Correlation Between Transepithelial Na+ Transport and Transepithelial Water Movement Across Isolated Frog Skin (Rana esculenta)

In the present work the coupling under short-circuited conditions between the net Na⁺-influx across isolated frog skin and the transepithelial transport of water was examined i.e., the short-circuit current (I _sc ) and the transepithelial water movement (TEWM) were measured simultaneously. It has been shown repeatedly that the I _sc across isolated frog skin is equal to the net transepithelial Na⁺ transport. Furthermore the coupling between transepithelial uptake of NaCl under open-circuit conditions and TEWM was also measured. The addition of antidiuretic hormone (AVT) to skins incubated under short-circuited conditions resulted in an increase in the I _sc and TEWM. Under control conditions I _sc was 9.14 ± 2.43 and in the presence of AVT 45.9 ± 7.3 neq cm⁻² min⁻¹ (n= 9) and TEWM changed from 12.45 ± 4.46 to 132.8 ± 15.8 nL cm⁻² min⁻¹. The addition of the Na⁺ channel blocking agent amiloride resulted in a reduction both in I _sc and TEWM, and a linear correlation between I _sc and TEWM was found. The correlation corresponds to that 160 ± 15 (n= 7) molecules of water follow each Na⁺ across the skin. In another series of experiments it was found that there was a linear correlation between I _sc and the increase in apical osmolarity needed to stop the TEWM. The data presented indicate that the observed coupling between the net transepithelial Na⁺ transport and TEWM is caused by local osmosis. Received: 16 October 1996/Revised: 6 March 1997     

Effects of D2O and Osmotic Gradients on Potential and Resistance of the Isolated Frog Skin

Exposure of the outside surface of isolated frog skin (R. pipiens and R. catesbeiana) to sulfate solution made up with D₂O decreased skin potential and resistance. Exposure of the inside surface to D₂O solution decreased the potential slightly but increased the resistance. The changes were linearly related to the D₂O concentration. Since D₂O acts like a hyperosmotic solution, the skin potential and resistance were studied upon exposure to solution made hyperosmotic by addition of sucrose, mannitol, acetamide, urea, thiourea, Na₂SO₄, or K₂SO₄. Skin potential and resistance decreased when the outside solution was made hyperosmotic. The changes depended upon the concentration and the nature of the solute. Thiourea and urea solutions were the most effective. Treatment of the inside surface gave relatively small decreases in potential; the resistance either increased or remained unchanged. These effects appeared to depend upon the direction of the osmotic gradient across the skin rather than upon the value of the osmolarity compared to normal body fluids. Experiments with a series of six polyhydric alcohols from methanol to mannitol and the polysaccharides, sucrose and raffinose, showed adonitol with 5 carbons to decrease the potential the most. Smaller and larger compounds of this set gave lesser effects. As yet no consistent explanation of the effects is forthcoming, but their demonstration calls for caution in the indiscriminate use of solutes such as mannitol or sucrose "to make up the osmolality" and in the neglect of urea because "it penetrates freely."     

Frequency Spectrum of Transepithelial Potential Difference Reveals Transport-Related Oscillations

How epithelia transport fluid is a fundamental issue that is unresolved. Explanations offered include molecular engines, local transcellular osmosis, local paracellular osmosis, and paracellular fluid transport. On the basis of experimental and theoretical work done on corneal endothelium, a fluid transporting epithelium, we suggest electroosmotic coupling at the level of the intercellular junctions driven by the transendothelial electrical potential difference as an explanation of paracellular fluid transport. We collect frequency spectra of that potential difference in real-time. For what we believe is the first time for any epithelium, we report that, unexpectedly, the potential difference displays oscillations at many characteristic frequencies. We also show that on both stimulating cell activity and inhibiting ion transport mechanisms, there are corresponding changes in the oscillations amplitudes that mirror changes known previously in rates of fluid transport. We believe these findings provide a novel tool to study the kinetics of electrogenic elements such as channels and transporters, which from this evidence would give rise to current oscillations with characteristic periods going from 150 ms to 8 s.     

The Effects of Alkali Metal Cations and Common Anions on the Frog Skin Potential

The effects on the potential difference across isolated frog skin (R. catesbeiana, R. pipiens) of changing the ionic composition of the bathing solutions have been examined. Estimates of mean values and precision are presented for the potential changes produced by substituting other alkali metal cations for Na at the outside border and for K at the inside border. In terms of ability to mimic Na at the outside border of bullfrog skin, the selectivity order is Li > Rb, K, Cs; at the outside border of leopard frog skin, Li > Cs, K, Rb. In terms of ability to mimic K at the inside border of bullfrog and leopard frog skin: Rb > Cs > Li > Na. Orders of anion selectivity in terms of sensitivity of the potential for the outside border of bullfrog skin are Br > Cl > NO₃ > I > SO₄, isethionate and of leopard frog skin are Br, Cl > I, NO₃, SO₄. An effect of the solution composition (ionic strength?) on the apparent Na-K selectivity of the outside border is described. The results of the investigation have been interpreted and discussed in terms of the application of the constant field equation to the Koefoed-Johnsen-Ussing frog skin model. These observations may be useful in constructing and testing models of biological ionic selectivity.     

Effect of the Putative Ca2+-Receptor Agonist Gd3+ on the Active Transepithelial Na+ Transport in Frog Skin

In this communication we show that Gd3+ acts as an activator of the apical sodium channel (ENaC) in frog skin epithelia. Application of Gd3+ to the apical solution of frog skin epithelia increased the Na+ absorption measured as the amiloride-inhibitable short-circuit current (Isc). The stimulation was dose dependent with a concentration for half-maximal stimulation (EC50) of 0.023 mM. The change in Isc was found to correlate with the net Na+ flux, confirming that Gd3+ enhances Na+ absorption. By monitoring the cellular potential (Vsc) with microelectrodes during addition of Gd3+, it was found that Vsc depolarized as Isc rose, indicating that Gd3+ affects apical Na+ permeability (PNa). This was confirmed by measuring the I/V relations of the apical membrane. In the presence of benzimidazolylguanidin (BIG), a drug known to abolish the Na+ self-inhibition, Gd3+ had no effect on Isc. The Na+ self-inhibition was investigated using fast changes of the apical Na+ concentration on K+-depolarized epithelia. BIG was found to abolish the Na+ self-inhibition and to activate the basal Na+ transport, whereas Gd3+ only activated the basal Na+ transport but had no effect on the self-inhibition. These results indicate the existence of an alternative nonhormonal mechanism to Na+ self-inhibition, via which both Gd3+ and BIG act, possibly components of the Na+ feedback inhibition system.     

Nuclear Magnetic Resonance Studies of Sodium Ions in Isolated Frog Muscle and Liver

Nuclear magnetic resonance (NMR) was used to determine Na⁺ complexing in muscle and liver (at 23°C) from bullfrogs (Rana catesbeiana) and to study the influence of temperature on Na⁺ complexing in muscle from leopard frogs (Rana pipiens). The Na⁺ complexed in muscle and liver was found to be 36.6 ± 4.6% and 66.1 ± 3.5% respectively. A temperature decrease from +34°C to -2°C results in a 20% decrease in the mobility of the free Na⁺ in the fresh muscle. This 20% decrease in mobility results in about 50% of the free Na⁺ at 34°C being complexed at the lower temperature.     

The Influence of Sodium-Free Solutions on the Membrane Potential of Frog Muscle Fibers

The membrane potential of frog sartorius muscle fibers in a Cl- and Na-free Ringer's solution when sucrose replaces NaCl is about the same as that in normal Ringer's solution. The K⁺ efflux is also about the same in the two solutions but muscles lose K and PO₄ in sucrose Ringer's solutions. The membrane potential in sucrose Ringer's solution is equal to that given by the Nernst equation for a K⁺ electrode, when corrections are made for the activity coefficients for K⁺ inside and outside the fiber. For a muscle in normal Ringer's solution, the measured membrane potential is within a few millivolts of E_K. This finding is incompatible with a 1:1 coupled Na-K pump. It is consistent with either no coupling of Na efflux to K influx, or a coupling ratio of 3 or greater.     

Exposure of the Isolated Frog Skin to High Potassium Concentrations at the Internal Surface : II. Changes in epithelial cell volume,resistance and response to antidiuretic hormone

Isolated frog skin epithelia undergo marked, but reversible swelling when the external skin surface is bathed with conventional NaCl Ringer''s and the internal surface with KCl Ringer''s solutions. In 2 hours, epithelial thickness increased by over twofold. When NaCl Ringer''s was replaced on both sides of the skin, volume returned to control levels in less than 1 hour. When sulfate, rather than chloride, was the predominant anion, exposure of the internal surface to high potassium concentrations did not evoke changes in epithelial cell volume. With both KCl and K₂SO₄ Ringer''s, an immediate drop in DC resistance across the skin occurred. This was followed by partial recovery. Both the immediate drop and partial recovery were unrelated to changes in volume. A slow, sustained secondary drop in resistance was observed with KCl but not K₂SO₄ Ringer''s. This slower drop was associated temporally with swelling. When epithelial cell swelling occurred (i.e. with KCl Ringer''s), the characteristic response of the skin to vasopressin was abolished. However, with sulfate as anion, vasopressin elicited an increase in short-circuit current and/or in cell volume despite high internal potassium concentrations. It is concluded that epithelial swelling increased the permeability of the sodium-selective barrier at the external surface of the cells; and the possibility exists that stretching of cell membranes altered dimensions of pathways through which Na and water move, thereby mimicking the effects of vasopressin.     

Properties of the Isolated Intact Chloroplast at Cytoplasmic K Concentrations : I. Light-Induced Cation Uptake into Intact Chloroplasts is Driven by an Electrical Potential Difference

Photosynthesis, stroma-pH, and internal K⁺ and Cl⁻ concentrations of isolated intact chloroplasts from Spinacia oleracea, as well as ion (K⁺, H⁺, Cl⁻) movements across the envelope, were measured over a wide range of external KCl concentrations (1-100 millimolar).

Isolated intact chloroplasts are a Donnan system which accumulates cations (K⁺ or added Tetraphenylphosphonium⁺) and excludes anions (Cl⁻) at low ionic strength of the medium. The internally negative dark potential becomes still more negative in the light as estimated by Tetraphenylphosphonium⁺ distribution. At 100 millimolar external KCl, potentials both in the light and in the dark and also the light-induced uptake of K⁺ or Na⁺ and the release of protons all become very small. Light-induced K⁺ uptake is not abolished by valinomycin suggesting that the K⁺ uptake is not primarily active. Intact chloroplasts contain higher K⁺ concentrations (112-157 millimolar) than chloroplasts isolated in standard media. Photosynthetic activity of intact chloroplasts is higher at 100 millimolar external KCl than at 5 to 25 millimolar. The pH optimum of CO₂ fixation at high K⁺ concentrations is broadened towards low pH values. This can be correlated with the observation that high external KCl concentrations at a constant pH of the suspending medium produce an increase of stroma-pH both in the light and in the dark. These results demonstrate a requirement of high external concentrations of monovalent cations for CO₂ fixation in intact chloroplasts.

     

The Origin of the Short-Circuit Current in the Isolated Skin of the South American Frog Leptodactylus ocellatus

In isolated skins of Leptodactylus ocellatus the short-circuit current is smaller than the sodium net flux and this difference disappears when the skins are bathed in solutions in which the chloride ions have been replaced by sulfate or methylsulfate ions. There is a net movement of chloride ions from outside to inside of the skins in the short-circuit condition with chloride Ringer's solutions bathing the skins. The addition of ouabain to the inside solution markedly reduced not only sodium net flux but also the chloride net influx found. Copper ions added to the outside solutions produced a rise in short-circuit current, as well as the known increase in potential difference. In sodium-free Ringer's (sodium replaced by choline) the orientation of the potential difference across the skins was reversed, the inside being negative instead of positive. The results are interpreted as direct or indirect indications of the presence of a net transfer of chloride ions from outside to inside of these frog skins.     

Effects of Sodium Azide on Sodium Fluxes in Frog Striated Muscle

Unidirectional Na fluxes from frog''s striated muscle were measured in the presence of 0 to 5 mM sodium azide. With azide concentrations of 2 and 5 mM the Na efflux was markedly stimulated; the Na efflux with 5 mM azide was about 300 per cent greater than normal. A similar increase was present when all but the 5.0 mM sodium added with azide was replaced by choline. 10^-5 M strophanthidin abolished the azide effect on Na²⁴ efflux. Concentrations of azide of 1.0 mM or less had no effect on Na efflux. The Na influx, on the other hand, was only increased by 41 per cent in the presence of 5 mM NaN₃. From these findings it is concluded that the active transport of Na is stimulated by the higher concentrations of azide. The hypothesis is advanced that the active transport of Na is controlled by the transmembrane potential and that the stimulation of Na efflux is produced as a consequence of the membrane depolarization caused by the azide.     

The Influence of the Intracellular Potential on Potassium Uptake by Beetroot Tissue

Intracellular potentials were measured in beetroot tissue during the steady-state uptake of K⁺ from various solutions. In solutions containing bicarbonate, the membrane potential becomes up to 70 mv more negative than the estimated equilibrium potential for K⁺. The uptake of K⁺ from such solutions is correlated with variations in the potential, both when the bicarbonate concentration is changed and also when the metabolic activity of the tissue is changed by washing in water for various periods. However, the estimated permeability to K⁺ varies from 0.4 x 10^-7 to 1.5 x 10^-7 cm·sec^-1. It is postulated that the change of potential arises from the metabolic transport of HCO₃^- into the cell or H⁺ outwards, and that the associated uptake of K⁺ is partly or entirely by passive diffusion across the cell membrane. In contrast, K⁺ uptake from KCl solutions is not accompanied by any significant change in the membrane potential, which remains relatively close to the K⁺ equilibrium potential. In solutions containing both KHCO₃ and KCl, it appears that an amount of K⁺ equal to the influx of Cl^- is taken up independently of the potential, while the component of K⁺ uptake which is not balanced by Cl^- uptake is related to the potential in the manner described. These results suggest that K⁺ uptake is linked to Cl^- uptake in an electrically neutral active transport process.     

Energetics of Sodium Transport in Frog Skin : II. The effects of electrical potential on oxygen consumption

Studies were made of the dependence of the rate of oxygen consumption, J_r, on the electrical potential difference, Δψ, across the frog skin. After the abolition of sodium transport by ouabain the basal oxygen consumption was independent of Δψ. In fresh skins J_r was a linear function of Δψ over a range of at least ±70 mv. Treatment with aldosterone stimulated the short-circuit current, I_o, and the associated rate of oxygen consumption, J_ro, and increased their stability; linearity was then demonstrable over a range of ±160 mv. Brief perturbations of Δψ (±30–200 mv) did not alter subsequent values of I_o. Perturbations for 10 min or more produced a "memory" effect both with and without aldosterone: accelerating sodium transport by negative clamping lowered the subsequent value of I_o; positive clamping induced the opposite effect. Changes in J_ro were more readily detectable in the presence of aldosterone; these were in the same direction as the changes in I_o. The linearity of J_r in Δψ indicates the validity of analysis in terms of linear nonequilibrium thermodynamics—brief perturbations of Δψ appear to produce no significant effect on either the phenomenological coefficients or the free energy of the metabolic driving reaction. Hence it is possible to evaluate this free energy.     

脯氨酸对黑麦原生质体膨胀势的影响

从未经抗寒锻炼的黑麦(Secale cereale cv.Puma)叶片中分离的原生质体,悬浮于山梨醇或NaGl,CaCl_2(10:1)溶液中,其可忍受的表面积增加值(TSAI_(50))为1000μm~2左右,该值不受原生质体收缩程度的影响。悬浮于脯氨酸溶液中的原生质体,其TSAI_(50)随收缩程度而改变。等渗脯氮酸溶液(0.53Osm)中的原生质体若在低渗溶液中膨胀,TSAI_(50)为1000μm~2左右,而在0.83,1.20和2.0 Osm高渗脯氨酸溶液中收缩后的原生质体再膨胀,它们的TSAI_(50)分别增加到1710±85,1873±91μm~2和2200±283μm~2。这些数值与经抗寒锻炼后的原生质体在高渗收缩后的TSAI_(50)相似。脯氨酸增加未经锻炼的原生质体膨胀势的效应发生在收缩过程中,与膨胀时的介质种类无关。     

Influence of Potential Difference and Current on the Electrotaxis of Caenorhaditis elegans

C. elegans responds directionally to a DC current. The response may be to the anode or cathode, depending on the current, potential difference, and ion concentration of KC1, Tracks of the responding nematodes show that electrotaxes are genuine orientation phenomena. The directional movement is not due to the passive movement of nematodes or to the influence of currents on the muscular physiology; electrotaxes are mediated sensorily. Details of the response are described.     

Malate Uptake into Isolated Vacuoles from Catharanthus roseus Cells: Role of the Membrane Potential

The mechanisms involved in the transport of malate into isolated vacuoles of Catharanthus roseus (L.) cells were investigated with special reference to the effects of induced changes in membrane potential and surface charges of the tonoplast. For this purpose, thiocyanate (SCN^?), a highly permeant anion often used as a membrane potential probe, was extensively exploited. In the absence of Mg-ATP, the low accumulation ratio of ¹⁴C SCN^? could be related to the presence of negative charges at the outer surface of the tonoplast exerting a screening effect on the displacement of lipophilic anionic species. Nevertheless, malate was taken up continuously by vacuoles supporting the concept of a transport component which facilitates its transfer through the tonoplast. From experiments showing the pH dependence of malata uptake, it is suggested that the protonated form of the transporter is implicated in this process. Moreover, when the vacuoles are energized by Mg-ATP, the study of the equilibrium distribution of ¹⁴C SCN^? indicated an inside positive membrane potential difference. Advantage was taken of these results to modulate the membrane potential with high levels of thiocyanate. The data obtained demonstrate that malate uptake results from electrophoretic movement in response to the positive potential difference.     

Peculiarities of Signal Transduction from Arginine-Vasopressin Receptors Located on the Apical Surface of Epithelial Cells of the Frog Urinary Bladder and on the Amoeba Outer Membrane

Mechanisms of action of arginine-vasopressin (AVP) on water transport across the wall of urinary bladder of the frog R. temporaria L. and the outer membrane of the amoeba A. proteus were considered. AVP and its functional analogs were added to the basolateral and apical surfaces of the frog urinary bladder membrane and to the amoeba outer surface. In amoeba the AVP effect was evaluated from action on frequency of contractions of contractile vacuole that represents its organ of water—electrolyte homeostasis. It was shown that the system of signal transduction from the apical AVP receptors differed considerably from that located on the basolateral membrane and had a marked similarity with the system of signal transduction from AVP-sensitive structures on the outer membrane of amoeba. The urinary bladder apical membrane as well as the amoeba outer membrane has a mixed type of sensitivity to AVP with unidirectional (not antagonistic like on the basolateral membrane) involvement of systems coupled to receptors of the V₁ and V₂ type. We suggest that the system of signal transduction from apical AVP receptors corresponds to the earlier stage of evolutionary development.     

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.     

The Relationship between the Cell Electrical Potential Difference and Salt Uptake in the Roots of Helianthus annuus

The effect of ringing the stem on the electrical potential difference(PD) in the root cortical cells of H. annuus was studied. PDand salt transport were followed simultaneously. By ringingit was possible to separate the PD from K⁺, , and Cl^– uptake and H⁺ efflux. The uptake of phosphatehowever was found to be closely connected with a component ofthe PD. It was concluded that there is an electrogenic pumpfor phosphate in these roots which generates 60–80 mV.