The Effect of Aldosterone in vitro on the Active Sodium Transport and Moulting of the Frog Skin

Acta physiol. scand. 1969. 77. 85-94 From the Institute of Biological Chemistry, University of Copenhagen, Denmark

The Effect of Aldosterone in vitro on the Active Sodium Transport and Moulting of the Frog Skin

BY

ROBERT NIELSEN

Received 9 December 1968

Abstract

NIELSEN, R. T h e effect of aldosterone in v i t 7 o on the active sodium transport and moulting of frog skin. Acta physiol. scand. 1969. 77. 85-94.

I t is shown that aldosterone in concentrations higher than 1.5. 10." M induces a moult in vitro, and that the moult is accompanied by characteristic changes in the potential and the short- circuit current. The sequence of changes can be divided into four parts: the first constant period, the inhibition period, the spontaneous activation period and the second constant period. At a concentration of 7 . M aldosterone the inhibtion starts 2-4 hrs and the activation 4-7 hrs after the addition. Furthermore it has been shown that the short-circuit current is higher in the second constant period as compared to the control skin half. During the spontaneous activation period the net sodium flux exceeds the short-circuit current by 25 %. The changes in chloride flux and sodium efflux during the moulting cycle were rather small. Both the moult and the activation of the short-circuit current were abolished by actinomycin D. A hypothesis which could explain the behavior during the moulting cycle and the activation of the sodium transport is given.

The in vitro stimulation by aldosterone of the sodium transport in the toad bladder is well established (CrabbC 1961, Porter and Edelman 1964, Sharp and Leaf 1964). The stimulation which occurs after a latent period of 40-90 min, is characterized by a gradual increase in the short-circuit current. I t has been shown that this stimulation is dependent upon protein synthesis (Fanestil and Edelman 1964). The action of aldosterone on the sodium transport of the frog skin has not been so fully in- vestigated. CrabbC (1964) described experiments in which the skins of Rana ridi- bunda and Rana esculenta were bathed on the outside with Ringer's solution con- taining choline instead of sodium. After 3 hrs incubation with 10 ,LAM d-aldosterone on the inner side of the skin, the choline-Ringer's solution was replaced by normal Ringer's solution. An increased short-circuit current was observed in the aldosterone treated skins as compared to the control skins.

CrabbC and De Weer (1964) have shown that the injection of aldosterone into Bufo marinus resulted in a stimulation of the active sodium transport across the

85

86 ROBERT N I E L S E N

isolated bladder and skin. Jerrgensen and Larsen (1964) and Stefan0 and Donoso (1964) have shown: "Extirpation of the pars distalis of the hypophysis inhibits shedding, whereas the formation of new sloughs continues at an increased rate. A slough was most often found to form within 2 or 3 days after the operation. The premature formation of a slough after hypophysectomy could be further accelerated by injection of ACTH or corticosteroids (aldosterone) about 18 hrs, or a little more, after the operation. After such injections a complete molt could be produced within about 6 9 hours". The present experiments show that aldosterone added to the isolated frog skin produces a moult and a stimulation of the active sodium transport.

Materials and methods The experiments were performed on male and female frogs ( R a n a temporaria) . The frogs were kept partially immersed in tap water at about 4". The skin was dissected from pitched animals and divided in two symmetrical halves, one of which was used as a control and the other for the experiment. The skins were mounted in perspex chambers and bathed in aerated Ringer's solution (Na+= 113.6, K-=2.0, Ca++=l.O, C1-=115.2, HCO-=2.4, mhl, pH=8.4) . The short-circuit experiments were performed according to the method of Ussing and Zerahn (1951), with an automatic voltage clamp apparatus which disconnected the short-circuit current every five minutes, allowing the potential to be measured for 15 sec. Two types of chambers were used, one with an area of 7 cm2, the other with an area of 5.6 cm2. The 5.6 cm2 chamber was open to the exterior so that it was possible to have access to the outside of the skin, which was supported on a nylon mesh (Andersen and Zerahn 1963). Radio- nuclides used were chloride-36, sodium-22 and sodium-24. Sodium-22 and sodium-24 were counted with a Selectronic autogamma spectrometer, and the chloride-36 with a Packard liquid scintillation spectrometer using a naphthalene-dioxane solution as scintillator. The aldosterone (Aldocorten, Ciba) was added to both sides of the skin unless otherwise stated.

Result

T h e effect of aldosterone on the short-circuit current and potential in the frog skin. Fig. 1A shows a typical result of adding 7 . 10 M aldosterone to the medium bath- ing the frog skin, while fig. 1B shows the corresponding control. The graphs de- picting the short-circuit current and the potential after addition of aldosterone are divided into four parts. A-B is called the first constant period, B-C the inhibition period, C-D the spontaneous activation period, and D-E the second constant period. At a concentration of 7 - M aldosterone the inhibition started in most cases 2 4 hrs and the activation 4-7 hrs after the addition. The initial decline in the short-circuit current during the constant period is the usual decrease seen at the commencement of short-circuiting conditions and is not due to the addition of the aldosterone (cf . Fig. 1B). In other experiments in which the short-circuit current was steady the addition of aldosterone produced no initial decrease. I n 7 out of 17 expts. there was a small but significant increase in the short-circuit current starting 40-90 min after the addition of the aldosterone (Fig. 2 ) . 3 of these 7 expts. did not show any subsequent inhibition and activation period. With a lower concentration of 1.5 * M (6 expts.) there was normally no clear indication of when the inhibition period started, and the activation commenced 7-12 hrs after addition of hormone. Four attempts to demonstrate activity at 7 - 10-l" M were unsuccessful. Experiments with a higher concentration of 7 * M (4 expts.) showed the same

T H E EFFECT OF ALDOSTERONE ON T H E FROG S K I N 87

u A , m V k n/crn'

-

-

.

-

.

-

-

Fig. 1A. Effect of aldosterone on the short-circuit current and potential across the frog skin. At zero time aldosterone was added on both sidess to give a concentration of 7.10-'M.

short-circuit current (,uA/7 cmz)

resistance (ohm/cme) 0- 0 potential (mV) 0- - - - -0

4.9

4 2

3.5

2.9

21

I 4

0.7

0 0 1 2 3 L 5 6 7 8

010 9

-

-

-

-

-

-

-

-

UP

301 5.6

49

4.2

3.5

2.8

21

1.L

0.7

0

2 01

101

Fig. 1B. Control skin half without added aldosterone; symbols as in Fig. 1A.

Hours

"V knfcrn'

0 . 0 1 2 3 4 5 6 7 8

Hours

effect as those performed with 7 - 10.' M. The concentrations which provide minimal and maximal response are therefore about 1.5 - M to 7 - 10.' M, respectively. The hormone elicited its effect both from the inside and the outside.

Effect of mechanical action. During the latter part of the inhibition period the skin is very sensitive to the mechanical action of changing the Ringer's solution or applying a pressure difference. Fig. 3 shows the result of applying a pressure of 50 cm of water for 20 sec to the inside of the skin. Immediately a large increase in short-circuit current was observed in the aldosterone-treated half-skin, whereas there was little change when a pressure difference was applied to the control. This effect was absent when a pressure difference was applied in the first constant period, or in the first part of the inhibition period. Further investigation showed that the same or even a higher activation could be obtained if the stratum corneum were removed (Fig. 4 A ) . Experiments with an open chamber showed that this effect was due to the removal of the stratum corneum and not to the mechanical dis- turbance resulting from draining and refilling the chamber. The stratum corneum

88

uA mV 7 . 1 0 - ~ M

Aldosterone

ROBERT h-IELSEN

200--100

100-50

0 1 2 3 4 5 6 0’ t 8 ’ ’ .

Hours

Fig. 2. Early activation by aldosterone in the short- circuit current and potential observed in 7 out of 17 expts.

short-circuit current; aldosterone treated skin.

0- 0 potential; aldosterone treated skin. - - - - - - - - short-circuit current; control skin half. 0- - - - -0 potential; control skin half.

was removed by gentle rubbing of the skin with a cotton-wool tampon, and i t slipped off rather easily in the aldosterone-treated skins whereas it could not be removed in the control half (Fig. 4B). Again, no effect was observed in the first constant period or in the first part of the inhibition period; attempts to remove the corneum during these periods were unsuccessful, and were always accompanied by a decrease in the potential. In only a few experiments was there any activation of the short-circuit current and it was of a small magnitude.

T h e effect of aldosterone on the sodium flux. T o ascertain whether the changes in the short-circuit current following aldosterone treatment were due to an effect on the active transport or on the passive flux, or on the combination of both, measurements were performed using the double-labelling technique of Levi and Ussing (1949). Two series of experiments were performed. I n one the stimulation was allowed to proceed spontaneously (Table I ) ; in the other the stimulation was induced by removal of the stratum corneum (Table 11). There was good agreement between the net sodium flux and the short-circulit current during the inhibition period (B-C) and the second constant period (D-E). However during the activation period (C-D), there was a difference between the two series of experiments. When the activation was spontaneous the short-circuit current was about 25 higher than the net sodium flux (Table I ) . When the activation was induced by the removal of the stratum corneum, there was a tendency for the short-circuit current to be lower than the net sodium flux (Table 11), even though the sodium efflux was doubled by the treatment (c f . Table I and 11). The cause of the discrepancy between the net sodium flux and the short-circuit current during the activation period were short lived, because good agreement between the two measurements was observed again during the one hour period following activation.

THE EFFECT OF ALDOSTERONE ON THE FROG SKIN 89

Y A mV

"A

LO(

3M

2 01

010 , 5 6 7 8 9 10 11

n w r s

Fig. 3

S omc cum removed "V

7.10.' M Atdostcrone

. . . . . ___--- - _ _ . . . . . -. .

100

50

a Scorncum rubbcd

.. ----- ------

50

0 1 2 3 L 5 6 7 0 9

HO"r5

Fig. 4 Fig. 3. Effect of applying a pressure of 50 cm of water to the inside for 20 sec.

At zero time aldosterone was added on both sides to give a concentration of 7.10-'M. Pressure applied 8 hrs after the addition of aldosterone, when the aldosterone treated skin was in the inhibition period.

0- 0 - - - - - - - - 0- - - - -0 Fig. 4A. Effect of removing the stratum corneum. At zero time aldosterone was added to both sides to give a concentration of 7.10-'M. The stratum corneum was removed 5 + hrs after the addition of aldosterone.

0- 0 potential.

Fig. 4B. Control skin haIf without added aldosterone; symbols as in Fig. 4A.

short-circuit current; aldostorene treated skin. potential; aldosterone treated skin. short-circuit current; control skin half. potential; control skin half.

short-circuit current (,uA/7 cmz)

Table I11 shows the changes in the sodium efflux during the first constant period and the first and the second half of the inhibition period in 6 half-skins. It appears that the efflux was rather constant during this period.

T h e effect of aldosterone on the chloride flux. I n the previous section it was shown that the short-circuit current was higher than the net sodium flux in the spontaneous activation period. This could be due to an active transport of chloride from the inside to the outside, since Koefoed- Johnsen, Ussing and Zerahn ( 1952) have shown

90 ROBERT NIELSEN

TABLE I. Comparison of the sodium flux and the short-circuit current across the frog skin with spontaneous activation following aldosterone treatment.

A B

sodium short-circuit flux current

Period influx efflux Mean net Mean B-A 5 S . E . p

pA/7 cmz

(B,-C,) 118.6 11.9 106.6 101.7 - 4.9 3.6 0.2<p<0.25 (BZ-CZ) 62.1 9.9 52.2 49.1 - 3.1 2.4 0.1 <p<o.2 (C-D) 94.0 15.1 79.0 97.7 18.4 1.9 p<O.OOl (D-E) 169.8 11.8 158.0 155.6 - 2.4 2.2 0 . 3 < P t 0 . 4

The influx was measured by means of Na-22 and the efflux simultaneously with Na-24; the short- circuit current was measured and recorded automatically.The mean results from 6 expts are presen- ted, in which each period was about 1 hr. B,-C,: first half of inhibition period; B,-C,: second half of inhibition period; C-D: activation period; D-E: constant period. For an acutal pettern of an experiment see fig. 1A.

TABLE 11. Comparison of the sodium flux and the short-circuit current across the frog skin after the mechanical removal of the stratum corneum following aldosterone treatment.

A B

sodium short-circuit flux current

Period Influx efflux Mean net Mean B-,4 S.E. p

yA/7 cmz

Activation 301.7 39.0 262 238 -24 14.2 0.1 <p<o.2 Constant 248.0 29.0 218 22 1 3 4.3 0.3<p<0.4

~______ ~

The influx was measured by means of Na-22 and the efflux simultaneously with Na-24. The short- circuit current was measured and recorded automatically. The mean results from 6 expts are presen- ted, in which each period was about 1 hr. For an actual pattern of an experiment see Fig. 4. The isotopes were added immediately after the corneum was removed. The first samples were taken 15 min after the addition of the isotopes.

that the glands of the skin actively liberate chloride to the outside when the skin is treated with adrenaline. Six experiments were therefore performed in which the chloride influx and efflux were measured separately on symmetrical half-skins during aldosterone treatment with subsequent spontaneous activation (Table IV) . The chloride permeation was constant during the first constant period ('4-B) and the inhibition period (B-C), there was in five of the experiments an increase in the chloride permeation during the activation period (C-D) but influx as well as


TABLE 111. Effect of aldosterone on sodium efflux during the first constant period and the inhibition period.

Period: Constant first Inhibition first Inhibition second half half

pA/7 cm"

11.5 10.7 14.7 8.2 7.5

11.5 11.3 12.6 10.7 10.8 11.9 13.1 12.6 16.1 27.2 23.4 22.5

TABLE IV. Effect of aldosterone on chloride fluxes during spontaneous activation.

Exp.no. Influx Efflux

Period A-B B-C C-D D-E A-B B-C C-D D-E

pA/7 cm2

1 13.4 13.9 8.8 11.0 2 26.3 30.0 33.0 11.0 18.8 15.0 3 11.8 12.6 16.1 14.5 19.3 18.2 22.0 22.8 4 12.1 7.2 24.7 25.7 20.6 21.4 27.3 23.3 5 10.7 11.5 15.5 19.3 18.5 15.3 23.8 29.4 6 71.8 79.9 49.6 68.8 72.6 82.8 64.9 62.9

~~

The chloride influx and efflux were measured simultaneously on symmetrical halves of a frog skin, during aldosterone treatment.

A-B first constant period B-C inhibition period

C-D spontaneous activation period D-E second constant period

efflux was equally affected. Thus there was no net movement of chloride during the activation period which would account for the discrepancy of 18.7 pA/7 cm3 observed between the net sodium flux and the short-circuit current.

Effect of actinomycin D on the aldosterone treated frog skin. To examine whether the aldosterone effects was dependent on protein synthesis, 6 expts. were performed in which two skin halves were incubated with 7 - M aldosterone, but one half had in addition 6 pg/ml of the protein-synthesis inhibitor actinomycin D. The actinomycin D abolished both the inhibition period which commenced after about 3 hrs and the activation period which commenced after five hours (Fig. 5) in the control half.

ROBERT NIELSEN

0 1 2 3 L 5 6 1 8

Hours

Fig. 5. Effect of actinomycin D on the aldosterone treated frog skin.

short-circuit current (pA/7 ern.). At zero time aldosterone and actinomycin D were added on both sides to give concentrations of 7- 10- ?M and 6pg/ml respectively. - - - - - - - short-circuit current (,uA/7 cm2). At zero time aldosterone was added on both sides to give a concentration of i.IO-?M.

Discussion

It appears from the above observations that aldosterone induces a “moult” in vitro i.e. the separation of the stratum corneum from the stratum granulosum, and that the moult is accompanied by characteristic changes in the electrical potential and the short-circuit current, one of which is a late activation of the short-circuit current. The fact that both these processes occur only after a latent period and are abolished in the presence of actinomycin D indicates that they are dependent on protein synthesis. A comparison between Fig. 1A and Fig. 1B shows that the short- circuit current is higher in the second constant period (D-E) in the aldosterone treated skin than in the control. The same is seen from Fig. 3 where the activation is induced by pressure treatment. The experiment shown in Fig. 4 indicates that the transport system already is activated when the skin is in the inhibition period, since the removal of the stratum corneum immediately causes an activation of the short- circuit current and the potential. A hypothesis to account for these observations has to provide answers to the following questions:

1. Why do the short-circuit current and the potential exhibit the pattern shown in Fig. l A ? The experiments show that the inhibition is not due to an increase in passive fluxes, since the sodium efflux and the chloride fluxes are constant during the first constant period and the inhibition period (Table 111 and IV) . The inhibition must therefore bue due to a specific action on the active sodium transport.

2. Why is there a discrepancy between the short-circuit current and the net sodium flux during the spontaneous activation period (Table I, C-D)? From Table I V it is seen that this discrepancy is not due to an active transport of chloride.

3. Why does the removal of the corneum induce a sharp rise in the short-circuit current and the potential (Fig. 4 ) ? The following working hypothesis would ex-


plain these observations. During the first constant period an enzyme which attacks the material between the stratum corneum and the stratum granulosum is formed and released. During the break-down a subcorneal space is formed (Voute, Nielsen and Ussing 1968). The space contains a compound which inhibits the active sodium transport; this postulate is supported by the fact that the active transport is activated immediately if the cornified layer is stripped off, when it would be expected that the inhibitor would rapidly diffuse away. The inhibitor could be a product of the brokendown cement material. The spontaneous activation could then be due to the gradual diffusion away or chemical disentegration of the inhibitor. The discrepancy between the net sodium flux and the short-circuit current during the spontaneous activation period could be explained by the fact that the samples are not taken during steady state conditions. I t has been shown by Hoshiko and Ussing (1960) and Andersen and Zerahn (1963) that it needs 0.3 to 1.5 peq sodium/7 cm2 to bring a skin from an unlabelled state to the labelled state. During the latter part of the inhibition period the influx of sodium-22 through the skin is very low, and the skin would be in a nearly unlabelled situation with respect to sodium-22 if the influx barrier, as generally accepted, is close to the outside of the skin. During the spontaneous activation period the sodium-22 influx increases, which would bring the skin from a nearly unlabelled state to a labelled state. Thus the discrepancy observed (0.6 peq/7 cm') is used to bring the skin frcm a nearly unlabelled form to a labelled iorm .

The cycle is finished when the skin is in the second constant period. The corneum .has now become so leaky due to the action of the enzyme that the ions diffuse easily through it, and the skin is back in its normal transport situation. The above working hypothesis explains the qualitative behavior during the moulting cycle, but it does not explain why the short-circuit current is increased in the second constant period as compared to the control half. A possible explanation would be that the diffusion of sodium through the corneum and the material between the corneum and the stratum granulosum was the rate limiting step. The activation observed would then be a consequence of the breakdown of this material and the corneum during the moult. However in 7 out of 17 skins (Fig. 2 ) there was an increase in the short-circuit current 40-90 min after the addition of aldosterone, as in the toad bladder. These skins were probably skins which had just moulted since it was observed in 4 of the 7 that the corneum slipped off during mounting of the skin. The results of these 7 expts. may indicate that aldosterone has two effects on the frog skin: 1, it activates the sodium transport in a unknown manner, 2 it induces a moult. However the activation could be due to an additional effect on the outermost border of the stratum granulosum by the moult enzymr or enzymes. If it is assumed that an early activation normally is superimposed on the inhibition during the moult, a higher short-circuit current would be expected in the aldosterone treated skins when the inhibitor concentration was low enough not to abolish the activation. A low inhibitor concentration could be due to the lack of a corneum or to a corneum which is rather permeable for the inhibitor so that this does not pile up. Another possibility .

94 ROBERT NIELSEN

would be that the amount of precursor to the inhibitor is low. One of these 01- a combination of both possibilities may be the case in recently moulted skins. How- ever it is not yet possible from the experimental data to distinguish between these hypotheses.

References ANDERSEN, B. and K. ZERAMN, Method for non-destructive determination of the sodium

transport pool in frog skin with radiosodium. Acta physiol. scund. 1963. 59. 319-329. C R A B B ~ , J., Stimulation of active sodium transport by the isolated toad bladder with al-

dosterone in vitro. J . clin. Invest. 1961. 40. 2103-2110. C R A B B ~ , J., Stimulation by aldosterone of active sodium transport across the isolated ventral

skin of amphibia. Endocrinology 1964. 75. 809-81 1. C R A B B ~ , J . and P. DE WEER, Action of aldosterone on the bladder and skin of the toad.

Nature (Lond.) 1964. 202. 208-209. FANESTIL, D. D. and I. S. EDELMAN, O n the mechanism of action of aldosterone on the sodium

transport: effects of inhibitors of RN.4 and protein synthesis. Fed. Proc. 1966. 25. 912- 916.

HOSHIKO, T. and H. H. USSIXG, The kientics of Na2" flux across amphibian skin and bladder. Acta physiol. scand. 1960. 49. 74-81.

JBRGENSEN, c . B. and L. 0. LARSEN, Further observation on molting and its hormonal control in Bufo bufo (L.), Gen. cornp. Endocr. 1964. 4. 389-400.

KOEFOED-JOHNSEN, V., H. H. USSING and K. ZERAHN, The origin of the short-circuit current in the adrenaline stimulated frog skin. Acta physL'oE. scand. 1952. 27. 38-48.

LEVI. H. and H. H. USSING. Restine potential and ion movements in froq skin. Nature - _ (Lond.) 1949. 164. 928-929.

PORTER, G. A. and I. S. EDELMAN, The acticn of aldosterone and related rorticosteroids on sodium transport across the toad bladder. J . clin. Invest. 1964. 43. 611-620.

202. 1185-1 188.

Gen. cornp. Endocr. 1964. 4. 473-480.

the short-circuited isolated frog skin. Acta physiol. scand. 1951. 23. 110-127.

SHARP, G. W. G. and A. LEAF, Biological action of aldosterone in vitro. Nature (Lond.) 1964.

STEFASO, F. J. E. and A. 0. DONOSO, Hypophyso-adrenal regulation of moulting in the toad.

USSING. H. H. and K . ZERAHK, Active transport of sodium as the source of electric current i n

VOGTE. C. L., R . NIELSES and H. H. USSIX, To be puhl.

Documents

The Effect of Aldosterone in vitro on the Active Sodium Transport and Moulting of the Frog Skin