|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online February 15, 2006
Journal of Experimental Biology 209, 834-844 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02057
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Cardiovascular and behavioural changes during water absorption in toads, Bufo alvarius and Bufo marinus
1 Zoophysiological Laboratory, August Krogh Institute, University of
Copenhagen, Denmark
2 Department of Zoophysiology, University of Aarhus, Denmark
3 School of Dental Medicine and Department of Biological Sciences,
University of Nevada, Las Vegas, USA
* Author for correspondence (e-mail: alviborg{at}aki.ku.dk)
Accepted 21 December 2005
 |
Summary |
|---|
 TOP Summary IntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
|---|
Key words: toad, Bufo marinus, Bufo alvarius, water absorption, blood flow
|
Introduction |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
|---|
; Christensen,
1974). Using non-invasive Laser Doppler Flowcytometry, a rapid
rise in seat patch blood cell flux (BCF) associated with increased water
absorption was demonstrated (Viborg and
Rosenkilde, 2004) when previously dehydrated toads (Bufo
bufo) were given access to water. In contrast, hydrated toads injected
with arginine vasotocin absorbed water faster without increasing BCF. BCF and
water absorption by dehydrated Bufo woodhouseii and Bufo
punctatus were higher than in hydrated toads, but the two were not
correlated (Viborg and Hillyard,
2005). Thus, the relationship between BCF and water absorption
remains unclear. B. punctatus and B. woodhouseii inhabit
dryer environments than B. bufo, and some of the differences in water
absorption may relate to the more extensive vascularization in the ventral
skin of arid-adapted toads (Roth,
1973). Specifically, B. alvarius that inhabit deserts of
the south western USA and northern Mexico have a denser array of blood vessels
than the more mesic B. marinus. This provided the opportunity to
determine whether BCF is correspondingly larger in B. alvarius vs B.
marinus and if it results in a greater rate of water absorption by
dehydrated toads.
Despite the reduced osmotic gradient, dehydrated toads, B.
punctatus and B. marinus rehydrate faster when immersed in 50
mmol l–1 NaCl than in deionised water
(Sullivan et al., 2000;
Hillyard and Larsen, 2001).
Hillyard and Larsen suggested that increased blood flow to the seat patch is
responsible for the elevated water uptake during immersion in 50 mmol
l–1 NaCl (Hillyard and
Larsen, 2001). We tested this hypothesis using a chamber in which
BCF and water absorption can be measured simultaneously
(Viborg and Hillyard, 2005).
This chamber also allowed us to evaluate the effect of different immersion
levels on BCF and water absorption from deionised water (DI) and salt
solutions. Because the toads were conscious and unrestrained, we were able to
observe whether the time course for behaviours associated with water
absorption (Hillyard et al.,
1998) corresponded to the physiological response to water contact
(increase in BCF).
The increase in BCF was characterized as a reflex stimulated by water
potential receptors in the skin (Viborg
and Rosenkilde, 2004). The degree to which internal factors might
affect this reflex was investigated
(Viborg and Hillyard, 2005),
and it was shown that hydrated toads (B. punctatus) whose bladders
had been emptied had a significantly greater BCF than toads that were allowed
to retain bladder water. However, B. punctatus is a small toad and
the process of emptying the bladder resulted in mild dehydration, so the
effect of bladder content alone was not established. Bladders of larger toads
used in the present study could be emptied without appreciable dehydration.
This permitted a more conclusive demonstration that the reflexive increase in
BCF is sensitive to the presence or absence of stored bladder water.
A rise in BCF requires diversion of blood from other organs or a rise in
cardiac output. Earlier studies with anesthetized anurans have used
measurement of central arterial blood flow (CAF) to infer levels of BCF
(Parsons and Schwartz, 1991).
In some cases, the heart was stopped by direct injection of MS222, so BCF was
abruptly stopped. Here we measure the relationship between CAF and BCF in
conscious B. marinus that were outfitted with flow probes placed
around the dorsal aorta to determine whether changes in CAF and heart rate
(fH) correlate with the increase in BCF as dehydrated
toads were presented with a hydration surface.
|
Materials and methods |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
|---|
; Jorgensen,
1994). Hydration status was evaluated relative to the standard
weight (Ruibal, 1962), which
is the weight of a fully hydrated toad with an empty urinary bladder.
Measurements of skin blood flow
BCF was measured using the technique described
(Viborg and Hillyard, 2005),
with a Periflux PF 2B 2 mW He-Ne laser (Perimed, Sweden) connected to a PF 313
Integrating Probe (Perimed AB, Sweden) that is designed for measurement of
skin blood flow (Salerud and Nilsson,
1986). BCF is linearly related to the product of the number of
blood cells and their average velocity in the explored volume of tissue. The
measurements can not be calibrated to absolute values, but can be expressed in
relative terms as voltage (V), and have frequently been used to assess
microvascular flow, including skin blood flow (Perimed Literature Reference
list;
http://www.lisca.se/).
During recording of seat patch skin BCF, toads were placed individually in a
Lucite chamber measuring 12.5 cmx16 cmx6.5 cm containing a water
reservoir at the bottom to allow the seat patch region of the skin to have
direct contact with water. A port in the centre of the water reservoir held
the laser Doppler probe, so BCF could be recorded prior to (dry) and after
(wet) water was added to the reservoir. The diagrams showing the increase in
BCF in dehydrated toads (Figs
1B,
2B) were constructed by
averaging BCF over 20 s (2000 points) intervals every 1 or 2 min. Data points
10 s prior to and after the minute were averaged to give mean BCF values for
each interval.
|
|
Water absorption behaviour
When given access to a wet surface, dehydrated toads will abduct the hind
limbs and press the seat patch towards the surface. This behaviour is termed
the `water absorption response' (WR)
(Stille, 1958;
Hillyard et al., 1998). In
addition to WR, toads will display a series of moves and body oscillations to
reposition the seat patch on the hydration surface
(Brekke et al., 1991). The
experimental chamber allowed observation of the WR and related behaviours that
could be quantified with respect to the increase in BCF.
Experimental protocol
BCF, water absorption and WR behaviour on deionised water vs 50 mmol l–1 NaCl
The urinary bladders of toads obtained from the maintenance terraria were
emptied by inserting a polyethylene cannula into the cloaca combined with
gentle abdominal pressure. The resulting standard weights were recorded and
the toads placed overnight in a dry terrarium to obtain a level of dehydration
(12–20% relative to the standard weight) that is sufficient to
consistently stimulate the toads to initiate water absorption behaviour
(Maleek et al., 1999;
Nagai et al., 1999). Following
dehydration, BCF and water uptake read from the pipette were measured for 40
min with the reservoir containing either deionised water (DI) or 50 mmol
l–1 NaCl (NaCl). At the end of the trials the toads were
blotted on paper tissue and weighed so that rates of water uptake measured by
the pipette could be compared with the gravimetrically measured increase.
In this and subsequent experiments, we observed that differing levels of
immersion affect rehydration rates from water vs dilute NaCl
solutions. Because factors such as barometric pressure
(Hoff and Hillyard, 1993)
affect hydration behaviour, we elected to serially evaluate different levels
of immersion on a given day to keep conditions as similar as possible. To do
this, toads that had completed a rehydration period at one level of immersion
were placed for 2 h in a dry terrarium with a fan to circulate air over the
animal. This procedure, which we term a `dehydration interval', produced a
level of dehydration that was comparable to that following the overnight
dehydration used for the initial measurements of BCF and water absorption,
i.e. toads lost the water that had been absorbed during the previous
trial.
After the initial dehydration interval, the toads were placed in a 2 l glass beaker holding 200 ml of either DI or NaCl. This produced a level of immersion that increased the skin area available for water absorption relative to that of toads absorbing water from the chamber reservoir, across the seat patch. Water gain was determined gravimetrically after immersion for 20 min. We termed this `full immersion', for comparison with toads placed in the chamber with increased water levels but not enough to allow the toads to float freely.
Except where noted, three trials were performed with each of the five toads on DI and with NaCl, giving a total of 15 trials for each group. For repetitive trials the toads were allowed to recover for a week between dehydrations.
Effects of immersion level on BCF, WR behaviour and rates of water uptake
Toads were again dehydrated overnight and SW recorded. For these
experiments, the chamber was initially filled to a depth of 1.5 mm with either
DI or 50 mmol l–1 NaCl. WR behaviour and BCF were monitored
in the chamber for 20 min. Then the toads were carefully blotted in paper
tissue and body weight were recorded for gravimetric determination of water
gain. After a dehydration interval, body weights were recorded and the toads
were again placed individually in the chamber that was now filled to a depth
of 6 mm with either DI or NaCl. BCF and WR behaviour were monitored for 20
min, the toads were blotted in paper tissue and body weights were again
recorded for gravimetric measurement of water gain. Finally, a second
dehydration interval was observed and the toads were fully immersed for 20 min
in 200 ml DI or NaCl in a 2 l glass beaker, for comparison with the first set
of experiments. As before, water absorption was measured gravimetrically.
Regional water absorption from DI vs NaCl
Only B. marinus were available for the third set of experiments.
Toads were dehydrated overnight as previously described and water absorption
was measured gravimetrically following immersion for 15 min in either 200 ml
DI or NaCl in a 2 l glass beaker. After a dehydration interval this procedure
was repeated to establish a baseline level for water absorption from DI and
NaCl. A second dehydration interval was then observed and one of two separate
procedures were conducted in which water absorption was measured in toads
having defined areas of the skin covered with a mixture of beeswax dissolved
in vegetable oil (0.14 g ml–1). In the first procedure, the
ventral and lateral skin was covered from a point 2 cm caudal from the
forelimbs to the posterior margins of the thighs at a level just below the
cloaca. This area includes the seat patch, where most water absorption is
believed to occur (Christensen,
1974). Rehydrating toads are also known to draw water from the
ventral to the dorsal surface of the skin by way of capillarity in grooves and
channels that Lillywhite and Licht referred to as epidermal sculpturing
(Lillywhite and Licht, 1974).
In the second procedure, the skin on the lateral sides, including the anterior
part of the thigh, was covered by the mixture to prevent this transfer but
keep the ventral skin available for absorption. After applying the mixture,
toads from both treatment groups were immersed for third and fourth 15 min
periods separated by a dehydration interval. The toads were allowed to moult
at least once between trials to ensure that all beeswax and vegetable oil had
disappeared.
Effects of bladder water on BCF
Body weights of B. alvarius obtained from their maintenance
terraria were recorded, prior to removing bladder urine. The volume of bladder
urine voluntarily retained by toads has previously been termed `ad
libitum bladder urine' (Tran et al.,
1992). The toads were placed individually in the chamber and BCF
was recorded for 6 min with no water in the reservoir (dry), and then for 6
min after the addition of deionised water to the reservoir (wet; hydrated
toads, ad libitum bladder). The urinary bladders were emptied,
standard weights (SW) were recorded and the toads were left in dry terraria
(40 cmx40 cmx40 cm) for 10 min. This provided a standardized
interval following the stress of handling so that toads could be positioned in
the chamber. The bladder volumes for each toad were assumed to be the
difference between the initial weight and the SW. Because B. marinus
spontaneously voided their bladders when handled, the initial weighing was the
SW. BCF for both species (hydrated, empty bladder toads) was measured for 6
min before and after the addition of water to the reservoir. The toads were
then transferred to the dry terraria for 3–4 h with a ventilating fan,
to induce a state of mild dehydration that may correspond to brief activity
periods in the field (Stille,
1952). Body weights were recorded and BCF was again measured for 6
min before and after the addition of deionised water to the reservoir (mild
dehydration). The toads were then transferred to the dry terraria and left
without access to water overnight to produce a greater degree of dehydration.
The following day, body weights were recorded and the percent dehydration
calculated. Then BCF was measured for 6 min with empty reservoir, and for 40
min with deionised water added to the reservoir. For repetitive experiments,
the toads were allowed to recover for 1 week between dehydrations.
Central arterial flow and BCF
Central arterial blood flow (CAF) and BCF were measured simultaneously in
five B. marinus (body mass 253–371 g). One or two trials were
performed for each toad, resulting in a total of 9 measurements. Prior to
surgery, the toads were anesthetized by immersion in a 2
solution of
benzocaine, until the corneal reflex disappeared. During surgery, the toads
were placed on water-saturated paper tissue. A 3 cm incision was made on the
dorsal side approximately 1 cm lateral to the vertebrae in regio lumbalis. The
muscles were gently separated to access the abdominal cavity and expose aorta
abdominalis. A Transonic blood flow probe (Transomic Systems Inc., Ithaca, NY,
USA) was placed around the aorta abdominalis and two holding sutures in the
muscles secured the probe. The incision was closed in two layers (muscles and
skin) by nylon sutures. The toads were allowed to recover for 2 full days in a
plastic cage (50 cmx35 cmx25 cm) containing 2 cm of tapwater
(Andersen and Wang, 2002). One
day before the experiment, the urinary bladder was emptied, SW was recorded
and the toad was placed in a dry plastic cage for dehydration. The next day,
body weight was recorded and toads were transferred individually to the
chamber after filling the reservoir with deionised water. CAF and BCF were
measured for 40 min with water in the reservoir.
In a separate experiment handling was avoided and only CAF was measured in four B. marinus (210–434 g). After emptying the urinary bladder and overnight dehydration, water was added gently to the dehydration chamber through a silicon tube in order to disturb the toads as little as possible, and CAF was measured over a 40 min period.
Analysis of data
A serial protocol with paired trials was used for the experiments. The
serial design was less time consuming and allowed trials for an individual
toad to be completed within a day. This served to reduce effects of
environmental factors like barometric pressure, which have been shown to
affect rehydration behaviour (Hoff and
Hillyard, 1993). Paired trials were chosen to minimize effects of
individual variation, as both rates of water absorption and BCF may vary
considerably between individual animals.
Two-way analysis of variance (ANOVA) applied to the randomized block design
was used to compare treatment groups where toads served as a blocking factor
with 3 replications for each toad. In order to check the model assumptions of
normally distributed residuals and subsequently apply ANOVA, the
Kolmogorov–Smirnov test was performed on the model residuals. An ANOVA
was used to compare BCF between species and the 
2-test was
used to compare the occurrence of oscillations in B. alvarius on DI
or NaCl. The linear regressions take account for variation due to differences
between individual toads.
Averages were calculated for each individual toad, an average representing all toads was then calculated and s.e.m. determined from this average with N=number of toads.
|
Results |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
|---|
BCF, WR behaviour and water absorption on NaCl vs DI
The temporal change of BCF and its magnitude was not affected by 50 mmol
l–1 NaCl compared to DI for either species; however, BCF
remained higher for B. alvarius compared to B. marinus.
Although B. marinus gradually adopted the WR posture rather than
abducting the hind limbs in a distinct movement, full abduction of the
hindlimbs generally occurred after maximal BCF had been achieved. B.
alvarius also settled into the WR posture well after maximal BCF had been
attained (mean settling time on DI was 10.5 min and on NaCl it was 10.6 min.
The time and the BCF when WR was initiated were not affected by NaCl. However,
the number of moves during the first 40 min was greater on NaCl than on DI
(Table 1). Further,
oscillations of the body following a move, when skin contact with the
reservoir was re-established, were observed in 9 of 12 trials on NaCl but in
only 2 of 15 trials on DI (P<0.01, N=27,
2-test). The lower number of trials on NaCl was due to one
toad, which consistently rejected water uptake from NaCl by leaving the
chamber.
|
For both species, the rate of water uptake from the reservoir was significantly lower from 50 mmol l–1 NaCl compared to DI (Fig. 3). In contrast, water uptake was significantly greater when the toads were fully immersed in 50 mmol l–1 NaCl compared to DI by toads immersed in the beakers (Fig. 3). The two species absorbed water at similar rates in any of the experimental conditions and the values obtained from the pipette were not different from weight change.
|
|
Rates of water uptake by B. alvarius immersed in 1.5 mm, 6 mm or fully immersed in DI were not significantly different from each other (Fig. 5A). In contrast, water absorption from NaCl increased significantly when the immersion level was increased from 1.5 mm to 6 mm, and a further significant increase was observed with full immersion. A similar pattern was observed with B. marinus (Fig. 5B): rates of water uptake from DI were not different among immersion levels while the rate of water uptake from NaCl increased significantly with 6 mm vs 1.5 mm immersion, and with full immersion vs 6 mm.
|
|
Effects of bladder content and dehydration on seat patch BCF
For hydrated B. alvarius with ad libitum bladder water,
BCF did not increase following water contact
(Fig. 7A, group A). In
contrast, removal of bladder water resulted in a highly significant
stimulation of BCF upon water contact (Fig.
7A, group B). Mild dehydration (mean 3.6±0.2% of SW)
resulted in a similar stimulation of BCF following water contact
(Fig. 7A, group C) that was not
different from empty bladder toads. Overnight dehydration (mean
15.0±0.44% of SW) caused a further significant increase in BCF
following water contact compared to mild dehydration
(Fig. 7A, groups C and D). The
gradual increase in BCF values on the dry surface as the level of dehydration
increased was not significant.
|
B. alvarius maintained in terraria with access to water retained amounts of bladder water ranging from less than 5% to nearly 25% of the body mass (group A in Fig. 7). A linear regression of BCF vs retained bladder volumes for the individual trials showed a significant correlation (R2=0.7403; P<0.001).
Central arterial blood flow and BCF
Typical traces of CAF and BCF are shown in
Fig. 8A for a dehydrated toad
transferred to the wet chamber. Heart rate (fH) was also
obtained from these records. The increase in BCF
(Fig. 8B) was similar to that
of dehydrated toads placed on water (Fig.
2B). In contrast, CAF was elevated after transfer and declined
from 15.2±2.0 to 9.4±1.0 ml min–1
kg–1 during the first 3 min followed by a steady rise (to
12.5±1.0 ml min–1 kg–1 at 10 min)
that was maintained throughout the following 40 min. Heart rate followed a
similar pattern.
|
The addition of water to the dehydration chamber without handling the toads elicited a steady rise in CAF (Fig. 8C) that followed a time course similar to that seen for BCF in Fig. 8B. CAF and fH were normalized as the fractional changes relative to the values recorded before water exposure. CAF increased by approximately 80% over a 10 min period and remained at this elevated level for the next 30 min. Heart rate was initially elevated but remained almost unchanged over most of the 40 min period.
|
Discussion |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
|---|
), as well as B. alvarius (present study),
had BCF in the range 3.5–4.0 V, while BCF ranged between 1.5 and 2.0 V
in B. bufo (Viborg and
Rosenkilde, 2004) and B. marinus (present study) that
inhabit more mesic environments. The lower BCF of dehydrated B.
marinus compared to B. alvarius is consistent with the much
lower vascularisation of the pelvic skin of B. marinus, and the
suggestion that differences in seat patch skin vascularization reflect
adaptations to different habitats (Roth,
1973).
Despite the much higher BCF in B. alvarius, rates of water uptake,
whether from DI or NaCl in the reservoir or during full immersion, were
similar between the two species. Furthermore, within each of the two species,
there was no correlation between BCF and rates of water uptake. These inter-
and intraspecific observations support the hypothesis
(Viborg and Hillyard, 2005)
that the increase in BCF is facultative once a favourable osmotic gradient has
been detected although internal factors, such as handling stress and bladder
reserve, are able to affect the magnitude of BCF. Nevertheless, a weak
correlation between BCF and water uptake in B. bufo was reported
(Viborg and Rosenkilde, 2004),
but this correlation was based on a rehydration period of 120 min during which
the toads were immersed in water between BCF and weight measurements and were
returning to a fully hydrated condition. This contrasts with the present study
where efforts were made to maintain a uniform dehydration state at the
beginning of each trial. As evidence that serial treatments reflect
reproducible physiological effects, BCF values obtained initially and after a
dehydration interval were not significantly different. Similarly, water
absorption by B. alvarius immersed in DI was similar when the
experiment was run before or after a dehydration interval (Figs
3 and
5).
Because the seat patch is highly vascularized
(Roth, 1973;
Christensen, 1974) and the rate
of capillary ultrafiltration of plasma proteins in the amphibian skin is very
high (Conklin, 1930), the
circulation continuously adds solutes to the interstitial fluid that maintains
an osmotic gradient favouring water gain
(Christensen, 1975;
Parsons et al., 1993). A high
rate of transcutaneous water gain also depends on the water permeability of
the skin, which may limit water movement to the cutaneous capillaries.
Recently, AQP-2 and 3 were demonstrated in the stratum granulosum of the
ventral pelvic skin of amphibians (Tanii
et al., 2002; Hasegawa et al.,
2003; Willumsen et al.,
2003). In response to AVT stimulation, both AQP-2 and 3 are
translocated to the apical membrane of the stratum granulosum
(Hasegawa et al., 2003). While
AQP-2 and AQP-3 are only present in the ventral pelvic skin, AQP-1 is
associated with the vascular system both inside and outside the seat patch
(Willumsen et al., 2003).
Aquaporins 3, 2 and 1 thus form a serial pathway from the exterior through the
skin into the capillaries. The results of the present study could be due to a
lower capillary density in B. marinus that is compensated for by a
greater expression of AQP-1 in the capillary endothelial cells or AQPs 2 and 3
in the stratum granulosum. Viborg and Rosenkilde also proposed this hypothesis
(Viborg and Rosenkilde, 2004),
noting that the increase in water absorption by B. bufo was
stimulated by AVT without an increase in BCF.
Behavioural correlations
The initiation of WR behaviour by B. alvarius consistently
occurred after maximal BCF values were attained, regardless of the fluid level
or salinity of rehydration medium in the chamber. B. marinus appeared
to behave similarly even though WR posture was adopted more gradually. The
rise in BCF appears to be a sympathetic reflex that is mediated by water
potential receptors in the skin (Viborg
and Rosenkilde, 2004). The delay in WR initiation indicates
further integration of the sensory information before a large area of skin is
committed to a rehydration source and that the lower water potential of the
NaCl solution (vide supra) did not affect the time course for WR
initiation despite the lower rehydration rate. Both B. alvarius and
B. marinus moved and resettled more often when absorbing water from
the NaCl solution. However, the number of moves decreased, significantly so in
B. marinus, when the immersion level was increased from 1.5 to 6 mm,
where the latter condition enhanced water uptake. Toads appear to be able to
evaluate not only the osmotic content of a hydration source but also the
efficacy of water absorption.
The time course for changes in BCF and the initiation of WR appear to
depend on body size. In the large toads of both species (300–400 g), BCF
reached maximal values within 3–4 min and WR was initiated well after
maximal BCF was attained. For B. woodhouseii weighing about 75 g, WR
was initiated within 1 min, before BCF was maximal, while 15–20 g B.
punctatus initiated WR within 20–30 s when BCF had already attained
a maximal value (Viborg and Hillyard,
2005). The differences as to whether WR is initiated during or
after the increase in BCF may relate to accumulation of solutes required for
water absorption, so WR behaviour is not initiated until a favourable osmotic
gradient can be sustained. The larger toads have a greater volume to surface
ratio so the additional contact area presented by WR behaviour will be less
beneficial for water absorption, compared to small toads.
BCF and water absorption from NaCl vs DI
Rehydration rates of both species from NaCl were 30–40% lower than
from DI when fluid contact was limited to the seat patch and became
significantly greater only when the immersion level was raised to 6 mm or in a
beaker. Lymph osmolality of B. marinus dehydrated by 10–15% is
approximately 260 mOsm kg–1
(Hillyard and Larsen, 2001).
Assuming ideal osmotic behaviour for NaCl, the osmotic gradient should be
reduced by a factor of 0.62 (160/260) relative to DI, which corresponds well
to the observed ratios (0.58 for B. alvarius and 0.67 for B.
marinus).
Coating the lateral skin with the oil/wax mixture abolished the rise in
water uptake upon full immersion in NaCl, but there was no effect when toads
were immersed in DI. As a control, coating the entire ventral and lateral skin
reduced rehydration from both DI and NaCl, which is consistent with numerous
studies showing that the seat patch accounts for most water absorption
(McClanahan and Baldwin, 1969;
Christensen, 1974;
Marrero and Hillyard, 1985).
The oil/wax mixture will interfere with water transfer to the lateral and
dorsal skin via epidermal sculpturing, which suggests that these
regions of the skin contribute to the greater rehydration rates from dilute
NaCl solutions. The mechanism whereby the lateral and dorsal skin might couple
salt and water absorption is not known. There was no effect of amiloride on
the stimulation of water absorption by 10 and 50 mmol l–1
NaCl (Hillyard and Larsen,
2001), but rehydration from 50 mmol l–1 sodium
gluconate was reduced, as predicted from the osmotic gradient.
Effect of stored bladder water
Arid-adapted toads appear to be more sensitive to hydration status. An
empty bladder stimulated BCF in hydrated B. alvarius (present study).
The empty bladder condition, in combination with mild dehydration (2.7% of
SW), elicited a large increase in seat patch BCF in B. punctatus
(Viborg and Hillyard, 2005).
In contrast, a greater level of dehydration (5–10%) was necessary to
elicit a pronounced increase in BCF of B. bufo
(Viborg and Rosenkilde, 2004),
and in B. marinus neither the empty bladder condition nor mild
dehydration elicited a substantial increase in seat patch BCF when the toads
were exposed to deionised water (present study). Marked differences were also
observed in the behaviour displayed by the two species in the holding
terraria. B. alvarius remained in their shelters and were never
encountered in the hydration tray during the day, while B. marinus
were usually taken from the water trough. B. marinus that remain in
water are known to have lymph osmolality that is more dilute than the plasma
(Hillyard and Larsen, 2001).
B. marinus had to be kept dry for 2 h to obtain equilibration between
lymph and plasma (Hillyard and Larsen,
2001). The surplus of water temporarily stored in the lymph spaces
of B. marinus could delay dehydration when toads are transferred to a
dry environment and thus the minimal response of BCF to mild dehydration
observed in the present study. However, overnight dehydration greatly exceeds
the small volume contained in the diluted lymph and we observed no appreciable
difference in BCF values for toads dehydrated over a range of 12–25%
dehydration.
The negative correlation between BCF and the ad libitum bladder
volume retained by B. alvarius indicates that the amount of stored
water reserve contributes to the regulation of pelvic skin BCF in this
species. The pathway for cutaneous water absorption remains controversial. It
has been proposed that water moves first to the lymphatic pathway and is then
returned to the circulation (Carter,
1979; Toews and Wentzell,
1995). More recently, Word and Hillman provide evidence that water
movement is primarily via the blood capillaries
(Word and Hillman, 2005). If
this is the case, the generally observed increase in BCF is sufficient to
transport all of the water molecules absorbed. Alternatively, the lack of
correlation between BCF and water absorption could result from variable
utilization of either pathway in response to the ensemble of sensory cues that
conscious animals receive. With an empty bladder but no osmotic stress, the
primary need is to fill the bladder, which requires water absorption by the
circulation and subsequent filtration by the kidneys.
The present study allowed us to quantify the relationship between CAF and
BCF in conscious, unrestrained animals. The high initial values seem to be
caused by handling stress, as indicated by the low CAF values recorded in
toads that remained unhandled in the dehydration chamber. When handling was
avoided CAF increased by approximately 80% over a 10 min period following
water contact. Heart rate was initially elevated by about 30% but declined as
CAF rose to stable elevated values, in contrast with the six- to sevenfold
increase in BCF following water exposure in the rehydration chamber. It
appears that the increase in BCF is not just the result of increased cardiac
output but is due to a redistribution of blood to arteries supplying the seat
patch skin, and further may involve local opening of capillaries in the skin
(Krogh, 1919). Increased CAF
and decreased BCF in response to stressful stimuli can be explained in terms
of a fight or flight response. Turning off seat patch perfusion corresponds to
a decrease in peripheral circulation, while increased CAF combined with
shunting of blood to the muscles will meet increased oxygen demands in
response to muscle activity. These observations support the hypothesis that
regulation of seat patch perfusion is mediated by the autonomic nervous
system, as suggested by Viborg and Rosenkilde
(2004).
|
List of abbreviations |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
|---|
|
Acknowledgments |
|---|
|
References |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
|---|
Andersen, J. B. and Wang, T. (2002). Effects of anaesthesia on blood gases, acid-base status and ions in the toad Bufo marinus. Comp. Biochem. Physiol. 131A,639 -646.[CrossRef]
Brekke, D. R., Hillyard, S. D. and Winokur, R. M. (1991). Behaviour associated with the water absorption response by the toad Bufo punctatus. Copeia 1991,393 -401.[CrossRef]
Carter, D. B. (1979). Structure and function of the subcutaneous lymph sacs in the anura (Amphibia). J. Herpetol. 13,321 -327.
Christensen, C. U. (1974). Adaptations in the water economy of some anuran amphibia. Comp. Biochem. Physiol. 47A,1035 -1049.[Medline]
Christensen, C. U. (1975). Correlation between net water flux, osmotic concentration of the interstitial fluid and osmotic water permeability of the isolated skin of Bufo bufo bufo. J. Comp. Physiol. 96,95 -100.
Conklin, R. E. (1930). The formation and
circulation of lymph in the frog. I. The rate of lymph production.
Am. J. Physiol. 95,79
-90.
Hasegawa, T., Tanii, H., Suzuki, M. and Tanaka, S.
(2003). Regulation of water absorption in frog skins by two
vasotocin-dependent water channel aquaporins, AQP-h2 and AQP-h3.
Endocrinology 144,4087
-4096.
Hillyard, S. D. and Larsen, E. H. (2001). Lymph osmolality and rehydration from NaCl solutions by toads, Bufo marinus.J. Comp. Physiol. B 171,283 -292.[Medline]
Hillyard, S. D., Hoff, K. S. and Propper, C. R. (1998). The water absorption response: a behavioral assay for physiological processes in terrestrial amphibians. Physiol. Zool. 71,127 -138.[Medline]
Hoff, K. and Hillyard, S. D. (1993). Inhibition of cutaneous water absorption in dehydrated toads by saralasin is associated with changes in barometric pressure. Physiol. Zool. 66, 89-98.
Jorgensen, C. B. (1991). Water economy in the life of a terrestrial anuran, the toad Bufo bufo. Biol. Skr. Dan. Vid. Selsk. 39,1 -30.
Jorgensen, C. B. (1994). Water economy in a terrestrial toad (Bufo bufo), with special reference to cutaneous drinking and urinary bladder function. Comp. Biochem. Physiol. 109A,311 -324.[CrossRef]
Krogh, A. (1919). The supply of oxygen to the tissues and the regulation of the capillary circulation. J. Physiol. 52,1919 .
Lillywhite, H. B. and Licht, P. (1974). Movement of water over toad skin: the role of epidermal sculpturing. Copeia 1974,165 -171.[CrossRef]
Maleek, R., Sullivan, P., Hoff, K., Baula, V. and Hillyard, S. D. (1999). Salt sensitivity and hydration behaviour of the toad, Bufo marinus. Physiol. Behav. 67,739 -745.[CrossRef][Medline]
Marrero, M. B. and Hillyard, S. D. (1985). Differences in c-AMP levels in epithelial cells from pelvic and pectoral regions of the toad skin. Comp. Biochem. Physiol. 82C, 69-73.[CrossRef]
McClanahan, L. L., Jr and Baldwin, R. (1969). Rate of water uptake through the integument of the desert toad, Bufo punctatus. Comp. Biochem. Physiol. 28,381 -389.[Medline]
Nagai, T., Koyama, H., Hoff, K. and Hillyard, S. D. (1999). Desert toads discriminate salt taste with chemosensory function of their ventral skin. J. Comp. Neurol. 408,125 -136.[CrossRef][Medline]
Parsons, R. H. and Schwartz, R. (1991). Role of circulation in maintaining Na+ and K+ concentration in the pelvic patch in Rana catesbeiana. Am. J. Physiol. 261,R686 -R689.[Medline]
Parsons, R. H., McDevitt, V., Aggerwal, V., LeBlang, T., Manley, K., Kim, N., Lopez, J. and Kenedy, A. (1993). Regulation of pelvic patch water flow in Bufo marinus: role of bladder volume and Ang II. Am. J. Physiol. 264,R1260 -R1265.
Roth, J. J. (1973). Vascular supply to the ventral pelvic skin of anurans as related to water balance. J. Morphol. 140,443 -460.[CrossRef]
Ruibal, R. (1962). The adaptive value of bladder water in the toad, Bufo cognatus. Physiol. Zool. 35,218 -223.
Salerud, E. G. and Nilsson, G. E. (1986).An integrating probe for tissue Laser Doppler Flowmetry . Medical Dissertation (No 216), Linköping University.
Stille, W. T. (1952). The nocturnal amphibian fauna of the Southern Lake Michigan beach. Ecology 33,149 -162.
Stille, W. T. (1958). The water absorption response of an anuran. Copeia 1958,217 -218.[CrossRef]
Sullivan, P. A., Hoff, K. and Hillyard, S. D.
(2000). Effects of anion substitution on hydration behaviour in
the red spotted toad, Bufo punctatus: is there an anion paradox in
amphibian skin? Chem. Sens.
25,167
-172.
Tanii, H., Hasegawa, T., Hirakawa, N., Suzuki, M. and Tanaka, S. (2002). Molecular and cellular characterization of a water channel protein, AQP-h3, specifically expressed in the frog ventral skin. J. Membr. Biol. 188,43 -53.[CrossRef][Medline]
Toews, D. P. and Wentzell, L. A. (1995). The role of the lymphatic system for water balance and acid base regulation in the amphibian. In Advances in Comparative and Environmental Physiology (ed. N. Heislerp), pp.201 -214. Berlin: Springer.
Tran, D., Hoff, K. V. and Hillyard, S. D. (1992). Effects of angiotensin II and bladder condition on hydration behaviour and water uptake in the toad Bufo woodhousei.Comp. Biochem. Physiol. 103A,127 -130.[Medline]
Viborg, A. L. and Rosenkilde, P. (2004). Water potential receptors in the skin regulate blood perfusion in the ventral pelvic patch of toads. Physiol. Biochem. Zool. 77, 39-49.[Medline]
Viborg, A. L. and Hillyard, S. D. (2005). Ventral skin blood flow in two species of desert toads, Bufo woodhouseii and Bufo punctatus. Physiol. Biochem. Zool. 78,394 -404.[CrossRef][Medline]
Willumsen, N. J., Amstrup, J., Nejsum, L. N., Larsen, E. H., Nielsen, S. and Hillyard, S. D. (2003). Differential localization of aquaporins 1-3 in amphibian skin. FASEB J. 17,A919 .
Word, J. M. and Hillman, S. S. (2005). Osmotically absorbed water preferentially enters the cutaneous capillaries of the pelvic patch in the toad Bufo marinus. Physiol. Biochem. Zool. 78,40 -47.[Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  S. D. Hillyard, V. Baula, W. Tuttle, N. J. Willumsen, and E. H. Larsen Behavioral and Neural Responses of Toads to Salt Solutions Correlate with Basolateral Membrane Potential of Epidermal Cells of the Skin Chem Senses, October 1, 2007; 32(8): 765 - 773. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
