The relationship between anion exchange and net anion flow across the human red blood cell membrane

The conductive (net) anion permeability of human red blood cells was determined from net KCl or K2SO4 effluxes into low K+ media at high valinomycin concentrations, conditions under which the salt efflux is limited primarily by the net anion permeability. Disulfonic stilbenes, inhibitors of anion exchange, also inhibited KCl or K2SO4 efflux under these conditions, but were less effective at lower valinomycin concentrations where K+ permeability is the primary limiting factor. Various concentrations of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) had similar inhibitory effects on net and exchange sulfate fluxes, both of which were almost completely DIDS sensitive. In the case of Cl-, a high correlation was also found between inhibition of net and exchange fluxes, but in this case about 35% of the net flux was insensitive to DIDS. The net and exchange transport processes differed strikingly in their anion selectivity. Net chloride permeability was only four times as high as net sulfate permeability, whereas chloride exchange is over 10,000 times faster than sulfate exchange. Net OH-permeability, determined by an analogous method, was over four orders of magnitude larger than that of Cl-, but was also sensitive to DIDS. These data and others are discussed in terms of the possibility that a common element may be involved in both net and exchange anion transport.     

Urea permeability of human red cells

The rate of unidirectional [14C]urea efflux from human red cells was determined in the self-exchange and net efflux modes with the continuous flow tube method. Self-exchange flux was saturable and followed simple Michaelis-Menten kinetics. At 38 degrees C the maximal self-exchange flux was 1.3 X 10(-7) mol cm-2 s-1, and the urea concentration for half-maximal flux, K1/2, was 396 mM. At 25 degrees C the maximal self-exchange flux decreased to 8.2 X 10(-8) mol cm-2 s-1, and K1/2 to 334 mM. The concentration-dependent urea permeability coefficient was 3 X 10(-4) cm s-1 at 1 mM and 8 X 10(-5) cm s-1 at 800 mM (25 degrees C). The latter value is consonant with previous volumetric determinations of urea permeability. Urea transport was inhibited competitively by thiourea; the half-inhibition constant, Ki, was 17 mM at 38 degrees C and 13 mM at 25 degrees C. Treatment with 1 mM p-chloromercuribenzosulfonate inhibited urea permeability by 92%. Phloretin reduced urea permeability further (greater than 97%) to a "ground" permeability of approximately 10(-6) cm s-1 (25 degrees C). This residual permeability is probably due to urea permeating the hydrophobic core of the membrane by simple diffusion. The apparent activation energy, EA, of urea transport after maximal inhibition was 59 kJ mol-1, whereas in control cells EA was 34 kJ mol-1 at 1 M and 12 kJ mol-1 at 1 mM urea. In net efflux experiments with no extracellular urea, the permeability coefficient remained constantly high, independent of a variation of intracellular urea between 1 and 500 mM, which indicates that the urea transport system is asymmetric. It is concluded that urea permeability above the ground permeability is due to facilitate diffusion and not to diffusion through nonspecific leak pathways as suggested previously.     

Hyperosmolarity and the Net Transport of Nonelectrolytes in Frog Skin

The permeability of frog skin to a series of nonelectrolytes (thiourea, urea, mannitol, and sucrose) under the influence of 2.5 times normal osmolarity in the outer bathing solution has been investigated. Although the flux of the tracer nonelectrolytes across the skin in either direction is greatly increased by hyperosmolarity, the influx is found to be increased to a significantly greater extent than the outflux. Flux ratios as high as 3:1 can be observed. The net inward movement of the nonelectrolyte proceeds in spite of a sizeable bulk flow of water in the opposite direction. Possible driving forces for this phenomenon are discussed.     

Dog Red Blood Cells : Adjustment of salt and water content in vitro

Dog red blood cells (RBC) lack a ouabain-sensitive sodium pump, and yet they are capable of volume regulation in vivo. The present study was designed to find in vitro conditions under which dog RBC could transport sodium outward, against an electrochemical gradient. Cells were first loaded with sodium chloride and water by preincubation in hypertonic saline. They were then incubated at 37°C in media containing physiologic concentrations of sodium, potassium, chloride, bicarbonate, glucose, and calcium. The cells returned to a normal salt and water content in 16–20 h. Without calcium in the medium the cells continued slowly to accumulate sodium. Removal of glucose caused rapid swelling and lysis, whether or not calcium was present. The net efflux of sodium showed a close relationship to medium calcium over a concentration range from 0 to 5 mM. Extrusion of salt and water was also demonstrated in fresh RBC (no hypertonic preincubation) when calcium levels in the media were sufficiently raised. The ion and water movements in these experiments were not influenced by ouabain or by removal of extracellular potassium. Magnesium could not substitute for calcium. It is concluded that dog RBC have an energy-dependent mechanism for extruding sodium chloride which requires external calcium and is quite distinct from the sodium-potassium exchange pump.     

A component of fluid absorption linked to passive ion flows in the superficial pars recta

We studied salt and water absorption in isolated rabbit superficial proximal straight tubules perfused and bathed with solutions providing oppositely directed transepithelial anion gradients similar to those which might obtain in vivo. The perfusing solution contained 138.6 mM Cl- 3.8 mM HCO-3 (pH 6.6) while the bathing solution contained 113.6 mM Cl- and 25 mM HCO-3 (pH 7.4); the system was bubbled with 95% O2-5% CO2. At 37 degrees C, net volume absorption (Jv nl min-1 mm-1) was 0.32 +/- 0.03 (SEM); Ve, the transepithelial voltage (millivolts; lumen to bath), was +3.1 +/- 0.2. At 21 degrees C, Ve rose to +3.7 +/- 0.1 and Jv fell to 0.13 +/- 0.01 (significantly different from zero at P less than 0.001); in the presence of 10(-4)M ouabain at 37 degrees C, Ve rose to +3.8 +/- 0.1 and Jv fell to 0.16 +/- 0.01 (P less than 0.001 with respect to zero). In paired experiments, the ouabain- and temperature-insensitive moieties of Jv and Ve became zero when transepithelial anion concentration gradients were abolished. Titrametric determinations net chloride flux at 21 degrees C or at 37 degrees C with 10(-4) M ouabain showed that chloride was the sole anion in an isotonic absorbate. And, combined electrical and tracer flux data indicated that the tubular epithelium was approximately 18 times more permeable to Cl- than to HCO-3. We interpret these results to indicate that, in these tubules, NaCl absorption depends in part on transepithelial anion concentration gradients similar to those generated in vivo and in vitro by active Na+ absorption associated with absorption to anions other than chloride. A quantitative analysis of passive solute and solvent flows in lateral intercellular spaces indicated that fluid absorption occurred across junctional complexes when the osmolality of the lateral intercellular spaces was equal to or slightly less than that of the perfusing and bathing solutions; the driving force for volume flow under these conditions depended on the fact that sigmaHCO3 exceeded sigmaCl.     

Volume regulation by flounder red blood cells in anisotonic media

The nucleated high K, low Na red blood cells of the winter flounder demonstrated a volume regulatory response subsequent to osmotic swelling or shrinkage. During volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation after osmotic swelling is referred to as regulatory volume decrease (RVD) and was characterized by net K and water loss. Since the electrochemical gradient for K is directed out of the cell there is no need to invoke active processes to explain RVD. When osmotically shrunken, the flounder erythrocyte demonstrated a regulatory volume increase (RVI) back toward control cell volume. The water movements characteristic of RVI were a consequence of net cellular NaCl and KCl uptake with Na accounting for 75 percent of the increase in intracellular cation content. Since the Na electrochemical gradient is directed into the cell, net Na uptake was the result of Na flux via dissipative pathways. The addition of 10(-4)M ouabain to suspensions of flounder erythrocytes was without effect upon net water movements during volume regulation. The presence of ouabain did however lead to a decreased ration of intracellular K:Na. Analysis of net Na and K fluxes in the presence and absence of ouabain led to the conclusion that Na and K fluxes via both conservative and dissipative pathways are increased in response to osmotic swelling or shrinkage. In addition, the Na and K flux rate through both pump and leak pathways decreased in a parallel fashion as cell volume was regulated. Taken as a whole, the Na and K movements through the flounder erythrocyte membrane demonstrated a functional dependence during volume regulation.     

A study of passive potassium efflux from human red blood cells using ion-specific electrodes

Summary Using ion-specific electrodes, the potassium leakage induced by ouabain in human erythrocytes can be measured continuously and precisely near physiological conditions. Upon small additions of isotonic sucrose solution to a suspension of red cells in physiological saline the passive potassium efflux increases proportionally to the chloride ratio. The same result is obtained upon addition of hypertonic sucrose solution, suggesting that neither osmolarity nor intracellular concentrations have any influence on the passive potassium efflux. The independence of the potassium efflux and osmolarity can be verified by addition of a penetrating substance like glucose to the cell suspension. Adding water or hypertonic sodium chloride solution shows that the potassium efflux increases slightly in more concentrated salt solutions. Inasmuch as it can be interpreted as a pure ionic strength effect, this result supports the hypothesis of independence of potassium efflux and intracellular concentrations. The results of this investigation together with other studies show that the passive permeability of the human red blood cell to potassium depends uniquely on the membrane potential near physiological conditions, while it depends on parameters such as pH or concentrations for large membrane potentials. This suggests that two different mechanisms of transport might be involved: one would control the permeability under normal conditions; the other would represent a leak through the route normally used by anions and become important only under extreme conditions.     

Salt excretion by the perfused head of trout adapted to sea water and its inhibition by adrenaline

Summary Isolated heads of trout (Salmo gairdneri) were used to study the unidirectional flux of sodium and chloride across the gills in salt water.Two perfusion techniques were employed. Under constant pressure perfusion, the addition of adrenaline during the perfusion causes an increase in the flow-rate. Under constant flow-rate conditions, adrenaline provokes a decrease in pressure. A comparison of influx determination made with these two techniques of perfusion shows that variations in flow-rate of perfusion do not affect the assessment of these fluxes.A net efflux of sodium, but not of chloride, is demonstrated in sea water. The effluxes of sodium and chloride observed in sea water are decreasedd about 50% during a rapid transfer to fresh water. The addition of potassium to this medium stimulates the effluxes of sodium and chloride, suggesting a Na/K exchange participating in the chloride excretion.Adrenaline causes an inhibition of sodium and chloride efflux in sea water which persists after transfer to fresh water and the addition of potassium. Only the influx of chloride is inhibited at a concentration of 10^–5 M whereas the sodium influx is unaffected. The presence of adrenaline results in a net influx of both sodium and chloride.The differential action of adrenaline on the influxes of sodium and chloride suggests that the hemodynamic modifications provoked by this catecholamine occur independently of its aforementioned ion exchange effects.     

Net uptake of chloride across the posterior gills of the Chinese crab (Eriocheir sinensis)

Two methods are commonly used for the determination of transbranchial net fluxes of Na+ and Cl-: direct analysis of changes in ion concentrations in the external medium using flame spectrophotometry or titration (net flux method), and measurement of unidirectional ion fluxes by means of radioactive tracers (tracer method). When we applied both methods in the same preparation, the isolated perfused posterior gill of freshwater-acclimated Eriocheir sinensis, to determine net fluxes of Cl-, the results differed substantially. In artificial fresh water (AFW) containing NaCl, the net flux method yielded a net uptake, but the tracer method showed a net efflux of Cl-. The net uptake of Cl- was abolished in Na(+)-free AFW indicating that Cl- uptake is coupled with the uptake of Na+. Applying the tracer method, net efflux of Cl- remained almost unchanged in Na(+)-free AFW. This suggests the opposite mechanism, i.e. uncoupled uptake of Na+ and Cl-. The discrepancy in the results obviously depends on the method employed. Since the data obtained with the net flux method explain the osmoregulatory performance of crabs living in fresh water, we consider this method as appropriate for determining net transbranchial ion fluxes.     

Role of AQP2 during apoptosis in cortical collecting duct cells

Background information. A major hallmark of apoptosis is cell shrinkage, termed apoptotic volume decrease, due to the cellular outflow of potassium and chloride ions, followed by osmotically obliged water. In many cells, the ionic pathways triggered during the apoptotic volume decrease may be similar to that observed during a regulatory volume decrease response under hypotonic conditions. However, the pathways involved in water loss during apoptosis have been largely ignored. It was recently reported that in some systems this water movement is mediated via specific water channels (aquaporins). Nevertheless, it is important to identify whether this is a ubiquitous aspect of apoptosis as well as to define the mechanisms involved. The aim of the present work was to investigate the role of aquaporin‐2 during apoptosis in renal‐collecting duct cells. We evaluated the putative relationship between aquaporin‐2 expression and the activation of the ionic pathways involved in the regulatory volume response. Results. Apoptosis was induced by incubating cells with a hypertonic solution or with cycloheximide in two cortical collecting duct cell lines: one not expressing aquaporins and the other stably transfected with aquaporin‐2. Typical features of apoptosis were evaluated with different approaches and the water permeability was measured by fluorescence videomicroscopy. Our results show that the rate of apoptosis is significantly increased in aquaporin‐2 cells and it is linked to the rapid activation of volume‐regulatory potassium and chloride channels. Furthermore, the water permeability of cells expressing aquaporin‐2 was strongly reduced during the apoptotic process and it occurs before DNA degradation. Conclusions. These results let us propose that under apoptotic stimulation aquaporin‐2 would act as a sensor leading to a co‐ordinated activation of specific ionic channels for potassium and chloride efflux, resulting in both more rapid cell shrinkage and more rapid achievement of adequate levels of ions necessary to activate the enzymatic apoptotic cascade.     

Theoretical Analysis of Net Tracer Flux Due to Volume Circulation in a Membrane with Pores of Different Sizes : Relation to solute drag model

When osmotic pressure across an artificial membrane, produced by a permeable electrically neutral solute on one side of it, is balanced by an external pressure difference so that there is no net volume flow across the membrane, it has been found that there will be a net flux of a second electrically neutral tracer solute, present at equal concentrations on either side of the membrane, in the direction that the "osmotic" solute diffuses. This has been ascribed to solute-solute interaction or drag between the tracer and the osmotic solutes. An alternative model, presented here, considers the membrane to have pores of different sizes. Under general assumptions, this "heteroporous" model will account for both the direction of net tracer flux and the observed linear dependence of unidirectional tracer fluxes on the concentration of the osmotic solute. The expressions for the fluxes of solutes and solvent are mathematically identical under the two models. An inequality is derived which must be valid if the solute interaction model and/or the heteroporous model can account for the data. If the inequality does not hold, then the heteroporous model alone cannot explain the data. It was found that the inequality holds for most published observations except when dextran is the osmotic solute.     

Ion Transport in Hydrodictyon africanum

The concentrations of K, Na, and Cl in the cytoplasm and vacuole, the tracer fluxes of these ions into and out of the cenocyte, and the electrical potential difference between bathing solution and vacuole and cytoplasm, have been measured in Hydrodictyon africanum. If the ions were acted on solely by passive electrochemical forces, a net efflux of K and Cl and a net influx of Na would be expected. Tracer fluxes indicate a net influx of K and Cl and efflux of Na in the light; these net fluxes are consequently active, with an obligate link to metabolism. The effects of darkness and low temperature indicate that most of the tracer K and Cl influx and Na efflux are linked to metabolism, while the corresponding tracer fluxes in the direction of the free energy gradient are not. Ouabain specifically inhibits the metabolically linked portions of tracer K influx and Na efflux. Alterations in the external K concentration have similar effects on metabolically mediated K influx and Na efflux. It would appear that K influx and Na efflux are linked, at least in the light.     

Mechanism of thiyl radical-catalyzed isomerization of unsaturated fatty acid residues in homogeneous solution and in liposomes

Taurine is an important osmolyte involved in cell volume regulation. During regulatory volume decrease it is released via a volume-sensitive organic osmolyte/anion channel. Several molecules have been suggested as candidates for osmolyte release. In this study, we chose three of these, namely ClC-2, ClC-3 and ICln, because of their expression in rat astrocytes, a cell type which is known to release taurine under hypotonic stress, and their activation by hypotonic shock. As all three candidates were also suggested to be chloride channels, we investigated their permeability for both chloride and taurine under isotonic and hypotonic conditions using the Xenopus laevis oocyte expression system. We found a volume-sensitive increase of chloride permeability in ClC-2-expressing oocytes only. Yet, the taurine permeability was significantly increased under hypotonic conditions in oocytes expressing any of the tested candidates. Further experiments confirmed that the detected taurine efflux does not represent unspecific leakage. These results suggest that ClC-2, ClC-3 and ICln either participate in taurine transport themselves or upregulate an endogenous oocyte osmolyte channel. In either case, the taurine efflux of oocytes not being accompanied by an increased chloride flux suggests that taurine and chloride can be released via two separate pathways.     

Volume regulation in salt-acclimated toad (Bufo viridis): the role of urea and the urinary bladder

Body water (weight) was studied in the euryhaline toad Bufo viridis during high salt (500 mOsm NaCl) acclimation. Plasma osmolality was greatly increased upon salt acclimation mainly by urea, and was always hyperosmotic to the ambient solution. Water content was regulated quite efficiently in slowly acclimated undisturbed toads. Repeatedly catheterized toads behaved like osmometers when transferred to hyperosmotic solutions. Total urea loss was greatly reduced in salt acclimated toads, suggesting urine was not voided under these conditions. It is concluded that urea accumulation, inhibition of the urine voiding response and the urine in the bladder are the principal factors involved in volume regulation under conditions of salt acclimation.     

The effect of ethanol on ion transport in frog skin.

Frog skin has been used as a model epithelial sodium-transporting system to study the effect of ethanol on ion transport. Treatment of the outside of frog skin with ethanol decreased the net sodium transport due to inhibition of 22Na+ influx. Ethanol did not alter sodium outflux when bathin the outside of the skin. The inhibition was in proportion to the concentration of ethanol, 0.25 M resulting in 50% inhibition. The chloride permeability of the skin was increased several-fold when the skin was exposed to ethanol in either bathing solution. With 0.4 M ethanol in the inner bathing solution, all the unidirectional fluxes of Na+ and C1- were increased. The movement of C1- was evaluated by comparison of C1- flux with urea flux, since urea is thought to move passively across frog skin via an extracellular (shunt) pathway. Chloride flux was increased to a greater extent than urea flux. These experiments indicate that ethanol affects chloride permeability beyond an increase in extracellular ion flow and independent of its effect of Na+ transport.     

Tracer Diffusion and Unidirectional Fluxes

Available experimental data have been utilized to examine the effects of cross-coefficients on tracer diffusion and on the estimation of unidirectional fluxes from observations on tracer flow. In free solution or in a nonselective membrane, the interaction between the flows of tracer and the unlabelled substance are small at concentrations of biological interest for the nonelectrolytes urea, alanine, and β-alanine, and for sodium and chloride ions. Under these conditions, measurement of tracer flow can be used to predict flow of the bulk substance to an accuracy of a few per cent.     

Ion tracer flows and flux ratios in heterogeneous membranes

Ion tracer flows and flux ratios at various electrical potentials were studied in heterogeneous membranes comprising parallel pathways of different intrinsic resistance. The total resistance to net flow exceeded the tracer exchange resistance, and the flux ratio was abnormal, as in exchange diffusion commonly attributed to a mobile carrier.     

Effect of osmolarity on potassium transport in isolated cerebral microvessels

Potassium transport in microvessels isolated from rat brain by a technique involving density gradient centrifugation was studied in HEPES buffer solutions of varying osmolarity from 200 to 420 mosmols, containing different concentration of sodium chloride, choline chloride, or sodium nitrate. The flux of 86Rb (as a tracer for K) into and out of the endothelial cells was estimated. Potassium influx was very sensitive to the osmolarity of the medium. Ouabain-insensitive K-component was reduced in hypotonic medium and was increased in medium made hypertonic with sodium chloride or mannitol. Choline chloride replacement caused a large reduction in K influx. Potassium influx was significant decrease when nitrate is substituted for chloride ion in isotonic and hypertonic media, whereas a slight decrease was found in hypotonic medium. The decrease of K influx in the ion-replacement medium is due to a decrement of the ouabain-insensitive component. Potassium efflux was unchanged in hypotonic medium but was somewhat reduced in hypertonic medium. The marked effect of medium osmolarity on K fluxes suggests that these fluxes may be responsible for the volume regulatory K movements. The possible mechanism of changes of K flux under anisotonic media is also discussed.     

Silicon deposition in the root reduces sodium uptake in rice (Oryza sativa L.) seedlings by reducing bypass flow

Sodium chloride reduces the growth of rice seedlings, which accumulate excessive concentrations of sodium and chloride ions in their leaves. In this paper, we describe how silicon decreases transpirational bypass flow and ion concentrations in the xylem sap in rice (Oryza sativa L.) seedlings growing under NaCl stress. Salt (50 mM NaCl) reduced the growth of shoots and roots: adding silicate (3 mM) to the saline culture solution improved the growth of the shoots, but not roots. The improvement of shoot growth in the presence of silicate was correlated with reduced sodium concentration in the shoot. The net transport rate of Na from the root to shoot (expressed per unit of root mass) was also decreased by added silicate. There was, however, no effect of silicate on the net transport of potassium. Furthermore, in salt-stressed plants, silicate did not decrease the transpiration, and even increased it in seedlings pre-treated with silicate for 7 d prior to salt treatment, indicating that the reduction of sodium uptake by silicate was not simply through a reduction in volume flow from root to shoot. Experiments using trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS), an apoplastic tracer, showed that silicate dramatically decreased transpirational bypass flow in rice (from about 4.2 to 0.8%), while the apparent sodium concentration in the xylem, which was estimated indirectly from the flux data, decreased from 6.2 to 2.8 mM. Direct measurements of the concentration of sodium in xylem sap sampled using Philaenus spumarius confirmed that the apparent reduction was not a consequence of sodium recycling. X-ray microanalysis showed that silicon was deposited in the outer part of the root and in the endodermis, being more obvious in the latter than in the former. The results suggest that silicon deposition in the exodermis and endodermis reduced sodium uptake in rice (Oryza sativa L.) seedlings under NaCl stress through a reduction in apoplastic transport across the root.     

Adrenergic-agonist-induced Ca2+ fluxes in rat parotid cells are not Na+-dependent.

We investigated the hypothesis that extracellular Na+ is required for the rapid mobilization of Ca2+ by rat parotid cells after adrenergic stimulation. When Na+ salts in the media were osmotically replaced with either choline chloride (+atropine) or sucrose, efflux of 45Ca2+ from preloaded cells, caused by 10 microM-(-)-adrenaline, was unchanged. Similarly adrenaline stimulated 45Ca2+ uptake into cells under nonsteady-state conditions in the presence or absence of Na+. Monensin, a Na+ ionophore, was able to elicit a modest increase in 45Ca2+ efflux, compared with controls. Studies of net 45Ca2+ flux, performed under near-steady-state conditions, showed that adrenaline caused net 45Ca2+ accumulation, whereas monensin caused net 45Ca2+ release. The effect of monensin required the presence of Na+ in the incubation medium. Both 1 mM-LaCl3 and 0.1 mM-D-600 prevented adrenaline-stimulated 45Ca2+ uptake into cells, but had no effect on monensin-induced changes. We conclude that (1) the rapid mobilization of Ca2+ by adrenergic agonists seen in rat parotid cells does not require a Na+out greater than Na+in gradient and (2) the nature of the monensin effect is quite different from the adrenergic-agonist-induced response.