The interplay of cutaneous water loss, gas exchange and blood flow in the toad, Bufo woodhousei: adaptations in a terrestrially adapted amphibian
1 Department of Biological Sciences, University of North Texas, PO Box
305189, Denton, TX 76203, USA
2 Biotechnology Laboratory, University of British Columbia, 6174 University
Boulevard, Vancouver BC V6T 1Z3, Canada
* Author for correspondence (e-mail: burggren{at}unt.edu)
Accepted 18 October 2004
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Summary |
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Cutaneous gas exchange
(O,
CO) and C18O
diffusing capacity (DSkinC18O) were then
examined in unanesthetized toads under different states of body hydration.
Blood gases and hematocrit were measured separately but under identical
conditions. In fully hydrated toads at 23-25°C, cutaneous gas exchange
values were:
O =
1.43±0.47 µmol g-1 h-1,
CO = 1.75±0.85
µmol g-1 h-1, and the respiratory exchange ratio R =
1.36±0.56 (N=6, mean + 1S.D.).
DSkinC18O was 0.48±0.03 µmol g body
mass-1 h-1 kPa. Following an enforced 20-25% loss of
body water, DSkinC18O fell by nearly 50% to
0.28±0.09 µmol g-1 h-1 kPa. However, cutaneous
O,
CO and R were unchanged at
1.48±0.15 µmol g-1 h-1, 1.72±0.29
µmol g-1 h-1 and 1.13±0.08 µmol
g-1 h-1, respectively. Partial pressure of arterial
(sciatic) oxygen, PaO2, normally about 12-13
kPa, remained unchanged by dehydration, but
PaCO2 increased about 250% from
0.93±0.27 up to 2.27±0.93 kPa. The fall in
DSkinC18O during dehydration presumably results
at least in part from decreased cutaneous blood flow, possibly in an attempt
to reduce the transcutaneous water loss that would otherwise result during
dehydrating conditions. Concurrently, cutaneous
CO is maintained under
dehyrdating conditions by a greatly increased
PaCO2 diffusion gradient across the skin. Thus,
Bufo woodhousei appears able to restrict cutaneous blood flow without
compromising vital cutaneous CO2 loss.
Key words: skin gas exchange, blood flow, dehydration
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Introduction |
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Despite the uncertain role of blood perfusion in regulating water loss
across terrestrial amphibian skin, it is equally well appreciated that
cutaneous perfusion in amphibians, particularly of the ventral patch, is under
exquisite hormonal and neural control and that skin blood flow in terrestrial
amphibians can show enormous ranges (e.g. Burggren and Moali, 1984;
West and Van Vliet, 1992;
Malvin, 1993
;
Rea and Parsons, 2001
;
Viborg and Rosenkilde, 2004
;
Viborg and Hillyard, 2004). Such changes in skin perfusion have been presumed
to regulate environmental water uptake across the skin (especially the ventral
patch) rather than limiting water loss. Yet, a recent study by Viborg and
Hillyard (2004) shows no relationship between cutaneous blood flow and water
uptake in bufonid toads, nor could these researchers find an effect on blood
flow produced by Angiotensin II (A II), in contrast with previous studies by
Parsons and Schwartz (1991
),
Parsons et al. (1993
) and
Slivkoff and Warburton
(2001
).
Investigation of transcutaneous water fluxes in toads is further
complicated by the skin's highly regional morphological and anatomical
characteristics, with the bulk of the skin surface area on the dorsal and
lateral surfaces responding quite differently than the ventral `seat patch'
(see Viborg and Hillyard, 2004). Moreover, behavioral modifications may reduce
`functional' skin surface area, especially of the ventral surface. Such
changes are certainly an important part of terrestrial amphibians' suite of
responses aimed at conserving body water (see
Hillyard et al., 1998;
Viborg and Rosenkilde,
2001
).
Against this backdrop of complex, interrelated and sometimes contradictory findings, it is perhaps of little surprise that few studies have concurrently examined both cutaneous gas exchange, and cutaneous water loss and uptake, in terrestrial toads. Consequently, we have used pharmacological tools to probe the relationships between cutaneous gas exchange, body water loss, cardiovascular performance and cutaneous perfusion in Bufo woodhousei, a terrestrial anuran that is widely distributed across North America, including the arid conditions of the Mojave Desert in southwestern United States. Specifically, we test the hypothesis that dehydration elicits physiological and/or behavioral responses that compromise cutaneous gas exchange.
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Materials and methods |
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All measurements described below were performed on undisturbed toads handled carefully to prevent bladder venting, thus allowing them voluntarily to carry an undetermined volume of bladder water according to their physiological and behavioral status.
Blood flow and water loss
Toads were anesthetized (0.5% MS 222 buffered to pH 7.6; Sigma Co., St
Louis, MO, USA) and then chronically implanted with a sciatic artery catheter,
through which blood pressure could be monitored and drugs injected following
recovery from surgery and anesthesia.
After surgery, each toad was placed on wet paper toweling in a tared
container (volume 1 l) through which humidified air was passed at a rate
of 1 l min-1. The catheter was connected to a NARCO pressure
transducer and bridge amplifier for recording of arterial blood pressure and
heart rate. After a 24 h recovery period, the paper toweling was gently
removed from the toad's container. The fully hydrated toads were then weighed
by measuring the weight of the toad in the tared container. Care was taken to
not disturb the toad during weighing, which could have resulted in bladder
venting, but bladder volume was otherwise not controlled for (i.e. `body
weight' in all measurements included an indeterminant bladder weight). Toads
were then weighed again at hourly intervals during a period of continual
exposure to a stream of dehydrating air (R.H.=0%) until each toad reached
80% of its hydrated weight (8-12 h). Dehydration to 80% of control body
mass was completely reversible without ill effect in these toads. Wet paper
towels were then placed back into the containers, allowing the dehydrated
toads to rehydrate fully. Body mass was weighed every 30 min during
rehydration, since rehydration occurred at a more rapid rate than dehydration.
Although specific behaviors were not recorded, each toad was free to move
about the chamber and adjust body posture.
On the first day a dehydration run under control conditions was conducted
for each toad. To mimic the small blood volume increase caused by drug
injections during subsequent dehydration runs, the control run was conducted
following injection of a 200 µl volume of saline injected as a sham. After
a 24 h recovery period in hydrating conditions, the experiment was repeated on
the second day using pharmacological stimulants of cutaneous blood flow. Each
toad was given an intra-arterial dose of phentolamine (5 mg kg-1;
Sigma), a powerful -blocker leading to vasodilation, and the entire
experiment repeated as for the control run. On the third day, each toad was
given an intra-arterial dose of isoproterenol (2 mg kg-1; Sigma), a
powerful cardiac ß-stimulant, and the entire experiment repeated a third
time. Both drugs were injected in a 200 µl carrier volume of saline that,
like the control saline injection, would have a negligible effect on blood
volume during the course of the experiment.
Blood pressure and heart rate was measured at hour 1, 3 and 5 h after injection of saline, phentolamine or isoproterenol. Preliminary experiments indicated that the cardiovascular effects of both drugs were in force for approximately 16-18 h, waning quickly thereafter until the return to control values within 24 h of treatment.
Cutaneous carbon dioxide elimination and carbon monoxide diffusing capacity
A face mask for each toad was constructed by the method of Glass et al.
(1978). Toads were
anesthetized (0.5% MS222) prior to making the plaster impression of their
head. Animals were allowed to recover from anesthesia for several days, during
which time they had free access to water.
Fully hydrated toads were then weighed and their customized face masks
secured in place with cyanoacrylate glue (3M, St Paul, MN, USA). Each toad was
placed into a gas-tight glass respirometer (volume 1 l). The face mask
was attached to a tube vented to atmosphere through a port in the wall of the
respirometer. This arrangement effectively separated pulmonary gas exchange
(with the air from outside the respirometer) from cutaneous gas exchange (with
the gas inside the respirometer). A 20 ml mixture of 8.7% He, 26.0%
O2, 9.3% CO, 3.7% C18O and balance N2 was
then introduced into the respirometer. We used an isotope of CO, because the
mass of CO and N2 are identical and cannot be distinguished by a
quadrupole mass spectrometer (see below). The C18O used to produce
the injected gas mixture came in the form of 28% C18O balanced with
CO, accounting for the presence of nonisotopic CO in the injected mixture.
After injection of the gas mixture into the respirometer, the gas in the
respirometer was fully mixed using alternating filling and emptying of the two
glass syringes attached to the lid. Gas was sampled from the respirometer for
30 s every 30 min and analyzed with a quadrupole mass spectrometer (Mediflex
V.G. Instruments; Beverly, MA, USA) that had a sample flow rate of 1 ml
min-1. The decrease in volume of the system due to gas sampling was
accommodated by allowing a decrease in volume of one of the syringes.
Consequently, the respirometer remained at atmospheric pressure throughout the
experiments. The respirometer volume was determined from the He dilution.
Cutaneous CO2 excretion was calculated from the rate of
accumulation of CO2 into the respirometer. Cutaneous diffusing
capacity and O2 consumption were calculated from the rate of
disappearance of C18O and O2 from the respirometer.
Measurements were made every 30 min for a total of three measurements per toad
and a mean value was computed. After 90 min, toads were removed from the
respirometer. They were immediately placed in a dry air stream to begin a
period of dehydration to 80% of hydrated weight. The toads were then returned
to their respirometer and the measurements were repeated as described above.
This technique determined the integrated gas fluxes across entire exposed
surface area, rather than fluxes across specific regions of the skin (e.g.
dorsal surface, ventral seat patch).
Blood gas determinations
A separate group of six toads were anesthetized and their sciatic artery
occlusively cannulated with PE 50 tubing filled with heparinized saline. After
a 24 h recovery period in hydrating conditions, a 300 µl blood sample was
then drawn from each animal. Arterial PaO2 and
PaCO2 was measured with a Cameron Blood Gas
Cell equipped with CO2 and O2 electrodes
(Microelectrodes; Bedford, NH, USA) whose input was displayed on a Radiometer
PHM 73 meter (Radiometer, Copenhagen, Denmark). Electrodes were calibrated
with precision gas mixtures. The hematocrit was also determined. Toads were
then dehydrated to 75% body weight and blood gas and hematocrit measurements
were repeated.
Data analysis
Cardiovascular and gas exchange data were tested for treatment effects with
an ANOVA followed by paired t-tests (each animal served as its own
control). The level of significance chosen was P<0.05. All values
are presented as means ± 1 S.D.
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Results |
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Dehydration, cutaneous blood flow and body water loss
Isoproterenol and phentolamine each produced a large and significant
increase in heart rate, with isoproterenol having the greatest effect on both
parameters (Fig. 1A). Blood
pressure was decreased slightly following -adrenergic blockade with
phentolamine, whereas an increase in blood pressure accompanied treatment with
isoproterenol (Fig. 1B). The
combined increase in heart rate, and systolic and diastolic blood pressure,
with no decrease in pulse pressure strongly suggests an increase in blood flow
from isoproterenol, as would be predicted from the known actions of this drug.
The increase in heart rate and decrease in blood pressure caused by
phentolamine is probably a consequence of peripheral vasodilation leading to
increased blood flow rather than central cardiovascular adjustments.
Importantly, the skin of the toad following either drug treatment showed the
very obvious blushing expected from cutaneous capillary recruitment,
especially in the pelvic patch area. Thus, although we were unable to measure
cutaneous blood flow directly, the observed cardiovascular effects
collectively suggest a large increase in cutaneous blood flow.
|
Control (saline-treated) toads placed in dehydrating conditions required a mean of 10.1±0.7 h to dehydrate to 80% body mass, equivalent to a dehydration rate of 1.98% of body mass h-1. Toads treated with isoproterenol and phentolamine required mean values of only 7.2±0.8 h and 7.4±0.9 h, respectively, reflecting a dehydration rate about 40% faster than in control toads (Table 1). Fig. 2 shows the time course of rehydration and dehydration in each of the conditions. Note that, compared with control toads, the time course of rehydration was also more rapid in the phentolamine-treated and, to a lesser extent, the isoproterenol-treated toads.
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Dehydration effects on gas exchange and diffusion
Following an enforced 20-25% loss of body water, dehydration was
accompanied by a 42% decrease in DSkinC18O from
0.49±0.03 µmol g body mass-1 h-1 kPa in fully
hydrated toads down to 0.28±0.09 µmol g-1 h-1
kPa in dehydrated toads (Table
2). Our hypothesis would thus predict a concomitant decrease in
cutaneous gas exchange. However, there were no significant differences
(P>0.05) from cutaneous gas exchange values measured in fully
hydrated toads, with
O2 remaining at
about 1.4 µmol g-1 h-1,
CO2 remaining at
about 1.7 µmol g-1 h-1, and R remaining between
1.1-1.4 (Table 2).
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Dehydration and blood, and blood gases
Dehydration produced a significant increase in hematocrit by nearly 25%
(Table 3), presumably due to a
decline in blood volume from plasma loss due to dehydration. The
PaCO2 was 0.97 kPa in hydrated toads, rising
significantly (P<0.01) by about 2.5 times to 2.27 kPa following
dehydration. PaO2, with mean values in the
range of 12-13 kPa, was as expected more variable than
PaCO2, but showed no significant change
(P>0.05) following hydration.
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Discussion |
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Mechanisms for altering cutaneous gas permeability
A reduction in overall cutaneous gas permeability, as indicated by a
profound decrease in skin CO-diffusing capacity, might comprise an important
physiological response to minimize evaporative water loss. Although a
prevailing view originally based on frog skin holds that blood flow to skin of
amphibians has no effect on skin water loss (e.g.
Adolph, 1931), most toads have
a highly sculptured skin that potentially presents a significantly greater
surface area than the relatively flat skin of aquatic frogs. Toads also tend
to have a smaller surface area to volume ratio. These anatomical differences
between toads and frogs raise the possibility that active control of cutaneous
perfusion might have effects on gas exchange and water loss that might not be
anticipated from earlier studies. For example, toads may be able to modify the
physiological (as opposed to anatomical) surface area of the skin, across
which not only gas exchange but also water fluxes could occur, by reduced
capillary recruitment. Amphibians facing dehydration can alter skin gas
exchange by decreasing the number of perfused skin capillaries (e.g.
Brown, 1972
;
Burggren and Moalli, 1984
;
Malvin and Hlastala, 1989
;
Slivkoff and Warburton, 2001
).
Indeed, Malvin et al. (1991
)
showed that, in Bufo woodhousei dehydrated to 75% of hydrated body
weight, blood cell flux (an index of blood flow) through the capillaries of
the pelvic patch decreased by more than 80%. At the same time, red blood cell
velocity fell by more than 75%. Although the responses are more profound in
the seat patch, similar changes specifically attributable to decreased
capillary recruitment also occur over the dorsal surface of Bufo
woodhousei, (W.W.B. and G. M. Malvin, unpublished).
Collectively, our data implicate regulated changes in cutaneous blood flow
with dehyration/hydration in toads. We conclude that a decrease in capillary
blood flow, probably combined with capillary decruitment contributes to, if
not causes, the observed decrease in DSkinC18O
during dehydration in Bufo woodhousei. Such decreases in cutaneous
blood flow reduce the `functional' or `physiological' surface area of the skin
- not only for water exchange but also for gas exchange (see
Burggren and Moalli, 1984;
Malvin, 1988
).
It is important to indicate that while a linkage between cutaneous blood
flow and cutaneous gas exchange appears likely, the link between cutaneous
blood flow and transcutaneous water flux remains ambiguous. Recent
measurements of cutaneous blood flow in Bufo alvarius and Bufo
marinus by Viborg and Hillyard (2004) reveal no significant correlation
between ventral skin blood flow and cutaneous water uptake. Viborg and
Rosenkilde (2004) have
demonstrated that, during rehydration in toads, arginine vasotocin (AVT)
stimulates an inward water flux across skin that is independent of changes in
skin blood flow. Recently Hasegawa et al.
(2003
) localized aquaporins in
the entire plasma membrane of the granular cells beneath the outermost layer
of skin in the pelvic patch of the frog Hyla japonica. These
aquaporins were upregulated in response to vasotocin, suggesting a mechanism
independent of blood flow that could account for the potent endocrine control
of water permeability at important sites for water flux in amphibians.
Clearly, additional experiments are required to examine potential antagonistic
and synergistic interactions between cutaneous blood flow and hormonally
induced changes in skin permeability.
Rehydration and cutaneous blood flow
Rehydration experiments in B. woodhousei revealed several
interesting features. Whereas dehydration involves a phase change from liquid
to gas associated with evaporation from the skin, rehydration involves osmotic
or bulk flow, so different rates of dehydration and rehydration might be
anticipated. Indeed, evident in Table
1 and Fig. 2 are
much greater rates of rehydration than dehydration for all three experimental
conditions. Toads exposed to phentolamine showed an `overshoot' during
rehydration, quickly increasing their body mass by 10% over control values.
One possible explanation is that the toads in this particular group were
somehow not fully hydrated at the start of the experiment. However, given that
all toads were treated identically, and that all toads were kept in hydrating
conditions, it is highly unlikely that the stimulation by phentolamine of
fluid uptake can be accounted for by different initial states of hydration.
Alternative mechanisms are manifold. For example, this rapid rehydration and
extra fluid accumulation could relate to an increase in skin perfusion.
Increased skin blood flow with relatively high osmolality blood created by
dehydration would maintain a steep osmotic gradient for inward water flux
across the skin, at least until full normal rehydration levels had been
reached. However, as noted earlier, Viborg and Hillyard (2004) failed to find
a relationship between ventral seat patch perfusion and water uptake. Another
possible mechanism for enhanced water uptake is that phentolamine increased
skin water permeability independent of skin blood flow changes, perhaps
through water channel insertion as discussed above.
Respiratory implications of reduced cutaneous gas permeability
A reduction in skin water permeability that accompanies a reduction in skin
permeability to gases would permit an anuran like Bufo woodhousei,
whose range extends into relatively (xeric) dry habitats, to spend more time
foraging away from water. By reducing skin water permeability and rates of
water loss, body water can be conserved and dehydration slowed when water
availability is reduced. However, reduced skin permeability also impacts
cutaneous gas exchange, a major route in amphibians for oxygen uptake and
carbon dioxide elimination (for reviews see Feder and Burggren,
1985,
1992
).
Why does carbon dioxide elimination and oxygen uptake across the skin not
decline equally precipitously in dehydrated toads? The dramatic increase in
PaCO2 after 20% dehydration provides a
substantially increased gradient for gas exchange across the skin. Since
cutaneous gas exchange is traditionally viewed as having a large diffusion
limitation (see Feder and Burggren,
1985; Piiper,
1988
), a large increase in the PCO2
gradient across the skin could easily compensate for the documented decrease
in the skin's gas diffusing capacity. Additionally, the measured increase in
blood PaCO2 during dehydration may well have
stimulated lung ventilation (see Smatresk
and Smits, 1991
; Branco et al.,
1993
; Wang et al.,
2004
). While only about 1/3 of CO2 elimination normally
occurs via the lungs in bufonids, the enhanced CO2 partial pressure
gradient across the pulmonary membranes under dehydration conditions would
probably act in concert with the enhanced PCO2
gradient across the skin, with both phenomena ensuring ongoing CO2
elimination despite reduce skin permeability to this gas.
The oxygen partial pressure gradient between arterial blood and air
covering the skin did not increase during dehydration, unlike the
CO2 gradient. This apparent inconsistency can be explained by the
fact that sciatic blood may not provide an accurate measure of cutaneous
arterial PaO2, for two reasons. First,
intracardiac and/or central vascular shunting may direct more venous blood to
the pulmocutaneous artery than to the sciatic artery (see
Burggren, 1988;
Hedrick et al., 1999
;
Gamperl et al., 1999
;
Anderson, 2003). Such shunts could easily result in a
PaO2 lower in the cutaneous artery, serving
large numbers of capillaries over major regions of the trunk, than in the
sciatic artery, which provides blood to a relatively small number of skin
capillaries on the legs (Moalli et al.,
1980
). Under this scenario, toads could maintain overall cutaneous
O2 uptake even with no change in sciatic
PaO2. A second reason why sciatic
PaO2 may not reflect the
PaO2 of blood entering the skin capillaries
stems from intermittent lung ventilation. Discontinuous patterns of air
breathing produce large (5-18 kPa) fluctuations in
PaO2 in amphibians (see
Feder and Burggren, 1992
).
This makes PaO2 a poor indicator of the
magnitude of the gradients between animal and environment. In such a situation
PaCO2 in the sciatic artery would remain a
useful indicator of overall blood CO2 levels because both
arterial-venous differences and fluctuations due to the discontinuous nature
of breathing are far smaller for CO2 than O2.
The increase in hematocrit in dehydrated toads could also play a role in
maintaining gas exchange across the skin, provided that hematocrit increases
were not so large as to compromise bulk delivery of blood by the
cardiovascular system (see Hillman et al.,
1985; Hillman,
1987
). For example, with the greatly lengthened capillary
residence times evident during dehydration
(Malvin et al., 1991
), blood
could become O2 saturated long before leaving the capillary. Under
these conditions an increased hemoglobin concentration in skin capillary blood
would facilitate additional diffusion of oxygen from air to blood by acting as
an enhanced O2 sink to lower the quantity of free oxygen molecules
dissolved in solution in plasma and thus lower capillary blood
PaO2. Increased hematocrit could also
facilitate CO2 excretion by providing more intracellular carbonic
anhydrase per unit volume of blood to cycle CO2 held in plasma
bicarbonate, although it is not clear whether carbonic anhydrase is indeed
rate limiting under any circumstances. Additionally, increased hematocrit (and
the attendant increased hemoglobin concentration) would provided greater
hemoglobin buffering of the increased [H+] that might attend the
elevated blood PaCO2 levels. Given the large
extent of water loss associated with the measured 25% increase in hematocrit
during dehydration, the most parsimonious explanation for the hematocrit
increase is a simple decrease in blood volume through plasma loss. A release
of red blood cells sequestered in the spleen might also contribute to the
elevated hematocrit, though this process is poorly understood in
amphibians.
Finally, we did not measure bladder water volume nor interfere with its retention, as one of our goals was to examine the effects of adrenergic drugs on heart rate and blood flow independent of any physiological or behavioral defenses against dehydration that could be mounted. Yet, the apparent similarity of the proportional increase in hematocrit and the proportional decrease in body water during dehydration suggests that these toads were either not using any retained bladder water to defend body tissue dehyration, or such use of bladder water use was ineffective.
Conclusions
Bufo woodhousei reduces skin gas-diffusing capacity during
dehydration. Although the mechanism is uncertain, there is a related reduction
in transcutaneous water loss that correlates with a reduction in skin blood
flow. The physiological conflict between the need to continue cutaneous gas
exchange while potentially lowering evaporative water loss through reduced
skin flow in Bufo woodhousei appears to be circumvented by
compensatory increases in gas exchange gradients and possibly fortuitous
changes in hematocrit. So long as Bufo woodhousei can tolerate the
acid-base and other implications of a considerably elevated blood and tissue
PCO2, then it can simultaneously achieve both
reduced water loss and maintained CO2 elimination across the skin
during dehydrating conditions. Such circumvention of perceived physiological
conflicts may in fact be more common than previously supposed, but requires
multi-faceted experiments that are specifically designed to test such
hypotheses.
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Acknowledgments |
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