Acta Physiol Scand 1979, 107: 189-191

A 3 to 2 coupling of the Na-K pump responsible for the transepithelial Na transport in frog skin disclosed by the effect of Ba

ROBERT NIELSEN

Institute of Biological Chemistry A , Aug. Krogh Institute, Copenhagen, Denmark

According to the two-membrane hypothesis (Koefoed-Johnsen & Ussing 1958) one would ex- pect an increase in the K+ permeability of the out- ward facing membrane to result in an active out- ward transport of K+ (Fig. 4). Polyene antibiotics increase the permeability of cell membranes for K + and other ions (De Kruijff & Demel 1974). Addition of polyene antibiotics (filipin and amphotericin B) to the outside bathing solution of the isolated frog skin did in fact result in an active outward transport of K’ (Nielsen 1971, Nielsen 1972). In a recent paper (Nielsen 1979) it is shown that the filipin-induced active outward K’ transport is coupled to the active inward Na+ transport, and the data suggest that the coupling ratio between the active transepithelial Na+ and K+ transport is 1.5 (3Na/2K). If the active Na+ transport across the isolated frog skin is carried

0 30 60 90 i i i l i i

Fig. 1. Effect of Ba++ on the short-circuit current across isolated frog skin. At the arrow, Ba-+ ( 5 mM) is added to the inside bathing solution. -, Short-circuit current pa/cmL; ---- . transepithelial resistance Knxcm’.

out by a Na-K pump with a coupling ratio of I S , then the addition of a component which completely inhibits the passive K+ current across the inward facing membrane should in the absence of filipin initially result in 66.6% inhibition of the short-cir- cuit current (SCC) (see below).

The experiments were performed on male and female frogs (Krrun temportiria) as described previ- ously (Nielsen 1977). The epithelia were dissected from collagenase-treated skins (Johnsen & Nielsen 1978) and mounted in perspex chambers (area 0.7 cmL) and bathed in stirred Ringer’s solution (Na+: 115, K+: 2.5, Ca”: 1, Mg‘+: I , CI-: 117, CO”: 2.5, PO’;: 1, glucose 5 mM, pH 7.8).

Addition of 5 mM Bai.+ to the inside bathing solution (IBS) resulted in an inhibition of the SCC and in an increase in the transepithelial resistance (Fig. 1 ) . About 2-5 min after the addition of Ba++ the SCC started to recover and the resistance started to decrease. The recovery phase was com- pleted 20-90 min after the addition of Ba++. The recovery varied from 30 % to complete recovery. In the present paper only the initial effect of Ba++ will be discussed. When Ba++ is added to the IBS of isolated epithelia the same result is obtained as in whole skins (Fig. 2 ) , but the inhibition is faster, because the thickness of the unstined layer on the inside has been reduced by removal of the con- nective tissue.

The primary inhibitory effect of Bat+ is much faster than the secondary effect (the recovery) of B a t + , so the two effects are separated in time. Therefore it is possible to measure the Ba++ in- duced initial inhibition without getting interference from the subsequent recovery. The initial Bat- in- duced inhibition is plotted against the Ba+’ con- centration in the IBS (Fig. 3), the Ba++ induced inhibition of the SCC increases with increasing Ba++ concentration until a maximum inhibition is reached. Addition of Bat’ ( 5 mM) to the IBS, a

190 R . Nielseri

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concentration which gave maximum inhibition, re- sulted in a 65.0f1.9% (n=10) inhibition of the SCC; this inhibition is not significantly different from 66.6%. Ba++ has been shown to reduce the K+ conductance in frog heart (Hermsmeyer & Spere- lakis 1970), in frog muscle (Henderson 1974), and in frog skin (Nagel 1978, Nielsen 1979).

According to the two-membrane hypothesis (Koefoed-Johnsen & Ussing 1958), the active Na+ transport across the isolated frog skin occurs in two steps: passive diffusion across the outward facing

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Fig. 3. Maximum percentage reduction of the short-circuit current elicited by various concentrations of Ba++. Values are the mean 2 S.E. , figures at the curve are number of expts.

membrane of the epithelium followed by an active extrusion across the inward facing membrane via the Na-K pump. If the active Na+ transport is car- ried out by a Na-K pump with a coupling ratio of 1.5, then according to this model (Fig. 4) f of the SCC across the inward facing membrane is carried by K+ ions and + by Na+ ions. Addition of a compo- nent (Ba++) which completely blocks the K channel would abolish the K+ current via the K+ channel, so the K+ which is pumped into the cell via the Na-K pump cannot leave the cells. In order to maintain

Fig. 4 . The two-membrane hypothesis. P, Na-K pump with a coupling ratio of 1.5 (3Na/2K).

Coirplrd Na und K trcinsport 191

electroneutrality in the cells the net Na+ flux across the outward facing membrane has to be reduced by the amount of K' which is pumped into the cells. If the coupling ratio for the Na-K pump is 1.5, a complete blocking of the K+ flux via the Kf channel will initially result in a 66.6% inhibition of the SCC. Thereafter the SCC will change, probably because the presence of Ba++ causes secondary increase in the K permeability of the membranes, as shown in cardiac muscle (Hermsmeyer & Sperelakis 1970). The observations that maximum Ba+'-induced ini- tial inhibition of the SCC is not significantly differ- ent from 66.6% (Fig. 3), supports the idea that the transepithelial Na' transport in frog skin is carried ou t by a Na-K pump with a coupling ratio of 1.5. In symmetrical cells (such as erythrocytes, muscle, and nerve) it is generally accepted that the coupling ratio for the Na-K pump is 1.5 (Thomas 1972).

R E F E R E N C E S

DE KRUIJFF, B. & DEMEL, R. A. 1974. Polyene an- tiobiotic-sterol interactions in membranes of achole- plasma laidlawii cells and lecithin liposomes. Biochim Biophys Acta (Amst.) 339: 57-70.

HENDERSON, E. C. 1974. Strophanthidin sensitive elec- trogenic mechanisms in frog sartorius muscles exposed to barium. Pliigers Arch Ges Physiol 350: 81-95.

HERMSMEYER, K. & SPERELAKIS, N. 1970. De- crease in K+ conductance and depolarization of frog cardiac muscle produced by Ba++. Am J Physiol 219: 1108-1114.

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

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

NAGEL, W. 1978. Ba++ decreases C, in frog skin. Fed Proc 37: 569.

NIELSEN, R. 1971. Effect of amphotericin B on the frog skin in vitro. Evidence for outwards active potassium transport across the epithelium. Acta Physiol Scand 83: 106-114.

NIELSEN, R. 1972. The effect of polyene antibiotics on the aldosterone induced changes in sodium transport across the isolated frog skin. J Ster Biochem 3: 121- 128.

NIELSEN, R. 1977. Effect of the polyene antibiotic filipin on the permeability of the inward- and the outward-fac- ing membrane of the isolated frog skin. Acta Physiol Scand 99: 39941 1.

NIELSEN, R. 1979. Coupled transepithelial Na and K transport across isolated frog skin. Effect of ouabain, amiloride, and the polyene antibiotic filipin. J Mem- brane Biol. In press.

THOMAS, R. C. 1972. Electrogenic sodium pump in nerve and muscle cells. Physiol Rev 52: 563-594.