Functional heterogeneity in the hamster medullary thick ascending limb of Henle's loop

Cellular heterogeneity was examined in the hamster medullary thick ascending limb (MAL) perfused in vitro by electrophysiological measurements with an intracellular microelectrode. Random measurements of fractional resistance of basolateral membrane (Rf _B) revealed two cell populations, high basolateral conductance (HBC) cells havingRf _B of 0.05±0.01 (n=24) and low basolateral conductance (LBC) cells havingRf _B of 0.80±0.03 (n=32). Basolateral membrane potentials (V _B) were not different between HBC cells and LBC cells (–72.6±1.2,n=43 vs. –70.0±1.2,n=35). Addition of 2 mmol/l Ba²⁺ to the bath depolarized the basolateral membrane in the HBC cells from –70.4±3.2 to –20.9±5.9 mV (n=8) but not in the LBC cells (from –74.4±1.9 to –72.0±2.1 mV). Increasing K⁺ or decreasing Cl^– in the bathing solution caused marked positive deflection ofV _B in the HBC cells but little or no change inV _B in the LBC cells. Elimination of Cl^– from the lumen or addition of furosemide to the lumen enhanced the potential response of the HBC cells to basolateral application of Ba²⁺. Accordingly, with Ba²⁺ present in the bath, the potential response of the HBC cells to a decrease in bath Cl^– concentration was enhanced. These observations suggest that a K⁺ conductance exists in the basolateral membrane of HBC cells in paralled with a Cl^– conductance. The basolateral cell membrane of LBC cells also contains a Cl^– conductance. In these cells, but not in HBC cells, the potential response to decreasing bath Cl^– concentration increased when bath pH was decreased from 7.4 to 6.0 Apparent K⁺ transference numbers of the luminal membrane were higher in LBC cells (0.74±0.05,n=7) than in HBC cells (0.20±0.02,n=5). From these data, we conclude: (1) there are two distinct cell types in the hamster medullary thick ascending limb; (2) there is a low Cl^– conductance in basolateral membrane of LBC cells which is stimulated by low pH.     

The luminal K+ channel of the thick ascending limb of Henle's loop

In vitro perfused rat thick ascending limbs of Henle's loop (TAL) were used (n=260) to analyse the conductance properties of the luminal membrane applying the patch-clamp technique. Medullary (mTAL) and cortical (cTAL) tubule segments were dissected and perfused in vitro. The free end of the tubule was held and immobilized at one edge by a holding pipette kept under continuous suction. A micropositioner was used to insert a patch pipette into the lumen, and a gigaohm seal with the luminal membrane was achieved in 455 instances out of considerably more trials. In approximately 20% of all gigaohm seals recordings of single ionic channels were obtained. We have identified only one single type of K⁺ channel in these cell-attached and cell-excised recordings. In the cell-attached configuration with KCl or NaCl in the pipette, the channel had a conductance of 60±6 pS (n=24) and 31±7 pS (n=4) respectively. In cell-free patches with KCl either in the patch pipette or in the bath and with a Ringer-type solution (NaCl) on the opposite side the conductance was 72±4 pS (n=37) at a clamp voltage of 0 mV. The permeability was 0.33±0.02 · 10^±12 cm³/s. The selectivity sequence für this channel was: K⁺=Rb⁺=NH ₄ ⁺ =Cs⁺>Li⁺Na⁺=0; the conductance sequence was K⁺Li⁺Rb⁺=Cs⁺= NH ₄ ⁺ =Na⁺=0. In excised patches Rb⁺, Cs⁺ and NH ₄ ⁺ when present in the bath at 145 mmol/l all inhibited K⁺ currents out of the pipette. The channel kinetics were described by one open (9.5±1.5 ms, n=18) and by two closed (1.4±0.1 and 14±2 ms) time constants. The open probability of this channel was increased by depolarization. The channel open probability was reduced voltage dependently by Ba²⁺ (half maximal inhibition at 0 mV: 0.07 mmol/l) from the cytosolic side. Verapamil, diltiazem, quinine and quinidine inhibited at approximately 1 mol/l ±0.1 mmol/l from either side. Similarly, the amino cations lidocaine, tetraethylammonium and choline inhibited at 10–100 mmol/l. The channel was downregulated in its open probability by cytosolic Ca²⁺ activities > 10^±7 mol/l and by adenosine triphosphate 10^±4 mol/l. The open probability was downregulated by decreasing cytosolic pH (2-fold by a decrease in pH by 0.2 units). The described channel differs in several properties from the K⁺ channels of other epithelia and of renal cells and TAL cells in culture. It appears to be responsible for K⁺ recycling in the TAL segment.Preliminary reports of the present study have been given at the following conferences: Tagung der Deutschen Physiologischen Gesellschaft, Würzburg, October 1988; Membranforum, Frankfurt, April 1989; 3rd Int. Conf. Diur., Mexico City, April 1989; 3rd Nephrology Forefront Symposium, Arrola, July, 1989; IUPS meeting, Helsinki, July 1989. This study has been supported by Deutsche Forschungsgemeinschaft Grant No. Gr 480/9     

pH dependence of K+ conductances of rat cortical collecting duct principal cells

The K⁺ channels of the principal cells of rat cortical collecting duct (CCD) are pH sensitive in excised membranes. K⁺ secretion is decreased with increased H⁺ secretion during acidosis. We examined whether the pH sensitivity of these K⁺ channels is present also in the intact cell and thus could explain the coupling between K⁺ and H⁺ secretion. Membrane voltages (V _m), whole-cell conductances (g _c), and single-channel currents of K⁺ channels were recorded from freshly isolated CCD cells or isolated CCD segments with the patch-clamp method. Intracellular pH (pH_i) was measured using the pH-sensitive fluorescent dye 2-7-bis(carboxyethyl)-5-6-carboxyfluorescein (BCECF). Acetate (20 mmol/l) had no effect on V _m, g _c, or the activity of the K⁺ channels in these cells. Acetate, however, acidified pH_i slightly by 0.17±0.04 pH units (n=19). V _m depolarized by 12±3 mV (n=26) and by 23±2 mV (n=66) and g _c decreased by 26±5% (n=13) and by 55±5% (n=12) with 3–5 or 8–10% CO₂, respectively. The same CO₂ concentrations decreased pH_i by 0.49±0.07 (n=15) and 0.73±0.11 pH units (n=12), respectively. Open probability (P _o) of all four K⁺ channels in the intact rat CCD cells was reversibly inhibited by 8–10% CO₂. pH_i increased with the addition of 20 mmol/l NH₄ ⁺/NH₃ by a maximum of 0.64±0.08 pH units (n=33) and acidified transiently by 0.37±0.05 pH units (n=33) upon NH₄ ⁺/NH₃ removal. In the presence of NH₄ ⁺/NH₃ V _m depolarized by 16±2 mV (n=66) and g _c decreased by 26±7% (n=16). The activity of all four K⁺ channels was also strongly inhibited in the presence of NH₄ ⁺/NH₃. The effect of NH₄ ⁺/NH₃ on V _m and g _c was markedly increased when the pH of the NH₄ ⁺/NH₃-containing solution was set to 8.5 or 9.2. From these data we conclude that cellular acidification in rat CCD principal cells down-regulates K⁺ conductances, thus reduces K⁺ secretion by direct inhibition of K⁺ channel activity. This pH dependence is present in all four K⁺ channels of the rat CCD. The inhibition of K⁺ channels by NH₄ ⁺/NH₃ is independent of changes in pH_i and rather involves an effect of NH₃.     

Torasemide inhibits NaCl reabsorption in the thick ascending limb of the loop of Henle

Torasemide (1-isopropyl-(4-(3-methylphenylamino)pyrid-3-yl)urea) is a new diuretic. The present study examines the effects of this substance in the isolated perfused thick ascending limb (TAL) of mouse and rabbit kidney. In cortical TAL segments of the rabbit, torasemide added to the lumen perfusate led to a fall in equivalent short circuit current (= transepithelial voltage divided by transepithelial resistance, which corresponds to the rate of chloride reabsorption) with a half maximal inhibition concentration of 3 · 10^–7 mol/l. This effect was accompanied by a hyperpolarization of the luminal and basolateral membrane from –78 to –81 mV and from –72 to –81 mV, respectively. A similar hyperpolarization of both membrane voltages was also observed in medullary TAL segments of the mouse. Torasemide, added to the basolateral perfusate of cortical TAL segments of the rabbit, also inhibited the equivalent short circuit current. However, 3 · 10^–5 mol/l were necessary for a half maximal inhibition. The fall in the equivalent short circuit current was accompanied by a significant increase in transepithelial resistance from 34 to 38 cm², by an increase in the fractional resistance of the basolateral membrane, and by a hyperpolarization mainly of the basolateral membrane. Again, similar results were obtained in the medullary TAL segment of the mouse.The strong inhibitory effect of torasemide from the lumen side can be explained by an interference with the Na⁺ 2Cl^–K⁺ carrier in the luminal membrane. In fact, torasemide apparently is structurally related to furosemide. The weaker effect of torasemide from the peritubular side can, at least in part, be explained as an interference with chloride channels present in the basolateral membrane. Torasemide is also structurally related to chloride channel blockers such as diphenylamine-2-carboxylate.Supported by Deutsche Forschungsgemeinschaft DFG Gr 480/6-4 and 6-5     

Piretanide-dextran and piretanide-polyethylene glycol interact with high affinity with the Na+ 2 Cl− K+ cotransporter in the thick ascending limb of the loop of Henle

Piretanide blocks the Na⁺ 2Cl^– K⁺ cotransporter protein in the thick ascending limb (TAL) of the loop of Henle reversibly. When tested from the luminal side in isolated perfused cTAL segments it leads to a half maximal inhibition (IC₅₀) of the equivalent short circuit current (I_sc) at a concentration of 10^–6 mol/l. From the basolateral side it has no effect on I_sc up to 10^–4 mol/l. The present study was designed to search for high affinity blockers of the Na⁺ 2Cl^– K⁺ cotransporter with large molecular weight in an attempt to use these macromolecules for antibody-labelling or affinity separation of this transport-protein. Amino-ethyl-dextran or amino-ethyl-polyethylene glycol (M.W. 5kd) were coupled to isothiocyanato-piretanide (ISO-PIR) at room temperature in DMSO. The resulting compounds dextran-sulfonylurea-piretanide (PIR-DEX) and polyethylene glycol-sulfonylurea-piretanide (PIR-PEG) (M.W. 5.38kd) were purified and tested in isolated perfused cTAL segments. IC₅₀ values for ISO-PIR, PIR-DEX and PIR-PEG were estimated from dose response curves after their addition to the lumen or bath perfusate, respectively. ISO-PIR, PIR-DEX and PIR-PEG acted from the lumen side at 3·10^–6, 6·10^–6 and 2·10^–6 mol/l. The inhibitory effect was easily reversible. From the basolateral side no effect for any compound was seen at up to 10^–4 mol/l. In clearance experiments PIR-DEX was given to female Wistar rats as an i.v. bolus (25 mol/kg) and the diuretic urine was collected. After dialysis (exclusion limit 2.5kd) the dialysed urine and the dialysate were tested in isolated perfused cTAL segments. The dialysates had no effect on I_sc, but the dialysed urine inhibited I_sc by 35% from the luminal side. The present data show: High molecular derivatives of piretanide with dextran or polyethylene glycol moieties block the Na⁺ 2Cl^– K⁺ cotransporter in cTAL segments at roughly the same low concentration as piretanide itself. Our data exclude a metabolism of these piretanide compounds in the kidney. Since these macromolecular probes can probably not enter the cell their inhibitory effect indicates that the binding site for piretanide diuretics on the Na⁺ 2Cl^– K⁺ cotransporter is exposed on the surface of the luminal cell membrane.This study was supported by Deutsche Forschungsgemeinschaft Gr 480/9     

Active NaCl transport in the cortical thick ascending limb of Henle's loop of the mouse does not require the presence of bicarbonate

The aim of the present study was to investigate whether bicarbonate buffer (CO₂ + HCO ₃ ^– ) is required to sustain maximal NaCl transport in the cortical thick ascending limb of Henle's loop (cTAL) of the mouse. Transepithelial Na⁺ and Cl^– net fluxes (J _Na, J _Cl, pmol min^–1 mm^–1), measured by electron microprobe analysis, were similar irrespective of the presence or absence of CO₂ + HCO ₃ ^– in luminal and bathing solutions J _NaCl with CO₂ + HCO ₃ ^– =203±25 pmol min^–1 mm^–1; J NaCl without CO₂ + HCO ₃ ^– =213±13 pmol min^–1 mm^–1, n=14). Furthermore the transepithelial potential difference, V _te, the transepithelial resistance, R _te, and the basolateral membrane potential, V _bl, were unaffected by CO₂ + HCO ₃ ^– . In the absence of CO₂ + HCO ₃ ^– , V _te was +17.0±1.7 mV(n=9) (lumen positive), R _te was 28±2 cm² (n=9) and V _bl was –76±4 mV (n=6). In the presence of CO₂ + HCO ₃ ^– , V _te, R _te and V _bl were +15.9±1.5 mV, 29±1 cm² and –73±5 mV, respectively. 4-Acetamido-4-isothiocyanatostilbene-2,2-disulphonic acid (SITS; 0.1 mmol l^–1) and amiloride (1 mmol l^–1) added to the (CO₂ + HCO ₃ ^– )-containing lumen perfusate were without effect on V _te and R _te. Finally, the effect of furosemide (0.1 mmol l^–1) on V _te and V _bl in the presence of CO₂ + HCO ₃ ^– was investigated. Furosemide reversibly decreased V _te from +13.7±1.1 mV to +1.7±0.7 mV (n=6) and hyperpolarized V_bl from –70±1 to –89±3 mV (n=5), suggesting passive distribution of Cl^– across the basolateral membrane. In conclusion, these data suggest that active NaCl transport in the cTAL of the mouse does not require the presence of CO₂ + HCO ₃ ^– .     

Potassium activity in cells of isolated perfused cortical thick ascending limbs of rabbit kidney

The Na⁺2Cl^–K⁺ cotransporter in the apical membrane of the cortical thick ascending limb of the Henle's loop (cTAL) of rabbit nephron utilizes the electrochemical gradient for Na⁺ to transport K⁺ and Cl^– against an unfavorable electrochemical gradient from lumen to cell interior. In the present study attempts are made to measure intracellular K⁺ activity ( ) under control conditions and after inhibition of the cotransport system by furosemide (50·10^–6 mol·l^–1). 70 cTAL segments of 55 rabbits were perfused in vitro. Conventional Ling-Gerard and K⁺-selective microelectrodes were used to measure the PD across the basolateral membrane (PD_bl) as well as the PD sensed by the single barrelled K⁺-selective electrode ( ). PD_bl was –64±1 (n=65) mV and +15±1 (n=32) mV under control conditions. The positive value, significantly different from zero, indicates that is higher than predicted for passive distribution. The estimate for obtained from PD_bl and was 113±8 mmol·l^–1. Furosemide lead to the previously reported hyperpolarization of PD_bl by 17±4 (n=13) mV and to a reduction of from 15±1 to 5±1 (n=20) mV. The , obtained from this set of data, was 117±9 mmol·l^–1, and was not different from the control value. The present data indicate that is significantly above Nernst equilibrium under control conditions. The source for this above equilibrium accumulation of K⁺ stems from the carrier mediated uptake of Na⁺2Cl^– and K⁺. Consequently, the electrochemical gradient for K⁺ is rapidly reduced when the carrier is blocked by furosemide. The electrochemical gradient for K⁺, under control conditions, energizes the back leak of K⁺ from cell to lumen. This K⁺ flux is one component responsible for the lumen positive transepithelial PD.Parts of this study have been presented at the 58th Tagung Deutsche Physiologische und Deutsche Pharmakologische Gesellschaft, Mainz 1983; 67th Federation Meeting, Chicago 1983. This study was supported by Deutsche Forschungsgemeinschaft Gr. 480/5-7     

Regulation of inwardly rectifying K+ channels by intracellular pH in opossum kidney cells

The effects of intracellular pH on an inwardly rectifying K⁺ channel (K_in channel) in opossum kidney (OK) cells were examined using the patch-clamp technique. Experiments with inside-out patches were first carried out in Mg²⁺-and adenosine triphosphate (ATP)-free conditions, where Mg²⁺-induced inactivation and ATP-induced reactivation of K_in channels were suppressed. When the bath (cytoplasmic side) pH was decreased from 7.3 to either 6.8 or 6.3, K_in channels were markedly inhibited. The effect of acid pH was not fully reversible. When the bath pH was increased from 7.3 to 7.8, 8.3 or 8.8, the channels were activated reversibly. The channel activity exhibited a sigmoidal pH dependence with a maximum sensitivity at pH 7.5. Inside-out experiments were also carried out with a solution containing 3 mM Mg-ATP and a similar pH sensitivity was observed. However, in contrast with the results obtained in the absence of Mg²⁺ and ATP, the effect of acid pH was fully reversible. Experiments with cell-attached patches demonstrated that changes in intracellular pH, which were induced by changing extracellular pH in the presence of an H⁺ ionophore, could influence the channel activity reversibly. It is concluded that the activity of K_in channels can be controlled by the intracellular pH under physiological conditions.     

Analogues of torasemide — structure function relationships —experiments in the thick ascending limb of the loop of Henle of rabbit nephron

The aim of the present study was to examine compounds related to torasemide with respect to their ability to block the equivalent short circuit current, corresponding to the rate of chloride reabsorption, in isolated in vitro perfused cortical thick ascending limbs of Henle of the rabbit. The torasemide molecule was modified with respect to the anionic sulfonylurea group, and the secondary amine linked to the pyridine ring. Our results indicate that only few of the tested 48 torasemide-related compounds were able to inhibit from both epithelial sides like torasemide. Only few of the tested compounds were equally effective as torasemide from the lumen side. Some analogues were acting only from the luminal side and some only from the peritubular side. The correlations between structure and potency of inhibition from the luminal side allow the following conclusions: a) The secondary amine moiety linked to the pyridine ring (toluidine in case of torasemide) can be replaced by a cycloalkylamine or, with some loss of inhibitory potency, by alkylamines. The inhibitory potency is increased with the number of C-atoms in the cycloalkylamine substituted compounds (optimum C₇ to C₈), and is also depending on the length of the alkylamines (optimum C₄). b) The secondary amine seems to be required since nitrogen cannot be replaced by –S- or –SO₂-. c) The sulfonylurea group cannot be substituted by other anionic groups such as –SO ₃ ^– or –COO^–. d) If the pyridine ring is replaced by a NO₂-substituted phenyl ring, the inhibitory potency from the luminal side is lost. However, these compounds act still (with some loss of potency) from the peritubular side. The data indicate that several of the conclusions drawn from our previous systematic surveys of chloride channel blockers and loop diuretics of the furosemide type, i.e. blockers of the Na⁺2Cl^–K⁺ carrier, hold also true for compounds related to torasemide. In addition, the pyridine ring is responsible for some specific structure activity correlations.Supported by Deutsche Forschungsgemeinschaft Gr 480/6     

cAMP increases the basolateral Cl−-conductance in the isolated perfused medullary thick ascending limb of Henle's loop of the mouse

The effect of cAMP on transepithelial and transmembrane potential differences and resistances was examined in isolated in vitro perfused mouse medullary thick ascending limbs of Henle's loop (mTAL). The effects of furosemide and barium were tested. Stimulation of NaCl transport by ADH 10^–9+dbcAMP 4·10^–4+forskolin 10^–6 mol·l^–1 (paired experiments) resulted in: a) an increase in transepithelial potential difference, referenced to the grounded bath, from +6.7±0.3 mV to +12.0±0.4 mV (n=47); b) a decrease in transepithelial resistance from 25±1 cm² to 20±1 cm² (n=47); c) a depolarization of the basolateral membrane by 12 mV and of the apical membrane by 7 mV (n=36); d) a decrease in the fractional resistance of the basolateral membrane from 0.27±0.005 to 0.15±0.06 (n=12). Furosemide (10^–4 mol·l^–1) abolished the active transepithelial transport potential and hyperpolarized the basolateral membrane potential to values which were similar in both control and cAMP treated mTAL segments. Barium increased the transepithelial resistance and depolarizedPD _bl to similar values in both functional states. An increase in the fractional conductance of the basolateral membrane was also seen, if, prior to the cAMP treatment, the luminal Na⁺2Cl^–K⁺ contransport was inhibited by furosemide. Thus, we propose that stimulation of active NaCl reabsorption in the mTAL segment of the mouse by ADH, mediated via cAMP, increases primarily the basolateral chloride conductance.Supported by Deutsche Forschungsgemeinschaft Gr 480/6-2Parts of this study have been presented at the 59th Meeting of the German Physiological Society in Dortmund 1984 and at the 69th FASEB Meeting in Anaheim 1985     

Dual effect of N-ethylmaleimide on Cl− transport across the thin ascending limb of Henle's loop

Effects of SH reagents on Cl^– transport were studied in the isolated hamster thin ascending limb of Henle's loop (TAL) perfused in vitro. Parachloromercuribenzene sulfonate (PCMBS) at 10^–4 M in the bath decreased the relative permeability for Cl^–/Na⁺ (P _Cl/P _Na), as determined by the transmural diffusion voltage (V _T) generated under a NaCl concentration gradient, from 2.71±0.16 to 1.11±0.09 (P<0.001). The effect of PCMBS was prevented by the pretreatment with 10^–3 M dithiothreitol (DTT). N-Ethylmaleimide (NEM) at 10^–3 M in the bath exhibited a dual action on Cl^– permeability of the TAL: It inhibited the Cl^– permeability in fresh preparations, whereas it stimulated the Cl^– permeability in the preparations pretreated with SH reagents including NEM, maleimide and PCMBS. The inhibitory effect was irreversible but the stimulatory effect was reversible. Both responses were prevented by DTT. Since dextranmaleimide did not show any inhibitory effect onP _Cl/P _Na, the SH site responsible for the inhibition may be located inside of the cell. The stimulatory effect of NEM onP _Cl/P _Na was markedly reduced when bath pH was reduced to 5.8. On the other hand, when the bathing fluid was made nominally Ca²⁺ free, the stimulatory effect of NEM was unaffected, although the basal level ofP _Cl/P _Na was reduced. These observations suggest that the conductive Cl^– pathway in the TAL is either stimulated or inhibited by modifying two distinct SH sites. The site of modulation by proton binding may exist distally to these SH sites. The regulatory mechanism involving Ca²⁺ may be independent of the SH regulatory sites.     

Mechanisms of calcium transport across the basolateral membrane of the rabbit cortical thick ascending limb of Henle's loop

Although net Ca²⁺ absorption takes place in the thick ascending limb of Henle's loop, detailed mechanisms are unknown. Because it has been reported that the Ca²⁺ entry step across the luminal membrane is mediated by Ca²⁺ channels inserted by stimulation with parathyroid hormone, we studied the mechanism of Ca²⁺ transport across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) perfused in vitro by using microscopic fluorometry of cytosolic Ca²⁺ ([Ca²⁺]_i) with fura-2. The resting [Ca²⁺]_i in this segment was 49.8±4.5 nmol/l. Neither Na⁺ removal from the bathing solution nor addition of ouabain (0.1 mmol/l) to the bath increased [Ca²⁺]_i, indicating that a Na⁺/Ca²⁺ exchanger in the basolateral membrane may not contribute in any major way to [Ca²⁺]_i of CTAL. To confirm our technical accuracy, similar protocols were conducted in the connecting tubule, where the existence of a Na⁺/Ca²⁺ exchanger has been reported. In this segment, Na⁺ removal from the bath increased cell Ca²⁺ from 148.6 ±6.4 nmol/l to 647.6±132.0 nmol/l, confirming the documented fact. [Ca²⁺]_i in the CTAL was markedly increased when 1 mmol/l NaCN was added to the bath in the absence of glucose. Calmodulin inhibitors (trifluoperazine or W-7) increased [Ca²⁺]_i. When the bath pH was made alkaline, [Ca²⁺]_i was also increased. This response was abolished when Ca²⁺ was eliminated from the bath, indicating that the Ca²⁺ entry across the basolateral membrane is dependent on bath pH. Increase in [Ca²⁺]_i induced by an alkaline bath was inhibited by increased the bath K⁺ from 5 nmol/l to 50 mmol/l, suggesting that the Ca²⁺ entry system is voltage-dependent. However, the pH-dependent [Ca²⁺]_i increase was unaffected by 0.1–10 mol/l nicardipine in the bath. We conclude that Ca²⁺ transport across the basolateral membrane of CTAL is mediated by a pump-and-leak system of Ca²⁺ rather than a Na⁺/Ca²⁺ exchanger secondarily linked to a Na⁺, K⁺ pump.     

Involvement of Na and Cl in ouabain-induced cell swelling in thick ascending limb of rat kidney

Changes in the volume of isolated segments of rat medullary thick ascending limb (MAL) were studied by a photographic technique, after tubule incubation in isotonic solutions in the absence or presence of ouabain and/or K. When segments were incubated at 30°C in NaCl solution, their volume increased by 75% after removal of external K, and by 170% after removal of external K plus addition of 1 mmol/l ouabain. At steady state, tubular volume was a function of the external K concentration. Resting volume was obtained with external K concentrations higher than 0.1 and 1.0 mmol/l in the absence and presence of ouabain respectively. When MAL samples were incubated in isotonic K-free Na₂SO₄ or K-free choline Cl solution, their volume per unit of length was similar to that determined in NaCl medium, but there was no swelling after the addition of ouabain. The ouabain-induced swelling was shown to depend on both the Na and Cl concentrations in the incubate (apparentK _m of 87 and 80 mmol/l for Na and Cl respectively). Swollen tubules recovered their resting volume when ouabain, Na or Cl was removed from the incubation medium. Recovery of resting volume was also observed after addition of K into the incubation medium. These observations indicate that rat MAL cell volume is the result of coupled passive net fluxes of Na and Cl, which depend on the respective electrochemical gradients for Na or Cl across the cell membranes and the Na-pump activity which continuously extrudes Na.     

Effect of high NaCl intake on Na+ and K+ transport in the rabbit distal convoluted tubule

Morphological studies have demonstrated that a chronic increase in distal Na⁺ delivery causes hypertrophy of the distal convoluted tubule (DCT). To examine whether high NaCl-intake also causes functional changes in the well defined DCT, we measured transmural voltage (V _T), lumen-to-bath Na⁺ flux (J _Na(LB)), and net K⁺ secretion (J _K(net)) in DCTs obtained from control rabbits and those on high NaCl-intake diets. The lumen negativeV _T was significantly greater in the high NaCl group than in the control group. The net K⁺ secretion (pmol mm^–1 min^–1) was greater in the high NaCl-intake group (54.1±13.0 vs 14.7±5.6). The K⁺ permeabïlities in both luminal and basolateral DCT membranes, as assessed by the K⁺-induced transepithelial voltage deflection inhibitable with Ba²⁺, were increased in the experimental group. The lumen-to-bath²²Na flux (pmol mm^–1 min^–1) was also greater in the experimental group (726±119 vs 396±65). TheV _T component inhibitable with amiloride was also elevated in the high NaCl-intake group. Furthermore, Na⁺–K⁺-ATPase activity of the DCT was higher in the experimental than in the control group. We conclude that high NaCl intake increases both Na⁺ reabsorption and K⁺ secretion by the DCT. This phenomenon is associated with an increased Na⁺–K⁺-ATPase activity along with increased Na⁺ and K⁺ permeabilities of the luminal membrane, and an increase in the K⁺ permeability of the basolateral membrane. Cellular mechanisms underlying these functional changes remain to be established.     

Relationship between cell volume and cation content in thick ascending limb of rat kidney

We examined the relationship between the cell volume and cation concentration ([Na_i] and [K_i]) of isolated segments of rat medullary thick ascending limb (MAL) after incubation at 30°C in various isotonic solutions. When the tubules were incubated in a normal NaCl solution containing 5 mmol/l K⁺, addition of 1 mmol/l of ouabain increased [Na_i] and decreased [K_i] but did not change the total ([Na_i]+[K_i]) concentration (about 90 mEq/l) or tubular volume. After incubation in various K⁺-free solutions, the tubules were almost fully K⁺-depleted; their volume per unit of length was similar in the three solutions, although the choline Cl-treated tubules had a very low sodium content compared to the NaCl-and Na₂SO₄-treated tubules (8 vs. 97 and 95 mEq/l respectively). Ouabain altered neither volume nor [Na_i] of tubules incubated in choline Cl or Na₂SO₄ solution. Transfer of tubules from K⁺-free Na₂SO₄ or K⁺-free choline Cl solution into K⁺-free NaCl solution resulted in an increase in [Na_i] (by 29 and 97 mEq/l respectively) without much increase in tubular volume. A marked swelling of the tubules was only observed when the K⁺-free NaCl solution contained also ouabain. Under this condition, [Na_i] was comparable to the Na⁺ concentration of the incubation medium. After washing and incubation in a normal NaCl solution containing K⁺, the swollen tubules recovered their initial volume and restored Na⁺ and K⁺ concentration gradients across the cell membranes. The ([Na_i]+[K_i]) concentration centration measured in the tubules preincubated in choline Cl solution was always smaller than that of the tubules preincubated either in NaCl or Na₂SO₄ solutions, an observation suggesting that choline ions enter rat MAL cells. Barium (3 mmol/l) prevented tubular swelling. This inhibition corresponded to a smaller increase in [Na_i] than that observed in control tubules. Furosemide or bumetanide (even at 0.1 mmol/l) did not alter the increases in tubular volume and in Na⁺ content induced by ouabain. The data provide additional evidence that the isoosmotic swelling of MAL cells requires an almost full inhibition of Na⁺-pump activity and involves coupled net fluxes of Na⁺ and Cl^– ions.     

Effect of extracellular volume expansion on renal cortical and medullary Na+−K+-ATPase

Summary The role of renal Na⁺–K⁺-ATPase in the acute changes in sodium reabsorption caused by isotonic volume expansion was evaluatedin vivo andin vitro in the rat and the dog. Duringin vivo volume expansion with isotonic saline in the rat, renal medullary Na⁺–K⁺-ATPase specific activity increased, while the simultaneously determined cortical Na⁺–K⁺-ATPase specific activity and kinetics remained unchanged. Furthermore, experimentsin vitro failed to demonstrate a circulating inhibitor of renal Na⁺–K⁺-ATPase both in plasma dialysates from volume-expanded rats and in plasma dialysates concentrated 20-fold by ultrafiltration from volume-expanded dogs. These results suggest that the decreased proximal tubular reabsorption of sodium during volume expansion is not mediated by inhibition of renal cortical Na⁺–K⁺-ATPase. The acute increment in medullary Na⁺–K⁺-ATPase observed could represent an adaptive response to increased sodium reabsorption by the loops of Henle, and raises the possibility that this enzyme may participate in relatively rapid adjustments in the transport of sodium by the renal tubule.     

pH regulation in HT29 colon carcinoma cells

The pH regulation in HT₂₉ colon carcinoma cells has been investigated using the pH-sensitive fluorescent indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Under control conditions, intracellular pH (pH_i) was 7.21±0.07 (n=22) in HCO ₃ ^– -containing and 7.21±0.09 (n=12) in HCO ₃ ^– -free solution. HOE-694 (10 mol/l), a potent inhibitor of the Na⁺/H⁺ exchanger, did not affect control pH_i. As a means to acidify cells we used the NH ₄ ⁺ /NH₃ (20 mmol/l) prepulse technique. The mean peak acidification was 0.37±0.07 pH units (n=6). In HCC ₃ ^– -free solutions recovery from acid load was completely blocked by HOE-694 (1 mol/l), whereas in HCO3 ₃ ^– -containing solutions a combination of HOE-694 and 4,4-diisothiocyanatostilbene-2, 2-disulphonate (DIDS, 0.5 mmol/l) was necessary to show the same effect. Recovery from acid load was Na⁺-dependent in HCO ₃ ^– -containing and HCO ₃ ^– -free solutions. Removal of external Cl^– caused a rapid, DIDS-blockable alkalinization of 0.33±0.03 pH units (n=15) and of 0.20±0.006 pH units (n=5), when external Na⁺ was removed together with Cl^–. This alkalinization was faster in HCO ₃ ^– -containing than in HCO ₃ ^– -free solutions. The present observations demonstrate three distinct mechanisms of pH regulation in HT₂₉ cells: (a) a Na⁺/H⁺ exchanger, (b) a HCO ₃ ^– /Cl^– exchanger and (c) a Na⁺-dependent HCC ₃ ^– transporter, probably the Na⁺-HCO ₃ ^– /Cl^– antiporter. Under HCO ₃ ^– — free conditions the Na⁺/H⁺ exchanger fully accounts for recovery from acid load, whereas in HCO ₃ ^– -containing solutions this is accomplished by the Na⁺/H⁺ exchanger and a Na⁺-dependent mechanism, which imports HCO ₃ ^– . Recovery from alkaline load is caused by the HCO ₃ ^– /Cl^– exchanger.This study was supported by DFG Gr 480/10     

Copper toxicity in cultured human skeletal muscle cells: the involvement of Na+/K+-ATPase and the Na+/Ca2+-exchanger

Copper (Cu²⁺) intoxication has been shown to induce pathological changes in various tissues. The mechanism underlying Cu²⁺ toxicity is still unclear. It has been suggested that the Na⁺/K⁺-ATPase and/or a change of the membrane permeability may be involved. In this study we examined the effects of Cu²⁺ on the Na⁺ and Ca²⁺ homeostasis of cultured human skeletal muscle cells using the ion-selective fluorescent probes Na⁺-binding benzofuran isophtalate (SBFI) and Fura-2, respectively. In addition, we measured the effect of Cu²⁺ on the Na⁺/K⁺-ATPase activity. Cu²⁺ and ouabain increase the cytoplasmic free Na⁺ concentration ([Na⁺]_i). Subsequent addition of Cu²⁺ after ouabain does not affect the rate of [Na⁺]_i increase. Cu²⁺ inhibits the Na⁺/K⁺-ATPase activity with an IC₅₀ of 51 M. The cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) remains unaffected for more than 10 min after the administration of Cu²⁺. Thereafter, [Ca²⁺]_i increases as a result of the Na⁺/Ca²⁺-exchanger operating in the reversed mode. The effects of Cu²⁺ on the Na⁺ homeostasis are reversed by the reducing and chelating agent dithiothreitol and the heavy metal chelator N,N,N,N,-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). In conclusion, SBFI is a good tool to examine Na⁺ homeostasis in cultured human skeletal muscle cells. Under the experimental conditions used, Cu²⁺ does not modify the general membrane permeability, but inhibits the Na⁺/K⁺-pump leading to an increase of [Na⁺]_i. As a consequence the operation mode of the Na⁺/Ca²⁺-exchanger reverses and [Ca²⁺]_i rises.The authors thank staff and coworkers of the Department of Neurology of the University Hospital Nijmegen, Nijmegen for their kind cooperation in obtaining muscle biopsies. Mr. Arie Oosterhof is gratefully acknowledged for culturing of the human muscle cells. The Prinses Beatrix Fonds and the Dutch-Chinese scientific exchange program contributed financial support for this study.     

The localization of the Na+−K+-ATPase in the cells of rat kidney cortex

Summary Plasma membrane fractions of rat kidney cortex were subdivided by centrifugation on a continuous and a discontinuous sucrose gradient and by carrier free continuous electrophoresis. In the different fractions the activity of alkaline phosphatase and aminopeptidase, enzymes which are present in the brushborder membrane, as well as Mg⁺⁺-ATPase, Na⁺–K⁺-ATPase, 5-nucleotidase, acid phosphatase and glucose-6-phosphatase were determined.The distribution of alkaline phosphatase, aminopeptidase and 5-nucleotidase is identical, indicating the localization of these enzymes in the brushborder membrane. Na⁺–K⁺-ATPase does not show an identical distribution with any of the enzymes studied.Using carrier free continuous electrophoresis fractions can be obtained which are enriched in alkaline phosphatase by a factor of 15 when compared to the cortex homogenate, whereas the specific activity of Na⁺–K⁺-ATPase is reduced to one third of the starting material. On the other hand fractions can be isolated in which the specific activity of Na⁺–K⁺-ATPase is 16 times higher than in the homogenate. No enrichment of alkaline phosphatase occurs in these fractions.It is therefore concluded that the Na⁺–K⁺-ATPase is not present in the brushborder membrane nor in the lysosomes or endoplasmatic reticulum. The most probable localization of the Na⁺–K⁺-ATPase are the basal infoldings of the plasma membranes of the cells.A preliminary report has been published by Kinneet al. [28, 29].Major part of this work was done by J. E. Schmitz for his degree of M. D.     

Anion and cation channels in the basolateral membrane of rabbit parietal cells

Ion channels in the basolateral membrane of rabbit parietal cells in isolated gastric glands were studied by the patch clamp technique. Whole-cell current-clamp recordings showed that the membrane potential (E _m ) changed systematically as a function of the chloride concentrations of the basolateral bathing solution ([Cl^–]₀), and of the pipette (intracellular) solution. The relationship betweenE _m and [Cl^–]₀ was not affected by additions of histamine, dibutyryl-cAMP, 4-acetoamido-4-isothiocyanostilbene-2,2-disulfonic acid and diphenylamine-2-carboxylate. The whole-cell Cl^– conductance was insensitive to voltage. In cell-attached and cell-free patch membranes, however, single Cl^– channel opening events could not be observed. The value ofE _m depended little on the basolateral K⁺ concentration, but inward-rectifier K⁺ currents were observed in the whole-cell configuration, activated by hyperpolarizing pulses and inhibited by extracellular Ba²⁺. In cell-attached and cell-free patches, openings of single inward-rectifier K⁺ channels and non-selective cation channels were infrequently recorded. Neither cAMP nor Ca²⁺ activated these cation channels. The single K⁺ channel conductance was about 230 pS under the symmetrical high K⁺ conditions and was inhibited by intracellular tetraethylammonium ions (TEA). The non-selective cation channel had a voltage-independent single conductance of 22 pS and was not inhibited by TEA.