Gen. Pharmac. Vol. 21, No. 5, pp. 617~20, 1990
0306-3623/90 $3.00 + 0.00 Copyright © 1990 Pergamon Press plc
Printed in Great Britain. All rights reserved
THE EFFECT OF ADH AND INSULIN ON ACTIVE SODIUM TRANSPORT ACROSS FROG SKIN IN THE PRESENCE OF ALTERNARIOL MYCOTOXIN S. MICELLI, A. BOTTALICOl and E. GALLUCCI Laboratorio di Fisiologia Generale, Dipartimento Farmaco-Biologico, Universit~i di Bari and qstituto tossine e micotossine dei Parassiti Vegetali del Consiglio Nazionale delle Ricerche, Bail, Italy
(Received 14 September 1989) Abstract--l. The effect of vasopressin and insulin on active sodium transport across frog skin in the presence of internal alternariol mycotoxin was studied, using the short-circuit technique. 2. Vasopressin stimulates sodium transport across frog skin by decreasing the resistence to sodium entry into the epithelial cells, thus partially removing the inhibition on the short-circuit current due to the action of Alternariol mycotoxin. 3. Even insulin which is known to increase the short-circuit current by a different mechanism, determines a rapid reversal effect on the inhibition due to Alternariol. 4. These data confirm the different action of the two hormones on active sodium transport across frog skin, and furthermore are indicative of an inhibition of transepitbelial sodium transport by Alternariol mycotoxin probably via the sodium pump.
INTRODUCTION
Frog skin re-absorbs sodium ions from the external environment and transports them into the blood. This transport is active, because sodium overcomes its electrochemical gradient in order to pass from the outer to the inner medium. The short-circuit current is a measure of the net sodium transport across isolated amphibian skin and bladder (Ussing and Zerahn, 1951; Leaf et al., 1958). In frog skin and in urinary bladder transepitheliai sodium transport is controlled by the antiduiretic hormone ( A D H ) which increases sodium re-absorption by a cAMP-mediated process (Aceves 1977; De Sousa, 1981; Sapirstein et al., 1973). Previous work has shown that insulin produces an increase in the short-circuit current across isolated frog skin and urinary bladder (Herrera et al., 1963; Herrera, 1976; Wiesman et al., 1976) probably as a result of the stimulation o f the active sodium transport step at the serosal surface. This active sodium transport may be considered as an index o f the ability of the cell to function, for this reason it is a useful tool in the testing of substances which impair the metabolism such as toxins, particularly those present in vegetable foods. In the present paper we focus our interest on the effect of Alternariol mycotoxin (AOH), whose fungal sources are dlternaria tenuis, d. dauci, d. cucumerins etc., on the active sodium transport across frog skin in the absence and in the presence of different hormones or combinations of hormones. A preliminary report of this study has already been published (Boll. Soc. It. Biol. Sper. LXIV, 25). MATERIALS AND METHODS
Experiments were performed on half symmetrical frog skin of Rana esculenta, that had been kept in tanks with
access to tap water at room temperature prior to the experiments. The AOH employed was produced, and purified according to Chu and Bennet (1981) using the approximately modified procedure of Visconti (unpublished data). The ADH was by Sigma, the Insulin was by Eli Lilly. Following the method described by Ussing and Zerahn (1951) paired hemiskins were mounted between Lucite chambers with an exposed tissue area of 1.14cm 2 and a 7 ml volume of each chamber filled with a solution containing: l l 0 m M NaCl; 2.5mM CaCl2; 2mM KCl; 1.7mM Nail: PO 4; 3.7 mM Na 2HPO 4(pH = 7. l; 230-240 mOsm/kg; temperature 22 ± 2°C); electric measurements of the short circuit current (I,~) were monitored after the membrane had been mounted and allowed to equilibrate.
RESULTS
Figure I reports the dose-response relationship between different concentrations of A O H and active sodium transport. It can be seen that micromolar concentrations of A O H inhibit the short-circuit current (I,¢). Indeed I~0 is reached when concentrations of 4.5-5.5/~M are used. Figure 2 reports the effect of internal A O H (5.5/zM) and washing out treatment on sodium transport across frog skin. It can be seen that A O H (in 2 or 3 hr) reduces the sodium transported by about 50%, washing out does not restore the sodium transport, which falls to 75% of the initial value in the space of 1 hr after mycotoxin removal. To investigate the possibility that an increase in the intracellular sodium pool may overcome or partially remove A O H inhibition the l~c was evaluated after stimulation of sodium transport by A D H . (Fig. 3A). Under these conditions the I~ showed a small but not significant increase. In half symmetrical skin (Fig. 3B) A D H causes, as usual, an increase in the short-circuit current. 617
618
S. M I C E L L I
et al. 1 -
AOH
~°°i-
ADH
I l
x(3)
;(5)
0
I
I
I
I
I
I
I
1
2
3
4
5
6
7
ALt.ernorioL
rnycotoxin (p,M)
Fig. I. Relationship between internal Alternariol concentrations and active sodium transport in frog skin. Action time of mycotoxin 2 hr. The sodium transport at equilibrium was 0.84 + 0.12/aequiv./cm 2 hr. (eleven experiments). In brackets are the number of experiments for each Alternariol concentration used.
It is known that the A O H effect is irreversible as the I,~ is not restored after washing out the treated tissue. This is indicative either of a strong binding of the A O H to the membrane receptors or of a penetration of the A O H into the cell. As in a previous paper it was reported that insulin increases the short-circuit current across frog skin, by acting on active sodium transport at the serosal surface, we decided to compare the effect of both insulin and A D H using two half-symmetrical frog skins• Figure 4 reports this test. It can be seen that the addition of insulin immediately determines a dramatic increase in sodium transport. Then the transport decreases below the equilibrium value; on the other hand the addition of a maximal concentration of A D H determins a slower but longer increase in sodium transport. The same experiment was conducted on symmetrical half-skin pretreated with AOH, In this case, when the A O H reduced the I= by about 50%, the insulin and A D H were added (Fig. 5). The insulin rapidly removed the AOH inhibition on the sodium transport and temporarily stimulated it. Then the inhibition
0
--
30 6 0 9 0 120 t 5 0 1 ~ ) 210 240 270
1
2 T
0
0
30 6 0 9 0 420 Time
Fig. 3. Effect of internal Alternariol (5.5#M) and of Alternariol plus ADH (45 mUI/ml) (top) and of internal ADH (45mUI/ml) (bottom) on transepithelial sodium transport in half-symmetrical frog skin. Mean values + SE of four experiments.
3
I
I I
2
6 6
~" 100~ E
(mini
W.O. 1(
o
t
t
t 0
~ z
o
I
I
1
2
J
3
I
I
I
I
30
60
90
12O
Time
(rnin)
4
Time (h)
Fig. 2. Effect of internal Alternariol (5.5/~M) and washing out treatment on active sodium transport across frog skin. Mean + SE of four experiments. The sodium transport at equilibrium (zero-time) was 1.07 + 0.21 ~equiv./cm2/hr.
Fig. 4. Effect of internal A D H (45mUl/m]) ( 0 ) and of
internal and external insulin (3.10 -~ M) ( 1 ) on transepithelial sodium transport in half-symmetrical frog-skin. Mean values + SE of the ratio ¢,t/¢,, of 5 experiments (¢~,= flux of sodium at equilibrium; ¢,, = flux of sodium of different times of treatment).
Mycotoxin inhibits sodium transport returned. Whereas ADH caused a small revitalization of I~ as previously shown (Fig. 3). Finally, with the same experimental protocol another series of experiments were conducted in which in one half-skin ADH was added after 10 min of insulin action. Figure 6 reports one of those experiments. It is evident that the successive ADH additions strengthen the insulin effect, in fact sodium transport maintains high levels, due presumably to the instantaneous insulin action and to the slower ADH action.
619
"~ 1.0 r-
• • • AOH •
,~
0
l
0
•
•
•
•
. eoo
•
In~Un
30
dO
90 Time
•
:*
.-
:
•
,2o
" ,'lo
laO
2;0
2,~0
(rain)
Fig. 6. Effect of internal ADH (45 mUl/ml) (A) and of internal and external insulin (3.10 -s M) plus internal ADH (45 mUI/ml) (Q) on transepithelial sodium transport in half symmetrical frog skin pre-treated with internal Alternariol (5.5 #M). One experiment representative of a series.
DISCUSSION The data presented show that serosal AOH causes a reduction in the active sodium transport across frog skin. It should also be noted that this effect is not a temporary effect, as the washing out of the AOH does not restore the short-circuit current (Barbarossa et al., 1988). It is known that active sodium transport is directly dependent on the sodium pump located on the serosal surface of the cell, so AOH could influence this pump. To investigate the possibility that an increase in transport per se may change the properties of the sodium pump in the presence of AOH, the short-circuit current was evaluated before and after stimulation of the sodium transport by ADH. The intracellular sodium increase due to ADH action (mediated by the endocellular second messanger AMPc) on the external barrier of the cell, shows its tendency to remove the inhibition due to AOH. As we are not in a position to say that AOH directly influences the sodium pump instead of, for example, the apical barrier of the membrane to sodium; experiments were performed using another hormone (insulin) to stimulate the sodium pump, as has been reported (Wiesmann et al., 1977). In this study we have compared the effect on the I~c of ADH and of the insulin on half-symmetrical skins (Fig. 4). The two hormones increase the active sodium transport but the rate at which they do so and the time taken differ, probably indicating a different mechanism of action on the frog skin.
~OH
•
]flsuLin
0.*
• • • • • e
|
°
e
°
i
,
,
•
•
o
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,
,
,
.
,
o
e
e
i
.
,
o
,0
AOH .
.
i
,
r..I
9015010
,
60
120
i
150
Time (rain) Fig. 5. Effect of internal ADH (45 mUl/ml) (O) and of internal and external insulin (3' l0 -s M) (II) on transepithelial sodium transport in symmetricalfrog skin after pretreatment with internal Alternariol (5.SgM). One experiment representative of a series is reported.
When the insulin was added after AOH inhibition, the I,~ value returned to its basal value or even more. (Fig. 5), then the inhibition returned. Furthermore, if ADH is added while the insulin is still working, the increase of the I,~ is maintained for a longer time before falling again. These last two experiments are indicative of the action of AOH on the active step of sodium transport. In fact insulin, which is known to positively influence active sodium transport by acting on the pump, removes the inhibition of AOH. The successive action of ADH in these conditions suggests that the apical membrane and the pathway of AMPc are not affected by AOH. In the crude intestinal membranes of frog AOH (6.4.10- s M) inhibits the Na +, K*-ATPase activity by 50% (unpublished data). The observations reported here are in line with this. Whether AOH acts on the enzyme units located on the serosal surface of the cell, or on the inner side of the cell or on the metabolism of the cell remains to be established. Research is in progress. REFERENCES
Barbarossa L., Gallucci E., Bottalico A. and Micelli S. (1988) The effect of Alternariol Mycotoxin on active sodium transport across frog skin (Rana esculenta). Boll. Soc. It. Biol. Sper. LXlV (9), 825-829. Chu F. S. and Bennet S. C. (1981) High performance liquid chromatography preparation of Alternariol methyl ether, and altenuene. J. Ass. Off. Analyt. Chem. 64, 950-954. De Sousa R. C. and Grosso A. (1981) The mode of the action of vasopressin: membrane microstructure and biological transport. J. Physiol. (Par/s) 77, 643~69. Herrera D. (1976) Effect of insulin on short-circuit current and sodium transport across toad urinary bladder. Am. J. Physiol. 209, 819-824. Herrera F., Whittembury G. and Planchart A. (1963) Effect of insulin on short-circuit current across isolated frog skin in the presence of calcium and magnesium. B.B.A. 66, 170-172. Koefoed-.Iohnsen W. and Ussing H. H. 0958) The nature of the frog skin potential. Acta Physiol. Scand. 42, 298-308. Leaf A., Anderson J. and Page L. B. (1958) Active sodium transport by the isolated toad bladder. J. Gen. Physiol. 41, 657-668. Ussing H. H. and Zerahn K. (1951) Active transport of sodium as the source of electrical current in the shortcircuited isolated frog skin. Acta Physiol. Scand. 23, 110-127.
620
S. MtCELLIet al.
Wiesmann W. P., Shina S. and Klahr S. (1976) Insulin stimulates active sodium transport in toad urinary bladder by two mechanism. Nature (Lond) 206, 546 547.
Wiesmann W. P., Sinha S., Klahr S. (1977) Effects of insulin, ADH, and cyclic AMP on sodium transport in the toad bladder. Am. J. Physiol 232 (Renal fluid electrolyte physiol.) 1, F307-F314.
0306-3623/90 $3.00 + 0.00 Copyright © 1990 Pergamon Press plc
Printed in Great Britain. All rights reserved
THE EFFECT OF ADH AND INSULIN ON ACTIVE SODIUM TRANSPORT ACROSS FROG SKIN IN THE PRESENCE OF ALTERNARIOL MYCOTOXIN S. MICELLI, A. BOTTALICOl and E. GALLUCCI Laboratorio di Fisiologia Generale, Dipartimento Farmaco-Biologico, Universit~i di Bari and qstituto tossine e micotossine dei Parassiti Vegetali del Consiglio Nazionale delle Ricerche, Bail, Italy
(Received 14 September 1989) Abstract--l. The effect of vasopressin and insulin on active sodium transport across frog skin in the presence of internal alternariol mycotoxin was studied, using the short-circuit technique. 2. Vasopressin stimulates sodium transport across frog skin by decreasing the resistence to sodium entry into the epithelial cells, thus partially removing the inhibition on the short-circuit current due to the action of Alternariol mycotoxin. 3. Even insulin which is known to increase the short-circuit current by a different mechanism, determines a rapid reversal effect on the inhibition due to Alternariol. 4. These data confirm the different action of the two hormones on active sodium transport across frog skin, and furthermore are indicative of an inhibition of transepitbelial sodium transport by Alternariol mycotoxin probably via the sodium pump.
INTRODUCTION
Frog skin re-absorbs sodium ions from the external environment and transports them into the blood. This transport is active, because sodium overcomes its electrochemical gradient in order to pass from the outer to the inner medium. The short-circuit current is a measure of the net sodium transport across isolated amphibian skin and bladder (Ussing and Zerahn, 1951; Leaf et al., 1958). In frog skin and in urinary bladder transepitheliai sodium transport is controlled by the antiduiretic hormone ( A D H ) which increases sodium re-absorption by a cAMP-mediated process (Aceves 1977; De Sousa, 1981; Sapirstein et al., 1973). Previous work has shown that insulin produces an increase in the short-circuit current across isolated frog skin and urinary bladder (Herrera et al., 1963; Herrera, 1976; Wiesman et al., 1976) probably as a result of the stimulation o f the active sodium transport step at the serosal surface. This active sodium transport may be considered as an index o f the ability of the cell to function, for this reason it is a useful tool in the testing of substances which impair the metabolism such as toxins, particularly those present in vegetable foods. In the present paper we focus our interest on the effect of Alternariol mycotoxin (AOH), whose fungal sources are dlternaria tenuis, d. dauci, d. cucumerins etc., on the active sodium transport across frog skin in the absence and in the presence of different hormones or combinations of hormones. A preliminary report of this study has already been published (Boll. Soc. It. Biol. Sper. LXIV, 25). MATERIALS AND METHODS
Experiments were performed on half symmetrical frog skin of Rana esculenta, that had been kept in tanks with
access to tap water at room temperature prior to the experiments. The AOH employed was produced, and purified according to Chu and Bennet (1981) using the approximately modified procedure of Visconti (unpublished data). The ADH was by Sigma, the Insulin was by Eli Lilly. Following the method described by Ussing and Zerahn (1951) paired hemiskins were mounted between Lucite chambers with an exposed tissue area of 1.14cm 2 and a 7 ml volume of each chamber filled with a solution containing: l l 0 m M NaCl; 2.5mM CaCl2; 2mM KCl; 1.7mM Nail: PO 4; 3.7 mM Na 2HPO 4(pH = 7. l; 230-240 mOsm/kg; temperature 22 ± 2°C); electric measurements of the short circuit current (I,~) were monitored after the membrane had been mounted and allowed to equilibrate.
RESULTS
Figure I reports the dose-response relationship between different concentrations of A O H and active sodium transport. It can be seen that micromolar concentrations of A O H inhibit the short-circuit current (I,¢). Indeed I~0 is reached when concentrations of 4.5-5.5/~M are used. Figure 2 reports the effect of internal A O H (5.5/zM) and washing out treatment on sodium transport across frog skin. It can be seen that A O H (in 2 or 3 hr) reduces the sodium transported by about 50%, washing out does not restore the sodium transport, which falls to 75% of the initial value in the space of 1 hr after mycotoxin removal. To investigate the possibility that an increase in the intracellular sodium pool may overcome or partially remove A O H inhibition the l~c was evaluated after stimulation of sodium transport by A D H . (Fig. 3A). Under these conditions the I~ showed a small but not significant increase. In half symmetrical skin (Fig. 3B) A D H causes, as usual, an increase in the short-circuit current. 617
618
S. M I C E L L I
et al. 1 -
AOH
~°°i-
ADH
I l
x(3)
;(5)
0
I
I
I
I
I
I
I
1
2
3
4
5
6
7
ALt.ernorioL
rnycotoxin (p,M)
Fig. I. Relationship between internal Alternariol concentrations and active sodium transport in frog skin. Action time of mycotoxin 2 hr. The sodium transport at equilibrium was 0.84 + 0.12/aequiv./cm 2 hr. (eleven experiments). In brackets are the number of experiments for each Alternariol concentration used.
It is known that the A O H effect is irreversible as the I,~ is not restored after washing out the treated tissue. This is indicative either of a strong binding of the A O H to the membrane receptors or of a penetration of the A O H into the cell. As in a previous paper it was reported that insulin increases the short-circuit current across frog skin, by acting on active sodium transport at the serosal surface, we decided to compare the effect of both insulin and A D H using two half-symmetrical frog skins• Figure 4 reports this test. It can be seen that the addition of insulin immediately determines a dramatic increase in sodium transport. Then the transport decreases below the equilibrium value; on the other hand the addition of a maximal concentration of A D H determins a slower but longer increase in sodium transport. The same experiment was conducted on symmetrical half-skin pretreated with AOH, In this case, when the A O H reduced the I= by about 50%, the insulin and A D H were added (Fig. 5). The insulin rapidly removed the AOH inhibition on the sodium transport and temporarily stimulated it. Then the inhibition
0
--
30 6 0 9 0 120 t 5 0 1 ~ ) 210 240 270
1
2 T
0
0
30 6 0 9 0 420 Time
Fig. 3. Effect of internal Alternariol (5.5#M) and of Alternariol plus ADH (45 mUI/ml) (top) and of internal ADH (45mUI/ml) (bottom) on transepithelial sodium transport in half-symmetrical frog skin. Mean values + SE of four experiments.
3
I
I I
2
6 6
~" 100~ E
(mini
W.O. 1(
o
t
t
t 0
~ z
o
I
I
1
2
J
3
I
I
I
I
30
60
90
12O
Time
(rnin)
4
Time (h)
Fig. 2. Effect of internal Alternariol (5.5/~M) and washing out treatment on active sodium transport across frog skin. Mean + SE of four experiments. The sodium transport at equilibrium (zero-time) was 1.07 + 0.21 ~equiv./cm2/hr.
Fig. 4. Effect of internal A D H (45mUl/m]) ( 0 ) and of
internal and external insulin (3.10 -~ M) ( 1 ) on transepithelial sodium transport in half-symmetrical frog-skin. Mean values + SE of the ratio ¢,t/¢,, of 5 experiments (¢~,= flux of sodium at equilibrium; ¢,, = flux of sodium of different times of treatment).
Mycotoxin inhibits sodium transport returned. Whereas ADH caused a small revitalization of I~ as previously shown (Fig. 3). Finally, with the same experimental protocol another series of experiments were conducted in which in one half-skin ADH was added after 10 min of insulin action. Figure 6 reports one of those experiments. It is evident that the successive ADH additions strengthen the insulin effect, in fact sodium transport maintains high levels, due presumably to the instantaneous insulin action and to the slower ADH action.
619
"~ 1.0 r-
• • • AOH •
,~
0
l
0
•
•
•
•
. eoo
•
In~Un
30
dO
90 Time
•
:*
.-
:
•
,2o
" ,'lo
laO
2;0
2,~0
(rain)
Fig. 6. Effect of internal ADH (45 mUl/ml) (A) and of internal and external insulin (3.10 -s M) plus internal ADH (45 mUI/ml) (Q) on transepithelial sodium transport in half symmetrical frog skin pre-treated with internal Alternariol (5.5 #M). One experiment representative of a series.
DISCUSSION The data presented show that serosal AOH causes a reduction in the active sodium transport across frog skin. It should also be noted that this effect is not a temporary effect, as the washing out of the AOH does not restore the short-circuit current (Barbarossa et al., 1988). It is known that active sodium transport is directly dependent on the sodium pump located on the serosal surface of the cell, so AOH could influence this pump. To investigate the possibility that an increase in transport per se may change the properties of the sodium pump in the presence of AOH, the short-circuit current was evaluated before and after stimulation of the sodium transport by ADH. The intracellular sodium increase due to ADH action (mediated by the endocellular second messanger AMPc) on the external barrier of the cell, shows its tendency to remove the inhibition due to AOH. As we are not in a position to say that AOH directly influences the sodium pump instead of, for example, the apical barrier of the membrane to sodium; experiments were performed using another hormone (insulin) to stimulate the sodium pump, as has been reported (Wiesmann et al., 1977). In this study we have compared the effect on the I~c of ADH and of the insulin on half-symmetrical skins (Fig. 4). The two hormones increase the active sodium transport but the rate at which they do so and the time taken differ, probably indicating a different mechanism of action on the frog skin.
~OH
•
]flsuLin
0.*
• • • • • e
|
°
e
°
i
,
,
•
•
o
o
o
o
i
,
,
,
.
,
o
e
e
i
.
,
o
,0
AOH .
.
i
,
r..I
9015010
,
60
120
i
150
Time (rain) Fig. 5. Effect of internal ADH (45 mUl/ml) (O) and of internal and external insulin (3' l0 -s M) (II) on transepithelial sodium transport in symmetricalfrog skin after pretreatment with internal Alternariol (5.SgM). One experiment representative of a series is reported.
When the insulin was added after AOH inhibition, the I,~ value returned to its basal value or even more. (Fig. 5), then the inhibition returned. Furthermore, if ADH is added while the insulin is still working, the increase of the I,~ is maintained for a longer time before falling again. These last two experiments are indicative of the action of AOH on the active step of sodium transport. In fact insulin, which is known to positively influence active sodium transport by acting on the pump, removes the inhibition of AOH. The successive action of ADH in these conditions suggests that the apical membrane and the pathway of AMPc are not affected by AOH. In the crude intestinal membranes of frog AOH (6.4.10- s M) inhibits the Na +, K*-ATPase activity by 50% (unpublished data). The observations reported here are in line with this. Whether AOH acts on the enzyme units located on the serosal surface of the cell, or on the inner side of the cell or on the metabolism of the cell remains to be established. Research is in progress. REFERENCES
Barbarossa L., Gallucci E., Bottalico A. and Micelli S. (1988) The effect of Alternariol Mycotoxin on active sodium transport across frog skin (Rana esculenta). Boll. Soc. It. Biol. Sper. LXlV (9), 825-829. Chu F. S. and Bennet S. C. (1981) High performance liquid chromatography preparation of Alternariol methyl ether, and altenuene. J. Ass. Off. Analyt. Chem. 64, 950-954. De Sousa R. C. and Grosso A. (1981) The mode of the action of vasopressin: membrane microstructure and biological transport. J. Physiol. (Par/s) 77, 643~69. Herrera D. (1976) Effect of insulin on short-circuit current and sodium transport across toad urinary bladder. Am. J. Physiol. 209, 819-824. Herrera F., Whittembury G. and Planchart A. (1963) Effect of insulin on short-circuit current across isolated frog skin in the presence of calcium and magnesium. B.B.A. 66, 170-172. Koefoed-.Iohnsen W. and Ussing H. H. 0958) The nature of the frog skin potential. Acta Physiol. Scand. 42, 298-308. Leaf A., Anderson J. and Page L. B. (1958) Active sodium transport by the isolated toad bladder. J. Gen. Physiol. 41, 657-668. Ussing H. H. and Zerahn K. (1951) Active transport of sodium as the source of electrical current in the shortcircuited isolated frog skin. Acta Physiol. Scand. 23, 110-127.
620
S. MtCELLIet al.
Wiesmann W. P., Shina S. and Klahr S. (1976) Insulin stimulates active sodium transport in toad urinary bladder by two mechanism. Nature (Lond) 206, 546 547.
Wiesmann W. P., Sinha S., Klahr S. (1977) Effects of insulin, ADH, and cyclic AMP on sodium transport in the toad bladder. Am. J. Physiol 232 (Renal fluid electrolyte physiol.) 1, F307-F314.
