Effects of standard diuretics and RPH 2823 on transepithelial Na+ transport in isolated frog skin

Summary Short-circuit current (SCC) techniques were used to monitor the effects of various diuretic agents on Na⁺ transport in isolated frog skin, a model for the late distal tubule and the collecting duct of the mammalian kidney. Acctazolamide, hydrochlorothiazide, torasemide, and ethacrynic acid did not affect sodium transport (as indicated by the SCC) or transepithelial electrical resistance when added either to the apical (outer) or to the inner (basolateral, corial) bathing solution of the tissue. However, Na⁺ transport was sensitive to amiloride, the triamterene derivate dimethylamino-hydroxypropoxytriamterene (RPH 2823), and to furosemide. Whereas apical amiloride, and RPH 2823 induced a dose-dependent decrease in SCC and increase in transepithelial electrical resistance, apical furosemide resulted in a dose-dependent increase in SCC and a decrease in electrical resistance. None of the three diuretic agents caused a significant change in SCC when applied to the inner bathing Ringer's solution. The small furosemide-induced decrease in resistance compared with the huge increase in SCC suggests that furosemide affects Cl^– permeability as well as Na⁺ permeability. Evidence for this notion was achieved by the following findings: (a) The decrease in resistance after furosemide was more pronounced in tissues bathed in Cl^–-free solutions compared with Cl^–-containing solutions. (b) In contrast, SCC stimulation by apical furosemide is Cl^–-ion independent, but strongly Na⁺-ion dependent. (c) SCC stimulation by furosemide is amiloride-sensitive. With respect to the onset, locus, and reversibility of action, it seems reasonable to assume that amiloride, RPH 2823, and furosemide all influence transepithelial Na⁺ transport by interacting with the Na⁺ channel or a regulator site of it within the apical membrane. The stoichiometry of the amiloride (RPH 2823)-receptor site interaction revealed Hill-coefficient(s) of less than 1, indicating a negative cooperativity among the receptor sites. The interaction between Na⁺ ions and amiloride or RPH 2823 displayed mixed competitive-noncompetitive inhibition. Taken together, these results support the hypothesis that amiloride and Na⁺ as well as RPH 2823 and Na⁺ may act at different loci on the apical entry mechanism inRana esculenta skin.Abbreviations R Transepithelial electrical resistance - R _x /R₀ Transepithelial electrical resistance at timex (the time after manipulation) divided by the value at time 0 (the time before manipulation) - SCC Short-circuit current - SCC _x /SCC₀ Short-circuit current at timex (the time after manipulation) divided by the value at time 0 (the time before manipulation) Dedicated to Prof. Dr. F. Krück on the occasion of his 65th birthday     

The key role of the mitochondria-rich cell in Na+ and H+ transport across the frog skin epithelium

We have investigated the possibility that the mitochondria-rich (MR) cells participate in sodium and proton transport, when the frog skin epithelium is bathed on its apical side with solutions of low Na⁺ concentration, by comparing transport rates with morphological observations (MR cell number and MR cell pit surface area). Frogs were adapted to various salinities or the isolated skins were treated with the following hormones, deoxycorticosterone acetate (DOCA), arginine vasotocin (AVT) and oxytocin in order to modify the transport of sodium and hydrogen ions. Adaptation of the frogs (either 3–4 days or 7–10 days) to distilled water, NaCl (50 mmol/l), KCl (50 mmol/l) or Na₂SO₄ (25 mmol/l) solutions modified the Na⁺ transport rate and the morphology of the epithelium. The highest Na⁺ transport rates were found for the animals adapted to the Na⁺ free solutions and were correlated with an increase in the total MR cell pit-surface area (number of MR cells x individual cell pit-surface area). The KCl adaptated group showed the largest increase in sodium and proton transport and also presented a metabolic acidosis as reflected by plasma acidification (pCO₂ increase and HCO ₃ ^– decrease). Proton secretion and sodium absorption were also found to be stimulated by either serosal DOCA addition (10^–6 M) or during acidification of the epithelium by serosally applied CO₂. Na⁺ transport was enhanced by AVT (10^–6 M) or oxytocin (100mU/ml) when the skin was bathed on its apical side with a high Na⁺ containing solution (115 mmol/l), whereas these hormones did not exert any effect on Na⁺ transport when the apical solution was low in Na⁺ (0.5mmol/l). It is concluded that MR cells play a key role in Na⁺ and H⁺ transport through the frog skin epithelium when bathed on its apical side with a low Na⁺ containing solution. Distinct pathways for sodium transport through two cell types (MR cells and granular cells) are proposed depending on the Na⁺ concentration of the solution bathing the apical side of the epithelium.     

Multicompartment kinetic analysis of the amiloride block of Na+ fluxes in frog skin

Previous studies have shown that the flow of Na⁺ in a multicompartment, dual Na⁺ pump frog skin model is in all hitherto tested kinetic and thermodynamic properties consistent with observations made on skin. It is shown here, that this is also true for a simulated amiloride block by which entrance of Na⁺ into a transport compartment via two parallel pathways is stopped. By the method of computer simulation, the following results were obtained.Before amiloride, steady state transmembrane influx (nEq×cm^–2×min^–1)J ⁱ=21. EffluxJ ^e=0.5 in a tight model, and 3.9 in a model which is leaky in its paracellular pathway. Total epidermal Na⁺ pool (under net flux conditions (nEq/cm²)P ⁿ=330, or 160 in a model with a stronger Na⁺ maintenance pump. The steady state time was nearly 1 h.After amiloride, rapid decrease of uptake of Na⁺ from the outside, slower release of Na⁺ to the inside under influx conditions; half time (min)t _h=2.2 ifP ⁿ=330; 0.9 ifP ⁿ=160. A new steady state was reached in 12–15 min.J ^e decreased by 45% in the tight model. In the leaky model,J ^e increased by 3%. Loss of Na⁺ from the model under net flux conditions, P ⁿ=30 nEq/cm² ifP ⁿ=330, P ⁿ=16 ifP ⁿ=160. The kinetic analysis suggests new laboratory experiments, and detailed layouts of speculative, but plausible Na⁺ current fields. These include for all compartments, values for [Na⁺]varying from 21 (P ⁿ=160) to 63 (P ⁿ=330) mM. In the Na⁺ transport compartment, [Na⁺] decreased from 5.5 to 1.4 mM after the simulated amiloride block.     

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.     

Procaine has opposite effects on passive Na and K permeabilities in frog skin

Procaine has opposite effects on the active transport of Na⁺ when applied on the mucosal side of the frog skin [where it produces a stimulation of the short-circuit current (I _sc)] or when added on the serosal side (where it produces an inhibition ofI _sc). In an attempt to reveal and localize the primary effect of procaine on either the apical or latero-basal membranes of the epithelial cells, we have tried to chemically dissect both membrane functions with inhibitors and ionophores. When applied on the apical side of the latero-basally depolarized epithelium, 25 mmol/l procaine increasesI _sc andV _oc (transepithelial open-circuit potential), while decreasing the transepithelial resistance. TheE ₁–E ₂ linearity domain of the I–V curves is narrowed. On the serosal side of the depolarized epithelium, the same concentration of procaine does not affectI _sc andV _oc (which are already inhibited) but it produces an increase in the transepithelial resistance (R _t). Procaine influence on the passive K⁺ permeability was studied by using the ionophore nystatin, which is assumed to form channels permeable to K⁺, when applied on the amiloride blocked apical membrane. In nystatin-treated epithelia, 25 mmol/l procaine on the apical side decreaseI _sc,V _oc andR _t. In parallel experiments during Cl^– substitution by SO ₄ ^2– , the procaine effects onI _sc andV _oc are no longer maintained, but transient. The results suggest that procaine positively influences the Na⁺ transient through the apical Na⁺-channels, and inhibits the epithelial permeability for K⁺, possibly by reducing K⁺-ions accessibility to the K⁺-channels.     

Deviating flux rations for Na+ in ouabain-treated frog skin

Summary Unidirectional Na⁺ fluxes across ouabain-treated frog skins were measured at different applied voltages. The calculated influx/efflux ratios, appear to deviate markedly from Ussing's flux-ratio equation. This means that interactions of Na⁺ ions with some component in the system occur. Possible mechanisms, responsible for this phenomenon, are indicated.with technical assistance of Anneke Adams-Schrijen     

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     

Effect of Cs+, Li+ and Na+ on the potassium conductance and gating kinetics in the frog node of Ranvier

The effects of monovalent internal cations Cs⁺, Li⁺ and Na⁺ on potassium channel conductance in the frog node of Ranvier were studied by means of the voltage clamp. As previously reported, when 10–80% of the internal K⁺ was replaced by one of the above cations, the steady-state current-voltage relationship was significantly modified. The main effect was a voltage-dependent attenuation of the currents. We demonstrate that the current attenuation is associated with a change in the channel gating kinetics. For small depolarizations the kinetics can be described by the usual potassium conductance activation time constant, τ _n . However, under certain experimental conditions (e.g. substitution of the intracellular K⁺ with 10% Cs⁺), during larger depolarizations, stepping the membrane potential to values above 40–60 mV, the conductance develops with two time constants: τ _n and a new, slower time constant that, in contrast to τ _n , grows with membrane potential. These results can be explained by assuming that the catins may occupy two different sites in the channel; when the first site is occupied the channel is blocked, while occupation of the second site results in slowing of the gating kinetics in the affected channels.     

Regulation of the cytosolic pH set point for activation of the Na+/H+ antiport in human platelets: the roles of the Na+/Ca2+ exchange,the Na+-K+-2Cl− cotransport and cellular volume

To explore further the mechanisms that regulate the Na⁺/H⁺ antiport in human platelets, we examined the effect of Na⁺ pump inhibition by ouabain and K⁺ removal from the extracellular medium on parameters of this transport system. Treatment with ouabain resulted in increased cytosolic free Ca²⁺ and Na⁺, coupled with an alkaline shift in the cytosolic pH set point for the Na⁺/ H⁺ antiport. Inhibition of the Na⁺ pump by the removal of K⁺ from the medium increased the cytosolic Na⁺ but not the cytosolic Ca²⁺; yet this treatment also produced a substantial alkaline shift in the cytosolic pH set point for the Na⁺/H⁺ antiport. This effect appeared to relate to a decline in cellular volume and it was attenuated by the Na⁺-K⁺-2Cl^– cotransport inhibitor, bumetanide. These findings indicate: (a) a link between the Na⁺ pump and the Na⁺/H⁺ antiport, mediated by the Na⁺/Ca²⁺ exchange and the cytosolic free Ca²⁺, and (b) a link between the Na⁺/H⁺ antiport and the Na⁺-K⁺-2Cl^– cotransport through cellular volume.This work was supported by grants from the National Heart, Lung, and Blood Institute (HL34807, HL42856) and the American Diabetes Association. M. Kimura is a postdoctoral research fellow of the American Heart Association, New Jersey Affiliate     

The effects of ouabain on resting membrane potential and hyperpolarization-activated current in neonatal rat nodose ganglion neurons

To determine whether the responses of resting membrane potential (RMP) and hyperpolarization-activated current (IH) are altered by the application of ouabain, one of the Na+-K+ pump inhibitors, in neonatal rat small-diameter (<30microm) nodose ganglion (NG) neurons, we examined the effects of 1microM ouabain on those responses using perforated patch-clamp techniques. In current-clamp mode, the RMP was 40.2+/-1.6mV (n=31). Twenty of 31 cells tested were depolarized by ouabain application, and these responses were associated with an increase in the cell input resistance. In the remaining 11 cells studied, 3 showed hyperpolarization in response to ouabain and 8 showed no effect on RMP. In voltage-clamp mode, 1muM ouabain application enhanced the IH in all of 10 neurons examined. These results suggest that ouabain application at 1microM is capable of setting both the RMP level and the neuronal excitability in small-diameter NG neurons.     

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.     

A novel synergistic stimulation of Na+-transport across frog skin (Xenopus laevis) by external Cd2+- and Ca2+-ions

Isolated skin of the clawed frogXenopus laevis was mounted in an Ussing-chamber. The transcellular sodiumcurrent (I _Na) was identified either as amiloride-blockable (10^–3 mol/l) short-circuit current (I _SC), or by correctingI _SC for the shunt-current obtained with mucosal Tris. A dose of 10 mmol/l Cd²⁺ applied to the mucosal side increased the current by about 70%. The half-maximal effect was reached at a Cd²⁺-concentration of 2,6 mmol/l (in NaCl-Ringer). The quick and fully reversible effect of Cd²⁺ could not be seen when 10^–3 mol/l amiloride was placed in the outer, Na⁺-containing solution, nor when Na⁺ was replaced by Tris. This suggests that Cd²⁺ stimulatesI _Na. Cd²⁺ intefered with the Na⁺-current self-inhibition, and therefore with the saturation ofI _Na by increasing the apparent Michaelis constant (K _Na) of this process. The I _Na recline after stepping up mucosal [Na⁺] was much reduced in presence of Cd²⁺. Ca²⁺-ions on the mucosal side had an identical effect to Cd²⁺, and 10 mmol/l Ca²⁺ increaseI _Na by about 100%. The half-maximal effect was obtained with 4.4 mmol/l Ca²⁺. The mechanism ofI _Na-stimulation by Ca²⁺ did not seem to differ from that of Cd²⁺. Thus, although of low Na⁺-transport capacity,Xenopus skin appears to be as good a model for Na⁺-transporting epithelia asRanidae skin, with the exception of the calcium effect which, so far, has not been reported forRanidae.     

Experimental verification of a multicompartment Na+ -flow model of frog skin epidermis.

A re-investigation was undertaken to consider the rates of transepidermal Na⁺-influx and Na⁺-backflux in the undamaged, isolated normal and Ouabain poisoned skins of Rana pipiens. The experimentally obtained rate values agreed with predictions derived from a computer model of skin epidermis. Both laboratory results and data computed by the use of a multicompartment epidermis model led to the conclusion that Ouabain greatly affects the flow of Na⁺ via the cellular, and not via the extracellular pathways. This study is presented to demonstrate the usefulness of the computer simulation technique in the analysis of the kinetics of flow of material across an epithelial membrane of considerable anatomical complexity.     

Anemonia sulcata toxins modify activation and inactivation of Na+ currents in a crayfish neurone

1. The effects of three toxins (ATX I, II, III) isolated from the sea anemoneAnemonia sulcata were studied in the soma membrane of a crustacean neurone under voltage-clamp conditions. 2. All three toxins affected the action potentials and the Na⁺ currents in a similar manner. The lowest concentrations tested (10 nM, 20 nM and 50 nM for AtX I, II and III, respectively) had pronounced selective effects on the Na⁺ current. No effect on K⁺ or Ca²⁺ currents was observed with concentrations up to 5 M. 3. In the presence of ATX the Na⁺ inactivation was incomplete even with pulses of 700 ms length or strong depolarizing prepulses. 4. Besides the effects on the inactivation process ATX affected also the activation of the Na⁺ current. 5. In cells treated with ATX the negative resistance branch of the peak Na⁺ current voltage relation was shifted by –5 mV to –20 mV. 6. The time to peak was increased for small depolarizations (up to –30 mV) and the rate of rise (I/t) was enlarged by ATX. A slow activating current component was also observed after depolarizing prepulses or if the Na⁺ current was outward. 7. The decay of the Na⁺ tail currents was considerably prolonged after the application of ATX if the membrane was repolarized to potentials more positive than about –60 mV. 8. Repetitive stimulation led to a shortening of the action potential in ATX II treated neurones. A simultaneous and parallel decrement of the peak and plateau current was observed with depolarizing voltage steps.     

Activation of luminal Na+/H+ exchange in distal nephron of frog kidney

Increased chronic intake of K⁺ induced H⁺ and K⁺ secretion in amphibian distal tubule, paralleled by an elevation of plasma aldosterone. The present experiments test whether the mineralocorticoid hormone is responsible for the alteration of ion transport. The blood capillaries of the isolated kidneys of NaCl-adapted (i.e. aldosterone-suppressed)Rana pipiens were perfused with HEPES-buffered amphibian Ringer solution (pH 7.8). Limiting intraluminal pH (pH_1u) was measured continuously with pH-sensitive microelectrodes while aldosterone (3·10^–7 to 3·10^–6 mol/l) was applied in the peritubular perfusate. Concomitant with a decrease of the lumen-positive transepithelial potential (V _te) from 8.5±1.1 mV to 4.0±0.6 mV pH_1u dropped from 7.73±0.02 to a new steady-state value of 7.17±0.05 within 60 to 180 min of aldosterone administration. Significant luminal acidification occurred already 20 min after application of aldosterone. Luminal addition of 10^–3 mol/l amiloride reversed luminal acidification to a pH_1u of 7.68±0.04; at the same timeV _te recovered partially. Pretreatment of the distal tubules with spironolactone prevented the aldosterone-induced acidification of the tubule fluid. We conclude that in early distal tubule of the amphibian kidney aldosterone — after interaction with cytoplasmic receptors — activates the luminal, amiloride-inhibitable Na⁺/H⁺ exchanger. This mechanism could explain enhanced H⁺ secretion found in the K⁺ adapted animal.     

Modification of cardiac Na+ channels by anthopleurin-A: effects on gating and kinetics

We used the whole cell patch clamp technique to investigate the characteristics of modification of cardiac Na⁺ channel gating by the sea anemone polypeptide toxin anthopleurin-A (AP-A). Guinea pig ventricular myocytes were isolated enzymatically using a retrograde perfusion apparatus. Holding potential was –140 mV and test potentials ranged from –100 to + 40 mV (pulse duration 100 or 1000 ms). AP-A (50–100 nM) markedly slowed the rate of decay of Na⁺ current (I _Na) and increased peak I _Na conductance (g _Na) by 38±5.5% (mean±SEM, P < 0.001, n = 12) with little change in slope factor (n = 12) or voltage midpoint of the g _Na/V relationship after correction for spontaneous shifts. The voltage dependence of steady-state I _Na availability (h ) demonstrated an increase in slope factor from 5.9±0.8 mV in control to 8.0±0.7 mV after modification by AP-A (P < 0.01, n = 14) whereas any shift in the voltage midpoint of this relationship could be accounted for by a spontaneous time-dependent shift. AP-A-modified I _Na showed a use-dependent decrease in peak current amplitude (interpulse interval 500 ms) when pulse duration was 100 ms (–15±2%, P < 0.01, n = 17) but showed no decline when pulse duration was 100 ms (–3±1%). This use-dependent effect was probably the result of a decrease in the rate of recovery from inactivation caused by AP-A which had a small effect on the fast time constant of recovery (from 4.1±0.3 ms in control to 6.0±1.1 ms after AP-A, P < 0.05) but increased the slow time constant from 66.2±6.5 ms in control to 188.9±36.4 ms (P< 0.002, n = 19) after exposure to AP-A. Increasing external divalent cation concentration (either Ca²⁺ or Mg²⁺) to 10 mM abolished the effects of AP-A on the rate of I _Na decay. These results demonstrate that modification of cardiac Na⁺ channels by AP-A markedly slowed I _Na inactivation and altered the voltage dependence of activation; these alterations in gating characteristics, in turn, caused an increase in g _Na presumably by increasing the number of channels open at peak I _Na. AP-A slows the rate of recovery of I _Na from inactivation which is probably the basis for a use-dependent decrease in peak amplitude. Finally, AP-A binding is sensitive to external divalent cation concentrations. Thus, increasing [Mg²⁺]_o or [Ca²⁺]_o displaces AP-A from binding, suggesting that they share related binding sites on the external surface of the Na⁺ channel.     

Na+/K+ pump and endothelial cell survival: [Na+]i/[K+]i-independent necrosis triggered by ouabain, and protection against apoptosis mediated by elevation of [Na+]i

Recent studies have demonstrated the tissue-specific effect of Na⁺/K⁺ pump inhibition by ouabain and other cardiac glycosides on cell viability. The vascular endothelium is an initial target of cardiac glycosides employed for the management of congestive heart failure as well as circulating endogenous ouabain-like substances (EOLS), the production of which is augmented in volume-expanded hypertension. This study examined the role of the Na⁺/K⁺ pump in the survival of cultured porcine aortic endothelial cells (PAEC). Complete Na⁺/K⁺ pump inhibition with ouabain led to PAEC death, indicated by cell detachment and decreased staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Based on cell swelling and resistance to benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk) a pan-caspase inhibitor, this type of cell death was classified as necrosis. In contrast to ouabain, Na⁺/K⁺ pump inhibition in K⁺-free medium did not affect PAEC viability and sharply attenuated apoptosis triggered by ³H decay-induced DNA damage. Necrosis evoked by ouabain was preserved after dissipation of the transmembrane gradient of K⁺ and Na⁺, whereas dissipation of the Na⁺ gradient abolished the antiapoptotic action of K⁺-free medium. Comparative analysis of these results and modulation of intracellular Na⁺ and K⁺ content by the above-listed stimuli showed that interaction of ouabain with Na⁺/K⁺-ATPase triggered necrosis independently of inhibition of Na⁺/K⁺ pump-mediated ion fluxes and inversion of the [Na⁺]_i/[K⁺]_i ratio, whereas protection against apoptosis under Na⁺/K⁺ pump inhibition in K⁺-depleted medium was mediated by [Na⁺]_i elevation. The role of Na⁺/K⁺ pump-mediated regulation of endothelial cell survival and vascular remodelling seen in hypertension should be investigated further in context of EOLS and chronic treatment with digitalis.     

Evidence for a Na+/Ca2+ exchange mechanism in frog skin epithelium

 In the present study we investigated the possible existence of a Na⁺/Ca²⁺ exchange mechanism in the basolateral membrane of the frog skin epithelium and whether such a mechanism plays a role in the regulation of transepithelial Na⁺ transport. Cytosolic calcium ([Ca²⁺]_i) was measured with the probe fura-2 in a set-up in which pieces of tissue were mounted on the stage of an epifluorescence microscope. Na⁺ transport was measured as the amiloride-sensitive short-circuit current (I _sc) using a conventional voltage clamp. Basal [Ca²⁺]_i was 65±6 nM (n=15). Removal of Na⁺ from the mucosal solution had no effect on [Ca²⁺]_i. When Na⁺ was removed from the serosal solution, [Ca²⁺]_i increased biphasically to a peak of 220±38 nM (n=8, P=0.006). Readdition of Na⁺ to the serosal solution returned [Ca²⁺]_i to control level. The serosal Na⁺ gradient and changes in [Ca²⁺]_i were closely correlated; stepwise changes in serosal Na⁺ were followed by stepwise changes in [Ca²⁺]_i. These observations indicate the existence of a Na⁺/Ca²⁺ exchange mechanism in the basolateral membrane of the frog skin epithelium. The transepithelial Na⁺ transport decreased from 13.2±1.8 to 9.2±1.5 μA cm^–2 (n=8, P=0.049) when Na⁺ was omitted from the serosal solution. When this protocol was repeated in the absence of serosal Ca²⁺, Na⁺ transport decreased similarly from 16.7±1.7 to 11.6 ±1.8 μA cm^–2 (n=6, P=0.004). We conclude that it is unlikely that the observed decrease in I _sc after removal of serosal Na⁺ is due to an increase in [Ca²⁺]_i per se. Received: 10 July 1998 / Received after revision: 23 September 1998 / Accepted: 25 September 1998     

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.     

Kinetic properties of Na+/H+ exchange in cultured bovine pigmented ciliary epithelial cells

Uptake studies with²²Na were performed in cultured bovine pigmented ciliary epithelial cells, in order to characterize mechanisms of Na⁺ transport. A large part of Na⁺ uptake was sensitive to amiloride, quinidine and harmaline. Na⁺ uptake was stimulated by intracellular acidification (using the NH ₄ ⁺ prepulse technique), and was inhibited with increasing extracellular proton concentration. Decreasing extracellular pH from 7.5 to 7.0 increased the apparentK _M for Na⁺ from 38 to 86 mM without considerable changes inV _max. In the presence of 5 mM Na⁺ half maximal inhibition of amiloride sensitive Na⁺ uptake by extracellular protons was observed at a hydrogen concentration of 50 nM. In the presence of 50 mM Na⁺ the proton concentration necessary for 50% inhibition was 139 nM. Thus, the mode of inhibition of extracellular H⁺ seemed to be competitive with aK _i of 20–40 nM. 10 M amiloride increased the apparentK _M for Na⁺ from 33 mM to 107 mM, whileV _max remained nearly unchanged. IC₅₀ for amiloride was 6 M at 5 mM Na⁺ and 36 M in the presence of 150 mM Na⁺. Thus, amiloride behaves as a competitive inhibitor with aK _i of about 5 M. The affinities of Na⁺ to the transport site (K _M16 mM), to the inhibitory site for protons (K _M21 mM), and to the inhibitory site for amiloride (K _M26 mM) were in the same order of magnitude.In summary, we have presented evidence for the presence of a Na⁺/H⁺ exchanger in cultured bovine pigmented ciliary epithelial cells. The kinetic data suggest the presence of only one common extracellular binding site for Na⁺, H⁺ and amiloride.