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Water transport response of toad bladder to prolactin
Camp. Biochem. Physiol,. 1975, Vol. 52A, pp. 75 to 76. Pergamon Press. Printed in Great Britain
WATER TRANSPORT RESPONSE OF TOAD BLADDER TO PROLACTIN E. S. DEBNAM AND R. S. SNART Department of Zoology, The University, Sheffield S10 2NT, England
(Received 28 June 1974) Abstract--I. Ovine prolactin increases the osmotic water flow across isolated toad bladder. 2. A dose-response characteristic has been determined. 3. The effects of theophylline (10 -3 M) and o-hydroxymercuribenzoic acid (10 -3 M), on this response, have been investigated. 4. The response has been studied in relation to vasopressin-stimulated osmotic water transport.
INTRODUCTION
RESULTS
PROLACTIN has been shown (Hirano, Johnson & Bern, 1971) to reduce osmotic water flow but increase sodium reabsorption across the urinary bladder of the euryhaline teleost Platichthys stellatus. They suggest that such effects may have particular adaptive significance in fresh water. Water movement across the bladder is believed to follow active sodium transport when the animal is in sea water. Other reports have described prolactin-stimulated electrolyte metabolism in isolated toad (Bufo marinus) bladder (Dalton & Snart, 1969) and in teleost gills, bladder and kidney (Ensor & Ball, 1972). The present work has involved a study of the effect of similar prolactin preparations on the osmotic water transport across the isolated toad bladder.
It was found that prolactin increased water permeability across the toad bladder (Fig. 1) with a time course that corresponds to the hormone-stimulated sodium transport (Snart & Dalton, 1973) and similar to vasopressin-stimulated water transport effects. The response is immediate but subject to a self inhibition; the resulting peak response occurs 30 min after hormone addition. Although theophylline (10 -3 M) potentiates the prolactin response neither the maximum prolactin-stimulated water transport nor the theophylline potentiated responses match those stimulated by vasopressin (Fig. 2). The prolactin osmotic water transport response measured in the presence of vasopressin appears to be completely additive. The response is insensitive to doses of sulfhydryl reagent (o-hydroxymercuribenzoic acid) that caused an 839/o inhibition of the vasopressin (10 mu/ ml) re,, )onse.
MATERIALS
AND
METHODS
Bufo marinus were kept in 0.1 M sodium chloride solu-
s0
tion for 24 hr, a standard procedure designed to reduce endogenous release of mineralocorticoids. They were rapidly pithed and the bladders excised and stretched across a double transport chamber for measurement of the osmotic water flow (Wright & Snart, 1971). The tissue was incubated for 1 hr with aerated bicarbonate Ringer's solution (Na* 113"5 m-equiv/1; K* 3"5 m-equiv/l; CI116.5 m-equiv/l; HCO3- 2-4 m-equiv/1; Ca 2+ 0-98 mmoles/l) bathing the serosal surface and a ten-fold dilution of Ringer's solution bathing the mucosal surface. The osmotic water flow was measured across an exposed area of 0"7 cm 2 at 10 min intervals over a 90 min period. The effects of serosal additions of 7500 mu/ml ovine prolactin (NIHP SI0), prolactin (7500 mu/ml) plus 10 mu/ml pitressin (Parke-Davis Co. Ltd.), prolactin (7500 mu/ml) plus theophylline (10 -3 M), pitressin (10 mu/ml) or bovine serum albumin (10 -~ M), on the osmotic water flow was measured in several repeat experiments. A dose response characteristic for the prolactin-stimulated water transport was obtained, using concentrations of prolactin over a 75 mu/ml to 75,000 mu/ml range, by measurement of the peak water transport response in several repeat experiments. In final experiments the effect of o-hydroxybenzoic acid (10 -3 M) on the osmotic water transport stimulated by pitressin (10 mu/ml) was measured. This experiment was repeated 3 times.
A ¢=
s.0 E o ~z, v 4o
(7)
(3)
~ 20
(7)
0 0
10
20
30 -
40
50
60
70
80
90
tirn¢ (rains)
Fig. 1. Shows the effect of ovine prolactin (7500 mu/ml) on the osmotic water flow across isolated toad bladders
in the absence (--O--O--) or presence of 1 mM theophylline (--O---O---) or l mM o-hydroxymercuribenzoic acid (--A--&--). The failure of bovine serum albumin (10 -5 M) to stimulate a water transport response is shown ( - - A - - A - - ) . The number of experiments is shown in brackets. Each point represents the mean +S.E.M. 75
76
E.S. DEBNAMAND R. S. SNART
7 m
~6 o
~.
s
(8) 3 3
(8)
2 " "G- . . . 0 _ . . . . 0 ..d 10
, 20
30
40 50 time ( m i n s )
60
70
80
s0
Fig. 2. The effect of vasopressin (10 mu/ml) on the osmotic water transport response across toad bladder, in the presence (--Q---O---) and absence (--O--O--) of ovine prolactin (7500 mu/ml). The difference curve ( - - O - - O - - ) shows the additive effect of the prolactin response. The number of experiments is shown in brackets. Each point represents the mean + S.E.M. The dose-response characteristic of the prolactinstimulated water transport is shown (Fig. 3) as a plot of peak response against prolactin concentration. This dose-response curve does not parallel that obtained for the prolactin-stimulated sodium transport in toad bladder (Snart & Dalton, 1973). Very high concentrations of prolactin were required to achieve a maximum water-flow response but no similar response could be obtained with high concentrations of bovine serum albumin (Fig. 1). DISCUSSION The distinctive character of the prolactin permeability effect in toad bladder was demonstrated in our earlier studies on sodium transport. The present work has confirmed this distinction in relation to the water
permeability. The response is potentiated by theophylline so probably involves a cyclic A M P mediated process. It is insensitive to mercurials but in many respects resembles the antidiuretic hormone response. This is in sharp contrast to its diuretic effect in teleost bladder in which its physiological activity appears to have particular adaptive significance. However, the mesodermal teleost bladder must be distinguished from the endodermal cloacal bladder of the amphibia. The antidiuretic activity of prolactin in amphibian bladder may be relevant to the evolutionary development of a cloacal bladder, for water conservation in the amphibia. The water transport response in toad bladder does not parallel the increased Na ÷ transport (Snart & Dalton, 1973) and doses used are much higher than those found to affect teleost bladder (Doneen & Bern, 1974). Prolactin effects on electrolyte metabolism in the cloacal bladder may therefore be considered more significant than any antidiuretic effect. REFERENCES
DALTON T. & SNART R. S. (1969) Effect of prolactin in the movement of Na ÷ across toad bladder. J. Endocr. 43, 6. DONEEN B. A. & BERN H. A. (1974) In vitro effects of prolactin and cortisol on water permeability of the urinary bladder of the teleost Gillicythys mirabilis. J. exp. Zool. 187, 173-179. ENSOR D. M. & BALLJ. N. (1972) Prolactin and osmoregulation in fishes. Fedn Proc. Fedn Am. Socs exp. Biol. 31, 1615-1623. HIRANO T., JOHNSON D. W . 8/. BERN H . A. (1971) Control of water movement in flounder urinary bladder by prolactin. Nature, Lond. 230, 469-471. SNART R. S. 8/. DALTON Z . 0973) Response of toad bladder to prolactin. Comp. Biochem. Physiol. 45A, 307-311. W R I G H T D. W . t~ SNART R. S. (1971) Simultaneous measurement of the effect of vasopressin on sodium and water transport across toad bladder. Life Sci. 10, 301308. Key Word hldex--Toad bladder; prolactin; theophylline; o-hydroxymercuribenzoic acid; Vasopressin.
1100 1000
9OO
8OO
(5)
°~ 700 3:
.c_ o w
6OO
5OO 400
o
5)
z0o
100 (5)
i
i
75
750 Proloctin
7.500 concentration
( IU. Iliit¢
75.000 )
Fig. 3. Dose-response characteristic of ovine prolactin-stimulated water transport across the isolated toad bladder. Each point represents the mean _+ S.E.M. '5o increase in water flow, measured at the 30 min peak. The number of experiments is shown in brackets and the prolactin concentration plotted on a log~0 scale.
WATER TRANSPORT RESPONSE OF TOAD BLADDER TO PROLACTIN E. S. DEBNAM AND R. S. SNART Department of Zoology, The University, Sheffield S10 2NT, England
(Received 28 June 1974) Abstract--I. Ovine prolactin increases the osmotic water flow across isolated toad bladder. 2. A dose-response characteristic has been determined. 3. The effects of theophylline (10 -3 M) and o-hydroxymercuribenzoic acid (10 -3 M), on this response, have been investigated. 4. The response has been studied in relation to vasopressin-stimulated osmotic water transport.
INTRODUCTION
RESULTS
PROLACTIN has been shown (Hirano, Johnson & Bern, 1971) to reduce osmotic water flow but increase sodium reabsorption across the urinary bladder of the euryhaline teleost Platichthys stellatus. They suggest that such effects may have particular adaptive significance in fresh water. Water movement across the bladder is believed to follow active sodium transport when the animal is in sea water. Other reports have described prolactin-stimulated electrolyte metabolism in isolated toad (Bufo marinus) bladder (Dalton & Snart, 1969) and in teleost gills, bladder and kidney (Ensor & Ball, 1972). The present work has involved a study of the effect of similar prolactin preparations on the osmotic water transport across the isolated toad bladder.
It was found that prolactin increased water permeability across the toad bladder (Fig. 1) with a time course that corresponds to the hormone-stimulated sodium transport (Snart & Dalton, 1973) and similar to vasopressin-stimulated water transport effects. The response is immediate but subject to a self inhibition; the resulting peak response occurs 30 min after hormone addition. Although theophylline (10 -3 M) potentiates the prolactin response neither the maximum prolactin-stimulated water transport nor the theophylline potentiated responses match those stimulated by vasopressin (Fig. 2). The prolactin osmotic water transport response measured in the presence of vasopressin appears to be completely additive. The response is insensitive to doses of sulfhydryl reagent (o-hydroxymercuribenzoic acid) that caused an 839/o inhibition of the vasopressin (10 mu/ ml) re,, )onse.
MATERIALS
AND
METHODS
Bufo marinus were kept in 0.1 M sodium chloride solu-
s0
tion for 24 hr, a standard procedure designed to reduce endogenous release of mineralocorticoids. They were rapidly pithed and the bladders excised and stretched across a double transport chamber for measurement of the osmotic water flow (Wright & Snart, 1971). The tissue was incubated for 1 hr with aerated bicarbonate Ringer's solution (Na* 113"5 m-equiv/1; K* 3"5 m-equiv/l; CI116.5 m-equiv/l; HCO3- 2-4 m-equiv/1; Ca 2+ 0-98 mmoles/l) bathing the serosal surface and a ten-fold dilution of Ringer's solution bathing the mucosal surface. The osmotic water flow was measured across an exposed area of 0"7 cm 2 at 10 min intervals over a 90 min period. The effects of serosal additions of 7500 mu/ml ovine prolactin (NIHP SI0), prolactin (7500 mu/ml) plus 10 mu/ml pitressin (Parke-Davis Co. Ltd.), prolactin (7500 mu/ml) plus theophylline (10 -3 M), pitressin (10 mu/ml) or bovine serum albumin (10 -~ M), on the osmotic water flow was measured in several repeat experiments. A dose response characteristic for the prolactin-stimulated water transport was obtained, using concentrations of prolactin over a 75 mu/ml to 75,000 mu/ml range, by measurement of the peak water transport response in several repeat experiments. In final experiments the effect of o-hydroxybenzoic acid (10 -3 M) on the osmotic water transport stimulated by pitressin (10 mu/ml) was measured. This experiment was repeated 3 times.
A ¢=
s.0 E o ~z, v 4o
(7)
(3)
~ 20
(7)
0 0
10
20
30 -
40
50
60
70
80
90
tirn¢ (rains)
Fig. 1. Shows the effect of ovine prolactin (7500 mu/ml) on the osmotic water flow across isolated toad bladders
in the absence (--O--O--) or presence of 1 mM theophylline (--O---O---) or l mM o-hydroxymercuribenzoic acid (--A--&--). The failure of bovine serum albumin (10 -5 M) to stimulate a water transport response is shown ( - - A - - A - - ) . The number of experiments is shown in brackets. Each point represents the mean +S.E.M. 75
76
E.S. DEBNAMAND R. S. SNART
7 m
~6 o
~.
s
(8) 3 3
(8)
2 " "G- . . . 0 _ . . . . 0 ..d 10
, 20
30
40 50 time ( m i n s )
60
70
80
s0
Fig. 2. The effect of vasopressin (10 mu/ml) on the osmotic water transport response across toad bladder, in the presence (--Q---O---) and absence (--O--O--) of ovine prolactin (7500 mu/ml). The difference curve ( - - O - - O - - ) shows the additive effect of the prolactin response. The number of experiments is shown in brackets. Each point represents the mean + S.E.M. The dose-response characteristic of the prolactinstimulated water transport is shown (Fig. 3) as a plot of peak response against prolactin concentration. This dose-response curve does not parallel that obtained for the prolactin-stimulated sodium transport in toad bladder (Snart & Dalton, 1973). Very high concentrations of prolactin were required to achieve a maximum water-flow response but no similar response could be obtained with high concentrations of bovine serum albumin (Fig. 1). DISCUSSION The distinctive character of the prolactin permeability effect in toad bladder was demonstrated in our earlier studies on sodium transport. The present work has confirmed this distinction in relation to the water
permeability. The response is potentiated by theophylline so probably involves a cyclic A M P mediated process. It is insensitive to mercurials but in many respects resembles the antidiuretic hormone response. This is in sharp contrast to its diuretic effect in teleost bladder in which its physiological activity appears to have particular adaptive significance. However, the mesodermal teleost bladder must be distinguished from the endodermal cloacal bladder of the amphibia. The antidiuretic activity of prolactin in amphibian bladder may be relevant to the evolutionary development of a cloacal bladder, for water conservation in the amphibia. The water transport response in toad bladder does not parallel the increased Na ÷ transport (Snart & Dalton, 1973) and doses used are much higher than those found to affect teleost bladder (Doneen & Bern, 1974). Prolactin effects on electrolyte metabolism in the cloacal bladder may therefore be considered more significant than any antidiuretic effect. REFERENCES
DALTON T. & SNART R. S. (1969) Effect of prolactin in the movement of Na ÷ across toad bladder. J. Endocr. 43, 6. DONEEN B. A. & BERN H. A. (1974) In vitro effects of prolactin and cortisol on water permeability of the urinary bladder of the teleost Gillicythys mirabilis. J. exp. Zool. 187, 173-179. ENSOR D. M. & BALLJ. N. (1972) Prolactin and osmoregulation in fishes. Fedn Proc. Fedn Am. Socs exp. Biol. 31, 1615-1623. HIRANO T., JOHNSON D. W . 8/. BERN H . A. (1971) Control of water movement in flounder urinary bladder by prolactin. Nature, Lond. 230, 469-471. SNART R. S. 8/. DALTON Z . 0973) Response of toad bladder to prolactin. Comp. Biochem. Physiol. 45A, 307-311. W R I G H T D. W . t~ SNART R. S. (1971) Simultaneous measurement of the effect of vasopressin on sodium and water transport across toad bladder. Life Sci. 10, 301308. Key Word hldex--Toad bladder; prolactin; theophylline; o-hydroxymercuribenzoic acid; Vasopressin.
1100 1000
9OO
8OO
(5)
°~ 700 3:
.c_ o w
6OO
5OO 400
o
5)
z0o
100 (5)
i
i
75
750 Proloctin
7.500 concentration
( IU. Iliit¢
75.000 )
Fig. 3. Dose-response characteristic of ovine prolactin-stimulated water transport across the isolated toad bladder. Each point represents the mean _+ S.E.M. '5o increase in water flow, measured at the 30 min peak. The number of experiments is shown in brackets and the prolactin concentration plotted on a log~0 scale.