Mechanism of hydrogen ion transport in the diluting segment of frog kidney

Transepithelial H⁺ transport was studied in diluting segments of the isolated-perfused kidney ofrana esculenta. The experiments were performed in controls as well as in K⁺-adapted and Na⁺-adapted animals (exposed to 50 mmol/l KCl or NaCl, resp. for at least 3 days). Conventional and single-barreled, liquid ion-exchanger H⁺-sensitive microelectrodes were applied in the tubule lumen to evaluate transepithelial H⁺ net flux (J _te ^H ) as well as limiting transepithelial electrical and H⁺ electrochemical potential differences (PD _te ,E _te ^H ) and luminal pH at zero net flux conditions. The measurements were made in absence (control) and presence of furosemide (5·10^–5 mol/l) or amiloride (10^–3 mol/l). E _te ^H (lumen positive vs ground) was 19±3 mV in controls, 43±3 mV in K⁺ adapted but about zero in Na⁺ adapted animals. Using the correspondingPD _te -values, steady state luminal pH of 7.63±0.05, 7.13±0.05 and 8.02±0.02 was calculated for the respective groups of animals (peritubular pH 7.80). In parallel, significant secretoryJ _te ^H (from blood to lumen) was found in controls (14±2 pmol·cm^–2·S^–1) which was stimulated by K⁺ adaptation (61±8 pmol·cm^–2·s^–1) but reversed in direction by Na⁺-adaptation (–8±1 pmol·cm^–2·s^–1). Amiloride inhibited secretoryJ _te ^H . Elimination of the lumen positivePD _te by furosemide did not affect significantlyE _te ^H andJ _te ^H in control and K⁺ adapted animals but abolished reabsorptiveJ _te ^H in Na⁺ adapted animals.We conclude that in frog diluting segment H⁺ secretion is an active, amiloride-sensitive, furosemide-insensitive transport process. The data are consistent with luminal Na⁺/H⁺ exchange. The activity of this system depends critically on the metabolic state of the animal.Parts of the data were presented at the 16th Ann. Meeting of the Am. Soc. Nephrol., Washington (1983)This work was supported by österr. Forschungsrat, Proj. No.: 4366 and by Dr. Legerlotz Stiftung     

Intracellular pH in diluting segment of frog kidney

Chronic exposure to high potassium (K⁺ adaptation) stimulates H⁺ net secretion in the diluting segment of the frog kidney. In order to investigate the cellular mechanism of the H⁺ secretory process intracellular pH (pH_i) measurements were performed in cells of the diluting segment of the isolated doubly-perfused kidney of K⁺ adaptedRana esculenta. pH_i changes were monitored by pH-sensitive microelectrodes while the tubule lumen was rapidly perfused with various solutions. With control solutions (extracellular pH=7.80) pH_i averaged 7.60±0.05. Luminal application of furosemide (5 · 10^–5 mol/l) or reduction of luminal Cl^– (from 104 mmol/l to 9 mmol/l) hyperpolarized the cell membrane potentials but pH_i was not altered. Reduction of luminal Na⁺ (from 98 mmol/l to 3 mmol/l) depolarized the cell membrane potentials but pH_i remained constant. Complete removal of luminal Na⁺, however, led to a significant decrease of pH_i from 7.61±0.08 to 7.18±0.08. Luminal application of amiloride (1 · 10^–3 mol/l) also decreased pH_i significantly (pH_i=0.15±0.02).The results indicate that an amiloride-sensitive H⁺ extrusion mechanism exists in the luminal cell membrane of the K⁺ adapted frog diluting segment. The data are consistent with Na⁺/H⁺ exchange which maintains a constant pH_i even at extreme experimental conditions.Parts of the data were presented at the 16th Ann. Meeting of the Am. Soc. Nephrol., Washington (1983)     

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

The dependence of intracellular pH (pH_i) and transepithelial H⁺ secretion on the cell membrane potential (V _m) 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 decreasedV _m from –49±4 to –29±1 mV while pH_i increased from 7.44±0.04 to 7.61±0.06. Addition of 1 mmol/l Ba²⁺ depolarizedV _m from –45±3 to –32±2 mV, paralleled by an increase of pH_i from 7.46±0.04 to 7.58±0.03. Application of 0.05 mmol/l furosemide hyperpolarizedV _m from –48±3 to –53±3 mV and decreased pH_i 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 HCO ₃ ^– exit step across the basolateral cell membrane. HCO ₃ ^– accumulates in the cytoplasm and pH_i increases. An alkaline pH_i inactivates the luminal Na⁺/H⁺ exchanger. This diminishes transepithelial H⁺ secretion. Cell hyperpolarization leads to the opposite phenomenon. Thus, pH_i serves as signal transducer between cell voltage and Na⁺/H⁺ exchange.     

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.     

Effect of parathyroid hormone on acid/base transport in rabbit renal proximal tubule S3 segment

The effect of parathyroid hormone (PTH) on acid/base transport in isolated rabbit renal proximal tubule S3 segment was investigated with double-barreled and conventional microelectrodes. PTH (10 nM) induced a small depolarization and enhanced the initial rates of cell pH (pH_i) increase and cell Cl^– ([Cl^–]_i) decrease in response to bath Cl^– removal by 28.0±2.1% and 31.0±6.4% respectively. The calculated initial HCO₃ ^– influx to bath Cl^– removal was also enhanced by 28%. On the other hand, PTH reduced the initial rate of pH_i decrease to luminal Na⁺ removal in the absence of HCO₃ ^–/CO₂ by 20.4±3.9%. The PTH-induced depolarization was not accompanied with changes in steadystate pH_i or [Cl^–]_i levels, but was greatly attenuated in the presence of ouabain (0.1 mM). Either dibutyrylcAMP (0.1 mM) plus theophylline (1 mM) or forskolin (10 M) alone could reproduce all the effects of PTH. These results indicate that (a) PTH inhibits the luminal Na⁺/H⁺ exchanger but stimulates the basolateral Cl^–/HCO₃ ^– exchanger in the S3 segment; (b) the PTH-induced depolarization largely results from inhibition of Na⁺/K⁺-ATPase and (c) all these effects are at least partly mediated by a cAMP-dependent mechanism.     

Thiazide-sensitive Na-Cl cotransport mediates NaCl absorption in amphibian distal tubule

To find out the mechanism(s) underlying NaCl absorption in the distal tubule of Necturus, we devised a variant of the split-drop technique. Following injection an oil column, subsequently split by a NaCl solution isotonic to plasma, a double-barrelled microelectrode (conventional/selective to Na⁺ or to Cl^– ions) recorded Na⁺ ( _Na) or Cl^– ( _Cl) activity and transepithelial potential (V _te). Paired control/low-Na⁺ solutions yielded reabsorptive half-times (t _1/2) of 0.68±0.11 min and 7.6±1.8 min respectively; corresponding V _te values were –22.2±4.0 mV and –7.6±1.9 mV. t _1/2 values of control versus low-Cl^– solutions were 0.77±0.32 min and 6.5±1.7 min respectively, whereas respective V _te values were not different from one another: –23.8±4.3 mV versus –18.8±5.5 mV. Nominally K⁺-free solutions or bumetanide, 10 mol/l, did not alter t _1/2 or V _te, with regard to the paired control. Amiloride, 5 mol/l or 2 mmol/l, failed to decrease t _1/2 or to lower V _te; apparently, the role of a Na⁺/H⁺ antiport does not contribute significantly to NaCl absorption. Furosemide, 0.1 mmol/l, reduced t _1/2 by 54% with regard to the control state. Determination of t _1/2 as a function of increasing hydrochlorothiazide concentrations revealed apical high- and low-affinity sites, estimated at 0.56 mol/l and 0.115 mmol/l respectively. Taken together these observations indicate that NaCl absorption is predominantly carried out by an electroneutral Na⁺-Cl^– cotransport.     

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

In frog diluting segment transepithelial K⁺ net flux (J _te ^K ) occurs via trans- and paracellular transport routes. Inhibition of transcellular K⁺ transport disclosesJ _te ^K across the shunt-pathway. By means of K⁺-sensitive microelectrodes we have measured secretoryJ _te ^K induced by an acute K⁺ load, in the diluting segment of the isolated and doublyperfused 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 the Na⁺/K⁺ pump inhibitor ouabain or by inducing an acute acid load. All experimental maneouvers led to a reduction of secretoryJ _te ^K to about 50% of the controlJ _te ^K . The apparent permeability coefficient for K⁺ of this nephron portion after inhibition of transcellular secretoryJ _te ^K was reduced to a similar extent. We conclude: In frog diluting segment the ratio of trans- over paracellularJ _te ^K 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.     

Mechanisms of intracellular pH regulation in the hamster inner medullary collecting duct perfused in vitro

To examine the mechanisms of H⁺ transport in the mid-inner medullary collecting duct of hamsters, we measured the intracellular pH (pH_i) in the in vitro perfused tubules by microscopic fluorometry using 2,7-bis(carboxyethyl)-carboxyfluorescein (BCECF) as a fluorescent probe. In the basal condition, pH_i was 6.74±0.04 (n=45) in HCO ₃ ^– -free modified Ringer solution. Either elimination of Na⁺ from the bath or addition of amiloride (1 mM) to the bath produced a reversible fall in pH_i After acid loading with 25 mM NH₄Cl, pH_i spontaneously recovered with an initial recovery rate of 0.096±0.012 (n=23) pH unit/min. In the absence of ambient Na⁺, after removal of NH ₄ ⁺ , the pH_i remained low (5.95±0.10, n=8) and showed no signs of recovery. Subsequent restoration of Na⁺ only in the lumen had no effect on pH_i. However, when Na⁺ in the bath was returned to the control level, pH_i recovered completely. Amiloride (1 mM) in the bath completely inhibited the Na⁺-dependent pH_i recovery. Furthermore, elimination of Na⁺ from the bath, but not from the lumen, decreased pH_i from 6.97±0.07 to 6.44±0.05 (n=12) in the HCO ₃ ^– /Ringer solution or 6.70±0.03 to 6.02±0.05 (n=8) in the HCO ₃ ^– free solution. pH_i spontaneously returned to 6.76±0.08 with a recovery rate of 0.017±0.5 pH unit/min in the presence of CO₂/HCO ₃ ^– , whereas it did not recover in the absence of CO₂/HCO ₃ ^– . Although elimination of ambient Na⁺ depolarized the basolateral membrane voltage (V _B) from –78±1.2 to –72 ±0.6 mV (n=5, P<0.01), the level of V _B was not sufficient to explain the pH_i recovery solely by HCO ₃ ^– entry driven by the voltage. These results indicate that (a) pH_i of the inner medullary collecting duct is regulated mainly by a Na⁺/H⁺ exchanger in the basolateral membranes, (b) no apparent Na⁺-dependent H⁺ transport system exists in the luminal membranes and (c) Na⁺-independent H⁺ transport may also operate in the presence of CO₂/HCO ₃ ^– Preliminary data were reported at the Conference on Bicarbonate, Chloride, and Proton Transport Systems, New York, USA, in January 1989     

Electrophysiological characterization of rabbit distal convoluted tubule cell

The distal convoluted tubule (DCT) from rabbit kidney were perfused in vitro to study the conductive properties of the cell membranes by using electrophysiological methods. When the lumen and the bath were perfused with a biearbonate free solution buffered with HEPES, the transepithelial voltage (V _T) averaged –2.8±0.6 mV (n=20), lumen negative. The basolateral membrane voltage (V _B) averaged –77.8±1.1 mV (n=33) obtained by intracellular impalement of microelectrodes. Cable analysis performed by injecting a current from perfusion pipette revealed that the transepithelial resistance was 21.8±1.7 ·cm² and the fractional resistance of the luminal membrane was 0.78±0.03 (n=8), indicating the existence of ionic conductances in the luminal membrane. Addition of amiloride (10^–5 mol/l) to the luminal perfusate or Na⁺ removal from the lumen abolished the lumen negativeV _T and hyperpolarized the apical membrane. An increase in luminal K⁺ concentration from 5 to 50 mmol/l reduced the apical membrane potential (V _A) by 37.5±2.6 mV (n=7), whereas a reduction of Cl^– in the luminal perfusate did not changeV _A significantly (0.5±0.5 mV,n=4). Addition of Ba²⁺ to the lumen reducedV _A by 42.6±1.0 mV (n=4). When the bathing fluid was perfused with 50 mmol/l K⁺ solution, the basolateral membrane voltage (V _B) fell from –76.8±1.5 to –31.0±1.3 mV (n=18), and addition of Ba²⁺ to the bath reducedV _B by 18.3±4.8 mV (n=7). Although a reduction of Cl^– in the bathing fluid from 143 to 5 mmol/l did not cause any significant fast initial depolarization (1.8±1.7 mV,n=8), a spike like depolarization (14.0±2.5 mV,n=4) was observed, upon Cl^– reduction in the presence of Ba²⁺ in the bath. From these results, we conclude that the apical membrane of DCT has both K⁺ and Na⁺ conductances and the basolateral membrane has a K⁺ conductance and a small Cl^– conductance.     

Effects of glucagon on Na+, Cl−, K+, Mg2+ and Ca2+ transports in cortical and medullary thick ascending limbs of mouse kidney

The effects of glucagon on transepithelial Na⁺, Cl^–, K⁺, Ca²⁺ and Mg²⁺ net fluxes were investigated in isolated perfused cortical (cTAL) and medullary (mTAL) thick ascending limbs of Henle's loop of the mouse nephron. Transepithelial ion 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. Simultaneously the transepithelial voltage (PD_te) and the transepithelial resistance (R _te) were recorded. In cTAL-segments (n=8), glucagon (1.2×10^–8 mol · l^–1) stimulated significantly the reabsorption of Na⁺, Cl^–, Ca²⁺ and Mg²⁺J _Na ⁺ increased from 204±20 to 228±23 pmol · min^–1 · mm^–1,J _Cl ^– from 203±18 to 234±21 pmol · min^–1 · mm^–1,J _Ca ²⁺ from 0.52±0.13 to 1.34±0.30 pmol · min^–1 · mm^–1 andJ _Mg ²⁺ from 0.51±0.08 to 0.84±0.08 pmol · min^–1 · mm^–1.J _K+ remained unchanged: 3.2±1.3 versus 4.0±1.9 pmol · min^–1 · mm^–1. Neither PD_te (16.3±1.5 versus 15.9±1.4 mV) norR _te (22.5±3.0 versus 20.3±2.6 cm²) were changed significantly by glucagon. However, in the post-experimental periods a significant decrease in PD_te and increase inR _te were noted. In mTAL-segments (n=9), Mg²⁺ and Ca²⁺ transports were close to zero and glucagon elicited no significant effect. The reabsorptions of Na⁺ and Cl^–, however, were strongly stimulated:J _Na ⁺ increased from 153±17 to 226±30 pmol · min^–1 · mm^–1 andJ _Cl ^– from 151±23 to 243±30 pmol · min^–1 · mm^–1. The rise in NaCl transport was accompanied by an increase in PD_te from 10.3±1.1 to 12.3±1.2 mV and a decrease inR _te from 19.1±2.7 to 17.8±2.0 cm². No net K⁺ movement was detectable either in the absence or in the presence of glucagon. A micropuncture study carried out in hormone-deprived rats indicated that glucagon stimulates Na⁺, Cl^–, K⁺, Mg²⁺ and Ca²⁺ reabsorptions in the loop of Henle. In conclusion our data demonstrate that glucagon stimulates NaCl reabsorption in the mTAL segment and to a lesser extent in the cTAL segment whereas it stimulates Ca²⁺ and Mg²⁺ reabsorptions only in the cortical part of the thick ascending limb of the mouse nephron. These data are in good agreement with, and extend, those obtained in vivo on the rat with the hormone-deprived model.This study was supported by the Commission des Communautés Européennes, Grant no. ST 23, 00951F (CD) and by Wissenschaftsausschuß der Nato über den DAAD     

Chloride and potassium conductances of cultured human sweat ducts

The purpose of this study was to characterize the ion conductances, in particular those for Cl^– and K⁺, of human sweat duct cells grown in primary culture. Sweat duct cells from healthy individuals were grown to confluence on a dialysis membrane, which was then mounted in a mini-Ussing chamber and transepithelial and intracellular potentials were measured under open-circuit conditions. Under control conditions the epithelia developed mucosa-negative transepithelial potentials, V _te, of about –10mV. The apical membrane potential, V _a, was –25 mV to –30 mV (n=97) in most cells, but several cells had a higher potential of about –55 mV (n=29). Mucosal amiloride (10 mol/l) hyperpolarized V _a from –31±1 mV to a new sustained level of –46±2 mV (n=36). These changes were accompanied by increase in the fractional resistance of the apical membrane, fR _a, and decreases of V _te and the equivalent short-circuit current, I _sc. In amiloride-treated tissues an increase in mucosal K⁺ concentration (5 mmol/l to 25 mmol/l) depolarized V _a by 5±1 mV (n=8), while the same step on the serosal side depolarized V _a by 20±2 mV (n=8). A Cl^– channel blocker 3,5-dichloro-diphenylamine-2-carboxylate DCl-DPC; 10 mol/l) depolarized V _a by 5±1 mV (n=6), an effect that was lost after amiloride application. The blocker had no effect from the serosal side. Reduction of mucosal Cl^– (from 120 to 30 or 10 mmol/l) depolarized V _a by 9–11 mV (n=35), an effect that was often followed by a secondary hyperpolarization of 10–30 mV (n=27). Isoproterenol (5 mol/l) increased the V _a responses to low Cl^– such that the depolarizing response was increased from 10±1 mV to 19±2 mV (n=8); the hyperpolarizing response seemed to be reduced. With changes in Cl^– concentration on the serosal side, V _a remained relatively constant at –25 mV, while V _te decreased from –8 mV to–3 mV; hence, V _bl depolarized by about 5 mV. Taken together, our results show that the human sweat duct epithelium possesses Na⁺, K⁺ and Cl^– conductances on the luminal membrane and Cl^– and K⁺ conductances on the basolateral membrane. The Cl^– conductances on the luminal membrane is sensitive to DCl-DPC, and can be activated by isoproterenol. The small K⁺ conductance on the luminal membrane could account for some K⁺ secretion in sweat glands.     

Mechanism of NaCl secretion in the rectal gland of spiny dogfish (Squalus acanthias)

Rectal gland tubules (RGT) of spiny dogfish were dissected and perfused in vitro. Transepithelial PD (PD_te), resistance (R_te), the PD across the basolateral membrane (PD_bl) and intracellular chloride and potassium activities (a _Cl– ^cell ,a _K+ ^cell ) were measured. In a first series, 67 RGT segments were perfused with symmetric shark Ringers solution. The bath perfusate contained in addition db-cAMP 10^–4, forskolin 10^–6, and adenosine 10^–4 mol · l^–1. PD_te was –11±1 (n=67) mV lumen negative, R_te 27±2 (n=47) cm². PD_bl –75±0.4 (n=260) mV.a _K+ ^cell anda _Cl– ^cell were 109±22 (n=4) and 38±4 (n=36) mmol · l^–1 respectively. These data indicate that Cl^– secretion across the RGT must be an uphill transport process, whereas secretion of Na⁺ could be driven by the lumen negative PD_te. Intracellular K⁺ is 14 mV above equilibrium with respect to the basolateral membrane PD and Cl^– is 23 mV above equilibrium across the apical membrane. In series 2, the conductivity properties of the apical and basolateral membrane as well as that of the paracellular pathway were examined in concentration step experiments. Decrease of the basolateral K⁺ concentration led to a rapid hyperpolarization of PD_bt with a mean slope of 19 mV per decade of K⁺ concentration change. Addition of 0.5 mmol · l^–1 Ba²⁺ to the bath solution lead to a marked depolarization and abolished the response to K⁺ concentration steps. In the lumen a Cl^– concentration downward step led to a depolarization of the lumen membrane; resulting in a mean slope of 18 mV per decade of Cl^– concentration change. When dilution potentials were generated across the epithelium, the polarity indicated that the paracellular pathway is cation selective. In series 3 the equivalent short circuit current (I_sc=PD_te/R_te) was determined as a function of symmetrical changes in Na⁺ concentration, with Cl^– held at 276 mmol · l^–1, and as a function of symmetrical changes in Cl^– concentration, with Na⁺ held at 278 mmol · l^–1 I_sc was a saturable function of Na⁺ concentration (Hill coefficient 0.9±0.1,K _1/2 4.4 mmol · l^–1,n=7) and also a saturable function of Cl^– concentration (Hill coefficient 2.0±0.1,K _1/2 75 mmol · l^–1,n=11). These data are compatible with the assumption that the carrier responsible for NaCl uptake has a 1 Na⁺ per 2 Cl^– stoichiometry. In series 4, the effect of a K⁺ concentration downward step on PD_bl anda _Cl– ^cell transients was followed with high time resolution in the presence and absence of basolateral furosemide (5 · 10^–5 to 10^–4 mol · l^–1) in an attempt to examine whether K⁺ reduction on the bath side inhibits Na⁺Cl^– uptake by the carrier system as does e.g. furosemide. The data indicate that removal of K⁺ from the bath side exerts an effect comparable to that of furosemide, i.e. it inhibits the carrier. We conclude that NaCl secretion in the RGT cell comprises at the least the following components: In the basolateral membrane, the (Na⁺+K⁺)-ATPase, probably the Na⁺ 2 Cl^–K⁺ carrier, and a K⁺ conductance. In the apical membrane a Cl^– conductance; and a Na⁺ conductive paracellular pathway.Supported by Deutsche Forschungsgemeinschaft DFG-Gr 480/8-1. Parts of this study have been presented at the 3rd International Symposium on Ion Selective Electrodes, Burg Rabenstein 1983, 16th Annual Meeting American Society of Nephrology, Washington DC 1983, 49th Tagung der Deutschen Physiologischen Gesellschaft, Dortmund 1984. A summary of the present study was published in Bulletin Mount Desert Island Biological Laboratory (Vol. 83)     

Phasic changes in intracellular pH during action potentials of sheep Purkinje fibres

Regulation of intracellular pH (pH_i) and the relationship between H⁺ and Ca²⁺ may vary during activity. Ion-selective microelectrodes were used to record pH_i during action potentials of sheep Purkinje fibres prolonged by low temperature (21°C) and elevated CO₂ content. Intracellular pH also was measured during changes in extracellular calcium concentration, [Ca²⁺]_o. Cytosolic alkalinization (peak pH_i change, 0.03–0.05) was observed during the long action-potential plateau and transient acidification (0.01–0.02 units) upon repolarization. Potassium-induced depolarization to plateau potentials (i.e. to –15±2 mV) simulated the peak magnitude of the alkalinization. However, compensation for the alkalinization occurred at a faster rate during the action potential (8.9±4.3 nM/min) than during K⁺ depolarization (1.2±0.5 nM/min). In comparison, the cytoplasm acidified in resting fibres (0.06–0.07 log units) during changes of [Ca²⁺]_o thought to increase intracellular calcium concentration. Alterations of pH_i were translated into changes of proton concentration ([H⁺]_i). Ten-to twenty-fold elevation of [Ca²⁺]_o evoked a comparable change in [H⁺]_i (mean increase, 5.7 nM) but oppositely directed from that during the plateau (mean decrease, 8.8 nM). The findings in resting fibres seem consistent with displacement of bound protons by Ca²⁺. In contrast, the initial change in pH_i during the plateau is proposed to be consequent to Ca²⁺-release from sarcoplasmic reticulum and/or phosphocreatine hydrolysis coupled to ATP regeneration.     

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.     

Proton transport mechanism in the cell membrane of Xenopus laevis oocytes

Mechanisms of H⁺ transport across the plasma cell membrane of prophase-arrested oocytes of Xenopus laevis were investigated by testing the effect of ion substitutions and inhibitors on cytoplasmic pH (pH_i), membrane potential (V _m) and membrane resistance (R _m). During superfusion with control solution of pH=7.4, pH_i was 7.49±0.12 (n=15), V _m was –61.9±7.8 mV (n=34) (cytoplasm negative), and R _m was 2.9±1.5 M (n=19). These data confirm that H⁺ ions are not distributed at electrochemical equilibrium. By following pH_i during recovery of the oocytes from an acid load (20 mmol/l NH₄Cl) in the presence and absence of extracellular Na⁺ or amiloride (1 mmol/l), a Na/H exchanger was identified. On the basis of the known Na⁺ gradient across the cell membrane, this transporter could suffice to generate the observed H⁺ disequilibrium distribution. Utilizing blockers or ion-concentration-step experiments no evidence was obtained for an ATP-driven H⁺ pump or for passive acid/base transporters such as H⁺ conductances or Na⁺ (HCO ₃ ^– )₃ cotransport. The membrane depolarization observed in response to extracellular acidification appeared to result from a pH-dependent, Ba²⁺-inhibitable K⁺ conductance.     

Effects of dinitrophenol on active-transport processes and cell membranes in the Malpighian tubule of Formica

In Formica Malpighian tubules KCl secretion is driven by a V-type H⁺ ATPase in the luminal membrane in parallel with a H⁺/K⁺ antiporter. The effect of the protonophore dinitrophenol (DNP) was investigated on the isolated, symmetrically perfused tubule. DNP was applied in two different concentrations: 0.2 mmol/l and 1 mmol/l. The effects were fast and rapidly reversible. The equivalent short-circuit current (I _sc) was reduced significantly to respectively 25±3% Cn=4) and –3±7% (n=11) of the control value when 0.2 mmol/ l or 1 mmol/l was added to the bath. When 1 mmol/l DNP was applied the transepithelial resistance (R _te) decreased significantly to 74±11% of the control value (n=11), and the luminal over basolateral voltage divider ratio (VDR), providing an estimate of luminal over basolateral membrane resistance, decreased to 37±12% of the control (n=6). A concentration of 1 mmol/l DNP was also applied from the lumen. The decrease in I _sc was significant, but much less pronounced (74±5% of control; n=6) and no significant changes in R _te and VDR were observed. It is argued that, when the concentration in the bath is high enough, DNP may cross the cell and have a protonophoric effect not only on the mitochondria but also across the luminal cell membrane explaining the drop in transepithelial and in relative luminal membrane resistance. The diminished effectiveness of DNP, when applied from the luminal side, suggests that the luminal membrane is somehow less permeable to toxic substances, but that DNP very rapidly enters the cell via the basolateral membrane and may bring about an initial protonophoric effect across this membrane.     

Mechanism of NaCl secretion in rectal gland tubules of spiny dogfish (Squalus acanthias)

Segments of rectal gland tubules (RGT) the spiny dogfish (Squalus acanthias) were perfused in vitro to study the cellular mechanism by which NaCl secretion is stimulated. Transepithelial PD (PD_te), transepithelial resistance (R_te), the PD across the basolateral membrane (PD_bl), the fractional resistance of the lumen membrane (FR₁), and the cellular activities for Cl^–, Na⁺, and K⁺ (a _x ^cell ) were measured. In series 1 the effects of stimulation (S) (dbcAMP 10^–4, adenosine 10^–4, and forskolin 10^–6 mol · l^–1) on these parameters were recorded and compared to nonstimulated state (NS). PD_te increased from –1.9±0.2 mV to –11.0±0.9 mV (n=51). PD_bI depolarized from –86±1 to –74±1.4 mV (n=52). R_te fell from 29±2.8 to 21±2 cm² (n=23), and FR₁ fell from 0.96±0.005 to 0.79±0.04 (n=9).a _K+ ^cell was constant (123±13 versus 128±17 mmol · 1^–1) (n=6), buta _Cl– ^cell -fell significantly from 48±4 to 41±3 mmol · l^–1 (n=7).a _Na+ ^cell increased from 11±2.1 to 29.5±6.6 mmol · l^–1 (n=4). In series 2 the conductivity properties were examined by rapid K⁺, and Cl^– concentration steps on the basolateral and luminal cell side respectively in NS and S states. In NS-segments reduction of bath K⁺ led to a hyperpolarization of PD_bI with a mean slope of 28±1.3 mV/decade (n=9) (as compared to 19 mV/decade for S-state). Reduction of lumen Cl^– led to very little depolarization of the lumen membrane PD in NS-state: 6.5±2.3 mV/decade (n=4) (as compared to 13 mV/decade for S-state). In series 3 the effects of furosemide (7 · 10^–5 mol l^–1, bath) were examined in NS and S tubules. In NS RGT segments furosemide had no effect on PD_bI or PD_te;a _Cl– ^cell fell slowly after furosemide with an initial rate of 0.33 mmol · l^–1 s^–1, as compared to 1.5 mmol · l^–1 · s^–1 for S-state. The increase ina _Cl– ^cell after removal of furosemide from NS to S-states was examined in the presence of furosemide. Despite the presence of furosemide stimulation was accompanied by a fall in R_te, FR₁, anda _Cl– ^cell . From these data we conclude that (a) stimulation by cyclic AMP increases the Cl^–-conductance of the apical cell membrane at least by a factor of 10, that (b) in the NS-state the Na⁺2Cl^–K⁺ carrier can be triggered to work at rates similar to the S state by loweringa _Cl– ^cell , and that (c) the increase in apical Cl^–-conductance is the primary event in cyclic AMP mediated stimulation of NaCl secretion.Supported by Deutsche Forschungsgemeinschaft Gr 480/8-1, and by NIH Grant AM 34208     

Electrophysiological study of transport systems in isolated perfused pancreatic ducts: properties of the basolateral membrane

In order to study the mechanism of pancreatic HCO ₃ ^– transport, a perfused preparation of isolated intra-and interlobular ducts (i.d. 20–40 m) of rat pancreas was developed. Responses of the epithelium to changes in the bath ionic concentration and to addition of transport inhibitors was monitored by electrophysiological techniques. In this report some properties of the basolateral membrane of pancreatic duct cells are described. The transepithelial potential difference (PD_te) in ducts bathed in HCO ₃ ^– -free and HCO ₃ ^– -containing solution was –0.8 and –2.6 mV, respectively. The equivalent short circuit current (I_sc) under similar conditions was 26 and 50 A·cm^–2. The specific transepithelial resistance (R_te) was 88 cm². In control solutions the PD across the basolateral membrane (PD_bl) was –63±1 mV (n=314). Ouabain (3 mmol/l) depolarized PD_bl by 4.8±1.1 mV (n=6) within less than 10 s. When the bath K⁺ concentration was increased from 5 to 20 mmol/l, PD_bl depolarized by 15.9±0.9 mV (n=50). The same K⁺ concentration step had no effect on PD_bl if the ducts were exposed to Ba²⁺, a K⁺ channel blocker. Application of Ba²⁺ (1 mmol/l) alone depolarized PD_bl by 26.4±1.4 mV (n=19), while another K⁺ channel blocker TEA⁺ (50 mmol/l) depolarized PD_bl only by 7.7±2.0 mV (n=9). Addition of amiloride (1 mmol/l) to the bath caused 3–4 mV depolarization of PD_bl. Furosemide (0.1 mmol/l) and SITS (0.1 mmol/l) had no effect on PD_bl. An increase in the bath HCO ₃ ^– concentration from 0 to 25 mmol/l produced fast and sustained depolarization of PD_bl by 8.5±1.0 mV (n=149). It was investigated whether the effect of HCO ₃ ^– was due to a Na⁺⁺-dependent transport mechanism on the basolateral membrane, where the ion complex transferred into the cell would be positively charged, or whether it was due to decreased K⁺ conductance caused by lowered intracellular pH. Experiments showed that the HCO ₃ ^– effect was present even when the bath Na⁺ concentration was reduced to a nominal value of 0 mmol/l. Similarly, the HCO ₃ ^– effect remained unchanged after Ba²⁺ (5 mmol/l) was added to the bath. The results indicate that on the basolateral membrane of duct cells there is a ouabain sensitive (Na⁺+K⁺)-ATPase, a Ba²⁺ sensitive K⁺ conductance and an amiloride sensitive Na⁺/H⁺ antiport. The HCO ₃ ^– effect on PD_bl is most likely due to rheogenic anion exit across the luminal membrane.     

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     

Cell pH of rat renal proximal tubule in vivo and the conductive nature of peritubular HCO 3 − (OH−) exit

Intracellular pH (pH_c) was measured on surface loops of rat kidney proximal tubules under free-flow conditions in vivo using fine tip double-barrelled pH microelectrodes based on a neutral H⁺ ligand. The microelectrodes had Nernstian slopes and a resistance of the order of 10¹² . By using a driven shield feed back circuit the response time to pH jumps was lowered to around 1 s. At a peritubular pH of 7.42 and a luminal pH of 6.68 ± 0.13 (n=27), pH_c was 7.17 ± 0.08 (n=19). Perfusing the peritubular capillaries suddenly with bicarbonate Ringer solutions of plasma-like composition which were equilibrated with high or low CO₂ pressures, acidified or respectively alkalinized the cells rapidly as expected from the high CO₂ permeability of the cell membranes. Such data allowed us to calculate the cytoplasmic buffering power of the tubular cells. Sudden peritubular perfusion with Ringer solution containing only 3 mmol/l of HCO ₃ ^– at constant physiological CO₂ pressure led to a similar fast cell acidification which indicated that the peritubular cell membrane is also highly permeable for bicarbonate or OH^– (H⁺). The latter response was completely blocked by the stilbene derivative SITS at the concentration of 10^–3 mol/l. The observations indicate first that pH_c of rat proximal tubule is more acidic than was previously thought on the basis of distribution studies of weak acids, second that intracellular bicarbonate concentration is around 13 mmol/l and third that bicarbonate exit across the peritubular cell membrane is a passive rheogenic process via a conductive pathway which can be inhibited by SITS. The latter point confirms the conclusion which we had derived previously from membrane potential measurements in response to changing peritubular bicarbonate concentrations.