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.     

Evidence for a Na+/H+ exchanger at the basolateral membranes of the isolated frog skin epithelium: Effect of amiloride analogues

We have investigated the possible existence of a Na⁺/H⁺ ion exchanger in the frog skin epithelium by using isotopic methods and two amiloride analogues: 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and phenamil. We found phenamil to be a specific blocker of sodium entry to its cellular transport compartment since it inhibited both the transepithelial Na⁺ influxes (J ₁₃) with aK _I of 4·10^–7 mol/l and the Na⁺ pool (control: 77±4 neq·h^–1·cm^–2; phenamil: 21±1 neq·h^–1·cm^–2). On the contrary EIPA (10^–5 mol/l) had no effect onJ ₁₃ nor on the apical Na⁺ conductance. Acidification of the epithelium by passing from a normal Ringer (25 mmol/l HCO ₃ ^– , 5% CO₂, pH 7.34) to a HCO ₃ ^– -free Ringer (5% CO₂, pH 6.20) while blocking the Na⁺ conductance with phenamil, produced a large stimulation of Na⁺ influxes exclusively across the basolateral membranes (J ₃₂), after return to a normal Ringer (J ₃₂=706±76 and 1635±199 neq·h^–1·cm^–2 in control and acid-loaded epithelia respectively). The stimulation ofJ ₃₂ was initiated when the epithelia were acid-loaded with Ringer of pH lower than 6.90 and was blocked by amiloride (K _I=7·10^–6 mol/l) and EIPA (K _I=5·10^–7 mol/l) whereas phenamil had no effect. In na⁺-loaded epithelia (ouabain treated) the Na⁺ efflux across the basolateral membranes was stimulated by an inwardly directed proton gradient and was blocked by EIPA (10^–5 mol/l) or amiloride (10^–4 mol/l), a result suggesting reversibility of the mechanism. We conclude that a Na⁺ permeability mediated by a Na⁺/H⁺ ion exchanger exists in the basolateral membranes, which is stimulated by intracellular acidification and is sensitive to amiloride or EIPA. This exchanger is proposed to be involved in intracellular pH regulation.     

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.     

Stimulation of colonic anion secretion by monochloramine: action sites

During inflammatory bowel disease, reactive oxygen metabolites are released by phagocytes reacting with intraluminal NH₃ to produce monochloramine (NH₂Cl). NH₂Cl is assumed to play role in the pathogenesis of inflammation-associated diarrhoea, as it is able to induce intestinal secretion. The aim of the present study was to determine the action sites of NH₂Cl in rat colonic epithelium with Ussing chamber and fura-2 experiments. In intact mucosa, NH₂Cl (5·10^–6–10^–4 mol·l^–1) evoked a concentration-dependent increase in short-circuit current (I_sc), consistent with the induction of anion secretion, as demonstrated by anion substitution and transport blocker experiments. When the apical membrane was permeabilised by the ionophore nystatin, two basolateral action sites of NH₂Cl (5·10^–5 mol·l^–1) could be identified, i.e. an increase in the K⁺ conductance and a stimulation of the Na⁺–K⁺ pump. When tissues were basolaterally depolarised by a high K⁺ concentration, the stimulation of an apical Cl^– conductance by NH₂Cl was observed. In isolated colonic crypts loaded with the Ca²⁺-sensitive fluorescent dye fura-2, NH₂Cl (5·10^–5 mol·l^–1) evoked an increase in the intracellular Ca²⁺ concentration. This increase was independent from the presence of Ca²⁺ in the extracellular medium, but was inhibited by blockade of intracellular sarcoplasmatic, endoplasmatic Ca²⁺-ATPases with cyclopiazonic acid (10^–5 mol·l^–1). The NH₂Cl-evoked Ca²⁺ release was sensitive against inhibition of ryanodine receptors with ruthenium red (5·10^–5 mol·l^–1) and against inhibition of inositol-1,4,5-trisphosphate (IP₃) receptors with 2-aminoethoxydiphenylborate (10^–4 mol·l^–1). Both blockers also inhibited the NH₂Cl-induced increase in I_sc. These results indicate that an intracellular Ca²⁺ release via ryanodine and/or IP₃ receptors is involved in oxidant stimulation of anion secretion in rat colon.     

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.     

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.     

Na+-Ca2+ exchange in the isolated cochlear outer hair cells of the guinea-pig studied by fluorescence image microscopy

The outer hair cell isolated from the guinea-pig was superfused in vitro and the cytosolic calcium concentration ([Ca²⁺]_i) and sodium concentration ([Na⁺]_i) were measured using fluorescence indicators. Under the resting condition, [Ca²⁺]_i and [Na⁺]_i were 91±9 nM (n = 51) and 110±5 mM (n = 12), respectively. Removal of external Na⁺ by replacing with N-methyl-D-glucamine (NMDG⁺) increased [Ca²⁺]_i by 270±79% (n = 27) and decreased [Na⁺]_i by 23±4 mM (n = 6). Both changes in [Ca²⁺]_i and [Na⁺]_i were totally reversible on returning external Na⁺ to the initial value and were inhibited by addition of 0.1 mM La³⁺ or 100 M amiloride 5-(N,N-dimethyl) hydrochloride. Elevation of external Ca²⁺ ions to 20 mM reversibly decreased [Na⁺]_i by 8±6 mM (n = 5). Moreover, the chelation of the intracellular Ca²⁺ with 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) exerted an inhibitory action on the NMDG⁺-induced reduction in [Na⁺]_i. Exposure to 5 mM NaCN for 2 min significantly and reversibly increased [Ca²⁺]_i by 290±37% (n = 5), but did not affect the [Ca²⁺]_i elevation induced by the NMDG⁺ solution. The rise in [Ca²⁺]_i induced by the NMDG⁺ solution was not enhanced by ouabain pretreatment. Addition of ouabain did not alter the [Na⁺]_i. The present results are best explained by the presence of an Na⁺-Ca²⁺ exchanger in cell membrane and indicate that the activity of Na⁺/K⁺ pump is poor in outer hair cells.     

Effect of olsalazine and mesalazine on human ileal and colonic (Na+ + K+)-ATPase

Summary Olsalazine (azodisalicylate) and mesalazine (5-aminosalicylic acid) have recently been developed as new treatment modalities for inflammatory bowel disease to avoid sulfasalazine-related side effects. However, there are reports regarding new and hitherto unexpected side effects in some patients receiving olsalazine or mesalazine, such as watery diarrhea. Since sodium pump activities play an important role in the pathogenesis of water and electrolyte disturbances, we investigated the influence of olsalazine and mesalazine on human ileal and colonic (Na⁺ + K⁺)-ATPase and its specific [³H]-ouabain binding. We found a concentration-dependent inhibition of ileal and colonic (Na⁺ + K⁺)-ATPase by olsalazine with an IC₅₀ of 4.1 mM and 4.8 mM, respectively. Mesalazine inhibited this enzyme in the ileum with an IC₅₀ of 4.5 mM and in the sigmoid colon with an IC₅₀ 3.5 mM. In addition, [³H]-ouabain binding was inhibited by mesalazine with an IC₅₀ of 3.6 mM. The maximal inhibition, however, did not exceed 80% under any conditions (up to 10 mM drug concentration). Olsalazine and mesalazine induce inhibition of the ileal and colonic sodium pump activities that may (in addition to other possible mechanisms) mediate impaired water and electrolyte absorption. This is possibly of clinical relevance in patients with severely damaged mucosa. In patients with milder forms of mucosal inflammation, this inhibition most likely is of minor importance because of the great capacitiy of the (Na⁺ + K⁺)-ATPase and the incomplete inhibition leaving at least 20% of the enzyme activity intact.Abbreviations 5-ASA 5-aminosalicylic acid - EDTA ethylenediaminetetracetic acid - IBD inflammatory bowel disease     

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     

Effects of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells

Kinetic properties of the Na⁺-H⁺ antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pH_i) and Na⁺ activity (aNa_i) with the aid of double-barreled H⁺- and Na⁺-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pH_i was 7.12±0.01 and aNa_i was 6.7±0.6 mmol/l. The cells were acid-loaded by exposure to an NH ₄ ⁺ solution followed by an Na⁺-free N-methyl-d-glucamine (NMDG⁺) solution. Intracellular Na⁺ and H⁺ concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na⁺ solution rapidly increased pH_i. This Na⁺-induced increase in pH_i was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNa_i. The rate of pH_i recovery induced by the standard Na⁺ solution increased in a saturable manner as pH_i decreased, and was negligible at pH_i 7.2–7.3, indicating an inactivation of the Na⁺-H⁺ antiport. The apparent K _m for intracellular H⁺ concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNa_i to less than 1 mmol/l by prolonged exposure to NMDG⁺ solution significantly increased the rate of Na⁺-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na⁺ concentration increased following Michaelis-Menten kinetics (V _max was 0.55 pH/min and the apparent K _m was 75 mmol/l at pH_i 6.88). The results clearly showed that the Na⁺-H⁺ antiport activity is dependent on the chemical potential gradient of both Na⁺ and H⁺ ions across the basolateral membrane, and that the antiporter is asymmetric with respect to the substrate affinity of the transport site. The data agree with the current model of activation and inactivation of the antiporter by an intracellular site through changes in the intracellular Na⁺ and H⁺ concentrations.     

Occurrence and properties of a (Na+−K+)-activated ATPase in the mucosa of the rat intestine

Summary The existence of an ouabain-sensitive (Na⁺–K⁺)-activated ATPase system has been demonstrated in the total intestine of the rat. The (Na⁺–K⁺)-ATPase activity was about 10–15% of the total ATPase in 4 equal parts of the small intestine; in the colon about 35% of the total ATPase was (Na⁺–K⁺)-activated ATPase. The highest (Na⁺–K⁺)-ATPase activity has been observed in the first and second part of the small intestine, while in the colon the activity was 2–4 times higher than in the ileum.The (Na⁺–K⁺)-ATPase of rat colon required both Na⁺ (K _m=8.3 mmoles/l) and K⁺ (K _m=0.6 mmoles/l). Maximal activation of the (Na⁺–K⁺)-ATPase system required 2 mM Mg²⁺ at an ATP concentration of 2 mM. The pH optimum for (Na⁺–K⁺)-ATPase of rat colon was 7.5, while the Mg²⁺-activated ATPase activity had a pH optimum of 8.6. The (Na⁺–K⁺)-ATPase was inhibited by ouabain (pI ₅₀=3.6).The relation between the differences in (Na⁺–K⁺)-ATPase activity and Na⁺-absorption on different parts of the intestine is discussed.     

(Na+K+)-activated ATPase in human cornea

Summary Distribution and principal characteristics of (Na⁺K⁺)-activated ATPase in human cornea were investigated.(Na⁺K⁺)-ATPase was present in both epithelium and endothelium, whereas the corneal stroma did not exhibit significant enzyme activity.In homogenates specific activity of the (Na⁺K⁺)-ATPase was 2.3-fold higher in endothelium than in epithelium. Calculation of total enzyme activity revealed a 6.1-fold higher content of (Na⁺K⁺)-ATPase in the epithelium.In the epithelium a 7-fold enrichment of (Na⁺K⁺)-ATPase compared to the homogenate was obtained in the 150–1500×g _av fraction. Maximum enrichment in the endothelium was 3.5-fold and was achieved in the 1500–2500×g _av fraction. Both fractions showed, however, the same specific activity.The pH-optimum of (Na⁺K⁺)-ATPase in the 150–1500×g _av fraction ranged from 8.0–8.2 in both epithelium and endothelium.In the epithelial 150–1500×g _av fraction the apparentK _m-values were 4.0 mM for Na⁺, 2.8 mM for K⁺ and 0.12 mM for Mg²⁺ · ATP in equimolar concentrations.The inhibition constant of epithelial (Na⁺K⁺)-ATPase for ouabain was determined asK _i=3.3×10^–7 M.The present data support the view that control of corneal hydration in man is a function of both endothelium and epithelium.     

Sodium movement into and out of corneal endothelium

Rabbit corneal endothelial cells mounted in vitro were impaled simultaneously with Na⁺-selective and conventional KCl-filled microelectrodes. The membrane potential (V _m) was –30.4±0.8 mV (mean ±SEM, n = 55) and the intracellular [Na⁺]_i (calculated from the Na⁺-selective electrode potential, V_Na) was 13.7 ±1.9 mM (mean±SEM, n = 16). When ouabain was added to the perfusate the cell depolarised, causing both V _m and V_Na to increase with a very similar time course. Final V _m was –6.3±0.6 mV (mean ±SEM, n = 15), and the final [Na⁺]_i was 114±6.9 mM (mean ± SEM, n = 5). The parallel increase in V _m and rise in [Na⁺]_i suggest that a component of the ouabain-induced depolarisation of the cell (increase in V _m) is due to Na⁺ entry into the cell down its concentration gradient. The lateral and basal location of the Na⁺/K⁺-ATPase in bovine endothelial cells was confirmed (for the first time at the electron-microscopic level) using a monoclonal antibody specific for the 1 subunit of Na⁺/K⁺-ATPase. The absence of a net Na⁺ flux across these cells combined with the basolateral location of the ATPase suggest that Na⁺ exit from the cell, and its re-entry take place across the same membrane (i. e. the basolateral).     

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)     

The effect of ouabain on intracellular activities of K+, Na+, Cl−, H+ and Ca2+ in proximal tubules of frog kidneys

Using conventional and ion selective microelectrodes, the effect of ouabain (10^–4 mol/l) on peritubular cell membrane potential (PD_pt), on intracellular pH (pH_i) as well as on the intracellular ion activities of Cl^– (Cl _i ^– ), K⁺ (K _i ⁺ ), Na⁺ (Na _i ⁺ ) and Ca²⁺ (Ca _i ²⁺ ) was studied in proximal tubules of the isolated perfused frog kidney. In the absence of ouabain (PD_pt=–57.0±1.9 mV), the electrochemical potential difference of chloride (apparent {ie6-1} and of potassium {ie6-2} is directed from cell to bath, of H⁺ {ie6-3}, of Na⁺ {ie6-4} and of Ca²⁺ {ie6-5} from bath to cell. Ouabain leads to a gradual decline of PD_pt, which is reduced to half (PD_{pt, 1/2}) within 31±4.6 min (in presence of luminal glucose and phenylalanine), and to a decline of the absolute values of apparent {ie6-6}, of {ie6-7}, {ie6-8} and {ie6-9}. In contrast, an increase of {ei6-10} is observed. At PD_{pt, 1/2} apparent Cl _i ^– increases by 6.2±1.0 mmol/l, pH_i by 0.13±0.03, Ca _i ²⁺ by 185±21 nmol/l, and Na _i ⁺ by 34.2±4.6 mmol/l, whereas K _i ⁺ decreases by 37.7±2.2 mmol/l. The results suggest that the application of ouabain is followed by a decrease of peritubular cell membrane permeability to K⁺, by an accumulation of Ca²⁺, Na⁺ and HCO ₃ ^- in the cell and by a dissipation of the electrochemical Cl^– gradient.Supported by Österr. Forschungsrat, Proj. No. 4366     

Effects of dopamine on ion transport across the rat distal colon

Dopamine (5·10^–6–5·10^–4 M) induced a concentration-dependent decrease in short-circuit current (I_sc) across the rat distal colon. This response was preceded by a transient and inconsistent increase in I_sc. The -adrenoceptor blocker phentolamine and the inhibitors of dopamine-2-like (D₂-like) receptors L-741,626 and L-745,870 inhibited the dopamine response, suggesting a contribution of adrenergic and dopaminergic receptors. The decrease in I_sc evoked by dopamine was inhibited by bumetanide, an inhibitor of the basolateral Na⁺-K⁺-2 Cl^– cotransporter responsible for the uptake of K⁺, and by quinine, a blocker of apical K⁺ channels, indicating that stimulation of K⁺ secretion contributes to the measured change in I_sc. In patch-clamp experiments dopamine hyperpolarized the membrane and increased cellular K⁺ current. This response was not concomitant with a change in the intracellular [Ca²⁺] as demonstrated in parallel fura-2 experiments. These results demonstrate that dopamine, like other catecholamines, stimulates colonic K⁺ secretion.     

Ba2+ and amiloride uncover or induce a pH-sensitive and a Na+ or non-selective cation conductance in transitional cells of the inner ear

The membrane potential V _m the cytosolic pH (pH_i), the transference numbers (t) for K⁺, Cl^– and Na⁺/ non-selective cation (NSC) and the pH-sensitivity of V _m were investigated in transitional cells from the vestibular labyrinth of the gerbil. V _m, pH_i, , and the pH_i sensitivity of V _m were under control conditions were –92±1 mV (n=89 cells), pH_i 7.13±0.07 (n=11 epithelia), 0.87±0.02 (n=22), 0.02±0.01 (n=19), 0.01±0.01 (n=24) and –5 mV/pH unit (n=13 cells/n=11 epithelia), respectively. In the presence of 100 mol/l Ba²⁺ the corresponding values were: –70±1 mV (n=32), pH_i 7.16±0.08 (n=6), 0.31±0.05 (n=4), 0.06±0.01 (n=6), 0.20±0.03 (n=10) and -16 mV/pH-unit (n=15/n=6). In the presence of 500 mol/l amiloride the corresponding values were: –72±2mV (n=34), pH_i 7.00±0.07 (n=5), 0.50±0.04 (n=6), 0.04±0.01 (n=11), 0.28±0.04 (n=9) and –26 mV/pH-unit (n=20/n=5). In the presence of 20 mmol/l propionate plus amiloride the corresponding values were: –61±2 mV (n=27), pH_i 6.72±0.06 (n=5), 0.30±0.02 (n=6), 0.06±0.01 (n=5) and 0.40±0.02 (n=8), respectively. V _m was depolarized and and pH_i decreased due to (a) addition of 1 mmol/l amiloride in 150 mmol/l Na⁺ by 38±1 mV (n=8), from 0.82±0.02 to 0.17±0.02 (n=8) and by 0.13±0.01 pH unit (n=6), respectively; (b) reduction of [Na⁺] from 150 to 1.5 mmol/l by 3.3±0.5 mV (n=30), from 0.83±0.02 to 0.75±0.04 (n=9) and by 0.33±0.07 pH unit (n=4), respectively and (c) addition of 1 mmol/l amiloride in 1.5 mmol/l Na⁺ by 20±1 mV (n=11) and from 0.83±0.03 to 0.53±0.02 (n=5), respectively. These data suggest that the K⁺ conductance is directly inhibited by amiloride and Ba²⁺ and that Ba²⁺ and amiloride uncover or induce a pH-sensitive and a Na⁺/NSC conductance which may or may not be the same entity.Some of the data have been presented at various meetings and appear in abstract form in [31, 35, 37]     

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     

Inhibition of voltage-dependent Na+ and K+ currents by forskolin in nodes of Ranvier

The effect of forskolin on voltage-activated Na⁺ and K⁺ currents in nodes of Ranvier from the toad, Bufo marinus, has been examined using the vaseline-gap voltageclamp technique. Peak Na⁺ currents (I _Na) were reduced by 35% and the rate of decline of Na⁺ current during continuous depolarization was accelerated following treatment with 450 M forskolin. However, the voltage-dependence of steady-state inactivation as well as the rate of recovery from fast inactivation remained unchanged. Upon repetitive depolarization at 1–10 Hz, a further inhibition of I _Na (60%) was observed. This use-dependent or phasic inhibition recovers slowly at -80 mV ( 13 s) and had a voltage-dependence like that of activation of the Na conductance. Near maximal steady-state phasic inhibition occurred with depolarizing pulse durations of only 4 ms, consistent with a direct involvement of the open Na⁺ channel in the blocking process. Inhibition of the delayed K⁺ current (I _K) was characterized by a concentration-dependent reduction in steady-state current amplitude (IC₅₀ 80 M) and a concentration-independent acceleration of current inactivation. A similar inhibition of I _K was obtained with 1,9-dideoxyforskolin, a homolog which does not activate adenylate cyclase (AC). The results suggest that the inhibition of I _K and perhaps I _Na follows directly from drug binding and is not a consequence of AC activation.     

Na+-Li+ countertransport and electrolyte composition in erythrocytes of patients with essential hypertension before and after antihypertensive treatment

Summary The effect of antihypertensive treatment with 6–12 mg of the loop diuretic piretanide over 12 weeks on Na⁺-Li⁺ countertransport, and on extra- and intracellular electrolyte composition was studied in 10 previously untreated patients with essential hypertension. These data were compared with 10 sex- and age-matched controls. Blood pressure fell from 180±18.3/110.5±9.8 to 154.7±9.7/92.5±10.9 mmHg during treatment. Na⁺-Li⁺ countertransport was significantly higher in hypertensives (0.36±0.13 mmol×l _rbc ^–1 ×h^–1) compared with controls (0.25±0.05;P<0.05).Na⁺-Li⁺ countertransport and intracellular electrolyte composition remained unchanged whereas the extracellular potassium concentration fell from 4.28±0.51 to 3.98±0.36 mmol/l (P<0.05). No increase in the intracellular Na⁺ content as compared with normotensive controls was found. It is concluded that the intracellular Na⁺ concentration is not a marker for essential hypertension. The Na⁺-Li⁺ countertransport does not seem to be directly related to elevated blood pressure but seems to be a general marker for diseases associated with an increased risk for the development of hypertension.Supported by the DFG (Er 65/4-4)