Calcium transport across the rabbit thick ascending limb of Henle's loop perfused in vitro

Ca²⁺ transport across the cortical thick ascending limb of Henle's loop (TALH) was studied on the isolated rabbit renal tubule perfused in vitro. Both the efflux (Ke) and influx coefficient (Ki) of Ca²⁺ were determined with⁴⁵Ca at three different levels of the transtubular electrical potential difference (PDt) caused by varying the transtubular Na⁺ concentration gradient. The flux ratios,Ke/Ki, always exceeded those predicted from observed PDt by simple passive diffusion, and the net Ca²⁺ efflux was shown to occur against an electro-chemical potential. An increase in Ca²⁺ concentration in the perfusate was associated with a decrease inKe. Efflux of Ca²⁺, therefore, tended to be saturated as luminal Ca²⁺ concentration was increased.Ke (10⁻⁷ cm²/s) decreased from 4.13±0.56 to 2.02±0.30 (P<0.02) along with a decrease in PDt when 10⁻³ M NaCN was added to the bathing fluid. By contrast, 10⁻³ M iodoacetamide did not affectKe in spite of a significant decrease in PDt. Similarly, neither 10⁻⁵ M ouabain added to the bathing fluid nor 10⁻⁴ M ruthenium red, added either to the bathing fluid or the perfusate, affectedKe despite significant decreases in PDt. Addition of 10⁻⁵ M furosemide in the lumen caused a decrease inKe from 4.31±0.43 to 3.17±0.25 (P<0.01) in association with a decrease in PDt from 6.3±1.04 to 1.7±0.37 mV. These findings suggest; (a) Ca²⁺ transport across the TALH is mainly an active process requiring aerobic metabolism; (b) dissociation of Ca²⁺ and Cl⁻ transport may occur under some experimental conditions.     

Stimulation of NaCl reabsorption by antidiuretic hormone in the cortical thick ascending limb of Henle's loop of the mouse

The effect of antidiuretic hormone (ADH) on transepithelial Na⁺, Cl^–, Ca²⁺ and Mg²⁺ net fluxes (J_Na, J_Cl, J_Mg, J_Ca) was investigated in isolated perfused cortical thick ascending limb segments (cTAL) of the mouse nephron, using the microperfusion technique and the electron microprobe analysis to determine the ionic composition of the collected tubular fluid. Simultaneously, the transepithelial potential difference (PD_te) and the transepithelial resistance (R_te) were recorded. Prior to the flux measurements cTAL segments were perfused for one hour. During this equilibration period PD_te decreased significantly from +19.9±1.6 to +14.9±1.l mV and R_te increased from 30.6±3.5 cm² to 38.8±2.4 cm² (n=7), reflecting a decline in NaCl transport. After ADH was added to the bath solution at 10^–10 mol.l^–1, PD_te increased from +14.4±1.1 to +18.0±1.5 mV, accompanied by a rise in J_Na and J_Cl from 205±11 to 273±19 and from 216±12 to 283±21 pmol.min^–1.mm^–1 (n=7), respectively. J_Ca and J_Mg also increased from 0.81±0.07 to 1.50±0.12 and from 0.43±0.11 to 0.76±0.08 pmol.min^–1.mm^–1 (n=7), respectively. All these effects were fully reversible after withdrawal of the hormone. In conclusion our data indicate that ADH stimulates divalent cation transport and NaCl transport in the cortical thick ascending limb of Henle's loop of the mouse.     

Presence of luminal K+, a prerequisite for active NaCl transport in the cortical thick ascending limb of Henle's loop of rabbit kidney

Previous data from our laboratory have shown that active transport in the cortical thick ascending limb of Henle's loop (cTAL), as measured by the short circuit current (I_SC, A · cm^–2), requires the presence of Na⁺ and Cl^–. The data were compatible with the model of secondarily active Cl^– reabsorption involving the cotransport of Na⁺ and Cl^– across the luminal membrane. The data suggested, furthermore, that 1 Na⁺ and 2 Cl^– interact with the luminal carrier. In the present study it was tested whether this reabsorptive mechanism also requires the presence of luminal K⁺. Isolated cTAL segments (n=40) were perfused at high flow rates with a modified Ringer's solution. Removal of K⁺ from the lumen reduced I_SC significantly from 215 to 133 A·cm^–2. Addition of Ba²⁺ (10^–3 mol·l^–1) which blocks the K⁺ conductance of the luminal membrane, to the K⁺-containing lumen perfusate decreased I_SC significantly from 234 to 141 A·cm^–2. Combination of both manoeuvres: perfusion with a K⁺-free and Ba²⁺-containing solution almost abolished I_SC from a control of 237 to 56 A · cm^–2. The results are compatible with the view that in rabbit cTAL the luminal carrier interacts with all 3 ions, possibly 1 Na⁺, 2 Cl^–, and 1 K⁺. K⁺ recycles across the luminal membrane through its conductive pathway.This study was supported by Deutsche Forschungsgemeinschaft Gr. 460/5-6-2     

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 ^– .     

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     

Properties of the basolateral membrane of the cortical thick ascending limb of Henle's loop of rabbit kidney

The present study utilizes the transepithelial and transmembrane electrophysiological approach to study the properties of the basolateral membrane of the in vitro perfused cortical thick ascending limb of Henle's loop (cTAL) of rabbit kidney. Eight different series were performed in a total of 119 tubules. The key observations are: 1. A K⁺ concentration upward step in the bath from 3.6 to 18.6 mmol · l^?1 depolarizes the basolateral membrane by 19±2 mV. 2. This depolarization can be abolished when Ba²⁺ (3 mmol · l^?1) is added to the bath: The depolarization by Ba²⁺ alone is equal to that by Ba²⁺ plus the K⁺ concentration upward step (21.7 versus 22.4 mV). 3. This effect of Ba²⁺ is not accompanied by any change in transepithelial resistance nor in the fractional resistance of the basolateral membrane. 4. A Cl^? concentration downward step in the bath from 150 to 50 mmol · l^?1 leads to a depolarization between 8–15 mV. We conclude that the K⁺ exit at the basolateral membrans is mainly electroneutral and that Cl^? leaves the cell both electroneutrally (KCl) and diffusionally. The present data, together with previous findings from our laboratory, are used to draw a tentative model for the NaCl reabsorption in the cTAL segment. In this model the (Na⁺+K⁺)-ATPase provides the primary driving force. Na⁺, 2 Cl^?, K⁺ are cotransported luminally, K⁺ recycles across the lumen membrane. Cl^? leaves the cell in part in conjunction with K⁺, and thus utilizing the chemical gradient for K⁺, and the remainder leaves the cell through the Cl^? conductive pathway. The discrepancy of the conductivity properties of both cell membranes, the lumen membrane K⁺ conductive, and the basolateral membrane Cl^? conductive, is the main source for the lumen positive transepithelialPD. ThePD, in turn, drives a seizable fraction of the Na⁺ through the paracellular shunt pathway.     

Hormonal stimulation of Ca2+ and Mg2+ transport in the cortical thick ascending limb of Henle's loop of the mouse: evidence for a change in the paracellular pathway permeability

Recent studies from our laboratory have shown that in the cortical thick ascending limb of Henle's loop of the mouse (cTAL) Ca²⁺ and Mg²⁺ are reabsorbed passively, via the paracellular shunt pathway. In the present study, cellular mechanisms responsible for the hormone-stimulated Ca²⁺ and Mg²⁺ transport were investigated. Transepithelial voltages (PD_te) and transepithelial ion net fluxes (J _Na, J _Cl, J _K, J _Ca, J _Mg) were measured in isolated perfused mouse cTAL segments. Whether parathyroid hormone (PTH) is able to stimulate Ca²⁺ and Mg²⁺ reabsorption when active NaCl reabsorption, and thus PD_te, is abolished by luminal furosemide was first tested. With symmetrical lumen and bath Ringer's solutions, no Ca²⁺ and Mg²⁺ net transport was detectable, either in the absence or in the presence of PTH. In the presence of luminal furosemide and a chemically imposed lumen-to-bath directed NaCl gradient, which generates a lumen-negative PD_te, PTH slightly but significantly increased Ca²⁺ and Mg²⁺ net secretion. In the presence of luminal furosemide and a chemically imposed bath-to-lumen-directed NaCl gradient, which generates a lumen-positive PD_te, PTH slightly but significantly increased Ca²⁺ and Mg²⁺ net reabsorption. In view of the observed small effect of PTH on passive Ca²⁺ and Mg²⁺ movement, a possible interference of furosemide with the hormonal response was considered. To investigate this possibility, Ca²⁺ and Mg²⁺ transport was first stimulated with PTH in tubules under control conditions. Then active NaCl reabsorption was abolished by furosemide and the effect of PTH on J _Ca and J _Mg measured. In the absence of PD_te and under symmetrical conditions, no Ca²⁺ and Mg²⁺ transport was detectable, either in the presence or absence of PTH. In the presence of a bath-to-lumen-directed NaCl gradient, Ca²⁺ and Mg²⁺ reabsorption was significantly higher in the presence than in the absence of PTH. Finally, when active NaCl transport was not inhibited by furosemide, but reduced by a bath-to-lumen-directed NaCl gradient, PTH strongly increased J _Ca and J _Mg, whereas only a small increase in PD_te was noted. In conclusion, these data suggest that PTH exerts a dual action on Ca²⁺ and Mg²⁺ transport in the mouse cTAL by increasing the transepithelial driving force for Ca²⁺ and Mg²⁺ reabsorption through hormone-mediated PD_te alterations and by modifying the passive permeability for Ca²⁺ and Mg²⁺ of the epithelium, very probably at the level of the paracellular shunt pathway.     

Role of Na+/Ca2+ exchange in transcellular Ca2+ transport across primary cultures of rabbit kidney collecting system

Cells from connecting tubule and cortical collecting duct of rabbit kidney were isolated by immunodissection with mAb R2G9 and cultured on permeable filters. Confluent monolayers developed an amiloride-sensitive transepithelial potential difference of –50±1 mV (lumen negative) and a transepithelial resistance of 507±18 cm². Transepithelial Ca²⁺ transport increased dose-dependently with apical [Ca²⁺] and, in solutions containing 1 mM Ca²⁺, the active transcellular Ca²⁺ transport rate was 92±2 nmol h^–1 cm^–2. Transcellular Ca²⁺ transport was dependent on basolateral Na⁺ (Na _b ⁺ ). Isoosmotic substitution of Na _b ⁺ for N-methylglucamine resulted in a concentration-dependent decrease in Ca²⁺ absorption, with maximal inhibition of 67±5%. A Hill plot of the Na⁺-dependence yielded a coefficient of 1.9±0.4, indicating more than one Na⁺ site on a Na⁺-dependent Ca²⁺ transport system. In addition, the absence of Ca _b ²⁺ resulted in a significant increase in Ca²⁺ transport both in the presence and absence of Na _b ⁺ . Added basolaterally, ouabain (0.1 mM) inhibited Ca²⁺ transport to the same extent as did Na⁺-free solutions, while bepridil (0.1 mM), an inhibitor of Na⁺/Ca²⁺ exchange, reduced Ca²⁺ transport by 32±6%. Methoxyverapamil, felodipine, flunarizine and diltiazem (10 M) were without effect. Depolarisation of the basolateral membrane, by raising [K⁺]_b to 60 mM, significantly decreased transcellular Ca²⁺ transport, which is indicative of electrogenic Na⁺/Ca²⁺ exchange. In conclusion, active Ca²⁺ transport in the collecting system of rabbit kidney is largely driven by basolateral Na⁺/Ca²⁺ exchange. However, a residual Ca²⁺ absorption of about 30% was always observed, suggesting that other Ca²⁺ transport mechanisms, presumably a Ca²⁺-ATPase, participate as well.     

Membrane transport in the proximal tubule and thick ascending limb of Henle's loop: Mechanisms and their alterations

Summary Over the past few years, our knowledge on renal tubular transport mechanisms has increased considerably. Due to new technical developments, it is now possible to understand in part transepithelial transport and its pathological and pharmacological alterations at the level of the cell membranes. Different membrane transport mechanisms are discussed in this article, whereby sodium coupled solute transport in the proximal tubule and sodium chloride transport in the thick ascending limb of Henle's loop are taken as examples. It is indicated that an altered function of the kidney can often be equated with an alteration of the membrane transport.     

Sodium-chloride transport in the thick ascending limb of Henle's loop

Isolated cells were prepared from the medullary thick ascending limb of Henle's loop (TALH) and the response of oxygen consumption was correlated with the active chloride transport system found in these cells. Oxygen consumption was 31.6 l O₂/mg protein·h and inhibited 50% by the absence of either sodium or chloride in the incubation medium. The absence of both sodium and chloride produced no further inhibition of oxygen consumption. Ouabain (10^–4 M) inhibited oxygen consumption by 50% and the inhibitory effect depended on the presence of both sodium and chloride in the incubation medium. Further, furosemide inhibited oxygen consumption by a maximum of 50% at 10^–3 M and also had no inhibitory effect if either sodium or chloride were absent. Furosemide had no effect on the Na, K-ATPase activity or ATP levels of the TALH cells. Thus, the data suggest that 50% of the oxygen consumption of the TALH cells is related to the movement of sodium and chloride into the cell and that the ions may be transported in a coupled manner.In addition the effect of various diuretics on oxygen consumption in the isolated TALH cells was tested. The diuretics could be grouped in three categories: (1) highly effective in inhibiting chloride-dependent oxygen consumption with an apparent inhibitory constant (K _i) of around 10^–6 M, including the diuretics furosemide, bumetanide, ethacrynic acid-cysteine and piretanide, (2) diuretics which were less effective in inhibiting oxygen consumption with an apparentK _i of around 10^–4 M, HOE 740 and ethacrynic acid, and (3) diuretics which were ineffective in inhibiting chloride-dependent oxygen consumption, amiloride and hydrochlorothiazide.     

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.     

Effect of NH4 +/NH3 on cytosolic pH and the K+ channels of freshly isolated cells from the thick ascending limb of Henle's loop

The conductance properties of the luminal membrane of cells from the thick ascending limb of Henle's loop of rat kidney (TAL) are dominated by K⁺. In excised membrane patches the luminal K⁺ channel is regulated by pH changes on the cytosolic side. To examine this pH regulation in intact cells of freshly isolated TAL segments we measured the membrane voltage (V _m) in slow-whole-cell (SWC) recordings and the open probability (P _o) of K⁺ channels in the cell-attached nystatin (CAN) configuration, where channel activity and part of V _m can be recorded. The pipette solution contained K⁺ 125 mmol/l and Cl^– 32 mmol/l. Intracellular pH was determined by 2,7 bis(2-carboxyethyl)-5,(6)-carboxyfluorescein (BCECF) fluorescence. pH changes were induced by the addition of 10 mmol/l NH₄ ⁺/NH₃ to the bath. In the presence of NH₄ ⁺/NH₃ intracellular pH acidified by 0.53±0.11 units (n=7). Inhibition of the Na⁺2Cl^– K⁺ cotransporter by furosemide (0.1 mmol/l) reversed this effect and led to a transient alkalinisation by 0.62±0.14 units (n=7). In SWC experiments V _m of TAL cells was -72±1 mV (n=70). NH₄ ⁺/NH₃ depolarised V _m by 22±2 mV (n=25). In 11 SWC experiments furosemide (0.1 mmol/l) attenuated the depolarising effect of NH₄ ⁺ from 24±3 mV to 7±3 mV. Under control conditions the single-channel conductance of TAL K⁺ channels in CAN experiments was 66±5 pS and the reversal voltage for K⁺ currents was 70±2 mV (n=35). The P _o of K⁺ channels in CAN patches was reduced by NH₄ ⁺/NH₃ from 0.45±0.15 to 0.09±0.07 (n=7). NH₄ ⁺/NH₃ exposure depolarised the zero current voltage of the permeabilised patches by-9.7±3.6 mV (n=5). The results show that TAL K⁺ channels are regulated by cytosolic pH in the intact cell. The cytosolic pH is acidified by NH₄ ⁺/NH₃ exposure at concentrations which are physiologically relevant because Na⁺2Cl^–K⁺(NH₄ ⁺) cotransporter-mediated import of NH₄ ⁺ exceeds the rate of NH₃ diffusion into the TAL. K⁺ channels are inhibited by this acidification and the cells depolarise. In the presence of furosemide TAL cells alkalinise proving that NH₄ ⁺ uptake occurs by the Na⁺2Cl^–K⁺ cotransporter. The findings that, in the presence of NH₄ ⁺/NH₃ and furosemide, V _m is not completely repolarised and that K⁺ channels are not activated suggest that the respective K⁺ channels may in addition to their pH regulation be inhibited directly by NH₄ ⁺/NH₃.     

Differential effects of ADH on sodium,chloride, potassium,calcium and magnesium transport in cortical and medullary thick ascending limbs of mouse nephron

The effect of antidiuretic hormone (arginine vasopressin, AVP) on transepithelial Na⁺, Cl^–, K⁺, Ca²⁺ and Mg²⁺ net transports was investigated in medullary (mTAL) and cortical (cTAL) segments of the thick ascending limb (TAL) of mouse nephron, perfused in vitro. Transepithelial net fluxes (J _Na ⁺,J _Cl ^–,J _K ⁺,J _Ca ²⁺,J _Mg ²⁺) were determined by electron probe analysis of the collected tubular fluid. Transepithelial potential difference (PD_te) and transepithelial resistance (R_te) were measured simultaneously. cTAL segments were bathed and perfused with isoosmolal, HCO ₃ ^– containing Ringer solutions, mTAL segments were bathed and perfused with isoosmolal HCO ₃ ^– free Ringer solutions. In cTAL segments, AVP (10^–10 mol·l^–1) significantly increasedJ _Mg ²⁺ andJ _Ca ²⁺ from 0.39±0.08 to 0.58±0.10 and from 0.86±0.13 to 1.19±0.15 pmol·min^–1 mm^–1 respectively. NeitherJ _Na ⁺ norJ _Cl ^–, (J _Na ⁺: 213±30 versus 221±28 pmol·min^–1 mm^–1,J _Cl ^–: 206±30 versus 220±23 pmol·min^–1 mm^–1) nor PD_te (13.4±1.3 mV versus 14.1±1.9 mV) or R_te (24.6±6.5 cm² versus 22.6±6.4 cm²) were significantly changed by AVP. No significant effect of AVP on net K⁺ transport was observed. In mTAL segments, Mg²⁺ and Ca²⁺ net transports were close to zero and AVP (10^–10 mol·l^–1) elicited no effect. However NaCl net reabsorption was significantly stimulated by the hormone,J _Na ⁺ increased from 107±33 to 148±30 andJ _Cl ^– from 121±33 to 165±32 pmol·min^–1 mm^–1. The rise inJ _NaCl was accompanied by an increase in PD_te from 9.0±0.7 to 13.5±0.9 mV and a decrease in R_te from 14.4±2.0 to 11.2±1.7 cm². No K⁺ net transport was detected, either under control conditions or in the presence of AVP.To test for a possible effect of HCO ₃ ^– on transepithelial ion fluxes, mTAL segments were bathed and perfused with HCO ₃ ^– containing Ringer solutions. With the exception ofJ _Ca ²⁺ which was significantly different from zero (J _Ca ²⁺: 0.26±0.06 pmol·min^–1 mm^–1), net transepithelial fluxes of Na⁺, Cl^–, K⁺ and Mg²⁺ were unaffected by HCO ₃ ^– . In the presence of AVP,J _Mg ²⁺ andJ _Ca ²⁺ were unaltered whereasJ _NaCl was stimulated to the same extent as observed in the absence of HCO ₃ ^– . In conclusion our results indicate heterogeneity of response to AVP in cortical and medullary segments of the TAL segment, since AVP stimulates Ca²⁺ and Mg²⁺ reabsorption in the cortical part and Na⁺ and Cl^– reabsorption in the medullary part of this nephron segment.This study was supported by the Commission des communautés européennes, grant no. ST2J 00951 F(CD), and by Wissenschafts-ausschuß der Nato über den DAAD     

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     

Antidiuretic hormone acts via V1 receptors on intracellular calcium in the isolated perfused rabbit cortical thick ascending limb

The effect of antidiuretic hormone ([Arg]vasopressin, ADH) on intracellular calcium activity [Ca²⁺]_i of isolated perfused rabbit cortical thick ascending limb (cTAL) segments was investigated with the calcium fluorescent dye fura-2. The fluorescence emission ratio at 500–530 nm (R) was monitored as a measure of [Ca²⁺]_i after excitation at 335 nm and 380 nm. In addition the transepithelial potential difference (PD _te) and transepithelial resistance (R _te) of the tubule were measured simultaneously. After addition of ADH (1–4 nmol/l) to the basolateral side of the cTAL R increased rapidly, but transiently, from 0.84±0.05 to 1.36±0.08 (n = 46). Subsequently, within 7–12 min R fell to control values even in the continued presence of ADH. The increase in R evoked by the ADH application corresponded to a rise of [Ca²⁺]_i from a basal level of 155±23 nmol/l [Ca²⁺]_i up to 429±53 nmol/l [Ca²⁺]_i at the peak of the transient, as estimated by intra- or extracellular calibration procedures. The electrical parameters (PD _te and R _te) of the tubules were not changed by ADH. The ADH-induced Ca²⁺ transient was dependent on the presence of Ca²⁺ on the basolateral side, whereas luminal Ca²⁺ had no effect. d(CH₂)₅[Tyr(Me)²]²,Arg⁸vasopressin, a V₁ antagonist (Manning compound, 10 nmol/l), blocked the ADH effect on [Ca²⁺]_i completely (n = 5). The V₂ agonist 1-desamino-[d-Arg⁸]vasopressin (10 nmol/l, n=4), and the cAMP analogues, dibutyryl-cAMP (400 mol/l, n = 4), 8-(4-chlorophenylthio)-cAMP (100 mol/l, n = 1) or 8-bromo-cAMP (200 mol/1, n = 4) had no influence on [Ca²⁺]_i. The ADH-induced [Ca²⁺]_i increase was not sensitive to the calcium-channel blockers nifedipine and verapamil (100 mol/l, n = 4). We conclude that ADH acts via V₁ receptors to increase cytosolic calcium activity transiently in rabbit cortical thick ascending limb segments, possibly by an initial Ca²⁺ release from intracellular stores and by further Ca²⁺ influx through Ca²⁺ channels in the basolateral membrane. These channels are insensitive to L-type Ca²⁺ channel blockers, e.g. nifedipine and verapamil.Supported by DFG GR 480/10     

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     

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     

Properties of single K+ channels in the basolateral membrane of rabbit proximal straight tubules

The basolateral membrane of rabbit straight proximal tubules, which were cannulated and perfused on one side, was investigated with the patch clamp technique. Properties of inward and outward directed single K⁺ channel currents were studied in cell-attached and insideout oriented cell-excised membrane patches. In cell-attached patches with NaCl Ringer solution both in pipette and bath, outward K⁺ currents could be detected after depolarization of the membrane patch by about 20–30 mV. The current-voltage (i/V) relationship could be fitted by the Goldman-Hodgkin-Katz (GHK) current equation, with the assumption that these channels were mainly permeable for K⁺ ions. A permeability coefficientP _K of (0.17±0.04) · 10^–12 cm³/s was obtained, the single channel slope conductance at infinite positive potentialg(V ) was 50±12 pS and the single channel conductance at the membrane resting potentialg(V _bl) was 12±3 pS (n=4). In cell-excised patches, with NaCl in the pipette and KCl in the bath, the data could also be fitted to the GHK equation and yieldedP _K = (0.1 ±0.01) ·10^–12 cm³/s,g(V ) = 40 ± 4 pS andg(V _bl) = 7 ± 1 pS (n=8). In cell-attached patches with KCl in the pipette and NaCl in the bath, inward K⁺ channels occurred at clamp potentials 60 mV, whereas outward K⁺ channel current was detected at more positive voltages. The current-voltage curves showed slight inward rectification. The single channel conductance, obtained from the linear part of the i/V curve by linear regression, was 46±3 pS and the reversal potential was 59±6 mV (n=9). In cell-excised patches with KCl in the pipette and NaCl in the bath, inward directed K⁺ channel currents could again be described by the GHK equation. The single channel parameters were similar to those recorded for outward K⁺ currents (see above). In inside-out oriented cell-excised patches with NaCl in the pipette and KCl in the bath, reducing bath (i.e. cytosolic) Ca²⁺ concentration from 10^–6 mol/l to less than 10^–9 mol/l did not affect the open state probability of single channel currents. These results demonstrate that the observed channels are permeable for K⁺ ions in both directions and that these basolateral K⁺ channels in rabbit proximal straight tubule are not directly dependent on Ca²⁺ ions.     

Properties of the lumen membrane of the cortical thick ascending limb of Henle's loop of rabbit kidney

Previous data suggest the cotransport of Na⁺, Cl^?, and K⁺ across the lumen membrane of the cortical thick ascending limb (cTAL) of rabbit nephron. For this cotransporter to operate K⁺ recycling across the lumen membrane has to be postulated. The present data focus on the conductivity properties of the lumen membrane. Methods for impaling individual cells of in vitro perfused cTAL segments are described. The meanPD across the lumen membrane (PD ₁) is 76 mV (lumen positive). Rapid increase in lumen perfusate K⁺ concentration (3.6→18.6 mmol · l^?1) leads to a depolarization ofPD ₁ by 20 mV. Ba²⁺ (3 mmol · l^?1) added to the lumen perfusate inhibits the K⁺ conductive pathway of the lumen membrane, and consequently increases the voltage divider ratio (current pulse induced voltage deflection across the lumen membrane divided by that across the basolateral membrane) from 2 to 36 as well as transepithelial resistance from 34 to 46 Ω cm². From these changes, and with the use of simultaneous equations of the VDR and of Kirchhoff's law, the resistances of the lumen membrane (88 Ω cm²), of the basolateral membrane (47 Ω cm²), and of the paracellular shunt pathway (47 Ω cm²) can be calculated. Using these estimates of the individual resistances and using the observed change inPD ₁ in the K⁺ concentration step experiments (29±3 mV per decade in K⁺ concentration change) an apparent transference number of the lumen membrane for K⁺ in the order of 0.9–1.0 can be calculated. This indicates that the lumen membrane is essentially K⁺ conductive. This conclusion is strengthened further by the results of another series in which no evidence for a Cl^? conductive pathway in the lumen was obtained. The data of this study can be used to calculate the intracellular K⁺ activity of some 90–100 mmol ·l^?1. For this K⁺ activity the K⁺ diffusion from cell to lumen equals the carrier mediated uptake from lumen to cell. This indicates that an essentially complete recycling of K⁺ across the lumen membrane of the cTAL segment is feasible.