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Determination of transepithelial (mucosal and serosal) electrical potentials in toad skin. action of chemical agents
Camp. Biochem. Physiol. Printed in Great Britain
Vol. 102A,
No. 4, pp. 687-691,
1992 0
0300-9629/92 $5.00 + 0.00 1992 Pergamon Press Ltd
DETERMINATION OF TRANSEPITHELIAL (MUCOSAL AND SEROSAL) ELECTRICAL POTENTIALS IN TOAD SKIN. ACTION OF CHEMICAL AGENTS B. C. NORRIS, J. B. CONCHA and G. M. CONTRERAS Department
of Physiology, Faculty of Biological and Natural Sciences, University of Concep&n, Concepcibn, Chile (Received 7 January 1991)
Abstract-l. Potential differences across the mucosal or outer, and the serosal or inner, membranes of the toad skin (M and S) were recorded separately. Total potential difference across the skin (T) and the short-circuit current (XC) were recorded by means of the classical Ussing method. 2. The independent determination of the M and the S is of importance in the elucidation of the mechanism of action of agents which alter ion Auxes across the skin. 3. The percentage values of the M and the S obtained in toad skins during the summer were similar to the percentage values obtained by microelectrode impalement of cells. 4. Angiotensin II (A,,) and antidiuretic hormone (ADH) increased T with a notable rise in M and a slight increase in S. These agents act mainly by increasing mucosal membrane permeability to Na+ since M is principally affected. 5. Amiloride and ouabain reversed M, decreased T and increased S above T. The reversal of M might be explained by the flow of a cation to the mucosal aspect or of an anion to the cell interior. 6. These results show that the effects of several agents on the toad skin potential may be analysed independently across the mucosal and serosal membranes and reflect the behaviour of the entire tissue rather than of a single cell.
techniques. Prior to the use of these methods, injury potentials were recorded to estimate the transmembrane potential in tissues such as skeletal and smooth muscle. Recordings were made by means of an electrode placed over the injured site and another over undamaged tissue. By this procedure it is possible, in transporting epithelial cells, to monitor the effects of agents which alter either the apical membrane potential (M) or the basolateral membrane potential (S) as mentioned by Kristensen (1978). The aim of the present work was to analyse separately the effects of several agents on the transporting cells of apical and basolateral membranes of the toad skin by means of independent recording of the potential differences across each of these membranes.
INTRODUCTION Studies carried out using microelectrodes for the measurement of intracellular potentials in toad skin (Whittembury, 1964) and in frog skin (Cereijido and Curran, 1965) have established that when the skin is penetrated with a microelectrode from the outer or mucosai surface (reference electrode in the outer solution) two potential steps are obtained; the sum of these steps is approximately equivalent in value to the total transepithelial potential difference (T). The first potential jump is dependent on Na+ diffusion across the mucosal membrane of the transporting cells and the second potential jump is due to the K+ diffusion potential across the basolateral border together with active Na+ extrusion by the Na+-K+ pump. According to Ussing and Zerahn (1951) the transtissue potential was equivalent to the sum of both diffusion potentials. The determination of these potentials is of importance in the study of the mechanism of action of hormones, drugs and several chemical mediators which influence movement of ions across high resistance epithelia such as Henle’s thick ascending limb epithelium, and the renal distal and collecting tubules. Given the diffic~ties of working on renal tubular epithelium, experiments using the toad skin model are an alternative approach to the investigation of ion transport across epithelia such as that of the kidney tubule. Up to the present time, research into the mechanism involved in the effects of hormones and other substances in the toad skin has depended on skin impalements with microelectrodes and patch clamp
MATERlALS AND METHODS Animals and experimental procedures
Experiments were performed on pieces of the abdominal skin of pithed Pleurodema thaul toads (8-20g). Skins were mounted between two halves of a modified Ussing chamber, with an exposed area of 1.33 cm*. Both sides of the chamber were bathed in 3 ml Ringer’s solution with the following composition (mM): NaCl 113; KC1 2.5; NaHCO, 2.5; glucose II and phosphate buffered to pH 7.5. An air pump maintained adequate oxygenation on both sides of the skin. Electrical measurements The T was monitored with calomel electrodes. The shortcircuit current (SCC) was measured with a microamperimeter connected to a voltage-clamp circuit (G. Metraux
687
688
B. C.
NORRIS et
al
M was found to be 42.6 + 3.2% and the S was 59.0 If: 3.4% in 11 skins. The value of the XC was 46.0 t_ 5.2 p A/cm*. Effect ofA,,. Addition of A,, to the inner bathing solution was followed by a significant increase in the T, M and SCC, and a non-significant increase in the S in 11 skins (Fig. 3A, Table 1). Effect of ADH. Skins used for the examination of the effect of ADH added to the inner bathing solution showed a slightly greater value for the M than for the S. The increase in T, M and SCC, was significantly greater (P < 0.001) than the increase in S (P < 0.01). Values are shown in Fig. 3B and in Table I. Experiments on toad skin bioelectric parameters in late autumn and early winter
+ f 101
1
see
J
Voltage clamp device
Fig. 1. Sketch of the system for recording transepithelial (T), mucosal (M) and serosal (S) potential differences across the isolated skin of Pleurodema thaul. 1, 2 and 3 are calomel electrodes: 3 is a reference electrode in contact with injured skin through a 3 M KC1 solution; 1 and 2 are potential difference recording electrodes. R, Normal toad Ringer’s solution. SCC. Short-circuit current.
Electronique) which could bring the T of the skin to zero when reading the current in microamperes. The currentcarrying electrodes were non-polarizable Ag-AgCl wires. The bioelectric parameters were recorded on a 2-channel Cole-Parmer recorder. The M and the S were recorded by means of a calomel electrode placed in the outer and then in the inner bathing solutions; for both measurements, the reference electrode was in contact with damaged skin through a 3 M KC1 solution (Fig. 1). Experiments were started when the preparation had reached a stable value for at least 30min. Drugs The following drugs were added to the solution bathing the skin in a volume sufficient to give the final concentrations mentioned in the text: (Asp’-Phe*) angiotensin II (A,,), antidiuretic hormone (ADH), ouabain (Sigma Chemical Co., St Louis, MO) and amiloride (Merck, Sharp and Dohme). The experiments were performed throughout the year and seasonal differences in the value of the T and of the SCC were observed. Statisticalanalysis The results are expressed as means k SEM and statistical analysis of the values obtained in the experiments was carried out according to Student’s paired values technique. RESULTS
Experiments on the toad skin bioelectric parameters in late summer and early autumn Figure 2A shows the values of the T, M and S in late summer skins. Considering the T as lOO%,
Figure 2B shows that the values of T, M and S were reduced during the cold season; the decrease in M was especially marked. The SCC was 27.0 + 5.5 p A/cm*, a value less than half that found for the summer skins. Efl^ect of A,,. A,, added to the inner solution significantly increased the T, M and SCC, whereas the increase in S was not significant in 10 skins (Fig. 3C and Table 2). Effect of amiIoride. As in the group of skins treated with A,, , the winter skins treated with amiloride showed very small control values for the M (Fig. 3D and Table 2); following the addition of this agent to the outer solution, an inversion of the M and an increase in the S to values above those for the T was observed, as well as the decrease in SCC in 10 skins (Table 2). EfSect of ouabain. As in the previous groups of winter skins, the values for the M were very small in comparison with those for summer skins. Ouabain applied in the inner solution reversed the M. increased the S to a value above that for the
LO
Z
30
d a 20
10
01
I w s
Fig. 2. Experiments showing values of total transepithelial, mucosal and serosal potential differences (T, M and S), in (A) late summer and (B) early winter toad skins. PD, Potential difference. Each bar represents means f SEM for 11 skins.
689
E 0
control
q Ouabain 60p0-
50-
po-
LO-
30-
30-
20-
20-
10.
lo-
o-
T MS
TMS
- r1
O-
-10’
-10’
representative of the effect of several agents on total transepithelial, mucosal and serosal potential differences (T, M and S) in summer (A and B) and in winter (C, D and E) toad skins. PD, Potential difference (A) 4.8 x IO-‘M angiotensin II (A,,), inner solution, summer skin. (B) 8.6 x 10m7 M antidiuretic hormone (ADH), inner solution, summer skin. (C) 4.8 x lo-’ M An, inner solution, winter skin. (D) 1 x 10e6 M amiloride, outer solution, winter skin. (E) 1 x 10m4 M ouabain, inner solution, winter skin. Fig.
3. Single experiments,
T and decreased Table 2).
the
SCC in 10 skins
(Fig. 3E and
DISCUSSION
As stated in the introduction, injury potentials were recorded prior to the use of microelectrodes, to examine the properties of the transmembrane potentials in tissues such as skeletal and smooth muscle. Amphibian skin ion-transporting cells are bipolar, i.e. they possess an entry step at the mucosal surface and an exit step at the serosal surface. By means of the technique used in this work, the potential differences of the mucosal and the serosal surfaces of the skin could be monitored independently through calomel electrodes placed in the respective solution. This procedure is of interest because it allows a direct approach to the study of agents which alter either the apical membrane potential dependent on Na+ influx, or the basolateral membrane potential dependent on Na+ extrusion followed by passive K+ efflux as proposed by Ussing and Zerahn (1951), KoefoedJohnsen and Ussing (1958) and further analysed by Turnheim (1991). Although frog skin epithelium is considered a functional syncytium, Eskesen and Ussing (1986), and Nagel and Diirge (1990) point out
Table 1. Effect of angiotensin
that in Rana esculenta, mitochondria-rich cells (MR) represent a very inhomogeneous compartment. Furthermore, the transporting cells are in continual dynamic changes; microelectrode and patch clamp techniques do not reflect the change in outer and inner membranes in whole skin, as admitted by Turnheim (1991), whereas the values in this work are representative of all the cells. If we consider the work of Whittembury (1964) and of Cereijido and Curran (1965), we may conclude that the per cent values of the potential jumps they obtained were similar to the per cent values of the M and the S obtained by the method described in this paper in toad skins during the summer months. In winter skins, the M was much smaller, indicating a reduced mucosal Na+ influx. Similarly, the T was much smaller than in summer toad skins. Voute and Meier (1978) state that in frogs, the main functional difference between winter and summer skins is found in their conductance for chloride and water; on the morphological side they had observed a seasonal dependence on the number of MR cells. These cells, which are abundant only in spring and summer skins, are important for inward Cl- transport. In summer skins, the finding that A,, and ADH markedly increased the T and the M with a negligible
II (A,,) and of antidiuretic hormone (ADH) applied in the inner solution, parameters in late summer and early autumn Control
on toad skin biolectric
Experimental M S (mVj (mV\
T (mV)
M (mV)
S (mV)
37.5k5.5 (4.8 x lo-’ M) ADH 45.Ok4.0 (8.6 x IO-‘M)
15.0+5.0
22.Ok5.8
43.Ok4.5
58.0+4.5**
29.0 f 4.2**
27.0 f 4.0NS
26.0+
21.0+2.5
62.0*
74.0+
45.0 f 2.8”
26.0 _+2.2*
Aeent
A,,
3.0
see (u A/cm’ )
7.0
T, total potential difference; M, mucosal potential difference; Values are means + SEM, N = 11. l, **Significantly different from control values, Student’s paired
T fmV)
3.6’.
S, serosal
potential
difference;
SCC,
see (u A/cm’ 1
short
71.0 f 7.5;’ 125.0 + 13.0.. circuit
r-test. *P -z 0.01; l*f <: 0.001; NS, not significant.
current.
B. C. NORRIS et al.
690 Table 2. Effect of angiotensi”
II (A,,) and of ouabain in the inner solution, and of amiloride applied toad skin biolectric parameters in late autumn and early winter Control
Agent A,, (4.8 x IO ‘M) Amiloride (1x10 ‘M) Ouabain (I x IO-‘M)
T (mV)
0%
(ZV)
(m’v) 39.5 f 3.9’
4.0 + I.5
28.0 1_ 2.0
29.0 f 6.0
27.5 & 3.0
2.0 + 0.5
25.0 + 3.3
29.5 & 4.0
9.5 * 1.5’1
26.5 + 2.4
31 0 + 6.1
12.0 i 1.7**
I .5
on
Experimental see (PA/C”+)
31.0 + 3.6
34.5 i 2.5 7.5 k
in the outer solution
(m”v)
(m”v)
10.0 + _ 2.S** 29.0 k 5.0NS -8.0
+ l.O**
-5.Ok
1.3”
18.0 t l.O*’ lS.Oi_ l.B**
XC 1 ___@A/cm’ ~~~ 46.0 i 6.5** 10.0 i 3.0” 7.0 5 2.7’*
T. total potential difference; M. mucosal potential difference; S, serosal potential difference; SCC, short circuit current. are means i SEM, N = IO. *. **Significantly different from control values. Student’s paired I-test. lP < 0.01, **P < 0.001, NS, not significant
increase in S, indicates that both agents act mainly by enhancing permeability of the apical membrane to Nat. This effect was also observed in winter skins exposed to A,,: the M increased and the rise in S was not significant, indicating that A,, increased apical membrane permeability to Nat. Unexpected results were obtained when examining the effect of amiloride; the marked decrease in the T and in the SCC was accompanied by an inversion of the M and the S increased to a value over that of the T. This finding might be explained by the flow of a cation from the cell to the mucosal aspect or by the flow of an anion from the mucosal solution to the cell interior; in this context, it is of interest that Kristensen (1978) found that amiloride inhibits both influx and efllux of Cl- in frog skins, with a possible net reduction in Cl- efflux. Nielsen (1984) demonstrated that amiloride not only blocked Nat transport but also reduced the K+ transport. Ouabain had a similar effect; it is well known that this cardiac glycoside not only inhibits the Nat-K+ pump but also reduces outer carrier Naf permeability, according to Helman et al. (1979) and Lacaz-Vieira et al. (1979). Although Ussing and Zerahn (1951) set forth the hypothesis that the T is equal to the sum of the Nat diffusion potential across the apical membrane, and the K+ diffusion potential across the basolateral membrane, Chowdhury and Snell (196.5) reported data which suggest the presence of factors which might alter the simplicity of this potential sum and that Nat- and K+-sensitive regions are rather more continuously distributed through several layers of frog skin. However, in the analysis of two-membrane models, changes in the properties of one membrane lead to changes of the properties of the other membrane; therefore experimental results may be falsely taken as evidence against the two-membrane hypothesis (Kristensen, 1978). The finding that ADH and A,, increase the M without significantly affecting the S is in accordance with previous works showing that these agents increase intracellular CAMP, thus increasing apical membrane permeability to Na+ and enhancing the active Na+ transport system (Concha et al., 1988; Norris et al., 1988). Bevevino and Lacaz-Vieira (1982) reported that ouabain transiently increases Na+ efflux and suggested that this may be ascribed to depolarization of the outer barrier following ouabain block of the electrogenic Nat pump. In the toad Bufo marinus, Varanda and Lacaz-Vieira (1979) and Lacaz-Vieira
Values
et al. (1979) reported that K+ efflux also increased under the effect of ouabain. We have presently no explanation for the effect of amiloride and ouabain on the S (increase above the value of the T). However, the algebraic sum of M + S is equal to the value of T. Whether other ions such as Ca*+, K+ or Cl- are partly responsible for some of the effects described, remains to be investigated in this species of toad.
Acknowledgements-This work received financial support from grants 20.33.30 and 20.33.58, University of Concepci6n. The authors are indebted to Mrs Maria Cecilia Nova for excellent technical assistance.
REFERENCES
Bevevino L. H. and Lacaz-Vieira F. (1982) Control of sodium permeability of the outer barrier in toad skin. J. Membrane Biol. 66, 97-107. Cereijido M. and Curran F. F. (1965) Intracellular electrical poientials in frog skin. J. gen. Physiol. 48, 543-557. Chowdhurv T. K. and Snell F. M. (1965) A microelectrode study of electrical potentials-in f&g skin and toad bladder. Biochim. biophys. Acta 94, 461477. Concha J., Norris B. and Contreras G. (1988) Posible mecanismo de acci6n de angiotensina II en piel aislada de sapo. Bol. Sot. Biol. Concepcidn (Chile) 59, 23-36. Eskesen K. and Ussing H. H. (1986) Single file diffusion through K+ channels in frog skin epithelium. J. Membrane Gel. 91, 245-250. _ Helman S. 1.. Navel W. and Fisher R. S. (1979) Ouabain on active transepithelial sodium transport in frog skin. J. gen. Physiol. 74, 105-127. Koefoed-Johnsen V. and Ussing H. H. (1958) The nature of the frog skin potential. Acta physiol. &and. 42, 293-308. Kristensen P. (1978) Effect of amiloride on chloride transport across amphibian epithelia. J. Membrane Biol. 40 (Special Issue), 167-185. Lacaz-Vieira F., Varanda W. A., Bevevino L. H. and Femandez D. R. (1979) Rheogenic sodium pump in the toad skin. In Cation Flux across Biomembranes (Edited by Nukonata Y. and Packer L.), pp. 51-52. Academic Press, New York. Nagel W. and Diirge A. (1990) Analysis of cation conductance in frog skin. P@tigers Arch. 416, 5341. Nielsen R. (1984) Active transepithelial potassium transport in frog skin via specific potassium channels in the apical membrane. Acta physiol. Sand. 120, 287-296. Norris B., Concha J., Contreras G. and GonzLlez C. (1988) Stimulatory effect of angiotensin II on the electrical properties of the isolated toad skin. Biochem. Pharmac. 37, 300-3010. I
,
Chemical agents on toad skin potentials Turnheim K. (1991) Intrinsic regulation of apical sodium entry in epitheha. P/rysio/. Rev. 71, 429445. Ussing H. H. and Zerahn K. (1951) Active transport of sodium as the source of electrical current in the short-circuited frog skin. Acta physiol. &and. 23, 1l&127. Varanda W. A. and Lacaz-Vieira F. (1979) Transient
691
potassium fluxes in toad skin. J. Membrane Biol. 49, 199-233. Vdute C. L. and Meier W. (1978) The mitochondria-rich cell of frog skin as hormone-sensitive “shunt path”. J. Membrane Biol. 40 (special issue), 151-165. Whittembury G. (1964) Electrical potential profile of the toad skin epithelium. J. gen. Physiol. 47, 795-808.
Vol. 102A,
No. 4, pp. 687-691,
1992 0
0300-9629/92 $5.00 + 0.00 1992 Pergamon Press Ltd
DETERMINATION OF TRANSEPITHELIAL (MUCOSAL AND SEROSAL) ELECTRICAL POTENTIALS IN TOAD SKIN. ACTION OF CHEMICAL AGENTS B. C. NORRIS, J. B. CONCHA and G. M. CONTRERAS Department
of Physiology, Faculty of Biological and Natural Sciences, University of Concep&n, Concepcibn, Chile (Received 7 January 1991)
Abstract-l. Potential differences across the mucosal or outer, and the serosal or inner, membranes of the toad skin (M and S) were recorded separately. Total potential difference across the skin (T) and the short-circuit current (XC) were recorded by means of the classical Ussing method. 2. The independent determination of the M and the S is of importance in the elucidation of the mechanism of action of agents which alter ion Auxes across the skin. 3. The percentage values of the M and the S obtained in toad skins during the summer were similar to the percentage values obtained by microelectrode impalement of cells. 4. Angiotensin II (A,,) and antidiuretic hormone (ADH) increased T with a notable rise in M and a slight increase in S. These agents act mainly by increasing mucosal membrane permeability to Na+ since M is principally affected. 5. Amiloride and ouabain reversed M, decreased T and increased S above T. The reversal of M might be explained by the flow of a cation to the mucosal aspect or of an anion to the cell interior. 6. These results show that the effects of several agents on the toad skin potential may be analysed independently across the mucosal and serosal membranes and reflect the behaviour of the entire tissue rather than of a single cell.
techniques. Prior to the use of these methods, injury potentials were recorded to estimate the transmembrane potential in tissues such as skeletal and smooth muscle. Recordings were made by means of an electrode placed over the injured site and another over undamaged tissue. By this procedure it is possible, in transporting epithelial cells, to monitor the effects of agents which alter either the apical membrane potential (M) or the basolateral membrane potential (S) as mentioned by Kristensen (1978). The aim of the present work was to analyse separately the effects of several agents on the transporting cells of apical and basolateral membranes of the toad skin by means of independent recording of the potential differences across each of these membranes.
INTRODUCTION Studies carried out using microelectrodes for the measurement of intracellular potentials in toad skin (Whittembury, 1964) and in frog skin (Cereijido and Curran, 1965) have established that when the skin is penetrated with a microelectrode from the outer or mucosai surface (reference electrode in the outer solution) two potential steps are obtained; the sum of these steps is approximately equivalent in value to the total transepithelial potential difference (T). The first potential jump is dependent on Na+ diffusion across the mucosal membrane of the transporting cells and the second potential jump is due to the K+ diffusion potential across the basolateral border together with active Na+ extrusion by the Na+-K+ pump. According to Ussing and Zerahn (1951) the transtissue potential was equivalent to the sum of both diffusion potentials. The determination of these potentials is of importance in the study of the mechanism of action of hormones, drugs and several chemical mediators which influence movement of ions across high resistance epithelia such as Henle’s thick ascending limb epithelium, and the renal distal and collecting tubules. Given the diffic~ties of working on renal tubular epithelium, experiments using the toad skin model are an alternative approach to the investigation of ion transport across epithelia such as that of the kidney tubule. Up to the present time, research into the mechanism involved in the effects of hormones and other substances in the toad skin has depended on skin impalements with microelectrodes and patch clamp
MATERlALS AND METHODS Animals and experimental procedures
Experiments were performed on pieces of the abdominal skin of pithed Pleurodema thaul toads (8-20g). Skins were mounted between two halves of a modified Ussing chamber, with an exposed area of 1.33 cm*. Both sides of the chamber were bathed in 3 ml Ringer’s solution with the following composition (mM): NaCl 113; KC1 2.5; NaHCO, 2.5; glucose II and phosphate buffered to pH 7.5. An air pump maintained adequate oxygenation on both sides of the skin. Electrical measurements The T was monitored with calomel electrodes. The shortcircuit current (SCC) was measured with a microamperimeter connected to a voltage-clamp circuit (G. Metraux
687
688
B. C.
NORRIS et
al
M was found to be 42.6 + 3.2% and the S was 59.0 If: 3.4% in 11 skins. The value of the XC was 46.0 t_ 5.2 p A/cm*. Effect ofA,,. Addition of A,, to the inner bathing solution was followed by a significant increase in the T, M and SCC, and a non-significant increase in the S in 11 skins (Fig. 3A, Table 1). Effect of ADH. Skins used for the examination of the effect of ADH added to the inner bathing solution showed a slightly greater value for the M than for the S. The increase in T, M and SCC, was significantly greater (P < 0.001) than the increase in S (P < 0.01). Values are shown in Fig. 3B and in Table I. Experiments on toad skin bioelectric parameters in late autumn and early winter
+ f 101
1
see
J
Voltage clamp device
Fig. 1. Sketch of the system for recording transepithelial (T), mucosal (M) and serosal (S) potential differences across the isolated skin of Pleurodema thaul. 1, 2 and 3 are calomel electrodes: 3 is a reference electrode in contact with injured skin through a 3 M KC1 solution; 1 and 2 are potential difference recording electrodes. R, Normal toad Ringer’s solution. SCC. Short-circuit current.
Electronique) which could bring the T of the skin to zero when reading the current in microamperes. The currentcarrying electrodes were non-polarizable Ag-AgCl wires. The bioelectric parameters were recorded on a 2-channel Cole-Parmer recorder. The M and the S were recorded by means of a calomel electrode placed in the outer and then in the inner bathing solutions; for both measurements, the reference electrode was in contact with damaged skin through a 3 M KC1 solution (Fig. 1). Experiments were started when the preparation had reached a stable value for at least 30min. Drugs The following drugs were added to the solution bathing the skin in a volume sufficient to give the final concentrations mentioned in the text: (Asp’-Phe*) angiotensin II (A,,), antidiuretic hormone (ADH), ouabain (Sigma Chemical Co., St Louis, MO) and amiloride (Merck, Sharp and Dohme). The experiments were performed throughout the year and seasonal differences in the value of the T and of the SCC were observed. Statisticalanalysis The results are expressed as means k SEM and statistical analysis of the values obtained in the experiments was carried out according to Student’s paired values technique. RESULTS
Experiments on the toad skin bioelectric parameters in late summer and early autumn Figure 2A shows the values of the T, M and S in late summer skins. Considering the T as lOO%,
Figure 2B shows that the values of T, M and S were reduced during the cold season; the decrease in M was especially marked. The SCC was 27.0 + 5.5 p A/cm*, a value less than half that found for the summer skins. Efl^ect of A,,. A,, added to the inner solution significantly increased the T, M and SCC, whereas the increase in S was not significant in 10 skins (Fig. 3C and Table 2). Effect of amiIoride. As in the group of skins treated with A,, , the winter skins treated with amiloride showed very small control values for the M (Fig. 3D and Table 2); following the addition of this agent to the outer solution, an inversion of the M and an increase in the S to values above those for the T was observed, as well as the decrease in SCC in 10 skins (Table 2). EfSect of ouabain. As in the previous groups of winter skins, the values for the M were very small in comparison with those for summer skins. Ouabain applied in the inner solution reversed the M. increased the S to a value above that for the
LO
Z
30
d a 20
10
01
I w s
Fig. 2. Experiments showing values of total transepithelial, mucosal and serosal potential differences (T, M and S), in (A) late summer and (B) early winter toad skins. PD, Potential difference. Each bar represents means f SEM for 11 skins.
689
E 0
control
q Ouabain 60p0-
50-
po-
LO-
30-
30-
20-
20-
10.
lo-
o-
T MS
TMS
- r1
O-
-10’
-10’
representative of the effect of several agents on total transepithelial, mucosal and serosal potential differences (T, M and S) in summer (A and B) and in winter (C, D and E) toad skins. PD, Potential difference (A) 4.8 x IO-‘M angiotensin II (A,,), inner solution, summer skin. (B) 8.6 x 10m7 M antidiuretic hormone (ADH), inner solution, summer skin. (C) 4.8 x lo-’ M An, inner solution, winter skin. (D) 1 x 10e6 M amiloride, outer solution, winter skin. (E) 1 x 10m4 M ouabain, inner solution, winter skin. Fig.
3. Single experiments,
T and decreased Table 2).
the
SCC in 10 skins
(Fig. 3E and
DISCUSSION
As stated in the introduction, injury potentials were recorded prior to the use of microelectrodes, to examine the properties of the transmembrane potentials in tissues such as skeletal and smooth muscle. Amphibian skin ion-transporting cells are bipolar, i.e. they possess an entry step at the mucosal surface and an exit step at the serosal surface. By means of the technique used in this work, the potential differences of the mucosal and the serosal surfaces of the skin could be monitored independently through calomel electrodes placed in the respective solution. This procedure is of interest because it allows a direct approach to the study of agents which alter either the apical membrane potential dependent on Na+ influx, or the basolateral membrane potential dependent on Na+ extrusion followed by passive K+ efflux as proposed by Ussing and Zerahn (1951), KoefoedJohnsen and Ussing (1958) and further analysed by Turnheim (1991). Although frog skin epithelium is considered a functional syncytium, Eskesen and Ussing (1986), and Nagel and Diirge (1990) point out
Table 1. Effect of angiotensin
that in Rana esculenta, mitochondria-rich cells (MR) represent a very inhomogeneous compartment. Furthermore, the transporting cells are in continual dynamic changes; microelectrode and patch clamp techniques do not reflect the change in outer and inner membranes in whole skin, as admitted by Turnheim (1991), whereas the values in this work are representative of all the cells. If we consider the work of Whittembury (1964) and of Cereijido and Curran (1965), we may conclude that the per cent values of the potential jumps they obtained were similar to the per cent values of the M and the S obtained by the method described in this paper in toad skins during the summer months. In winter skins, the M was much smaller, indicating a reduced mucosal Na+ influx. Similarly, the T was much smaller than in summer toad skins. Voute and Meier (1978) state that in frogs, the main functional difference between winter and summer skins is found in their conductance for chloride and water; on the morphological side they had observed a seasonal dependence on the number of MR cells. These cells, which are abundant only in spring and summer skins, are important for inward Cl- transport. In summer skins, the finding that A,, and ADH markedly increased the T and the M with a negligible
II (A,,) and of antidiuretic hormone (ADH) applied in the inner solution, parameters in late summer and early autumn Control
on toad skin biolectric
Experimental M S (mVj (mV\
T (mV)
M (mV)
S (mV)
37.5k5.5 (4.8 x lo-’ M) ADH 45.Ok4.0 (8.6 x IO-‘M)
15.0+5.0
22.Ok5.8
43.Ok4.5
58.0+4.5**
29.0 f 4.2**
27.0 f 4.0NS
26.0+
21.0+2.5
62.0*
74.0+
45.0 f 2.8”
26.0 _+2.2*
Aeent
A,,
3.0
see (u A/cm’ )
7.0
T, total potential difference; M, mucosal potential difference; Values are means + SEM, N = 11. l, **Significantly different from control values, Student’s paired
T fmV)
3.6’.
S, serosal
potential
difference;
SCC,
see (u A/cm’ 1
short
71.0 f 7.5;’ 125.0 + 13.0.. circuit
r-test. *P -z 0.01; l*f <: 0.001; NS, not significant.
current.
B. C. NORRIS et al.
690 Table 2. Effect of angiotensi”
II (A,,) and of ouabain in the inner solution, and of amiloride applied toad skin biolectric parameters in late autumn and early winter Control
Agent A,, (4.8 x IO ‘M) Amiloride (1x10 ‘M) Ouabain (I x IO-‘M)
T (mV)
0%
(ZV)
(m’v) 39.5 f 3.9’
4.0 + I.5
28.0 1_ 2.0
29.0 f 6.0
27.5 & 3.0
2.0 + 0.5
25.0 + 3.3
29.5 & 4.0
9.5 * 1.5’1
26.5 + 2.4
31 0 + 6.1
12.0 i 1.7**
I .5
on
Experimental see (PA/C”+)
31.0 + 3.6
34.5 i 2.5 7.5 k
in the outer solution
(m”v)
(m”v)
10.0 + _ 2.S** 29.0 k 5.0NS -8.0
+ l.O**
-5.Ok
1.3”
18.0 t l.O*’ lS.Oi_ l.B**
XC 1 ___@A/cm’ ~~~ 46.0 i 6.5** 10.0 i 3.0” 7.0 5 2.7’*
T. total potential difference; M. mucosal potential difference; S, serosal potential difference; SCC, short circuit current. are means i SEM, N = IO. *. **Significantly different from control values. Student’s paired I-test. lP < 0.01, **P < 0.001, NS, not significant
increase in S, indicates that both agents act mainly by enhancing permeability of the apical membrane to Nat. This effect was also observed in winter skins exposed to A,,: the M increased and the rise in S was not significant, indicating that A,, increased apical membrane permeability to Nat. Unexpected results were obtained when examining the effect of amiloride; the marked decrease in the T and in the SCC was accompanied by an inversion of the M and the S increased to a value over that of the T. This finding might be explained by the flow of a cation from the cell to the mucosal aspect or by the flow of an anion from the mucosal solution to the cell interior; in this context, it is of interest that Kristensen (1978) found that amiloride inhibits both influx and efllux of Cl- in frog skins, with a possible net reduction in Cl- efflux. Nielsen (1984) demonstrated that amiloride not only blocked Nat transport but also reduced the K+ transport. Ouabain had a similar effect; it is well known that this cardiac glycoside not only inhibits the Nat-K+ pump but also reduces outer carrier Naf permeability, according to Helman et al. (1979) and Lacaz-Vieira et al. (1979). Although Ussing and Zerahn (1951) set forth the hypothesis that the T is equal to the sum of the Nat diffusion potential across the apical membrane, and the K+ diffusion potential across the basolateral membrane, Chowdhury and Snell (196.5) reported data which suggest the presence of factors which might alter the simplicity of this potential sum and that Nat- and K+-sensitive regions are rather more continuously distributed through several layers of frog skin. However, in the analysis of two-membrane models, changes in the properties of one membrane lead to changes of the properties of the other membrane; therefore experimental results may be falsely taken as evidence against the two-membrane hypothesis (Kristensen, 1978). The finding that ADH and A,, increase the M without significantly affecting the S is in accordance with previous works showing that these agents increase intracellular CAMP, thus increasing apical membrane permeability to Na+ and enhancing the active Na+ transport system (Concha et al., 1988; Norris et al., 1988). Bevevino and Lacaz-Vieira (1982) reported that ouabain transiently increases Na+ efflux and suggested that this may be ascribed to depolarization of the outer barrier following ouabain block of the electrogenic Nat pump. In the toad Bufo marinus, Varanda and Lacaz-Vieira (1979) and Lacaz-Vieira
Values
et al. (1979) reported that K+ efflux also increased under the effect of ouabain. We have presently no explanation for the effect of amiloride and ouabain on the S (increase above the value of the T). However, the algebraic sum of M + S is equal to the value of T. Whether other ions such as Ca*+, K+ or Cl- are partly responsible for some of the effects described, remains to be investigated in this species of toad.
Acknowledgements-This work received financial support from grants 20.33.30 and 20.33.58, University of Concepci6n. The authors are indebted to Mrs Maria Cecilia Nova for excellent technical assistance.
REFERENCES
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