Ba2+-induced changes in the Na+- and K+-permeability of the isolated frog skin

Acta Physiol Scand 1985, 124, 61-70

Ba2+-induced changes in the Na+- and K+-permeability of the isolated frog skin

R O B E R T N I E L S E N

Institute of Biological Chemistry, University of Copenhagen, Denmark

NIELSEN, R. 1985. Baz+-induced changes in the Na+- and K+-permeability of the isolated frog skin. Acta Physiol Scand 124, 61-70. Received 10 May 1984, accepted 9 October 1984. ISSN 0001-6772. Institute of Biological Chemistry, University of Copenhagen, Denmark.

Addition of the K+-channel blocking agent Baz+ to the basolateral solution (in a concentration which is assumed to block the K+-flux via the K+-channels completely) resulted initially in a two-thirds reduction in the short-circuit current (SCC), followed by a complete recovery of the SCC. To examine the reason for this recovery, experiments were carried out which made it possible to calculate the Na+-permeability of the apical membrane (PNa) and the K+-permeability of the basolateral membrane (Pk) . The presence of BaZ+ had no significant effect on the cell volume and the cellular Na+- and K+-concentration. Addition of Ba2+ resulted in a depolarization of the intracellular potential ( Vscc) from a control value of - 76.3 f 2.8 mV to - I 5. I f I .7 mV. Although a complete recovery in the SCC was observed, Vscc did not recover. The K+-flux across the basolateral membrane was estimated from washout experiments. The washout of 4zK+ (the K+-efflux) could be described by a single exponential component with a half time of 3-70 min. The addition of BaZ+ during the washout resulted in a transient decrease in 4ZK+-efflux from the epithelium. From V,,, and the cellular K+ and Na+-concentration and the coupling ratio of the Na-K pump, it was found that Na+-permeability of the apical membrane was 6.5.10-7 crn.s-' before the addition of BaZ+ and I .7.10-6 cm . s-1 when the SCC had recovered after the addition of BaZ+ and Pk changed from 8.8.100 cm.s-I to 1.5.10-~ cm.s-'. Thus, the observed recovery in SCC was due to a considerable increase in Na+-permeability of the apical membrane and the presence or appearance of a small Baz+-insensitive K+-permeability in the basolateral membrane.

Key words: barium, frog skin, K+-permeability, Na+-permeability.

T h e absorption of Na+ appears to be similar in electrically ' tight' epithelia, such as frog skin, toad and rabbit bladder, cortical collecting tubule, and mammalian colon. I n these epithelia, the two-membrane hypothesis has served as an excellent model in the study of Na+-transport. According to this hypothesis (Fig. I a), the apical membrane is permeable to Na+ but impermeable to K+, whereas the basolateral membrane, which contains the Na-K pump, is permeable to K+

Correspondence : Dr Robert Nielsen, Institute of Biological Chemistry A, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen 0,

Denmark.

(Koefoed-Johnsen & Ussing 1958). Recent experiments have shown that the Na-K pump in isolated frog skin, turtle colon, and rabbit urinary bladder is a coupled electrogenic Na-K pump with a coupling ratio of I : 5 (3 N a : 2 K ) (Nielsen 1979a, b, Kirk et al. 1980, Lewis & Wills 1981).

Previous experiments (Nielsen 1979a, 1984, Nagel 1979, Van Driessche & Zeiske 1980) have shown that BaZ+ is a blocker of K+-channels in frog skin. Addition of BaZ+ (0.1-4.5 mM) to the basolateral solution of isolated frog skin results in a transient decrease in the short-circuit current (SCC) (Nielsen 1979a, b).

T h e effect of total blockade of the K+-channels

61

62 R . iVielsen

( a ) r--+

/FT+ '. . TO0+ 2K+ -

I - T 2 C I -

A B A B A B A B

Fig. I. (a) The two-membrane hypothesis. P, Na-K pump with a coupling ratio of I : 5 (3 Na: 2 K). (A) Apical membrane. (B) Basolateral membrane, (b) the CI--permeability of the apical and basolateral membrane is low, (c) The Cl--permeability of the basolateral membrane is high, (d) The Cl--permeability of the apical membrane is high.

on the SCC depends, according to the two- membrane hypothesis, on the C1--permeability of the membranes. Figure I(M) shows three extreme situations. In model b, it is assumed that the C1--permeability of the membranes is much smaller than the Na+ and K+-permeabilities; this model predicts that a sudden total blockade of K+-channels would reduce the SCC to one-third of its original value, because the current carried by K+-ions across the basolateral membrane is switched off. Thereafter, the SCC would continue to decrease because the cellular Na+-concentration decreases (3 Na+ are pumped out of the cell and z K+ + I Na+ enter the cell). In model c, it is assumed that the C1--permeability of the basolateral membrane is very high (Cl- is in equilibrium). Blockade of the K+-channel in this situation has no effect on the SCC, because serosal C1- would follow the pumped K+ into the cell and the cell would swell but maintain the current. In model d, it is assumed that the Cl--permeability of the apical membrane is high (Cl is in equilibrium). Blockade of the K+-channel in this system would reduce the SCC to one-third of the value it had before the addition of the K+-channel blocking agent (because z C1- follow 3 Na+ into the cell) and the cell would swell.

Addition of BaZ+ to the isolated frog skin in a concentration which is assumed to block the K+-flux via the K+-channel completely results initially in a two-thirds reduction of the SCC, but after the initial inhibition a complete recovery in the SCC was often observed (Nielsen 1979b). None of the models shown above predicts that the SCC should recover. The observed recovery must therefore be due to secondary changes in the Na+ and the K+-permeabilities of the membranes

or, the addition of BaZ* may turn the Na-K pump into a pure electrogenic Na-pump. T o get information about the nature of the observed recovery in the SCC, the changes in intracellular potential and ion composition, and the effect of Ba2+ on the K+-flux across the basolateral membrane were investigated.

From the data obtained it is concluded that the observed recovery in the SCC is caused by an increase in the Na+-permeability of the apical membrane and the presence or appearance of a small Baz+-insensitive K+-permeability in the basolateral membrane.

MATERIALS AND METHODS The experiments were performed on skins or isolated epithelia from male and female frogs (Rana temporaria). The skins were mounted in perspex chambers and bathed in stirred C1--Ringer's solution (Na+ = I I j.0, K' = 2.5, CaZ' = 1.0, HCO; = 2.5, and C1- = 117.0 mM, pH = 8.2). The epithelia were isolated as described by Johnsen & Nielsen (1978) and were incubated in a modified Ringer's solution (Na+ = 115.0, K+ = z . j , Caz+ = 1.0, Mg2+ = 1.0, HCO; = 2.5, HPO; = 1.0, C1- = 116.0, pH = 7.8 and glucose 5 mM): '4C-mannitol was added as a marker for extracellular space.

After the experiment, the epithelium was blotted on filter paper and weighed (wet weight); it was then dried for IZ h in a vacuum oven at 70 "C and weighed (dry weight). The dry epithelium was then extracted for at least 12 h with 2 ml0 .1 M HNO,, and the amount of K' and Na+ in the extract was determined by flame photometry. The amount of mannit~l-'~C was measured with a liquid scintillation counter. The short-circuit experiments were performed according to the method of Ussing & Zerahn (1951) with an automatic voltage clamp.

Efect of Ba on N a and K permeability 63

Table I. Comparison of the net Na+-flux and short-circuit current across frog skin before and after the addition of BaZ+ (4.53 mM)

Mean net Mean short- Efflux Sodium flux circuit

Minutes Influx (n mol . cmz .min-') (n mol . cmz. min-1) current

Control A 0 10.46 & 0.48 0.16fo.12 10.30fo.45 10.61 fo.50

Baz+ 125-185 10.43 f 1.26 0.34 f 0.09 10.09f1.23 10.41 f 1.42 Baz+ 65-125 7.71+0.49 0.41 k0.06 7.31 k0.45 7.49fo.79

The flux period was 60 min. The control values are the steady-state values found before the addition of Baz+. The influx was measured with z4Na+ and the efflux simultaneously with zzNa+. Values are the mean f SE of four experiments.

K washout

The isolated epithelium was mounted in Ussing-type chambers with an area of 1.5 cmz. The Ringer's solution containing 4zK+ was added to the basolateral side of the epithelium and was left there for 2 h. Ferreira (1979) has shown that the epithelia reach equilibrium with the 4*K+ of the loading solution during that period. After 2 h the loading solution was removed and both sides of the chamber, were washed three times in 3 min, and the washout of 4zK+ was started and carried out under short-circuited conditions. The solution bathing the apical and the basolateral side of the epithelium was changed every 10 min and replaced by a new solution (2.5 ml).

The intracellular potential was measured as described by Helman & Fisher (1977) using microelectrodes pulled from I .z mm fibreglass capillaries. The microelectrodes were filled with 0.5 M KCI and had a resistance between 40 and 60 MQ. The skins were punctured blindly from the outside with an impalement angle of 90'.

R E S U L T S

It is mentioned above that addition of the K+-channel blocking agent BaZ+ to the basolateral solution of the isolated frog skin results in a transient decrease in the SCC; for an actual experiment (see, for example, Fig. 3 or 4). The observed changes in the SCC after addition of BaZ+ are due to changes in the active Na+-transport (Table I) except for a short period (about 5 min) after the addition of BaZ+, where the net Na+-flux is higher than the SCC (Nielsen 1982b).

Water and ion content. The cellular water and ion contents were measured in isolated epithelia halves incubated under short-circuited conditions, one half in the absence of BaZ+ (the

control), and the other half in the presence of BaZ+ (1.57 mM). The epithelia were incubated until the SCC in the epithelia halves, to which BaZ+ was added, had reached a new steady state (60-90 min). The addition of Ba2+ resulted in a slight increase of the total K+-content and a slight decrease of the Na+-content, but had no effect on the C1- and water content. During the period after Baz+ addition and until the SCC had reached a new steady state, the total amount of Na+ transported was 483 82 nmol * mg dry weight-'. If the Na-K pump has a coupling ratio of I .5 (3 Na: 2 K) and theK+-channels are closed, then the net K+-uptake via the Na-K pump should amount to (483/1.5) = 322 nmol.mg dry weight-', and as C1- has to follow, it should result in an uptake of 2.5 mg cell water.mg dry weight-'. The K+-uptake was much smaller and no significant change in the cell water content was observed (Table 2). Therefore, the addition of Ba2+ to the isolated frog skin must result either in the formation of a K+-pathway with no or low affinity for Ba2+ or the Na-K pump has to change coupling ratio, such that it becomes more electrogenic.

The cellular Na+-concentration shown in Table 2 was estimated by measuring the wet weight, dry weight and mannitol space of the isolated epithelia, and the Na+-content in the epithelial extract with atomic absorption. I t is well known that this method gives too high values for the cellular Na+-concentration (Macknight & Leaf 1978, Nielsen 1982a): The cellular Na+- concentration can, however, be estimated by measuring the Na+-transport pool and cell water in isolated epithelia: cellular Na+ -concentration = (Na+-transport pool. cmz)/(cell water .cm-z).

(1)

64 R. Nielsen

50

40

- I 5 3 0 - E

5 5 2 0 -

I

- 0

10

Table 2. Effect of BaZ+ ( I .57 mM) on cell volume and cellular Na+, K+, and Cl--concentration of isolated frog skin epithelia

-

-

Cell volume Ion content (nmol.mg dry wt-I) Ion concentration (mM) (mg water.mg -

dry wt-I) Na+ K+ c1- Na+ K+ c1- Control 2.34 k O . 1 2 180f17 374+14 2 1 o f 4 4 1 . 9 k 6 . 2 1 5 8 f 5 53 .5k1.6 1.57 mM Ba2+ 2.49fo .14 1 5 6 f 1 8 436f17 213f13 31 .5f8 .7 173+4 52 .0k3.4 n 8 6 8 6 6 8 6

The isolated epithelia were incubated short-circuited in Cl--Ringer's solution in presence of absence of Ba2+. Values are the means+SE

The Na+-transport pool is equal to the amount of Nat in the cells which participates in the transepithelial Na+-transport and can be estimated in isolated epithelia by measuring the build-up of the transepithelial tracer influx (Nielsen 1982a). When the cellular Na+- concentration was estimated in this way, the relationship between the SCC and the cellular Na+-concentration could be described by equation 2 :

SCC = SCC,,,/(I +K,,/NaJn (2)

where n is the number of Na+-binding sites per pump unit, K,, the apparent dissociation constant of the Na+-sites of the Na+-pump complex, and SCC,,, the maximum Na+-pump flux, and Na, the cellular Na+-concentration: n was found to be equal to 3, KNa equal to 3.6 mM, and SCC,,, equal to 90 nmol .cm-z. min-' (Nielsen 1982a). Equation (2) was found to hold, when the SCC was reduced by addition of amiloride and activated by addition of antidiuretic hormone. T o investigate whether one could use equation ( 2 ) to obtain an estimate of the cellular Na+-concentration in the presence of BaZ+, the cellular Na+-transport pool and cell water were measured under steady-state conditions in isolated epithelia before and after the addition of BaZ+. The full line in Fig. 2 is drawn according to equation 2 , the broken line connects the individual experiments. The data presented in Fig. 2 show that one can get a reasonably good estimate of the cellular Na+-concentration by measuring the SCC under steady-state conditions and then using equation 2. From the data in Fig. 2 it was found that the cellular Na+- concentration under control conditions was 9.75 _+ I .43 mM and 5.32 + 0.74 mM after the addition of Ba2+.

K t - . u x . The Kt-efflux across the basolateral

membrane (the flux from the cells to the solution bathing the basolateral side of the epithelium) was estimated from washout experiments. Of the total amount of 4*K+ washed out from the epithelia only 0.34 0.05 yo was washed out to the apical solution; the rest (99.6%) was washed out to the basolateral solution. The washout of 42K+ from the cells to the basolateral solution

/ 5 0 5 10 15

mmol

Fig. 2. Steady-state SCC as function of the cellular Na+-concentration. The cellular Na+-concentration is estimated as shown in equation ( I ) . The full line has been calculated from equation 2. The broken line connects the separate experiments, x , Absence of BaZ+; 0, presence of 1.57 mM BaZ+ in the basolateral bathing solution.

Effect of Ba on N a and K permeability 65

Table 3. K+-efflux across the basolateral membrane of isolated frog skin epithelia in the presence and absence of Ba2+ (1.57 mM) in the basolateral solution

Rate constant K-content K-efflux SCC (min-I) (nmol . cm-z) (nmol . c m P . minP) (nmol . cmP. min-1) SCC/K,,,,,,

Control 0.0164+0.0018 674f23 10.gko.g 25.8 f 4.0 2.46 k 0.47 BaZ+ 0.01 10 fo.0017 760 t 9 2 7.7 k0.6 27.5 t 5.3 3.77 k0.89

The K+-efflux was calculated from the K+-content in the cells and the washout of 42K+ from the cells to the basolateral solution. The washout was carried out under short-circuited conditions. The data are the means f SE of five experiments.

Table 4. Uptake of 42K+ from the basolateral solution into isolated frog skin epithelia incubated in the presence and absence of BaZ+ (I .57 mM) in the basolateral solution

Uptake of 4zK+ SCC (nmol . cm-z. min-I) (nmol . cm-2. min-I) SCC/uptake of 42K+

Control 4 8.32t1.33 20.1 f6 .5 2.17f0.43 1.57 mM BaZ+ 6 8.02+0.65 17.5 f 3 . 6 2.21 f0.4

The epithelia were incubated under short-circuited conditions and 4zK+ was added to the basolateral solution 10 min before the epithelia were removed from the chamber.

could be described as consisting of a single exponential component with a half time of 3-70 min corresponding to a rate constant of 0.0164fo.0018 min-' (n = 5). To investigate the effect of BaZ+ on the K+-efflux across the basolateral membrane the epithelia were incubated in the presence of BaZ+ (1.57 mM) in the basolateral solution, until the SCC had reached a new steady state (about 70 min). The washout of 4zK+ was then carried out in the presence of BaZ+ in the basolateral solution. 1.95 fo.31 yo (n = 6) of the 42K+ in the cells was washed out to the apical solution, whereas the rest (98 yo) was washed out to the basolateral solution. The washout of 4*K+ from the cells to the basolateral solution in the presence of BaZ+ could be described as a single exponential with a rate constant ofo.0110 +0.0017 min-I. From the rate constant and the K+-content in the epithelial cells, the K+-efflux across the basolateral membrane was calculated (Table 3). It is seen that addition of BaZ+ in a concentration which is supposed to block theK+-fluxvia the K+-channels completely, resulted only in a small reduction in the K+-efflux. In another series of experiments the uptake of 4zK+ into the epithelial cells from the basolateral solution (the K+-influx) was measured in epithelia which were incubated under short-circuited conditions in the presence or the absence of BaZ+. When the SCC had

3

reached steady state, 4zK+ was added to the basolateral solution, and after 10 min of incubation the epithelia were punched out of the chambers and the uptake of 4zK+ into the epithelial cells was measured. From the data in Table 4 it is seen that the presence of BaZ+ had no effect on the 42K+-uptake. Thus, when epithelia were incubated with Baz+, until the SCC had reached the new steady state, BaZ+ had only a small effect on the 4ZK+-influx across the basolateral membrane.

However, if BaZ+ was added to the solution bathing the basolateral side of the isolated epithelium during the washout of 4zK+, a significant decrease in the washout of 4zK+ to the basolateral solution was observed (Fig. 3). Thus, just after the addition of BaZ+ to isolated epithelia, a highly significant decrease in both the SCC and the K+-efflux was observed, whereas, after about 70 min incubation of the epithelia in the presence of BaZ+, a nearly complete recovery of both the SCC and the K+-efflux was observed. The K+-fluxes given in Tables 3 and 4 are only quantitatively correct if there is isotope equilibrium between K+ in the interspaces and the basolateral solution, However, the volume of the interspaces is small compared with the volume of the cells, and the K+-concentration in the cells is much higher than the K+-concentration in the interspaces; therefore it is very likely that some

ACT 124

66 R . Mie hen

I , movement of K+ via the K+-channels (Ussing

20

I c E 0.0 Y

-

Under control conditions, the coupling ratio of the Na-K pump is 1.5 (Nielsen 1982~) . Thus,

0 20 40 60 80

Minutes

Fig. 3. Effect of BaZ+ (1.57 mM) on the short-circuit current and the rate constant for the 42K+-efflux into the basolateral solution. The rate constant was calculated for each sampling period (10 min) and was plotted against the midpoints for each period. (-), SCC, nmol.cm-Z.min-'. (---) Rate constant, k, min-I.

of the K+ ions leaving the cells via the K+-channels are pumped into the cells via the Na-K pump. Thus, K+ might recycle across the basolateral membrane (Harris & Burn 1949, Nielsen 1982~) . A recycling of K+ would cause the K+-efflux across the basolateral membrane to be higher than the efflux found by measuring the appearance of 42K+ in the basolateral solution.

Under steady-state conditions, the net K+-influx into the cells via the Na-K pump is equal to the net K+-flux from the cells via the K+-channels to the basolateral solution (Fig. I a). If the coupling ratio of the Na-K pump (p) is known, the net K+-flux via the Na-K pump and the K+-channels can be calculated from SCC/p.

1949). The value of Kef f /Kin is 3.2 under control conditions. Thus, Kin = Keff /3 .z . From equation (3) one can calculate that SCC/Keff should be about I . From the data in Tables 3 and 4 it is seen that SCC/Keff is about 2.2-2.5. Thus a considerable recycling of K+ takes place under these experimental conditions. After the addition of BaZ+ Kef f /Kin is 38.5. If BaZ+ has no effect on the coupling ratio of the Na-K pump, SCC/Keff should be 1.46.

The addition of BaZ+ to isolated epithelia had only a small effect on the cell volume and the cellular K+-concentration (Table 2). As both the SCC and the thickness of the epithelia are about the same before and after addition of Baz+, one would expect the degree of recycling of K+ across the basolateral membrane to be the same in these two situations. The thickness of the epithelia used in the experiments shown in Table 3 was 68.9+2.2pm in the absence of BaZ+ and 68.5 k7 .1 p m in the presence of BaZ+. The value of SCC/Keff in the control experiments was 2.46 (Table 3), whereas it should be one according to the arguments presented above. Thus, each K+-ion crosses the basolateral membrane 2.46 times during the washout of 4zK+. In order to get a correct 4ZK+-efflux in the presence of BaZ+ the K-efflux given in Table 3 should be multiplied by the recycling factor (2.46). If, in the presence

Table 5 . Effect of BaZ+ and Baz+ plus amiloride on the electrical parameters of frog skin

SCC (nmol . cm-z. min-' 1 VSCC (mV) FRa PDa (mV) PD, (mV)

- __ ~ ~ ~~ . ~~~

Control I3.9f 1.8 -76.3 f2.8 o.84f0.05 2.5f5.1 -97.5 & 1.5 BaZ+, 70 min 15.3k2.6 - 1 5 . 1 f1 .7 0.60f0.04 25.3 f 4 . 2 -45.9 k 5.6 BaZ' + amil o.3fo.1 -43.9k3.1 0.93f0.02 -43.3f7.2 -43.3f7.2

The experiments were performed as illustrated in Fig. 6 and the abbreviations are the same as used in Fig. 6. Values are the means & SE of five experiments.

Eflect of Ba on N a and K permeability 67

- 100 > E

- 50

Amil

Minutes

Fig. 4. Effect of Ba2+ and BaZ+ plus amiloride on short-circuit current (SCC), the cellular potential under short-circuit conditions ( Vscc), the fractional resistance of the apical membrane FRa = R,/(R, + Rb), where R, is the resistance of the apical membrane and R, is the resistance of the basolateral membrane. At the arrow marked Baz+, Ba2+ (1.57 mM) was added to the basolateral bathing solution and at the arrow marked amil, amiloride (0.1 mmol) was added to the apical bathing solution.

of BaZ+ (Table 3) Keff is multiplied by 2.46, one finds that SCC/Kef, is 1.46 f 0.36, the value one would get, if the coupling ratio of the Na-K pump is 1.5 in the presence of BaZ+.

Effect of Bat+ on the intracellular potential. In the previous section it was shown that after 70 min of incubation with Bat+ the SCC and the passive K+-efflux had recovered the values they had before the addition of Bat+. To evaluate the effect of BaZ+ on the permeabilities of the membranes, the intracellular potential under short-circuiting conditions ( V,,,) in the presence and absence of BaZ+ was measured. The value of V,,, in isolated frog skins was - 76.3 & 2.8 mV (Table 5); the addition of Baz+ to the basolateral solution resulted in a prompt decrease in V,,, (Fig. 4). This observation confirms the data obtained by Nagel (Nagel 1979). Although the SCC recovered completely, no recovery in V,,, was observed (Fig. 4, Table 5). Furthermore, as one would expect for a blocker ofthe K+-channels, addition of Baz+ resulted in a decrease in the fractional resistance of the apical border (FR,) and in a depolarization of the open-circuit potential across the basolateral membrane (PD,) (Fig. 4, Table 5).

Addition of amiloride resulted in a nearly total inhibition of the SCC and V,,, changed from - 15.1 to -43.9 mV (Table 5, Fig. 4).

As BaZ+ had no effect on the cellular K+-concentration (Table z), and as the cellular Na+-concentration did not increase (Fig. 2), the observed recovery in the SCC must be due to an increase in the Na+-permeability of the apical membrane and the presence or the appearance of Baz+-insensitive K+-channels in the basolateral membrane.

D I S C U S S I O N

If the epithelial cells were permeable for C1-, a blockade of the K+-channels would result in a swelling of the cells (Fig. I). Addition of the K+-channel blocking agent Bat+ to the basolateral solution had no effect on the cell volume (Table z ) , so one can conclude that the C1--permeability of the epithelial cells is low. Under these circumstances, one would expect that a total closure of the K+-channels (if the coupling ratio of the Na-K pump is 3 Na+:2 Ka+) would initially result in a two-thirds inhibition of the SCC, followed by a slower decrease (Fig. I b). After the expected Baz+-induced inhibition of the SCC an increase, and not the expected decrease, was observed (Nielsen 1979 b). This observed recovery in the SCC must be due to Bat+-induced secondary changes in the transport system, such as formation of Bat+-insensitive K+-channels, or changes in the Na-K pump coupling ratio

3-2

68 R. n;ielsen

A B A B Fig. 5. P, Na-K pump. (a) Apical membrane. (b) Basolateral membrane.

making it a pure electrogenic pump. If the latter is the case, the active transepithelial Na+-transport before BaZ+ addition is brought about as shown in Fig. 5a and after BaZ+ addition and SCC recovery as shown in Fig. j b. If the Na-K pump becomes electrogenic, comparison of 5 a with 5 b shows that both K+-uptake and K+-efflux would be expected to decrease. Ba2+ had no effect on the steady-state K+-uptake and K+-efflux (Tables 3 and 4). Thus, the recovery in SCC after addition of Ba2+ is not explained by a change in the coupling ratio of the Na-K pump.

This statement is supported by previous experiments (Nielsen 1979a), where it was shown that the active transepithelial K+-transport (measured in skins, where the apical membrane has been made permeable for K+ by addition of the polyene antibiotic, filipin) was activated by adding BaZ+. In Na+-transporting epithelia, such as frog skin, toad bladder, and rabbit urinary bladder, the Na+-permeability of the apical membrane appears to be much higher than that of the basolateral membrane, and the K+- permeability of the apical membrane is much lower than that of the basolateral membrane.

Therefore, under steady-state, short-circuit conditions, the net flux across the apical membrane (the SCC) is equal to the net Na+-flux via the Na-K pump. The net K+-flux into the cells via the Na-K pump is equal to the SCC divided by the coupling ratio of the Na-K pump (SCC/I.5), and the net K+-flux into the cells via the Na-K pump is equal to the net K+-flux out of the cells via the K+-channels.

From the data presented in Table 5 it is seen that the steady-state SCC was 13.9 nmol . cmpz. min-' before the addition of BaZ+, and Vscc was -76.3 mV. When the SCC had recovered after addition of BaZ+ (and was in a new steady state), the SCC was I 5.3 nmol.cm-Z.min-l and V,,, was - I j.1 mV. Addition of Ba*+ had only a slight effect on the cellular K+-concentration (Table 2). These data are put together in Table 6A. The net K+-flux via the K+-channels has been calculated from S C C / I . ~ (as shown above). By using this data and the Goldman-Hodgkin-Katz equation (4), one can calculate the K+-permeability of the basolateral membrane (Pk) (Table 6a):

3i. R . T [ I - exp (zi F A $ / R T ) zi . F . A$ cp - cE (exp zi F A $ / R T )

pi = -

(4) Pi is the permeability of the ion i, A@ is equal to Vscc, cp is the concentration of the ion in the bathing solution, cE is the concentration of the ion in the cells, and zi, R , T, and F have their usual meaning. T i e data in Table 6 b is used to calculate the Na+-permeability of the apical membrane (PaNa). The net Na+-flux across the apical membrane is equal to the SCC. The cellular Na+-concentration was calculated from the relationship between the SCC and the cellular Na+- concentration (Fig. 2, eqn 2) (Nielsen 1982a). From the data in Tables 6a and b it is seen that, although the SCC was ofnearly thesamemagnitude under steady-state conditions before and after addition of the K+-channel blocking agent BaZ+,

Efect of Ba on Na and K permeability 69

Table 6. Data used for the calculation of the K+-permeability of the basolateral membrane ( P k ) and the Na+-permeability of the apical membrane (paNa)

SCC Net K+-efflux K+-cell p; (nmol.cm-2.min-') (nmol .cm-2.min-2) (mM) V,,, (mV) (cm.s-')

(a) Control I3.9f 1.8 9.27 1 5 8 f 5 -76.3 f 2.8 8.8 I O - ~ 1.57 Ba2+ 15.3f2.6 '0.2 '73 +4 - 15. I f I .7 I .5 . I O - ~

(nmol . ern-*. min-I) (mM) (cm . s-I)

Control 13.9f1.8 '3.9 4.16 -76.3f2.8 6.5.10-7 1.57 Ba2+ 15.3f2.6 '5.3 4.47 - 1 5 . 1 f 1 . 7 1 . 7 . 1 0 - ~

The net K+-efflux via the K+-channel in the basolateral membrane was calculated from S C C / I . ~ . The cellular

net Nat-influx Na+-cell P N a

(b)

Nat-concentration was calculated from the SCC by using equation 2.

both P$ and PNa had changed significantly. Thus, the observed recovery in the SCC is due to a highly significant increase in the Na+-permeability of the apical membrane and the presence of a small Ba2+-insensitive Kf-permeability in the basolateral membrane.

Cell volume and Cl-. I f C1- is in equilibrium across the cell membrane, the C1--concentration in short-circuited epithelial cells (V,,, - 76.3 mV) should be about 6 mM in the absence of Bat+ and about 65 mM in the presence of Bat+ (V,,, - 15.1 mV). T h e cellular Cl--concentration was 53.5 mM in the absence of Ba2+ and 52.0 mM in the presence of Ba2+ (Table 2) . I n isolated frog skin, electron microprobe analyses have shown that the ClF-concentration was 36 5 mmol . kg wet mass-' (Rick et al . 1978). Thus, C1- is not distributed at equilibrium across the membranes. The high cellular C1--concentration is supposed to be maintained by a co-transport of NaCl or NaKzCl (Nellans et al. 1973, Geck & Heinz 1980, Ussing 198z).The cellular C1- is in a steady state, when the net Cl--flux into the cells via the co-transport system is equal to the net Cl--flux out of the cells via the Cl--channel or 'leak'. Thus, a depolarization of V,,, should result in a swelling of the cells, because a depolarization of V,,, per se has no effect on the driving force for the co-transport system, but it reduces the net Cl--flux out of the cells. However, if, as shown by Ussing (1982), the co-transport mechanism is virtually dormant, unless the cells have lost C1-, the changes in the cellular C1--content might proceed so slowly that they could not be detected in the period used for

the experiment. Alternatively, Bat+ per se might have an effect on the C1--transport system. However, the mechanism remains obscure and is now under investigation.

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JOHNSEN, A.H. & NIELSEN, R. 1978. Effects of the antidiuretic hormone, arginine vasotocin, theophyl- line, filipin and A23187 on cyclic AMP in isolated frog skin epithelium (Rana temporaria). Acta Physiol Scand 102, 281-289.

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KOEFOED-JOHNSEN, V. & USSING, H.H. 1958. The nature of the frog skin potential. Acta Physiol Scand 42, 298-308.

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Ba2+-induced changes in the Na+- and K+-permeability of the isolated frog skin