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The water balance response of the pelvic “patch” of Bufo punctatus and Bufo boreas
Comp. Biochem. Physiol.,
1974,Vol.
47A, pp. 1285 to
1295.Pwgamon
Press. Printed in Great Britain
THE WATER BALANCE RESPONSE OF THE PELVIC “PATCH” OF BUFO PUNCTATUS AND BUFO BOREAS ROGER Department
A. BALDWIN*
of Biology, California State University,
Fullerton,
California 92631, U.S.A.
(Receiwed 15 May 1973) Abstract-l. Net transfer of water was measured in oitro across ventral pelvic and ventral pectoral integument of Bufo punctatus and B. boreas. 2. Arginine vasopressin (AVP) increased net transfer of water across ventral pelvic integument but not ventral pectoral integument. 3. Cyanide and dinitrophenol made ventral pelvic integument unresponsive to AVP. 4. Adenosine 3’,5’-monophosphate (cyclic AMP) increased net transfer of water in ventral pelvic integument of B. punctatus. 5. Pretreatment of ventral pelvic integument with dinitrophenol made the integument unresponsive to cyclic AMP. INTRODUCTION
THE PELVIC“patch” or ventral pelvic integument of Bufopumtatus has been shown to be the major site of water uptake in dehydrated toads while the ventral pectoral integument accounts for comparatively little water uptake (McClanahan & Baldwin, 1969). It has been suggested that the pelvic “patch” is an integumental area specialized for water uptake. The rate of water movement through the isolated skin of some anurans has been shown to increase following the application of neurohypophysial hormones (Sawyer, 1951, 1960; Bentley, 1958, 1963). These observations suggest that the pelvic “patch” may be more responsive to antidiuretic hormone than other integumental areas. This investigation compares the in vitro response of ventral pelvic and ventral pectoral skin of two species of toad, B. pumtatus and B. boreas. The effect of cyanide, dinitrophenol (DNP) and adenosine 3’,5’-monophosphate (cyclic AMP) on the pelvic skin response to arginine vasopressin is also investigated. MATERIALS
AND METHODS
Specimens of Bzrfo boreas were collected in Orange County, California, during the fall of 1971 and 1972. Specimens of B. punctatus were collected near Palm Desert, Riverside County, California, in early April of 1972. Toads were maintained in aquaria filled to a depth of about 6 cm with moist sand. Pieces of clay pot were placed in the aquaria to provide cover for the animals. Toads were fed Tenebrio larvae twice a week. * Present address: Neurochemistry Hospital, Sepulveda, California 91343.
Laboratory, 1285
Sepulveda Veterans Administration
1286
ROGER A. BALDWIN
The net rate of water transfer across isolated toad skin in response to an osmotic gradient was measured volumetrically. Specimens of ventral pelvic skin were taken from the “patch” Specimens of pectoral skin were area of the lower abdomen and groin of both species. Toads were first killed by a sharp blow on the back of taken from between the forelegs. the head and skin from both areas was rapidly removed and placed in separate S-6-ml aliquots of Ringer’s solution contained in lo-ml beakers. The Ringer’s solution consisted of 65 g NaCl, 0.14 g KCl, 0.12 g CaCl,, 0.20 g NaHCO, and 0.20 g glucose brought to 1 1. with distilled water. The osmolarity of the Ringer’s solution was 205 mOsmole/l. The pieces of skin were then tied tautly over the ends of slightly flared glass tubes of known cross-sectional area (about 0.2 cm”). The outer surface of the skin faced the inside of the tubes. The tubes were then rinsed twice and filled with a one to ten dilution of the Ringer’s solution. The osmolarity of the one to ten dilution of Ringer’s was 25.6 mOsmole/l. Lengths of rubber tubing about 4 cm long were used to attach the glass tubes to Hamilton microliter syringes. The syringes had the needles removed and were of 50 ~1 capacity graduated in 1 yl units. Syringes and tubing were filled with the one to ten diluted Ringer’s solution. A lOO-ml beaker containing 50 ml of normal Ringer’s solution was clamped in a The Ringer’s solution was aerated for 15-20 water-bath which was maintained at 20°C. min before the skin specimens with attached tubing and syringes were placed in it. This period of time was always sufficient for temperature equilibration. The assembled microliter syringes, glass and rubber tubing and skin specimens were suspended over the beaker of Ringer’s solution so that the skin specimens were below the surface of the Ringer’s solution, but not touching the bottom or sides of the beaker. Air from an aquarium pump was bubbled through the Ringer’s solution throughout an experiment to mix and aerate the Ringer’s solution. Evaporation from the Ringer’s solution during 1 hr of aeration changed the osmolarity from 205 to 212 mOsmole/l. An approximate tenfold difference in the concentration of water (205 vs 25.6 mOsmole/l.) existed across the skin. The net transfer of water along an osmotic gradient from the syringes through the skin and into the beaker could be read directly from the syringes. Net transfer rates were determined by timing net transfer to the nearest minute. To determine the effect of arginine vasopressin (AVP) on the net transfer rate of water, specimens of pelvic and pectoral skin were assembled onto syringes as described above After an initial period of 40-60 and immersed in the same beaker of Ringer’s solution. min, during which the net transfer rate of water across the specimens became relatively constant, 100 ~1 of a 50.7 unit/ml solution of AVP was added to the Ringer’s solution bathing the specimens. The final concentration of AVP was 0.101 units/ml. AVP was obtained from Parke-Davis as a pitressin powder prepared solely from bovine sources. The powder was dissoled in one to four hundred (1 : 400) glacial acetic acid and distilled water. Preliminary experiments indicated that 100 ~1 of 1 : 400 dilution of glacial acetic acid had no effect on net transfer rates. Following the addition of AVP, net transfer was again monitored for an additional 40-60 min. The high level of AVP used to stimulate net transfer in these experiments was chosen because animals maintained in the laboratory for varying periods of time showed great Preliminary attempts to establish a dose variability in their response to the hormone. response curve for B. punctatus pelvic skin showed that while some newly collected animals responded to 5 ~1 of the AVP solution, some animals which had been maintained in the laboratory for 3 or more months would not respond until 50 ~1 of the AVP solution had been added. Since these animals were difficult to obtain, excess hormone was used so that all animals collected could be used. The effect of this non-physiological level of hormone was investigated in vivo by subcutaneous injection of AVP. The in vitro AVP level of 0.101 units/ml was approximated by the injection of 0.101 units of AVP for each g of body water in normally hydrated toads. Total body water was calculated by assuming that 80 per cent of a normally hydrated toad’s body weight is water (see McClanahan, 1967). Following injection, animals were placed
WATER BALANCE RESPONSE OF BUFO PUNCTATUS
AND BUFO
BOREAS
1287
in tap water 1 cm deep and water uptake determined gravimetrically as described by McClanahan & Baldwin (1969). In order to study the effects of cyanide on the pelvic skin’s response to AVP, duplicate specimens of skin were taken from the pelvic area of either B. punctatus ox B. b~reusand assembled on syringes as described above. Each specimen from the same animal was piaced, however, in separate 50-ml beakers containing 25 ml of Ringer’s solution. A stock solution of 6 mM NaCN was made up in Ringer’s solution as well as a stock NaCl Ringer’s containing an additional 6 m-moles/l. of NaCl. After an initial period of 35-40 min, sufficient NaCN solution (5 ml) was added to one beaker to bring the final concentration of cyanide ion to 1 mM. An equal amount (5 ml) of the stock NaCl Ringer’s was added to the other beaker to maintain approximately equal osmotic conditions for the two skin specimens. Net transfer was monitored for 20 min, which was sufficient for the rates to become relatively constant and then 50 ~1 of the AVP solution described above was added to each beaker. Net transfer was monitored for 30-40 min following which, in some cases, 10 mg of NaCN crystals were added to the skin specimen which had not previously received cyanide. Net transfer was again monitored. To determine the effect of adenosine triphosphate (ATP) on net transfer rates in skin which had been poisoned with cyanide, duplicate skin specimens from the pelvic patch of the same animal were assembled on the syringes and immersed in separate beakers of Ringer’s solution to which the stock solutions of NaCN and NaCl Ringer’s had already been added, Following an initial period of 30-40 min, 50 ~1 of the AVP solution was added to each beaker. After about 20 min, net transfer rates had become relatively constant and 38.0 mg of ATP and 50.8 mg of MgC&,.6H,O were added to the beaker containing the cyanide poisoned specimen. The final concentration of ATP was 3 mM and the final concentration of magnesium ion was 10 mM. Following the addition of ATP and magnesium, net transfer was monitored for 20-40 min. The effect of ATP on pelvic skin poisoned with 2,4-dinitrophenol (DNP) was determined by a procedure similar to that for cyanide. To one beaker of the pair 2.3 mg of DNP was added, resulting in a O-5 mM concentration of DNP. Nothing was added to the other beaker since the increase in osmotic pressure due to this quantity of DNP was assumed insignificant. ATP and magnesium were added and net transfer monitored as in the cyanide poisoned responses. To test if cyclic AMP could mimic the effect of AVP, pelvic skin specimens from B. punctutuswere assembled on syringes as usual, except that the specimens were suspended in 10 ml of Ringer’s solution contained in a 20-ml beaker. Following an initial 40-min period of monitoring net transfer, 32.9 mg of cyclic AMP was added to the Ringer’s solution, resulting in a 10 mM concentration of cyclic AMP. Net transfer was again monitored for 40 min. The effects of DNP on the pelvic skin’s response to cyclic AMP was studied by setting up apparatus and skin specimens from B. punctutus as in the preceding experiment, except that 1 mg of DNP was added to the 10 ml of Ringer’s solution prior to placing the skin specimens in the Ringer’s The concentration of DNP was approximately 0.5 mM. Net transfer was measured for 30-min periods before and after the application of 32.9 mg of cyclic AMP. RESULTS
In both B. punctatus and B. boreas the movement of water across the pelvic skin increases following the application of AVP (Table 1). Net transfer of water across the pelvic skin of B. pumtatus increased by a factor of approximately 13 while B. boreas increased by a factor of approximately 3. These increases are statistically signi&ant at the @OOS per cent level using the Student’s t-test. In
ROGERA. BALDWIN
1288
TABLE ~----RATEOF NET TRANSPORT OF WATERACROSSINTEGUMENT OF B. punctatus AND B. boreas Rate of net transport of water &l/cm2 per hr) Experimental toad No.
Integument area
Before AVP
After AVP
B. punctatus
1 2 3 4 5 6
218 231 196 208 140 257
17.3 15.2 9.9 14.3 16.6 18.2
Pelvic Pelvic Pelvic Pelvic Pelvic Pelvic z = S.D. =
15.3 3.0 11.0 9.6 13,o 7.3 10.2 8.8
Z = 208 S.D. = 39.3 10.7 7.3 5.1 6.9 9.2 6.2
z = SD. =
10.0 1.9
2 = S.D. =
Pectoral Pectoral Pectoral Pectoral Pectoral Pectoral
7.6 2.0
B. boreas 1 2 3 4 5 6
1 2 3 4 5 6
14.8 llO*O 93.9 95,3 40.2 22.9
Pelvic Pelvic Pelvic Pelvic Pelvic Pelvic D= S.D. =
62.3 41.6 115.0 37.2 17.4 38.8 22.9 16.3
Ci!= S.D. =
41.3 37.4
Pectoral Pectoral Pectoral Pectoral Pectoral Pectoral
195 183 221 200 128 101 5 = 171.3 S.D. = 46.6 94.8 32.2 20.2 30.6 14.3 14.3 Lz= S.D. =
34.4 30.6
contrast to this the movement of water across pectoral skin of both species showed a slight decrease. The values for B. punctatus are from newly collected animals while the values for B. boreas are from animals which had been maintained in the laboratory for 3-9 months. Because of the tendency for responsiveness to AVP to
WATERBALANCERESPONSE OF EUFO PVNCTATUS ANDBUFO
BOREAS
1289
decline in the 1aborator.y the observed net transfer rates of B. pmctatus and B. boreas are not strictly comparable. The rk &JO rates of water uptake that were observed in AVFGnjected B. punctatus show a mean gain of 17 per cent of their standard weight (normally hydrated body weight without bladder water) during the first hour of hydration (Table 2). These rates are not significantly different from uptake rates observed TABLE ~-RATE OF WATERUPTAKEOF AVP INJBCTJZDB. punctatus
Experimental toad No. 1 2 3 4
weight (g)
Injection (Unita)
11.10 6.82 7.60 9.28
0.094 O*OS6 0.062 0.089
Rate of water uptake (% St. wt. gained per hr) 10.8 16-S 18.4 22.2 I = SD. =
17.0 4*8
by McClanahan & Baldwin (1969) for B. pwzctatus dehydrated to 80 per cent of their standard weight and presumably circulating native antidiuretic hormone. This suggests that the high dosage of AVP used in these experiments causes TABLE
3-
Tm KPPKCT OF CYANIDE ON NET TRANSFER OF WATERACROSS B. punctutus PELVIC SKIN Rate of net transfer of water following successive additions of cyanide or chloride, AVP and NaCN Cl-y&S
Experimental toad No. 1 2 3 4
42
Shin specimen and ion added
Q.d/cm* per hr)
A-ClB-CNA-ClB-CNA-ClB-CNA-CIB-CN-
Before ion
After ion
After AVP
After NaCN
15.5 19.4 17.4 17.4 8.5 45.4 17.0 20.8
17.7 25.8 20.6 14.2 3.3 35.2 9.9 19.8
43.2 17.3 39.9 12.5
68.2 56.0 -
95.3 36.7
125.8 -
83.4 13.0
99.7 -
65.4 28.1 19.9 11.4
83.5 28.6 -
A-CI-
d= SD. =
14.6 4.1
12.8 7.8
B-CN-
3 = S.D. =
25.7 13.2
23.8 9.0
1290
ROGER
A.
BALDWIN
changes in skin permeability to water which are quantitatively similar to those caused by physiologic levels of native antidiuretic hormone in dehydrated toads. One response of Anuran skin to AVP and other neurohypophysial hormones is the increase in permeability to water as indicated by increased net transfer rates (Sawyer, 1960; Bentley, 1963). The addition of cyanide to the Ringer’s solution prevents this response in pelvic skin from B. punctatus, while cyanide itself causes only a slight decrease in net transfer from 25.7 to 23.8 $/cm2 per hr. (Table 3). Since cyanide is a strong inhibitor of the ATP generating electron transport system, the effect of ATP on net transfer of water across cyanide poisoned pelvic skin of B. punctatus and B. boreas was investigated. Table 4 shows that while cyanide will prevent the permeability change, 3 mM ATP and 10 mM magnesium ion will not initiate the change in permeability. Because cyanide also inhibits other enzymes besides those involved in the production of ATP (Dixon & Webb, 1964), the effect of DNP and ATP on net TABLE~-THEEFFECTOFATPONTHENETTRANSFEROFWATERACROSSCYANIDE POISONED PELVIC SKIN Rate of net transfer of water following Skin
Experimental toad No.
1 2 3
1 2 3
specimen and ion in medium
A-Cl B-CNA-Cl B-CNA-Cl B-CN-
successive additions of AVP and ATP &l/cma per hr) Before AVP
After AVP
B. boreas 21.7 12.4 11.2 7.8 15.9 15.7
69.2 12.4 59.7 7.9 65.3 26.1
A-Cl-
2 = S.D. =
16.2 5.3
64.7 4.8
B-CN-
z = S.D. =
12.0 4.0
15.6 9.5
A-ClB-CNA-Cl B-CNA-Cl B-CN-
B. punctatus 26.3 30.6 28-8 16.3 20.8 25.2
141 61.2 207 21.4 57.1 33.9
A-CI-
Z= S.D. =
25.3 4.1
135.0 75.1
B-CN-
Z= S.D. =
24.0 7.2
38.8 20.3
After ATP
14.5 -
6.4
13.7
72.6 13.3 26.7
37.5 31.1
WATER
BALANCE
RESPONSE OF BUFO PUNCTATUS
1291
AND BUFO BOREAS
transfer across pelvic skin was determined. For both species pretreatment with DNP prevents initiation of the response (Table 5). One determination for each species also indicates that DNP applied after initiation of the response by AVP does not greatly change net transfer rates. Application of ATP and magnesium failed to initiate the response. The effect of DNP is very similar to that of cyanide.
TABLE
S-THE
EFFECT OF DNP
AND ATP ON THE RATE OF NET TRANSFER OF WATER PELVIC SKIN
ACROSS
Rate of net transfer ofwater following successive
additions of DNP, AVP and ATP (&xn* per hr) Experimental toad No.
1 2
Skin specimen
A-no DNP B-DNP A-no DNP B-DNP A B
1 2
Before AVP
3 = z =
z = d=
A B
TABLE &THB NET TRANSFER
After ATP
After DNP
-
116 -
B. boreas 18.8 14.3 30.3 23.2
123 12.2 86.7 15.8
24.5 18.7
104.8 14.0
-
91.4 10.4 89.3 9.8
-
90.3 10.1
-
B. punctatus 26.0 14.5 12.5 14.7
A-no DNP B-DNP A-no DNP B-DNP
After AVP
19.2 14.6
12.2 21.6 16.9
13.2 12.2 12.9
EFFBCT OF CYCLIC AMP ON THE RATE OF OF WATER ACROSS B.~~~ctatus PELVIC SKIN
Rate of net transfer of water
Experimental toad No.
Before application of cyclic AMP @l/cm* per hr)
After application of cyclic AMP (~~l/cn? per hr)
12.2 29.9 24.5
72.0 92.3 99.8
22.2 9.1
88.0 14.4
1 2 3 3 = SD. =
69.5 -
ROGERA. BALDWIN
1292
The application of cyclic AMP to pelvic skin of B.punctatus (Table 6) results in a significant change (PC 0.005, Student’s t-test) in net transfer rates from a mean of 22.2 to 88.0 &cm2 per hr. Pretreatment with DNP, however, prevents the increase in permeability caused by cyclic AMP (Table 7). Application of cyclic AMP to DNP poisoned pelvic skin results in a slight decrease in net transfer from a mean of 27.3 to 18.7 $/cm2 per hr. TABLE ~--THE EFFECTOF CYCLICAMP ON DNP POISONED PELVICSKINOF B. @4?ZCtdUS Rate of net transfer of water
Experimental toad No.
Before application of cyclic AMP ($/cm2 per hr)
After application of cyclic AMP &l/cm2 per hr)
24.2 31.9 25.5
17.7 19.5 18.8
27.3 4.1
18.7 0.9
1 2 3 2 = SD. =
DISCUSSION
The difference in response of pelvic and pectoral skin to AVP presented here suggests a partial explanation for the in vivo difference in net transfer rates of these areas observed by McClanahan & Baldwin (1969). Using B. punctatus dehydrated to 80 per cent of their standard weight, they observed a net transfer rate of water across pelvic skin of 423 &cm2 per hr, while net transfer across pectoral skin was in most cases not measurable. By other experiments, they found that the dorsal integument was not an important area of water uptake. Data presented in Table 1 suggest that in vitro pectoral skin of B.punctatw (and B. boreus) does not respond to AVP. Since AVP levels used in these experiments produce changes in water uptake quantitatively similar to those observed for in vivo dehydrated animals, the pectoral skin probably does not respond to native antidiuretic hormone. Since the dorsal integument is not important in water uptake, the permeability changes involved in the anuran Brunn effect are, for this species, restricted to the pelvic “patch”. Although similar in vmo data are not available for B. boreas, the existence of a pelvic “patch” in this species, as well as the unresponsiveness of the pectoral skin to AVP, suggest a similar condition. The mean net transfer rate of 208 ,u1/cm2 per hr for AVP stimulated B.punctatus pelvic skin is less than half the rate observed in vivo. This difference is probably due to a “steeper” diffusion gradient across in vivo skin. The Ringer’s solution used in these experiments had an osmolarity of 205 mOsmole/l. Dehydrated animals could be expected to have higher plasma and lymph osmolarities and thus
WATER
BALANCE
RESPONSE
OF
BUFO PVNCTATVS
AND
BVFO BOREAS
1293
a “steeper” diffusion gradient across the skin. Capillary circulation in vivo would also serve to maintain a steeper gradient. Rapid removal of water by capillaries in the dermis would confine the total diffusional path to the thinner epidermis. The in vitro skin, of course, has no capillary circulation and the total diifusional path is the entire thickness of the integument. Neurohypophysial hormones are known to enhance the permeability of a number of epithelial membranes to water. The evidence that this effect is mediated by cyclic AMP is very substantial and is reviewed by Orloff & Handler (1964, 1967). The effects of cyclic AMP on frog skin (Ranu esculenta) has been tested by Bastide & Jard (1968). Their study demonstrates that exogenous cyclic AMP, as well as theophyline, produce changes in frog skin similar to those produced by vasopressin. The concept of cyclic AMP as second messenger (Robison et al., 1971) stresses a receptor unit in conjunction with an adenyl cyclase system located in the cell membrane. Upon stimulation of the receptor by hormone, the adenyl cyclase system catalyzes the conversion of ATP to cyclic AMP and pyrophosphate. The cyclic AMP then acts as messenger for initiation of the hormonal response. Cyclic AMP is ultimately converted to 5’-AMP by the soluble enzyme phosphodiesterase. The effects presented here of cyanide, DNP, ATP and cyclic AMP on pelvic skin of both species are consistent with the cyclic AMP mechanism described by Orloff & Handler (1964, 1967) and Robison et al. (1971). The failure of cyanide and DNP to significantly change net transfer rates indicates that the movement of water across the skin is not directly linked to oxidative phosphorylation. The increase observed in net transfer following the addition of NaCN crystals (Table 3) is probably the result of an increased osmotic gradient. The inhibition of the permeability change by cyanide and DNP may be the result of lowered ATP levels. Lowered ATP levels would decrease the conversion of ATP to cyclic AMP. The failure of exogenous ATP to initiate a cyanide or DNP poisoned response may be due to the low permeability of ATP and phosphorylated compounds in general (Robison et al., 1971), as well as possible ATPase activity present in the outer surface of cell membranes. Bastide & Jard (1968), using 1 mM cyclic AMP and 10 mM theophyline, were able to produce the effects of oxytocin in skin of R. escuzenta. Bouquet (1968) has produced similar results working with the bladder of R. esculentu. Orloff & Handler (1964, 1967) were able to produce large increases in net transfer across B. marinus bladder by applying 10 mM cyclic AMP and also by applying 20 mM theophyline. The application of 10 mM cyclic AMP to B. punctatus pelvic skin produces a large increase in net transfer of water, thus implicating cyclic AMP as an intermediate in the pelvic patch’s response to AVP. According to Robison et aZ. (1971), the intracellular content of cyclic AMP in non-stimulated cells is in the order of lo-’ M, though possibly varying greatly, due to compartmentilization or protein binding. Preliminary experiments with pelvic skin from B. pmctatw indicated that 1 and 5 mM cyclic AMP produced little change in net transfer of water. The high concentration of cyclic AMP
1294
ROGERA. BALDWIN
required to initiate the permeability response is probably due to the low membrane permeability of phosphorylated compounds as well as the phosphodiesterase system, which was not inhibited in these experiments. The failure of cyclic AMP to initiate a permeability change in DNP poisoned specimens suggests blockage in the mechanism at some point beyond cyclic AMP. Since DNP would maintain lowered intracellular levels of ATP, there may exist another ATP consuming step beyond the formation of cyclic AMP. SUMMARY
Evidence
has been presented suggesting that the skin water balance response of B. punctatus is confined to the pelvic patch. A similar condition probably exists in B. boreas. Changes in pelvic patch permeability alone are able to account for about half of the in viva water uptake rate of dehydrated animals with circulatory effects and differing osmotic gradients contributing to the other half. The inhibition of the water balance response in pelvic skin by cyanide and DNP as well as the initiation of the response by cyclic AMP is consistent with the hypothesis that the response is mediated by cyclic AMP. Some evidence is also presented that suggests a second ATP requring step in the mechanism beyond the formation of cyclic AMP. Acknoruoleu’gements-This investigation was performed in the Biology Department, California State University at Fullerton. I would like to thank my committee members for their advice and suggestions during the investigation, and particularly my major advisor, Dr. Lon McClanahan, not only for his advice and suggestions but also for his help in many situations throughout my graduate career. Special thanks should go to my wife, Kay, for tolerating my Saturday absences from home and also for the major part of the typing of this paper. Tim Daly and Ray Kogut, Jr. should also be mentioned for their help in collecting toads. This research was in part supported by N.S.F. Grant Nos. GB-7684 and GB-19084. REFERENCES BASTIDEF. & JARD J. (1968) Action of noradrenalin (norepinephrine) and oxytocin on the active transport of Naf and the permeability of frog skin to water. Role of cyclic AMP. Biochim. biophys. Acta 150, 113-123. BENTLEY P. (1958) The effects of neurohypophysial extracts on water transfer across the wall of the isolated urinary bladder of the toad Bufo marinus. J. Endocr. 17, 201-212. BENTLEY P. (1963) Neurohypophysial function in amphibians, reptiles, and birds. Symp. Zool. Sot. Lond. 9, 141-152. BOURGUETJ. (1968) Kinetic analysis of the effect of oxytocin on the water permeability of frog bladder. Role of cyclic adenosine monophosphate (AMP). Biochim. biophys Acta 150,104-112. DIXON M. & WEBB E. (1964) Enzymes, 2nd Edn, p. 950. Academic Press, New York. MCCLANAHANL. (1967) Adaptations of the spadefoot toad, Scaphiopus couchi, to desert environments. Comp. Biochem. Physiol. 20, 73-99. MCCLANAHANL. & BALDWINR. (1969) Rate of water uptake through the integument of the desert toad, Bufo punctatus. Comp. Biochem. Physiol. 28, 381-389. ORLOFF J. & HANDLERJ. (1964) The cellular mode of action of antidiuretic hormone. Am. J. Med. 36, 686-697.
WATER BALANCE RESPONSE OF
BUFO PUNCTATUS
AND
BUFO
BOREAS
1295
ORLOFF J. & HANDLER J. (1967) The role of adenosine 3’,5’-phosphate in the action of antidiuretic hormone. Am. J. Med. 42, 757-768. ROBISON G.,BUTCHER R.& SUTHERLAND E.(1971) Cyclic AMP, p. 531. Academic Press, New York. SAWYER W. (1951) Effect of posterior pituitary extract on permeability of frog skin to water. Am. J, Physiol. 164, 44-48. SAWYER W. (1960) Increased water permeability of the bullfrog (Runa cutesbeiuna) bladder in vitro in response to synthetic oxytocin and arginine vasotocin and to neurohypophysial extracts from nonmammalian vertebrates. Endocrinology 66, 112-l 20. Key Word Index-Net AMP; Bnmn response.
transfer of water; pelvic “patch”;
arginine vasopressin; cyclic
1974,Vol.
47A, pp. 1285 to
1295.Pwgamon
Press. Printed in Great Britain
THE WATER BALANCE RESPONSE OF THE PELVIC “PATCH” OF BUFO PUNCTATUS AND BUFO BOREAS ROGER Department
A. BALDWIN*
of Biology, California State University,
Fullerton,
California 92631, U.S.A.
(Receiwed 15 May 1973) Abstract-l. Net transfer of water was measured in oitro across ventral pelvic and ventral pectoral integument of Bufo punctatus and B. boreas. 2. Arginine vasopressin (AVP) increased net transfer of water across ventral pelvic integument but not ventral pectoral integument. 3. Cyanide and dinitrophenol made ventral pelvic integument unresponsive to AVP. 4. Adenosine 3’,5’-monophosphate (cyclic AMP) increased net transfer of water in ventral pelvic integument of B. punctatus. 5. Pretreatment of ventral pelvic integument with dinitrophenol made the integument unresponsive to cyclic AMP. INTRODUCTION
THE PELVIC“patch” or ventral pelvic integument of Bufopumtatus has been shown to be the major site of water uptake in dehydrated toads while the ventral pectoral integument accounts for comparatively little water uptake (McClanahan & Baldwin, 1969). It has been suggested that the pelvic “patch” is an integumental area specialized for water uptake. The rate of water movement through the isolated skin of some anurans has been shown to increase following the application of neurohypophysial hormones (Sawyer, 1951, 1960; Bentley, 1958, 1963). These observations suggest that the pelvic “patch” may be more responsive to antidiuretic hormone than other integumental areas. This investigation compares the in vitro response of ventral pelvic and ventral pectoral skin of two species of toad, B. pumtatus and B. boreas. The effect of cyanide, dinitrophenol (DNP) and adenosine 3’,5’-monophosphate (cyclic AMP) on the pelvic skin response to arginine vasopressin is also investigated. MATERIALS
AND METHODS
Specimens of Bzrfo boreas were collected in Orange County, California, during the fall of 1971 and 1972. Specimens of B. punctatus were collected near Palm Desert, Riverside County, California, in early April of 1972. Toads were maintained in aquaria filled to a depth of about 6 cm with moist sand. Pieces of clay pot were placed in the aquaria to provide cover for the animals. Toads were fed Tenebrio larvae twice a week. * Present address: Neurochemistry Hospital, Sepulveda, California 91343.
Laboratory, 1285
Sepulveda Veterans Administration
1286
ROGER A. BALDWIN
The net rate of water transfer across isolated toad skin in response to an osmotic gradient was measured volumetrically. Specimens of ventral pelvic skin were taken from the “patch” Specimens of pectoral skin were area of the lower abdomen and groin of both species. Toads were first killed by a sharp blow on the back of taken from between the forelegs. the head and skin from both areas was rapidly removed and placed in separate S-6-ml aliquots of Ringer’s solution contained in lo-ml beakers. The Ringer’s solution consisted of 65 g NaCl, 0.14 g KCl, 0.12 g CaCl,, 0.20 g NaHCO, and 0.20 g glucose brought to 1 1. with distilled water. The osmolarity of the Ringer’s solution was 205 mOsmole/l. The pieces of skin were then tied tautly over the ends of slightly flared glass tubes of known cross-sectional area (about 0.2 cm”). The outer surface of the skin faced the inside of the tubes. The tubes were then rinsed twice and filled with a one to ten dilution of the Ringer’s solution. The osmolarity of the one to ten dilution of Ringer’s was 25.6 mOsmole/l. Lengths of rubber tubing about 4 cm long were used to attach the glass tubes to Hamilton microliter syringes. The syringes had the needles removed and were of 50 ~1 capacity graduated in 1 yl units. Syringes and tubing were filled with the one to ten diluted Ringer’s solution. A lOO-ml beaker containing 50 ml of normal Ringer’s solution was clamped in a The Ringer’s solution was aerated for 15-20 water-bath which was maintained at 20°C. min before the skin specimens with attached tubing and syringes were placed in it. This period of time was always sufficient for temperature equilibration. The assembled microliter syringes, glass and rubber tubing and skin specimens were suspended over the beaker of Ringer’s solution so that the skin specimens were below the surface of the Ringer’s solution, but not touching the bottom or sides of the beaker. Air from an aquarium pump was bubbled through the Ringer’s solution throughout an experiment to mix and aerate the Ringer’s solution. Evaporation from the Ringer’s solution during 1 hr of aeration changed the osmolarity from 205 to 212 mOsmole/l. An approximate tenfold difference in the concentration of water (205 vs 25.6 mOsmole/l.) existed across the skin. The net transfer of water along an osmotic gradient from the syringes through the skin and into the beaker could be read directly from the syringes. Net transfer rates were determined by timing net transfer to the nearest minute. To determine the effect of arginine vasopressin (AVP) on the net transfer rate of water, specimens of pelvic and pectoral skin were assembled onto syringes as described above After an initial period of 40-60 and immersed in the same beaker of Ringer’s solution. min, during which the net transfer rate of water across the specimens became relatively constant, 100 ~1 of a 50.7 unit/ml solution of AVP was added to the Ringer’s solution bathing the specimens. The final concentration of AVP was 0.101 units/ml. AVP was obtained from Parke-Davis as a pitressin powder prepared solely from bovine sources. The powder was dissoled in one to four hundred (1 : 400) glacial acetic acid and distilled water. Preliminary experiments indicated that 100 ~1 of 1 : 400 dilution of glacial acetic acid had no effect on net transfer rates. Following the addition of AVP, net transfer was again monitored for an additional 40-60 min. The high level of AVP used to stimulate net transfer in these experiments was chosen because animals maintained in the laboratory for varying periods of time showed great Preliminary attempts to establish a dose variability in their response to the hormone. response curve for B. punctatus pelvic skin showed that while some newly collected animals responded to 5 ~1 of the AVP solution, some animals which had been maintained in the laboratory for 3 or more months would not respond until 50 ~1 of the AVP solution had been added. Since these animals were difficult to obtain, excess hormone was used so that all animals collected could be used. The effect of this non-physiological level of hormone was investigated in vivo by subcutaneous injection of AVP. The in vitro AVP level of 0.101 units/ml was approximated by the injection of 0.101 units of AVP for each g of body water in normally hydrated toads. Total body water was calculated by assuming that 80 per cent of a normally hydrated toad’s body weight is water (see McClanahan, 1967). Following injection, animals were placed
WATER BALANCE RESPONSE OF BUFO PUNCTATUS
AND BUFO
BOREAS
1287
in tap water 1 cm deep and water uptake determined gravimetrically as described by McClanahan & Baldwin (1969). In order to study the effects of cyanide on the pelvic skin’s response to AVP, duplicate specimens of skin were taken from the pelvic area of either B. punctatus ox B. b~reusand assembled on syringes as described above. Each specimen from the same animal was piaced, however, in separate 50-ml beakers containing 25 ml of Ringer’s solution. A stock solution of 6 mM NaCN was made up in Ringer’s solution as well as a stock NaCl Ringer’s containing an additional 6 m-moles/l. of NaCl. After an initial period of 35-40 min, sufficient NaCN solution (5 ml) was added to one beaker to bring the final concentration of cyanide ion to 1 mM. An equal amount (5 ml) of the stock NaCl Ringer’s was added to the other beaker to maintain approximately equal osmotic conditions for the two skin specimens. Net transfer was monitored for 20 min, which was sufficient for the rates to become relatively constant and then 50 ~1 of the AVP solution described above was added to each beaker. Net transfer was monitored for 30-40 min following which, in some cases, 10 mg of NaCN crystals were added to the skin specimen which had not previously received cyanide. Net transfer was again monitored. To determine the effect of adenosine triphosphate (ATP) on net transfer rates in skin which had been poisoned with cyanide, duplicate skin specimens from the pelvic patch of the same animal were assembled on the syringes and immersed in separate beakers of Ringer’s solution to which the stock solutions of NaCN and NaCl Ringer’s had already been added, Following an initial period of 30-40 min, 50 ~1 of the AVP solution was added to each beaker. After about 20 min, net transfer rates had become relatively constant and 38.0 mg of ATP and 50.8 mg of MgC&,.6H,O were added to the beaker containing the cyanide poisoned specimen. The final concentration of ATP was 3 mM and the final concentration of magnesium ion was 10 mM. Following the addition of ATP and magnesium, net transfer was monitored for 20-40 min. The effect of ATP on pelvic skin poisoned with 2,4-dinitrophenol (DNP) was determined by a procedure similar to that for cyanide. To one beaker of the pair 2.3 mg of DNP was added, resulting in a O-5 mM concentration of DNP. Nothing was added to the other beaker since the increase in osmotic pressure due to this quantity of DNP was assumed insignificant. ATP and magnesium were added and net transfer monitored as in the cyanide poisoned responses. To test if cyclic AMP could mimic the effect of AVP, pelvic skin specimens from B. punctutuswere assembled on syringes as usual, except that the specimens were suspended in 10 ml of Ringer’s solution contained in a 20-ml beaker. Following an initial 40-min period of monitoring net transfer, 32.9 mg of cyclic AMP was added to the Ringer’s solution, resulting in a 10 mM concentration of cyclic AMP. Net transfer was again monitored for 40 min. The effects of DNP on the pelvic skin’s response to cyclic AMP was studied by setting up apparatus and skin specimens from B. punctutus as in the preceding experiment, except that 1 mg of DNP was added to the 10 ml of Ringer’s solution prior to placing the skin specimens in the Ringer’s The concentration of DNP was approximately 0.5 mM. Net transfer was measured for 30-min periods before and after the application of 32.9 mg of cyclic AMP. RESULTS
In both B. punctatus and B. boreas the movement of water across the pelvic skin increases following the application of AVP (Table 1). Net transfer of water across the pelvic skin of B. pumtatus increased by a factor of approximately 13 while B. boreas increased by a factor of approximately 3. These increases are statistically signi&ant at the @OOS per cent level using the Student’s t-test. In
ROGERA. BALDWIN
1288
TABLE ~----RATEOF NET TRANSPORT OF WATERACROSSINTEGUMENT OF B. punctatus AND B. boreas Rate of net transport of water &l/cm2 per hr) Experimental toad No.
Integument area
Before AVP
After AVP
B. punctatus
1 2 3 4 5 6
218 231 196 208 140 257
17.3 15.2 9.9 14.3 16.6 18.2
Pelvic Pelvic Pelvic Pelvic Pelvic Pelvic z = S.D. =
15.3 3.0 11.0 9.6 13,o 7.3 10.2 8.8
Z = 208 S.D. = 39.3 10.7 7.3 5.1 6.9 9.2 6.2
z = SD. =
10.0 1.9
2 = S.D. =
Pectoral Pectoral Pectoral Pectoral Pectoral Pectoral
7.6 2.0
B. boreas 1 2 3 4 5 6
1 2 3 4 5 6
14.8 llO*O 93.9 95,3 40.2 22.9
Pelvic Pelvic Pelvic Pelvic Pelvic Pelvic D= S.D. =
62.3 41.6 115.0 37.2 17.4 38.8 22.9 16.3
Ci!= S.D. =
41.3 37.4
Pectoral Pectoral Pectoral Pectoral Pectoral Pectoral
195 183 221 200 128 101 5 = 171.3 S.D. = 46.6 94.8 32.2 20.2 30.6 14.3 14.3 Lz= S.D. =
34.4 30.6
contrast to this the movement of water across pectoral skin of both species showed a slight decrease. The values for B. punctatus are from newly collected animals while the values for B. boreas are from animals which had been maintained in the laboratory for 3-9 months. Because of the tendency for responsiveness to AVP to
WATERBALANCERESPONSE OF EUFO PVNCTATUS ANDBUFO
BOREAS
1289
decline in the 1aborator.y the observed net transfer rates of B. pmctatus and B. boreas are not strictly comparable. The rk &JO rates of water uptake that were observed in AVFGnjected B. punctatus show a mean gain of 17 per cent of their standard weight (normally hydrated body weight without bladder water) during the first hour of hydration (Table 2). These rates are not significantly different from uptake rates observed TABLE ~-RATE OF WATERUPTAKEOF AVP INJBCTJZDB. punctatus
Experimental toad No. 1 2 3 4
weight (g)
Injection (Unita)
11.10 6.82 7.60 9.28
0.094 O*OS6 0.062 0.089
Rate of water uptake (% St. wt. gained per hr) 10.8 16-S 18.4 22.2 I = SD. =
17.0 4*8
by McClanahan & Baldwin (1969) for B. pwzctatus dehydrated to 80 per cent of their standard weight and presumably circulating native antidiuretic hormone. This suggests that the high dosage of AVP used in these experiments causes TABLE
3-
Tm KPPKCT OF CYANIDE ON NET TRANSFER OF WATERACROSS B. punctutus PELVIC SKIN Rate of net transfer of water following successive additions of cyanide or chloride, AVP and NaCN Cl-y&S
Experimental toad No. 1 2 3 4
42
Shin specimen and ion added
Q.d/cm* per hr)
A-ClB-CNA-ClB-CNA-ClB-CNA-CIB-CN-
Before ion
After ion
After AVP
After NaCN
15.5 19.4 17.4 17.4 8.5 45.4 17.0 20.8
17.7 25.8 20.6 14.2 3.3 35.2 9.9 19.8
43.2 17.3 39.9 12.5
68.2 56.0 -
95.3 36.7
125.8 -
83.4 13.0
99.7 -
65.4 28.1 19.9 11.4
83.5 28.6 -
A-CI-
d= SD. =
14.6 4.1
12.8 7.8
B-CN-
3 = S.D. =
25.7 13.2
23.8 9.0
1290
ROGER
A.
BALDWIN
changes in skin permeability to water which are quantitatively similar to those caused by physiologic levels of native antidiuretic hormone in dehydrated toads. One response of Anuran skin to AVP and other neurohypophysial hormones is the increase in permeability to water as indicated by increased net transfer rates (Sawyer, 1960; Bentley, 1963). The addition of cyanide to the Ringer’s solution prevents this response in pelvic skin from B. punctatus, while cyanide itself causes only a slight decrease in net transfer from 25.7 to 23.8 $/cm2 per hr. (Table 3). Since cyanide is a strong inhibitor of the ATP generating electron transport system, the effect of ATP on net transfer of water across cyanide poisoned pelvic skin of B. punctatus and B. boreas was investigated. Table 4 shows that while cyanide will prevent the permeability change, 3 mM ATP and 10 mM magnesium ion will not initiate the change in permeability. Because cyanide also inhibits other enzymes besides those involved in the production of ATP (Dixon & Webb, 1964), the effect of DNP and ATP on net TABLE~-THEEFFECTOFATPONTHENETTRANSFEROFWATERACROSSCYANIDE POISONED PELVIC SKIN Rate of net transfer of water following Skin
Experimental toad No.
1 2 3
1 2 3
specimen and ion in medium
A-Cl B-CNA-Cl B-CNA-Cl B-CN-
successive additions of AVP and ATP &l/cma per hr) Before AVP
After AVP
B. boreas 21.7 12.4 11.2 7.8 15.9 15.7
69.2 12.4 59.7 7.9 65.3 26.1
A-Cl-
2 = S.D. =
16.2 5.3
64.7 4.8
B-CN-
z = S.D. =
12.0 4.0
15.6 9.5
A-ClB-CNA-Cl B-CNA-Cl B-CN-
B. punctatus 26.3 30.6 28-8 16.3 20.8 25.2
141 61.2 207 21.4 57.1 33.9
A-CI-
Z= S.D. =
25.3 4.1
135.0 75.1
B-CN-
Z= S.D. =
24.0 7.2
38.8 20.3
After ATP
14.5 -
6.4
13.7
72.6 13.3 26.7
37.5 31.1
WATER
BALANCE
RESPONSE OF BUFO PUNCTATUS
1291
AND BUFO BOREAS
transfer across pelvic skin was determined. For both species pretreatment with DNP prevents initiation of the response (Table 5). One determination for each species also indicates that DNP applied after initiation of the response by AVP does not greatly change net transfer rates. Application of ATP and magnesium failed to initiate the response. The effect of DNP is very similar to that of cyanide.
TABLE
S-THE
EFFECT OF DNP
AND ATP ON THE RATE OF NET TRANSFER OF WATER PELVIC SKIN
ACROSS
Rate of net transfer ofwater following successive
additions of DNP, AVP and ATP (&xn* per hr) Experimental toad No.
1 2
Skin specimen
A-no DNP B-DNP A-no DNP B-DNP A B
1 2
Before AVP
3 = z =
z = d=
A B
TABLE &THB NET TRANSFER
After ATP
After DNP
-
116 -
B. boreas 18.8 14.3 30.3 23.2
123 12.2 86.7 15.8
24.5 18.7
104.8 14.0
-
91.4 10.4 89.3 9.8
-
90.3 10.1
-
B. punctatus 26.0 14.5 12.5 14.7
A-no DNP B-DNP A-no DNP B-DNP
After AVP
19.2 14.6
12.2 21.6 16.9
13.2 12.2 12.9
EFFBCT OF CYCLIC AMP ON THE RATE OF OF WATER ACROSS B.~~~ctatus PELVIC SKIN
Rate of net transfer of water
Experimental toad No.
Before application of cyclic AMP @l/cm* per hr)
After application of cyclic AMP (~~l/cn? per hr)
12.2 29.9 24.5
72.0 92.3 99.8
22.2 9.1
88.0 14.4
1 2 3 3 = SD. =
69.5 -
ROGERA. BALDWIN
1292
The application of cyclic AMP to pelvic skin of B.punctatus (Table 6) results in a significant change (PC 0.005, Student’s t-test) in net transfer rates from a mean of 22.2 to 88.0 &cm2 per hr. Pretreatment with DNP, however, prevents the increase in permeability caused by cyclic AMP (Table 7). Application of cyclic AMP to DNP poisoned pelvic skin results in a slight decrease in net transfer from a mean of 27.3 to 18.7 $/cm2 per hr. TABLE ~--THE EFFECTOF CYCLICAMP ON DNP POISONED PELVICSKINOF B. @4?ZCtdUS Rate of net transfer of water
Experimental toad No.
Before application of cyclic AMP ($/cm2 per hr)
After application of cyclic AMP &l/cm2 per hr)
24.2 31.9 25.5
17.7 19.5 18.8
27.3 4.1
18.7 0.9
1 2 3 2 = SD. =
DISCUSSION
The difference in response of pelvic and pectoral skin to AVP presented here suggests a partial explanation for the in vivo difference in net transfer rates of these areas observed by McClanahan & Baldwin (1969). Using B. punctatus dehydrated to 80 per cent of their standard weight, they observed a net transfer rate of water across pelvic skin of 423 &cm2 per hr, while net transfer across pectoral skin was in most cases not measurable. By other experiments, they found that the dorsal integument was not an important area of water uptake. Data presented in Table 1 suggest that in vitro pectoral skin of B.punctatw (and B. boreus) does not respond to AVP. Since AVP levels used in these experiments produce changes in water uptake quantitatively similar to those observed for in vivo dehydrated animals, the pectoral skin probably does not respond to native antidiuretic hormone. Since the dorsal integument is not important in water uptake, the permeability changes involved in the anuran Brunn effect are, for this species, restricted to the pelvic “patch”. Although similar in vmo data are not available for B. boreas, the existence of a pelvic “patch” in this species, as well as the unresponsiveness of the pectoral skin to AVP, suggest a similar condition. The mean net transfer rate of 208 ,u1/cm2 per hr for AVP stimulated B.punctatus pelvic skin is less than half the rate observed in vivo. This difference is probably due to a “steeper” diffusion gradient across in vivo skin. The Ringer’s solution used in these experiments had an osmolarity of 205 mOsmole/l. Dehydrated animals could be expected to have higher plasma and lymph osmolarities and thus
WATER
BALANCE
RESPONSE
OF
BUFO PVNCTATVS
AND
BVFO BOREAS
1293
a “steeper” diffusion gradient across the skin. Capillary circulation in vivo would also serve to maintain a steeper gradient. Rapid removal of water by capillaries in the dermis would confine the total diffusional path to the thinner epidermis. The in vitro skin, of course, has no capillary circulation and the total diifusional path is the entire thickness of the integument. Neurohypophysial hormones are known to enhance the permeability of a number of epithelial membranes to water. The evidence that this effect is mediated by cyclic AMP is very substantial and is reviewed by Orloff & Handler (1964, 1967). The effects of cyclic AMP on frog skin (Ranu esculenta) has been tested by Bastide & Jard (1968). Their study demonstrates that exogenous cyclic AMP, as well as theophyline, produce changes in frog skin similar to those produced by vasopressin. The concept of cyclic AMP as second messenger (Robison et al., 1971) stresses a receptor unit in conjunction with an adenyl cyclase system located in the cell membrane. Upon stimulation of the receptor by hormone, the adenyl cyclase system catalyzes the conversion of ATP to cyclic AMP and pyrophosphate. The cyclic AMP then acts as messenger for initiation of the hormonal response. Cyclic AMP is ultimately converted to 5’-AMP by the soluble enzyme phosphodiesterase. The effects presented here of cyanide, DNP, ATP and cyclic AMP on pelvic skin of both species are consistent with the cyclic AMP mechanism described by Orloff & Handler (1964, 1967) and Robison et al. (1971). The failure of cyanide and DNP to significantly change net transfer rates indicates that the movement of water across the skin is not directly linked to oxidative phosphorylation. The increase observed in net transfer following the addition of NaCN crystals (Table 3) is probably the result of an increased osmotic gradient. The inhibition of the permeability change by cyanide and DNP may be the result of lowered ATP levels. Lowered ATP levels would decrease the conversion of ATP to cyclic AMP. The failure of exogenous ATP to initiate a cyanide or DNP poisoned response may be due to the low permeability of ATP and phosphorylated compounds in general (Robison et al., 1971), as well as possible ATPase activity present in the outer surface of cell membranes. Bastide & Jard (1968), using 1 mM cyclic AMP and 10 mM theophyline, were able to produce the effects of oxytocin in skin of R. escuzenta. Bouquet (1968) has produced similar results working with the bladder of R. esculentu. Orloff & Handler (1964, 1967) were able to produce large increases in net transfer across B. marinus bladder by applying 10 mM cyclic AMP and also by applying 20 mM theophyline. The application of 10 mM cyclic AMP to B. punctatus pelvic skin produces a large increase in net transfer of water, thus implicating cyclic AMP as an intermediate in the pelvic patch’s response to AVP. According to Robison et aZ. (1971), the intracellular content of cyclic AMP in non-stimulated cells is in the order of lo-’ M, though possibly varying greatly, due to compartmentilization or protein binding. Preliminary experiments with pelvic skin from B. pmctatw indicated that 1 and 5 mM cyclic AMP produced little change in net transfer of water. The high concentration of cyclic AMP
1294
ROGERA. BALDWIN
required to initiate the permeability response is probably due to the low membrane permeability of phosphorylated compounds as well as the phosphodiesterase system, which was not inhibited in these experiments. The failure of cyclic AMP to initiate a permeability change in DNP poisoned specimens suggests blockage in the mechanism at some point beyond cyclic AMP. Since DNP would maintain lowered intracellular levels of ATP, there may exist another ATP consuming step beyond the formation of cyclic AMP. SUMMARY
Evidence
has been presented suggesting that the skin water balance response of B. punctatus is confined to the pelvic patch. A similar condition probably exists in B. boreas. Changes in pelvic patch permeability alone are able to account for about half of the in viva water uptake rate of dehydrated animals with circulatory effects and differing osmotic gradients contributing to the other half. The inhibition of the water balance response in pelvic skin by cyanide and DNP as well as the initiation of the response by cyclic AMP is consistent with the hypothesis that the response is mediated by cyclic AMP. Some evidence is also presented that suggests a second ATP requring step in the mechanism beyond the formation of cyclic AMP. Acknoruoleu’gements-This investigation was performed in the Biology Department, California State University at Fullerton. I would like to thank my committee members for their advice and suggestions during the investigation, and particularly my major advisor, Dr. Lon McClanahan, not only for his advice and suggestions but also for his help in many situations throughout my graduate career. Special thanks should go to my wife, Kay, for tolerating my Saturday absences from home and also for the major part of the typing of this paper. Tim Daly and Ray Kogut, Jr. should also be mentioned for their help in collecting toads. This research was in part supported by N.S.F. Grant Nos. GB-7684 and GB-19084. REFERENCES BASTIDEF. & JARD J. (1968) Action of noradrenalin (norepinephrine) and oxytocin on the active transport of Naf and the permeability of frog skin to water. Role of cyclic AMP. Biochim. biophys. Acta 150, 113-123. BENTLEY P. (1958) The effects of neurohypophysial extracts on water transfer across the wall of the isolated urinary bladder of the toad Bufo marinus. J. Endocr. 17, 201-212. BENTLEY P. (1963) Neurohypophysial function in amphibians, reptiles, and birds. Symp. Zool. Sot. Lond. 9, 141-152. BOURGUETJ. (1968) Kinetic analysis of the effect of oxytocin on the water permeability of frog bladder. Role of cyclic adenosine monophosphate (AMP). Biochim. biophys Acta 150,104-112. DIXON M. & WEBB E. (1964) Enzymes, 2nd Edn, p. 950. Academic Press, New York. MCCLANAHANL. (1967) Adaptations of the spadefoot toad, Scaphiopus couchi, to desert environments. Comp. Biochem. Physiol. 20, 73-99. MCCLANAHANL. & BALDWINR. (1969) Rate of water uptake through the integument of the desert toad, Bufo punctatus. Comp. Biochem. Physiol. 28, 381-389. ORLOFF J. & HANDLERJ. (1964) The cellular mode of action of antidiuretic hormone. Am. J. Med. 36, 686-697.
WATER BALANCE RESPONSE OF
BUFO PUNCTATUS
AND
BUFO
BOREAS
1295
ORLOFF J. & HANDLER J. (1967) The role of adenosine 3’,5’-phosphate in the action of antidiuretic hormone. Am. J. Med. 42, 757-768. ROBISON G.,BUTCHER R.& SUTHERLAND E.(1971) Cyclic AMP, p. 531. Academic Press, New York. SAWYER W. (1951) Effect of posterior pituitary extract on permeability of frog skin to water. Am. J, Physiol. 164, 44-48. SAWYER W. (1960) Increased water permeability of the bullfrog (Runa cutesbeiuna) bladder in vitro in response to synthetic oxytocin and arginine vasotocin and to neurohypophysial extracts from nonmammalian vertebrates. Endocrinology 66, 112-l 20. Key Word Index-Net AMP; Bnmn response.
transfer of water; pelvic “patch”;
arginine vasopressin; cyclic