Electrolyte distribution and active salt uptake in frog skin

1. The "chloride space" in frog skin was determined and found to be 69.7 per cent by weight of wet skin. The chloride space occupies about 94 per cent of the total water space of skin. From this and other information, it appears that the "non-chloride space" measures only a part of the space occupied by the structural elements of skin. This space is referred to here as the intracellular compartment and the remainder as the extracellular compartment of frog skin. On this basis, potassium and sodium in skin are distributed as follows: total sodium, 60 to 75 µeq./gm. of wet skin; all sodium is probably extracellular; total potassium, 39 to 49 µeq./gm.; intracellular potassium, 37 to 47 µeq./gm. 2. Skins were immersed in solutions differing from each other in their sodium and potassium concentrations. Three levels of NaCl were studied: 48, 119, and 169 µeq./ml. For each of these solutions (referred to below as diluted, physiological, and concentrated saline), the potassium levels were varied from 0.1 to 20 µeq./ml. For skins in solutions low in potassium and high in sodium, it was found that an exchange of intracellular potassium against extracellular sodium occurs. The ratio for the number of potassium ions lost/number of sodium ions gained was 4:1,4:6, and 4:8 for skin in K⁺-free diluted, physiological, and concentrated saline, respectively. 3. Uptake of NaCl by the epithelium of frog skin is dependent on the potassium concentration of the environment. For skins in physiological saline, net uptake of NaCl was optimal (0.90 µeq. x cm.^–2 x hr.^–1) at 1 to 5 µeq. K₊/ml. For skins in diluted and concentrated saline optimal NaCl uptake was seen at potassium concentrations of approximately 5 and 10 µeq. K₊/ml., respectively. Net uptake of NaCl by the skin is also discussed, with relation to the potassium balance of skin. 4. Skin potentials decreased with increasing extracellular potassium concentration when diluted saline solutions were used. The opposite of this was found for skins in concentrated saline. For skins in physiological saline, skin potentials rose sharply from rather low values, when placed in solutions very low in potassium, to relatively high values, when immersed in solutions containing 1 to 5 µeq. K⁺/ml. Further increase in potassium concentration of the bath led to slight reductions in skin potentials. The highest potentials observed were of the order of 40 mv. In all cases studied, the inside was positive with relation to the outside. 5. It can be shown that values for intracellular potassium concentration as a function of extracellular potassium concentration satisfy, at a first but good approximation, Freundlich's isotherm. A modification of Freundlich's isotherm, recently introduced by Sips, may also be used to correlate the experimental data quantitatively. Since the latter isotherm has a rational interpretation, it is suggested that this be used, rather than Freundlich's isotherm, to express quantitatively the dependence of intracellular on extracellular potassium in frog skin.     

Potential, Current, and Ionic Fluxes across the Isolated Retinal Pigment Epithelium and Choroid

A flux chamber was utilized for in vitro studies of a membrane formed by the retinal pigment epithelium and choroid of the eye of the toad (Bufo arenarum and Bufo marinus). A transmembrane potential of 20 to 30 mv was found, the pigment epithelium surface positive with respect to the choroidal surface. Unidirectional fluxes of chloride, sodium, potassium, and calcium were determined in the absence of an electrochemical potential difference. A net transfer of chloride from pigment epithelium to choroid accounted for a major fraction of the mean short-circuit current. A small net flux of sodium from choroid to pigment epithelium was detected in Bufo marinus. In both species of toads, however, about one-third of the mean short-circuit current remained unaccounted for. Manometric determinations of bicarbonate suggested an uptake of this ion at the epithelial surface of the membrane but did not provide evidence of a relationship between this process and the short-circuit current.     

Potassium Exchange and Afterpotentials in Frog Sartorius Muscles Treated with Glycerol

The potassium exchange properties of glycerol-treated sartorius muscles of the frog were determined. Potassium (⁴²K) uptake, efflux, and net flux were measured in the presence of glycerol and at various times after exposure to glycerol and return to isotonic Ringer solution. Potassium uptake was not altered by the presence of glycerol but was reduced on the average 53% after glycerol treatment. Efflux transiently increased in the presence of glycerol and was reduced 37% after glycerol removal. Consequently, there was a net loss of intracellular potassium as well as a gain of sodium. In contrast to the irreversible alterations of potassium exchange induced by glycerol treatment, action potentials with normal negative afterpotentials (N.A.P.) were elicited 4–5 hr after glycerol removal. The reappearance of the N.A.P. was associated with a return of the membrane potential to normal values (90 ± 2 mv). However, the response of these muscles to reduced extracellular potassium was anomalous. In K⁺-free Ringer solution the average resting membrane potential was 74 ± 3 mv and a positive afterpotential of 11 ± 3 mv was associated with the action potential.     

Sodium Flux in Necturus Proximal Tubule under Voltage Clamp

Na transport and electrical properties of Necturus renal proximal tubules were analyzed, in vivo, by a voltage clamp method which utilizes an axial electrode in the tubule lumen for passage of current and simultaneous determination of net fluid (or Na) flux by the split droplet method. When the average spontaneous transepithelial potential difference of –8 mv (lumen negative) was reduced to zero by current passage, net Na flux doubled from a mean of 107 to 227 pmoles/cm² per sec. The relationship between flux and potential over the range –25 to +10 mv was nonlinear, with flux equilibrium at –15 mv and droplet expansion at more negative values. Calculated Na permeability at flux equilibrium was 7.0 x 10^–6 cm/sec. Voltage transients, similar to those caused by intraepithelial unstirred layers, were observed at the end of clamping periods. Tubular electrical resistance measured by brief square or triangle wave pulses (<100 msec) averaged 43 ohm cm². The epithelial current-voltage relationship was linear over the range –100 to +100 mv, but displayed marked hysteresis during low frequency (<0.04 Hz) triangle wave clamps. The low transepithelial resistance and large opposing unidirectional ion fluxes suggest that passive ionic movements occur across extracellular shunt pathways, while the voltage transients and current-voltage hysteresis are consistent with the development of a local osmotic gradient within epithelium.     

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.     

Changes in ionic conductances and in sensitivity to amiloride during the natural moulting cycle of toad skin (Bufo viridis,L.)

Summary The resistance of the apical membranes of toad skin (Bufo viridis) was measured during its natural moulting cycle using a fast flow technique. The skin behaved in all periods of the moulting cycle as a nearly perfect sodium electrode. In the presence of amiloride (10^–4 M), the total resistance of the same skin was identical with solutions which contained either sodium or potassium. The resistance of the skin with potassium was sensitive to amiloride in the period just after moulting. The resistance of skins which were made shunted by treating them with urea on the outside was insensitive to amiloride in solutions containing potassium; a small effect was still observed with sodium. It is suggested that the transient sensitivity to amiloride, with potassium, is the result of differentiation of the sodium specific sites at the apical membranes of the skin.     

Charges and Potentials at the Nerve Surface : Divalent ions and pH

The voltage dependence of the voltage clamp responses of myelinated nerve fibers depends on the concentration of divalent cations and of hydrogen ions in the bathing medium. In general, increases of the [Ca], [Ni], or [H] increase the depolarization needed to elicit a given response of the nerve. An e-fold increase of the [Ca] produces the following shifts of the voltage dependence of the parameters in the Hodgkin-Huxley model: m_∞, 8.7 mv; h_∞, 6.5 mv; τ_n, 0.0 mv. The same increase of the [H], if done below pH 5.5, produces the following shifts: m_∞, 13.5 mv; h_∞, 13.5 mv; τ_n, 13.5 mv; and if done above pH 5.5: m_∞, 1.3 mv; h_∞, 1.3 mv; τ_n, 4.0 mv. The voltage shifts are proportional to the logarithm of the concentration of the divalent ions and of the hydrogen ion. The observed voltage shifts are interpreted as evidence for negative fixed charges near the sodium and potassium channels. The charged groups are assumed to comprise several types, of varying affinity for divalent and hydrogen ions. The charges near the sodium channels differ from those near the potassium channels. As the pH is lowered below pH 6, the maximum sodium conductance decreases quickly and reversibly in a manner that suggests that the protonation of an acidic group with a pK_a of 5.2 blocks individual sodium channels.     

Acidification and sodium entry in frog skin epithelium

Acidification of the external medium by isolated frog skin epithelium (Rana catesbeiana, Rana temporaria, and Caudiververa caudiververa) and its relationship to Na⁺ uptake was studied. Acidification was measured by the pH-stat technique under short-circuit or open-circuit conditions. The results of this study demonstrate that (a) acidification by these species of in vitro frog skins is not directly coupled to Na⁺ or anion transport; (b) acidification can be inhibited by the diuretic drug amiloride, but only at high external Na⁺ concentrations; (c) acidification rate in these species of frog skin is controlled in part by the metabolic production of CO₂; and (d) the positive correlation between net Na⁺ absorption and net acidification observed in whole animal studies could not be replicated in the in vitro skin preparation, even when the frogs were first chronically stressed by salt depletion, a physiological state comparable to that used in the in vivo experiments.     

THE ELECTRICAL CONDUCTIVITY OF SODIUM AND POTASSIUM GUAIACOLATES IN GUAIACOL

The data of the author and Uhlig, and new data, on the conductivity of sodium and of potassium guaiacolates in guaiacol at 25° have been computed with an improved conductance equation which is valid to somewhat higher concentrations than the equations formerly used. The new constants are, Λ₀ = 9.0, K = 2.8 x 10^–5 for sodium guaiacolate and Λ₀ = 9.5, K = 3.4 x 10^–5 for potassium guaiacolate.     

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.     

Control of the Ca2+-triggered bioluminescence of Veretillum cynomorium lumisomes

Calcium ions can trigger an emission of light from Veretillum cynomorium lumisomes (bioluminescent vesicles) under conditions where they are not lysed. This process does not require a metabolically-linked source of energy, but is dependent upon the nature of the ions present inside and outside the vesicles. The Ca²⁺-triggered bioluminescence is stimulated by an asymmetrical distribution of cations or anions. Either high internal sodium or high external chloride is required for the maximal effect. When sodium is present outside the structure and potassium inside, the slow inward diffusion of calcium is decreased. Unbalanced diffusion of internal cations also stimulates the bioluminescence, suggesting control of the calcium influx by an electrochemical gradient. It is assumed that rapid outward diffusion of sodium or inward diffusion of chloride generates an electrical potential difference (inside negative) which drives the Ca²⁺-influx. With purified lumisomes it has been shown that Ca²⁺-triggered bioluminescence and calcium uptake (presumably net uptake) were correlated. In two instances uptake of the lipophilic cation dibenzyldimethylammonium has given direct evidence for the existence of a potential difference. With NaCl-loaded vesicles, it has not been possible to demonstrate an uptake of lipophilic cations but experiments with ²²Na and ⁴²K indicated a higher rate of sodium efflux, in accord with the proposed hypothesis.     

Nonosmotic Effects of Polyethylene Glycols upon Sodium Transport and Sodium-Potassium Selectivity by Rice Roots

Addition of polyethylene glycol (PEG) as an osmotic agent (at −230 kilopascals) dramatically lessened the toxicity of NaCl (at 50 moles per cubic meter) to rice (Oryza sativa L.) seedlings. This was explained by a reduction in the uptake of NaCl. This reduction was much greater than could be accounted for by the lowered transpiration rate resulting from the solute potential changes due to the PEG.

Low concentrations of PEG (−33 kilopascals and less) had no effect upon transpiration rate but reduced sodium uptake (from 10-50 moles per cubic meter NaCl) by up to 80%. PEG (at −33 kilopascals) also reduced chloride uptake but had no effect upon the uptake of potassium from low (0.5-2.0 moles per cubic meter) external concentrations. However, the increased uptake of potassium occurring between 2 and 10 moles per cubic meter external concentration was abolished by PEG. Similar concentrations of mannitol had no effect upon sodium uptake in rice. PEG, in similar conditions, had much less effect upon sodium uptake by the more salt-resistant species, barley.

²²Na studies showed that PEG reduced the transport of sodium from root to shoot, but had a long half time for maximal effect (several days).

¹⁴C-labeled PEG was shown to bind to microsomal membranes isolated from rice roots; it is suggested that this is due to multipoint attachment of the complex ions of PEG which exist in aqueous solutions. It is argued that this reduces passive membrane permeability, which accounts for the large effect of PEG on sodium influx in rice and the different effects on sodium influx and (carrier-dependent) potassium influx.

     

Apical sodium entry in split frog skin: Current-voltage relationship

Summary Apical Na⁺ entry into frog skin epithelium is widely presumed to be electrodiffusive in nature, as for other tight epithelia. However, in contrast to rabbit descending colon andNecturus urinary bladder, the constant field equation has been reported to fit the apical sodium current (N _Na)-membrane potential (^mc) relationship over only a narrow range of apical membrane potentials or to be inapplicable altogether. We have re-examined this issue by impaling split frog skins across the basolateral membrane and examining the current-voltage relationships at extremely early endpoints in time after initiating pulses of constant transepithelial voltage. In this study, the rapid transient responses in ^mc were completed within 0.5 to 3.5 msec. Using endpoints to 1 to 25 msec, the Goldman equation provided excellent fits of the data over large ranges in apical potential of 300 to 420 mV, from approximately –200 to about +145 mV (cell relative to mucosa). Split skins were also studied when superfused with high serosal K⁺ in order to determine whether theI _Na-^mc relationship could be generated purely by transepithelial measurements. Under these conditions, the basolateral membrane potential was found to be –10±3 mV (cell relative to serosa, mean±se), the basolateral fractional resistance was greater than zero, and the transepithelial current was markedly and reversibly reduced. For these reasons, use of high serosal K⁺ is considered inadvisable for determining theI _Na-^mc relationship, at least in those tissues (such as frog skin) where more direct measurements are technically feasible. Analysis of theI _Na-^mc relationships under baseline conditions provided estimates of intracellular Na⁺ concentration and of apical Na⁺ permeability of 9 to 14mm and of 3 × 10^–7 cm · sec^–1, respectively, in reasonable agreement with estimates obtained by different techniques.     

PERMEABILITY OF ERYTHROCYTES TO RADIOACTIVE POTASSIUM

The diffusion coefficients for the exchange of potassium across the membrane of erythrocytes of humans, rats, and rabbits have been determined by the use of artificially radioactive potassium, both into and out of the erythrocytes both in vitro and in vivo. The diffusion coefficients found in minutes^–1 were 0.2 to 0.25 x 10^–3 for human, 0.32 to 0.665 x 10^–3 for rabbits, and 1.0 x 10^–3 for rat erythrocytes. Rabbit erythrocytes appear to be more permeable in vivo. Reasons are advanced to explain the failure of earlier workers to demonstrate appreciable exchange of potassium in erythrocytes.     

Electrical Characteristics and Activation Potential of Bufo Eggs

Electrical characteristics and their changes during activation were studied with the microelectrodes on the oocytes and eggs of the toad, Bufo vulgaris formosus Boulenger. In young oocytes, the membrane characteristics had some similarities to those of nerve and muscle, except for a relatively large resistance of 25 KΩcm.² and an absence of the action potential in the former. After maturation, however, the membrane characteristics became entirely different from those of oocytes and other excitable tissues. In the mature eggs the membrane resistance was measured to be as high as 200 KΩcm.², and no specific permeability of the membrane to potassium ions was observable. A slow monophasic change in the membrane potential was recorded in every activation produced by mechanical stimulation, and termed "activation potential." In fresh water, its amplitude was as large as 80 to 90 mv. with an overshoot of about 50 mv. The activation potential might be comparable to the action potential of nerve and muscle, but was fundamentally different in ionic mechanism from the latter, since the former was caused by a marked increase in permeability to chloride ions.     

Potassium Ion Current in the Squid Giant Axon: Dynamic Characteristic

Measurements of the potassium current in the squid axon membrane have been made, after changes of the membrane potential to the sodium potential of Hodgkin and Huxley (HH), from near the resting potential, from depolarizations of various durations and amplitudes, and from hyperpolarizations of up to 150 mv. The potassium currents I given by I = I_∞ {1 - exp [- (t + t₀)/τ]}²⁵, where t₀ is determined by the initial conditions, represent the new data and approximate the HH functions in the regions for which they are adequate. A corresponding modification for the sodium current does not appear necessary. The results support the HH assumptions of the independence of the potassium and sodium currents, the dependence of the potassium current upon a single parameter determined by the membrane potential, and the expression of this parameter by a first order differential equation, and, although the results drastically modify the analytical expressions, they very considerably extend the range of apparent validity of these assumptions. The delay in the potassium current after severe hyperpolarization is used to estimate a potassium ion mobility in the membrane as 10^-5 of its value in aqueous solutions.     

Electrical Properties of Neurospora crassa : Effects of external cations on the intracellular potential

Glass micropipette electrodes have been employed to study the transsurface potential difference of Neurospora crassa. For mature hyphae grown in agar cultures, the internal potential is large and negative, often exceeding -200 mv. The potential is sensitive to the concentrations of extracellular potassium, sodium, hydrogen, and calcium ions, but does not vary in a manner which is readily explained by ionic diffusion potentials. With extracellular solutions containing only potassium chloride (or sulfate) and sucrose, the internal potential shifts toward zero (becomes less negative) at 45 mv per tenfold increase of potassium, over the range 0.1 to 10 mM. A similar result has been found with sodium, though the slope is only 33 mv/log unit. Calcium (1 mM) diminishes the influence of potassium and sodium by 60 to 70 per cent. As potassium or sodium is raised above 20 mM, the slope of the internal potential increases sharply to 85 to 90 mv/log unit, both in the presence and absence of calcium. With increasing hydrogen ion concentration, too, the internal potential shifts toward zero; in this case the slope is about 12 mv/pH unit at pH 9 and rises smoothly to 33 mv/pH unit at pH 3. All these phenomena are probably properties of the plasma membrane. The polysaccharide cell wall contains few fixed negative charges, has a low transverse resistance, and supports very little potential difference when separated from the plasma membrane.     

Effect of FeCl3 on ion transport in isolated frog skin

Summary The effect of addition of FeCl₃ to the media bathing the isolated skin ofRana pipiens was studied by measuring short-circuit current, transepithelial potential, and resistance, and by determining the influx and efflux of sodium (J ₁₃ ^Na andJ ₃₁ ^Na , respectively) and the influx and efflux of chloride (J ₁₃ ^Cl andJ ₃₁ ^Cl , respectively) across the epithelium. With normal Ringer's solution on both sides of the skin, addition of 10^–3 m FeCl₃ to the external medium resulted in nearly complete inhibition of active Na transport (J ₁₃ ^Na decreased from 1.30±0.14 to 0.10±0.04 eq/cm² hr (N=8)) and in appearance of active chloride transport in outward direction due to an 80% increase inJ ₃₁ ^Cl . Average (J ₃₁ ^Cl –J ₁₃ ^Cl ) obtained from means of 8 skins in 6 consecutive control and last 3 experimental periods was –0.17±0.04 and 0.38±0.05 eq/cm² hr, respectively. FeCl₃ added to external medium also induced substantial net chloride movement in outward direction when external medium contained Na-free choline chloride Ringer's or low ionic strength solution. Under the latter condition net Na movement was virtually eliminated by external FeCl₃. After addition of FeCl₃ to serosal medium there was delayed inhibition ofJ ₁₃ ^Na but no change in chloride fluxes. Immediate and profound changes in Na and Cl transport systems seen after external application of FeCl₃ indicate charge effects of Fe³⁺ on surface of apical cell membranes, possibly close to or in ion channels.     

K Fluxes in Frog Skin

A method has been developed for measuring K influx into the epithelial cells of frog skin from the inside solution. Diffusion delay in the connective tissue has been taken into account. Ninety-four per cent of skin K was found to exchange with K⁴² in the inside solution with a single time constant. K influx showed saturation with increasing K concentration, was not altered by imposing a potential difference of ±200 mv across the skin, and was inhibited by dinitrophenol, fluoroacetate, and ouabain. Relatively low concentrations of dinitrophenol (5 x 10^-5 M) and fluoroacetate (10^-10 M) had no effect on k influx but caused a 40 per cent decrease in net Na flux. There was no correlation between the rate of K uptake at the "inner barrier" and the rate of net Na transport. Reduction of net Na transport by lowering Na concentration in the outside solution caused little change in K uptake. These observations indicate that there is not a significant Na-K exchange involved in active transport of Na across the skin. K influx was found, however, to require Na in the inside bathing solution.     

The penetration of sodium into the epithelium of the frog skin

The aim of this paper is twofold. First, to describe a method for the measurement of the unidirectional flux of Na from the outer bathing solution into epithelium (J_OT), and second, to describe the use of this method under a variety of experimental conditions in order to obtain some insight into the nature of this flux. The method developed is based on the exposure of a frog skin to a Ringer solution containing ²²Na. The exposure is made so that neighboring points along the surface remain in contact with the ²²Na solution for gradually longer periods, ranging from 0 to 46 sec. Some 8 to 10 samples of the exposed part are used to obtain the time course of the uptake of ²²Na and this time course is used, in turn, to evaluate J_OT. This flux is then studied in skins mounted between two identical Ringer solutions with 115 mM Na (11.25 ± 0.10 [18] µmole·hr^-2 cm^-2), and in skins mounted with Ringer with 1 mM Na on the outside and 115 mM Na on the inside (0.43 ± 0.05 [18] µmole·hr^-1·cm^-2. From the observations that the flux is much larger than the net Na flux across the whole skin, that it is inhibited by K⁺, and is unaffected by ouabain, it is concluded that the penetration of Na⁺ into the epithelium does not occur by simple diffusion and is not directly dependent on an ouabain-sensitive mechanism. In the course of these experiments it was observed that when the skin was crushed between two chambers the uptake of Na in the neighboring exposed areas was decreased.