The Frog Skin Potential

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The Frog Skin Potential

HANS H. USSING

From the Institute of Biological Chemistry, University of Copenhagen, Denmark

I N T R O D U C T I O N

For many years the isolated surviving frog skin has been among the favorite objects of electrophysiologists and students of ion transport. This interest has turned out to be rather rewarding. Not only is the frog skin preparation inter- esting in its own rights, but there is mounting evidence that it can serve as a model system for the study of properties found in many epithelial and glandu- lar tissues. In fact, certain features like the active sodium transport which is so conveniently studied in the frog skin, seem to be characteristic of most animals cells and of many plant cells as well.

Since the time of D u Bois Reymond (1848) it has been known that the isolated frog skin maintains a potential difference between its outside and inside, the inside solution becoming sometimes more than 100 my positive relative to the outside.

In the beginning of this century Galeotti (1904, 1907) demonstrated that the maintenance of the potential depends on the presence of sodium ions (or lithium ions), and he proposed that the potential arises from the fact that the skin is more permeable for these ions in the inward direction than in the outward direction. As this seems to violate the second law of thermodynamics, the theory was, however, discarded by his contemporaries. Nevertheless, he was not far from the truth as will appear from the following.

Besides maintaining the potential difference, even when in contact with identical sodium-containing solutions, the amphibian skin also has another striking property, namely that of transporting sodium chloride from the outside to the inside solution (Huf, 1935, Krogh, 1937, 1938). That the sodium transfer is due to active transport is evidenced by the fact that it can take place against the electric as well as the chemical potential gradient (Ussing, 1949a). The chloride transfer, on the other hand, always takes place down the electrochemical potential gradient. Indeed, the r a t i o between the inward and outward fluxes of chloride ions is exactly what would be predicted from the simple flux equation (Ussing 1949b) for an ion moving passively through the skin, influenced only by the combined effect of concentration and electric gradients (Koefoed-Johnsen, Levi, and Ussing, 1952).

135

The Journal of General Physiology

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136 Pavs~oLoov oF CELs M~M~RAN~

Active Sodium Transport as Source of Short-circuit Current

T h e fact that the sodium transport is bound to be active whereas the chloride m o v e m e n t is passive obviously suggests that the potential driving the chloride ions through the skin is created by the active sodium transport (Ussing 1949b), and conclusive proof of this hypothesis was obtained by way of the short- circuiting technique (Ussing and Zerahn, 1951). When the potential across a skin with identical sodium-containing solutions on both sides is maintained at zero by an applied external ~..M.F., the current generated by the frog skin is exactly equal to the net flux of sodium ions transported from the outside to the inside. Chloride ions, under the same conditions, pass through the skin in both directions at exactly the same rate, thus giving no contribution to the flow of electric current. These findings were fully confirmed by Linderholm (1952).

T h e transfer of sodium ions quite clearly is active, since the inward flux is from 20 to 100 times greater than the outward flux (as evidenced by simul- taneous determination of the two fluxes with the two tracers for sodium, Na 2~- and Na24). Thermodynamically, the work involved when sodium ions are transported between solutions of equal composition and equal potential is formally nil, but it should be remembered that, during the transport, the sodium ions must overcome the frictional resistance of the cell membranes and other structures in the skin. T h e overcoming of these resistances represents the work done in the short-circuited skin.

Thus qualitatively the total electric asymmetry of the isolated surviving frog skin (when in contact with identical solutions) comes from the active transport of sodium ions from the inside to the outside. T h e chloride (and other passively diffusing ions) presents a shunt, tending to lower the voltage of the total system below the ideal ~..M.F. of the "sodium battery."

Sodium Transport and Oxygen Consumption

Quantitatively we have observed that, during short-circuit, there is a one to one relationship between active sodium transport and output of

electric

current. Another important correlation exists between the active sodium transport and the oxygen consumption of the skin. (Zerahn 1956, Leaf and Ren- shaw, 1957). T h e rate of oxygen consumption at an arbitrary rate of sodium transport minus the oxygen consumption in the absence of sodium transport (blank value) is proportional to the rate of sodium transport. For each mole- cule of oxygen (02) used over and above the "blank value" there is a net transport of 18 sodium ions. This relationship holds whether the sodium is being transported between identical solutions at zero potential difference or whether it is being transported "uphill" an electrochemical potential gradi-

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Ussx~o

The Frog Skin Potential

i37 ent. Thus it seems that the sodium transport is linked to the metabolism in a stoichiometric way.

T h e same relationship has been found in skins of m a n y amphibian species and seems to be independent of temperature. T h e link between oxygen consumption and sodium transport is not a direct one, however. According to Andersen (unpublished), the oxygen consumption adjusts to changes in the rate of ion transport with a delay of about 15 minutes, indicating that there is some energy store in the skin which can be drawn upon when the d e m a n d increases, and which can be built up again in periods of lowered demand.

Probably this energy store is identical with the cellular store of energy-rich phosphate esters. This view gains support from the fact that the sodium transport is strongly inhibited by D N P and other uncoupling agents.

Lithium Transport

T h e transport mechanism is highly specific to sodium ions. Only lithium can to some extent replace sodium (Zerahn, 1955). It seems, however, that al- though Li can enter the epithelium from the outside about as fast as sodium, it cannot be transported onward as effectively. Thus, Li piles u p in the epithelium cells and ultimately brings about an inhibition of the ion transport mechanism.

Necessity of K for Current Output

Potassium ions, even in high concentration, do not contribute to the short- circuit current as judged from its contribution to the net current generated by the skin. Nevertheless, K is absolutely necessary for the generation of current. If K is left out of the ions of the bathing solution, the potential and the short-circuit current are completely abolished. (Huf, 1955, see also Koefoed-Johnsen and Ussing, 1958).

The Potential Development in the Absence of Diltusible Anions

When the skin is in contact with Ringer and other chloride-containing solutions the skin potential is a rather involved function of the active sodium transport and the shunt presented by the permeating chloride ions. If, however, this shunt is removed, either by replacing the chloride by a non-penetrating anion like sulfate or by making the skin practically impermeable to G1 by treating the outside with a highly diluted (10-~ molar) solution of GuSO4, the potential turns out to be a rather simple function of the concentrations of K and N a in the bathing solutions. In short, the potential behaves as if the inward facing side of the skin were a potassium electrode, whereas the outward

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z38 P H Y S I O L O G Y O F C E L L M E M B R A N E facing side were a sodium electrode (Ussing and Koefoed-Johnsen, 1956, Koefoed-Johnsen and Ussing, 1958). Fig. 1 shows the potential as a function of the logarithm of the sodium concentration in the outside medium. It is seen that proportionality between increase in potential and log (Nao) exists over a wide range of concentrations. T h e slope of the line is almost the ideal one for a sodium electrode. T h e concentration of K can be varied extensively on the outside without giving rise to the slightest change in potential. T h u s

m v /20 II0 too 90 7O 60 5o 30 20 I0 O 0.1

/

.2kin in A(~S~.Rin9er / ~/

/

///

/////

~ , . I i , , ~ | . . . i a e a s i a 5 t o e o s o t o o [Not~J m. e~./litet.

Fxoum~ 1. Frog skin (Rana temporaria) with Na~70~ on both sides. Ordinate: skin potential. Abscissa: sodium concentration of the outside bathing solution.

one gets the impression that the outward facing side of the skin is passively permeable to Na, b u t practically impermeable to K, as well as other cations (apart from Li).

If the dependence of the potential upon the composition of the inside bathing solution is studied in a similar way, it turns out that now the potential is practically independent of the sodium concentration, as long as the latter remains higher than I0 n ~ / l i t e r . O n the other h a n d the potassium concentration becomes of overwhelming importance. As shown in Fig. 2, which gives the potential as a function of the log K inside, the potential changes have the right magnitude for a potassium electrode, suggesting that the inward facing m e m b r a n e of the epithelium cell must be m u c h more permeable

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U~mG The Frog Skin Potential I39 to K than to all other ions present. I n equation form the total potential seems to be given by the equation:

E = R T/F(In(Nao/Na~) Jr ln(K,/K,))

in which Nao is the outside N a concentration and Na~ the cellular N a concen-' tration, K~ the cellular K concentration and K~ the K concentration in the inside bathing solution. We thus have the apparently paradoxical situation

m y I$o too s o x ~ Cu÷tfreo&d ~l~in 0 I l I . . * ' t l [ I I I I ~ i i 2 ¥ 6 B f O 20 ~0 6 0 B O

F m u ~ 2. Frog skin (Rana temporaria) with Ringer solution on both sides. Outside bathing solution made lO-6M with respect to Cu ++. Ordinate: skin potential, Ab- sci.~a: potassium concentration of the inside bathing solution.

that the potential can be fully accounted for on the basis of simple diffusion potentials, and yet we have equally good evidence that the potential is due to active transport of sodium. It is clear, however, that the contradiction is only apparent. This becomes obvious the m o m e n t we allow the anion to penetrate the skin. This will result in leakage of NaCI into the epithelium cells from the outside medium, whereas KCI w o u n d flow from the cells to the inside bathing solution, depleting the cells of K. Thus the potential would r u n down, and, as a matter of fact, the cells would swell and probably burst if there were not an active transport mechanism located in the inward facing cell m e m b r a n e which maintained the cellular sodium low and the potassium high (see Fig. 3). T h u s the electric and osmotic properties of the

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z4 o P H Y S I O L O G Y O F C E L L M E M B R A N E

epithelium cells could be accounted for if we make the following three assumptions: (1) T h e inward facing cell m e m b r a n e has properties similar to those of nerve a n d muscle fibres, viz., it is highly permeable to K and CI but slightly permeable to Na (and sulfate); (2) the o u t w a r d facing m e m b r a n e is highly permeable to N a and also permeable to C1, but practically impermeable to K ; and (3) the inward facing cell m e m b r a n e only is provided with an active cation transport mechanism which maintains the cellular sodium low and the potassium high. Practically speaking, N a passes this m e m b r a n e only b y w a y of the pump. T h e potential profile obtained by puncturing the skin with micro-electrodes is in accord with these concepts (Hoshiko and Engbaek,

1956; Engbaek and Hoshiko, 1957). t /°MI

Q~,.~. Ze.m..

FmURE 3. Scheme illustrating the origin of the skin potential. The stratum germinativum cell has an outward facing cell membrane, O.¢.m., which is selectively permeable to Na, and an inward facing cell membrane, Lc.m., which is selectively permeable to K. Further the inward facing membrane is provided with the ion pump, P, which pumps Na from the cell to the inside solution in return for K.

Osmotic Properties of the Epithelial Cells

So far the explanation of the potential development might be considered an

ad ho¢ hypothesis, constructed so as to account for the electric properties of the skin. It is, however, possible to m a k e predictions from the hypothesis which c a n be verified by i n d e p e n d e n t means. Thus, from the assumption that the i n w a r d facing m e m b r a n e is permeable to C1 and K, b u t not to Na, it can be predicted that the epithelium should swell osmotically w h e n the NaC1 of the inside solution is partly replaced by KCI, whereas no swelling, b u t r a t h e r a slight shrinkage, should ensue if a similar replacement takes place in the outside solution. These and several other predictions have been verified in experiments in which the thickness of the epithelium has been measured u n d e r the microscope in a special setup u n d e r which the inside a n d outside solutions could be varied independently. T h e experiments were initiated by the a u t h o r d u r i n g a stay at the National H e a r t Institute, Bethesda, in 1957, and have been continued by Dr. M a c R o b b i e in our laboratory. T h e setup is shown in Fig. 4,

The skin (S) is tied onto a plastic ring (R). The cup formed by the ring and the skin is placed upon a thin mat of glass wool (G) on top of a glass or lucite plate (L). The outside of the skin is facing upward whereas the inside is facing the glass wool. Solutions can be perfused though the glass wool sheet by running them in through a

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Ussmo The Frog Skin Potential ~4~

pipette (P) and sucking them off through the pipette (S), connected to the suction pump. Thus the inside of the skin can be effectively flushed, and the solutions can be changed quite rapidly by having a battery of inflow tubes connected to solutions of different composition. The outside of the skin (which forms the bottom of the cup) is covered by solution which flows in through (I) and out through tube (0). The whole assembly is placed on a microscope stand and a water immersion lens (Leitz; magnification 60 or 80) dips into the solution in the cup. The skin thus can be observed from above, light being admitted through the glass wool layer. By turning the fine screw of the microscope it is possible to focus sharply either on the cornified layer (which shows up as a characteristic "chicken fence") or on a suitably chosen melanin granule in the melanophore layer immediately beneath t h e epithelium. Thus the thickness of the epithelium can be estimated from the calibration of the

w

\\

FIGURE 4. Setup for measuring volume changes in the frog skin epithelium. L: lucite plate; G:

glass wool mat; S: frog skin; R: plastic ring; W:

water immersion lens; P: inflow pipette for "inside bathing solution"; ~': pipette for sucking off "inside bathing solution"; I: inlet for "outside bathing solution"; O: outlet for "outside bathing solution." S and 0 are connected to water suction pump. B l a n d B2 KCI agar bridges connecting the outside and inside bathing solutions to calomel half cells for measuring the skin potential.

fine screw. If, for any reason, the epithelium cells change their volume, this manifests itself in a change in thickness, since the glass wool mat keeps the tissue from chang- ing its area. Measurements of the thickness can be reproduced with an accuracy of a few micra.

T h e applicability of the m e t h o d obviously depends on the assumption that the cells are relatively readily p e r m e a b l e to water and that they do n o t mechanically resist attainment of osmotic equilibria or steady states. T h a t these assumptions are most likely to be correct is clearly seen in experiments in which the sole anion of the m e d i u m is the non-penetrating sulfate ion. After allowing a suitable time for equilibration, one can assume that all diffusible salts in the cells have been leached out, so that only salts of non- diffusible anions like phosphate esters and proteins remain. F r o m then on- w a r d no salt can m o v e in or out and only water should be able to cross the membrane. Fig. 5 shows an experiment of this type in which the m e d i u m on the inside of the skin is first sulfate-Ringer, which is then changed stepwise to half sulfate-Ringer and b a c k to sulfate-Ringer. It is seen that the v o l u m e changes are rapid, well defined, and reversible. T h u s the inward facing m e m - brane is readily p e r m e a b l e to water. If, however, the tonicity of the outside

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I42 P H Y S I O L O G Y O F C E L L M E M B R A N E

55 5 0

45

40

_d

Fxoux~ 5. Volume changes of frog skin epithelium

(Rana temporaria)

in response to

changes in tonicity of an inside bathing solution with a non-penetrating anion (sulfate). Outside medium sulfate-Ringer, i.e., Ringer where chloride is replaced by the equiva- lent amount of sulfate. Ordlnate: Thickness of the epithelium. Abscissa: time in minutes. At the beginning of the experiment the inside medium is also sulfate-Ringer. At the first arrow the inside bathing solution is changed to ~'~ sulfate-Ringer, at the next arrow to half-sulfate-Ringer, at the third arrow back to ~ sulfate-Ringer, and at the fourth arrow to sulfate-Ringer again.

b a t h i n g s o l u t i o n is c h a n g e d t h e v o l u m e response is v i r t u a l l y nil. T h u s t h e o u t - w a r d f a c i n g b o u n d a r y o f t h e e p i t h e l i u m is m u c h less p e r m e a b l e to w a t e r t h a n is t h e inside. I n passing it m a y b e m e n t i o n e d t h a t a d d i t i o n of a n t i d i u r e t i c h o r m o n e o r o x y t o c i c h o r m o n e to t h e inside b a t h i n g s o l u t i o n increases t h e w a t e r p e r m e a b i l i t y of t h e o u t w a r d f a c i n g b o u n d a r y of t h e e p i t h e l i u m , so t h a t a f t e r t h e h o r m o n e t r e a t m e n t t h e e p i t h e l i u m does r e s p o n d t o o s m o t i c c h a n g e s of t h e outside m e d i u m . T h i s m e a n s t h a t t h e c h a n g e in p o r e size s o m e w h e r e 20

/

8 0 , ~ / , 0 R R ! ,, . e : _

F m v ~ 6. Volume changes (lower curve) and potential changes (upper curve) of the

frog skin epithelium

(Rana teraporaria),

associated with changes in K-- concentration of

the inside bathing solution. Ordinate: lower curve, thickness of epithelium (/~), upper curve potential in m;llivolts (inside solution positive). Abscissa: time in hours. Outside medium: ~'~0 Ringer. Inside medium at the beginning of the experiment normal Ringer. At the first arrow a modified Ringer is introduced in which 80 per cent of the Na is replaced with K. At the second arrow ordinary Ringer is again introduced.

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Ussmo The Frog 8kin Potentid z43 in the skin which was postulated previously (Koefoed-Johnsen and Ussing,

1953; Ussing a n d Andersen, 1956; Andersen and Ussing, 1957) on the basis of penetration kinetics to u n c h a n g e d molecules, can now be located at the outer border of the epithelium.

T h e swelling in dilute sulfate solutions can be used to determine the osmotically effective thickness of the epithelium since, obviously, the content of osmotically active w a t e r in the cells should double w h e n the tonicity is re- d u c e d to half. It will be noticed that with an original thickness of 38 m i c r a the skin swells by 19 m i c r a in half sulfate-Ringer. T h u s n e a r l y half the

sR /,o my t20 IO0 8O 6O 40 2O o .... $ o do i o

FIOURE 7. Volume changes (upper curve) and potential changes (lower curve) of the frog skin (Rana teraporaria) associated with changes in the inside and outside bathing solutions. Ordinate: upper curve, thickness of the epithelium, lower curve skin potential (inside solution positive). Abscissa: time in minutes. Arrows pointing down- wards indicate times of change of inside solution, arrows pointing up indicate times of change of outside solution. At the beginning of the experiment both outside and inside solution are sulfate-Ringer. For explanation, see text.

thickness seems osmotically inert. It is highly suggestive t h a t the s t r a t u m g e r m i n a t i v u m ceils which are assumed to be the seat of the active transport and the potential also occupy about half the thickness of the epithelium, the remaining thickness being occupied by cells which have lost their foothold on the basement m e m b r a n e a n d which are in the process of cornifying. These cells probably are not in osmotic contact with the inside bathing solution.

According to the hypothesis outlined above, KCI should penetrate the in- w a r d facing m e m b r a n e whereas the outside of the skin should be impermeable to KCI. This in fact t u r n e d out to be correct. N o penetration of K C I could be demonstrated from outside. If, however, p a r t of the NaCI of the inside solutions was replaced b y KCI the epithelium responded b y swelling correspondingly. This is shown in Fig. 6. T h e thickness of the epithelium be-

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I44 P H Y S I O L O G Y O F C E L L M E M B R A N E

fore application of a high K solution on the inside was 80 micra, but after replacement of 80 per cent of the NaCI (on a molar basis) with KC1, the epithelium started swelling and reached a quasi-equilibrium in about one hour. O n returning to normal Ringer in the inside solution, the volume re- verted to its original value, indicating that the treatment with high K h a d not d a m a g e d the tissue.

If, instead of chloride as the dominating anion, sulfate is used there is no volume response whatsoever when K replaces N a in the inside solution, in accord with the assumption that the inward facing m e m b r a n e is impermeable to sulfate ions.

I

4O 30 , , . , . , , , _ t0 20 30 40 SO 60 70 80 90 IIW 9o ~0 ~0 SO

FIGU~ 8. Volume changes (upper curve) and potential changes (lower curve) of the frog skin epithelium (Rang temporaria), associated with dilution of the Ringer solution bathing the inside of the skin. Outside solution ~'~0 Ringer. At the first arrow the inside bathing solution is changed to half-Ringer. At the second arrow the inside solution is changed back to Ringer.

Due to the presence of the effective sodium pump, the cell sodium is practically always kept relatively low. T h u s even if sodium chloride is supposed to be able to pass the outside boundary of the epithelium, one cannot expect drastic changes in the volume when the outside NaCI concentration is changed. Nevertheless the volume does change measurably under such conditions, and the changes are of the right direction and magnitude. This is shown in Fig. 7. T h e skin is first in sulfate-Ringer and the inside solution is then changed to half sulfate-Ringer in order to obtain from the swelling the effective thickness of the epithelium. After the return to normal sulfate-Ringer, the inside solution is next changed to chloride-Ringer. There is a slight shrink-

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UssmG The Frog Skin Potential x45

age (because the osmolarity of the chloride-Ringer is higher than that of sulfate-Ringer), b u t the volume increases again, apparently due to entry of chloride into the ceils. If now a modified Ringer, containing both Na(57m~) and CI(ll7.5mM), is used as outside medium, there is a further swelling which can only be due to the entry from the outside of sodium chloride. Thus we have demonstrated that the outward facing boundary of the epithelium is permeable to NaC1 b u t not to KC1, whereas the opposite is true for the inward facing membrane, all in agreement with the conclusions drawn from purely electrical measurements.

It was mentioned above that with solutions having sulfate as the sole anion, changes in tonicity of the m e d i u m lead to simple osmotic responses of the epithelium, indicating that only water passes the membranes. If chloride- Ringer is used, changes in osmolarity by dilution of the inside m e d i u m no longer result in such simple behavior of the system. This can be seen from Fig. 8. Going from Ringer to half-Ringer, one observes a rapid swelling, b u t it is followed by a shrinkage which brings the volume almost half the way back to its original value.

These responses are of considerable interest, since they provide evidence that the epithelium cells possess a potassium p u m p besides, or possibly coupled with the sodium p u m p . T h e argument goes as follows: W h e n the Ringer bathing the inside of the skin is suddenly diluted to half, water starts moving into the cells, b u t the ensuing shrinkage indicates that they must contain some solute which can leave the cells by diffusion into the inside solution. J u d g i n g from the secondary shrinkage, the a m o u n t of this diffusible sub- stance is so large that it is bound to be an inorganic salt. F r o m the foregoing we know that the inward facing m e m b r a n e is impermeable for all the ions present except K and C1. T h u s it seems that an a m o u n t of KC1 corresponding to at least 30 per cent of the osmolarity of the cells before swelling can be given off after dilution of the inside medium. This, however, means that the cells were not in D o n n a n equilibrium with the inside m e d i u m as far as KC1 is concerned. I n the Ringer solution the ionic product K X C1 (using milli- moles as the unit) is 2.5 X 115 = 287.5. We know that the cellular K concentration is about as high as the CI concentration of the Ringer. T h u s the C1 concentration of the cell interior ought not to be more than 2.5 millimolar, b u t that obviously cannot be reconciled with the fact that at least 25 per cent of the osmolarity of the ceils seems to be due to KCI. T h e conclusion is that KC1 must be present in the cells over and above the concentration required for D o n n a n equilibrium. We know, however, that chloride behaves completely passively in the frog skin, and the conclusion therefore is that it is the potassium ion which is present in a concentration (and electrochemical ac- tivity) higher than that in the bathing solution. I n other words, potassium

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146 P H Y S I O L O G Y O F C E L L M E M B R A N E

must be p u m p e d into the cells, just as seems to be the case for nerve, muscle, a n d red cells. T h e simplest m e c h a n i s m thus seems to be a coupled N a / K - p u m p .

C O N C L U S I O N

It will be seen that the hypothesis outlined above gives a unified description of salt transport, potential development, cellular electrolyte composition, a n d regulation of cell volume. T h e r e is m o u n t i n g evidence in the literature t h a t the same or very similar principles are at work in m a n y other systems. It is one of the advantages of the hypothesis that it seems to bridge the gap between ionic regulation in individual cells and net transport of ions from one cell b o u n d a r y to the other.

R E F E R E N C E S

ANDERSEN, B., and UssrNo, H. H., 1957, Solvent drag on non-electrolytes during osmotic flow through isolated toad skin and its response to antidiuretic hormone,

Acta physiol, stand., 39,228.

Du Bots REY~OND, E., Untersuchungen fiber Tierisehe Elektrizit~it, Berlin, 1848. ENOBmK, L., and HosH~o, T., 1957, Electrical potential gradients through frog skin,

Acta physiol, stand., 39,348.

GAL~-Oaa'I, G., 1904, Concerning the E.M.F. which is generated at the surface of ani- mal membranes on contact with different electrolytes, Z. physik. Chem., 49,542. GALV.OTrI, G., 1907, Ricerche di elettrofisiologia secondo i criteri dell 'elettrochimica.

Z. allg. Physiol., 6, 99.

HuF, E., 1935, Versuche fiber den Zusammenhang zwischen Stoffwechsel, Potential- bildung und Funktion der Froschhaut, Arch. ges. Physiol., 235,655.

HuF, E., 1955, Ion transport and ion exchange in frog skin, in Electrolytes in Bio- logical Systems. (Shanes, A. M., editor), Am. Physiol. Sot., Washington, D. C. HosmKo, T., and ENGB~:, L., 1956, Microelectrode study of the frog skin potential,

Abstr. Com. 20th Internat. Physiol. Cong., Brussels, 1956, 443.

KOEFOED-JOm~S~N, V., LEvi, H., and Ussmo, H. H., 1952, The mode of passage of chloride ions through the isolated frog skin, Acta physiol, stand., 25, 150.

KOEFOr.D-JoHNS~N, V., and Uss~o, H. H., 1953, The contributions of diffusion and flow to the passage of D~O through living membranes, Acta physioL stand, 28, 60. KOEFOED-Jom~S~.N, V., and UsSlNO, H. H., 1958, The nature of the frog skin poten-

tial, Acta physiol, scand., 42,298.

KROOH, A., 1937, Osmotic regulation in the frog (R. esculenta) by active absorption of chloride ions, Stand. Arch. Physiol., 76, 60.

KROOH, A., 1938, The active absorption of ions in some fresh water animals, Z.

vergleich. Physiol., 25,335.

LEAr,, A., and RENSHAW, A., 1957, Ion transport and respiration of isolated frog skin,

Biochem. J., 65, 82.

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Ussmo The Frog Skin Potential x47

LINDERHOLM,

H., 1952, Active transport of ions through frog skin with special refer- ence to the action of certain diuretics, Acta physiol, scan&, 27, Suppl. 97, 1. Ussmo, H. H., 1949a, The active ion transport through the isolated frog skin in the

light of tracer studies, Acta physiol, scan&, 17, 1.

UssmG, H. H., 1949b, The distinction by means of tracers between active transport and diffusion, Acta physiol, scan&, 19,43.

Ussmo, H. H., KRtrH~F~ER, P., HESS THAX'SEN, J., and THORN, N. A., 1959, The alkali metal ions in biology, Handb. exp. Pharmakol., Erganzungswerk 13.

UssmG, H. H., and KO~FO~D-JOHNSE~, V., 1956, Nature of the frog skin potential,

Abstr. Corn. 20th Internat. Physiol. Cong., 568, Brussels, 1956.

Ussmo, H. H., and AND~.RSBN, B., 1956, The relation between solvent drag and active transport of ions, Proc. 3rd Internat. Cong. of Biochera., Brussels, 1955, 434.

Ussmo, H. H., and ZERAnN, K., 1951, Active transport of sodium as the source of electric current in the short-circuited isolated frog skin, Acta physiol, scan&, 23, 110. Z~RAm% K., 1956, Oxygen consumption and active sodium transport in the isolated

and short-circuited frog skin, Acta physiol, stand., 36,300.

ZsRta-m, K., 1955, Studies on the active transport of lithium in the isolated frog skin,

Acta physiol, scan&, 33,347.