Effects of Antidiuretic Hormone on Water Absorption in the American Tree Frog,
Hyla cinerea
Poupak Fakhrai and Julanar Selman
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
Terrestrial anurans absorb water through their pelvic region, which is affected
by the antidiuretic hormone, arginine vasotocin (AVT). It was predicted that AVT
would increase the rate of absorption in frogs subject to an isotonic medium and
decrease depletion in a hypertonic solution. Prior to measurements, frogs were
dehydrated to 90% of initial weight and bladders emptied. Control frogs were
injected with 0.1 mL/g bodyweight of amphibian Ringer’s and treatment condition
were given 0.01025 μg AVT per 0.1 mL administered. Frogs were placed into petri
dishes with hypertonic (425 mOsm) Ringer’s solution, taken out at 10 minute
intervals, blotted dry and weighed for 30 minutes. Thereafter, dishes were filled
with isotonic (204 mOsm) Ringer’s, and measured for an additional 30 minutes.
Control frogs lost water to the hypertonic medium, at a mean rate of 9.9 μg/minute
(n=6), while AVT treated frogs gained water at a mean rate of 0.5 μg/minute (n=6).
These results were significant (p= 0.040 one-tailed paired t-test). In isotonic solution,
treatment frogs gained water at a significantly faster rate, 9.7μg /minute than did
control, 6.3 μg/minute (p= 0.035 one-tailed paired t-test). These data suggest that
AVT regulates rate of water absorption and depletion.
Introduction
All animals function as aqueous systems with water as the primarily channel for
biochemical pathways. Amphibians adapted to land face particularly demanding osmotic
challenges, loosing many times more water than a comparatively sized reptile. Terrestrial
amphibians from order Anura have evolved a number of physiological adaptations to help
combat water loss and limited water availability. One such adaptation is the storage of
water as dilute urine, McClanahan et al., (1994) found many species to deposit as much
as 25% of their bodyweight in their urinary bladder. Hence, when faced with limited
hydration sources, terrestrial anurans prolong their activity by reabsorbing water from
their bladders (Hillyard et al., 1998).
Furthermore, terrestrial and arboreal species do not typically drink water but rather
maintain osmotic balance by soaking up water through their specialized pelvic region
labeled as their “seat patch”. The skin on this ventral surface is thinner and highly
vascularized, generating more blood flow when in contact with a hydration source
(Viborg and Hillyard, 2005). A supporting network of two different categories of
aquaporin’s has also been identified in the pelvic region (Ogushi et al., 2010). These
membrane proteins act as water channels and facilitate rapid water absorption into cells.
Arginine vasotocin (AVT), the amphibian antidiuretic hormone has been shown to
regulate these aquaporin’s by translocation from the cytoplasm to the outermost region of
the plasma membrane (Hasegawa et al., 2003). AVT is released by the pituitary gland in
response to dehydration and serves to decrease water loss by urine and alters the water
potential of the frog, promoting absorption (Tracy, 1976).
The present study aimed to examine the suggested nature of AVT and its mechanism
of decreasing water potential in tree frogs, thereby aiding in water absorption. It was
hypothesized that AVT would increase the rate of water absorption in an isotonic
medium and decrease the rate of water depletion in hypertonic solution of dehydrated tree
frogs.
Materials and Methods
Six tree frogs of species Hyla cinerea were purchased from the Reptile Zoo in
Fountain Valley, CA. They were initially housed in a ten-gallon aquarium filled with
branches and greenery to simulate their natural habitat. Temperature was maintained at
27 °C during the day and 21°C at night. Bottled mineral water was provided and
aquarium was misted daily, as well as live crickets administered three times a week.
During the experiments each frog was placed in separate holding tanks and numbered
accordingly. All experiments were carried out in the laboratory at room temperature 24 ±
2°C. To minimize individual variances in assessments, each frog served as its own
control, and experiments were carried out on alternate days with approximately 48 hours
of rest in between. All measurements were taken after frogs were dehydrated to 90% of
their standard weight; namely a fully hydrated frog with an empty urinary bladder.
Forceps were inserted into frog’s cloaca and urinary bladder emptied by applying light
pressure. Standard weight was recorded and frogs were placed in ventilated areas of the
laboratory and allowed to dehydrate for approximately 1 hour.
An isotonic (204 mOsm) amphibian Ringer’s solution was prepared (Wright, 2006)
and amount administered was measured to correspond to dosage of AVT. At 90% of
standard weight, frogs were injected with 0.1 mL/g of bodyweight of Ringer’s solution
into their dorsal lymph sacs, and placed into individual petri dishes filled with 14 mL of a
hypertonic (425 mOsm) Ringer’s solution. Frogs were contained within the hypertonic
medium and taken out at 10 minute intervals, blotted dry and weighed for a total of 30
minutes. Thereafter, petri dishes were filled with 14 mL of the isotonic Ringer’s solution
and frogs were measured for an additional thirty minutes according to the aforementioned
procedure. Following the control, frogs were given 48 hours of rest before carrying out
the treatment portion of experiment. To examine the effect of antidiuretic hormone on
water absorption, above-mentioned procedures were repeated with injections of arginine
vasotocin. A solution was prepared using isotonic Ringer’s, such that each frog was
injected with 0.1 mL/g of body weight, containing 0.01025 μg AVT per 0.1 mL
administered. All data collected was transferred to MS Excel (Microsoft Corporation,
Redmond, Washington) where statistical analysis was completed.
Results
Control frogs were found to lose water in hypertonic medium, at a mean rate of 9.9 ±
0.18 μg/minute (± SEM n=6). Frogs treated with AVT gained water against the osmotic
gradient, at a mean rate of 0.5 ± 0.13 μg/minute (± SEM n=6). The difference between
the two conditions was found to be significant (p= 0.040 one-tailed paired t-test) and is
presented in figure 1 and figure 2. In isotonic solution, control frogs gained water at a rate
of 6.3 ± 0.8 μg/minute (± SEM n=6), while treated frogs absorbed water at a rate of 9.7 ±
0.8 μg/minute (± SEM n=6). These results are shown in figure 3 and figure 4. AVT
injected frogs absorbed water at a significantly higher rate (p= 0.035 one-tailed paired ttest).
4.2
FrogWeight (gram)
4.1
4
3.9
3.8
3.7
Control
3.6
AVT
3.5
3.4
3.3
0
10
20
Time (minutes)
30
Figure 1. Water gained or lost by mean weight of frogs in hypertonic (425 mOsm)
amphibian Ringer’s solution (n=6). AVT injected frogs displayed a net gain of water
compared to control frogs that lost water (p= 0.040 one-tailed paired t-test). Error bars are
mean ± SEM.
Frog Weight (%)
94
92
90
88
Control
86
AVT
84
82
0
10
20
Time (minutes)
30
Figure 2. Water gained or lost by mean percentage of initial body weight of frogs in
hypertonic (425 mOsm) amphibian Ringer’s solution (n=6). AVT injection resulted in a
net gain in percent body weight (p= 0.0059 one-tailed paired t-test). Error bars are mean
± SEM.
4.3
Frog Weight (gram)
4.2
4.1
4
3.9
3.8
Control
3.7
AVT
3.6
3.5
3.4
3.3
0
10
20
Time (minutes)
30
Figure 3. Water absorption by mean weight of frogs in isotonic (204 mOsm) amphibian
Ringer’s solution (n=6). AVT injected frogs gained water at a faster rate than did control
frogs (p= 0.035 one-tailed paired t-test). Error bars are mean ± SEM.
100
98
Frog Weight (%)
96
94
92
90
Control
88
AVT
86
84
82
80
0
10
20
Time (minutes)
30
Figure 4. Water gained by mean percentage of initial body weight of frogs in isotonic
(204 mOsm) amphibian Ringer’s solution (n=6). Treated frogs absorbed water at a faster
rate than did control frogs (p= 0.018 one-tailed paired t-test). Error bars are mean ± SEM.
Discussion
Terrestrial anuras face particularly demanding challenges on land in regards to
desiccation, often roaming from aquatic to terrestrial habitats. As a result, various
adaptations have evolved to ensure their continuous success. Among these is the dynamic
skin on their seat patch with supporting aquaporin’s, with its permeability being modified
depending on the substrate suitability. The permeability of their seat patch and osmotic
balance has been shown to be regulated by the neurohypophyseal hormone, arginine
vasopressin (Duellman and Trueb, 1994). AVT is believed to merge vesicles holding
aquaporin’s with membranes of water absorbing tissues (Hasegawa et al., 2003).
Additionally, AVT is assumed to increase cutaneous blood flow in the pelvic region,
facilitating rapid absorption (Malvin, 1993). Moreover, the response to the mechanism of
AVT seems to correspond to the species habitat and has been used to determine
phylogenetic relationships (Wells, 2007).
As an arboreal species, Hyla cincerea was predicted to respond to the action of AVT
and decrease rate of dehydration in hypertonic solution. Results indicated not only an
ability to withstand water loss but a net gain of water despite the osmotic gradient. When
subject to the hypertonic medium, an initial rise in hydration levels cushioned against
further depletion, resulting in a net gain of water. It is likely that this was made possible
by relocating dilute fluids and ions to lower the osmotic potential of their seat patch,
promoting water absorption. As the capacity of doing so was reached, water loss was
imminent and increased over time. Nevertheless, the initial absorption buffered against a
large fall in hydration levels. Desert toads have been known to utilize this strategy
frequently by retaining urea from their stored urine (Cooke, 2004).
In isotonic solution, AVT injection resulted in a faster rate of absorption compared to
control. This is likely due to faster transportation of water into tissues by the fusion of
aquaporin’s to their membranes.
Acknowledgments
We would like to thank professor Teh for his support and guidance throughout this
experiment and also express our sincere gratitude to Saddleback’s Department of Biology
for provision of arginine vasopressin obtained from Sigma-Aldrich Co. LLC.
Literature Cited
Bentley, J. Peter. 2002. Endocrines and Osmoregulation: A Comparative Account in
Vertebrates. Springer-Verlag New York, LLC.
Brekke, R. Dona. Hillyard, D. Stanley and Winokur, M. Robert. 1991. Behavior
Associated with the Water Absorption Response by the Toad, Bufo punctatus. Copeia:
American Society of Ichthyologists and Herpetologists. Vol 2: p 393-401.
Cooke, Fred. 2004. The Encyclopedia of Animals: A complete Visual Guide. University
of California Press.
Duellman, E. William and Trueb, Linda. 1994. Biology of Amphibians. John Hopkins
University Press.
Duncan, RL. Watlington, CO. Biber, TU and Huf, EG. 1985. Sodium Transport and
Distribution of Electrolytes in Frog Skin. Physiological Chemistry and Physics. Vol 17
(2): p 155-172.
Hasegawa, Takahiro. Tsnii, Haruna. Suzuki, Masakazu. Tanaka, Shigeyasu. 2003.
Regulation of Water Absorption in the Frog Skins by Two Vasotocin-Dependent WaterChannel Aquaporins, AQP-h2 and AQP-h3. Endocrinology. Vol 144 (9): p 4087-4096.
Hillyard, D. Stanley. Von Seckendorff, Karin and Propper, Catherine. 1998. The Water
Absorption Response: A Behavioral Assay for Physiological Processes in Terrestrial
Amphibians. Physiological Zoology. Vol 71 (2): p 127-138.
Maejima, Sho. Yamada, Toshiki. Hamada, Takayuki. Matsuda, Kouhei. Uchiyama,
Minoru. 2008. Effects of Hypertonic Stimuli and Arginine Vasotocin (AVT) on Water
Absorption Response in Japanese Tree Frog, Hyla japonica. General and Comparative
Endocrinology. Vol 157: p 196-202.
Malvin, G.M. 1993. Vascular Effects of Arginine Vasotocin in Toad Skin. American
Journal of Physiology. Vol 265 (2): p 426-432.
McClanahan, L. Lon. Ruibal, Rodolfo. Shoemaker, H. Vaughan. 1994. Frogs and Toads
in Deserts. Scientific American. Vol 270 (3): p 82-88.
Ogushi, Y. Kitagawa, D. Hasegawa, T. Suzuki, M and Tanaka, S. 2010. Correlation
between Aquaporin and Water Permeability in Response to Vasotocin, Hydrin and βadrenergic Effectors in the Ventral Pelvic Skin of the Tree Frog Hyla japonica. Journal
of Experimental Biology. Vol 213: p 288-294.
Rouille, Y. Michael, G. Chauvet, M. T. Chauvet, J. Acher, R. 1989. Hydrins,
Hydroosmotic Neurohypophysial Peptides: Osmoregulatory Adaptation in Amphibians
through Vasotocin Precursor Processing. Proceedings of the National Academy of
Sciences of the United States of America/Biochemistry. Vol 86: p 5272-5275.
Sullivan, A. Polly. Von Seckendorff, Karin and Hillyard, D. Stanley. 2000. Effects of
Anion Substitution on Hydration Behavior and Water Uptake of the Red-spotted Toad,
Bufo punctatus: is there an Anion Paradox in Amphibian Skin? Oxford Journal of
Chemical Senses. Vol 25: p 167-172.
Tracy, Richard. 1976. A Model of the Dynamic Exchanges of Water and Energy between
a Terrestrial Amphibian and Its Environment. Ecological Society of America. Vol 46 (3):
p 293-326.
Tracy, Richard and Rubink, L. William. 1978. The Role of Dehydration and Antidiuretic
Hormone on Water Exchange in Rana Pipens. Comparative Biochemistry and
Physiology. Vol 61 (4): p 559-562.
Viborg, L.A and Hillyard, D. S. 2005. Cutaneous Blood Flow and Water Absorption by
Dehydrated Toads. Physiological and Biochemical Zoology: Ecological and
Evolutionary Approaches. Vol 78 (3): p 394-404.
Wells, D. Kentwood. 2007. The Ecology and Behavior of Amphibians. University of
Chicago Press.
Wright K. 2006. Important clinical aspects of amphibian physiology. Proceedings of the
North American Veterinary Conference.Vol. 20. Orlando, Florida.