 +/- 0.07 to 7.46 +/- 0.07. Addition of furosemide decreased luminal pH from 7.32 +/- 0.03 to 7.24 +/- 0.05. We conclude: cell depolarization reduces the driving force for the rheogenic HCO3- exit step across the basolateral cell membrane. HCO3- accumulates in the cytoplasm and pHi increases. An alkaline pHi inactivates the luminal Na+/H+ exchanger. This diminishes transepithelial H+ secretion. Cell hyperpolarization leads to the opposite phenomenon. Thus, pHi serves as signal transducer between cell voltage and Na+/H+ exchange.

Animals↗
PubMed1987

Evidence for Na+ dependent rheogenic HCO3- transport in fused cells of frog distal tubules.

The mechanism of HCO3- transport was studied applying microelectrodes in "giant" cells fused from single epithelial cells of the diluting segment of frog kidney. A sudden increase of extracellular HCO3- concentration from 10 to 20 mmol/l at constant pH hyperpolarized the cell membrane potential of the fused cell. This cell-voltage response was totally abolished by 10(-3) mol/l SITS and significantly reduced by 10(-4) mol/l acetazolamide or by omission of Na+ from the extracellular perfusate. Removal of Na+ from the perfusate caused a transient depolarization. Reapplication of Na+ induced a transient hyperpolarization. 10(-3) mol/l SITS abolished the cell-voltage response to removal and reapplication of Na+. In the intact diluting segment of the isolated perfused frog kidney peritubular perfusion of 10(-4) mol/l acetazolamide reduced the limiting transepithelial electrochemical gradient for H+ significantly from 30 +/- 4 mV to 14 +/- 3 mV. The results suggest: In the diluting segment of the frog kidney a Na+-dependent rheogenic HCO3- transport system exists across the peritubular cell membrane. This rheogenic peritubular Na+/HCO3- cotransporter cooperates with a Na+/H+ exchanger in the luminal membrane, thus driving HCO3- reabsorption. Reabsorption of HCO3- and secretion of H+ depend upon the presence of carbonic anhydrase.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1987

Fused cells of frog proximal tubule: I. Basic membrane properties.

Proximal tubular cells of the frog (Rana esculenta) kidney were fused within an isolated tubule portion to giant cells according to the polyethylene-glycol fusion method. Cell membrane potentials (Vm) were measured while cells were superfused with various experimental solutions. Rapid concentration step-changes of different ions allowed to calculate the respective transference numbers (tion). In some experiments the specific cell membrane resistances (Rm) were evaluated by measuring Vm induced by short current pulses injected into the cell with a second electrode. The experiments reveal: i) Fused cells of the proximal tubule exhibit a Vm of -49.5 +/- 1.6 mV (n = 65). ii) Addition of glucose to the perfusate yields a transient depolarization, consistent with a rheogenic Na/glucose cotransport system. iii) In absence of organic substrates the whole cell membrane conductance is made up of K+ and HCO3-. iv) There is a positive relationship between Vm and tK+ and a negative relationship between Vm and tHCO3-. v) HCO3--induced Vm changes are attenuated or abolished when Na+ is replaced with choline+, consistent with a rheogenic Na+/HCO3- cotransport system. vi) Replacement of Na+ by choline+ depolarizes Vm and increases Rm by about 50%; addition of 3 mmol/liter Ba2+ to the Na+-free perfusate increases Rm by about 58% compared to the initial control value. vii) There is no measurable cell membrane Cl- conductance. We conclude that fused cells of proximal tubule exert both luminal and peritubular membrane properties. In absence of organic substrates the cell membrane potential is determined by the HCO3- and K+ transport systems.

Animals↗
PubMed1987

Aldosterone activates Na+/H+ exchange and raises cytoplasmic pH in target cells of the amphibian kidney.

The hypothesis was tested if the mineralocorticoid hormone aldosterone stimulates Na+/H+ exchange in "giant cells" fused from individual target cells of the distal nephron of the frog kidney. By means of microelectrodes, steady-state intracellular pH (pHi) and pHi recovery from an acid load were recorded continuously while the fused cells were exposed to aldosterone. Twenty minutes after addition of the hormone, pHi started to rise and reached a new steady state after about 60 min (delta pHi = 0.28 +/- 0.01). After hormone treatment, pHi recovered significantly faster in response to an intracellular acid load. The diuretic drug amiloride blocked pHi recovery. Experiments in intact tubules showed that aldosterone induces H+ and K+ secretion. Thus, intracellular alkalosis, mediated by Na+/H+ exchange, could serve as a signal that activates pH-sensitive K+ channels of the luminal cell membrane.

Aldosterone↗
PubMed1986

Chloride transport in the diluting segment of the K+ adapted frog kidney: effect of amiloride and acidosis.

The hypothesis was tested whether amiloride and/or an acute acid load influence Cl- transport in the diluting segment of the isolated-perfused kidney of the K+ adapted frog (rana pipiens). Transepithelial resistance (luminal cable analysis) and Cl- net flux (Cl- sensitive microelectrodes) were evaluated at various concentrations of amiloride, at high pCO2 or low HCO-3 in the kidney perfusate. Amiloride or an acute acid load increase transepithelial resistance. The resistance-change at given concentrations of amiloride is markedly enhanced under static head conditions, i.e. at low luminal NaCl concentrations. Amiloride or acidosis (high pCO2) reduce Cl- net reabsorption; combination of both potentiates this inhibitory effect. We conclude: an acute acid load acidifies the cell cytosol. This effect is aggravated dramatically after amiloride-induced inhibition of the luminal Na+/H+ exchanger. The luminal pH-sensitive K+ conductance decreases. This results in a depolarization of the cell membranes. Consequently, the peritubular electrochemical driving force for the exit step of Cl- (from cell to blood) dissipates. Therefore, Cl- net reabsorption is blunted.

Adaptation, Physiological↗
PubMed1986

Fusion of renal epithelial cells: a model for studying cellular mechanisms of ion transport.

The investigation of epithelial ion transport at the cellular level by means of electrophysiological techniques is hampered by the small size of epithelial cells. Moreover, interpretation of experiments is complex due to poorly defined and highly variable paracellular leaks (shunt pathways). In search of a new experimental approach we developed a technique to isolate renal epithelial cells (diameter approximately equal to 10 micron) from diluting segments of the frog kidney and to fuse them to "giant" cells (diameter approximately equal to 100 micron). These cells generate membrane potentials of -54.1 +/- 1.6 mV (mean +/- SEM; n = 40). They are sensitive to the diuretic drugs furosemide and amiloride and to the K+- and Cl- -permeability blockers Ba2+ and anthracene-9-carboxylic acid. The experiments demonstrate membrane potential measurements in cells isolated from renal epithelium and fused to giant cells. The cells retain their specific membrane properties and could serve as a valuable experimental model in epithelial research.

Amiloride↗
PubMed1986

Amiloride reduces potassium conductance in frog kidney via inhibition of Na+-H+ exchange.

The existence of a carrier-mediated Na+-H+ exchange has been described recently in many epithelial and nonepithelial tissues including the diluting segment of the amphibian kidney. In this preparation the Na+-H+ exchanger is dramatically stimulated by so-called K+ adaptation (chronic exposure of animals to high potassium) and completely inhibited by the diuretic drug amiloride. We performed electrophysiological experiments in diluting segments of the isolated perfused frog kidney to investigate whether amiloride affects the conductance properties of this epithelium. Amiloride dramatically increased the transepithelial resistance and the ratio of lumen over peritubular cell membrane resistance. Cell membrane potential changes, induced by luminal K+ concentration steps, were blunted by luminal application of amiloride, by luminal Na+-free perfusates, or by acidification of the kidney perfusion solution. K+ secretory net flux, measured by K+-sensitive microelectrodes, decreased by half in presence of the diuretic. The experiments reveal that amiloride reduces the K+ conductance of the luminal cell membrane of frog diluting segment via inhibition of the luminal Na+-H+ exchanger. This decreases transepithelial K+ net secretion in this nephron segment.

Amiloride↗
PubMed1986

Electrophysiological heterogeneity of proximal convoluted tubules in Amphiuma kidney.

The present study was designed to identify functional differences between dark (early to mid) and white (late) proximal tubule segments in Amphiuma kidney. The potential difference across the peritubular cell membrane (Vb), the luminal cell membrane (Va), and the epithelium (Vte) are not significantly different between dark and white segments. Cellular and luminal cable analysis reveals that the resistance of the cell membranes in parallel is lower in dark (28.6 +/- 3.2 k omega X cm) than in white segments (63.2 +/- 5.0 k omega X cm) in contrast to the transepithelial resistance, which is higher in dark (26.6 +/- 5.5 k omega X cm) than in white (3.5 +/- 0.7 k omega X cm) segments. A step-increase of peritubular potassium (from 2.5 +/- 12.5 mmol/liter) depolarizes Vb more in white (20.1 +/- 1.2 mV) than in dark (7.2 +/- 0.4 mV) segments, whereas addition of bicarbonate to peritubular perfusate hyperpolarizes Vb more in dark (-22.4 +/- 1.6 mV) than in white (-5.9 +/- 0.7 mV) segments. An increase of luminal potassium depolarizes Va more in dark (21.3 +/- 2.0 mV) than in white (9.3 +/- 1.9 mV) segments. Similarly luminal glucose depolarizes Va more in dark (10.7 +/- 1.2 mV) than in white segments (3.2 +/- 1.4 mV). Partial peritubular replacement of NaCl and reduction of peritubular chloride polarize Vte more in white (9.6 +/- 1.0 and 28.9 +/- 2.9 mV) than in dark segments (7.0 +/- 0.5 and 15.5 +/- 1.9 mV). In conclusion, compared with white segments, dark segments have lower cell membrane and higher shunt resistances, lower potassium and higher bicarbonate conductances of the peritubular cell membrane, and a higher capacity to reabsorb glucose. Paracellular shunt chloride conductance is relatively high in both segments.

Animals↗